Virtual Library

Background:
ALK/RET/ROS and MET exon 14 skipping detection are important to guide of treatment in non-small cell lung cancer (NSCLC) patients. Next generation sequencing (NGS) platform has been implemented in the daily practice for the diagnosis. However, the validation of the NGS-based panel is still complicated and difficult.

Method:
We developed NGS-based fusion assay panel to detect 183 total fusion variants including ALK/RET/ROS as well as MET exon 14 skipping. The fusion call agreement between OCP-50 NGS assay and Nanostring was performed. The result was compared with the FISH and IHC results. This study describes the validation of the assay to define assay parameters, performance characteristics and reproducibility across laboratories.

Result:
For the 55 samples analyzed, the results are as follows;

	ALK Fusion			ROS1 Fusion			RET Fusion			METD14
	NGS(+)	NGS(-)	Total	NGS(+)	NGS(-)	Total	NGS(+)	NGS(-)	Total	NGS(+)	NGS(-)	Total
NS (+)	20	0	20	6	0	6	5	0	5	0	0	0
NS (-)	1	33	34	1	47	48	0	49	49	2	52	54
Total	21	33	54	7	47	54	5	49	54	2	52	54

Conclusion:
The results of our study will be of help in learning the process of establishing, validating and applying the fusion gene detection method in NSCLCs. Furthermore, NGS based OCP-50 assay for fusion detection is more accurate and reliable method for the diagnosis.

Background:
Non-small cell lung cancer (NSCLC) is a heterogeneous disease characterised by somatic mutations in many genes, some of which are actionable drivers. Modern management of NSCLC requires the identification such driver variants to predict sensitivity to targeted drug therapies. Current methods for mutation detection exhibit varying diagnostic accuracies and limitations. In this study, we determined the utility of a novel high-throughput assay by NanoString for mutation testing in comparison to an alternate platform. This is the first presentation of this combination of NanoString technologies in NSCLC.

Method:
Mutational status was evaluated using the nCounter SPRINT Profiler and the Vantage 3D DNA SNV Solid Tumour Panel in a cohort of 174 fresh-frozen NSCLC tumours, utilising digital counting of unique barcoded probes to detect 104 SNV, multi-nucleotide variants (MNVs) and InDels from 25 genes of clinical significance. 5ng of tumour-derived gDNA was subjected to multiplexed pre-amplification and hybridisation of variant-specific probes to unique fluorescent barcodes. Positive variant calls required raw digital count levels above 200, with a raw-count fold-change above the reference DNA sample greater than 2.0 and a statistical significance of p<0.01. SNV calls by the nCounter assay were made by comparison to a previous study using MALDI-TOF mass spectrometry. An agreement analysis was performed for variants common to both platforms to determine positive, negative and overall percentage agreement (PPA, NPA and OPA). A subset of discordant cases were validated using ddPCR.

Result:
The nCounter SNV assay detected at least one variant in 102/174 (58.6%) cases. Seven (4.0%) cases harboured two SNVs. KRAS variants were detected in 79 (45.4%) cases, EGFR in 6 (3.5%), PIK3CA in 3 (1.7%), TP53 in 3 (1.7%). Overall agreement analysis revealed PPA, NPA and OPA of 96.0%, 93.2% and 94.8% respectively. 5/9 discordant samples were available for validation using ddPCR. 4/5 validated cases favoured the nCounter assay, with one case harbouring a KRAS G12V variant confirmed at a fractional abundance of 1.15%.

Conclusion:
This study found the performance of the nCounter SNV assay and a contemporary platform to be highly concordant. The advantages of this technology include low DNA input, digital data output, reduced turn-around-time (<24hr) and customisability for inclusion of novel variants. The nCounter SNV assay is a robust and sensitive method for translational cancer research.

Background:
This study aimed to develop and assess a practical prognostic lncRNA signature for squamous cell carcinoma of the lung (LUSC).

Method:
RNA expression profile and clinical data from 388 LUSC patients were accessed and download from the Cancer Genome Atlas (TCGA) database. Differential lncRNA expression was compared and analyzed between normal tissue and tumor samples. By univariate and multivariate Cox regression analyses, a seven-lncRNA signature was developed and used for the purpose of survival prediction in LUSC patients. We applied receiver operating characteristic analysis to assess the performance of our model.

