Virtual Library

Background:
Depth of target lesion response to crizotinib has been associated with overall survival (OS) (J Clin Oncol 2016;34:abstract 2590). ALTA (NCT02094573) is an ongoing randomized phase 2 trial of brigatinib, an ALK inhibitor, in crizotinib-refractory advanced ALK+ NSCLC patients. As the ALTA primary endpoint of confirmed objective response rate (cORR), a binary outcome, might not fully capture clinical benefit, we examined the association of maximum decrease in target lesions with progression-free survival (PFS) and OS.

Method:
Patients were randomized to receive brigatinib at 90 mg qd (arm A; n=112) or 180 mg qd with a 7-day lead-in at 90 mg (arm B; n=110). Arms were pooled in this analysis. Patients with any target lesion shrinkage were sorted into 4 groups based on greatest decrease from baseline per RECIST v1.1; outcomes in these groups were compared with outcomes in patients with no shrinkage.

Result:
As of February 21, 2017, cORR in arm A/B (ITT population) was 46%/55% per investigators. 201/222 patients had ≥1 evaluable response assessment with 18.4-month median follow-up. Median age of these patients was 53 years; 57% were female. Patients with target lesion shrinkage (vs none) had numerically longer PFS (hazard ratios [95% CIs]: 0.61 [0.30–1.22], 1%–25% shrinkage; 0.47 [0.24–0.91], 26%–50%; 0.54 [0.28–1.05], 51%–75%; 0.30 [0.15–0.63], 76%–100%) and numerically higher estimated 1-year OS (Table). In a multivariable analysis, 76%–100% shrinkage (vs none) was independently associated with longer PFS/OS (hazard ratios [95% CIs]: 0.37 [0.18–0.76]/0.35 [0.14–0.89]); arm B (vs A) was independently associated with longer PFS.

Conclusion:
In this exploratory post hoc analysis, brigatinib-treated patients with target lesion shrinkage, including those without confirmed partial response, had improved PFS/OS vs patients without shrinkage. Patients with the deepest response (76%–100% shrinkage) appeared to have the longest PFS and higher estimated 1-year OS.

Best Target Lesion Shrinkage	n (%)[a]	Median PFS,[b,c] Months (95% CI)	Median OS,[b ]Months (95% CI)	1-year OS,[b ]% (95% CI)
None	18 (9)	3.7 (1.9–11.0)	8.3 (4.7–NR)	48 (22–99)
1%–25%	40 (20)	9.3 (4.0–21.2)	NR (14.5–NR)	75 (58–99)
26%–50%	60 (30)	12.8 (9.2–15.7)	NR (NR–NR)	82 (70–99)
51%–75%	44 (22)	11.1 (7.4–18.2)	27.6 (20.2–NR)	77 (62–99)
76%–100%	39 (19)	19.5 (12.6–NR)	NR (22.3–NR)	92 (78–99)
NR, not reached [a]Evaluable patients [b]Kaplan-Meier estimate [c]Per investigator

Background:
Advanced non-small-cell lung cancer patients who harbor anaplastic lymphoma kinase (ALK) rearrangement are sensitive to an ALK inhibitor (Crizotinib), but not all ALK-positive patients benefit equally from crizotinib treatment. We analyze the impact of TP53 mutations on response to crizotinib in patients with ALK rearrangement non-small cell lung cancer (NSCLC).

Method:
66 ALK rearrangement NSCLC patients receiving crizotinib were analyzed. 21 cases were detected successfully by the next generation sequencing validation FFPE before crizotinib. TP53 mutations were evaluated in 8 patients in relation to disease control rate (DCR), objective response rate (ORR), progression-free survival (PFS) and overall survival (OS).

Result:
TP53 mutations were observed in 2 (25.00%), 1 (12.50%), 1 (12.50%) and 4 (50.00%) patients in exons 5, 6, 7 and 8, respectively. The majority of patients were male (75.00%, 6/8), less than 60 years old (62.50%, 5/8) and never smokers (75.00%, 6/8). ORR and DCR for crizotinib in the entire case series were 61.90% and 71.43%, respectively. Statistically significant difference was observed in terms of PFS and OS between TP53 gene wild group and mutation group patients (P=0.038, P=0.021, respectively).

Conclusion:
TP53 mutations reduce responsiveness to crizotinib and worsen prognosis in ALK rearrangement NSCLC patients.

Background:
The c-ros oncogene 1 receptor tyrosine kinase (ROS1) rearrangement has been identified in 1%-2% of non-small cell lung cancer (NSCLC) cases, these patients would benefit from the inhibitor of anaplastic lymphoma kinase (ALK), crizotinib. But the resistance to crizotinib inevitably developed in the patients with ROS1 rearrangement NSCLC and shown a response to crizotinib initially. The mechanism of acquired resistance to crizotinib for the patients with ROS1 rearrangement NSCLC is not identified completely now.

Method:
A 66-year-old female diagnosed with adenocarcinoma, who shown EGFR wild and ALK negative detected by Polymerase Chain Reaction(PCR). According to the detection of ROS1 rearrangement by the next generation sequencing (NGS) in blood after the patient received chemotherapy twice (pemetrexed and carboplatin), the addition of bevacizumab to chemotherapy 4 times (pemetrexed, carboplatin and bevacizumab) and maintenance therapy 3 times (pemetrexed and bevacizumab), crizotinib was used. Disease progressed explosively 6 months later, although the patient shown a response to crizotinib initially. Then NGS was carried out on blood again, a novel point mutation (p.L531P)of the PIK3CA gene was detected.

Result:
This case was the second report for bypass activation conferred crizotinib resistance to the patient with ROS1 rearrangement NSCLC. And it also was the first report that confirmed mTOR signaling pathways activation would lead to acquired resistance to crizotinib in the clinical. And everolimus, the mTOR signaling pathway inhibitor, was used. However, the disease of the patient was too serious, and she still died of circulatory failure. In conclusion, progression-free survival was 5.0 months and overall survival was 16.0 months.

Conclusion:
Bypass activation is one of potential resistance mechanisms to ROS1 rearrangement NSCLC conferred crizotinib and regimen for mTOR signaling pathway inhibitor may be one of the treatment options.

Background:
The next-generation ALK inhibitor brigatinib received accelerated approval in the United States in April 2017 for the treatment of patients with metastatic ALK+ NSCLC who have progressed on or are intolerant to crizotinib. Hypertension has been identified as an adverse event of interest with brigatinib treatment based on prior clinical data; here, we report incidence, management, and outcomes of hypertension in ALTA (NCT02094573).

Method:
In ALTA, 222 patients were randomized 1:1 to receive brigatinib at 90 mg qd (arm A; n=112 randomized, n=109 treated) or 180 mg qd with a 7-day lead-in at 90 mg (arm B; n=110 randomized and treated). A medical history of hypertension was allowed, but patients with significant, uncontrolled, or active cardiovascular disease were excluded. Blood pressure (BP) was measured at screening, on days 1, 8, and 15 of the first 28-day cycle, and then every 4 weeks (starting on day 1 of cycle 2).

Result:
Median age was 50/57 years in treated patients in A/B; 22%/25% of treated patients in A/B had a history of hypertension at baseline. As of February 21, 2017, hypertension was reported as a treatment-emergent adverse event (TEAE; any grade) in 17%/27% of patients (A/B) and as a grade 3 TEAE in 6%/8%; no grade 4 hypertension was reported. Few patients had dose interruptions (1%/2%, A/B) or reductions (1%/1%) due to hypertension; no patients discontinued brigatinib due to hypertension. Among patients with hypertension, median time to onset of first hypertension TEAE was 5.8 months/2.1 months in A/B. Among patients with baseline systolic BP <120 mmHg (n=50/n=48, A/B), 20%/42% had a maximum shift to 140–159 mmHg postbaseline (6%/10%, <120 mmHg to ≥160 mmHg); among patients with baseline diastolic BP <80 mmHg (n=68/n=72, A/B), 29%/35% had a maximum shift to 90–99 mmHg postbaseline (10%/8%, <80 mmHg to ≥100 mmHg). Among patients with hypertension TEAEs (n=19/n=30, A/B), 84%/80% started a new antihypertensive medication during the study. Among patients with hypertension TEAEs and no medical history of hypertension (n=11/n=20, A/B), 73%/70% started a new antihypertensive medication during the study. Cardiovascular events in patients with hypertension TEAEs included: angina pectoris in 1 patient without a medical history of hypertension and, in patients with a medical history of hypertension, hypertensive retinopathy (n=1), intermittent claudication (n=1), and peripheral artery stenosis (n=1).

Conclusion:
Hypertension was observed frequently with brigatinib, and appeared dose-related, but was managed with antihypertensive therapy and rarely led to dose modification or discontinuation.

Background:
Brigatinib, a next-generation ALK inhibitor, has been associated with a median progression-free survival of approximately 16 months in ALTA, a randomized phase 2 trial (NCT02094573) investigating 2 brigatinib regimens in patients with crizotinib-refractory, advanced ALK+ NSCLC. This post hoc analysis explores the clinical benefit of continuing brigatinib beyond PD in ALTA.

Method:
Patients were randomized to arm A (brigatinib 90 mg qd; n=112) or B (brigatinib 180 mg qd with 7-day lead-in at 90 mg; n=110). Patients in arm A were permitted to escalate to 180 mg qd after RECIST PD. 107 patients who had PD on brigatinib, did not have PD as their best response, and survived ≥21 days post-PD were analyzed here.

Result:
As of February 21, 2017, 84 patients continued and 23 discontinued brigatinib treatment post-PD. Estimated 1-year OS after first PD and OS hazard ratios (HRs; unadjusted and adjusted) are shown in the table. Unadjusted HRs indicated significantly longer OS in patients who continued brigatinib vs those who did not (HR: 0.32 [95% CI, 0.17–0.62]), particularly those who continued at 180 mg. Patients who were more heavily pretreated, had longer duration of brigatinib therapy before PD, did not progress due to new lesions, had confirmed objective response, and had better ECOG performance status at PD were more likely to continue brigatinib post-PD. Adjusted HRs indicated numerically longer OS in patients who continued brigatinib (HR: 0.53 [95% CI, 0.26–1.08]). Number of prior regimens (2 vs ≥3), shorter time to investigator-assessed PD, PD due to new lesions only, and ECOG performance status at PD (≥2 vs 0) were independently associated with worse OS (P<0.05).

Conclusion:
Survival was better among patients who continued vs discontinued brigatinib after PD. Continuing brigatinib, especially at 180 mg, could be one of many factors associated with longer OS after PD in these patients.

Patients With PD on Brigatinib (n=107)	n	1-Year OS Post-PD,[a] % (95% CI)	Unadjusted HR (95% CI)	Adjusted HR[b] (95% CI)
Continued brigatinib	84	66(53–99)	0.32(0.17–0.62)	0.53(0.26–1.08)
At 90 mg qd (arm A without escalation)	23	62(38–99)	0.49(0.22–1.11)	0.61(0.25–1.46)
At 180 mg qd	61	68 (51–99)	0.26(0.13–0.54)	0.48(0.21–1.06)
Arm A with escalation	23	70(44–99)	0.29(0.12–0.72)	0.51(0.19–1.33)
Arm B	38	66(44–99)	0.25(0.11–0.57)	0.45(0.18–1.16)
Did not continue brigatinib	23	31(13–100)	Reference	Reference
[a]Kaplan-Meier estimate from time of first PD [b]Adjusted for duration of prior crizotinib exposure, number of prior treatment regimens (1, 2, or ≥3), time to investigator-assessed PD, PD due to new lesions only (yes/no), and ECOG performance status at PD (0, 1, or ≥2)

Background:
EML4-ALK fusion gene is a molecular subtype of non-small cell lung cancer (NSCLC), which is carcinogenic both in vitro and in vivo. Most of the EML4-ALK-positive NSCLC patients have effectively sensitivity with an ALK tyrosine kinase inhibitor (TKI), such as crizotinib. However, the treatment outcomes and duration of response are heterogeneous. EML4-ALK has several variants. The effects of ALK fusion variants on the efficacy of crizotinib is still unclear, although many scientists are committed to this work. In addition, we also unknown the effects of ALK variants allele fraction (AF) on the efficacy of crizotinib.

Method:
Among 54 patients with advanced NSCLC were treated with crizotinib as the first-line or further-line ALK-TKI between 2013 and 2017, eventually, we identified 48 patients whose the tumor samples were detected by IHC(38), FISH(2), NGS(5),PCR(1) and ARMS(2). Through retrospective analysis, we assumed the efficacy of crizotinib on the basis of the PFS according to the ALK variants and its allele fraction.

Result:
Among the 29 ALK-positive patients, the most common ALK variants was variant 1 in 13 patients (44.9%), followed by variant 3 in 7 patients (24.1%), variants 2 in 2 patients (6.9%), other variants in 7 patients (24.1%). We divided all variants into two subgroups: V1/3 and V2/others. We found 35.4% of the samples test results between the next generation sequencing (NGS) and hospital immunohistochemical were not concordence. Further analysis found that patients who did not match that PFS were shorter (p=0.036). By the NGS, we observed from the figure that the variant 2/others group, the median PFS had a longer trend than V1/3 group, although not statistically significant（p>0.05. The level of AF was no correlated with PFS (P=0.346).

Conclusion:
The above results show that next-generation sequencing (NGS) can identify ALK variants and AF, therefore, NGS can be used as a supplement to a detection method. The type of EML4-ALK fusion variants may has a certain correlation with PFS in patients who oral crizotinib treatment. Since the sample size of this study is small, we have not yielded accurate results and found only these phenomena. We believe that in the near future, most NSCLC patients can be detected by NGS detection of gene mutations, especially EML4-ALK fusion gene, and according the different of the fusion gene variant type which can be estimated the efficacy of the ALK-TKIs, to provide the basis of individualized treatment options for NSCLC patients.

Background:
The CAP/IASLC/AMP molecular testing guidelines recommend ALK testing on patients with lung adenocarcinoma, regardless of clinical characteristics. FISH is the recommended assay to detect ALK rearrangement, however other assays, such as NGS and IHC, are available. There have been limited published data to assess adherence to ALK testing guidelines using large real-world data sources. The objective of this study was to assess real-world ALK testing patterns among community practices in the United States.

Method:
The Flatiron database provides real-world clinical data collected from EHRs used by US cancer care providers. The Flatiron network comprises ~15% of US cancer patients and is geographically and demographically diverse. Patients with ≥2 visits within the Flatiron Network after Jan 1, 2011, >=18 years of age, and an stage IIIB/IV NSCLC diagnosis from 2011 through 2017 Q1 were included in this analysis. Logistic regression was used to identify patient characteristics associated with receiving ALK testing.

Result:
Of 29,903 patients identified from community-based clinics (mean age: 71.6, 52.2% male), ALK testing rates have steadily increased over time from 32.2% in 2011 to 61.0% in 2016 for all NSCLC patients, and 41.0% in 2011 to 74.0% in non-squamous patients. Patients that are younger, no history of smoking, women and living in the West region were more likely to be tested for ALK. Patients with Medicaid insurance, recurrent disease and squamous histology were less likely to be tested. The most common first assay to test for ALK was FISH (70%) followed by NGS (8%), PCR (4%) and IHC (1%). The median time from specimen receipt by lab to test result ranged from 6 days (FISH) to 11 days (NGS). Patients who had NGS testing were more likely to initiate chemotherapy prior to test result (34% of patients tested with NGS) than FISH (20%). 1235 patients had at least one FISH and another ALK test, with the percent agreement between FISH and other assays (NGS, PCR, IHC) ranging from 92% to 97%.

Conclusion:
Several patient characteristics predicted ALK testing indicating that some subgroups of patients may be under tested, according to guidelines. Consistent with guidelines, FISH was the most common assay and turnaround times from lab receipt to test result was under 2 weeks. There was a high agreement between FISH and NGS, indicating the potentially clinical utility of NGS, however NGS had also the longest turn around time and the highest proportion of patients initiating treatment prior to test results.

Background:
Based on clinical trials, ALK+ patients have been described as typically younger and never/former smokers, however patients enrolled in clinical trials may be different than those in the real-world. While the ALK positivity rate has been described as about 4% among all NSCLC patients, limited information is available on the positivity rates in patient subgroups. The objective of this study is to describe the real-world ALK positivity rates, patient characteristics and treatment patterns in ALK+ NSCLC patients.

Method:
The Flatiron database provides real-world clinical data collected from EHRs used by US cancer care providers. The Flatiron network comprises ~15% of US cancer patients and is geographically and demographically diverse. Patients with ≥2 visits within the Flatiron Network after Jan 1, 2011, ≥18 years of age, ≥1 ALK+ test result and an stage IIIB/IV NSCLC diagnosis from 2011 through 2017 Q1 were included in this analysis. Logistic regression was used to examine the association of ALK positivity and initiation of ALK inhibitor therapy based on patient characteristics. Survival model adjusting for censoring was used to estimate the time to ALK inhibitor order.

Result:
599 out of 15,551 ALK tested patients were identified to have an ALK positive test result, for a positivity rate of 3.9%. The ALK positivity rate varied by age (<65: 6.3% vs. ≥65: 2.9%), smoking status (no history of smoking: 11.6% vs. history of smoking: 2.3%), and histology (non-squamous: 4.0% vs. squamous: 1.8%). Factors associated with ALK positivity included younger age, academic practice, male, non-squamous histology, and no history of smoking. 78% of patients with ALK+ disease had evidence of an order for an ALK inhibitor after NSCLC diagnosis. The median time from test result to ALK inhibitor order was 24 days, with 42% of patients without an order for an ALK inhibitor within 90 days. Among patients with an order for an ALK inhibitor, 23% received chemotherapy prior to their ALK test result and 20% received chemotherapy after their test result but before the first order of ALK inhibitor. Patients diagnosed after 2014 and patients who received chemotherapy prior to the ALK test result were more likely to have an order for an ALK inhibitor.

Conclusion:
The ALK positivity rate and patient characteristics in this real-world NSCLC population are consistent with clinical trials, with some subgroups having higher positivity rates. ALK inhibitors were the most frequently ordered treatment, however many patients had a delayed time to ordering the ALK inhibitor.

Background:
Crizotinib is a standard of care in anaplastic lymphoma kinase(ALK)-positive advanced non-small-cell lung cancers (NSCLC).Undoubtedly,the resistance to crizotinib is a current bottleneck.Hence,it is necessary to explore the resistance mechanisms to ALK inhibitors.

Method:
From October 2010 to May 2017,225 ALK-positive NSCLC patients treated with crizotinib were reviewed at the Guangdong General Hospital in China.The status of ALK rearrangement was assessed by Lysis ALK Break Apart fluorescence in situ hybridization,reverse transcription polymerase chain reaction,or Ventana ALK immunohistochemistry.Next generation sequencing(NGS) was used to test the tissue or plasma from patients with resistance to crizotinib.Primary resistance to crizotinib occurred when Progression-free survival was less than 3 months for the patients treated with crizotinib.

Result:
Among enrolled patients,72.4%(163/225) gained secondary resistance,and 8.9%(20/225) had primary resistance.Molecular mechanisms of clinically primary resistance were shown in Figure a.The variants of ALK fusion were different between primary and secondary resistance patients.There were more variants of ALK fusion appeared in the group with primary resistance except E6-A20 and E13-A20.Among secondary resistant patients，non-EML4 partners fusion,such as DMD-ALK fusion,YWHAQ&TAF1B-ALK fusion,GALNT14-ALK fusion and SLC19A3-CCL20-ALK fusion were found,which responsed to crizotinib treatment.Acquired ALK L1196M/G1269A mutations were found in both primary and secondary resistant patients,and while ALK I1171T mutation was only found in secondary resistantpatients.Wnt signaling pathway was activated significantly after the treatment of crizotinib according to Kyoto Encyclopedia of Genes and Genomes(KEGG) and GeneOntology(GO) analyses.Moreover, AMER1 aberrance was inclined to appear in the primary resistance patients, which was significant different between the two groups in KEGG and GO analyses.Figure 1



Conclusion:
ALK mutations could exist in both primary and secondary resistance to crizotinib in ALK-rearranged NSCLC. Response to crizotinib was also observed in ALK-rearranged NSCLC patients with non-EML4 partners. NGS may facilitate precision treatment for both primary and secondary resistant patients though they have a few differences in molecular mechanisms of resistance.

Background:
Leptomeningeal metastases (LM) are more frequent in non-small cell lung cancer (NSCLC) patients with oncogenic drivers. Resistance mechanisms of LM with ALK rearrangement remained unclear due to limited access to leptomeningeal lesions.

Method:
Primary tumor, cerebrospinal fluid (CSF) and plasma in patients with suspected LM of NSCLC were tested by Next-Generation Sequencing.

Result:
In patents with ALK rearrangement, driver genes were detected in 66.7%, 50.0% and 28.6% patients of CSF cfDNA, CSF precipitates and plasma, respectively; and all of them had much higher allele fractions in CSF cfDNA than the other two media. The diagnosis criteria of LM were positive in brain MRI or CSF cytology, and driver genes were identified in CSF cfDNA of all patients with positive CSF cytology while in those CSF cytology negative all genes were negative. Resistance mutations including gatekeeper genes ALK G1202R and ALK G1269A were identified in CSF cfDNA but they were absent in their plasma. Moreover, tailor therapy based on CSF cfDNA obtained surprising outcomes, and genetic profiles of CSF cfDNA showed dynamic changes, suggesting the potential role of CSF for follow-up studies. Figure 1



Conclusion:
CSF cfDNA could reveal the driver and resistant genes of LM, and it may function as the media of liquid biopsy for LM in NSCLC with ALK rearrangement.

Background:
ALK rearranged non-small cell lung cancer (ALK+NSCLC) represents a unique sub-group of lung cancer. Multiple effective treatments have been investigated and reported with the optimal strategy to treat advanced disease evolving rapidly with new data. First, second and now third generation single agent ALK inhibitors (ALKi) achieve excellent objective response rates (ORR), superior to chemotherapy; however, drug resistance is inevitable and remains under ongoing evaluation. Further studies are underway incorporating combination treatments, particularly immunotherapy with ALKi. Overall survival data from clinical trials continues to mature, as few non-trial series have been reported. We report our overall survival (OS) experience in treating ALK+NSCLC in a real-world cohort.

Method:
All patients with advanced lung cancer and a diagnosis of ALK+ NSCLC treated until Jan 2017 in two tertiary referral centres in Sydney, Australia were pooled together for analysis. Baseline demographic, symptom, treatment and sequencing, ORR and central nervous system (CNS) ORR, survival, toxicity and cause of death data were collected. Data will be presented on updated survival via Kaplan-Meir plots with 95% confidence intervals and a swimmer plot of treatment sequencing and ORR via RECIST 1.1.

Result:
Between 18/2/2010 and 28/1/2017, 56 ALK-rearranged lung cancer patients were identified. Median age was 63 years, 41% were female; 62% never-smokers, 63% non-Asian and 66% managed on a clinical trial. At first data cut (March 31, 2017), 52% had died. Median OS in the whole cohort was 44.6 months (95%CI: 27.8-61.4mo). Two patients were not fit for active treatment; one did not receive CNS imaging. All current ALKi therapies, chemotherapy, brain directed therapy, treatment to oligo-progressive disease and combination ALKi/immunotherapy were represented. Sixty-one percent of patients received an ALKi first line with an ORR 87%; 85% of the 34 (61%) patients who received second line therapy received an ALKi, ORR 52%. Thirty-percent received at least two lines of ALKi; 44% who received only one line of ALKi remained on and are still responding at data cut-off. Median OS in the 59% of patients with CNS metastases was 44.6mo (95% CI 14.7-74.6 mo).

Conclusion:
Analysis of real world data from two ALK referral centres in Australia reveals an imposing survival, despite many patients being managed before next generation inhibitors were available in the early line setting. While CNS disease is common in ALK patients, with aggressive local therapy and evolving treatments, survival in this cohort was comparable to those without brain metastases.

Background:
In phase 1 ASCEND-1 study (NCT01283516), ceritinib 750 mg/day (fasted) demonstrated durable whole-body and intracranial responses in both anaplastic lymphoma kinase inhibitor (ALKi)-naïve and ALKi-pretreated patients with ALK-rearranged non-small cell lung cancer (NSCLC). Here, we report the efficacy and safety of ceritinib in patients who were pretreated with crizotinib only from the ASCEND-1 study.

Method:
Patients with ALK+ NSCLC who were enrolled globally received ceritinib 750 mg/day (fasted). Efficacy and safety were evaluated in a subset of patients who had received prior crizotinib only (no other prior antineoplastic therapy). Data cut-off was May 03, 2016.

Result:
Overall, 246 patients with ALK+ NSCLC (83 ALKi-naïve and 163 ALKi-pretreated) received ≥1 dose of ceritinib, of whom, 26 had received prior crizotinib only. Among the 26 crizotinib-pretreated patients, 11 (42.3%) had baseline brain metastases, of which, 7 received prior radiotherapy, 6 (23.1%) had an ECOG performance status of 0, and 24 (92.3%) patients had stage IV disease. The median time from initial diagnosis to ceritinib initiation was 10.5 months (range, 2.4-33.0). At data cut-off, the median duration of exposure (range) was 41.0 weeks (2.9-180.4). In the 26 crizotinib-pretreated patients, per investigator assessment, the overall response rate was 65.4% (95% confidence interval [CI]: 44.3, 82.8), and the disease control rate was 80.8% (95% CI: 60.6, 93.4) (Table). The most frequently reported grade 3/4 adverse events (AEs), regardless of study drug relationship, were ALT increased (30.8%), AST increased (15.4%), diarrhea (11.5%), nausea (7.7%), fatigue (7.7%), and blood alkaline phosphatase increased (7.7%). All 26 patients discontinued treatment due to disease progression (n=12), consent withdrawal (n=6), AEs (n=2), administrative problems (n=4), or death (n=2).

	Investigator Assessment N=26	Blinded Independent Review Committee Assessment N=26
Best overall response
Complete response (CR), n (%)	1 (3.8%)	1 (3.8%)
Partial response (PR), n (%)	16 (61.5%)	15 (57.7%)
Stable disease (SD), n (%)	4 (15.4%)	5 (19.2%)
Progressive disease (PD), n (%)	2 (7.7%)	1 (3.8%)
Unknown, n (%)	3 (11.5%)	4 (15.4%)
Overall response rate (ORR), % [95% CI]	65.4% [44.3-82.8]	61.5% [40.6-79.8]
Disease control rate (DCR), % [95% CI]	80.8% [60.6-93.4]	80.8% [60.6-93.4]
Median time to response*, weeks [95% CI]	6.1 [5.1-23.6]	6.4 [5.1-14.0]
Median DOR**, months [95% CI]	8.3 [4.2-11.2]	8.5 [3.0-13.6]
Median PFS**, months [95% CI]	8.5 [5.3-9.9]	8.2 [4.4-15.2]

*Median value derived from summary statistics; **Median value estimated by Kaplan-Meier method.

Conclusion:
Ceritinib demonstrated durable efficacy in crizotinib-pretreated patients with ALK-rearranged NSCLC. Safety was consistent with the overall ASCEND-1 study population.

Background:
Alectinib demonstrated superior efficacy versus chemotherapy in ALK+ NSCLC after crizotinib failure (ALUR; NCT02604342). We present PROs and safety in the ITT population and in patients with baseline CNS disease (C-ITT).

Method:
Patients (n=107) with pre-treated ALK+ NSCLC (randomised 2:1) received alectinib (600mg BID) or chemotherapy (pemetrexed 500mg/m[2] or docetaxel 75mg/m[2] q3w) until PD/death/withdrawal. Primary endpoint: investigator-assessed PFS. Secondary endpoints: safety and PROs. Symptoms, functioning, and HRQoL were reported using questionnaires: EORTC QLQ-C30; lung module QLQ-LC13; BN-20 (3 items, CNS symptoms). Pre-specified endpoints included time-to-deterioration (TTD) in lung cancer symptoms, longitudinal analyses of mean score changes from baseline, proportion of patients with clinically meaningful change (≥10-point change from baseline) during treatment.

Result:
High compliance with assessment completion (alectinib 91.7%, chemotherapy 88.6% at baseline); compliance remained ≥70% with alectinib, and decreased with chemotherapy (64.3%, Week 6; ≤70% thereafter). Deterioration of patient-reported fatigue (median TTD 2.7 vs 1.4 months) and arm/shoulder pain (median TTD 8.1 vs 1.9 months) was delayed with alectinib versus chemotherapy. Median TTD in composite symptom endpoint (cough, dyspnoea, chest-pain) was similar between arms. Alectinib patients reported improvement in lung cancer symptoms from baseline (least square [LS] mean) during treatment: fatigue (-6.2), single-item dyspnoea (-6.0), multi-item dyspnoea scale (-2.3), coughing (-4.9), chest pain (-4.2), pain in other parts (-5.3). More patients reported improvement in baseline symptoms (nausea/vomiting, diarrhoea, peripheral neuropathy) with alectinib versus chemotherapy, except constipation. More alectinib patients reported improvements in cognitive function versus chemotherapy (ITT 19% vs 3%; C-ITT 24% vs 4%); average change from baseline in cognitive function favoured alectinib (LS means difference 10.0, 95% CI 2.2–17.7). Median treatment duration: 20.1 weeks alectinib (95% CI 0.4–8.2), 6 weeks chemotherapy (95% CI 1.9–47.1). For alectinib versus chemotherapy: AEs leading to discontinuation, 5.7% vs 8.8%; dose reductions, 4.3% vs 11.8%; dose interruptions due to AEs, 18.3% vs 8.8%. AEs: 77.1% alectinib (grade 3–5, 27.1%); 85.3% chemotherapy (grade 3–5, 41.2%); one fatal AE (chemotherapy); grade ≥3 AEs: 41.2% chemotherapy versus 27.1% alectinib. TEAEs occurring in ≥10% patients: constipation (alectinib 18.6%, all grade 1–2; chemotherapy 8.8% [grade ≥3 2.9%]), nausea (alectinib 1.4%, all grade 1–2; chemotherapy 17.6% [grade ≥3 2.9%]) and fatigue (alectinib 5.7%, all grade 1–2; chemotherapy 26.5% [grade ≥3 8.8%]).

Conclusion:
Alectinib improved HRQoL, functioning, and symptom burden versus chemotherapy (except constipation). Safety of alectinib compared favourably to chemotherapy. Alectinib patients (ITT and C-ITT populations) derived benefit versus chemotherapy.

Background:
Fluorescence in situ hybridization (FISH) using tissue biopsy specimen is the gold standard for detection and confirmation of ALK rearrangement in non-small cell lung cancers (NSCLC), but it is time-consuming and labor-dependent procedure. Liquid biopsy using reverse-transcriptase polymerase chain reaction (RT-PCR) is expected to overcome these limitations, and provide an easy accessibility and frequent assessment of biomarkers. The aim of this study is to investigate the feasibility of liquid biopsy using plasma and platelets for detection of ALK rearrangement.

Method:
FISH was performed in 664 patients between January 2015 and May 2017. We retrospectively analyzed the formalin-fixed paraffin-embedded (FFPE) tissue and blood sample to detect ALK rearrangement using multiplex RT-PCR from 30 advanced NSCLC patients who had available tissue specimen and agreed with venous sampling. Total RNA were extracted from FFPE cell blocks, plasma and platelets, respectively. Echinoderm microtubule-associated protein-like 4 (EML4)-ALK, the most common translocation, fusion RNA was detected using PANAqPCR[TM] EML4-ALK fusion gene detection kit.

Result:
Twenty-eight patients were FISH positive and two were negative. In a validation data compared with FISH, RT-PCR using FFPE tissue demonstrated 57.1% sensitivity and 69.2% accuracy. Liquid biopsy (plasma or platelets-positive) had higher sensitivity (96.4%) and accuracy (93.3%). Among the specimen of liquid biopsy, platelets showed slightly higher sensitivity and accuracy than plasma (82.1 and 83.3% vs 78.6 and 76.7%). Compared with FFPE tissue using RT-PCR, liquid biopsy showed 100% sensitivity, 20.0% specificity and 69.2% accuracy. Median proportion of positive cells in FISH was higher in subgroups of liquid biopsy with positive result (Plasma, 30.0 vs 15.0%; Platelets 30.0 vs 20.0%), but it was not statistically significant (p=0.062 and 0.104). In 18 patients with crizotinib treatment, platelets-positive subgroup showed a tendency of longer duration of treatment (7.2 vs 1.5 months) and higher response rate (57.1 vs 0.0 %), but the difference was not significant (p=0.071 and 0.100). However, platelets-positive subgroup showed significantly higher disease control rate than platelets-negative subgroup when they were treated with crizotinib (85.7 vs 25.0%, p=0.044).

Conclusion:
Plasma and platelets are a valuable source for liquid biopsy using RT-PCR technique in detection of ALK rearrangement, and they could play a supplementary role in diagnosis of ALK-positive NSCLC. Furthermore, platelets, especially, may be useful for predicting the treatment outcome of crizotinib.

Background:
Crizotinib is the standard of care in NSCLC with ALK rearrangement. Recent data showed that the drug is dramatically effective in patients with ROS1 rearrangement (ROS1[+]), with promising activity also in individuals with MET exon 14 mutations (MET[Ex14]) or MET amplification (MET[FISH+]).

Method:
The METROS is an Italian multicenter prospective phase II trial designed to assess the efficacy and safety of crizotinib in ROS1[+ ]or MET[Ex1][4 ]or MET[FISH][+ ]advanced NSCLC patients who failed at least 1 standard chemotherapy regimen. The co-primary end-point was response rate (RR) in cohort A (ROS1+: centrally confirmed ROS1 rearrangement) and cohort B (MET+: centrally confirmed MET[FISH][+ ]defined as ratio MET/CEP7 >2.2 or locally confirmed MET[Ex1][4]). Eligible patients received crizotinib at the standard dose of 250 mg BID orally.

Result:
At the data cut-off of April 30[th], 2017, both cohorts completed accrual. Among 498 screened patients, 52 accounted for the intent-to-treat population (ITT) and received at least 1 dose of crizotinib. Among them, 26 resulted ROS1[+], 16 MET[FISH][+] and 10 MET[Ex1][4]. Notably, 3 MET[Ex1][4] cases had concurrent KRAS mutation and 1 had concurrent MET gene amplification. No concomitant driver event was detected in the ROS1 cohort. Cohort A included individuals with adenocarcinoma, median age of 55 years (range 29-86), predominantly female (61%) and never smokers (54%). Cohort B included older subjects (median age 68, range 39-78), predominantly male (65%), current/former smokers (77%) and with adenocarcinoma (92%). In both cohorts, the vast majority of patients (85%) presented > 2 metastatic sites and crizotinib was mainly offered as second line treatment (74%). Time from end of first line therapy to crizotinib was 4.1 and 1.6 months for cohort A and B, respectively. In ITT population RR, median progression free-survival (PFS) and overall survival (OS) were 61.5%, 17.2 months and not reached in cohort A and 26.9%, 3.1 months and 5.3 months in cohort B, respectively. For cohort B, responses were observed in both MET[FISH][+] and MET[Ex1][4] (25% and 30%, respectively), with evidence of rapid progression in patients carrying MET[Ex1][4][/KRAS]. At present, for 2 MET+ patients assessment is pending. Therapy was generally well tolerated with no unexpected adverse event.

Conclusion:
The METROS is the first prospective trial specifically conducted in ROS1+ or MET+ deregulated NSCLC. The study confirms remarkable efficacy of crizotinib in ROS1[+] NSCLC. Responses observed in the MET cohort were of short duration confirming aggressiveness of the disease and the urgent needs for innovative therapies.

Background:
Crizotinib (XALKORI®) is a small molecule ALK, ROS1, and c-MET tyrosine kinase inhibitor approved for the treatment of patients with ALK-positive or ROS1-positive metastatic NSCLC. PROFILE 1005 was a single arm phase 2 study of the safety and efficacy of crizotinib in previously treated patients with advanced NSCLC that is ALK-positive as determined by the investigational use only FISH test or on a case-by-case basis using a local FISH, IHC or RT-PCR laboratory developed test. In this study 54.1% of patients exhibited a confirmed complete or partial response to crizotinib (responders) by investigator assessment, while 9.9% had a best overall tumor response of progressive disease (progressors). The objective of this analysis was to investigate mechanisms of intrinsic resistance to crizotinib by comparing progressors with responders through a targeted cancer gene panel of next-generation sequencing (NGS).

Method:
Archival tumor tissue used to screen patients for enrollment was analyzed using the FoundationOne NGS panel (Cambridge, MA). Results of the analyses from tumor tissue positive by ALK FISH were compared for a subgroup of progressors (N=22) with a randomly selected subgroup of responders (N=25).

Result:
There was a higher proportion of patients who were ALK-negative by NGS in progressors (8 of 22; 36%) as compared to responders (3 of 25; 12%) (p=0.083), including 5 patients with oncogenic driver mutations in KRAS (G12S, Q61H, amp), EGFR (L858R) and BRAF (G469A). Among responders, 4 patients (16%) had non-EML4 ALK fusions (KIDINS220, EDC4, DTWD2, AFF2) while no such case was detected in progressors. TP53 mutations were detected in 10 progressors (45%) and 5 responders (20%) (p=0.115). Excluding NGS-negative patients, TP53 mutations were detected in 7 of 14 progressors (50%) and 3 of 22 responders (13%) (p=0.026).

Conclusion:
In the small percentage of patients with ALK-positive NSCLC with a best response of progression upon treatment with crizotinib, a higher proportion are ALK-negative by NGS, representing either a technical false-positive or an accurate FISH result reflecting a non-activating gene rearrangement that is not detected by NGS. TP53 mutations were observed at a higher frequency in progressors than in responders in patients with ALK-positive NSCLC by both FISH and NGS. Both technical and biologic factors thus may contribute to apparent intrinsic resistance in patients with ALK-positive NSCLC treated with crizotinib.

Background:
Patients with lung cancer are at increased risk for venous thromboembolism (VTE).About 8% to 15% of patients with advanced none small-cell lung cancer(NSCLC) experience a VTE in the course of their disease. However, the incidences of VTE in different molecular subtypes of NSCLC are rarely reported though they have big differentiation in clinical feature and therapeutic effect.Tissue Factor (TF) expressed in many solid tumors could bind and activate coagulation factor FVII and trigger the downstream coagulation cascade leading to thrombin generation and clot formation.

Method:
Here we extracted retrospective data from electronic medical records at Henan Cancer Hospital in China between January 2015 and January 2016. Lung adenocarcinomas with ALK-rearranged, EGFR mutation and both negative were classified. The incidence rate of VTE after diagnosed with lung adenocarcinoma was calculated and analyzed. Then we randomly selected ALK-rearranged and ALK-rearranged negative lung adenocarcinoma tissues and detected TF expression in them with imunohistochemistry.

Result:
At a median follow-up of 19 months, 5.85% (30 in 513) patients with advanced lung adenocarcinoma experienced VTE. Patients with different molecular subtypes put up different rate of VTE (P=0.0021). Among them ALK-rearranged were more likely to experience VTE (6 in 29, 20.69%). EGFR and both negative had lower rate of VTE and had no big difference between them (11 in 218, 5.05%; 13 in 266, 4.89% respectively).The expression of TF performed similar feature. TF high expression in ALK-rearranged tissues is 41.67% (10 in 24) dramatically higher than that in ALK-rearranged negative tissues (11.54%, 3 in 26, P=0.0152).

Conclusion:
The rate of VTE in ALK-rearranged advanced lung adenocarcinoma cohorts was about 4 fold higher than that in EGFR mutation and both negative patients. ALK-rearranged may promote that by increasing TF expression. The mechanism warrants further research.

Background:
MET exon14 skipping mutation has been regarded the driver mutation for MET activation, but with relatively low frequency of occurrence. MET overexpression can be a promising biomarker to predict the response to crizotinib. However, little is known about acquired resistance to treatments in tumors with de novo MET overexpression.

Method:
This prospective observational study included 33 NSCLC patients with MET IHC overexpression received crizotinib treatment From January 2013 to June 2017, 23 eligible patients evaluable for response . MET expression level were detected by immunohistochemistry (IHC) with antibody SP44, and ≥50% tumor cells with moderate to high intensity staining were defined as positive. Gene copy numbers were detected by FISH (Met probes from KREATECHTM.), and referring to Cappuzzo scoring system or MET/CEP7 ratio, ≥5 copies were positive or MET/CEP7 ratio ≥1.8 (low ≥1.8-≤2.2, Intermediate >2.2-<5 and High ≥5) was defined as MET amplification;. The status of EGFR, ALK, KRAS and ROS1 were also tested at baseline. Biopsy specimens obtained both at baseline and at the time of progression using targeted next-generation sequencing to assess for mechanisms of resistance.

Result:
Response were evaluable for 23 NSCLC patients with MET overexpression (4 female, 19 male). Fifteen of them achieved partial response (PR, 65.2%), 2 were stable disease (SD) and 6 were progressive disease (PD). All responders had high MET expression , and 12(52.2%) with FISH positive. The PFS and OS in the ITT population were 3.2 and 13.2 month respectively. Median PFS was 7.4m(95% CI,4.5-10.4) for MET IHC (100%+++) patients vs. MET IHC (50%++~100%+++) 1.9m (95% CI 0.9-2.9,P=0.053), For FISH positive patients, mPFS was 8.2 m(95% CI,5.2-11.1) m v.s. FISH negative 1.3m(95% CI,0.2-1.7,p=0.002). Two acquired resistance mechanisms were found after resistance, a 64 male patient with MET IHC 100%×3,FISH (+),crizotinib first line and the best response PR, rebiopsy after resistance showed the MET D1228N mutation by NGS, and the second patient was 50 years old male with MET IHC 100%×3,FISH (+),crizotinib first line and the best response was PR, EGFR amplification were found upon progression when rebiopsy after resistance. The patient acheived PR with subsequent treatment of cetuximab plus Taxel.

Conclusion:
Multiple mechanisms of acquired resistance to crizotinib were found in de novo MET overexpressed patients. A secondary mutation in the MET gene and EGFR amplification may be the two main mechanisms. MET overexpression could be as a biomarker for de novo MET positive NSCLC. FISH seems better in predicting efficacy for MET inhibitor.

Background:
This study describes the characteristics, treatment sequencing, and outcomes among locally advanced/metastatic crizotinib-treated ALK+ Non-small cell lung cancer (NSCLC) Hispanic patients.

Method:
From June 2014 to June 2017, a retrospective patient review was conducted among several centers from México (n=10), Costa Rica (n=4), Panamá (n=13), Colombia (n=16), Venezuela (n=10), and Argentina (n=20). Participating clinicians identified their ALK+ NSCLC patients who received crizotinib and reported their clinical characteristics, treatments, and survival using a pre-defined case report form. Kaplan-Meier analyses were used to describe overall survival (OS) and progression-free survival (PFS).

Result:
73 ALK+ NSCLC patients treated with crizotinib as a first line were included. Median age at diagnosis was 62 years (range, 34-77), 60.3% were female and histological distribution was adenocarcinoma in 93.2%, squamous cell carcinomas in 2.7%, NOS in 2.7% and adenosquamous in 1.4%. Sixty-five patients (89%) were never or former smokers, 52 (71.1%) had ≥2 sites of metastasis and 15 (20.5%) had brain metastasis at diagnosis. Median PFS to treatment with first line crizotinib was 12.3 months (95%CI 9.4-15.3) and overall response rate (ORR) was 52% (CR 6.8% and PR 45.2%). Of those who discontinued crizotinib, 26.1% had brain progression, 35.6% switched to chemotherapy, 14% switched to a different ALK inhibitor and 59% received no further therapy. After starting crizotinib, median OS was 32.5 months (95%CI 25.6-39.4), 42.6 months (95%CI 31.8-53.5) for those who received ceritinib or/and alectinib, and 23.8 months (95%CI 19.0-28.6) among those treated with second line platinum based chemotherapy (p=0.003).

Conclusion:
The ORR and PFS observed in Hispanic patients with ALK+ NSCLC treated with first-line crizotinib was similar to that previously described. Limited access to new-generation ALK inhibitors affects OS. Those patients exposed to ceritinib or alectinib demonstrated a significant improvement in OS versus those treated with second-line platinum-based chemotherapy.

Background:
Molecular analysis when managing a patient with lung cancer is important. This importance seems never stop increasing as the methods of testing are improving and our knowledge specially concerning therapeutic is expanding. In patients with non-small cell lung cancer (NSCLC) translocation anaplastic lymphoma kinase/echinoderm microtubule-associated protein-like 4 (ALK/EML4) is identified in 3-5% and those patients have their own epidemiology. Studies concerning ALK/EML4 are still few in Portugal but this is changing with new treatments, such as Crizotinib. The aim of our study was to analyse NSCLC ALK/EML4+ to find out their features and treatment responses to Crizotinib.

Method:
Retrospective study of 340 non-squamous NSCLC patients followed in our Thoracic Tumours Unit between 1 January of 2012 and 31 May of 2017. The translocation ALK/EML4 was analysed by FISH test. From ALK+ group an epidemiology characterization (age, gender, perform status and smoking habits) was made and were selected the ones treated with Crizotinib. From this last selection the adverse effects were registered and the progression free survival (PFS) was studied throw a Kaplan–Meier method.

Result:
The population included: 12% (41) patients ALK+, 68% (230) ALK- and 20% (69) inconclusive. Patients ALK+ were constituted by 51.2% women and 56% of non-smokers, with average age of 64 years old. Of this subgroup 20 patients were treated with Crizotinib: 45% as 1[st ]line, 45% as 2[nd] line, 5% as 3[rd] line and 5% as 4[th] line of treatment. The response obtained was: 10% with partial remission, 35% with stable disease, 20% with progressive disease and 35% non-evaluated; no one achieved complete remission. The median of PFS was 273 days (± 111 days). Lateral effects were registered in 70% of the patients, the main ones being: nausea (25%) and elevations of transaminases (25%). Other side effects were visual alterations (15%), bradycardia (5%) and others (25%). Because toxicity 3 patients suspended Crizotinib.

Conclusion:
Lung oncology is reaching more and more a molecular level. However, the inadequacy of the samples and other errors are limiting the tests, as here is stand out by 20% of inconclusive results. Our study revealed a prevalence of ALK+ higher than is described in literature (12%), although the epidemiology is in concordance (majority of women and non-smokers). Under Crizotinib PFS is similar as previous described data (9,1months). In sum, more is yet to be known and improved.

Background:
There is a small proportion of lung cancer patients who are ALK positive by IHC but negative by FISH, or vice versa. Outcome of this subset of patients when treated with crizotinib is not well known. This analysis was planned to study the FISH and IHC discordance in ALK rearranged NSCLC.

Method:
We retrospectively collected data from a prospectively maintained database in medical oncology department. We analyzed 257 patients who had been diagnosed with ALK rearranged NSCLC cancer. Patients who had discordant FISH and IHC findings for ALK were selected for this analysis. Their response rates, progression free survival and overall survival were calculated. Progression free survival and overall survival were estimated using Kaplan Meier method.

Result:
Out of 257 patients, 28 patients (10.9%) had discordance. 7 patients had IHC -ve status while had FISH positive status for ALK rearrangement. 21 patients had vice-versa. The response rate for crizotinib in IHC-/ FISH+ was 57.1% versus 71.4% in IHC+/FISH - group ( p-0.331). The overall median progression free survival was 8.7 months and median overall survival was not reached. There was no statistical difference in PFS and OS between the 2 groups.

Conclusion:
In around 1/10th of ALK rearranged NSCLC patients, FISH and IHC have discordant findings, however these patients also benefit from crizotinib.

Background:
Crizotinib has offered patients with non-small cell lung cancer (NSCLC) positive to ALK rearrangements a powerful therapeutical option. Despite the benefit of crizotinib, most patients develop resistance and progression with special emphasis on the central nervous system. Early identification of patients that will present brain metastases could potentially lead to additional interventions preventing relapse. The objective of this study was to identify patients who would present with future CNS relapse after initiation of crizotinib.

Method:
A random forest tree model was constructed. Data from Hispanic patients with NSCLC harboring ALK rearrangements treated with crizotinib were collected from the CLICaP database. Clinical variables including age at diagnosis, sex, smoking status, number of metastasis and location and objective response were included. Based on these parameters, progression to central nervous system was predicted.

Result:
66 patients were included in the analysis. Median age for the cohort was 55 years old (r, 33-85), 33 (59%) were women, 38 (58%) were never smokers and 29 (44%) presented disease progression during crizotinib treatment while 17 had central nervous system involvement. Median overall survival (OS) was 13.9 months (95%CI 11.6-19.3) in contrast to 8.3 months (95%CI 4.47-13.13) in terms of progression free survival (PFS) after crizotinib initiation. The best predictors for central nervous system progression were age, sex, number of metastasis, objective response to crizotinib and previous CNS involvement. With an AUC of 0.99, a sensitivity of 100% and a specificity of 88%, the model reached an overall accuracy of 97%.

Conclusion:
Central nervous system progression after crizotinib treatment can be accurately predicted. Validation for this model in larger cohorts is warranted.

Background:
ALK gene rearranged NSCLC are a rare subset of lung cancers. However, treatment with crizotinib leads to an improvement in outcomes. In this study, we have audited the outcomes of ALK-rearranged NSCLC at our institute.

Method:
This was a subset analysis of a prospective observational study. It was approved by the institutional ethics committee and was carried out in accordance with good clinical practice guidelines and declaration of Helsinki. For this analysis all Alk rearranged NSCLC patients, diagnosed at our center between November 2011- April 2017 were selected. The treatment received and the outcomes were noted. Progression-free survival and Overall survival were estimated using Kaplan-Meier method.

Result:
We had diagnosed 257 patients during this period. The median age was 50 years ( 23-77 years). There were 102 females (39.7%) and 155 males (60.3%). Only 49 patients were smokers (19.1%). The ECOG PS was 0-1 in 197 patients (76.7%), 2 in 28 patients (10.9%) and 3-4 in 32 patients (12.4%). The median number of sites of metastasis was 2 (0-7). Brain metastasis was seen in 36 patients (14.0%). The upfront treatment received was crizotinib in 168 patients (65.3%), pemetrexed -platinum doublet in 57 patients (22.2%), taxane-platinum in 4 patients (1.6%), gemcitabine-platinum in 4 patients (1.6%), others in 14 patients (5.4%). The crizotinib received was started upfront in 88 patients and after a few cycles chemotherapy in 80 patients. In these 80 patients, 53 patients were started as soon as the ALK rearrangement report was available while 27 patients were started after a few cycles once finances were available. The median PFS for crizotinib was 16.1 months versus 11.4 months in pemetrexed platinum (p-0.159) The median PFS in the patients receiving upfront crizotinib was 17.1 months versus 14.7 months in patients receiving crizotinib after the switch (p-0.399). The median overall OS was 39.9 months and it was similar between the two strategies of crizotinib(p-0.964). There were 57 patients (22.1%) who never received crizotinib. The median OS in patients who never received crizotinib was 11.0 months versus it was not reached in patients receiving crizotinib in any line (p-0.000). The 3 year OS in patients receiving crizotinib in any line was 67.0%.

Conclusion:
Crizotinib substantial improves outcomes in ALK-rearranged patients whether given upfront or post start of few cycles of chemotherapy.

Background:
Circulating cell free tumor DNA (ctDNA) from liquid biopsy is a potential source of tumor genetic material in absence of tissue biopsy for EGFR testing. NSCLC patients with detectable levels of oncogenic driver mutations in peripheral blood are known to be associated with more advanced disease and poorer prognosis. Liquid biopsy was performed on132 NSCLC patients with matched tumor tissue for genomic analysis. An EGFR mutation of one major molecular subtype in NSCLC was performed on massive parallel sequencing. The Study’s primary goals were to: (1) Derive high concordance between tissue biopsy and ctDNA for oncogenic driver mutations in Exon 19 and Exon 21 of the EGFR gene. (2) Establish and validate Liquid biopsy as a clinically useful surrogate for tissue biopsy in NSCLC whenever tissue biopsy is unavailable and (3) Treatment monitoring and detection of early recurrence.

Method:
Single gene EGFR mutation analysis was performed on the ctDNA by using ultra deep sequencing on the HiSeq platform. Then custom designed bioinformatics algorithms were used to detect somatic mutations at allele frequencies as low as 0.01%.

Result:
Overall concordance of mutation status between 132 pairs of tissue and plasma ctDNA samples for EGFR mutation status was about 97%. 34% (45/132) of the study subset was EGFR mutated on tissue typing and 31% (41/132) in ctDNA, with 100% specificity, 91.1% sensitivity. Positive predictive value was 100% and negative predictive value was 95.6% - with diagnostic accuracy of 97%. A false negative rate of 3% was observed in this study. 14 out of 132 (10%) samples which had rare Exon19 deletions and complex indels could be confidently detected by NGS methods. 6/31 patients (19%) who could not go for biopsy got the EGFR mutation testing on plasma alone, who were positive for Exon19/ Exon21 hotspot mutations could benefit from targeted therapy. An objective efficacy response rate for Gefitinib was estimated at 74%, with a disease control rate of 95.7%. Median period of follow-up was 13.9 months. Median PFS was 18.933 months (95% CI 11.168-26.198) and overall survival was 78.3%.

Conclusion:
10% of newly diagnosed NSCLC patients could get the additional benefit of targeted therapy, by using the NGS which detected recurrent novel HOTSPOT mutations. Liquid Biopsy based tests will soon be as widespread as “standard” biopsies and imaging techniques, offering invaluable diagnostic, prognostic, and predictive information and would promisingly move from research into clinical practice in lung cancer.

Background:
The past several years have seen a number of changes in standards of care for NSCLC, challenging oncologists to integrate evolving diagnostic paradigms into practice. A study was conducted using simulation-based technology to assess medical oncologists’ current performance in ordering biomarker testing and diagnosing advanced NSCLC.

Method:
A virtual patient simulation (VPS) consisting of 2 patient cases was made available online via a website dedicated to continuous professional development. The VPS platform allowed oncologists to assess the patients and choose from an extensive database of diagnostic possibilities matching the scope and depth of practice. Clinical decisions made by participants in the VPS were analyzed using a sophisticated decision engine, and instantaneous, formative clinical guidance employing the current evidence-base and expert faculty recommendations were provided. After CG, oncologists had a second chance to address errors of omission. Data were collected between 1/29/2016 and 11/29/2016.

Result:
197 oncologists fulfilled the participation criteria for completing the VPS. Assessment of their clinical decisions prior to CG revealed: · In a patient with newly diagnosed advanced NSCLC, 21% of oncologists did not order histopathology to determine subtype while 58% failed to order EGFR mutational testing Moreover, 30% ordered ROS1-translocation FISH testing and 39% PD-L1 IHC testing. Interestingly, although no approved targeted agent exists for MET amplified-, RET-translocated-, or BRAF-mutated NSCLC, 17%-28% ordered these molecular tests. · In a patient with EGFR-mutated NSCLC who had progressed on first line EGFR TKI, 40% did not order testing for T790M. Moreover, 40% ordered PD-L1 staining. Consistent with findings from the first case, between 11%-18% ordered testing for rarer mutations for which patients may qualify for a clinical trial.

Conclusion:
Histopathologic and biomarker testing are critical elements for selecting the most appropriate regimen for patients with advanced NSCLC across the continuum of care. Our analysis of current practice using a VPS platform that immerses and engages the clinician for an authentic, practical and consequence-free experience demonstrates that there is variability in biomarker testing by oncologists. In addition, our findings demonstrate a continued need to educate oncologists about prioritizing biomarker tests in order to select the most appropriate regimen for a patient with advanced NSCLC.

Background:
Cancer cachexia is a common problem found in advanced stage cases. Pathophysiology of cachexia is complicated, involving cytokines and regulator molecules such as microRNA (miRNA). MiR-206, a specific miRNA in skeletal muscle cells was thought to play important role in regulating skeletal muscle loss but have not been studied well in cachectic patients.

Method:
A cross-sectional study was performed in Dharmais Cancer Hospital, Jakarta between September and December 2015. Subjects were patients with advanced lung cancers. Cachexia was defined as body mass index less than 20 kg/m[2] calculated after treatment. MiR-206 expression was assayed using quantitative real-time polymerase chain reaction (RT-PCR), whereas miR-16 served as internal control. Blood from health subjects were also taken for comparison. The results were expressed as cycle threshold (C~T~) and fold change (FC) which was calculated using the 2[-ΔΔC]~T~ method.

Result:
Thirty-seven patients were enrolled; 27 (73.0%) were men. Patients’ mean age was 51.7+11.1 years. Most of the patients (91.9%) were in stage IV. There were 16 (43.2%) patients with cachexia after treatment. Compared to normal healthy subjects, circulating miR-206 expression level was 13.7 times higher in lung cancer patients (FC=13.699). Among cancer patients, miR-206 expression was slightly up-regulated in cachectic than non-cachectic patients (FC=1.355).

Conclusion:
Circulating miR-206 is overexpressed in advanced stage lung cancer patients. Increased circulating miR-206 in cachectic patients may reflect extensive skeletal muscle loss associated with cancer cachexia.

Background:
Programmed cell death ligand 1 (PD-L1) expression had been proposed as predictive biomarker to immune-checkpoint inhibitors. Yet treatment responses are not always consistent with this single agent in the second-line therapy of NSCLC. Whether combination of PD-L1 and clinicopathologic characters could circle out optimal beneficiaries are still unknown.

Method:
We performed a meta-analysis of randomized control trials that compared immune-checkpoint inhibitors against chemotherapy in second-line therapy. Data including smoking status, EGFR status, KRAS status and histology were extracted as subgroup analyse to estimate the potential predictor of efficacy for anti PD-1/L1.

Result:
Five clinical trials that compared immune-checkpoint inhibitors against chemotherapy for second-line therapy were included. Both PD-L1 positive (HR=0.64, 95%CI=0.56-0.73, P<0.00001) and PD-L1 negative (HR=0.88, 95%CI=0.78-1.00, P=0.05) favored anti PD-1/L1. Subgroup analyse indicated that adenocarcinoma (ADC) as well as squamous cell carcinoma (SCC) preferred anti PD-1/L1. Never smokers may not benefit from anti PD-1/L1 but current/ever smokers did (HR=0.70, 95%CI=0.63-0.79, P<0.00001). Patients with EGFR mutation could not gain benefit from anti PD-1/L1 while the EGFR wild type could (HR=0.67, 95%CI=0.60-0.76, P<0.00001). Both KRAS mutation (HR=0.60, 95%CI=0.39-0.92, P=0.02) and wild type/unknown (HR=0.81, 95%CI=0.67-0.97, P=0.02) were apt to anti PD-1/L1. Figure 1



Conclusion:
Regardless of PD-L1 status, immune-checkpoint inhibitors could achieve better efficacy than chemotherapy in second-line therapy. Current/ever smokers without EGFR mutations may benefit more from anti PD-1/L1. Combination of PD-L1 and strongly relevant clinicopathologic characters should be considered to tailor optimal patients for anti PD-1/L1.

Background:
A third generation EGFR tyrosine kinase inhibitor, osimertinib, is effective for T790M positive lung cancer patients. Although the tissue re-biopsy of primary or metastatic tumors was required to use osimertinib, cell free DNA (cfDNA) has been recently approved for detection of T790M in Japan. We have reported that cfDNA size distribution was different between lung cancer and healthy individuals using a capillary electrophoresis system, that is one peak around the size of 170 bp in healthy individuals, and two peaks, 170 bp and 5 kb in advanced lung cancer patients.　The purpose of this study is to clarify the clinical and biological characteristics of each sized cfDNA.

Method:
The plasma collected from 92 lung cancer patients, 18 benign pulmonary disease patients, 20 healthy individuals at Saga University Hospital were analyzed. cfDNA extraction was performed from 1000μl plasma by automated DNA extraction system using cellulose magnetic beads. We first compared the DNA concentrations and cfDNA size among three groups. The DNA concentrations were quantified by Quantus[®], the fluorescent measurement of dsDNA intercalated dye, and cfDNA size was analyzed by the Agilent 2100 Bioanalyzer[®]. We next separately isolated cfDNA 170 bp and 5 kb fragments, and detected the epidermal growth factor receptor (EGFR) L858R point mutation by mutation-biased PCR and quenched probe system (MBP-QP) method.

Result:
The DNA concentration was higher in lung cancer patients compared to those in benign pulmonary disease and healthy individuals. Divided by histological types, DNA concentrations were highest in small cell carcinoma, and were increased in patients with advanced stages. Especially, DNA concentrations were higher in presence of metastasis, and 5 kb fragments were significantly increased in these cases. L858R positive patients showed higher DNA concentration with more obvious difference in 5 kb fragments. L858R was detected in both cfDNA fragments, 170 bp and 5 kb, which were separately isolated, suggesting that both sized DNA fragments contain circulating tumor-derived DNA (ctDNA).

Conclusion:
cfDNA concentrations were associated with progression of lung cancers, and bimodal characteristic was observed in terms of cfDNA size. Although the 170 bp short fragments of cfDNA are well known as an apoptotic product, the origin of 5 kb long fragments is still unclear. We have continuously examined how ctDNA was released from tumor cell.

Background:
Chronic state of inflammation is an important factor in advanced cancer which is used by tumor cells for maintaining survival and growth. Hematological parameters such as neutrophil/lymphocyte ratio (NLR), thrombocyte/ lymphocyte ratio (TLR) and lymphocyte / monocyte ratio (LMR) are reliable indicators of systemic inflammation. We aimed to elucidate the effect of hematological parameters and clinical features of patients on the prognosis of advanced stage non-small cell lung cancer (NSCLC) .

Method:
We included 102 stage IV NSCLC patients who presented to the oncology clinic between 2010-2016. Pretreatment clinical parameters and NLR, TLR, and LMR were retrieved from the medical records. The cut off values, calculated with ROC analysis, for NLR, LMR , TLR were 2.5, 3 and 183 , respectively. All patients were divided into two groups according to cut off values and analyzed accordingly.

Result:
Median OS and PFS were 10 and 6 months respectively. In univariate analysis high NLR, high TLR and low LMR were found to be significantly associated with survival . Among clinical parameters having ECOG performance score 0-1 , older age (≥70 years ) single metastatic disease were prognostic. In multivariate cox regression analysis only the number of metastatic lesions and LMR were found to be independent predictors for survival.

Conclusion:
Although the interaction between tumor cells and the host immune system is a very complex process, LMR, NLR and TLR are hematological parameters that can be easily derived from total blood counts and can be used in daily clinical practice. Among these markers, we suggest that LMR holds the greatest potential as a viable prognostic factor in the setting of metastatic NSCLC.

Background:
Recognition of molecular targets, such as EGFR, ALK, ROS and MET involved in cell signaling have led to the development of new targeted therapies.Widespread use of predictive markers has generated new data on prevalence and clinicopathological correlates. However, there is a lack of comprehensive data from Indian subcontinent. This study identifies clinical, histomorphological and molecular corelates of biomarker positive NSCCl.

Method:
Archival data of NSCC patients diagnosed with stage IV diseasea was retrieved. Those who tested positive for one of the four biomarkers EGFR, ALK, MET, ROS from January 2010 to December 2016 were included. EGFR testing was done using Qiagen EGFR TherascreenRGQ PCR KIT. ALK-1 protein was tested by FDA approved immunohistochemistry.Ros-1 gene rearrangement was assessed using Dual Color Break Apart DNA probe (Zytolight). C- MET gene amplification assay was done using Zytolight labeled LSI MET DNA probe(green)and cen-7 probe(orange). Epidemiological patient profile and tumor histomorphology on small biopsies was correlated with molecular signature and assessed with treatment response and overall survival.

Result:
Of the total 1938 lung cancer patients included in our study, 347 patients were observed to exhibit positivity for either one among four molecular markers. Among 347 patients, 77.8% had EGFR mutation. Of these del 19 was commonest and observed in 55% cases, L858R in 27.6% cases,Exon 20 Insertion in 5% and G719X in 3% cases. 7% of these EGFR mutants showed dual mutation.ALK-1 protein overexpression was seen in 19.3% . ROS rearrangement was present in 0.8% . MET amplification was observed in 2%. Predominant histological type was adenocarcinoma (87.6%) with predominant solid pattern. The median overall survival for EGFR, ALK, ROS, MET were 13.44,11.5, 17.2 and 15.1 months respectively. Sex, age, histology, mutation type and performance status affected overall survival.

	EGFR	ALK	ROS	MET
	DEL 19 L858R EXON 20I G719X DUAL OTHERS(L861Q) (T790M)
CASES	151 76 14 10 19 01	67	03	07
%	55 27.6 5 3 7 0.3 19.3 0.8 2
OS	13.4 12.5 14.5 13 12.6 11 11.5 17.2 15.1

Conclusion:
Biomarker testing has improved outcome and provides new insight to cancer clinicopathological profile.

Background:
Actionable insights generated by FMI’s hybrid capture-based next-generation sequencing (NGS) comprehensive genomic profiling services are increasingly important for navigating cancer care in aNSCLC patients. FMI and other NGS platforms support treatment decisions by detecting a variety of genetic alterations implicated in oncogenesis. We describe: 1) the characteristics of aNSCLC patients receiving FMI testing and 2) the utilization patterns and timing of FMI testing in relation to treatment and other molecular tests using a real world oncology electronic health record (EHR) database.

Method:
Flatiron Health has a longitudinal, demographically and geographically diverse database containing EHR data, reflecting routine clinical practice, from over 265 cancer clinics in the US. Inclusion criteria were aNSCLC diagnosis and ≥2 clinic visits within the Flatiron network on or after January 1, 2011. Data pertaining to molecular testing was available on 5 biomarkers (EGFR, ALK, KRAS, ROS1, PDL1) and used to identify 3 mutually exclusive testing groups: FMI, other NGS and non-NGS.

Result:
As of March 31, 2017, the aNSCLC cohort included 33,473 patients. Of 1,395 patients with FMI testing, 738 (53%) also had ≥1 non-FMI test (43% EGFR, 40% ALK, 20% ROS1, 17% PDL1, 16% KRAS). In FMI-tested patients, 45% received results before starting a first line of therapy (vs. 57% of other NGS tested and 79% of non-NGS tested patients). Table 1 details patient and testing characteristics for FMI tested patients, along with first treatments received after FMI testing.

Table 1. Patient, Testing and Treatment Characteristics for Patients Receiving FMI testing
	FMI	Other NGS	Non-NGS	No tests
Patient count (%) (Total N=33,473)	1,395 (4%)	1,946 (6%)	17,002 (51%)	13,128 (39%)
Patient and Testing characteristics
Age at aNSCLC diagnosis (years) (median, [IQR])	67 [59, 73]	68 [60, 75]	69 [61, 76]
No history of smoking	335 (24%)	376 (19%)	2,731 (16%)
Squamous cell histology	194 (14%)	195 (10%)	1,532 (9%)
No. of tests
1	1,224 (88%)	1,587 (82%)	n/a
≥2	171 (12%)	359 (18%)
Year of testing[a]
<2011	0 (0%)	1 (0%)	137 (1%)
2011	0 (0%)	5 (0%)	1,215 (7%)
2012	2 (0%)	16 (1%)	2,368 (14%)
2013	59 (4%)	117 (6%)	2,894 (17%)
2014	185 (13%)	235 (12%)	3,263 (19%)
2015	377 (27%)	560 (29%)	3,099 (18%)
2016	634 (45%)	818 (42%)	2,921 (17%)
2017 (through March 31, 2017)	123 (9%)	186 (10%)	670 (4%)
Number and Timing of Other Tests in patients with an FMI test	FMI tested prior to start of 1st LoT	FMI tested during 1st and before start of 2nd LoT	FMI tested during 2nd and before start 3[rd] LoT	FMI tested during or after 3rd LoT
Patient count (%) (Total N=1,386)[b]	626 (45%)	489 (35%)	157 (11%)	114 (8%)
Other tests conducted before FMI testing[c,d]
ALK	111 (18%)	188 (38%)	100 (64%)	86 (75%)
EGFR	133 (21%)	204 (42%)	110 (70%)	86 (75%)
KRAS	51 (8%)	60 (12%)	25 (16%)	29 (25%)
PDL1	41 (7%)	44 (9%)	23 (15%)	22 (19%)
ROS1	53 (9%)	92 (19%)	40 (26%)	32 (28%)
Other tests conducted after FMI testing[c,d]
ALK	48 (8%)	34 (7%)	8 (5%)	3 (3%)
EGFR	53 (9%)	38 (8%)	9 (6%)	5 (4%)
KRAS	35 (6%)	30 (6%)	7 (5%)	2 (2%)
PDL1	49 (8%)	33 (7%)	7 (5%)	3 (3%)
ROS1	37 (6%)	28 (6%)	6 (4%)	1 (1%)
First Treatment immediately following FMI testing
Patient count (%) (Total N=802)[e]	424 (68%)	252 (52%)	77 (49%)	49 (43%)
NCCN-recommended targeted treatment for aNSCLC (N=196)[f,i]	105 (25%)	56 (22%)	20 (26%)	15 (31%)
NCCN-recommended immunotherapy (N=227)[g,i]	82 (19%)	110 (44%)	22 (29%)	13 (27%)
Non NCCN-recommended targeted treatment for aNSCLC (N=13)[h,i]	1 (0%)	7 (3%)	1 (1%)	4 (8%)
[a] If a patient had more than 1 FMI test, the year of the first FMI test is shown; similarly if a patient had more than 1 other NGS (ie. non-FMI), the first test is shown. If an FMI tested patient had both an FMI and non-FMI NGS test, the year of the first FMI test is shown; similarly if a other NGS tested patient has both an other NGS and non-NGS test, the year of the first other NGS test is shown. [b] 9 patients where the date of FMI test (ie. test result date or sample collection date) in relation to dates of treatment and line of therapy were missing or unknown are not included in this table. [c] Total of 738 patients (53% of all FMI tested patients) also received a non-FMI test in addition to their FMI test. [d] Percentages may not add up to 100% because of patients received more than one test. [e] Based on 802 total patients who received FMI testing and where treatment data was reported following their FMI test. [f ]NCCN-recommended targeted treatments received included the following: erlotinib (N=63), gefitinib (N=10), afatinib (N=56), crizotinib (N=36), ceritinib (N=4), alectinib (N=8), trastuzumab (N=1), vemurafenib (N=2), dabrafenib (N=2), osimertinib (N=19), cabozantinib (N=0); it may be possible for patients to receive regimens containing more than 1 NCCN-recommended targeted treatment. [g] NCCN-recommended immunotherapy (immune checkpoint inhibitors) received included the following: nivolumab (N=189), pembrolizumab (N=29), atezolizumab (N=9); it may be possible for patients to receive regimens containing more than 1 NCCN-recommended immunotherapy. In addition, 2 patients received ipilimumab, an immune checkpoint inhibitor currently not recommended by NCCN for aNSCLC. [h] Non NCCN-recommended targeted treatments received included the following: olaparib (N=2), necitumumab (N=2), cetuximab (N=2), palbociclib (N=1), pazopanib (N=1), temsirolimus (N=1), trametinib (with no dabrafenib) (N=4) ; it may be possible for patients to receive regimens containing more than 1 non NCCN-recommended targeted treatment. [i] Based on the National Comprehensive Cancer Network (NCCN) guidelines for NSCLC, version 6.2017 LoT: line of therapy as recorded in the Flatiron data, IQR: inter-quartile range. Patients with missing data are excluded from the table; Percentages are rounded to closest decimals.