Result:
Sixteen out of 1414 differentially expressed lncRNAs in the TCGA dataset were associated with the overall survival of LUSC patients. Risk score analysis was used to select 7 lncRNAs to be included in our model development and validation. The ROC analysis indicated that the specificity and sensitivity of this profile are high. Figure 1 Figure 1. Kaplan-Meier and ROC curves for the 7-lncRNA signature in the validation set. (A) The differences between the high-risk (n=103) and low-risk (n=91) groups were determined by the log-rank test (p<0.0001). Five year overall survival was 36.8% (95% CI: 26.1%-51.8%) and 61.9% (95% CI: 51.4%-74.6%) for the high-risk and low-risk groups, respectively. (B) ROC curves indicated that the area under receiver operating characteristic of 7-lncRNA model was 0.685.



Conclusion:
The current study identified a seven-lncRNA signature that predicts the outcome of LUSC, offering potentially novel therapeutic targets for the treatment of squamous cell carcinoma of the lung.

Background:
Primary lung enteric adenocarcinoma is a rare type of invasive lung carcinoma. Its morphology and immunohistochemistry is close to colorectal carcinoma, but there is no associated primary colorectal carcinoma. The purpose of this study is to assess the gene mutational feature in lung enteric adenocarcinoma by the next generation sequencing.

Method:
From Feb. 2013 to Dec. 2016, 11 lung enteric adenocarcinoma patients (5 males and 6 females) received treatment from three medical centers: including Beijing, Zhejiang and Fujian. All the patients were diagnosed by pathology. Analysis was used by the next generation sequencing.

Result:
ALK/ROS1 primary point mutations were confirmed in 5 patients (71.42%, 5/7). MSH2/MSH6 point mutations were confirmed in 3 patient (42.86%, 3/7). There was no case with drive genes changed, such as EGFR mutation, ALK rearrangement, ROS1 rearrangement, RET rearrangement, MET amplification or 14 exon skipping mutation. The median overall survival (OS) of 11 lung enteric adenocarcinoma patients was 9.0 months, Subgroup analysis the median OS of ALK/ROS1 primary point mutation patients was 6.5 months, the median OS of MSH2/MSH6 primary point mutation patients was 26.0 months.

Conclusion:
ALK/ROS1 primary point mutations or MSH2/MSH6 point mutations are the most frequent feature of heterozygous mutation in our study. The MSH2/MSH6 subgroup of the median OS is longer. Further investigations will be required to validate our findings.

Background:
The cases of pulmonary mucoepidermoid carcinoma (PMEC) are extremely rare. There is only limited data on treatment outcome for chemotherapy in PMEC, and less so for targeted therapy such as targeted therapy with icotinib. The aim of this study is to investigate the molecular characteristics of pulmonary mucoepidermoid carcinoma (PMEC).

Method:
From July 2013 to December 2016, 13 PMEC patients received treatment. All the patients were diagnosed by pathology. We retrospectively reviewed the clinical data and genetic state.

Result:
EGFR mutation rate was 15.38% (2/13), and 2 cases were both L861Q point mutations, the relationship between EGFR gene status and gender (P=1.000), age (P=1.000), smoking (P=0.848) and stage (P=1.000) were no significant, respectively; the positive rate of MAML2 fusion gene was 45.45%(5/11). the relationship between MAML2 fusion gene status and gender (P=0.521), age (P=0.521), smoking (P=1.000) and stage (P=0.924) were no significant, respectively.

Conclusion:
The most common form change of pulmonary mucoepidermoid carcinoma is EGFR gene L861Q point mutation, MALM2 fusion gene exist in the EGFR gene wild type patients. icotinib treatment may benefit from patients with EGFR L861Q point mutations and MALM2 fusion gene.

Background:
With the development of High-throughput DNA sequencing technologies, several tools have been developed aim at searching structural variations. However, most of available structural variation predication tools can only identity the abnormal connections, a systematically parsing the pattern of structure variation to obtain the length and connection type of SVs is still tough work.