Conclusion:
Patients with FMI testing tended to be younger, non-smokers, and have squamous histology compared to patients receiving non-FMI tests. Nearly 50% of all FMI testing occurred prior to first treatment. Patients receiving FMI testing earlier were less likely to have a non-FMI biomarker test beforehand. Regardless of when FMI testing occurred, ~20-30% of patients received a NCCN-recommended targeted therapy immediately after the FMI test.

Background:
All patients with advanced stage NSCLC should have their EGFR and ALK mutation status known prior to initiation of first line therapy. Multiple plasma-based technologies such as ARMS and ddPCR are available for rapid detection of EGFR mutation, while only the more laborious Next Generation Sequencing (NGS) may cover EGFR, ALK and other uncommon mutations in a single blood test. cSMART is a novel NGS-based technology with rapid turnaround time that can detect EGFR, ALK and KRAS mutations plus others less common lung cancer specific driver oncogenes (BRAF, ROS-1, HER-2, PIK3CA, RET, MET14skipping).

Method:
Objectives of this study is to investigate the clinical application of cSMART on patients with advanced NSCLC. In cSMART assay, each cfDNA single allelic molecule is uniquely barcoded and universally amplified to make duplications. The amplified products are circularized and re-amplified with target-specific back-to-back primers. These DNA are then ligated with sequencing adapters and pair-end sequenced (>40,000x) with illumine sequencers. The original cfDNA molecules are reconstituted by multi-step bioinformatics pipeline for censor and correction. The final products are quantified for calculation of allele frequencies

Result:
Out of the 1664 samples tested, total of 1469 were of advanced stage NSCLC. We detected EGFR mutations in 758 (51.6%), ALK translocation in 34 (2.3%) and KRAS mutation in 78 (5.8%) patients. Among the patients with activating EGFR mutations, 301(39.7%) have exon 19 deletion and 279 (36.8%) have exon 21 point-mutation. Total of 6 (0.8%) patients with EGFR mutation have concurrent presence of ALK translocation. Incidence and mean allele frequency of the less common target mutation is summarized in Table. Median sample turnaround time is 7 days.

Incidence (%) Median Mutation Allele frequency (%) BRAF 29 (1.97%) 0.08% ROS1 2 (0.14%) 0.77% HER-2 19 (1.29%) 0.20% PIK3CA 70 (4.77%) 0.17% RET 14 (0.95%) 0.57% MET14skipping 63 (4.29%) 0.08%

Conclusion:
cSMART is a novel plasma cfDNA-based technology that can detect the actionable target oncogenes for patients with advanced NSCLC. This is a sensitive method with capacity of detecting the uncommon targets at relatively low allele frequency.

Background:
For patients with NSCLC, the risk of thromboembolism (TE) is high but heterogeneous. We aimed to profile biomarkers among NSCLC patients receiving anticancer therapy to identify patients and time periods of high TE risk where intervention with proven preventative strategies is likely to achieve maximal benefit.

Method:
We assessed the association between baseline biomarker levels and longitudinal biomarker changes, with subsequent incidence of TE (venous (VTE) or arterial (ATE)), disease progression and overall survival. Biomarkers (thromboelastography, d-dimer, fibrinogen, haemoglobin, white cell count, platelet count, neutrophil/lymphocyte ratio (NLR), and platelet/lymphocyte ratio (PLR)) were sequentially assessed at commencement of anticancer therapy (baseline), weeks 1, 4 and 12, and 3-monthly until 12 months.

Result:
During study follow-up (median 22 months, range 6-31), 129 patients were sequentially assessed over median 5 time points (range 1-9). Patients underwent surgery (n=12), chemo-radiotherapy (CRT, n=47), palliative chemotherapy (CHT, n=36), and single modality radiotherapy (RT, n=34) – only surgical patients received thromboprophylaxis. 24 patients (19%) had documented TE, 19 (15%) VTE and 5 (4%) ATE; 79% occurred within the first 6 months with median time to TE 48 days (range 1-151). Among ambulatory patients (CHT/CRT/RT), an initial model identified as high TE risk those patients with baseline fibrinogen ≥4g/L and d-dimer ≥0.5mg/L, or d-dimer ≥1.5mg/L. Hazard ratio (HR) for TE was 8.0 (p=0.04) for high versus low risk CHT/CRT patients and 6.5 (p=0.07) for high versus low risk for CHT/CRT/RT patients. Comparatively, using an established risk score, HR for TE with Khorana score ≥3 vs. <3 was 1.3 (p=0.68) for CHT/CRT and 1.1 (p=0.91) for CHT/CRT/RT. Considering temporal changes (d-dimer ≥1.5mg/L at week 4), risk assessment was enhanced with 100% sensitivity and 34% specificity for CHT/CRT. Specificity reduced to 27% when including RT patients. NLR, PLR and platelet count were not associated with TE. High TE risk patients (Fib≥4 + d-dimer ≥0.5, d-dimer ≥1.5) also had increased risk of cancer progression (HR 2.3, p<0.01) and mortality (HR 2.5, p<0.01). Baseline NLR≥2.5 and platelet count ≥350 were associated with progression (HR 2.0, p=0.01 and HR 2.0, p<0.01 respectively) and mortality (HR 2.6, p=0.01 and HR 1.9, p=0.01 respectively); PLR was not.

Conclusion:
For ambulatory patients with NSCLC, d-dimer and fibrinogen were associated with TE, cancer progression, and prognosis and could easily be applied in a simple algorithm, for real-time decision-making. In spite of low specificity, consideration of thromboprophylaxis is warranted for high risk patients given substantial TE-associated adverse clinical and economic consequences.

Background:
Tumor heterogeneity in central nervous system (CNS) metastases may lead to poor response to treatment in some patients with non-small-cell lung cancer (NSCLC), and genotyping of cell-free DNA (cfDNA) from cerebrospinal fluid is a promising method to reveal CNS metastasis specific gene alterations.

Method:
We conducted deep-sequencing on a 168-gene panel in cfDNA from matched cerebrospinal fluid (CSF) and plasma among 32 advanced or progressive NSCLC patients with indicated CNS metastases.

Result:
We detected single nucleotide variants (SNV) in 68.8% (22/32) of CSF samples and 77.4% (24/31) of plasma samples. The SNV detection rate was 100% in cytology positive CSF samples (11/11) or samples with cfDNA above 20ng (8/8), while it dropped to 53.3% (11/21) in cytology negative CSF samples and 58.3% (14/24) in samples with cfDNA below 20ng. Enriched copy number variations were detected in 10 patients. We also found CNS metastases specific driver gene alterations in 10 patients, including gene mutations (EGFR, TP53, RB1) or amplifications (MET, ERBB2). Among them, 3 patients carried common gene alterations with plasma. In two patients with only intracranial progression after targeted therapy or chemotherapy, driver gene mutations were detected in CSF samples but not in paired plasma.

Conclusion:
CSF profiling could successfully reflect genetic alterations for the CNS metastatic sites for both cytology positive or negative patients. Copy number amplifications and mutations on TP53 and RB1 are unique events identified in CSF. NGS on CSF and plasma ctDNA reveals tumor heterogeneity in NSCLC patients with CNS metastasis.

Background:
The development of targeted therapies has revolutionized the treatment of non-small cell lung cancer. Interrogating the status of driver mutations has become routine practice. In this study, we applied next-generation sequencing to investigate the association between molecular signature and clinical benefit.

Method:
We performed capture-based targeted ultra-deep sequencing on 204 samples obtained from NSCLC patients at a single center, including 93 FFPE, 70 fresh tissue and 41 plasma samples. One hundred and twenty two samples were subjected to a panel consisting of 8 driver genes; 50 samples were subjected to a 56-gene panel. The remaining 32 samples were subject to a 168 gene panel.

Result:
In 159 TKI-naïve patients, driver mutation was identified in 95.2% of (79/83) patients using the 8-gene panel; among them, 65.1% (54/83) carried EGFR mutations. Larger panels identified mutations in 68.1% of patients; 21% carried mutations other than driver mutations. Treatment-naïve patients were primarily subject to the 8-gene panel; in contrast, patients progressed on chemotherapy were subject to larger panels. Seventy-two percent of patients (80/111) undergone matched targeted therapy (MTT) according to sequencing results had a significantly longer PFS than 29 patients who chose chemotherapies despite the fact of harboring driver (p=4.58x10[-4] HR=0.342, 95% CI: 0.158, 0.74). Next, we investigated whether the number of EGFR mutations a patient carries and the presence of concurrence EGFR amplification have an effect on PFS. Our data revealed that both parameters are not associated with PFS. Among 46 patients receiving chemotherapy, patients with KRAS mutations were associated with a shorter PFS, 133 days vs 207 days (p= 0.073, HR=2.06 95% CI; 0.791, 5.36). For TKI-naïve patients, primary tumor tissue was collected from 86 patients and tumor tissue from metastatic lymph nodes was collected from 35 patients. Interestingly, we observed that lymph node samples had a higher maximum mutation allelic fraction (MAF) than primary lung tumor samples in patients with distance metastasis, especially with visceral metastasis (p=0.0986); such trend was not observed in patients without distant metastasis. We also analyzed samples obtained after TKI-treatment. Among 36 TKI-treated patients, patients with visceral metastasis were more likely to harbor TP53 mutations (p=0.04), which were primarily missense mutations not loss of function mutations, primarily seen in tumorigenesis. TP53 missense mutations can potentially promote distant visceral metastasis after the development of resistance to TKIs.

Conclusion:
Our study highlighted the utility of sequencing-based screening technologies and characteristics in providing treatment guidance.

Background:
For metastatic non-small cell lung cancer (NSCLC), guidelines include molecular testing for actionable biomarkers and recommend broad profile testing. Yet previous studies indicate that not all patients with NSCLC are receiving testing, even for actionable mutations in EGFR, ALK, and ROS. We hypothesized that rates of molecular testing would be low for patients calling a community HelpLine and that we could potentially increase testing rates with one-on-one caller education and providing free precision medicine services.

Method:
Caller statistics were collected on the toll-free Lung Cancer Alliance (LCA) HelpLine from Sept 1, 2016 – May 31, 2017. Recruitment to the LungMATCH molecular testing program began Nov 10, 2017. Patients are recruited through conversations on the LCA HelpLine, then entered into Perthera Cancer Analysis (PCA) through consent into an IRB-approved registry protocol. PCA includes tissue acquisition, multi-omic molecular profiling, and medical review of testing results and clinical and treatment history. PCA reports are returned to both treating physicians and patients. Data is being collected longitudinally on treatment decisions, patient outcomes including progression-free and overall survival, and patient experience.

Result:
Data from the LCA Helpline identified a gap in molecular testing. 44% (100/228) of patients who were asked if they received any kind of molecular testing replied "No". Of 46 patients who were tested and knew the results, patients indicated changes in EGFR (25), PD-L1 (10), ALK (5), KRAS (4), MET(2), BRAF, and RET. Most of these alterations are potentially actionable. From Nov 10, 2016 – May 31, 2016, 63 interested patients were referred for PCA. Sixteen patients consented and eight more are currently in the consent process. Reasons for non-consent include: doctor refusal, initiation of testing at the treating institution, concern about financial implications, and seven deaths. Ten patients are actively undergoing PCA and six have received completed PCA reports. Of those six, three patients reported that treatment decisions were made using the molecular testing information. Updated results will be presented.

Conclusion:
Caller data indicate that patients with lung cancer are not receiving molecular testing in accordance to guidelines. To address this problem, we introduced a program through a nonprofit-corporate partnership that navigates patients and their physicians through a comprehensive precision therapy program. This type of program is feasible and there is patient interest.

Background:
Therapeutic selection has been shown to lead to marked clonal evolution, thus revealing limitations in imaging scan as a monitoring method, which does not reflect biological processes at a molecular level. However, currently, response assessment of patients with non-small cell lung cancer (NSCLC) primarily relies on imaging scans, necessitating the development of methodologies for dynamic monitoring of treatment response. We evaluated ctDNA as a tumor clonal response biomarker.

Method:
We screened 831 advanced NSCLC patients with a mixture of prior treatment exposure by performing capture-based ultra-deep targeted sequencing on plasma samples using a panel consisting of 168 NSCLC-related genes. Eighty-six patients with driver mutations and a minimum of 2 evaluation points in addition to baseline were included for further analysis.

Result:
At baseline, 79.9% patients harbored at least one mutation from this panel; the remaining 20.1% had no mutation detected. Sixty-nine percent of patients (570/831) harbored driver mutation. Patients harboring 2 mutations or fewer at baseline had a median progression-free survival (PFS) of 7.4 months; in contrast, patients harboring more than 2 mutations had a median PFS of 3.8 months (P=6.6x10[-5 ]HR=0.34), suggesting a significant inverse correlation between number of mutations at baseline and PFS. Next, we evaluated the ability of ctDNA as a tumor clonal response biomarker in 86 patients with a minimum of 2 follow-ups. After a median follow-up of 314 days, 64 patients (74.4%) reached disease progression. During treatment, 46 patients, treated with either matched targeted therapy (MTT) or chemotherapy, had a minimum of one time of ctDNA clearance, occurring from 1.6 months to 7.5 months after the commencement of treatment, with a median PFS of 8.07 months, an overall response rate (ORR) of 41% and a disease control rate (DCR) of 93%. Median overall survival (OS) for this group has not reached. In contrast, 40 patients who had consistent detectable ctDNA throughout the course of treatment had a median PFS of 3.47months, a median OS of 425 days, an ORR of 20% and a DCR of 53%. Our data revealed that patients with a minimum of one time ctDNA clearance are associated with a better ORR (p=0.05), DCR (p=5.9x10[-5]), a longer PFS (p=5.4x10[-10 ]HR=0.21) and OS (p=2.3x10[-5 ]HR=0.21), regardless the type of treatment commenced and the time of evaluation.

Conclusion:
This real world study comprising a heterogeneous population reveals the predictive and prognostic value of ctDNA and warrants further investigations to explore its clearance as a surrogate endpoint of efficacy.

Background:
Metastasis was found in most of lung cancer patients resulting in death. Patient-derived xenograft model using tumor tissues and circulating tumor cells provides the opportunity to assess the biological features of cancer repeatedly during the cancer evolution, enabling clinicians to react quickly to treat the patient with the most suitable specific targeted therapy.

Method:
For setting up ex vivo culturing CTCs and establishment of patient-derived CTC xenograft model obtained from liquid biopsy of lung cancer patient, we plan to collect liquid biopsy (n>100) samples from each patient either once or several times before and after treatment. This study will be approved by our institutional review board and local ethics committee. All patient agree their informed consent for inclusion in this study. After collecting mononuclear cells, CTCs is isolated. Patient-derived CTC xenograft model will established using ex vivo cultured CTCs. Using both ex vivo cultured CTCs and patient-derived CTC xenograft model, various target anti-cancer drugs will be screened. And we then examined genetic variations and expression profile of CTC cells.

Result:
Based on these results, we identify the drugable target somatic mutations and characterizing the biologic behaviors of CTCs cells based on the results of consequence network via activation of somatic mutation. In case of lung cancer cells expressing EGFR mutation at diagnosis, we found that EGFR mutation was existed during chemotherapy. At the stage of metastasis, clonal evolution of lung cancer cells having EGFR mutation was detected. After examining the cytotoxicity of CTC cells by treatment of 3[rd] generation anti-EGRF inhibitor (3 different types) in vitro and in vivo model, we found a set of somatic mutations were we identified a set of somatic mutations associated with relapse and chemoresistance. These somatic mutations were involved in impairing DNA repair and activating EGFR-mediated cell signaling. Since effective anti-cancer drugs could be selected through screening of target anti-cancer drugs in PDX model following by identifying somatic mutation and expression profiles of CTC lung cancer cells, we suggest that our screening biosystem is useful to maintain the status of complete remission.

Conclusion:
CTC analysis can support the delivery of precision anticancer treatments, as specific biomarkers of response to targeted drugs can be appraised. Assuming that CTC induces the recurrence and metastasis, isolated CTC isolated from blood of lung cancer patients may have characteristics of cancer stem cells. Subsequently, CTC could be ex vivo cultured and apply to xenograft model to confirm genetic signatures of CTC.

Background:
Next-generation sequencing (NGS) enables a comprehensive genomic analysis of lung cancer patients. It has uncovered many novel genetic abnormalities and identified actionable genomic alterations in lung tumors that previously tested "negative" by conventional non-NGS tests. In this study, we evaluated the clinical impact of NGS, performed at different stages of the oncologic management, on overall survival of advanced lung cancer patients.

Method:
In this retrospective study, 178 consecutive non-small cell lung cancer (NSCLC) patients who performed hybrid capturing NGS were enrolled at the Thoracic Cancer Unit at Rabin Medical Center, Israel, between 2011-2017. Hybrid capture-based NGS was performed by Foundation Medicine and Gaurdant 360[TM] if tissue was not available.

Result:
178 consecutive NSCLC patients were included in this study. Median age at diagnosis was 63±12.1 years. 83% had adenocarcinoma. NGS was performed upfront in 45.5% (81/178) and after 1[st] line failure in 54.5% (97/178). Treatment decision was taken toward targeted therapy subsequent to NGS analysis in 34% (61/178) of patients (29 and 32 respectively) with an objective response rate of 54%. Overall survival (OS) was evaluated for 51% (31/61) with a median of 12.2±14.1 months. For patients who performed upfront NGS, OS ranged between 1.8 to 32.5 months, with a median OS of 13.8 months. For patients who performed NGS on progression, OS ranged between 1.7 to 77.1, with a median OS of 12.7 months.

Conclusion:
Comprehensive tissue and liquid-based NGS have revealed targeted treatment options for one third of the patients. Overall Survival of patients treated with tailored therapy was positively impacted by earlier performed NGS.

Background:
The failure rate of tissue-based NGS in newly diagnosed NSCLCs is approximately 20-25 %, reaching 40 % in the case of tumor biopsy samples collected at disease progression. In this study, we are analyzing the clinical utility of plasma-based NGS using cell-free circulating tumor DNA (ctDNA) for advanced-stage lung adenocarcinoma patients, as a complement or alternative to tissue-based molecular profiling.

Method:
Eight Academic Spanish Institutions are participating in patient recruitment. We are stratifying patients in three cohorts: 1) Patients with advanced-stage lung adenocarcinomas with insufficient tumor tissue for EGFR, ALK or ROS1 analysis; 2) Patients with EGFR, ALK or ROS1 altered tumors with acquired TKI resistance; 3) Patients with EGFR T790M positive cancers progressing on third generation EGFR TKIs. Next-generation ctDNA sequencing is being performed using GUARDANT360 73-gene panel at a CLIA certified central laboratory facility (Redwood City, California). We are stratifying gene variant actionability into four levels according to the OncoKB website criteria.

Result:
We have currently included 97 patients (January-June 2017). Complete clinical and molecular data are available at present for the first 37 patients. Twelve, 19 and 6 patients have been enrolled in cohorts 1, 2 and 3 respectively. Never smoker patients were overrepresented (n = 21, 56 %), predominantly at cohorts 2 and 3. A total of 30 cases (81 %) had detectable ctDNA. We have detected potentially actionable genetic alterations involved in mitogenic pathways in 16 patients (43 %). Level 1 alterations (variants with matched approved drugs) were found in three patients’ tumors (25 %) from cohort 1 (two EGFR sensitizing mutations and one ROS1 rearrangement). Nine patients (36 %) from cohort 2 and 3 had tumors with potentially targetable acquired genetic alterations, including three cases with EGFR T790M mutations and one case with a ROS1 kinase domain mutation. Six patients (16 %) received matched targeted therapies, four (11 %) in genotype-driven clinical trials. Reasons for not receiving matched targeted therapies in patients with actionable tumors were clinical deterioration or death (n = 2), unavailability of matched clinical trials (n = 6), treatment with non-genotype-tailored therapies (n =1) or no disease progression to ongoing therapies (n =1). Final clinical and molecular data of the whole cohort will be provided at the meeting.

Conclusion:
On the basis of our preliminary data, next-generation ctDNA sequencing (GUARDANT 360) appears to detect actionable genetic alterations when tissue is unavailable, avoiding multiple biopsies and enabling rapid patient selection for genotype-tailored therapies.

Background:
It is not possible to properly target treatments in cases of relapse without knowing the nature of new lesions. Third-generation epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI) can overcome T790M-mediated resistance in non-small-cell lung cancer (NSCLC). But the re-biopsy to confirm T790M status is occasionally difficult. In Japan, transbronchial lung tissue biopsy (TBLB / TBB) is the most common sampling method used for re-biopsy to confirm patients eligible for treatment. We aimed to investigate the success rate of re-biopsy and re-biopsy status of patients with advanced or metastatic NSCLC completing either 1st line chemotherapy or EGFR-TKI therapy.

Method:
We initially screened 39 consecutive patients with NSCLC harboring EGFR-sensitive mutations who had experienced PD after any chemotherapy at Tokyo Medical University Hospital January 2014 and December 2016.

Result:
38 patients who had experienced PD after EGFR-TKI treatment were eligible. Among 30 patients, tumor progression sites included 3 pleural effusion, 9 thoracic primary/metastatic lesions, 2 hepatic metastases, 15 lymph node metastases. Of the 38 patients, 47.3% underwent rebiopsy sucessfully. Of the 38 biopsied patients, 18 (47.3%) were analyzed for EGFR mutation, using tissue or cytology samples; T790M mutations were identified in 10 (55%) of the 18 patients. Of the 38 biopsied patients, 18 (47.3%) were analyzed for EGFR mutation, using tissue or cytology samples; T790M mutations were identified in 10 (55%) of the 18 patients.

Conclusion:
Most re-biopsy samples were diagnosed with malignancy. T790M mutations were identified as much as same in previous studies. However, tissue samples were less available in previous studies. Skill and experience with re-biopsy and noninvasive alternative methods will be increasingly important.

Background:
It is difficult to identify tumor driving genes in advanced non-small cell lung cancer (NSCLC) patients especially for those who were unable to obtain cancer tissue samples and had developed treatment resistance. Circulating tumor DNA (ctDNA) has garnered much excitement over the past few years for its potential clinical utility as a tumor therapy monitoring tool. Our study aims to evaluate the usefulness of ctDNA for serial monitoring actionable genetic alterations in NSCLC patients.

Method:
34 pairs of matched cancer tissue and plasma samples were collected from patients with advanced NSCLC and applied to capture-based next generation sequencing (NGS) using the lung panel, consisting of critical exons and introns of 168 genes. Additional, serial monitoring of ctDNA was conducted in 11 NSCLC patients with actionable genetic alterations using the above NGS assay.

Result:
ctDNA yielded a close agreement of 85.3% (29/34) on actionable genetic alteration status when compared to cancer tissue at baseline in this study. Analysis of ctDNA at different time points showed a strong correlation to treatment efficacy. Interestingly, secondary genetic alterations such as EGFR mutation T790M were detected in 45.5% (5/11) of the patients during monitoring.

Conclusion:
ctDNA may be a potential alternative to conventional primary tissue based NGS assay. ctDNA assay by NGS could be clinically used to monitor the efficiency of personalized target therapy for NSCLC patients.

Background:
According to IALSC/CAP guidelines EGFR and ALK testing is recommended for all non-squamous advanced lung cancers. However, the real access to molecular test and treatment, especially in LATAN is unknown.

Method:
We conducted an online survey with medical oncologists from LATAN during May 2017. The survey has 20 questions about molecular test and target treatment, but also clinical practice in the management of advanced non-squamous NSCLC.

Result:
144 oncologists from 10 countries answer the survey, mostly of them (75%) from Brazil. Although 95% of the oncologists have access to EGFR mutation test and most of them can also test the ALK-fusion protein, only half of them test all patients. Usually these tests are supplied by the pharmaceutical industries (75% of EGFR and 78% of ALK). The mutation status are available in 2 weeks for the EGFR and in 3 weeks for the ALK. The main reason for not testing is lack of sufficient tissue (30% of oncologists), but also some difficulty in access and the long turn-around time where also an issue, 20% and 13% of the oncologist, respectively. Poor performance status and patient clinical characteristics were rarely considered a reason for not testing. Target therapy is available for mostly of the patients with private insurance, but only 50% in the public heath system have access to an anti-EGFR TKI and solely 20% can receive an anti-ALK TKI. New biopsies should be done in the progressive disease, but only 22% of the oncologists perform the procedure in more than 75% of their patient. Immunotherapy is a new treatment modality, especially in the develop countries, but it should be restricted as first line treatment to patients with high expression of PD-L1. In LATAN, immune checkpoints blockage is almost limited to the patients with private insurance (85%), being rare in the public heath system (15%). 83% of the oncologist considered to test the PD-L1 expression only after the results of EGFR /ALK are available.

Conclusion:
There are difficulties in the implementation of IALSC guidelines in LATAN. Mostly of the patients have access to EGFR mutation test, however the treatment is not available to everyone. It is clear the importance of the pharmaceutical industries in providing the molecular test by their voucher programs. The most important difficulty point out by the oncologists is the lack of tissue, but simple barriers as long time to get the results and access to the test should also be managed.

Background:
Previous studies on the clinical value of circulating tumor cells (CTCs) enumeration as a predictor of prognosis in non-small cell lung cancer (NSCLC) have yielded controversial results. The purpose of this study was to further evaluate the prognostic relevance of CTC count in patients with advanced-stage NSCLC before and after first line chemotherapy.

Method:
A total of 43 patients with chemotherapy-naïve, advanced NSCLC and at least one available measurement of CTCs were enrolled in this single-center retrospective study. The presence of CTCs was evaluated by the use of the CellSearch System; CTC positivity was defined as ≥2 CTC in 7.5 ml of whole blood. CTC status was assessed at two different time points, i.e. at baseline (before administration of chemotherapy) and after completion of the first chemotherapy cycle. Baseline CTC count and its dynamic change between the above time points were correlated with clinicopathological features of patients, treatment response and survival, using univariate and multivariate regression analysis.

Result:
Eight (18.6%) out of 43 patients (mean age 67.1 ± 9.9 years, male/female ratio 39/4) harbored CTCs at baseline (mean 22.75 CTCs/patient, range: 2 - 108). Positive baseline CTC count was significantly associated with the presence of five or more metastatic sites (p=0.018). Response to treatment was recorded in 40.6% of patients and disease stabilization or progression in 59.4%. No significant associations were found between response or progression rates and baseline CTC counts (p=0.067 and p=0.083, respectively). On the contrary, progressive disease status and metastatic disease burden were significantly associated with the change in CTC count, (p=0.033 and p=0.035, respectively). No significant associations were found between survival (PFS or OS) and CTC count or the dynamic change of CTC status. Worse performance status (PS) and the presence of five or more metastatic sites were recognized as independent predictors of reduced PFS [HR=2.7; 95% CI: 1.1-6.8; p=0.035 and HR=2.9; 95% CI: 1.1-8.1; p=0.042, respectively], while PS was the only variable independently associated with OS (HR=16.9; 95% CI: 4.3-65.8; p<0.001).

Conclusion:
In our study, CTC count and the dynamic change of CTC status following chemotherapy administration were both associated with increased metastatic disease burden but not with PFS or OS. These data support the suggested role of CTCs in the metastatic cascade and underline the need for further validation of this candidate biomarker before its implementation into routine oncology practice.

Background:
Success of a target therapy is directly correlated with the accuracy of its companion diagnostic test. Without a corresponding biomarker, target therapy may yield shorter survival, waste time, increase burdens and costs. As important as conducting cost-effectiveness studies for therapies, it is also valuable to compare different molecular tests. In lung cancer, the mutational status of EGFR and translocation of ALK and ROS-1 are commonly tested to offer the best intervention. Our objective is to evaluate the cost-effectiveness of a unique exam using NGS versus other routinely used tests such as the ones which involve RT-PCR and FISH.

Method:
Target population is NSCLC, adenocarcinoma, and candidates to first-line therapy. Strategy 1: test EGFR mutation if EGFR test negative, individual follow to FISH for ALK; if FISH negative, follow to FISH for ROS-1. Strategy 2: differs from 1 since FISH for ALK and ROS are requested together. Strategy 3: all individuals are submitted to NGS (multicomplex platform which include EGFR/ALK/ROS-1 and other genes). Prevalence of biomarker, test accuracy and proportion of unknown results were used to calculate each decision tree branch. Sensitivity and specificity was obtained from literature review using Sanger as reference standard for RT-PCR tests and NGS. The study was analyzed from a healthcare-payer perspective based on Brazilian private sector. Costs were based on data from diagnostic companies, ANS and AMB-CBHPM 2016. Survival time and utilities were based from randomized clinical trials. Decision tree model was designed to select an appropriate treatment regimen according to test results; and microsimulation model (M) simulate costs and survival from the corresponding treatment. If EGFR test was positive, M1: gefitinib 1ºline>pemetrexed+cisplatin>docetaxel; if ALK or ROS1 were positive, M2: crizotinib1ºline>pemetrexed+cisplatin>docetaxel; if EGFR/ALK/ROS negative or test unknow, M3: pemetrexed+cisplatin>docetaxel>gemcitabine.

Result:
The use of NGS added 24% extra cases correct identified as well as extra costs (US$ 800.76; PPP 2015) attributed to the molecular testing. The ICER comparing NGS with sequential tests was US$ 3,381.82/correct case detected. Comparing strategy 2:1, the ICER was US$937.86/correct case detected. Therefore, when treatment was incorporated into the model, the effectiveness was diluted between arms. The NGS improves slight gain in life years and quality-adjusted life years, but this could be explained by minor differences in global survival time between treatments.

Conclusion:
: this study is part of an effort to integrate companion diagnostics discussions on precision medicine and covered drugs in the Brazilian health system. Studies considering liquid biopsy could be worth value to highlight effectiveness between tests in clinical routine.

Background:
NSCLC patients with activating EGFR mutations benefit from 1[st] generation EGFR-TKIs. It eventually develops acquire resistance after 10-12 months during of response. Of note, approximately one-third of those patients develop brain metastases, which deteriorate their quality of life and survival. Few effective therapeutic options are currently available for BM patients. Several case studies have showed the well response with osimertinib in BM patients. BM model also found the high penetration rate of Osimertinib into blood-brain barrier. This study evaluated the feasibility of osimertinib treatment on BM patients after 1st generation EGFR-TKI resistance.

Method:
Patients with advanced or recurrent NSCLC who had progressed during EGFR-TKIs treatment were collected from our previous clinical trial (NCT02418234) from March 2015 to March 2016. Blood samples were drawn within two weeks from PD occurred. T790M mutations were evaluated by droplet digital PCR. We undertook follow-up every 3 months by phone until April 2017. The median follow-up time was 11 months (range, 2 to 22 months).

Result:
Fifty NSCLC patients with BM after EGFR-TKI resistance were collected from our previous trials. After TKI resistance, ten patients received subsequent osimertinib treatment. Finally, ten patients included three males and seven females were included in the study. The median age was 66.5 (56 to 73). Seven were detected acquired T790M mutation. The median survival was 15.3 months (95% CI, 10.1 to 20.6 mo), 15.3 mo for T790M negative and 12.9 mo for T790M positive patients.

Conclusion:
Our preliminary study showed the well efficacy of osimertinib on NSCLC patients with BM. It provides well survival benefit. Randomized control trials should be required before it is widely used.

Background:
Patients with non-small cell lung cancer（NSCLC）harboring epidermal growth factor receptor (EGFR) mutations benefit a great deal from EGFR tyrosine kinase inhibitors(TKIs). However, most patients get recrudesced within one or two years, and many of them progressed in central nerve system（CNS）.Owing to the blood– brain barrier, detecting tumor-derived cell-free DNA (cfDNA) in the blood of brain metastasis tumor patients is challenging. Detecting tumor-specific mutations in cerebral spinal fluid (CSF) became an alternative method.

Method:
41 initial or progressed lung adenocarcinoma patients with EGFR mutation and CNS metastasis from Henan Cancer Hospital were included in this study. 41 patients were all detected EGFR mutation status in CSF with dropplet digital PCR (ddPCR) and 37 in 41 patients were detected EGFR mutation status in blood with the same method. Their clinical informations were also collected at the same time.

Result:
The rate of EGFR mutations in CSF （15/41，36.6%）were obviously lower than that in blood（24/37，64.9%）（P=0.013), especially in those with EGFR exon 19del mutation patients (7/18 vs 13/16, P = 0.017), without TKIs treated patients (3/13 vs 8/11,P = 0.038) and less than 60 years patients (10/25 vs 17/22, P = 0.010). In patients with CNS symptoms positive, the rate of EGFR mutations in CSF was higher than those negative (13/27 vs 2/14 , P=0.033). In patients with MRI indicating leptomeningeal metastasis, the rate of EGFR mutations in CSF was higher than those not indicating(8/11 vs 7/30 , P=0.003).

Clinical characteristics, EGFR mutation status in CSF and plasma (n = 41)

Characteristic	n(%)
Gender
Male	23 (56.1)
Female	18 (43.9)
Age
≥ 60	16 (39)
< 60	25 (61)
Smoking status
Yes	12 (29.3)
No	29 (70.7)
Brain metastases (MRI)
Only metastatic tumors	30 (73.2)
With meningeal lesions	11 (26.8)
Brain metastatic tumor number
Single	10 (24.4)
Multiple	31 (75.6)
Neurological symptoms
Positive	15 (36.6)
Negtive	26 (63.4)
Prior TKIs treat
Yes	28 (68.3)
No	13 (31.7)
CSF EGFR mution
19	7 (17.1)
21	6 (14.6)
Both	2 (4.9)
Negtive	26 (63.4)
Blood EGFR mution
19	13 (31.7)
21	10 (24.4)
Both	1 (2.4)
Negtive	13 (31.7)

Conclusion:
The EGFR mutations status in CSF is different from that in blood in patients with EGFR mutation and CNS metastasis. This warrants confirmation in larger cohorts and further more studies are needed to find out the internal mechanism. Detecting EGFR mutations status in both blood and CSF will be helpful to make individualized treatment.

Background:
Though target agents like epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI) have shown activity in patients with brain metastasis, the impact of EGFR mutation on incidence of non-small cell lung cancer (NSCLC) with brain metastasis and treatment outcome remain inconclusive.

Method:
MEDLINE, PubMed, and EBSCO Libraries were systematically searched until August 31, 2015. Retrospective studies including investigating the correlation between EGFR mutation status with brain metastasis from NSCLC were included.

Result:
The result of the fourteen studies including 4432 patients indicated NSCLC patients with EGFR mutation have higher incidence of brain metastasis (Figure 1, odd ratio = 2.09, 95% CI: 1.72–2.53). And, EGFR mutations were associated with better survival in patients with brain metastasis from five studies though not statistically significant (Figure 2, hazard ratio = 0.47, 95% CI: 0.16–1.35). Figure 1. Meta-analysis of the association between EGFR mutation status and the risk of brain metastasis. Figure 1 Figure 2. Meta-analysis on mean overall survival among NSCLC patients with brain metastasis according to EGFR mutation status. Figure 2





Conclusion:
We found that EGFR mutation increased risk of brain metastasis but EGFR mutation might predict better survival with appropriate treatments.

Background:
Lung cancer: the leading cause of cancer-related deaths worldwide About 85% of lung cancers are NSCLC with Adenocarcinoma in predominance EGFR-TKI became a current standard of care in adenocarcinoma, requires EGFR mutation test Biopsy needed to obtain tumor tissue for EGFR mutation test, with a failure rate of about 10-50% due to inadequate tumor tissue Blood plasma which contains circulating-free tumor DNA might be used as alternative DNA source for the EGFR mutation test To conduct a preliminary study to see the suitability between EGFR mutation examination in plasma samples with samples of tumor tissue (biopsy or cytology)

Method:
Design: descriptive cross sectional Subject: 10 Patients; Naive NSCLC adenocarcinoma patients (>18 y.o.) at dr. H. A. Rotinsulu Lung Hospital, Bandung Indonesia Examination: Kalgen Laboratory Jakarta-Indonesia and Prodia Diagnostic Molecular Laboratory Jakarta-Indonesia, using therascreen EGFR Kit Analyzed descriptively by comparing EGFR mutation from plasma sample with tumor or cytology tissue, calculated the conformity in percentage

Result:

EGFR mutation test result between tumor tissue samples and blood plasma samples

EGFR mutation status of blood plasma samples	EGFR mutation status of tumor tissue samples
	Exon 19del	L858R	T790M	No Mutation detected	Inadequate sample
Exon 19del	2
L858R					1
T790M				1
No mutation detected				5	1

Success rate: Plasma 100%, tissue 80% Mutation detected: Plasma 40%, tissue 20% No mutation detected: Plasma 50%, tissue 60% concordance of tumor tissue samples with blood plasma samples

Blood plasma samples	Tumor tissue samples
	No mutation detected	Mutation detected	Inadequate sample
No mutation detected	5	0	1
Mutation detected	1	2	1
Conformity result: 70%

Conclusion:
This study has met the primary objective with conformity of 70% Needs further study with bigger population and design, although this study showed some trends favoring blood plasma samples

Background:
Pembrolizumab, a monoclonal antibody against PD-1, is approved by several regulatory agencies for first line treatment in metastatic NSCLC with a PD-L1 tumour proportion score (TPS) ≥50%. An economic model was developed to evaluate the cost-effectiveness of employing a biomarker (PD-L1) test-and-treat strategy (BTS), in which patients with TPS ≥50% are treated with pembrolizumab, and other patients receive standard-of-care (SoC) cytotoxic chemotherapies versus a non-BTS strategy with all patients receiving SoC. Patients with activating EGFR mutations and ALK translocations were excluded from the analysis.

Method:
The model was built with partitioned survival approach to estimate the incremental cost effectiveness ratio (ICER) expressed as cost per quality-adjusted life-year (QALY) gained. The clinical efficacy, utility and safety data used in this model were derived from the KN024 trial. The base case comparator in the model included five different platinum-based chemotherapy regimens used as SoC for advanced NSCLC in Hong Kong. The base-case time horizon for the model was 10 years with costs and health outcomes discounted at a rate of 3% per year. Utilities for the base case were based on utility data collected in KN024. Costs and disutility associated with grade 3-5 adverse effects of incidence rate 5%, including anaemia, neutropenia, pneumonia, thrombocytopenia and pneumonitis were considered in the model. Treatment was continued until disease progression or maximum 2 years for pembrolizumab. Local drug acquisition costs, PD-L1 testing costs, drug administration costs, disease management costs were applied. A series of sensitivity analyses were conducted to evaluate the uncertainty of cost-effectiveness results.

Result:
The BTS approach was projected to increase QALY by 0.29 with an additional total cost of HK$ 249,077 (USD 31,933) compared to non-BTS approach resulting in an incremental cost-effectiveness ratio (ICER) of HK$ 865,189 (USD 110,922) per QALY gained. This is lower than the World Health Organization (WHO) cost-effectiveness threshold of 3 times 2016 GDP per capita of Hong Kong, HK$ 1,017,819 (USD 130,490). Probabilistic sensitivity analysis showed 94.6% probability that the ICERs would be below this threshold. In a scenario analysis, a lower ICER of HK$ 859,284 (USD 110,165) was shown in comparison of pembrolizumab versus SoC among patients with TPS ≥50%.

Conclusion:
A BTS to identify a subset of NSCLC patients with PD-L1 TPS ≥50% to be treated with pembrolizumab in the first line setting can be considered cost-effective in Hong Kong.

Background:
Nivolumab, an immune checkpoint inhibitor, is approved for advanced non-small cell lung cancer (NSCLC) after platinum-based chemotherapy, based on results of 2 pivotal studies (CheckMate 017 and 057). This prospective observational study (CA209-118) compared outcomes with nivolumab versus chemotherapy as post-platinum therapy for NSCLC in a real-world community setting.

Method:
This analysis, as of May 16, 2017, included patients with advanced NSCLC who completed an initial course of platinum-based chemotherapy and started subsequent treatment prior to November 16, 2016, with a minimum of 6 months of potential follow-up. Patients receiving experimental therapy, immunotherapy other than nivolumab, or tyrosine kinase inhibitors for EGFR/ALK-mutated NSCLC were excluded. Patients in this non-randomized study were grouped by receipt of nivolumab or chemotherapy as first post-platinum therapy and followed until death, study discontinuation, or initiation of subsequent immunotherapy. Unadjusted and adjusted survival analyses were conducted. For adjusted analysis, multivariate regression was performed that included age, ECOG performance status score, time since stage IV diagnosis, smoking status, and squamous histology as covariates.

Result:
Of 383 eligible patients, 161 received post-platinum nivolumab and 222 received post-platinum chemotherapy, including 158 who received a regimen recommended by the National Comprehensive Cancer Network (docetaxel, pemetrexed, gemcitabine, ramucirumab + docetaxel, or erlotinib) and 40 who received a second platinum-based regimen. Baseline characteristics were well balanced between treatment groups, except that the percentage of men was higher in the nivolumab versus chemotherapy group (59% vs 49%). Mean age was 66 years, and 79% of patients had non-squamous histology. In all, 65% of patients in the nivolumab group and 69% in the chemotherapy group started post-initial platinum therapy ≤1 year after stage IV diagnosis. Median survival from the start of post-initial platinum therapy was 11.5 months (95% confidence interval [CI]: 7.9, not reached) in the nivolumab group and 8.3 months (95% CI: 6.1, 11.0) in the chemotherapy group. Unadjusted survival analyses showed a reduction in mortality risk of 20% with nivolumab versus chemotherapy (hazard ratio = 0.80; 95% CI: 0.59, 1.08); adjusted survival analyses yielded comparable results. In the nivolumab and chemotherapy groups, respectively, 9% and 18% of patients discontinued due to adverse effects; 41% and 49% discontinued due to death or disease progression.

Conclusion:
The results of this early survival analysis from a prospective study in a real-world community setting were similar to those seen in randomized trials, further supporting the benefit of nivolumab over chemotherapy in previously treated advanced NSCLC.

Background:
The phase III OAK study in NSCLC (NCT02008227) demonstrated prolonged overall survival with atezolizumab (an anti-programmed death-ligand 1 antibody) versus docetaxel (median 13.8 vs 9.6 months; HR:0.73, 95% CI 0.62–0.87; p=0.0003). PROs were collected to support documentation of clinical benefit. We report data regarding symptom burden, functioning, and health-related quality of life (HRQoL).

Method:
Patients (n=850) with squamous/non-squamous, previously treated NSCLC, ≥18 years, with measurable disease (RECIST), and ECOG PS 0–1 were randomized to receive atezolizumab 1200mg or docetaxel 75mg/m[2] q3w. PROs were collected using two questionnaires: EORTC QLQ-C30 and its lung module, QLQ-LC13. Analyses included time-to-confirmed-deterioration (TTD) in lung cancer symptoms, physical and role function, HRQoL, longitudinal analyses of mean scores change from baseline to Cycles 5 and 6, proportion of patients with clinically meaningful worsening (≥10-point change from baseline) by Cycles 5 and 6.

Result:
High completion rates were observed throughout treatment (>80% for most cycles). Atezolizumab delayed TTD in physical (HR:0.75, 95% CI 0.58–0.98) and role function (HR:0.79, 95% CI 0.62–1.00). Prolonged TTD in chest pain (HR:0.71, 95% CI 0.49–1.05) was observed with atezolizumab; no differences in TTD were seen for other lung cancer symptoms and HRQoL. Longitudinal analyses demonstrated average changes from baseline in favor of atezolizumab for lung cancer symptoms (Cycle 6: dyspnea, fatigue), domains of functioning (Cycle 6: physical function, social function), HRQoL (Cycle 5); see Table. Fewer atezolizumab-treated patients experienced clinically meaningful worsening in possible treatment-related symptoms during treatment (Cycle 6: diarrhea [OR:0.51, p<0.0001], sore mouth [OR:0.40, p<0.0001], dysphagia [OR:0.61; p=0.0052], peripheral neuropathy [OR:0.50, p<0.0001], alopecia [OR:0.02; p<0.0001]).

Conclusion:
In OAK, atezolizumab delayed the time until NSCLC patients experience limitations in physical and role functioning versus docetaxel. Patient-reported data indicate atezolizumab maintained/improved lung cancer symptom burden and HRQoL compared with baseline, while demonstrating improved tolerability, versus docetaxel.

	By Cycle 5		By Cycle 6
	LS means difference between treatment arms (average change from baseline)	P value	LS means difference between treatment arms (average change from baseline)	P value
EORTC QLQ-C30 Global Health Status and Function Scales (positive values indicate greater improvement with atezolizumab over docetaxel)
Global Health Status	4.32*	p=0.0151	3.08	p=0.1257
Physical Function	3.33*	p=0.0290	6.64*	p<0.0001
Role Function	2.93	p=0.1959	4.72	p=0.0542
Emotional Function	2.66	p=0.1110	1.92	p=0.2868
Cognitive Function	-0.67	p=0.6790	-1.08	p=0.5309
Social Function	3.25	p=0.1159	4.68*	p=0.0319
EORTC QLQ-C30 Symptom Scales (negative values indicate greater improvement with atezolizumab over docetaxel)
Fatigue	-6.27*	p=0.0015	-7.66*	p=0.0003
Nausea/Vomiting	-0.37	p=0.7824	-0.18	p=0.9040
Pain	1.44	p=0.5132	-1.67	p=0.4727
Dyspnea	-4.70*	p=0.0317	-5.92*	p=0.0138
Insomnia	3.50	p=0.1675	0.83	p=0.7564
Appetite Loss	-2.94	p=0.1994	-4.49	p=0.0586
Constipation	-0.31	p=0.8772	-0.33	p=0.8816
Diarrhea	-3.14*	p=0.0482	-2.05	p=0.1748
EORTC QLQ-LC13 Symptom Scales (negative values indicate greater improvement with atezolizumab over docetaxel)
Dyspnea	-1.66	p=0.3146	-4.80*	p=0.0140
Coughing	-2.60	p=0.2572	-1.38	p=0.5772
Sore Mouth	-7.29*	p<0.0001	-9.23*	p<0.0001
Dysphagia	-0.08	p=0.9595	-2.01	p=0.1575
Peripheral Neuropathy	-12.98*	p<0.0001	-15.71*	p<0.0001
Hemoptysis	-0.24	p=0.7365	-0.91	p=0.2080
Alopecia	-50.59*	p<0.0001	-47.04*	p<0.0001
Chest Pain	-0.91	p=0.6064	-0.58	p=0.7779
Arm/Shoulder Pain	-2.27	p=0.3177	-0.58	p=0.8109
Pain in Other Parts	0.94	p=0.7197	-1.05	p=0.7034
*Values that are significantly in favor of atezolizumab versus docetaxel

Background:
Among patients (pts) affected by non-squamous non-small cell lung cancer (non-Sq-NSCLC), those with secondary brain metastases are very common and are characterized by a poor prognosis. As they are usually excluded from clinical trials, the EAP offered an opportunity to evaluate nivolumab efficacy and safety in these patients outside of a controlled clinical trial in Italy.

Method:
Nivolumab was available upon physician request for pts aged ≥18 years with a diagnosis of non-Sq-NSCLC who had relapsed after a minimum of one prior systemic treatment for stage IIIB/stage IV non-Sq-NSCLC. Nivolumab 3 mg/kg was administered intravenously every 2 weeks to a maximum of 24 months. Pts included in the analysis had received ≥ 1 dose of nivolumab and were monitored for adverse events using Common Terminology Criteria for Adverse Events. Pts with brain metastasis were eligible if asymptomatic, neurologically stable and either off corticosteroids or on a stable dose or decreasing dose of ≤ 10 mg daily prednisone.

Result:
Out of 1588 patients with non-Sq-NSCLC participating in the EAP in Italy, 409 (26%) had asymptomatic and controlled secondary brain metastases. Pts received a median number of 7 doses (1-45) and had a median follow-up of 6.1 months (0.1-21.9). The disease control rate was 40%, including 3 pts with a complete response, 65 pts with a partial response and 96 with stable disease. Among these pts, 118 were receiving steroid therapy at baseline and 74 received concomitant radiotherapy. As of March 2017, median overall survival of this subpopulation was 8.1 months (6.2-10.1). Overall, among pts with brain metastasis, 337 discontinued treatment for any reason, with only 23 (7%) pts who discontinued treatment due to adverse events, in line with what observed in the general population and in previous studies.

Conclusion:
These data confirmed the activity of nivolumab in patients with non-Sq-NSCLC and brain metastases, supporting the use of nivolumab in this population with poor prognosis. Moreover, as already observed in other tumor types, safety results were consistent to what already reported and confirmed the favorable safety profile.

Background:
Immune checkpoint inhibitors (ICIs) have good treatment outcomes for non-small cell lung cancer (NSCLC) especially with smoking history. The drug-induced interstitial lung diseases (ILDs) are frequently seen in patients treated with ICIs. The risk assessment for ILDs before ICIs treatment is important, however the pulmonary toxicities of ICIs in patients with smoking related pulmonary diseases such as emphysema and fibrosis are not known. In this study, we retrospectively analyzed PD-L1 expression of NSCLC according to underlying pulmonary disease.

Method:
We tested PD-L1 expressions in NSCLC using 22C3 antibody as tumor proportion score (TPS). We then compared PD-L1 expression TPS according to underlying pulmonary diseases assessed by chest CT (normal, fibrosis, emphysema).

Result:
We reviewed 44 NSCLC patients at NTT Medical Center Tokyo. The median age of all the patients was 71 years (range 46-90). Thirty-eight patients were male (86%). Adenocarcinoma was the most frequent with 26 patients (59%), followed by squamous cell carcinoma with 16 patients (36%). As to PD-L1 expression, 7 patients (16%) were TPS more than 50%, 12 patients (27%) were TPS 1-49% and 22 patients (50%) were TPS less than 1%. Three patients (7%) did not have evaluable material. All the patients with TPS >50% and 1-49% had smoking history. For patients with TPS <1%, there were three patients (14%) without smoking history. As to histology, there were 4 patients (57%) with squamous cell carcinoma for patients with TPS >50%, 4 patients (33%) for TPS 1-49% and 8 patients (36%) for TPS <1%. Among patients with TPS >50%, 2 patients (29%) had emphysema, 5 patients (23%) fibrosis, and no normal lung. Among patients with TPS 1-49%, there were 4 patients (33%) with normal lung, 6 patients (50%) with emphysema and 2 patients (17%) with fibrosis. For patients with TPS <1, there were 9 patients (41%) with normal lung, 10 patients (45%) with emphysema and 2 patients (9%) with fibrosis.

Conclusion:
No patients with normal lungs showed TPS >50%, whereas more than half of patients with TPS <1% had normal lung. Our results show that patients with higher PD-L1 expression has higher rate of underlying pulmonary disease which might be a higher risk for drug-related ILDs. Further treatment strategy is needed for use of ICIs with higher PD-L1 expression with underlying pulmonary diseases.

Background:
While marked responses have been observed in patients with non-small-cell lung cancer (NSCLC) treated with PD-1 pathway inhibitors, anecdotal evidence indicates that rapid progression with dramatic tumor burden increase early in the course of therapy may be noted in a few patients. The study characterized the spectrum of early progression of advanced NSCLC treated with PD-1 inhibitors, and investigated quantitative imaging markers for poorer outcome.

Method:
The study included 134 patients (53 men, 81 women; median age: 66) with advanced NSCLC treated with commercially prescribed single-agent nivolumab or pembrolizumab, who had follow-up CT scans at 8 +/- 2 weeks of therapy. Tumor burden measurements were performed using RECIST1.1 on baseline and 8-week scans to characterize the spectrum of early progression during PD-1 therapy. Tumor burden changes at 8 weeks were studied for association with overall survival (OS), which was measured from the 8-week scan date.

Result:
The tumor burden changes at 8 weeks comparing to baseline ranged from -72.7% to +138.7% (median: +4.3%; the 90[th] percentile: +50.07%). OS of 15 patients with ≥50% increase of tumor burden at 8 weeks was significantly shorter compared to 119 patients with <50% increase at 8 weeks (median OS: 4.5 months [95%CI: 1.3-4.9] vs. 12.7 months [95%CI: 8.5-14.7]; log-rank p=0.0003). Among 42 patients who experienced tumor burden increase ≥20% (RECIST progression threshold) at 8 weeks, 15 patients with ≥50% increase had shorter OS than 27 patients with ≥20% but <50% increase (median OS: 4.5 months [95%CI: 1.3-4.9] vs. 6.8 months [95%CI: 5.4-20.1]; log-rank p=0.08), indicating that ≥50% increase threshold may identify a distinct group of early progressors with poorer prognosis. Never smokers were more likely to experience ≥50% increase at 8 weeks than former or current smokers (Fisher p=0.03).

Conclusion:
Tumor burden increase of ≥50% at 8 weeks of therapy was associated with significantly shorter OS in advanced NSCLC patients treated with commercial PD-1 inhibitors, indicating that it can serve as an imaging marker to identify a distinct subset of patients with poorer outcome of PD-1 inhibitor therapy, and may thus help guide treatment decisions.

Background:
We aimed to evaluate quality of life (QoL) and clinical outcomes of nivolumab (Nivo) as ≥ 2nd line treatment within the expanded access program in NSCLC pts. The QoL changes, response rates, survival and safety were studied within the multicenter prospective observational study.

Method:
Adult pts with advanced refractory NSCLC were enrolled in 7 centers in RF. All the pts received Nivo 3 mg/kg q2w. Tumor response was assessed using RECIST v. 1.1, adverse events (AEs) with NCI CTCAE v3.0; QoL by SF-36 and symptoms by ESAS-R at baseline, 4 and 12 weeks after treatment start. Overall survival (OS) and progression-free survival (PFS) curves were evaluated from the start of Nivo treatment by the Kaplan-Meyer method and compared by the log rank test. For QoL analysis Generalized Estimating Equations (GEE) method was used.

Result:
The interim analysis was performed in the group of 172 pts with the median follow-up – 4.7 mos (65% – males; median age – 62 (29−80); ECOG PS 0-1/2-3 – 81%/19%; former/current smokers – 71%; non squamous NSCLC – 65%; ≥2 lines of previous treatment – 51%). After 2 cycles of Nivo QoL improvement was registered in 53% pts, after 6 cycles – in 60% pts; mean QoL index increased by 59% and 51%, respectively. Upon GEE, significant improvement of QoL index during 6 cycles was revealed (p<0.05). The most severe and frequent baseline symptoms, fatigue and shortness of breath, decreased after 2 cycles in 44% and 33% of pts as compared to baseline, and after 6 cycles – in 54% and 46% pts, respectively.Efficacy was evaluated in 118 pts (median first evaluation – 2.2 mos): PR – 9%, SD – 44%, PD – 47%. 14 pts died before first efficacy evaluation. 40 pts were not evaluated for response on cut-off. In the group of pts who completed Nivo treatment (n=116) median PFS was 2.7 mos (95%CI 2.1–3.3), median OS – 8.4 mos (95%CI 6.2–10.7); median follow-up – 4.6 mos. Pts with brain mts (median OS 2.5 mos vs 9.0 mos) and pts with ECOG PS 2-3 (median PFS 2.5 mos vs 3.2 mos) had worse survival; p<0.05. AEs were registered in 54 pts; among them 14 had grades 3-4 AEs.

Conclusion:
Early data from this study supports the acceptable efficacy (53% pts had PR/SD) and safety of Nivo (11% pts with 3-4 grades AEs) in NSCLC pts. Nivo treatment leads to meaningful QoL improvement in this patient population.

Background:
Nivolumab, a monoclonal antibody against the Programmed Death 1 (PD-1) pathway, has been shown to improve outcome and safety compared to Docetaxel in second-line NSCLC. We evaluated the actual use of Nivolumab in routine clinical practice at a single center in patients with NSCLC.

Method:
Data from patients with a diagnosis of advanced NSCLC who were treated with Nivolumab at standard dose (3mg/kg every 14 days), between January 2016 and March 2017 in our Hospital, were retrospectively collected. We performed a descriptive analysis of multiple demographic, clinical and analytical variables, as well as seeking differences between progression-free survival (PFS) according to squamous (SqCC) and non-squamous (Non-SqCC) histology, ECOG Performance Status (PS) and prior lines received by log-rank, Kaplan-Meier methods and Cox proportional models.

Result:
Forty patients were treated. Median age was 67; 67.5% were male; 80% were smokers. Histologies: Non-SqCC 42.5%, SqCC 52.5%, 5% NOS. ECOG PS: 50% ECOG 2, 45% ECOG 1, 5% ECOG 0. Twenty patients were treated as second-line (50%), and 20 had received ≥ 2 prior systemic therapies (50%). Median PFS was 3 months. Response rate: 35% (2.5% of patients had complete response); 42.5% of patients had stable disease and 22.5% progression disease. Adverse events were mostly grade 1 and 2, as expected, just one patient discontinued treatment due to grade 4 inmuno-mediated colitis. Patients with SqCC achieved a longer median PFS than Non-SqCC patients (4.9 vs 2.8 months, HR 2.14, 95% CI 1.1-4.6, p <0.05). Median PFS in patients who received 1 prior line was 2 months vs 3.5 months in patients who received ≥2 prior lines (log-rank, p=0.5). PFS in ECOG PS 0-1 patients was 3 months vs 2 months in ECOG PS 2 (log-rank, p=0.2).

Conclusion:
Nivolumab in NSCLC routine clinical practice is a safe and active alternative to chemotherapy. Nivolumab achieve a good outcome in both histologies, SqCC and Non-SqCC, but we detected a longer PFS in SqCC. Adverse events were as expected, grade 1 and 2.

Background:
Nivolumab has improved overall survival (OS) in metastatic non-small cell lung cancer (NSCLC) patients. Analysis of the use of these drugs in real world provides more evidence about efficacy and toxicity. We describe here the experience of the use of nivolumab in NSCLC in Argentina.

Method:
NSCLC patients (pts) who received nivolumab between 6/2015 - 12/2016. Patients consented their respective physicians to be treated on a drug expanded access program. Data was collected retrospectively by the physician. Images and follow up were done according to physician´s discretion. Adverse events were classified according to CTC3.1. Responses were evaluated according to RECIST 1.1 criteria. DFS and OS was assessed. All pts who received at least one dose of Nivolumab were evaluated for toxicity.

Result:
N 109. Fup 8.83 m (IQR 3.4-12.67). 57.8% men, 29.4% current smoker, 78.0% non-squamous, 8.3% EGFR mutated. Chemotherapy lines before nivolumab 2 md (r 1-4), and 59.6% received radiotherapy. 89% received previously platinum based chemotherapy. Sites of relapse or progression before nivolumab were: lung (75.2%), lymph nodes (47.7%), bone (19.3%), liver (11.9%), central nervous system (11.0%), and adrenal gland (13.8%). PS 0 26.6%, 1 56.0%, 2 13.8% and 3 1.8%. Cycles of nivolumab 10 Md (IQR 3-18). Drug related toxicity 78.9%. Grade 2-3 28.4%. Corticoid use 33.9%. Responses were evaluated in 104 pts who had as best response CR 2/104 (2%), PR 28/104 (27. %), SD 33/104 (32%) and PD 41/104 (39.%). Time to the best response was 4.0 m (IQR 2.3-5.9). DFS 6.1 m (IQR 2.4-13.1) and OS 12.3 m (IQR 4.1-NR). Univariate analysis revealed that absence of corticoids use (p=0.034), toxicity grade 1-3 (p=0.0025), PS≤1 (p=0.049), age<=50 (p= 0.0011) were associated with longer DFS; PS≤1 (p<0.001) and toxicity grade 1-3 (p=0.001) were associated with longer OS. In multivariate cox regression analysis, toxicity grade 1-3 (HR 0.44 CI95% 0.24-0.81, p=0.008) and age<=50 (HR 0.28 CI95% 0.13-0.61, p=0.001) were associated with longer DFS while corticoids use was associated with shorter DFS (HR 2.06 CI95% 1.22-3.48, p=0.007); toxicity grade 1-3 (HR 0.28 CI95% 0.14-0.54, p<0.0001) and PS≤1 (HR 0.16 CI95% 0.08-0.31, p<0.0001) were associated with longer OS.

Conclusion:
The use of Nivolumab in a real world setting, in heavily pre-treated NSCLC patients was well tolerated and showed promising clinical efficacy. PS, the use of corticoids and immune-mediated toxicity seem to be conditions which could affect clinical outcomes.

Background:
Chemotherapy and immunotherapy have been shown to be beneficial for patients with non-small cell lung cancer (NSCLC) and performance status (PS) of 0 or 1; there still is debate, however, regarding its efficacy for patients with a PS of 2 which comprises approximately 30% of the NSCLC population. Pre-clinical data have demonstrated that low dose carboplatin/paclitaxel have resulted in superior immune efficacy compared to the maximum tolerated dose regimen. Given the significant unmet need for treatment options in this patient population, our study evaluated low-dose weekly carboplatin/paclitaxel combined with pembrolizumab in patients with NSCLC and a PS of 2.

Method:
Patients with metastatic or recurrent NSCLC and PS of 2 were randomized to single agent pembrolizumab at 200mg every 3 weeks or pembrolizumab plus weekly carboplatin AUC 1 and paclitaxel 25 mg/m[2] irrespective to PDL-1 status. Response was determined using immune-related RECIST, and toxicity was graded using CTCAE 4.0.

Result:
Between 6/2016 and 2/2017, 20 patients were enrolled, and 19 patients were evaluable for response. The median age was 69 years (54-83). All 19 patients (100%) had a PS of 2. Ten patients were randomized to the single agent arm and 9 patients to the combination arm. Six patients received the therapy as second line (2 combination arm and 4 single agent arm). Mean 3 week cycles per patient: 9 (4-16) in combination arm and 7 (2-14) in single arm group. Response at 9 weeks in the combination arm: partial response (PR) 6 (67%), stable disease (SD) 2 (22%), and progressive disease (PD) 1 (11%). Response at 9 weeks in the single agent arm, PR 2 (20%), SD 4 (40%), and PD 4 (40%). Adverse events in combination arm: 1 (11%) discontinued therapy due to grade 3 fatigue, 3 (33%) discontinued carboplatin due to allergic reactions at 7, 9, and 10 months of treatment but continued pembrolizumab and paclitaxel, and 1 (11%) on hormone replacement therapy due to treatment-induced hypothyroidism. Adverse events in single agent arm: 1 (10%) discontinued treatment due to complete A-V block successfully resolved with pacemaker insertion, and 2 (20%) are on hormone replacement therapy due to treatment-induced hypothyroidism.

Conclusion:
Combination pembrolizumab and weekly low dose carboplatin/paclitaxel is an active and well tolerated regimen in patients with advanced NSCLC with PS of 2.

Background:
In patients with advanced non–small-cell lung cancer (NSCLC), docetaxel has been established as the second line of treatment after progression to platinum-based regimens. Following the results of the CheckMate 017 and 057 studies, Nivolumab, an anti-PD-1, has been second in line approved. This has increased overall survival in comparison with Docetaxel. This paper analyzes the experience with Nivolumab in second and successive lines of advanced NSCLC within the service of Medical Oncology from the Complejo Asistencial Universitario of León, Spain.

Method:
25 patients diagnosed with advanced NSCLC were included in the period from July 2015 to November 2016. The clinical-pathological characteristics, efficacy, objective response rate (ORR), duration of response (DOR), progression-free survival (PFS), and safety were analyzed. The dose of Nivolumab used was 3 mg/kg every 15 days.

Result:
The median age was 64 years old. All subjects had an ECOG performance status <2. 84% had stage IV cancer. 96% were current smokers or former smokers, in the same proportion (Table 1). 72% received a single pre-treatment line. A median of 7 cycles were administered (range: 2-28). The ORR was 28% (CR 4%, PR 24%) with a disease control rate of 44%. The median DOR was not reached at the time of analysis (range: 2.5+ - 10.4+ months). The median PFS was 5.4 months (95% CI 4.86-6). The rate of immune-related adverse events (IR-AE) of any grade was 36%, with endocrinopathies (hypothyroidism and hyperthyroidism) being the most common IR-AE (24%). The rate of IR-AE G3 was 8% (1 patient pneumonitis, 1 patient hepatotoxicity). There were no toxic deaths. Figure 1



Conclusion:
Nivolumab represents a standard of treatment in advanced NSCLC. Its results of efficacy and safety in this retrospective real-time observational analysis (RWD), with 25 consecutive patients,are consistent with published studies. More patients and extended follow-up are required to draw more precise conclusions.

Background:
Non-small cell lung cancer (NSCLC) patients treated with standard chemotherapies experience progression rapidly. A novel therapy based on programed death 1 (PD-1) inhibitors showed an increasing potential in several malignancies including advanced NSCLC.This article is a meta-analysis aiming to systematically evaluate the efficacy and safety profiles of PD-1 agents in patients with NSCLC.

Method:
Data sources: Data were collected from eligible studies searched from PubMed, Embase, and Cochrane. Synthesis methods: Pooled hazard ratio (HR) for overall survival (OS) and progression-free survival (PFS) was estimated to assess the efficacy of PD-1 inhibitors versus docetaxel, pooled odds ratio (OR) was calculated for objective response rate (ORR). OR for occurrence of any grade and grade 3 to 5 treatment related adverse effect was calculated for evaluating the safety of PD-1 therapies.

Result:
Five studies were included in this analysis. The pooled HRs for OS and PFS were 0.66 (95% CI 0.60–0.74; P<0.00001) and 0.78 (95% CI 0.71–0.86; P<0.00001) respectively, the pooled OR for ORR was 2.12 (95% CI 1.50–2.98; P<0.0001), indicating a significant improvement in OS, PFS, and ORR. OR for occurrence was 0.78 (95% CI 0.63–0.96; P=0.02) in any grade treatment-related adverse effect and 0.27(95% CI 0.23–0.33; P<0.00001) in grade 3 to 5 treatment-related adverse effect, suggesting a superior safety profile of PD-1 inhibitors.

Conclusion:
The anti-PD-1 therapy can significantly prolonge the OS、PFS,improve the ORR and can also reduce the treatment-related adverse effect events versus standard chemotherapies.

Background:
AntiPD1/antiPDL1-based immunotherapy has changed dramatically the prognosis of LC p with a substantial improvement of overall survival (OS) and even presenting long lasting responses in a subset of p. Several factors have been associated with the likelihood of better survival, which include the smoking exposure and the presence of KRAS-mut according to data from randomized clinical trials that compared chemotherapy to these immunotherapeutic agents.

Method:
By reviewing the clinical records of all stage IV LC p treated with antiPD1/antiPDL1 agents, we identified p with KRAS-mut and evaluated their clinical outocomes.

Result:
129 p with advanced NSCLC were treated with nivolumab, pembrolizumab or atezolizumab (65.1%, 17.1% and 17.8 %, respectively) from November 2013 to April 2017. 14 p were identified as adenocarcinomas with KRAS-mut (20.3%) of all non-squamous NSCLC (60p), once squamous cell carcinoma (39 p), p with Kras status unknown (15p), other reasons (6p) were excluded. KRAS-mut subgroup include 28.5% of female, median (m) age of 62.3 years, 92.8% of ever smokers, and PS0-1. The immunotherapy consisted of nivolumab (71.4%) and pembrolizumab and atezolizumab (14.3% each) and was administered as 1[st], 2[nd] and >3[rd] therapy in 7.1,78.6 and 14.3% of p, respectively. 71.4% of p responded to therapy (64.3% partial response) and in 42.8% of p this response lasted >12 months (range 12-32). For this cohort of p m progression-free survival was 7.65 months and OS was 58 months. At the time of analysis 57.1% were still receiving treatment.