Method:
In this study, we present a tool, NovoSV, which can identify the abnormal connections precisely, and based on associated abnormal connections NovoSV will report the length and connection type of the structural variation.

Result:
NovoSV took a BWA mapped results as input and identified abnormal connections and pattern of chromosomal structure variations would be reported. For validation, NovoSV was applied to target sequencing data derived from 10 samples, NovoSV identified 8 abnormal connections, and 7 of these results could be validated by polymerase chain reaction (PCR). When applied to whole-genome sequencing data derived from 5 samples, NovoSV reported 46 SVs with their length and connection type. Of the 4 random selected identified results, 3 were validated by Sanger sequencing.

Conclusion:
NovoSV is an efficient tool for chromosomal variation detection, which can accurately identify abnormal connections and parse the pattern of chromosomal variations. NovoSV has been validated on GNU/Linux systems, and an open source PERL program is available.

Background:
STK11 is commonly mutated in non-small cell lung cancer (NSCLC). In light of recent experimental data showing that specific STK11 mutantion can acquire oncogenic activities due to the synthesis of a short STK11 isoform, The aim of this study is to investigate whether this new classification of STK11 mutants can help refine its role as a prognostic marker.

Method:
A total of 879 patients with NSCLC were recruited between July 2012 and December 2014. The status of STK11 mutation and other genes were detected by the next generation sequencing (NGS).

Result:
STK11 gene mutation rate was 0.91% (8/879) in NSCLC, including p.K269fs*18 (1 patient), p.K329stop (1 patient), c.464 plus 1G>T (1 patient), p.D194E (1 patient), p.D176V (1 patient), p.D53Tfs*11 (1 patient), p.D194A (1 patient) and p.Y118* (1 patient), and median overall survival (OS) for these patients was 22.2 months. Among them, all patients were STK11 gene with co-occurring mutations. Briefly, patients with (n=2) or without (n=6) co-occurring EGFR mutations had a median OS of 29.0 months and 22.2 months repectively (P=0.73); patients with (n=5) or without (n=3) co-occurring TP53 mutations had a median OS of 29.0 months and 22.2 months repectively (P=0.95); patients with (n=3) or without (n=5) co-occurring KRAS mutations had a median OS of not reached so far and 13.8 months repectively (P=0.02); patients with (n=2) or without (n=6) co-occurring SMARCA4 mutations had a median OS of 14.0 months and 37.0 months repectively (P=0.11); patients with (n=3) or without (n=5) co-occurring KEAP1 mutations had a median OS of not reached so far and 14.6 months repectively (P=0.20).

Conclusion:
STK11 mutations represent a distinct subset of NSCLC. NGS showed that STK11 mutations commonly co-existed with other driver genes. Our results show that STK11 mutations delineate an aggressive subtype of lung cancer for which a targeted treatment through STK11 inhibition might offer new opportunities.

Background:
Lung cancer patients often have poor prognosis, on account of high propensity for metastasis. Cancer-associated fibroblasts (CAFs) are the main type of stromal cells in lung cancer tissue, which are activated by tumor cells, play a significant role in tumor development. However, it is uncertain whether CAFs induce lung cancer cells metastasis and which pathway is involved. Snail1 is a transcriptional factor and its expression in the stroma associates with lower rates of survivors in cancer patients. Nevertheless, it has not been determined how Snail1 regulate the crosstalk between stromal and tumor cells when it expresses in stroma. Altered expression of microRNA (miRNA) correalates with lung carcinogenesis and metastasis. Our previous study of miRNAs showed that the level of miR-33b was obviously decreased in lung adenocarcinoma cell lines and lung cancer tissues, and when miR-33b was elevated, it can suppressed tumor cell growth and EMT in vitro and in vivo experiments.

Method:
(1) We co-culcured four different human lung cancer cells (A549, H1299, SPC-a-1, LTEP-a-2) with control medium, NFs and CAFs，then we examined lung cancer cells motility, migration, invasion, miR-33b expression level, relevant mRNAs and proteins expression levels. (2) A549 and H1299 cells was transfected with miR-33b mimics or with miR-NC prior to CAF stimulation, then subjected to wound healing assay, Transwell assay, qRT-PCR, immunoblotting assay. (3) Using coculture lung cancer cell lines with SNAI1 transfected CAFs models, we explore the role of Snail1 in CAFs cells.