Conclusion:
Although the number of p is small, KRAS-mut p represent a subgroup of p that seem to substantially benefit from antiPD1/PDL1 agents in terms of both response and survival.

Background:
Several randomized phase III trials with immune checkpoints inhibitors have accrued patients with metastatic non-small cell lung cancer (NSCLC). These trials employed strict patient selection criteria, and it is currently unknown how it represents the ‘real-world’ population.

Method:
From January 2011 to December 2016, all patients with metastatic NSCLC referred for first oncological evaluation at two University Hospitals in South of Brazil were identified by electronic database and included in the analysis. Twelve pre-defined eligibility criteria, all used in the recent first line phase III immunotherapy trial, were analyzed. OS and PFS were estimated by Kaplan-Meier curves. Multivariate analysis was performed to identify factors associated with survival. Statistical analysis was performed with SPSS 22.0.

Result:
Three hundred and nine patients were collected for this analysis. Patient characteristics revealed a mean age of 63.73 ± 09.47 years, 56% male and 65% had adenocarcinoma. One hundred ninety-seven (64%) patients did not meet one or more eligible criteria at first evaluation. ECOG performance status ≥2 (118 patients) and active brain metastasis (69 patients) accounted alone for 79.7% of non-eligibility cases. One hundred (50.76%), 53 (26.9%), 30 (15.22%) and 14 (7.1%) had 1, 2, 3 or 4 non-eligible criteria respectively. The median survival after the diagnosis of metastatic disease was 6.34 (95% IC, 5.59 to 7.08) months in the non-eligible group and 11.07 (95% IC, 8.65 to 13.48; p<0.001) in the eligible group. The hazard ratio of 1.90 (95% IC, 1.46 to 2.47) to mortality in the non-eligible group should reflect the worse baseline prognostic features in this group.

Conclusion:
To our knowledge, this is the first report of metastatic NSCLC patients analyzed regarding the eligible criteria of the phase III trials. It is clear that clinical trials do not represent the “real world” population and its outcomes have an important selection bias. Phase III clinical trials eligibility criteria should be reviewed to better represent the NSCLC population.

Background:
Immune checkpoint inhibitors are a novel group of immunotherapeutic agents. Antibodies to programmed death-1 (PD-1), such as nivolumab, have shown promising clinical activity in patients with advanced non-small-cell lung cancer (NSCLC), but their efficacy and safety in elderly patients with advanced NSCLC are unclear, because available major clinical trials involved a small number of elderly patients.

Method:
A total of 34 patients received nivolumab for advanced NSCLC from February 2016 to June 2017 in our hospital. We retrospectively reviewed the clinical medical records of these patients. They were divided into two groups; patients aged > 70 and < 70 years. Overall response rates (ORR), progression-free survival (PFS), major adverse effects and discontinuation rate due to adverse effects were compared between in two groups.

Result:
All of them were included in this study (median age was 64 years). Almost all patients had been previously treated with cytotoxic chemotherapy. They included 10 patients in aged > 70 years and 24 patients in aged < 70 years. There was no significant difference in histological type, performance status (PS), smoking history, line of therapy, and laboratory data between the two groups. Comparison between patients aged > 70 years and aged < 70 years in advanced NSCLC shows no statistically significant difference in median PFS (140 vs. 128, HR: 0.96, 95 % CI, 0.37-2.46, p = 0.93), ORR (40 % vs. 32 %, p = 0.70). The treatment was discontinued in 40 % (4/10) and 13 % (3/24) owing to adverse effect; however, there was no significant difference in two groups, and there was no adverse effect death in both groups.

Conclusion:
Nivolumab is tolerable and effective treatment for elderly patients with advanced NSCLC.

Background:
Because of the novel mechanism of action of immunotherapies like nivolumab, response patterns may differ from other therapies and may provide a rationale for considering treatment beyond progression. Immunotherapy protocols generally allow patients (pts) to continue treatment beyond investigator-assessed progressive disease (PD) as long as there is ongoing clinical benefit. Here we report the analysis of pts treated beyond PD in the Italian nivolumab EAP for pts with non-squamous non small cell lung cancer (Non-Sq-NSCLC).

Method:
Nivolumab was provided upon physicians’ request for pts aged ≥18 years who had relapsed after a minimum of one prior systemic treatment for stage IIIB/stage IV non-Sq-NSCLC. Nivolumab 3 mg/kg was administered intravenously every 2 weeks for <24 months. Pts included in the analysis received ≥1 dose of nivolumab and were monitored for adverse events (AEs) using Common Terminology Criteria for Adverse Events. Patients were allowed to continue treatment beyond initial PD as long as they met the following criteria: investigator-assessed clinical benefit, absence of rapid PD, tolerance of program drug, stable performance status and no delay of an imminent intervention to prevent serious complications of PD.

Result:
In total, 1588 Italian pts with advanced Non-Sq-NSCLC received at least one dose of nivolumab in the EAP across 168 sites and 1056 (66%) had PD. Of those, 274 pts (26%) were treated beyond progression. Before being treated beyond PD, the disease control rates (DCR) was 28%, with 1 complete response (CR), 27 partial responses (PR) and 49 stable diseases (SD). Post PD, 58 of all pts treated beyond PD achieved a non-conventional benefit, meaning a subsequent tumor reduction or stabilization in tumor lesions. With a median follow-up of 10.3 months (0.1-21.9) and a median of 11 (4-44) doses, median overall survival for pts treated beyond PD was 15.5 months (range: 13.1-17.9). Overall, among pts treated beyond PD, 200 discontinued treatment for any reason, with only 11 (5.5%) pts who discontinued treatment due to adverse events, suggesting no increased safety signals.

Conclusion:
As already observed in clinical trials, these preliminary EAP data seem to confirm that a proportion of pts who continued treatment beyond PD demonstrated sustained reductions or stabilization of tumor burden, with an acceptable safety profile. Further investigations are warranted in order to better define and identify pts who can benefit from this treatment strategy.

Background:
Immune checkpoint inhibitors provide a new treatment strategy for lung cancer. Therefore, microenvironment of tumor and interaction between immune cells and tumor cells become more important. Until now, the most frequently used marker to predict treatment response is immunohistochemical (IHC) stain of tumor PD-L1. However, the microenvironment of malignant pleural effusion is not clear. Weather we could use IHC stain of PD-L1 on cells of pleural effusion to predict treatment response have not been studied, either.

Method:
We retrospective enrolled patients who received malignant pleural effusion drainage and had cell blocks specimens from 2014-2016. IHC stain for PD-L1 was performed for tumor cells, immune cells, and macrophage of all cell block specimens. An experienced pathologist reviewed all the cell block cytology. The intensity of IHC stain was graded into grade 0, 1, 2, and 3. We also collected the clinicopathological characteristics of all patients.

Result:
PD-L1 expression of pleural effusion tumor cells was associated with the PD-L1 expression of pleural effusion macrophage (p=0.003) and immune cells (p<0.001). However, PD-L1 expression of immune cells is not associated with that of macrophages. PD-L1 expression of tumor cells is correlated with gender (p=0.012), smoking status (p=0.032), and ECOG performance status (p=0.017). Finally, PD-L1 expression of immune cells was associated with overall survival of our patients (p=0.004).

Conclusion:
These results suggested that there might be an immune interaction of pleural effusion tumor cells with macrophage/immune cells, and low expression of PDL1 expression of immune cell are associated with poor survival of lung cancer patients with malignant pleural effusion.

Background:
Lung cancer in younger patients (45 years of age and younger) is rarely found and has different characteristics with older patients. In this study we are looking for characteristics and survival rate of younger NSCLC patients at Indonesia.

Method:
NSCLC patients that came to Dharmais Cancer Center during January 2005 – December 2015 aged 45 years or younger were included in this study. We analyzed patients’ age, gender, history of smoking, histology type, stage, therapy and survival rate.

Result:
Out of 956 NSCLC patients, there were 134 young patients (13.9%). Median age of patient is 39 years old. The most common range of age is 41-45 years old (n=57, 42.5%) with more male patients compared to female patients (n=92, 68.7%). 108 young NSCLC patients (80.6%) did not have history of smoking. Adenocarcinoma is the most common histology type found (59%) and stage IV (52.2%) is most frequent in this study. There is no significant difference between gender and diagnosis (p=0.737), stage (p=0.170), history of smoking and type of histology (p=0.534) in younger NSCLC patients. Median survival rate of the younger patients is 12.2 months (95% CI: 11.045 – 13.355), compared with older patients being 13.2 months (95% CI: 11.547 – 14.853). There is no significant difference between survival rate of younger NSCLC patients and older patients (p=0.543). Patients with EGFR mutation does not have significant association with gender (p=0.07), history of smoking (p=0.259), amount of cigarettes per day (p=0.942), and Brinkman Index (p=0.366). There is a significant difference of survival rate between patients who have EGFR mutation and those who do not (27.4 months VS. 12.2 months; p=0.05). There is no significant difference between patients with EGFR mutation who received target therapy and those who did not (p=0.426). However, there is a significant difference between patients without EGFR mutation who received chemotherapy and those who did not (15.2 months vs. 11.5 months, p=0.05).

Conclusion:
NSCLC in younger patients have shorter survival rate compared with older patients. Survival rate in patients with EGFR mutation who received chemotherapy is better.

Background:
Lung cancer is the leading cause of cancer-related mortality in the United States. Over the last decade an improved understanding of the pathways that drive malignancy and disease progression have fundamentally altered the NSCLC treatment paradigm. We sought to determine if participating in a case-based online educational intervention related to NSCLC diagnosis and management improved the clinical decision-making of oncologists and pathologists in the US.

Method:
Oncologists participated in an innovative online CME activity using branching logic that assessed clinical decisions in management of patients with NSCLC and provided tailored feedback. Two patient cases were presented and clinicians were assessed on answers to multiple-choice questions pre- and post- education. If first attempt answers were incorrect, clinicians received feedback and clinical consequences for their choices, and provided a second opportunity to answer. The CME activity launched May 5, 2016 and data were collected through June 6, 2016.

Result:
For oncologists (n=149), between 52% and 70% answered clinical decision questions correctly on the first attempt, while 27% to 94% of pathologists (n=44), answered correctly . After consequence-based feedback, between 12% and 41% of both oncologists and pathologists improved their decision-making. Specific improvements seen include: 20% oncologists and 23% pathologists increased their ability to identify the need to order IHC staining to identify the tumor’s histopathology 23% more oncologists and 29% more pathologists selected the most appropriate regimen for a patient with adenocarcinoma without actionable mutations after 30% of oncologists and 41% pathologists increased in their ability to select the most appropriate therapy for a patient who has progressed on a first-line regimen based on prior treatment and the PD-L1 status of their tumor

Conclusion:
This innovative, case-based CME activity using branching logic with tailored consequence-based feedback improved clinical decision-making in management of patients with NSCLC to drive learning. It is anticipated that improved clinical decisions among oncologists and pathologists in diagnosis and management of NSCLC will lead to translation in practice and better patient outcomes. Future education using a similar design could be used to translate ongoing developments in NSCLC into clinical decisions for comprehensive management of NSCLC.

Background:
IBM Watson for Oncology (WFO) is a Memorial Sloan Kettering-trained cognitive computing system. Watson provides evidence-based treatment options and ranks them into three categories for oncologist decision making as "Recommended ","For Consideration" and "Not Recommended". We examined the concordance of treatment options in Lung cancer patients between WFO and tumor board in the Affiliated Hospital of Qingdao University, China.

Method:
33patients with stage I-IV lung cancer were enrolled in this study. By tumor stage, 15.1% (5 of 33) patients are Phase I or II, 15.1% (5 of 33) are Phase III and the rest of the patients (23 of 33) are Phase IV. By histology, 18.2% (6 of 33) are small cell lung cancer (SCLC) and 81.8% (27 of 33) are non-small cell lung cancer (NSCLC). Options were considered concordant when it was included in the “Recommended” or “For Consideration” categories.

Result:
Overall, treatment recommendations were concordant in 96.9% (32 of 33) of cases. By histology, 100% of SCLC patients’ therapy regimes were concordant with the recommended one, and treatment recommendations were concordant in 96.3% of NSCLC patients. By tumor stage, treatment recommendations were concordant in 100% of I- III and 96% of Phase IV lung cancer. Of the whole cases, 69.7% were recommended and 27.3% were for consideration (figure1). The majority reason for choosing consideration option is that the immunotherapy drugs targeted PD-1 or PD-L1 such as Nivolumab, Pembrolizumab and Atezolizumab recommended by Watson for Oncology had not been launched in China.Figure 1



Conclusion:
Treatment recommendations made by the Affiliated Hospital of Qingdao University and Watson for Oncology were highly concordant in lung cancer. We’ll make further analysis for this cognitive computing system in more cases and other types of carcinomas.

Background:
GM2 ganglioside is a tumor-associated antigen that is overexpressed in a high proportion of several malignancies, e.g. SCLC, NSCLC, mesothelioma, melanoma, neuroblastoma, multiple myeloma. BIW-8962 is a recombinant, humanized, non-fucosylated immunoglobulin G1 monoclonal antibody to GM2 ganglioside that shows pre-clinical activity towards lung cancer cell lines and in an animal model bearing SCLC xenografts. The aim of this study was to determine the safety and preliminary clinical efficacy of BIW-8962 administered as monotherapy in patients with previously treated lung cancer.

Method:
In phase I, patients (N=16) with advanced, recurrent lung cancer (8 each with SCLC and NSCLC) received increasing doses of BIW-8962 (1–10 mg/kg) intravenously every 3 weeks using a standard 3+3 design to determine the maximum tolerated dose (MTD). The highest dose (10 mg/kg) was administered to patients with advanced, recurrent SCLC (N=21) in phase II.

Result:
It was only possible to obtain pre-study biopsy samples for two patients, both of which showed cell surface GM2 overexpression of moderate intensity on immunohistochemistry testing. In phase I and II, all patients received the total planned dose. There were no dose-limiting toxicities in phase I and the MTD was not established. BIW-8982 10 mg/kg therefore used as the recommended phase II dose. The phase II study was prematurely terminated due to lack of efficacy. The objective response rate was 5.0% (95% CI, 0.1%–24.9%) in the efficacy evaluable population (N=20). Median overall survival was 304.0 days (95% CI, 70.0–406 days) and median progression free survival (PFS) was 43.0 days (95% CI, 38.0–43.0 days). One patient showed a durable partial response with PFS of 463 days and response duration of 382 days. There were a few patients with stable disease, which was generally not durable. No pattern of consistent toxicity was observed across the phases: there were no treatment-related adverse events (AEs) Grade ≥3, serious AEs, AEs leading to discontinuation of BIW-8962, or deaths. No unexpected trends or safety concerns were identified from laboratory parameter, vital sign, or electrocardiogram assessments. Anti-BIW-8962 antibodies were not detected in serum of any patient before or following treatment. Exploratory analysis of circulating tumor cells and other potentially predictive or pharmacodynamic markers did not reveal any results consistent with an effect from BIW-8962.

Conclusion:
This study was prematurely terminated due to lack of efficacy, for which the reason is unknown. Clinical development of BIW-8962 has been discontinued.

Background:
Ascorbic acid (AA) infusion and modulated electrohyperthermia (mEHT) are widely used by integrative cancer practitioners for many years. However, there are no sufficient data in safety, quality of life and clinical response of the above treatments in patients with stage III-IV Non-Small Cell Lung Cancer (NSCLC).We designed a Phase I trial to provide the evidence.

Method:
Blood ascorbic acid in the fasting state was obtained from 35 NSCLC patients; selecting from them 15 patients with stage III-IV entered the phase I -II study. They were randomized allocated into 3 groups, and received doses 1.0, 1.2, 1.5g/kgAA infusions. Pharmacokinetic profiles were obtained when they received solely IVAA at concentrations of 1g/kg, 1.2g/kg, and 1.5g/kg, and when IVAA in combination with mEHT. The process was applied 3 times a week (every other day, weekend days off) for 8 weeks. Participants in the first group received intravenous AA (IVAA) when mEHT was finished, in the second group IVAA was administered simultaneously with mEHT and in the third group IVAA was applied first, and followed with mEHT. DLT was defined as any reversible grade ≥3 adverse events, whether haematological or non-haematological. CT enhanced scan were collected from the patients before the treatment and one month after the treatment finished.

Result:
Fasting plasma AA levels were significantly correlated with stage of the disease. The overall toxicity was marginal.Thirstiness was the major symptom during all of the treatments.Peak concentration of AA were significantly higher in the simultaneous treatments than in other combinations with mEHT or in solely IV AA-managed groups. The average scores for the functioning scales continuously increased and the symptoms gradually decrease over the full cycle of the study.By using RECIST 1.1 criteria, two of four subjects diagnosed with lung squamous cell carcinoma had partial response (PR), two of four had stable disease(SD). Two of ten patients diagnosed with lung adenocarcinomahad had PR, three of ten had SD, five of ten had progressive disease.

Conclusion:
IVAA synergies with simultaneous mEHT were safe and can be well tolerated in patients. Patients diagnosed with squamous cell lung cancer were sensitive to the above two treatments.There’s a need for a phase II study with advanced NSCLC patients administering mEHT simultaneously with IVAA to assess for the efficacy and progression free survival time.

Background:
Lung cancer is the one of the highest incidence and fatality disease in the world wide. Although several of treatments have been found such as chemotherapy, radiotherapy and target therapy, lung cancer treatments remain many obstacles, for instance, drug resistant and gene mutant.

Method:
Epithelial-to-mesenchymal transition (EMT) process is a traditional pathway in cells program, during this process cells loss epithelial marker and acquire mesenchymal characteristic. EMT associates with cancer cell migration and invasion, more EMT process predicts poor prognosis and the resistant to chemotherapy even target treatment. More therapy target to EMT process such as some markers or signaling pathway. In this review, we discussed the new relationship of EMT and lung cancer. Some compounds which inhibited cancer cells acted with regulator of EMT or EMT process. Recent years, the target treatment and even the immune therapy were appeared in National Comprehensive Cancer Network guideline.

Result:
The effective treatment of lung cancer is correlated with EMT process. In vivo and in vitro research showed that occurrence of EMT process predicted poor prognosis and treatment failure in target and immune therapy.

Conclusion:
With the development of lung cancer treatment, EMT process may become a key research target.

Background:
This study aimed to evaluate the biological role of geranylgeranyl diphosphate synthase (GGPPS) in the progression of lung adenocarcinoma

Method:
GGPPS expression was detected in lung adenocarcinoma tissues by qRT-PCR, tissue microarray (TMA), and western blotting. The relationship between GGPPS expression and the clinicopathological characteristics and prognosis of lung adenocarcinoma patients was assessed. GGPPS was downregulated in SPCA-1, PC9, and A549 cells, using siRNA, and upregulated in A549 cells, using an adenoviral vector. The biological role of GGPPS in cell proliferation, apoptosis, migration, and invasion was determined by MTT and colony formation assays, flow cytometry, and transwell and wound-healing assays, respectively. In addition, the regulatory role of GGPPS on the expression of several EMT markers was determined

Result:
GGPPS expression was significantly increased in lung adenocarcinoma tissues compared to that in adjacent normal tissues. Over-expression of GGPPS correlated with patients with large tumors, high TNM stage, lymph node metastasis and poor prognosis. Knockdown of GGPPS inhibited migration and invasion of lung adenocarcinoma cells, but did not affect cell proliferation and apoptosis. Meanwhile, GGPPS inhibition significantly increased the expression of E-cadherin, and reduced the expression of N-cadherin and vimentin in lung adenocarcinoma cells

Conclusion:
Over-expression of GGPPS correlates with poor prognosis of lung adenocarcinoma, and contributes to metastasis through the regulation of EMT

Background:
The inherent challenges associated with lung tissue biopsies have spurred an enormous interest in the use of liquid biopsies. Pleural effusions are one such liquid biopsy which may enable lung cancer profiling and also assess if the patient suffers from a benign or malignant process in the lungs. Recently extracellular vesicles of endocytic origin, exosomes, have attracted interest as liquid biopsy of tumors since they can be used to look at tumor derived mutations as well as tumor cell activity represented by the RNA transcriptome. The RNA cargo carried in exosomes is known to resemble the RNA profile of the primary tumor. Here we aimed to analyze if microRNA and targeted cancer mRNA profiling of exosomes isolated from pleural effusions could decipher biomarkers associated with lung adenocarcinoma.

Method:
A systematic microRNA profiling of matured processed microRNAs along with targeted cancer mRNA profiling was carried out on extracellular vesicles, including exosomes, derived from 36 clinical pleural effusions, separated into 18 benign and 18 lung adenocarcinoma samples. Benign pleural effusion consisted of unspecific inflammation in the majority of cases. The two groups were well balanced with respect to age (median = 72Y) and smoking history (ever smokers in circa 70% of cases). However, males were overrepresented in the benign group (83% vs 44%). Both microRNA and mRNA profiling was conducted using TaqMan RT-qPCR Open Arrays (containing 800 genes each) followed by statistical ranking (Wilcoxon test) of differentially regulated transcripts between the two patient groups.

Result:
Systematic RNA profiling revealed a substantial, and highly significant (p<0.0001), elevated expression of all members from the extended miR-200 family in pleural effusions collected from patients with NSCLC adenocarcinoma. By cross-analyzing the obtained microRNA profiling data to the mRNA cancer panel expression, statistical enrichment between miR-200 family members and predicted miR-200 target genes, including PIK3CA, NOTCH1 and KRAS was observed (Fisher’s exact test, p=0.0105).

Conclusion:
Our study demonstrates the usage of exosomal RNA profiling from pleural effusions to define patients with lung adenocarcinoma and further highlights miR-200 microRNAs as diagnostic markers in lung cancer liquid biopsies. Acknowledgment: This study was supported from the following funding bodies: Swedish Cancer Society, Stockholm Cancer Society, Stockholm County Council, Knut and Alice Wallenberg Foundation and Erling Persson Family Foundation.

Background:
Assessment of several immune-genes and tumor drivers is critical for individualized treatment of non-small cell lung cancer (NSCLC). We have previously demonstrated the ability of the transcript-based nCounter Technology for the detection of ALK, ROS1 and RET gene fusions, using a customized codeset (Reguart et al. Clinical Chemistry 2017). Here, we present the first results of the validation in advanced NSCLC samples of a new CodeSet designed to simultaneously characterize clinically relevant gene fusions, MET alterations and the expression of immune genes.

Method:
We have designed an in-house custom set to detect driver fusions involving 4 genes (ALK, ROS, RET, NTRK), MET exon 14 skipping mutation, MET overexpression and immune genes (PD1, PDL-1, CD4, CD8, FOXP3, GZMM, IFNG). A panel of ALK-ROS-RET-NTRK positive cell lines (H2228, H3122, SU-DHL-1, HCC78, BaF3 pBABE, LC2/ad and a NTRK-positive cell line), Hs746T (METex14), EBC-1 (overexpressing MET) and a negative cell line (PC9) were used for the initial validation of the panel. Subsequently, 58 FFPE samples from advanced NSCLC patients, previously characterized by FISH, RT-PCR and IHC, have been analyzed. Total amount of 100-150 ng RNA was used for detection. Workflow includes RNA isolation, hybridization and digital counting with for a total turnaround of 3 days. Raw counts were normalized using positive controls, negative controls and house-keeping genes.

Result:
.Results obtained with the cell lines positive for ALK, ROS1, RET and NTRK1 fusion genes were exactly coincident with their genotypes, with fusion transcripts successfully detected in all cases by 3’/5’ imbalance and direct fusion probes. In addition, METex14 splicing transcripts were detected in the Hs746T cells at significant levels, higher than those of wt MET mRNA. In contrast, METex14 mRNA counts were almost undetectable in the rest of cell lines. Regarding FFPE samples from advanced patients, 46 could be successfully analyzed by nCounter. Among 13 patients positive for ALK and ROS1 fusions, 12 were confirmed by nCounter. Regarding the METex14 splicing variant, 5 out of 6 patients previously detected by RT-PCR were also positive by nCounter.

Conclusion:
Preliminary data suggest that multiplex detection of clinically relevant drivers can be successfully achieved using nCounter Technology. The assay is simple, requires short hands-on-time, needs low input RNA and is highly efficient in detecting gene rearrangements and METex14 splicing variants. Results will be prospectively validated in a larger cohort of advanced NSCLC patients and we will determine if clusters of different inmune-phenotypes exist among oncogenic-driven NSCLC tumors.

Background:
Recently, target therapy for cancer patients should be considered according to the individual mutational status. Therefore, detection of mutations is clinically important for patients’ outcome. Molecular testing of lung cancer is one of the most important standard of care and treatment. However, it is not always easy to get enough tumor tissue from patients. There is a promising novel mutation detection technology, which is PANA Mutyper. We compared effectiveness of both methods for the detection of KRAS mutation using tumor tissues, cell blocks and pleural effusions with patients with malignant pleural effusion.

Method:
: KRAS mutations were assessed by PANA Mutyper and PNA clamping using tumor tissues, cell blocks and pleural effusions. The diagnostic usefulness of two methods were analyzed.

Result:
A total of 104 patients with malignant pleural effusion were enrolled which includes 56 adenocarcinoma of lung, 11 squamous carcinoma of lung, 17 small cell lung cancer, 3 large cell lung cancer, 3 stomach cancer, 2 ovary cancer, etc. PANA Mutyper showed more superior detection rate than PNA clamping through tumor tissues, cell blocks and pleural effusions.

Conclusion:
PANA Mutyper had a diagnostic superiority for the detection of KRAS mutations in patients with malignant pleural effusion. This method can be used as more sensitive and accurate detection of KRAS mutations. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2014R1A2A1A11052422).

Background:
Never smoking Asian patients with lung adenocarcinoma are usually accompanied with recurrent occurring mutations of oncogenic drivers, such as EGFR, HER2, ALK fusions, ROS1 fusions etc. HER2 mutations were identified in approximately 2–4% of NSCLCs and these mutations were usually mutually exclusive with other driver mutations. Preclinical studies have suggested that overexpression of HER2 or mutations of the HER2 kinase domain are critical in oncogenic transformation and tumorigenesis. We found a novel HER2 755[PL] in-frame (also called HER2[PL]) mutation in a 52-year old never smoking lung adenocarcinoma patient. She did not have EGFR mutation. Patient was treated with a second generation of EGFR tyrosine kinase inhibitor (TKI), afatinib and had responded. However, the role of HER2[PL] mutation in lung tumorigenesis and its response to EGFR TKIs have never been addressed before.

Method:
We established a plasmid construct carrying a HER2 gene with HER2[PL] and transfected into normal murine fibroblasts, NIH/3T3 and human lung adenocarcinoma, NCI-H358 which express wide type EGFR. HER2 activation pathways were examined by western blots of phosphorylation of down-stream molecules with and without gefitinib and afatinib treatment.

Result:
Overexpression of HER2[PL] mutation can activate HER signaling pathways in both NIH/3T3 and NCI-H358. HER2[PL] mutation induces much higher phosphorylation of HER2 and downstream AKT signaling pathway compared to wide-type HER2. In addition, we found that HER2PL mutation can trigger HER2 signaling in ligand-independent manner and afatinib can significantly decrease HER2[PL] mutation-induced HER2 signaling pathway compared to a first generation of EGFR TKI, gefitinib. Furthermore, we found that the distribution of HER2[PL] mutation is in cytosol as well as on the membrane and the expression of p-HER2 (Tyr1221/1222) can be effectively attenuated with afatinib treatment in NCI-H358 stable lines using immunofluorescence assay. The cell growth and drug sensitivity to different generations of EGFR TKI in NCI-H358 lines transfected with HER2[PL] mutation are under investigation.

Conclusion:
This research may bring us new insights to understand the oncogenic significance of HER2[PL] mutation and be applied to relevant therapeutics.

Background:
This paper studies the quality of Life (QoL) of taiwan advanced non-small-cell lung cancer (NSCLC) patients receiving first-line target therapy; QoL data into sexual scores using the European Organization for Research and Treatment of Cancer Quality of Life-Core 30 questionnaire EORTC QLQ-C30 and the QLQ-13 in patients with NSCLC.

Method:
From March 2016 to March 2017, patients with in a teaching hospital in southern Taiwan were recruited as the research participants, the patients with advanced or metastatic EGFR mutation-positive NSCLC who received gefitinib, erlotinib, or afatinib as first-line treatment, were invited to complete the EORTC QLQ-C30 and QLQ-C13 on a 5 time visit, the data was analyzed by using SPSS 20.0 software.

Result:
A total of 30 patients with NSCLC, The global health status showed differences between QOL before Target therapy and 4 and 12 weeks after commencing therapy, compared to baseline. Quality of life-30 scores were 35.6 ± 8.3 before therapy, 38.1 ± 7 after 4 weeks and 43.4 ± 6.8 after 12 weeks (P <0.000)(Table 1). For the other scales, at 12 weeks, improvement of insomnia, Pain, Dyspnoea, Fatigue and Appetite loss, but worsening of diarrhea, Sore mouth and Dysphagia were observed (P <0.05).

Conclusion:
Longitudinal QoL assessments are important in advanced lung cancer patients because the data they provide could, for example, help to assess more patient areas and enable early recognition of arising symptom aggravation, there support for case management and health education.Figure 1

Abstract not provided

Background:
Although outcomes for early stage lung cancer are encouraging,overall survival with standard therapy is only 15-30% for nearly half of all patients with Stage III disease. The local recurrence is the main failure mode. We retrospectively analyzed the treatment of dose-painting radiotherapy with chemotherapy for the locally advanced non-small cell lung cancer.

Method:
Eligible patients had to have biopsy confirmed non-small cell lung cancer (NSCLC) and had stage III, T1-4N2-3M0 and T3N1M0 disease. All the patients received two 21-day cycles of cisplatin plus paclitaxel. The dose-painting target was defined using 18F-fluorodeoxyglucose (FDG) positron emission tomography in 47 patients with the non-small cell lung cancer. Doses of 70-80 Gy(3.5-4 Gy/fraction) and 60 Gy (3Gy/fraction) were prescribed to the PET-based planning target volume (PTVPET) and the union of PET and anatomical imaging-based PTV, respectively, in 20 fractions, using simultaneous integrated boost. After concurrent chemoradiotherapy, patients received an additional two cycles of consolidation chemotherapy.

Result:
Median follow-up time from the end of treatment was 62.1 months (range 37.6–111.5 months). All 47 patients completed treatment without interruptions and no incidents of early grade 4+ toxicity were observed. The incidence of Grade ≥2 radiation pneumonitis was 14.9% ,the incidence of Grade ≥ 3 esophagitis was 4.26%.the 1,3,5-year OS were 63.8% ,25.5%,12.1%. the 1,3,5 year disease-free survival rate were 48.9%,14.9%,8.5%. The1,3,5-year local-control rates were 64.7%, 32.6%,13%. Late radioactive esophagitis were seen in only 2 patients who experienced Swallowing obstruction. Only 1 patient was observed who has severe symptom of pulmonary fibrosis. Fatal bronchopulmonary hemorrhage did not occur.

Conclusion:
Dose-paiting radiation with concurrent chemotherapy is feasible and well tolerated. Local-control rates are encouraging.But further large-scale randomized controlled studies are needed to confirm it.

Background:
No predictive biomarker for cytotoxic chemotherapy is approved for clinical use. Schlafen family member 11 (SLFN11) protein is widely reported as sensitizing to DNA-damaging agents. Epigenetically mediated suppression of SLFN11 is associated with poor response to platinum in patients with ovarian and lung cancer. Pre-clinical lung cancer models suggest that SLFN11 expression may be a useful biomarker of response to cisplatin, PARP inhibitors and topoisomerase inhibitors. Tumor expression of SLFN11 is assessed by immunohistochemistry, RNA expression or DNA methylation; no standard method exists. We used mass spectrometry to quantify SLFN11 protein in archived samples of patients with early stage NSCLC treated with taxane plus platinum (TP) and correlated proteomic expression of SLFN11 with survival.

Method:
We obtained archived tissue sections representing 594 patients with lung cancers of multiple subtypes. A board-certified pathologist marked the tumor areas, which were microdissected and solubilized. In each liquefied tumor sample, 60 protein biomarkers including SLFN11 were quantified with selected reaction monitoring mass spectrometry. Patients were stratified by a SLFN11 cutoff of 100 amol/ug, based on the proteomic assay’s limit of quantification. Survival outcomes were assessed with Kaplan-Meier and Mantel-Cox log-rank analyses.

Result:
Among 86 TP-treated early stage NSCLC patients, those with SLFN11 protein levels above the cutoff (n=51) had better progression-free survival (PFS) than patients with SLFN11 levels below the cutoff (HR: 2.26; 95%CI: 1.08-4.72; p=0.052). Similar differences in PFS were found in the subset of patients with NSCLC (n=77) (HR: 2.79; 95%CI: 1.29-6.05; p=0.030). Differences in overall survival by SLFN11 expression were not statistically significant. In a group of untreated patients (n=440), there were no differences in PFS between patients with high and low expression of SLFN11.

Conclusion:
Mass spectrometric evaluation of SLFN11 retrospectively identified responders to platinum-containing chemotherapy and could be used to predict response for platinum-containing therapy and warrants further validation. Multiplexed proteomics can quantitate SLFN11 simultaneously with other therapeutically relevant proteins (eg, HER2, ALK, ROS1) to inform therapy selection at initial diagnosis and upon relapse.

Background:
Malignant mesothelioma is a highly aggressive asbestos related cancer with poor prognosis and its diagnosis and differentiatiation from various cancers is challenging. In addition to histological features, many positive and negative immunohisotchemical markers are needed to differentiate epitheioid mesothelioma from lung adenocarcinoma and/or squamous cell carcinoma. The positive mesothelial markers calretinin, WT-1, D2-40, CK5/6; positive lung adenocarcinoma markers, TTF-1, Napsin-A, Claudin-4, CEA; and positive squamous cell markers, P40, P63, CK5/6, MOC31 are routinely used. However, these markers are not sufficient and novel markers have to be identified.

Method:
Patients and Histologic Samples Pathological specimens (formalin-fixed paraffin-embedded tissue blocks) of 65 epithelioid mesothelioma and 60 lung adenocarcinoma and 57 squamous cell carcinoma were obtained from the archives of the Department of Pathology, Hiroshima University. All histological sections were reviewed and reclassified according to recent 2015 WHO classification and was confirmed by histologic findings and an immunohistochemical marker panel recommended by 2012 IMIG update to practical guidelines Immunohistochemical Procedures and Evaluation of Expression of MUC4 Immunohistochemical staining was performed using the Ventana Benchmark GX automated immunohistochemical station (Roche Diagnostics, Tokyo, Japan). Cells showing nuclear staining for calretinin, WT1, p40, p63, and TTF-1, cytoplasmic staining for MUC4 and napsin A, membranous staining for D2-40, MOC-31, and claudin-4 or membranous and/or cytoplasmic staining for CK5/6 and CEA were regarded as ‘positive’. Positive Immunoreactivity was semiquantified scored from 0 to 3+.