Result:
(1) Cocultivation of CAFs with lung cancer cells induced downregulation of miR-33b in lung cancer cells and promoted epithelial cells epithelial-mesenchymal transition (EMT). (2) MiR-33b overexpression by transfecting cancer cells with miR-33b mimics reverted CAFs induced EMT. (3) Transfection of CAFs with SNAI1 enhanced CAFs modulation on lung cancer cells EMT when CAFs co-cultured with A549 and H1299 cells, whereas si-SNAI1 attenuated CAFs modulation role.

Conclusion:
The induction of cancer cells EMT by CAFs is a key mechanism underlying the acquisition of cancer cells aggressive propensity. And CAFs induce lung cancer cells EMT in a miR-33b-mediated manner. Expression of Snail1 in fibroblasts was essential for the induction effect of CAFs on lung cancer cells EMT.

Background:
Metabolomics measures low weight molecules, generally called metabolites, and it is an effective technique to understand how metabolism is changed by various factors, including environment and disease, particularly malignant disease. Body fluids, for example sputum or urine, harvested non-invasively havebeen used in remarkable recent developments of omics analysis technology, yielding highly precise results for diagnosis of oral cancer, breast cancer, and pancreatic cancer. Metabolomic analysis has begun to be reported based on the pattern information of metabolites. It can be used for practical clinical early detection of carcinoma of various organs. However, practical metabolomic analysis regarding lung cancer has not been repored yet. We used surgically resected specimen of lung cancer to analyze and clarify metabolomics as an aspect of lung cancer.

Method:
We obtained resected specimens from patients with lung cancer after obtaining informed consent for this study, and compared the metabolism profile of the normal tissue portion with carcinoma tissue in 80 patients in terms of various clinical aspects. Metabolomic analysis was performed by capillary electrophoresis / time-of-flight mass spectrometry (CE-TOFMS) of metabolites of the lung tissue and analysed ionized tissue which contained the most main metabolites.

Result:
Analysis of serum and metabolite organization by CE-TOFMS revealed that the intermediate metabolite levels of several pathways changed markedly in lung cancer tissue. We can identify a characteristic metabolic marker in advanced lung cancer tissue with metabolomic clinical information by analysing the association with the overall metabolism profile.

Conclusion:
We identified metabolomic biomarkers which were characteristic of lung cancer using resected tissue in this study. At present, we are analysing various body fluids for analysis of lung cancer cases including prognostic implications. Applications to non-invasive, simple, easy and cheap cancer screening are expected in the future.

Background:
Non-Small-Cell Lung Cancer (NSCLC) is often diagnosed at an advanced stage and carries a poor prognosis. Greater knowledge of the molecular origins and progression of lung cancer may lead to improvements in the treatment and prevention of the disease. Although it is well-known the importance of desmosomes in cell-to cell adhesion, several evidences suggest that some of their structural proteins may have additional and relevant roles in cancer development. In this regard, Plakophilin 1 (PKP1) is up-regulated in primary NSCLC tumors (SCC and AD) suggesting an oncogenic role in tumor development that it is probably beyond their role into cell-to-cell adhesion.

Method:
In order to gain greater insight into the multifuntional role of PKP1 in NSCLC tumours, we have generated functional models in that express different levels of PKP1 protein using siRNA, Crispr Cas9 system or lentivirus-vectors. We have carried out several phenotypical assays to demonstrate the PKP1´s role in tumor development into NSCLC cell lines (proliferation, cell cycle, apoptosis, wound healing, BrdU and colony assays, etc.) and an in vivo xenograph assays using CrispR-Cas9.