Result:
MUC4 positivity was present in 50/60(83.3%) cases, case of adenocarcinoma and 50/56(89.3%) cases of squamous cell carcinoma but none of 65 epithelioid mesotheliomas (0%). Among lung adenocarcinoma cases, 21 cases showed score 3+, 9 cases 2+ and 20 cases score +1. In lung squamous cell carcinoma, 21 cases score 3+, 10 cases score 2+ and 19 cases score 1+. The sensitivity and specificity of MUC4 to differentiate epithelioid mesothelioma from lung adenocarcinoma were 100% and 83.3% respectively with accuracy rate of 92%. Similarly, sensitivity and specificity of MUC4 to differentiate epithelioid mesothelioma from lung squamous cell carcinoma 100% and 89.3% respectively with accuracy rate of 95%. MUC4 expression showed sensitivity of 100%, but lower specificity of 86.2% and accuracy rate of 91.2% than CEA or Claudin-4 expression. However, it showed better sensitivity, specificity and accuracy rate than that of MOC-31.

Conclusion:
MUC4 is an additional negative immunohistochemical marker to differentiate epithelioid mesothelioma from lung adenocarcinoma and/or squamous cell carcinoma.

Background:
Adriamycin (ADR), a potent anticancer chemotherapeutic agent, is used to treat a variety of human neoplasms. However, its clinical use is hampered by severe side effects including cardiotoxicity. It has been reported that ADR-induced cardiotoxicity is related to myocardial oxidative stress, disruption of cellular and mitochondrial Ca[2+] homeostasis and DNA damage. Nevertheless, the remedy for ADR cardiotoxicity is still not developed. Here we describe the effect of NAD[+]/NADH modulation by NQO1 enzymatic action on ADR-induced cardiotoxicity in mice.

Method:
C57BL/6 male mice were intraperitoneally injected with ADR. Before and after exposure to ADR, the mice were orally administrated with WK0202, a substrate of NQO1, (20 mg/kg body weight of mice). Cardiac biomarkers (CPK, Trop I, LDH and SGOT) in plasma levels, oxidative biomarkers and mRNA levels of pro-inflammatory cytokines were determined to compare cardiac toxicity of each experimental group.C57BL/6 male mice were intraperitoneally injected with ADR. Before and after exposure to ADR, the mice were orally administrated with WK0202, a substrate of NQO1, (20 mg/kg body weight of mice). Cardiac biomarkers (CPK, Trop I, LDH and SGOT) in plasma levels, oxidative biomarkers and mRNA levels of pro-inflammatory cytokines were determined to compare cardiac toxicity of each experimental group.

Result:
Cardiac biomarkers in sera, oxidative biomarkers, and mRNA levels of pro-inflammatory cytokines were significantly increased in ADR-treated mice. However, these increases were significantly alleviated by WK0202. We also demonstrated that the downfall in SIRT1 and SIRT3 activities is critically involved in ADR-induced cardiotoxicity through acetylation of NF-κB p65 and p53. However, increase of NAD[+]/NADH by WK0202 through NQO1 enzymatic action attenuated ADR-induced cardiotoxicity through recovery of SIRT1 and SIRT3 activities and subsequent deacetylation of NF-κB p65 and p53. .

Conclusion:
WK0202 has a protective effect against ADR-induced acute cardiotoxicity through NQO1 enzymatic action. Therefore, WK0202 might be a new therapeutic option for preventing chemotherapy-associated side effects.

Background:
Many studies have shown that long non-coding RNAs (lncRNAs) are implicated in cancer progress including lung cancer. In our previous studies, we screened the differentially expressed lncRNAs between lung adenocarcinoma with lymph node metastasis and lung adenocarcinoma without lymph node metastasis by microarray analysis. XLOC_000090, an lncRNA without a known function, was identified. Here, we investigated the functions of XLOC_000090 in lung cancer.

Method:
The expression of XLOC_000090 was detected by qRT-PCR in 96 pairs of NSCLC tissues and the adjacent normal lung tissues. Then, we investigated the correlation between XLOC_000090 expression and clinicopathological variables and prognosis. Cell invasion and migration assay was used to detect cell invasion and migration ability in vitro. Murine model of lung cancer was used to detect the effect of XLOC_000090 on pulmonary metastasis in vivo. Dual luciferase reporter assay was used to determine the direct binding between XLOC_000090 and miR-4505.

Result:
Compared with normal lung tissues, XLOC_000090 expression was higher in NSCLC tissues (P<0.05). XLOC_000090 expression was associated with lymph node metastasis (P<0.05) and pathological stage (P<0.05). Patients with high XLOC_000090 expression exhibited significantly poorer disease-free survival and overall survival (P<0.05). XLOC_000090 overexpression increased tumor cell migration and invasion ability, whereas downregulation of XLOC_000090 expression decreased tumor cell migration and invasion ability in both A549 and Calu3 lung cancer cells. Murine model of lung cancer also showed that XLOC_000090 promoted metastasis of lung cancer cells in vivo. Furthermore, a potential XLOC_000090-targeting miRNA, miR-4505, was identified by ceRNA regulatory network prediction analysis. miR-4505 expression was significantly downregulated in lung cells transfected with XLOC_000090, and significantly upregulated in lung cells transfected with sh-XLOC_000090. Dual-luciferase reporter assay showed the direct binding between miR-4505 and XLOC_000090. XLOC_000090-promoted cell migration and invasion ability was diminished in miR-4505 overexpressed cells.

Conclusion:
Our results demonstrated that XLOC_000090 promoted the migration and invasion of lung cancer cells through regulating miR-4505. XLOC_000090 could be served as a new molecular marker for the progression and prognosis of patients with NSCLC.

Background:
The analysis of clinically relevant human tissue preserved in optimal cutting temperature (OCT) medium with activity based proteomic approaches are promising for the discovery of novel druggable disease biomarkers for the diagnosis, prognosis and prediction of response to therapeutic interventions. Nonetheless, and for many different proteomic approaches, there are important signal interferences observed in the presence of the OCT compound.

Method:
We tested activity based multiplex profiling tyrosine kinase substrates in a large batch of neoplastic and non-neoplastic lung resection specimen embedded with or without OCT. Since January 2003 we collected fresh frozen matched pairs from malignant adenocarcinoma and non-neoplastic lung biopsies. In 2007, we started to embedded all our samples in OCT. We obtained all clinical characteristics of 47 patients with early TNM stage 1 lung adenocarcinoma. We observed significant differences in overall phosphorylation levels and searched for reasons explaining such a large effect.

Result:
We ruled out the implication of either short versus long storage time after sample extraction or of nonhomogeneous batch processing of samples. We documented that the clear downward shift in overall phosphorylation levels coincided with the introduction of OCT as an improved embedding medium for resection specimen. For all the kinomes extracted, we developed a corrective procedure where a median centering was performed on the values of each peptide, separately for the with or without OCT samples.

Conclusion:
We applied corrective filtering of data to the multiplex profiling approach of well characterised tyrosine kinase substrates obtained in lung resection specimen embedded with or without OCT. With the OCT correction parameters applied, the quantitation of molecular prognosis signature based on tyrosine kinase activity differences found in lung adenocarcinoma resection specimens may result in the identification of novel targets for future anti-lung cancer therapies.

Background:
Chronic exposure to arsenic leads to the onset of different diseases, including lung cancer. Arsenic-induced lung tumors have been associated with a high-frequency of lung squamous-cell carcinomas among never smokers (a rare epidemiological pattern), suggesting a unique underlying biology. Epigenetic alterations are known to play a role in this process; however, detailed mechanisms are not yet fully elucidated. Piwi-interacting RNAs (piRNAs), a novel class of small non-coding RNAs (sncRNAs), play a key role in epigenetic regulation and maintenance of genome integrity. Here, we examine the impact of different arsenic species in the human piRNA transcriptome, using lung cell models mirroring chronic, low dose exposure. We also investigate the interaction network of deregulated piRNAs and identified biological pathways potentially affected.

Method:
One normal lung (HBEC) and two lung cancer cell lines: A459 (adenocarcinoma) and H520 (squamous-cell carcinoma) were grown in 10 ppm of sodium arsenite (AsIII) or arsenate (AsV) for six passages. Total RNA was extracted at different time points and sequenced. piRNA expression was deduced using our custom sncRNA analysis pipeline, which interrogates >23K piRNA-encoding human loci. piRNA/DNA binding prediction was performed using two different algorithms (miRanda/ThermoBLAST). Network analysis was performed using Partek Pathways.

Result:
Overall, 691 piRNAs were expressed. Persistent changes in piRNA expression over time were identified, with specific patterns associated with the different arsenic species. In HBECs (non-malignant lung tissue), 14 piRNAs were persistently upregulated and 16 downregulated in response to AsIII. Similarly, 6 were up- and 11 downregulated when the same cells were exposed to AsV. Only 1 piRNA, DQ598008, was commonly upregulated in response to both arsenic species, while 4 piRNAs were commonly downregulated. Lung cancer cell lines follow the same arsenic species-specific trends, with a high subtype-specificity indicating these species maintain a role during lung tumor development. Remarkably, we found an enrichment of genes associated with methyltransferase activities predicted to be targeted by piRNAs altered by AsIII (a biologically-relevant form of arsenic), evidencing their role in arsenic-related carcinogenic mechanisms.

Conclusion:
Arsenic induces persistent alterations in the lung sncRNA transcriptome, particularly piRNAs, impacting pathways linked to epigenetic regulation. Together, these results provide insights into sncRNA-related mechanisms in arsenic-induced lung carcinogenesis. Moreover, different arsenic species induce distinct alteration patterns, highlighting the relevance of the source of exposure. piRNAs, as with other sncRNAs, are stable in biofluids, circulating tumour cells, and archival clinical materials. Therefore, piRNAs hold great promise as potential exposure and monitoring biomarkers for arsenic-related health effects.

Background:
TP53 and DNA repair genes polymorphisms have been proposed as clinically significant cancer risk factors. The TP53 gene, coding for a known tumor suppressor, is found to be mutated in over 30% of human cancers. Impaired DNA repair efficiency caused by differences in expression, methylation and polymorphisms of DNA repair genes XRCC1 and Rad51 is likely to affect cancer occurence. This study aimed to evaluate the association of the TP53 Arg72Pro single-nucleotide polymorphism (SNP) (rs1042522), XRCC1 Arg399Gln SNP (rs25487) and Rad51 G135C SNP (rs1801320) with the occurrence of lung adenocarcinoma in Serbia, both individually and in combination.

Method:
This case-control study included 65 advanced lung adenocarcinoma patients treated at two Institutes in Serbia, stage IIIB or IV, and ECOG performance status 0, 1 or 2, and 68 healthy matched controls. All subjects were of Caucasian descent. TP53, XRCC1 and Rad51 genotyping was done by polymerase chain reaction followed by restriction length polymorphism (PCR-RFLP). Statistical analysis was performed using the Chi-square test and descriptive analyses included genotype and allelic frequencies. Deviations of the genotype frequencies from those expected under Hardy-Weinberg equilibrium were assessed using the χ2 test. The odds ratio (OR) and 95 % confidence intervals (CI) were also calculated as an estimate of relative risk, with significance set at p < 0.05 for all analyses.

Result:
The frequencies of XRCC1 alleles in patients vs. controls were 0.75 vs. 0.6 for Arg, and 0.25 vs. 0.4 for Gln. XRCC1 Arg allele was significantly associated with the development of lung adenocarcinoma only in the recessive model [p=0.019; OR (95% CI) = 2.47 (1.21 - 5.05)]. The frequencies of Rad51 alleles in patients vs. controls were 0.78 vs. 0.76 for G, and 0.22 vs. 0.24 for C. The frequencies of TP53 alleles in patients vs. controls were 0.52 vs. 0.63 for Arg, and 0.48 vs. 0.37 for Pro. We found no statistically significant associations of Rad51 and TP53 polymorphisms with lung adenocarcinoma. Investigating all possible gene-gene interactions, we also found no statistically significant associations with lung adenocarcinoma.

Conclusion:
In this study, homozygous carriers of the XRCC1 Arg allele were found to be more susceptible to the development of lung adenocarcinoma during lifetime. Thus, XRCC1 genotyping might be useful as an additional tool for predicting individual lung cancer risk

Background:
Mismatch repair (MMR) pathway is a fundamental cellular process required for the maintenance of genomic stability. Recently, a growing body of evidence suggests that a non-canonical MMR (ncMMR) function may be present as a source of mutations in human cells, which could lead to tumorigenesis. The dual functions of MMR with anti-mutagenic and mutagenic activities complicate its role in disease. Given that the functional role of MMR in lung cancer was rarely reported and is still controversial, the current study aimed to address the clinical significance of MMR proteins and their associations with therapeutic biomarkers in lung adenocarcinoma.

Method:
A panel of tissue microarrays containing 442 lung adenocarcinomas was examined for the expression of MMR proteins MLH1, PMS2, MSH2 and MSH6 using immunohistochemistry. The associations between MMR expression and clinicopathological features, patients’ survival as well as therapeutic biomarkers including EGFR mutation and PD-L1 expression were analyzed.

Result:
MLH1, PMS2, MSH2 and MSH6 protein expression significantly correlated with one another in lung adenocarcinoma (p<0.001). Neither age nor sex nor predominant histological pattern correlated with their expression, except that only MSH2 expression positively associated with the solid-pattern growth (p<0.05). Survival analyses showed that high expression of each MMR protein statistically correlated with poor patients’ overall and progression-free survivals (p<0.05). For therapeutic biomarker analysis, expression of MMR or PD-L1 independently associated with poor patients’ overall survival, but no significant correlation between their expression was observed. High expression of MLH1, MSH2 and MSH6 (p<0.05) but not PMS2 (p=0.12) was linked to poor survival in EGFR mutation-positive patients. To examine EGFR-TKI treatment response, the outcomes of 50 patients with EGFR mutation-positive tumors treated with EGFR-TKI were included for further analysis. Interestingly, patients with high expression of MLH1 in tumors showed a worse progression-free survival after EGFR-TKI treatment (n = 23, 13.8 months, 95% CI: 6.5 to 21.1 months) than those with low expression (n =27, 25.6 months, 95% CI: 10.9 to 40.3 months).

Conclusion:
MMR proteins are overexpressed in lung adenocarcinoma and significantly associate with poor patients’ survival, which may have a prognostic value in predicting patients’ drug response and clinical outcomes. Our results also raise a possibility that ncMMR function may participate in the tumorigenesis of lung adenocarcinoma.

Background:
Accumulation of mutations in normal-appearing lung tissue is believed to be important for development of lung cancer, and to be heavily influenced by smoking. However, their very low levels have been hampering their measurement, and their links with cancer risk and smoking history have not been demonstrated. To overcome this limitation, we recently developed a novel method that can measure levels of somatic mutations at 10[-6]/bp levels [Yamashita et al., Cancer Lett, online].

Method:
Eleven healthy lung tissues (Group1:G1) were collected from the normal lungs of metastatic lung cancer patients without smoking history, and 11 exposed lung tissues (Group2:G2) were collected from those with smoking history. 11 high-risk lung tissues (Group3:G3) were collected from the lungs of lung cancer patients with smoking history. A sequence library (15,552 bases of 291 regions of 55 cancer-related genes) was prepared by multiplex PCR using 100 DNA molecules, and was sequenced using a next generation sequencer.

Result:
The accumulation levels of mutations were significantly higher in G3 (2.7 ± 0.8×10[-5] mutations/base) than those in G1 (1.8 ± 0.5×10[-5] mutations/base) (p = 0.0189). The accumulation appeared to be associated with smoking history (OR = 3.2; 95 % CI = 0.54–18.98), and the C>T mutation, a signature reported in cancer tissues [Alexandrov et al., Science, 354:2016], was significantly more frequent in G2 than in G1. The GCC>GTC and CCC>CTC mutations, a signature of exposure to the nitrosamines contained in tobacco smoke, were significantly more frequent in G2 and G3.

Conclusion:
The accumulation level of mutations was increased in exposed lung tissues, and the mutation accumulation was associated with cancer risk.

Background:
The base excision repair (BER) pathway is responsible for removing damaged or incorrectly incorporated uracil bases in the genome. Mismatched bases that persist in the genome and remain unrepaired may result in either lethal mutations or cytotoxic DNA double strand breaks. Previous studies have determined that hSSB1 is critical for the detection, signaling and repair of cytotoxic double strand DNA breaks and oxidized DNA lesions within the genome. The role of hSSB2 is, however, less clear. In this study, we have identified that the single stranded DNA binding proteins, hSSB1 and 2, are involved in the detection and removal of uracils within the genome and function as part of the BER pathway.

Method:
We identified a novel role for hSSB1 and hSSB2 in BER. EMSA and incision biochemical assays were used to determine the ability of hSSB1/2 to bind uracil containing mismatches. Incision assays were used to determine the effect hSSB2 and hSSB1 have on UNG2 activity. Two cytotoxic drugs (5-fluorouracil and pemetrexed), which induce uracil misincorporation in the genome, were used to determine the cell sensitivity in control and hSSB1/2-depleted cells using a live and dead cell assay. Immunoprecipitation, immunofluorescence and Protein-Protein interactions were carried out to determine whether hSSB2 and hSSB1 interacts with key regulatory proteins of the BER pathway.

Result:
This study demonstrates that hSSB1 and hSSB2 proteins can recognize and bind to double stranded DNA substrates containing a uracil mismatch. Interestingly, we have identified that hSSB1 and hSSB2 have a differential preference for uracil mismatches, with hSSB1 preferentially binding UA and hSSB2 UG mismatches. Furthermore, hSSB2 induces the incision activity of UNG2 by approximately two fold for a U:G mismatch but not a U:A mismatch. A549 lung adenocarcinoma cells depleted of both hSSB1 and hSSB2 are hypersensitive to 5-fluorouracil and pemetrexed. Loss of either hSSB1 or hSSB2 alone by siRNA results in a compensatory upregulation of hSSB2 or hSSB1 respectively, suggesting over-lapping functionality and substrate specificity.

Conclusion:
This study highlights the importance of hSSB2 and hSSB1 in the removal of uracil from the genome. Currently, pemetrexed and fluorouracil based agents are in use for treating lung cancer. This study raises the possibility that hSSB2 and hSSB1 may be biological indicators of response to fluorouracil and pemetrexed. Further, it may be possible to develop future hSSB2/hSSB1 inhibitors that could enhance the activity of these agents in the treatment of lung cancer.

Background:
Alterations in the DNA repair capacity of damaged cells is now recognised as an important factor in mediating resistance to chemotherapeutic agents.

Method:
DNA Repair Pathway RT[2 ]Profiler Arrays were used to elucidate key DNA repair genes implicated in chemoresistant NSCLC cells using cisplatin resistant (CisR) and corresponding parental (PT) H460 cells previously established in our laboratory. DNA repair genes significantly altered in CisR cells were validated at the mRNA and protein level, using RT-PCR and Western blot analysis, respectively. The translational relevance of differentially expressed genes was examined in a cohort of chemo-naïve matched normal and tumour lung tissues from NSCLC patients. Loss of function studies were carried out using siRNA technology. The effect of XRCC6BP1 gene knockdown on apoptosis was assessed by FACS using Annexin-V/PI staining. Cellular expression and localisation of XRCC6BP1 protein and H2AX foci in response to cisplatin were examined by immunofluorescence using the Cytell Imaging System. To investigate a role for XRCC6BP1 in lung cancer stem cells, Side Population (SP) studies were used to characterise stem-like cells in a panel of chemoresistant cell lines. XRCC6BP1 mRNA analysis was also examined in ALDH1[+] and ALDH1[- ]subpopulations. Immunohistochemistry analysis was carried out on a cohort of resected lung tumour tissues (n=20) and XRCC6BP1 expression was correlated with clinicopathological parameters.

Result:
We identified a number of critical DNA repair genes that were differentially regulated between H460 PT and CisR NSCLC cells, where XRCC6BP1 mRNA and protein expression was significantly increased (mRNA; 19.4-fold) in H460 CisR cells relative to their PT counterparts. Relative to matched normal lung tissues, XRCC6BP1 mRNA was significantly increased in lung adenocarcinoma patients. Gene silencing of XRCC6BP1 induced significant apoptosis of CisR cells and reduced the DNA repair capacity of these cells relative to scrambled (negative) controls. Immunofluorescence studies showed an increase in XRCC6BP1 protein expression and H2AX foci in CisR cells relative to their PT counterparts. While SP analysis revealed a significantly higher stem cell population in CisR cells, XRCC6BP1 mRNA expression was considerably increased in SKMES-1, H460 and H1299 ALDH1[+] CisR cells compared to ALDH1[-] cells. Data analysis of XRCC6BP1 immunohistochemistry is currently ongoing.

Conclusion:
We identified XRCC6BP1 as key DNA repair gene implicated in cisplatin resistant NSCLC. Our data highlight the potential of targeting components of the DNA repair pathway in chemoresistant lung cancer, in particular XRCC6BP1, either alone or in combination with conventional cytotoxic therapies.

Background:
We hypothesised that some lung cancers have DNA repair alterations that either are therapeutically targetable, or that result in resistance to particular DNA damaging therapies. Expression profiling of DNA repair genes may thus enable better matching of patients with the current chemotherapeutic options. Furthermore, profiling may identify tumours that will be more responsive to other DNA repair-directed therapies not normally used in treating NSCLCs e.g. PARP or CDKN4/6 inhibitors.

Method:
We tested RNA of more than 166 samples from tumour cores of 107 patients and 12 normals on the Nanostring platform. We developed a new approach to normalising Nanostring data based on the RUV (removing unwanted variation) method so that we could better identify differences between the patients.

Result:
Many interesting findings emerged indicating some new therapeutic options. The conclusions obtained from Nanostring analysis were verified by examination of the TCGA lung adenocarcinoma RNA-Seq data.

Conclusion:
This is the first systematic study of DNA repair gene deficiencies in NSCLC. There are important implications for the rational use of chemotherapy and radiotherapy. This work was funded by Cancer Australia.

Background:
Ataxia telangiectasia-mutated (ATM) is a critical first responder to DNA damage in the cell, but despite being one of the most mutated genes in lung cancer, no specific mutation hotspots have been linked with disease development. Our own quantitative analysis of ATM protein levels in patient samples suggests that ATM is lost in 20-25% of cases and that this loss correlates with poor overall survival and increased response to adjuvant chemotherapy treatments. We believe that this may be the result of increased genomic instability within the cancer cells caused by a lack of adequate DNA repair. Given that ATM-deficient cancers may have higher genetic instability, and that ATM is so highly mutated in lung cancer, we sought to quantify the relationship between ATM mutations and genomic instability, as measured by somatic mutation burden.

Method:
Using genomic and sequencing data available from publically available databases including the Broad Institute Cancer Cell Line Encyclopedia (CCLE) and the NIH Cancer Genome Atlas (TCGA), we correlated mutations in ATM and other genes involved with the DNA damage response with the total number of mutations annotated in ~900 cancer cell lines and ~200 lung adenocarcinomas.

Result:
We show that in cell lines across all cancer types, and particularly in lung, breast, and esophageal cancers, mutations in ATM correlate with a significantly higher number of total mutations. Only mutations in the direct damage response genes appeared to associate with total mutations, whereas p53 – while more commonly mutated – did not correlate with higher mutations in cell lines or patients. In lung cancer patients, however, neither ATM mutations nor ATM protein levels were similarly correlated with higher mutation burden.

Conclusion:
We have identified a potential relationship between ATM mutation and total somatic mutations in cancer cell lines which may be indicative of overall genetic instability. Analysis of the ATM mutations in both cell lines and patient samples clearly shows that there are no specific hotspots for mutation in ATM that correlate with increased total mutations. Thus screening for ATM mutations alone may not be sufficient to indicate loss of function or instability. However, this data may prove useful in developing panels of targets to screen as mutation hotspots of instability, and ultimately to help identify patients that may benefit from targeted or modified therapy options based on ATM-deficiency or higher genetic instability.

Background:
TGFalpha, one of the EGFR ligands (EGF, TGFA, AREG, EREG, HBEGF, BTC and EPGN), is known to be associated with poor survival in human lung adenocarcinoma (Tateishi et al., Cancer Res 1990). However, it remains unknown whether TGFalpha promotes EGFR-mutant lung adenocarcinoma and/or KRAS-mutant lung adenocarcinoma in vivo.

Method:
In order to understand the role of TGFalpha in lung cancer in vivo, we developed transgenic mice that conditionally induce mutant EGFR (Politi et al., Gene Dev 2006) or mutant KRAS (Fisher et al., Gene Dev 2001) along with TGFalpha (Hardie et al., Am J Physiol Lung Cell Mol Physiol 2004) in lung epithelium and analyzed the survival and histology of the mice. Based on the mouse study, we also investigated the association of TGFalpha with EGFR mutation or KRAS mutation in human lung cancer using TCGA databases (TCGA, Nature 2012; 2014), lung cancer cell lines and lung cancer specimens.

Result:
TGFalpha enhanced the growth of EGFR-mutant lung tumors but not that of KRAS-mutant lung tumors in the transgenic mice. The growth of EGFR-mutant lung tumors enhanced by TGFalpha was accompanied by the expression of two key tumor-promoting regulators p63 (a marker for airway basal cells and lung squamous cell carcinoma cells) and AGR2 (disulphide isomerase). TGFalpha was associated with poor survival in EGFR-mutant lung adenocarcinoma but not in EGFR wild-type adenocarcinoma (e.g., KRAS-mutant lung adenocarcinoma) in human lung cancer. Although osimertinib and brigatinib have been shown to be clinically and preclinically effective for the treatment of gefitinib and erlotinib-resistant EGFR-mutant lung adenocarcinoma, including EGFR.T790M or EGFR.C797S (Goss et al., Lancet Oncol 2016; Mok et al., N Engl J Med 2017; Uchidori et al., Nat Commun 2017), the history of moleculary-targeted therapy for EGFR-mutant lung adenocarcinoma indicates that lung tumor clones resistant to osimertinib and brigatinib would likely emerge. Our results suggest that blocking EGFR ligands (e.g., TGFalpha and/or EGF) may provide therapeutic benefit to treat such drug-resistant EGFR-mutant lung adenocarcinoma. However, such a strategy may not work for KRAS-mutant lung adenocarcinoma.

Conclusion:
TGFalpha (an EGFR ligand) promoted growth of EGFR-mutant lung tumors but not that of KRAS-mutant lung tumors in vivo. Importantly, the growth of EGFR-mutant lung tumors promoted by TGFalpha was accompanied by the expression of p63, which may suggest initiation of lung tumor lineage alteration from adenocarcinoma (p63 negative) to adenosquamous carcinoma (p63 positive).

Background:
Lung cancer is the most prominent cancer in human with high mortality rate. Although chemo-radiation therapies have been improved, the patient survival is still poor. Thus, not only developing therapeutic medications, but also biomarkers of early diagnosis have been desired. Several models of primary lung cancer research are in use, however, systematic evaluation and characterization of models are required to have suitability and relevance based on study aims. To provide research environment for lung cancer, we reappraise relevant models for lung cancer.

Method:
Three concepts of primary lung cancer models were evaluated: (I) Urethane induced lung tumor. (II) Cell line induced tumor model via intravenous (iv) or subcutaneous (sc) injection. (III) ex vivo 3D primary cell culture model. 20 weeks after urethane injection, the animals were harvested to analyze tumor incidence and tumor immunity. Lewis Lung Carcinoma (LLC) cell line was employed for the orthotopic development of lung tumor in 2 weeks after the injection. Hanging drop method was used for the 3D culture of primary cells from LLC sc induced tumor. Immunohistochemistry (IHC) of proliferation markers (pH3 and Ki67), tumor immunity (CD4, CD8, B220, F4/80, NKp46, and PDL-1) were performed for a finer characterization of tumors.

Result:
Urethane and iv injection of LLC cell line developed heterogeneously distributed tumors in lung. sc injection stably developed single tumor nodule. 3D cultured primary cells formed spheroids within 5 days. IHC revealed that all tumors were consistently proliferating with less extend in urethane and 3D model. F4/80[+ ]cells and CD4[+] cells infiltrated into tumors significantly more than CD8[+], B220[+], or NKp46[+] cells. T cell populations (CD4[+] and CD8[+]) were much more prominent in LLC iv model than other models. Interestingly the expression of PDL-1 was found only in 3D model.

Conclusion:
The urethane-induced primary lung tumor is reliable with a high rate of development, but needs longer time period to develop tumor compared to iv and sc models. iv injection model develops lung tumor in the original location. With relatively more convenience, sc model allows the analysis of tumor without adjacent tissue bias. 3D primary cell culture model enable for conferring characterization of individual tumor and strategic design of therapy, namely personalized medicine. The involvement and characteristics of immune cells found within tumors were comparable across all models. Injections by i.v. or s.c. of cell line to mouse can be considered as an alternative yet convenient model to develop various different types of lung cancers.

Background:
Effective non immune- oncology targeted therapies are available only for less than 25% (non-Asian) and 60% (East Asian) of lung adenocarcinoma (ADC) patients. ADC has one of the largest burdens of genetic abnormalities among all cancers. It is understood that ADC arise from the accumulation of abnormalities which dysregulate key cellular processes to permit a growth and survival. Further improvement in our ability to develop novel therapies requires additional lung cancer models that closer mimic the genetic alterations found in patient tumors. Three-dimensional organoid culture (“mini organs”) of tumor cells recently has generated great interest as such a novel preclinical model.

Method:
We experimented to develop novel media formulation to generate organoid models from 10 established ADC cell lines, 7 primary culture ADC cell lines developed from patient-derived xenograft (PDX) models, 8 ADC PDX models, and 20 resected patient ADC tissues. Organoid cultures that could be serially passaged for at least 5 passages were defined as long-term organoid models. Organoids were characterized for their histopathological features and immunomarker expression (p40, TTF-1, p53), growth rate and drug sensitivities.

Result:
Long-term organoid cultures were developed from 9/10 (90%) of established ADC cell lines, 3/7 (42%) of PDX-derived cell lines, 2/8 (25%) of ADC PDX models, and 1/20 (5%) of primary patient ADC tissues. Established organoid cultures recapitulated the histological features of ADC. We are currently collecting the data on growth rates and drug sensitivities of selected organoid cultures.

Conclusion:
Lung adenocarcinoma organoid cultures can be established for both established cell lines and patient tumor tissues but with variable success rates. Further studies are necessary to understand the discrepancy in the establishment rates from different sources of the tumor cells.

Background:
Previous studies demonstrated that freely floating cancer cells (FFCCs) in the lymph node sinuses were of prognostic significance for colorectal and gastric cancers. The goal of the study is to assess the clinical significance of detecting FFCCs using Fast Red staining for cytokeratin in both stage I/II lung cancer patients and hilar lymph node positive lung cancer patients who underwent curative resection.

Method:
Between 2002 and 2011, a total of 167 patients ( including 23 hilar lymph node positive patients) were enrolled. Resected lymph nodes were stained for cytokeratin in order to achieve a clear distinction from coal dust. An anti-cytokeratin antibody was labeled with a secondary antibody conjugated with alkaline phosphatase, which was detected by a reaction with Fast Red/naphthol that produced a red color. Patients were considered to be positive for FFCCs (FFCC+) if more than one freely floating cytokeratin-positive cell was detected in the lymph node sinuses.

Result:
Among all 167 patients, a significant difference was observed in Five-year relapse-free survival rates (5Y-RFS), with 75.9% and 31.6% being achieved by FFCC- and FFCC+ patients, respectively (P<0.001). Similarly, the 5-year overall survival rate (5Y-OS) was significantly lower in FFCC+ patients, with 85.4% being achieved by FFCC- and 62.3% by FFCC+ patients, respectively (P=0.007). Among 23 hilar lymph node positive patients, a significant difference was also observed in 5Y-RFS, with 50.0% and 0.0% being achieved by FFCC- and FFCC+ patients, respectively (P=0.013). The 5Y-OS tended to be lower in FFCC+ patients, with 64.3% being achieved by FFCC- and 53.3% by FFCC+ patients, respectively (P=0.595).

Conclusion:
The presence of FFCCs in stage I/II lung cancer patients was associated with a poor prognosis. In addition, FFCCs in hilar lymph node positive patients also have potential as a useful marker foreseeing the recurrence.

Background:
Patients with completely resected lung adenocarcinomas smaller than 2 cm have a good prognosis, though some develop recurrence. It has been shown that Noguchi’s classification and WHO pathologic type are correlated with prognosis. We examined the correlation of number of cancer cells/mm[2 ]in adenocarcinoma specimens with prognosis, Noguchi’s classification, and WHO pathologic type.

Method:
Primary tumors were obtained from 104 patients who underwent surgery from January 2006 through December 2010 and had pulmonary adenocarcinomas ≤2 cm in maximum diameter. This prognostic investigation was performed in November 2016. All specimens were stained with hematoxylin-eosin and evaluated using Noguchi’s classification and WHO pathologic type. We also determined the number of cancer cells/mm[2] in the specimens, with those findings evaluated using receiver operator characteristic (ROC) curve analysis and tumor size. Overall survival curves were produced using the Kaplan-Meier method and analyzed using a log rank test according to number of cancer cells, Noguchi’s classification, and WHO pathologic type. The relationship among those 3 parameters was investigated with Pearson’s correlation coefficient. A p-value <0.05 was considered to indicate significance.

Result:
At the time of the examination, 90 patients were alive and 14 had died due to lung cancer. The average number of cancer cells/mm[2] was 791.3 (range 129.4-1739.5) and that was strongly correlated with progression of Noguchi’s grade (correlation coefficient 0.519, p<0.001) and WHO pathologic type (correlation coefficient 0.436, p<0.001). ROC curve analysis established a cut-off of 992/mm[2]. In general, cases with cancer cells above the cut-off had worse prognosis (5-year survival 64.0% vs. 94.2%, p<0.001). Figure 1



Conclusion:
The number of cancer cells/mm[2] in lung adenocarcinoma specimens was found to be correlated with Noguchi’s classification and WHO pathologic type, and is considered useful for prognostic evaluation of affected patients.

Background:
Anomalies in the family of Notch receptors (1, 2, 3, and 4) have been implicated in a range of solid tumors, including lung cancer. There is growing evidence that Notch plays an oncogenic and tumor suppressor role in adenocarcinoma and lung squamous cell carcinoma, respectively. Gaining a complete understanding of the mechanisms underlying these opposing activities in lung cancer is key to the development of novel targeted therapy approaches.

Method:
The Cancer Genome Atlas (TCGA) datasets were used to look at gene co-expression patterns of Notch in lung adenocarcinoma and lung squamous cell carcinoma. Biological pathways implicated by gene families were assessed using functional annotation tools (DAVID, ToppGene, and IPA). In vitro and in vivo knockdown studies assessed the functional role of Notch in lung cancer.