Result:
On the one hand, when we inhibit PKP1 in cell lines with high PKP1 protein basal level, we observe an increment in migration in the wound-healing assays. This PKP1-function formally can be considered a tumour-suppressor activity. On the other hand, we have also observed pro-oncogenic activity after PKP1 expression inhibition. In this way, after the knock-down or knock-out PKP1, we have observed cell-growth and cell-cycle arrest and higher levels of apoptosis. Additonally, when we over-express PKP1 in a cell line with no PKP1 protein basal level,we detect cell growth and S phase increment, and apoptosis reduction; gainning new evidences that support pro-oncogenic role of this protein. To determine the results of these apparently opposing tumoral activity, we have developed and in vivo xenograph assay using CrispR-Cas9. The tumoral burden dynamics in the model clearly indicate that, although the loss of PKP1 promotes cell-migration, overall the role of PKP1 is pro-oncogenic. Currently, we are performing a PKP1 immunoprecipitation and an expression array in order to elucidate PKP1 tumoral functions that could help us to explain these new pro-oncogenic activities that remain unknown.

Conclusion:
In conclusion, our results indicate PKP1 protein has a contribution in NSCLC development and it may be a potential useful for NSCLC diagnosis, prognosis and treatment.

Background:
Lung cancer cell line data suggest FGFR1 status defined by FGFR1 mRNA levels and FGFR1 gene copy number can predict sensitivity to FGFR tyrosine kinase inhibitors (TKIs).

Method:
A phase II biomarker preselected trial of ponatinib, an FGFR1, FGFR2, and FGFR4 targeted TKI was designed. Patients with metastatic EGFR- and ALK-negative lung cancers (both NSCLC and SCLC) were pre-screened for FGFR1 mRNA levels by in-situ hybridization (ISH) and FGFR1 gene copy number by silver in-situ hybridization (SISH). Positivity criteria for ISH were defined as a score of 3 (Dot clusters seen in 1 to <10% tumor cells; otherwise >10 dots/cell in ≥ 10% tumor cells), or 4 (Dot clusters seen in ≥ 10% of tumor cells). Positivity for SISH was defined as an average of ≥ 4 FGFR1 signal clusters/nucleus or FGFR1/CEN8 ratio ≥ 2.0. Clinical factors including sex, histology, age and sites of metastases at diagnosis of stage IV disease, smoking status, status of other known molecular drivers, and response to initial platinum-doublet therapy for stage IV disease were collected.

Result:
From 11/2013 to 05/2017, the study has pre-screened 163 patient samples for FGFR1 ISH and SISH. Thirty-eight (23.3%) had insufficient tissue; four had incomplete clinical or FGFR1 information. Clinical variables according to FGFR1 ISH/SISH status (n=121) are summarized in Table 1. Impact of alternate positivity cut-points, outcomes of patients treated with ponatinib and survival analysis according to ISH/SISH subgroups will be presented. Figure 1



Conclusion:
Although the numbers were small, the FGFR1 ISH+/SISH+ subgroup had a greater percentage small cell histology, liver metastases at diagnosis and male sex compared to other FGFR1 subgroups. FGFR1 ISH and/or SISH positivity can overlap with other known oncogenic drivers suggesting that the initial cut-points for FGFR1 positivity used may be too low to identify a true FGFR1 addicted state.

Background:
Small cell lung cancer (SCLC) is a neuroendocrine tumor which account for 15% of all lung cancers. SCLC is characterized by rapid progression which led metastasis to distant organs and represented poor prognosis. Although there were various molecular targeted therapies in SCLC that were already under investigation, results have been disappointing. The focus on biological pathways has recently shifted from genetic to epigenetic regulations. Histone methylation is one of the epigenetic mechanism which has pivotal role in gene expression, cell cycle and differentiation. Lysine-specific demethylase 1 (LSD1)/KDM1 is a histone modifier that is expressed in certain human cancers including SCLC. LSD1+8a is one of the isoform of LSD1 that was reported to be restricted in neural tissues and it plays critical role in neuronal differentiation. The aim of this study is to elucidate the epigenetic role of LSD1+8a in SCLC.

Method:
The expression of LSD1 and LSD1+8a mRNAs were analyzed by qPCR in several cancer cell lines. Neuroendocrine markers were detected by qPCR in several SCLC cell lines. The Pearson correlation test was performed to investigate the correlation between LSD1+8a and neuroendocrine markers. siRNA against specific to exon 8a was designed and knockdown LSD1+8a isoform specifically. Cell proliferation assays following knockdown of LSD1+8a were performed in LSD1+8a expressed SCLC cell lines.