Result:
Co-expression analysis supports the hypothesis that Notch is co-expressed with different genes in lung adenocarcinoma and squamous cell carcinoma. Knockdown of Notch in vitro and in vivo support our in silico finding of opposing effects of Notch. Our analysis implicates genes associated with metabolic pathways, angiogenesis and cell cycle that may underlie the differential role of Notch in lung adenocarcinoma and squamous cell carcinoma.

Conclusion:
These results support the hypothesis differences in the Notch co-expression may underlie its opposing roles in lung adenocarcinoma and squamous cell carcinoma. These finding help unravel the context dependent role of Notch as an oncogene and tumor suppressor in subtypes of lung cancer. Understanding the similarities and differences in co-expression patterns can improve our understanding of the regulatory mechanisms of Notch and strategies for its clinical development as single agent or in combination.

Background:
Invasive mucinous adenocarcinoma (IMA) is a variant of lung adenocarcinoma. Growth pattern such as lepidic, acinar, papillary and micropapillary can be seen in IMA. However, no study regarding prognostic and clinicopathologic aspects of IMAs with different growth pattern has been reported.

Method:
From January 1999 to July 2011, of 2236 patients with newly diagnosed primary lung adenocarcinoma, 16 patients were identified as lepidic-predominant IMA and 10 patients as acinar-predominant IMA. Data regarding the clinicopathological characteristics, CT features and prognosis was collected.

Result:
No statistically significant difference was noted in gender, age as well as smoker proportion between lepidic-predominant and acinar-predominant IMA. There was no statistically significant difference in T classification. The proportion of lymph node metastasis was significantly higher in acinar-predominant IMA (P=0.046). Both the tumors shared many signs in CT findings. Air-bronchgram was a relatively specific sign for lepidic-predominant IMA. Survival analysis showed that lepidic-predominant IMA also have a more favorable outcome than acinar-predominant IMA (P=0.0294). Figure 1Figure 2





Conclusion:
Lepidic-predominant and acinar-predominant IMA are two different subtypes of IMA. Acinar-predominant IMA is associated with lymph node metastasis and has a poorer prognosis than lepidic-predominant IMA.

Background:
Tumor hypoxia is characterized by necrosis and a low microvessel density (MVD). Necrosis and MVD are invaluable tools for predicting survival outcomes in non-small-cell lung cancer (NSCLC). Furthermore, hypoxia increases the extent of resistance to gefitinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, in lung adenocarcinomas. However, the invasive nature of tumor sampling remains a major clinical obstacle. [18]F-fluorodeoxyglucose positron emission tomography ([18]F-FDG PET) accumulation is a well-validated in vivo measure of tissue glucose metabolism and hypoxia. We hypothesized that [18]F-FDG PET could identify tumor necrosis of, and quantify the MVD in lung adenocarcinoma. Therefore, we investigated the relationship between preoperative [18]F-FDG PET parameters and tumor necrosis and MVD. Hypoxia biomarkers including glucose transporter type 1 (GLUT1), carbonic anhydrase IX and vascular endothelial growth factor were also evaluated.

Method:
Data on 164 patients who underwent the surgical resection of pulmonary adenocarcinomas were retrospectively reviewed. Preoperative [18]F-FDG-PET data, the extent of tumor necrosis, and immunohistochemical measures of the expression of GLUT1, carbonic anhydrase IX, vascular endothelial growth factor, and CD31 for detecting MVD were evaluated. The associations between PET parameters and the levels of pathological markers, and the prognostic significance of necrosis, were evaluated.

Result:
The standardized uptake value (SUV), metabolic tumor volume (MTV), and total lesion glycolysis (TLG) level were significantly lower in patients exhibiting no necrosis compared to those with partial or diffuse necrosis. When we divided the patients into two groups based on high vs. low PET parameter values, elevated SUVmax, MTV, and TLG values were significantly more associated with partial or diffuse necrosis than were lower values (p<0.001). A negative correlation was evident between the MVD and SUVmax (p=0.002), MVD and MTV (p=0.038), and MVD and TLG (p=0.019). GLUT1 expression correlated with high PET parameter values, a low MVD, and the presence of necrosis. Patients without necrosis exhibited better 5-year recurrence-free survival and overall survival than patients with necrosis (p<0.001 and p=0.007, respectively). However, a multivariate analysis revealed that necrosis was not of prognostic significance.

Conclusion:
High-level FDG accumulation predicted tumor necrosis. Clinicians can thus predict prognosis and improve treatment policies by reference to PET parameters.

Background:
Primary pulmonary synovial sarcoma is rare, comprising 0.5% of all primary lung malignancies, and spontaneous regression, defined as tumor disappearance without treatment, is very unusual.

Method:
This is a case report of a primary pulmonary synovial sarcoma showing spontaneous regression. The clinical and pathologic records were reviewed, and histologic analysis of the resected specimens was performed.

Result:
Clinical summary: A 38-year-old woman had no history of smoking and no respiratory symptoms. Chest computed tomography revealed a well-demarcated peripheral part-solid nodule measuring 3.8cm in the right lower lobe. Transbronchial biopsy was performed and the diagnosis was synovial sarcoma (SYT-SSX1 variants). She underwent thoracoscopic right lower lobectomy and systematic lymph node dissection. Pathological findings: The cut surface of the resected specimen showed a smooth walled cyst measuring 2.7 × 2.0 cm containing necrotic tissue. The histological examination revealed a widespread coagulative necrosis of tumor cells with peripheral granulation. Only a few regenerated residual tumor cells were observed.

Conclusion:
This is the first report of spontaneous regression of primary pulmonary synovial sarcoma. Although the mechanism is unknown, blood flow obstruction after the transbronchial biopsy may affect the tumor regression.

Background:
About 90% of invasive lung adenocarcinoma cases contain several histologic subtypes and demonstrate heterogenous histologic patterns. How such histological heterogeneity is formed remains unclear. The histological subtypes are associated with the prognosis in lung adenocarcinoma patients. Understanding the molecular mechanisms contributing to the pathological subtypes may provide a basis for developing new therapeutic strategies. Tumor microenvironments (TME) including endothelial cells, immune cells, and fibroblasts, have all been recognized as key components regulating cancer progression. TME influences tumor cells via direct cell-cell contact or their products, such as cytokines and extracellular matrixes. In this study, we determined the role of TME in the histological heterogeneity of lung adenocarcinoma.

Method:
We inoculated GFP-labeled A549 human lung adenocarcinoma cells into tissues in four different sites of immunodeficient mice including; the pleural cavity, subcutaneous region, intracardial, and the renal capsule. We then compared the histopathological features of those xenograft tumors. We established immortalized αSMA-positive cancer associated fibroblasts (CAFs) from the xenograft tumors, and co-cultured them with A549 cells in 3D culture conditions so as to analyze the interaction between the tumor cells and the stromal cells.

Result:
We found that the xenografted A549 cells developed distinct histological types of tumors; solid types and acinar types, depending on the inoculated sites. The solid type tumors contained an abundance of acidic mucins stained with Alcian blue, and they expressed MUC5AC, which is one of the common mucin core proteins. The acinar type tumors showed gland-like structures encircled by stromal cells. We found that the phosphorylation of Smad3 were upregulated in the acinar type tumors, especially αSMA-positive CAFs. Smad3 is the downstream of the transforming growth factor-β (TGF-β) signal. These data indicate that the TGF-β/Smad pathway is activated in acinar type tumors. CAFs derived from acinar type tumors induced acinar formations of A549 cells under in vitro 3D culture conditions. We also found that the inhibitor of TGF-β receptor I suppressed such acinar formations, suggesting that TGF-β signaling is associated with the histological subtypes of lung adenocarcinoma.

Conclusion:
Our data show that the histological heterogeneity of lung adenocarcinoma is dependent on TGF-β signaling mediated by the αSMA-positive CAFs. Inhibition of TGF-β signaling might block interactions between cancer cells and αSMA-positive CAFs, and TGF-β signaling inhibitors might suppress tumor heterogeneity in lung adenocarcinoma.

Background:
Tumor size has been recognized as an important prognostic factor. In renal tumor, CT imaging generally overestimates pathological tumor size. However, systematic correlation of tumor size between pathology and radiology in lung cancer has not been studied yet. Herein, we compared the maximal tumor diameter between surgically resected fresh lung tissue and chest CT.

Method:
Our study included 75 surgically resected nonsmall cell lung cancer specimens submitted in a fresh state. Pathologic tumor size (PTS) was obtained by measuring the largest cross-sectional tumor diameters in the fresh specimen. Radiologic tumor size (RTS) was retrospectively measured in axial (RTSax) and reconstructed oblique (RTSrecon) image of chest CT. Tumors larger than 4 cm, tumors with uncertain margins owing to underlying lung fibrosis or pneumonia, and tumors sectioned in formalin-fixed state were excluded.

Result:
The mean PTS, RTSax, and RTSrecon with standard deviation were 2.1cm ± 0.815, 2.068cm ± 0.822, and 2.26cm ± 0.871, respectively. PTS and RTrecon was significantly different (PTS-RTSrecon: -1.179±0.404, p<0.001), while there was no statistically significant difference between PTS and RTSax. Scatter plot and linear regression analysis demonstrated a strong positive correlation between PTS and RTS (PTS = 0.395+0.824xRTSax, p<0.001; PTS=0.233+0.818xRTSrecon, p<0.001). The intraclass correlation coefficient (ICC) between PTS and RTSax was 0.832 (95% confidence interval, 0.747-0.890). The ICC between PTS and RTSrecon was 0.884 (95% confidence interval, 0.822-0.925). There was no significant difference between PTS and RTS associated with histologic subtype, specimen type, warm ischemic time, predominant lepidic histology, pleura invasion, and gross feature.

Conclusion:
Both RTSax and RTSrecon were significantly correlated with PTS. Reliability analysis showed that RTSrecon correlated with PTS slightly better than RTSax, although RTSrecom tended to overestimate PTS. Further study with larger sample size would be needed.

Background:
Primary epithelial-myoepithelial carcinoma (EMC) of the lung is extremely rare carcinoma of salivary type lung cancer. Only 16 cases were reported for peripheral pulmonary EMC in literatures in the world, at present.

Method:
We report a resected case of primary peripheral EMC of the lung existed in right S2 with considerations of literature.

Result:
The patient was 63-year old male, received medical check-up at nearby clinic and an abnormal shadow was pointed out on chest X-ray film. He was referred to our hospital for more detailed examinations and therapy. He had no past history, symptoms nor abnormal physical findings. Chest CT scan showed a 56 x 24 mm mass in right S2. Transbronchial lung biopsy (TBLB) was performed and the tumor was diagnosed as adenoid cystic carcinoma. There was no metastasis to other organs in detailed examinations. Right upper lobectomy and lymph node dissection (ND2a-2) was performed. He was discharged our hospital at 8 post operative days with no post-operative complications. Macroscopically the tumor was 32x30x22 mm in size, white-colored, homogeneous, and well-defined surrounding pulmonary substance. Microscopically the tumor was composed of double tubular layers. Inner tubular layer showed epithelial cell characteristics, whereas the outer layer showed myoepithelial cell characteristics. Immunohistochemical examinations revealed positive for cytokeratin AE1/3 at inner tubular layer, and positive for SMA, p63, and calponin at outer tubular layer. Finally, the diagnosis of the tumor was determined as peripheral pulmonary EMC. There were no metastases in dissected lymph nodes. Epidermal growth factor receptor (EGFR) was wild type and anaplastic lymphoma kinase (ALK) was negative. Pathological stage was IB because of T2aN0M0. He was followed up with no adjuvant therapy, and disease free for 2 years after operation.

Conclusion:
Primary salivary gland type tumors of the lung are rare. Among them, primary peripheral EMC is extremely rare. Differential diagnoses of pulmonary EMC include mucoepidermoid carcinoma, adenoid cystic carcinoma, pleomorphic adenoma and so on. There is a possibility of misdiagnosis in small specimens. EMC of the lung is regarded as a low-grade malignant neoplasm. For diagnosis of EMC, it is necessary to obtain large specimen and/or resect by operation. In this paper, we report a completely resected case of primary peripheral EMC. It is thought that a biological characteristic of EMC will be elucidated by the further accumulation of EMC case.

Background:
The aim of this study was to retrospectively analyze the clinical data of resected Adenosquamous lung cancer (ASLC) and to explore the influencing factors and clinicopathological characteristics of the metastasis lymph nodes.

Method:
A total of 1156 consecutive patients with surgically resected lung cancer in the three institutions from January 2009 to June 2014 were studied. Fifty-four previously diagnosed ASLC patients were re-evaluated by experienced pathologists. IHC and H&E staining were employed to examine the primary focus and metastasis lymph nodes. The relationship between lymph node metastasis and clinicopathological characteristics of ASLC patients was then analyzed and the pathological type of metastasis lymph node was also determined.

Result:
Thirty-seven cases of typical ASLC were included in the study. Of the 37 ASLC patients, 20 cases presented lymph node metastasis. Lymph node metastasis was not associated with gender, smoking, tumor distribution, histological type of primary focus, and preoperative CEA level, but was associated with age (p=0.023) and tumor size (p=0.012). Lymph node metastasis was more frequently (90% of lymph node metastasis patients) presented in patients <65 yrs, and only 2 patients aged ≥65 presented metastatic lymph node. Moreover, metastatic lymph nodes were more frequently presented in patients with ≥ 3 cm tumor size. In addition, 19 cases (95%) of metastasis lymph nodes were adenocarcinoma and only 1 patient presented with N1 adenosquamous carcinoma.

Conclusion:
Lymph node metastasis adenocarcinoma was the main type in ASLC patients, and was related to the age and tumor size of the primary focus. Further large sample studies are necessary to identify influencing factors and clinicopathological characteristics of the metastasis lymph nodes.

Background:
Minute pulmonary meningothelial-like nodules (MPMNs) are generally detected incidentally in resected lung specimens. Recently, with increased use of high-resolution computer tomography (HRCT), MPMNs have occasionally been detected before surgery. They may appear as mild restrictive lung disease or randomly distributed micronodules of ground-glass attenuation on HRCT.

Method:
In this study, we retrospectively evaluated a case in which multiple ground glass density nodules (GGNs) were detected incidentally and operated for diagnosis in our hospital with the final diagnosis of MPMNs.

Result:
A 58-year-old non-smoking woman was referred to our hospital for multiple GGNs in bilateral lower lobes detected on a chest CT scan. HRCT was obtained for further evaluation. Numerous tiny GGNs were seen in the both lower lungs with centrilobular, subpleural and gravitational distribution. Many of them showed cystic or cavitary changes. Three differential diagnoses were presented by HRCT findings. The first was multifocal adenocarcinoma in situ or adenocarcinoma, the second was multifocal micronodular pneumocyte hyperplasia, and the third was atypical manifestation of langerhans cell histiocytosis or respiratory bronchiolitis interstitial lung disease. A follow up HRCT was reobtained after 2 months to determine diagnostic strategy and there was no significant change or mild prominence. For pathologic confirmation, video-assisted thoracoscopic surgery (VATS) right lower lobe wedge resection was performed. Microscopically, the surgical lung biopsy specimen showed multifocal ovoid cell proliferation along alveolar interstitium. The cells were bland, and no mitotic activity was identified. Immunohistochemical analysis was performed, and the sample was positive for epithelial membrane antigen (EMA), progesterone (PR) and CD56. Cytokerain, thyroid transcription factor-1, and S100 were negative. Finally the diagnosis of MPMNs was established, and there was no evidence of malignancy. On the second postoperative day, the patient was discharged without any complications.

Conclusion:
MPMNs are not uncommon incidental pathologic findings but the HRCT findings are nonspecific. They can occasionally manifest as multiple GGNs on HRCT, mimicking multifocal adenocarcinoma in situ or interstitial lung disease. Although most cases do not require special treatment, when there is no confident clinical diagnosis, such as in our case, a pathological correlation could be performed. An awareness of MPMNs presenting as GGNs is important because it may simulate neoplastic or other nonneoplastic diseases.

Background:
Lung cancer is a heterogeneous disease and we have few good markers of therapy prediction for chemotherapy and even immunotherapy. The biological mechanisms driving the tumour growth of different tumours even within the same histology are likely to vary and cause the diversity in therapy response. Pathifier is an algorithm developed by Eaton Domany’s group (Drier Y et al. PNAS, 2013 ) to estimate the deregulation of pathways of tumour samples based on mRNA expression levels of the genes in the pathway. Briefly, the algorithm estimates the deviation of the pathway in the tumour samples compared with normal samples. We analyse pathways deregulated in early stage squamous cell carcinomas to identify pathways potentially linked to therapy response. Such analyses have been performed for breast cancer (Livshits A et al, MolOnc, 2015), but have so far not been applied to lung carcinomas.

Method:
A total of 198 patients undergoing surgery for squamous cell lung cancer were included in the study. mRNA was extracted from the surgically resected tumour from all patients and from adjacent normal lung tissue from 22 patients. An adjustment of the pathifier algorithm was developed to avoid over-estimation of pathway deregulation. The samples were clustered according to adjusted pathway deregulation. Extensive clinical information such as mutation status, smoking history and survival was available.

Result:
Hierarchial clustering of the adjusted pathway deregulation scores identified separate clusters of squamous cell carcinomas of the lung dominated by different biological pathway with putative difference in clinical behaviour. Based on the biological activity, subgroups of patients are suggested to respond to immunotherapy and cell cycle inhibitors. The clusters are linked with survival, smoking history and expression of immune-related genes including PD-L1.

Conclusion:
Subgroups of lung squamous cell carcinomas have different biology represented by deregulation of groups of pathways. These biological differences can be used to identify clinically relevant markers of prognosis and therapy prediction. The results should be validated in functional studies.

Background:
Pulmonary adenofibroma is a rare benign tumor with biphasic pattern resembling adenofibroma of female genital tract and fibroepithelial lesion of breast. Since Scarff and Gowar first described it as a fibroadenoma of lung in 1944, only 10 cases have been reported in English literature. It is generally detected in middle-aged patients with solitary subpleural nodule. Histogenesis of this lesion is controversial, whether it is hamartomatous lesion or benign neoplasm. We report a case of a pulmonary adenofibroma in a 77-year-old male, presented with a subpleural bulla.

Method:
Section not applicable

Result:
A 77-year-old male, an ex-smoker, presented with chronic cough. Chest computed tomography revealed a 7-cm sized bulla in right middle lobe. As the large bulla had a thick wall and increased with lapse of time, resection was done. Frozen diagnosis suggested a proliferative lesion which cannot exclude mesothelioma or a parenchymal epithelial neoplasm. Grossly, the tumor was subpleural cystic lesion with central solid portion. Histologically, the lesion was characterized by a leaf-like branching and glandular pattern composed of a single layer of bland ciliated cuboidal lining epithelium and fibrous stroma. Immunohistochemical analysis revealed epithelium that stained positively for cytokeratin 7 and TTF-1, and stroma stained positively for SMA and nonspecific for CD34. The patient was diagnosed as pulmonary adenofibroma and discharged without any complications.

Conclusion:
Pulmonary adenofibroma is a rare biphasic tumor that could be misinterpreted as other benign or malignant tumors. Pulmonary hamartoma and solitary fibrous tumor could contain entrapped bronchial epithelium in the periphery of the tumor, which is distinguished from diffusely distributed epithelial component of adenofibroma. Pulmonary blastoma is another biphasic tumor which has a distinctive primitive appearance of epithelial and mesenchymal components. Moreover, this case presented as a thick-walled bulla that the possibility of primary mucinous adenocarcinoma or cystic metastasis was suspected in the radiological examination. Although pulmonary adenofibroma is recommended minimal surgical resection, lobectomy was done in this case due to its diagnostic difficulty in radiology and intraoperative frozen pathology. To avoid any inappropriate treatment, pulmonary adenofibroma should be regarded as a differential diagnosis of a solitary pulmonary nodule showing biphasic appearance.

Background:
Inflammatory cytokines, including tumor necrosis factor-α (TNFα), interleukin(IL)-6, IL-8, and IL-18 in the blood are elevated in patients with chronic obstructive pulmonary disease (COPD), but the influence of COPD on the differentiation and function of cancer-associated fibroblasts (CAFs), which are the dominant stromal component in the tumor microenvironment, remains to be elucidated in non-small cell lung cancer (NSCLC) patients. The purpose of this study is to examined the relationship between degree of COPD and CAFs in NSCLC patients who had undergone the lung surgery.

Method:
The expression of αSMA in tumor tissue was analyzed by immunohistochemistry for 45 cases with COPD and 8 cases without COPD who had undergone lung cancer surgery between 2014 and 2015 to evaluate the frequency of CAFs. We evaluated the correlations between low attenuation area (LAA), Brinkman index (BI), FEV1/FVC, GOLD COPD Stages and frequency of CAFs in tumor tissue.

Result:
Univariate analysis showed a strong correlation between the frequency of CAFs and LAA (P＜0.001), BI (P＜0.001), FEV1/FVC (P＜0.001) except for GOLD COPD Stages (P=0.208). Multivariate analysis showed that LAA was significant predictors of frequency of CAFs in tumor tissue.

Conclusion:
Parameters obtained from the pulmonary function test and smoking index are often affected by the skill and memory of the patients. It was suggested that the LAA which objectively quantified the degree of emphysematous change was more related by the frequency of CAFs in tumor microenvironment.

Background:
Pulmonary pleomorphic carcinoma is a rare histologic type with poor prognosis, proposed in the third edition of the 1999 WHO histologic classification of lung tumors.

Method:
We performed surgical treatment for 16 cases with pulmonary pleomorphic carcinoma between April 2000 and April 2017, and investigated the patients’ characteristics and perioperative outcomes.

Result:
The male-to-female ratio was 14 to two. The median age was 68 years, ranging from 55 to 88. All cases had a smoking habit. The locations of tumors were RUL in 5 cases, RML in 3, RLL in 4, LUL in 4 and LLL in 2. Pathological stages were IA2 in1, IA3 in 1, IB in 2, IIA in 3, IIB in 1, IIIA in 6 and IIIB in 2, based on eighth of the TNM classification for lung cancer. Nodal status was classified as pN0 in 11, pN1 in 1 and pN2 in 4. Epithelial components were squamous cell carcinoma in 5 (31%), adenocarcinoma in 5 (31%), double-cell components in 2 (12.5%), no epithelial components in 2 (12.5%) and indistinct in 2 (12.5%), while sarcomatous elements, spindle cells in 8 (50%), giant cells in 1 (6%), double-cell elements in 5 (31%) and indistinct in 2 (13%). Eight cases received adjuvant treatments, including chemotherapy in 5, radiotherapy in 1 and chemoradiotherapy in 2. Postoperative recurrence was detected in 9 cases (56%). And 7 of the nine cases had the recurrence within 6 months after the surgical resection. On the contrary, no recurrence was detected in 7 cases comprised of pN0 in 5 cases and pN1-2 in 2. And 2 of the 7 cases have been disease-free for more than two and a half years.

Conclusion:
Although pulmonary pleomorphic carcinoma has poor prognosis, patients without lymph node metastasis have the possibility of having favorable prognosis compared to the patients with lymph node metastasis. It is important to observe the postoperative courses carefully during 6 months from surgical resection in order to detect the early recurrences promptly.

Background:
In the new 2015 WHO classification, invasive mucinous adenocarcinoma (IMA), formerly referred to as mucinous BAC, is categorized as variant of invasive adenocarcinoma. IMA has goblet or columnar cell feature with abundant intracytoplasmic mucin and may show the same heterogeneous mixture of histologic subtypes (lepidic, acinar, papillary, and micropapillary) as non-mucinous tumors. In the 8th edition of TNM classification, invasive tumor size is used for T factor, but few comprehensive studies about invasive components of IMA are reported.

Method:
We evaluated 460 patients with lung adenocarcinoma surgically resected at our facility from Jan 1, 2000 to Jan 31, 2017. In cases identified as IMA, invasive components (the predominant histologic subtypes, the size of invasive area and the presence of spread through air spaces (STAS)) were noted. We classified all cases with the 8[th] TNM system and investigated the clinical course retrospectively.

Result:
24 cases (5.2%) were diagnosed as IMA. Of 24 IMAs, 21 cases (91.3%) expressed CK20 with lack of TTF-1. KRAS mutation was found in 2 of the examined 3 cases. 23 cases (96.3%) were found histologic subtype other than lepidic pattern. All cases of IMA were classified into 16 cases of lepidic predominant IMA and 8 cases of non-lepidic predominant IMA (3 cases of acinar predominant, 5 cases of papillary predominant) depending on the predominant histologic subtype. The proportions of lepidic predominant and non-lepidic predominant IMA in each stages were as follows; IA: 68.8% vs 12.5%, ⅠB: 12.5% ​​vs 37.5%, II: 6.3% vs 25.0%, IIIA: 12.5% ​​vs 25.0%. Lymph node metastasis was observed in only one case of non-lepidic predominant IMA. The recurrence was observed 37.5% (6 of 16, included 3 relapsed cases of StageⅠ) and 37.5% (3 of 8) respectively in the period. STAS was found with a high probability in both category (71.4% vs 100%) and a total of 7 cases were pulmonary recurrence. 5-year DFS did not differ significantly (48.5% vs 51.4%, log rank test p=0.76). 5-year OS was a high tendency for lepidic predominant IMA, but no significant difference was observed (73.9% vs 43.8%, log rank test p=0.14).

Conclusion:
Lepidic predominant IMA seemed to be more frequent in early-stage cancer than non-lepidic predominant IMA, but the frequency of relapse in the clinical course was similar in both groups. These results suggest that IMA have a prognostic factor other than patterns of the histologic subtypes and invasive size.

Background:
In the 2011 IASLC/ATS/ERS classification, guidelines recommend that the major adenocarcinoma (ADC) subtypes should be classified according to the predominant histologic pattern. In published papers, the ADC classification has significant prognostic and predictive value regarding death or recurrence has been reported. The purpose of this study was to retrospectively evaluate characteristic differences between cytomorphological findings among histological subtypes in the preoperative bronchoscopic materials and radiologic features on high-resolution computed tomography (HRCT) and [(18)F]-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) /CT examinations in our database.

Method:
Forty-four consecutive lung cancer patients with peripheral lung nodules diagnosed by bronchoscopic biopsies underwent surgery in our hospital from April 2011 to May 2016. The cyotologic material obtained by brushing bronchoscopically was placed onto a glass slide, immediately fixed in 95% ethanol, and stained with Papanicolaou stain. The cytomorphological studies were retrospectively performed separately by two experienced cytotechnicians. Clinicopathological data with preoperative radiologic features on HRCT and 18F-FDG PET/CT in the patients was utilized for comparing to cytomorphological analyses.

Result:
Out of the forty-four patients with lung cancer, by excluding one typical carcinoid and four not otherwise specified (NOS), thirty-nine specimens in the patients consisted of ADC (n=32) and squamous cell carcinoma (SQCC, n=7) were analyzed. Thirty-two ADC were subclassified cytomorphologically into acinar (n=22), solid (n=8), papillary (n=1) and lepidic (n=1). The subtypes of ADC except for eight solids were categorized as nonsolid of ADC on comparisons of analyzed data. Specimens classified as solid pattern of ADC had a predominant 3D clusters (8 of 8 specimens, 100%) and conspicuous nucleoli (7 of 8 specimens, 87.5%) than nonsolid patterns of ADC. There were statistically significant differences between the nonsolid and the solid patterns about the two features (P=0.0011 and 0.0007, respectively). C/T ratio (a diameter of consolidation divided by a diameter tumor size) of the nonsolid pattern (0.9±0.1) was significantly lower than that for the solid pattern and the SQCC (1.0±0.0) (p=0.01). Maximum standardized uptake value (SUVmax) of the SQCC at 60 and 120 minutes (12.3±4.0 and 16.9±7.3) was significantly higher than that for the nonsolid pattern of ADC (7.0±3.6 and 9.0±4.6; P=0.003 and 0.004, respectively). Figure 1



Conclusion:
Preoperative cytomorphological subtyping might predict value of radiologic features on CT and PET examinations, therefore their features also could provide information about a degree of pathological invasiveness or biological malignancy of tumor with some decisions for treatments.

Background:
Ultrasound has been widely utilized in clinical to visualize the internal structure of the objective non-invasively. However ultrasound image can’t distinguish malignant lesion from the normal tissue. Spectrum analysis of ultrasound is a newly developed technology which may reflect on the histological feature. We examine if the spectrum analysis is able to distinguish malignant tissue from normal tissue.

Method:
Spectrum was measured using a prototype ultrasound processor EUME5 given by Olympus Japan. three parameters of spectrum such as Midband-fit(M), Intercept(I), and Slope(S) were measured for the objective tissue. In animal study, human lung cancer Xenograft were created in nude mice for each lung cancer cell line (A549, H460, HCC827, and H3122). In clinical setting, surgically excised lungs including lung cancers were examined spectrum analysis for both lung cancers (n=19, 106 slices) and normal lungs (n=17, 65 slices).

Result:
Four different Xenografts exhibited significant differences of spectrum data. In the clinical study, the mean value of M, I and S of both lung cancers and normal lungs were M: -43.22 ±4.09 vs -39.31±3.87(p<0.01,) I: -55.28±3.19 vs -54.13±2.4 (N.S), S: -1.43±0.35 vs -1.73±0.30 (p<0.01)

Conclusion:
Each lung cancer Xenograft of different histology showed different spectrum value. Spectrum analysis is likely to reflect the histological feature. In clinical, M and S showed statistically different values between lung cancer and normal lung. Based on spectrum value, a malignant tumor can be distinguished from the normal lung in the ultrasound image.

Background:
Lung cancer is the leading cause of cancer death worldwide. In 1979, Syrjänen suggested a role for human papillomavirus (HPV) infection in bronchial carcinoma. Many studies have found HPV on lung carcinoma, predominantly in squamous cell carcinoma (SCC). There seems to be a geographical factor determining prevalence rates. In Latin America, only 2 studies, altogether including 51 cases of lung SCC, examined this association. However, data from Argentina is lacking. The aim of this study is to asses the incidence of HPV infection in lung SCC of Argentinean population, and to correlate with p16[INK4a ]expression from an Argentine population.

Method:
The study was approved by CEMIC’s Ethics Committee. Informed consent was obtained. Materials consisted of formalin-fixed paraffin-embedded (FFPE) tissue from 29 surgically excised and 11 transbronchial biopsies of primary L-SCC evaluated between 2006-2016. HPV Genotyping: On 50μm-thick slides, tumor was microdissected and DNA was extracted (columns method). Wide-spectrum HPV DNA (L1-ORF) was amplified by PCR. Positive specimens were genotyped by PCR for types 16 and 18. Immunohistochemistry: All p16 staining’s were performed on VENTANA BenchMark GX using antibody CINtec® p16. Staining patterns were interpreted on a binary way (positive or negative). Only cases with diffusely intense cytoplasmic and/or nucleic staining on tumor cell (TC) were considered positive. Cases in which the normal bronchial epithelium resulted p16 positive but TC were negative, were also registered.

Result:
HPV was isolated in 10/40 cases (25%). The details of HPV infection and the clinicopathological data is depicted on table 1.

Clinicopathological features of SCC

	HPV positive (n=10)	HPV negative (n=30)
Gender
Female	5	11
Male	5	19
Age
<50	1	-
50-60	-	21
>60	9	9
Smoking
Never-smoker	-	-
Smoker	-	-
Unknown	10	30
Tumor cell differentiation
Well	1	-
Moderate	-	10
Poorly	9	20
Keratinizing
Non-keratinizing	9	27
Keratinizing	1	3
p16 positive on tumor cellls
Positive	3	2
Negative	7	28
p16 on bronchial epithelium
Positive	3	2
Negative	7	28
HPV type
HPV 16	3	-
HPV 18	5	-
Co-infection HPV 16 and 18	2	-
Specimen type
Transbronchial biopsy	3	8
Surgical excision	7	22

Conclusion:
We detected an HPV infection rate of 25%. HPV18 was the common genotype. On 7 cases, normal bronchial epithelium was both p16 and HPV positive, suggesting that adjacent tumor tissue may be HPV infected. p16 should not be used as a surrogate marker for HPV infection, since it is only positive on 60% of cases.

Background:
The diagnosis of lung cancer can be challenging due to different forms of disease presentation, coupled with institutional familiarity with specific techniques. Transthoracic fine needle aspiration (FNA) is relatively simple and inexpensive, yielding diagnostic material in 70-95% of cases. In the era of personalized oncology where immunohistochemical and molecular assays may be required, the role of FNA to provide enough material should be reassessed.

Method:
This is a prospective study designed to evaluate the feasibility and safety of FNA as a primary technique in the diagnosis of lung tumors. Patients were randomized in a 1:1 ratio into two arms, one using conventional FNA needle (control arm) and another using coaxial needle (experimental arm). Eligible patients were at least 18 years old and had a lung mass suspicious of lung cancer. The procedure was performed in an outpatient clinic, under local anesthesia, by an experienced thoracic surgeon in the presence of a pathologist. The primary endpoint was a positive cell block containing at least 40% of neoplastic cells, a surrogate for successful additional assays. We present the first interim analysis. This study was approved by the Ethics Committee.

Result:
Between January 2013 and May 2015, 34 patients were enrolled, 17 in each arm. The cohort was mostly comprised of males (62%) and smokers (91%), with a median age of 66 years (range 51-85). Most cases were assessed with a computed tomography (94%), with target tumor measuring a median of 8.2 cm (range 1-13 cm). Baseline characteristics were well balanced between the 2 arms, except for gender (82% males in the experimental arm, 41% in the control arm). A positive cell block was acquired in 9 (53%) and 7 (41%) cases in the experimental and control arms, respectively. Altogether, a positive cell block was acquired in 16 cases (47%). A diagnosis was obtained in 82% of cases, but histological subtyping was only possible in 73%. The cell block positivity rate was significantly associated with histological subtyping (p<0.001). Local pain was the only adverse event, reported in 2 cases in each arm.

Conclusion:
FNA was a safe procedure in both arms, but yielded enough tumor material in less than half patients and was less than optimal to determine histological subtyping. No relevant difference was observed between conventional and coaxial needle, neither for safety nor for efficacy. FNA alone should not be considered a standard procedure in most cases suspicious for lung cancer.

Background:
Division of typical (TC) and atypical (AC) carcinoids is based on the mitotic index and/or the presence of necrosis. A mitotic rate is a well-established and highly prognostic factor, but many studies indicate that assessment of proliferative activity based on Ki-67 value could be easier and more reproducible. The aim of the study was: •assessment of Ki-67 index in PC and its usefulness in distinguishing TC from AC, •to determine whether the mean Ki-67 index is significantly different in TC and AC, •selection the optimal cut-off Ki-67 value that would help predict overall survival in pulmonary carcinoids (PC).

Method:
The clinicopathological features from 329 resected PC were correlated and survival analysis were performed. Mitoses and the proliferative index (PI) were analyzed. For mitotic counting, a scale bar depicting an area of 2 mm[2 ]was provided and for PI assessment, the percentage of positively stained cells by Ki-67 in the area with the highest proliferative activity was counted.

Result:
There were 217 (66%) TC and 112 (34%) AC, with a median follow-up time of 7,6 ys (230 females and 99 males). The mean age at diagnosis was 52,8 years, median 55 y. Most tumors were localized centrally (73,7%). AC were larger than TC (2,54 vs 1,9cm) and more common located peripherally. Lymph nodes involvement was present in 49 cases (15%), N1-34 (10%) and N2-15 (5%), frequently in AC.The high cellular atypia, cartilage destruction, invasion of peribronchial tissue or adjacent lung parenchyma, presence of pleural, perineural and vascular invasion correlated with AC. ACs were associated with significantly higher Ki-67 indices (8,7%, median 7%) than TCs (3,1%, median 2%). There were significant correlations between high PI and: large tumor size, mitotic activity, vascular invasion and lymph node metastases.The Ki-67 index was a good factor differentiating TC from AC, with cut-off ≤ 4% for TC and > 4% for AC, with high specifity (90%) and sensitivity (70%) and with high likelihood ratio (2,97).The number of deaths among the patients with TC and AC was 7 (6,3%) and 31 (14,3%) respectively. The 5-,10- and 15 year overall survivals for the entire group were 92,8%, 86,8% and 78,6% respectively.

Conclusion:
Ki-67 is an effective grading tool for PC and for differentiation between TC from AC. Apart the mitotic count and necrosis, the Ki-67 index should be incorporated into the mandatory histological criteria for diagnosis PC. The optimal cut-off value of Ki-67 that helps distinguish TC from AC is 4%.

Background:
Both lymphangioleiomyomatosis (LAM) and diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH) are rare entities of diffuse intra-pulmonary cellular proliferation. Thus far, these have not been reported to occur synchronously in the same patient.

Method:
We investigated the lungs of a 54 year old woman who underwent bilateral lung transplantation for radiologically and histologically diagnosed sporadic LAM by routine histological and molecular diagnostic procedures.

Result:
Upon macroscopic inspection both explanted lungs had a multicystic appearance, with multiple nodules, with a max. diameter of 1.0 cm in the left lung, and a max. diameter of 2.9 cm in the right lung. Microscopically, both lungs contained extensive lesions consistent with LAM, with cystic change and surrounding monomorphic spindle cell proliferations immunohistochemically positive for SMA, ER and HMB-45. In addition, diffuse intraepithelial and nodular proliferations of neuro-endocrine cells were seen, positive for TTF-1, CD56, chromogranin A, and synaptophysin, amounting to DIPNECH with multiple neuroendocrine tumourlets and foci of typical carcinoid. There were no lymph node metastases and SSTR-2A staining was negative in neuro-endocrine cell proliferations. Upon mutation analysis by next generation sequencing using an extended diagnostic panel, no pathogenic mutations were found in the largest carcinoid, but the LAM component contained 2 different TSC2 gene splice variants (TSC2 c.599+1G>A; and TSC2 c.1119+1G>C). In view of these genetic findings the two different disease processes were thought not to be clonally related.

Conclusion:
In summary, we present the first case of bilateral combined pulmonary LAM and DIPNECH, which were histomorphologically intricately related but by mutation analysis deemed to be separate entities with presumably distinct histogenesis.

Background:
Id1 has been shown to be involved in cell viability and migration of lung cancer cell lines and confer poor prognosis in LAC-patients. The most frequently mutation in LAC is KRAS, but no targeted therapy has been successfully developed. Here we study the role of Id1 in a KM-LAC murine model.

Method:
The expression of Id1 was analyzed in a panel of human LAC cell lines by qPCR and Western-Blot. Several human cell lines with known mutations (H1792-604, H2009, H358, H1568, H1437, H1703, H2126) were selected to deplete Id1 expression by inducible short hairpin RNA (shRNA) regulated by doxycycline. Proliferation, cell cycle and apoptosis assays were performed to study the cellular mechanism underlying the effect of Id1 deficiency. Mouse xenograft models were generated by subcutaneous injection of KM-LAC cells (H1792-604 and H2009), both shId1 and shGFP cells, in flanks of immunodeficient mice treated with doxycycline (drinking water) from the time of inoculation or once the tumors were established.

Result:
Id1 overexpression was observed in 11 out of 12 cell lines as occurs in previously reported clinical data. Id1 inhibition was achieved in all cell lines compared to controls. In absence of Id1, proliferation assays showed a significant impairment of cell growth in KM-LAC cell lines [H1792-604 31.61% ± 3.96 (P < 0.001); H2009 52.73% ±4.74 (P < 0.001); H358 70.85% ± 8.01 (P < 0.001)]. In KM cells, a significant arrest in G2/M phase of cell cycle was observed when Id1 was inhibited whereas no significant changes were observed in wild type(WT) KRAS cells [KM 1.86 ± 0.28;WT 1.02 ± 0.05 (P < 0.001)]. KM-cells showed a significant apoptosis increase compared to WT-cells [KM-cells 1.66 ± 0.41;WT-cells 0.99 ± 0.13 (P = 0.001)]. In vivo, we observed a significant decrease in tumor volume in mice injected with H1792-604-shId1 cells (60% ± 32.39) compared to shGFP group (356.29% ± 115.32)(P < 0.001). Moreover, mice injected with H2009-shId1 cells did not develop tumors compared to control mice (168.35 ± 68.71)(P < 0.001). Activation of shId1 in established tumors induced a significant reduction of tumor volume in both xenograft models. The inhibition led to regression of 4 out of 10 tumors H1792-604 and all tumors in H2009 inoculated mice.

Conclusion:
These findings support a crucial role of Id1 in tumor development in KRAS-driven adenocarcinoma of the lung. Id1 targeting was proven effective in both, tumor prevention and treatment in our humanized murine model of KM LAC.

Background:
Compared with the numerous studies of somatic mutations using sporadic lung cancer, the research into germline mutations using familial lung cancer (FLC) is very limited. In the present study, we used FLC samples obtained from the Chinese population in highly air-polluted regions to screen for novel germline mutations in lung cancer.

Method:
Through a whole genome sequencing (WGS) analysis of the nine subjects (four lung cancer patients and five normal family members of FLC), we obtained a whole genome dataset of DNA alterations in FLC samples. A total of 1218 genes were identified with mutations of multiple types. Subsequently, the top 12 highly mutated genes were selected for validation by PCR and DNA sequencing in an expanded sample set including FLC, sporadic lung cancer, and healthy population.

Result:
Mutations of the five genes (ARHGEF5, ANKRD20A2, ZNF595, ZNF812, MYO18B) may be potential germline mutations of lung cancer. We also analyzed specific mutations within the 12 genes and found that some specific mutations within the MUC12, FOXD4L3 and FOXD4L5 genes showed higher frequencies in the samples of FLC and/or lung cancer tissue, compared with the healthy population. Moreover, some genes with copy number variation may be potentially associated with a predisposition to lung cancer. Furthermore, non-coding DNA alterations of the WGS data in FLC were systematically analyzed and arranged. Interestingly, we found that germline mutations also occurred in many non-coding genes.

Conclusion:
Our study uncovered the mutation spectrum in FLC of the Chinese population. The investigation of novel and known gene mutations detected by the present study may contribute to evaluate functional impacts of these mutations not only in FLC but in sporadic lung cancer as well.

Background:
Recently, components of the switch/sucrose non-fermenting (SWI/SNF) complex have emerged as novel tumor suppressors in non-small cell lung cancer (NSCLC). However, the detailed genotypic and phenotypic features of NSCLC with mutations of SWI/SNF subunits remain unclear. In this study, we intended to clarify detailed morphologic and phenotypic features of NSCLC with mutations of SWI/SNF subunits through analysis of a panel of NSCLC cell lines and primary NSCLC.

Method:
We used 38 NSCLC cell lines which had been analysed for mRNA profiles and major driver mutations in our previous studies (Matsubara et al. AJP 2010, JTO 2010). The genetic status of SWI/SNF subunits was interrogated and retrieved from the public databases (COSMIC, CCLE). Morphological features of these cell lines were examined by 3-D cell culture and xenografts in NOD/SCID mice. We also immunohistochemically analyzed the expressions of multiple SWI/SNF subunits (BRG1, BRM, BAF47, ARID1A, and ARID1B.) in primary 133 NSCLC consisting of 25 squamous cell carcinomas, 70 adenocarcinomas, 16 large cell carcinomas, and 22 pleomorphic carcinomas.

Result:
In a panel of 38 NSCLC cell lines, mutations of SWI/SNF subunits tended to be found in the cell lines without major driver mutations and with mesenchymal (E-cadherin-low, vimentin-high) phenotypes. Consistently, these cell lines formed grape-like or stellate spheroid in 3-D cell culture, and poorly differentiated, solid or medullary tumors in xenograft models. In the primary tumors, reduced expression of BRG1 and BRM was significantly more frequent in large cell carcinomas and pleomorphic carcinomas than in squamous carcinomas and adenocarcinomas. Loss of expression of ARID1A, ARID1B and BAF47 was only rarely found only in a fraction of NSCLC cases.

Conclusion:
Collectively, these results indicate that loss of the SWI/SNF complex components is found in driver mutation pauci tumors and correlates with dedifferentiation and mesenchymal phenotype in NSCLC.

Background:
The Period clock gene family has three family members, including Per1, Per2 and Per3. Among them, Per2 is an indispensable component of the circadian clock, which not only modulates circadian oscillations, but also has a decreased expression in many types of cancers and functions as a tumor suppressor gene. However, the expression and specific roles of Per2 in human non-small cell lung cancer(NSCLC) is still unknown. And the present study is aimed to investigate the roles of Per2 porced expression in NSCLC in vitro and in vivo.

Method:
Per2 expression level in tissues was examined by immunohistochemistry. mRNA and protein expression alterations were teste by RT-PCR and Western blot. To clarify the specific roles of Per2 in A549 cell line, we construct the stable overexpressed cell line by lentivirus transfection. And the roles of Per2 porced expression in tumor proliferation and migration were examined by CCK8, colony formation, cell wound, cell migration and invasion, and we also detected the alterations in cell cycle and apoptosis by flow cytometric analysis. Furthermore, we established subcutaneous tumor animal mode to test the tumorgenesis and migration ability of Per2 overexpressed A459 cell in vivo.

Result:
We found that Per2 has a commonly higher expression in para-carcinoma tissues than carcinoma tissues in 26 NSCLC patients(P=0.0001). And Per2 expression in NSCLC patients were correlated with some clinicopathological features(P=0.0000). Then, function assay showed that forced expression of Per2 in A549 cells markedly decreased the ability of cancer cell proliferation, migration and invasion in vitro(P<0.05), and also increased the apoptosis and the number of cells in G1/G0 phase(P<0.05). Furthermore, overexpression of Per2 altered the proliferation and migration related protein expression(P<0.05). Consistent with the in vitro study, we also showed that Per2 overexpression decreased the tumorigenicity of A549 cells in vivo(P<0.05).

Conclusion:
Per2 has a lower expression in NSCLC tissues and also funtions as a tumor supressor gene. It regulates the numerous important downstream tumor-related genes, which may be a novel molecular target for cancer treatment.

Background:
BRAF, one of the three members of the RAF kinase family, belongs to the group of serine-threonine kinases and plays a vital role in mitogen-activated protein kinase (MAPK) pathways. Mutations of BRAF have been found in 0.5-3% of non-small-cell lung cancer (NSCLC). Among the different mutations occurring in the BRAF gene, BRAF V600E is the most common. A number of BRAF inhibitors, including sorafenib, vemurafenib and dabrafenib, are under clinical development. Thus, the detection of genetic driver mutation in lung cancer patients has become the most important tool in clinical practice. The aim is to detect the consistency of the BRAF gene mutation in peripheral blood and tumor tissue of patients with NSCLC and discuss the clinical application value of BRAF gene mutation in peripheral blood.

Method:
Real-time fluorescent quantitative polymerase chain reaction (RT-PCR) was used to detect the tissues in 257 patients of NSCLC and the peripheral blood samples in 318 patients of NSCLC, of which 185 cases of peripheral blood specimens could match the tissue samples, and detected the BRAF gene mutation in them by comparison of mutations consistency in blood and tissue samples, and analyzed the correlation between BRAF gene mutations and clinical characteristics of patients.

Result:
The BRAF gene mutation rate was 7.23% in peripheral blood of 23 patients with NSCLC, and was 5.45% in 14 cancer tissues, the mutation consistency was 80.00% in peripheral blood tumor tissue matched samples. The consistency was statistically significant (κ=0.710, P<0.001).

Conclusion:
The consistency of the BRAF gene mutation in peripheral blood and tissue is high. BRAF gene mutations of peripheral blood could be used for clinical diagnosis and treatment in cases when tissue specimen is hard to get.

Background:
RET fusion gene is identified as a novel oncogene in a subset of non-small cell lung cancer (NSCLC). However, few datas are available about the prevalence and clinicopathologic characteristics in RET rearrangement lung adenocarcinoma patients. The aim of this study is to investigate mRNA expressions and relationship of RET rearrangement and thymidylate synthase (TYMS) genes in NSCLC tissues.

Method:
The positive rate of RET rearrangement and the mRNA expressions of of TYMS gene in NSCLC tissues of 642 patients were detected by using real-time fluorescent quantitative PCR method, and the relationship and its correlation between the expression and clinicopathological features were also analyzed.

Result:
The positive rate of RET rearrangement in NSCLC was 0.93% (6/642); High mRNA expression of TYMS gene was 63.55%(408/642). The expressions showed no relationship with gender, age, smoking, tumor size, lymph node metastasis and clinical stages (P>0.05). The mRNA expressions between RET rearrangement and TYMS genes showed positive correlation (P<0.05).

Conclusion:
Thymidylate synthase gene shows low expression level in NSCLC patients with positive RET fusion gene, which may benefit from pemetrexed of first-line chemotherapy drug.

Background:
PIK3CA mutation represents a clinical subset of diverse carcinomas. We explored the status of PIK3CA mutation and evaluated its genetic variability and prognosis in patients with lung adenocarcinoma. The aim of this study is to investigate mutations and prognosis of non-small-cell lung cancer(NSCLC) harboring PIK3CA mutations.

Method:
A total of 517 patients with NSCLC were recruited between July 2012 and December 2014. The status of PIK3CA mutation and other genes were detected by reverse transcription polymerase chain reaction(RT-PCR) or next generation sequencing.

Result:
PIK3CA gene mutation was detected in 3.09% (16/517) NSCLC patients, including H1047R (4 patients), E545A (2 patients), E453K (2 patients), H1065Y (2 patients), E545K (1 patient), E39K (1 patient), E542K (1 patient), C420R (1 patient), K111E (1 patient) and E545K plus L781F (1 patient), and median overall survival (OS) for these patients was 23.0 months. Among them, 12 patients with co-occurring mutations had a median OS of 28.0 months, and median OS of the 4 patients without complex mutations was 21.0 months. No statistically significant difference was found between the two groups (P=0.06). Briefly, patients with (n=5) or without (n=11) co-occurring EGFR mutations had a median OS of 28.5 months and 21.0 months repectively (P=0.45); patients with (n=4) or without (n=12) co-occurring TP53 mutations had a median OS of 30.6 months and 21.0 months repectively (P=0.51).

Conclusion:
There is no significant difference of molecular features in PIK3CA gene mutations in NSCLC. Patients with complex mutations benefited more from therapy than those with single mutations.

Background:
HER2 is a driver gene identified in non-small-cell lung cancer(NSCLC). The prevalence, clinicopathology and genetic variability of HER2 mutation non-small cell lung cancer patients are unclear. The aim of this study is to investigate mutational subtypes and prognosis of NSCLC harboring HER2 mutations.

Method:
A total of 781 patients with NSCLC were recruited between July 2012 and December 2014. The status of HER2 mutation and other genes were detected by reverse transcription polymerase chain reaction (RT-PCR) or next generation sequencing.

Result:
HER2 gene mutation rate was 1.92%(15/781) in NSCLC, including S310F (2 patients), A775_G776insYVM (2 patients), S280F (2 patients), P780_Y781insGSP (1 patient), C630Y (1 patient), L755P (1 patient), T327S (1 patient), K907R (1 patient), R70W (1 patient), E117D (1 patient), L970V (1 patient), and C965S (1 patient). Mutation rate of female was much higher than male(3.76% vs 1.23%, P=0.022), and current-smoker was much higher than no-smoker(3.17% vs 0.74%, P=0.027), and median overall survival (OS) for these patients was 42.6 months. Among them, 12 patients with co-occurring mutations had a median OS of 42.6 months, and median OS of the 3 patients without cpmplex mutations was 40.3 months. No statistically significant difference was found between the two groups(P=0.43). Briefly, patients with (n=8) or without (n=7) co-occurring EGFR mutations had a median OS of 50.6 months and 42.6 months repectively (P=0.19); patients with (n=9) or without (n=6) co-occurring TP53 mutations had a median OS of 40.4 months and 46.7 months repectively (P=0.39); patients with (n=2) or without (n=13) co-occurring SMARCA4 mutations had a median OS of 50.6 months and 42.6 months repectively (P=0.33); patients with (n=2) or without (n=13) co-occurring MTOR mutations had a median OS of 44.3 months and 42.6 months repectively (P=0.71); patients with (n=2) or without (n=13) co-occurring SMARCA4 mutations had a median OS of 50.6 months and 42.6 months repectively (P=0.33); patients with (n=2) or without (n=13) co-occurring APC mutations had a median OS of 39.0 months and 42.6 months repectively (P=0.92).

Conclusion:
There are some significant difference of molecular features in HER2 gene mutations with non-smoking women in NSCLC, along with the state of HER2 gene mutations little influence on prognosis. Afatinib treatment may displayed moderate efficacy in patients with HER2 mutations.

Background:
In non-small cell lung cancer (NSCLC) RAS-mutant status is a negative prognostic and predictive factor. The prevalence, clinicopathology and genetic variability of RAS mutation NSCLC patients are unclear. The aim of this study is to investigate mutations and prognosis of NSCLC harboring RAS mutations.

Method:
We retrospectively reviewed clinical features from 41 patients with RAS gene mutation NSCLC, and the survival rate was calculated by Kaplan-Meier method and log-rank test was used to compare the survival rates.

Result:
KRAS gene mutation rate was 8.00% (38/475) in NSCLC, including G12C (9 patients), G12D (8 patients), G12V (7 patients), G12A (2 patients), G12S (2 patients), G13D (2 patients), Q61H (2 patients), G12L (1 patient), G12 (1 patient), G12K (1 patient), G12fs*3 plus G12V (1 patient), G13C plus V14I(1 patient) and K5N(1 patient). Mutation rate of current-smoker was much higher than no-smoker(15.76% and 4.41%, P<0.01), and median overall survival (OS) for these patients was 18.3 months; NRAS gene mutation rate was 0.29% (1/346), G12D, and OS for these patients was 14.2 months; HRAS gene mutation rate was 0.63% (2/315), including H27N and N85I, and median OS for both patients was 19.2 months. Among them, 18 cases of the 41 RAS mutation patients with co-occurring mutations had a median OS of 28.0 months, and median OS of the 23 patients without cpmplex mutations was 21.0 months. No statistically significant difference was found between the two groups(P=0.06). Briefly, patients of KRAS mutations with (n=4) or without (n=34) co-occurring EGFR mutations had a median OS of 40.0 months and 16.3 months repectively (P=0.07); patients with (n=3) or without (n=35) co-occurring TP53 mutations had a median OS of 36.4 months and 18.3 months repectively (P=0.22); patients with (n=3) or without (n=35) co-occurring STK11 mutations had a median OS of not reached so far and 16.3 months repectively (P=0.22); patients with (n=2) or without (n=36) co-occurring KEAP1 mutations had a median OS of 43.6 months and 16.3 months repectively (P=0.06).

Conclusion:
Mutation rate of KRAS gene in current-smoker NSCLC patients was higher than no-smoker, there is no other significant difference of molecular features in RAS gene mutations in NSCLC. Patients with complex mutations benefited more from therapy than those with single mutations. Immunotherapy may displayed moderate efficacy in patients with TP53 and RAS co-exist mutations.

Background:
RB1 (retinoblastoma 1) was reportedly one of the major determinative factors for sensitivity to taxanes in previous studies. The dephosphorylated RB1 protein confers the higher sensitivity to chemotherapy drug, but the RB1 mutation non-small-cell lung cancer (NSCLC) genetic variability and prognosis is unclear. The aim of this study is to investigate mutations and prognosis of NSCLC harboring RB1 mutations.

Method:
A total of 728 patients with NSCLC were recruited between July 2012 and December 2014. The status of RB1 mutation and other genes were detected by next generation sequencing.

Result:
RB1 gene mutation was detected in 0.96% (7/728) NSCLC patients, including p.G449E (1 patient), p.L542stop (1 patient), p.R552* (1 patient), p.Y6511fs*7 (1 patient), c.2663+2T>C (1 patient), p.P23del (1 patient) and F684fs*7 plus R418T (1 patient), and median overall survival (OS) for these patients was 32.7 months. Among them, all patients were RB1 gene with co-occurring mutations. Briefly, patients with (n=3) or without (n=4) co-occurring EGFR mutations had a median OS of 34.9 months and 30.4 months repectively (P=0.95); patients with (n=5) or without (n=2) co-occurring TP53 mutations had a median OS of 32.7 months and 31.7 months repectively (P=0.90).

Conclusion:
EGFR or TP53 gene accompanied may have less correlation with RB1 mutation in NSCLC patients. Chemotherapy drugs may displayed moderated efficacy in patients with RB1 mutation, especially, accompanied with TP53. These data have implications for both clinical trial design and therapeutic strategies.

Background:
CDKN2A is the tumor suppressor, which regulates cell cycle progression by inhibiting cyclinD-CDK4 and cyclinD-CDK6 complexes responsible for initiating the G1/S phase transition. CDKN2A gene disruption happens by different types of mutations, such as the loss of heterozygosity, homozygous deletion, or promoter silencing. There is some clinical evidence for the use of CDKN2A mutations as prognostic and predictive biomarker. The aim of this study is to investigate mutations and prognosis of non-small cell lung cancer(NSCLC) harboring CDKN2A mutations.

Method:
A total of 1046 patients with NSCLC were recruited between July 2012 and December 2014. The status of CDKN2A mutation and other genes were detected by next generation sequencing.

Result:
CDKN2A gene mutation was detected in 0.77% (8/1046) NSCLC patients, including p.M53I (1 patient), p.R58* (2 patients), p.R80* (2 patients), c.193+2T>C (1 patient), p.A127T (1 patient) and p.D74Y (1 patient), and median overall survival (OS) for these patients was 29.8 months. Among them, all patients were CDKN2A gene with co-occurring mutations. Briefly, patients with (n=4) or without (n=4) co-occurring EGFR mutations had a median OS of 23.4 months and 33.6 months repectively (P=0.32); patients with (n=6) or without (n=2) co-occurring TP53 mutations had a median OS of 23.4 months and 34.8 months repectively (P=0.27).

Conclusion:
EGFR and TP53 gene accompanied may have less correlation with CDKN2A mutation in NSCLC patients. CDK4/6 inhibitor palbociclib drugs may displayed moderated efficacy in patients with CDKN2A mutation.The findings of this study could facilitate the identification of therapeutic target candidates for precision medicine of NSCLC

Background:
Genes expressed at the imprinted chromosome 14q32 locus play crucial roles in both fetal and adult development. The protein-coding genes expressed by the paternal allele are required for proper placentation and organogenesis; while the expression of non-coding genes encoded by the maternal allele were found to be temporally active in the brain. Previous reports identified few miRNAs at the locus that were able to stratify low and high risk patients with non-small cell lung carcinoma (NSCLC). However, a locus-wide analysis of miRNA deregulation across smoking-associated tumours still remains unexplored. To address this, we quantified the expression of 51 miRNAs in 10 smoking-associated cancer types and assessed their differential expression in unpaired normal and malignant tissues.

Method:
We analyzed the small RNA transcriptome in 10 cancer types from The Cancer Genome Atlas (TCGA) clinical cohorts (i.e. LUAD, LUSC, HNSC, kidney, stomach, esophagous, bladder, cervical, pancreas and liver). All small RNA sequencing reads were aligned on the human genome build 19 (hg19) and normalized using our in-house bioinformatics pipeline. miRNAs with expressions greater than or equal to 1.0 RPM in 10 percent of samples were included for further analysis. Zeta-score values were calculated and used for unsupervised hierarchical clustering to identify distinct patterns of deregulation across different cancer types. We validated our findings using 132 paired NSCLC samples from the British Columbia Cancer Agency (BCCA). Small RNA sequencing of the validation cohort was performed using Illumina Hi-seq 2000 platform. All samples were microdissected prior to RNA isolation.

Result:
We identified 39 miRNAs that are expressed in all cancer types included in this study. Unsupervised hierarchical clustering based on miRNA z-score values revealed a distinct pattern of deregulation in NSCLC compared to other smoking-associated malignancies. Many of these miRNAs were upregulated in both lung adenocarcinoma and squamous carcinoma while other cancers showed either repression or unchanged expression. We further assessed the expression of these miRNAs in metastatic (with lymph node/distant organ invasion) and non-metastatic lung adenocarcinoma (LUAD) cases using TCGA and BCCA clinical cohorts. From this analysis, we found miR-323b, miR-433 and miR-889 consistently unregulated (p value ≤ 0.01) in metastatic tumours.

Conclusion:
NSCLC tumours showed a unique pattern of deregulation in chromosome 14q32 small non-coding genes when compared with other smoking-associated malignancies. Also, we identified three miRNAs that were significantly upregulated in metastatic LUAD cases. Target prediction and thorough functional assays of these miRNAs may reveal novel pathways disrupted during the metastatic progression of LUAD.

Background:
Ground-glass nodule (GGN) is a type of nonspecific abnormality in lung parenchyma detected by computed tomography (CT) as hazy lesion with preservation of bronchial structures and vascular margins, which has a high correlation with lung adenocarcinoma. Different from typical lung cancer, malignant GGN appears a very early stage characteristic with long and indolent course. Large-scale radiological, pathological, and genetic studies on GGNs have broadened our knowledge to develop strategies of management. However, the molecular pathogenesis of GGNs remains unclear, leaving several questions of great clinical significance unsolved.

Method:
Motivated by this, we collected a cohort of 29 GGN patients diagnosed as early stage lung adenocarcinoma and performed whole exome sequencing (WES) to clarify the comprehensive genomic features and underlying molecular mechanism of GGNs. With the expectation of low purity of these samples, we adopted an ultra-deep sequencing depth to ~1000x, which is the deepest WES strategy so far in a single sample of single sequencing experiment to our knowledge, and got a high resolution landscape of genomic alterations in GGNs.

Result:
We found the extreme heterogeneity within each GGN patient, most of mutations manifesting as low frequency, indicating that GGNs grew under neutral evolutionary dynamics. Next we analyzed the mutation signature of these mutations and identified two novel signatures, Cp*C>A and Gp*CpC>T/Gp*CpG>T. These two signatures can reflect the mutation accumulating process within a growing tumor after initiation. Seven driver genes calculated in our cohort were all known lung cancer related genes, including EGFR，MGA，PIK3CA，PPP2R1A，RBM10，SETD and TP53. Of note, copy number alterations in GGNs were significantly less than other late stage lung cancers and this would result in the specific nature of GGNs.

Conclusion:
In summary, this analysis of exome sequencing data highlights the repertoire of cancer genes and mutational processes in GGN patients, and progresses towards a comprehensive account of the somatic genetic basis of GGNs. These results, combined with further study efforts, will accelerate the pace to the achievement of accurate diagnosis and treatment for GGN patients. Also, the endeavor here provides a framework for the research on early stage tumors and low purity tumors, which will become a subject of active investigation in the near future.

Background:
Malignant mesothelioma is a fatal malignant tumor. It is often difficult to diagnose and to differentiate from other carcinomas, especially pulmonary adenocarcinoma. As there are currently no absolute immunohistochemical positive markers for the definite diagnosis of epithelioid mesothelioma, the identification of additional “positive” markers that may facilitate this diagnosis becomes of clinical importance.

Method:
Gene Expression Analysis Formalin-fixed paraffin-embedded tissue sections from 6 epithelioid mesothelioma and 6 pulmonary adenocarcinoma cases were used for gene expression analysis. RNA extraction for gene expression analysis was performed from papillary or solid growth of tumor cells in each specimen. The Human Transcriptome 2.0 GeneChip Array containing gene transcript sets of 44,699 protein coding and 22,829 nonprotein coding clusters was used to analyze gene expression profiles. Validation by Real-time RT PCR & Western Blotting The increased mRNA expression of DAB2 and Intelectin-1 was validated by reverse transcriptase polymerase chain reaction of RNA from tumor tissue and protein expression was validated by Western blotting of 5 mesothelioma cell lines. Immunohistochemical Procedures and Evaluation of Expression of DAB2 and Intelectin-1 The utility of DAB2 and Intelectin-1 in the differential diagnosis of epithelioid mesothelioma and pulmonary adenocarcinoma was examined by an immunohistochemical study of 75 cases of epithelioid mesothelioma and 67 cases of pulmonary adenocarcinoma.

Result:
Differential Gene Expression Of the 44,699 protein coding and 22,829 nonprotein coding transcripts on the Human Transcriptome 2.0 GeneChip Array, 902 statistically significant mRNA transcripts were differentially expressed, with a greater than 1.3-fold difference, between epithelioid mesothelioma and pulmonary adenocarcinoma. Validation Realtime RT-PCR showed relative mRNA expression of DAB2 and Intelectin-1 was significantly higher in epithelioid mesothelioma than that in pulmonary adenocarcinoma. Western blot analysis showed DAB2 and Intelectin-1 protein expression in all 5 commercially available mesothelioma cells lines with anti-DAB2 and anti-Intelectin-1 antibody. Immunohistochemical Expression Profiles in Epithelioid Mesothelioma and Pulmonary Adenocarcinoma The expression of DAB2 and Intelectin-1 was localized in the cytoplasm of tumor cells in epithelioid mesothelioma cases. Positive DAB2 expression was observed in 60 of 75 epithelioid mesotheliomas (80.0%) and 2 of 67 pulmonary adenocarcinomas (3.0%). In half of epithelioid mesotheliomas, DAB2 immunoreactivity was generally strong and diffuse (score 3+). In contrast, pulmonary adenocarcinomas showing DAB2 expression was focal (score 1+).

Conclusion:
We identified 2 novel positive markers of epithelioid mesothelioma, DAB2 and Intelectin-1, by using gene expression microarray analysis and confirmed their utility to differentiate epithelioid mesothelioma from pulmonary adenocarcinoma by immunohistochemical study.

Background:
Adjuvant chemotherapy of lung cancer including cisplatin is recommended for patients with lung cancer p-stage II-IIIA in case of complete resection. However, at the present, treatment outcomes are not necessarily satisfactory, and there is not effective chemotherapy resume for each patient individually. Establishing effective chemotherapy resume for each patients individually and practicing personalized medicine of lung cancer are expected for improving treatment outcomes after operation of p-stage II-IIIA patients. We reported that anti-aging gene Klotho expression was a prognostic factor for small cell lung cancer and LCNEC so far. In this study, we aimed at revealing possibility of the Klotho gene expression as prognostic factor and effect prediction factor of the chemotherapy, and establishing personalized medicine for adjuvant chemotherapy of lung cancer.

Method:
We introduced the Klotho-plasmid into lung cancer cell A549 and established Klotho overexpressing cell, A549/Klotho. We examined the sensitivity test for various anticancer agents including pemetrexed, CDDP, paclitaxel, gefitinib, etc, using A549 cells and A549/Klotho cells.

Result:
We performed sensitivity test by MTT assay. A549/Klotho cells were more sensitive seven times against pemetrexed compared to A549 cells (IC50; 0.1 micro M). A549/Klotho cells were a little sensitive against docetaxel. There is no difference of sensitivity between A549/Klotho cells and A549 cells against molecular target drugs such as afatinib, alectinib, ceritinib, gefitinib, and osimertinib. In A549/Klotho cells expression of N-cadherin was completely inhibited by Western blot analysis.

Conclusion:
From these result, we conclude that overexpression of Klotho may regulate the sensitivity against pemetrexed through the inhibition of N-cadherin. In the future, we may establish a new strategy of adjuvant chemotherapy for lung cancer based on the expression of anti-aging gene Klotho.

Background:
Lung adenocarcinoma is a heterogeneous disease which can be challenging to classify accurately, yet precise histological subtyping is becoming increasingly important. Subgroup patterns have been shown to confer important prognostic information. In this study, we sort to identify gene expression profiles for the six predominant subtypes within adenocarcinoma, in a sequential cohort of resected tumours, to explore whether molecular markers could enhance standard histological diagnosis.

Method:
89 paired (fresh frozen tumour and normal tissue) lung adenocarcinomas were profiled both histologically and by global gene expression and correlated with multiple clinical parameters including stage, age, gender and smoking status. The tumour samples were reviewed by a thoracic pathologist to determine the predominant subtype and assigned into lepidic, acinar, papillary, micropapillary, solid or cribriform predominant groups. Gene expression was generated using Affymetrix Human gene 1.1ST arrays and the Genetitan platform. All RINs > 6. The data was rma treated using Affymetrix Power Tools and poor quality arrays were detected and excluded using Array Quality Metrics. Low expressed probes and control probes were removed. Differential gene expression of the adenocarcinoma predominant subtypes was evaluated using Limma.

Result:
Survival analysis confirms that age and stage are the most significant predictors of outcome. Application of a highly stringent threshold (adj. P value 0.0001) identified 4805 gene transcripts that were significantly differentially expressed among the six predominant adenocarcinoma subtypes. We determined that 3887 of these transcripts are unique to one of the adenocarcinoma subgroups and therefore have the potential to contribute to a predominant subtype defining transcriptional signature. These unique transcripts include functionally interesting genes such as transcriptional factors SOX2/4/7/13/18 involved in determination of cell fate and ROR1 a receptor tyrosine kinase-like orphan receptor among others. A pairwise comparison of the individual subgroups identified that the most significant gene variation is seen between lepidic predominant and solid predominant, indicating that these subgroups are transcriptionally the most disparate to one another.

Conclusion:
In this study multiple highly significant gene transcripts that allow differentiation between the adenocarcinoma subgroups have been identified. These adenocarcinoma subtype gene signatures have the potential to augment current histological diagnosis of lung adenocarcinoma and provide valuable insights into the different biological processes underpinning the six adenocarcinoma subtypes.