Result:
LSD1 mRNA was expressed in majority of SCLC cell lines, interestingly LSD1+8a mRNA was detected in small subset of SCLC cell lines. There were positive correlations of LSD1+8a and neuroendocrine marker genes such as chromogranin A (CHGA), enolase2 (ENO2), neural cell adhesion molecule (NCAM) and synaptophysin (SYP) in human cancer cell lines. The suppression of LSD1+8a by siRNA drove to decrease expression of neuroendocrine marker genes in SCLC and inhibited cell proliferation in LSD1+8a expressed cell.

Conclusion:
LSD1+8a could play an important role in SCLC.

Background:
Carcinogenesis may result from accumulation of molecular aberrations (molecular evolution) and escaping from host immune surveillance (immunoediting). It has been postulated that atypical adenomatous hyperplasia (AAH) represents preneoplastic lesion that may progress to adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA) and further to frankly invasive adenocarcinoma (ADC). However, due to lack of appropriate study materials, the molecular and immune landscape of AAH, AIS or MIA have not been well studied and the definition and management of these lesions remain controversial.

Method:
With the intent to delineate the pivotal molecular and immune events during early carcinogenesis of lung adenocarcinoma, we have collected 119 resected pre- and early neoplastic lung lesions including AAH (N=24), AIS (N=27), MIA (N=54) and ADC (N=14) from 53 patients including 41 patients presenting with multifocal lesions and 25 patients carrying more than one type of pathology. Two to five spatially separated regions from each lesion were subjected to whole exome sequencing and T cell receptor sequencing.

Result:
Mutation burden (average SNVs) was found to progressively increase from 1.32/Mb in AAH to 2.55/MB in AIS, 5.42/MB in MIA and 15.38/MB in ADC. Genomic heterogeneity has also become more complex with neoplastic progression with mean Shannon index of 1.53 in AAH, 1.78 in AIS, 1.56 in MIA and 1.79 in ADC. An increase in C>A transversions coincident with a decrease in A>G transitions and progressively increasing APOBEC enrichment scores (4.13 in AAH, 5.63 in AIS, 6.02 in MIA and 6.59 in ADC) were observed with neoplastic disease progression. Furthermore, phylogenetic analysis revealed varying evolutional processes in AAH, AIS, MIA and ADC with canonical cancer gene mutations in KRAS, ATM, TP53 and EGFR etc. as key drivers in a subset of patients. TCR sequencing demonstrated a progressive decrease in T cell density (average percent T cells among all nuclear cells: 12% in AAH, 8% in AIS, 7% in MIA and 4% in ADC) and a progressive decrease in productive TCR clonality (average productive TCR clonality: 0.0434 in AAH, 0.0427 in AIS, 0.0399 in MIA and 0.0395 in ADC) suggesting suppressive T cell repertoire in more advanced diseases.

Conclusion:
Our results provide molecular evidence supporting the model of early lung carcinogenesis from AAH, to AIS, MIA and ADC and demonstrated that with disease progression, genomic landscape of lung neoplastic lesions has become progressively more complex along with progressive immunosuppressive TCR repertoire.

Background:
Currently, assessment of several tumor drivers is critical for individualized treatment of non-small cell lung cancer (NSCLC). Tools for molecular profiling are based on DNA, RNA and protein (PCR, NGS, FISH, IHC). However, these tests have several disadvantages including hands-on-time and tissue mass requirements. Nanostring digital barcoding technology enables simultaneous assay of different analytes, DNA and RNA from a single sample with 3D Biology Technology.

Method:
The nCounter Vantage 3D SNV:Fusions Lung Assay was used to analyze a total of 36 formalin-fixed paraffin embedded (FFPE) samples from advanced NSCLC patients. Samples were known to harbor mutations (EGFR, KRAS, NRAS, PIK3CA, BRAF, P53) or gene-fusion rearrangements (ALK, ROS1, RET, NTRK1) as verified by sequencing (Ion Torrent, Gene Reader), nCounter Elements, IHC and/or FISH. Probes were designed to target 25 genes for SNVs (104 different point and InDel mutations) as well as four genes for fusion transcripts (ALK, ROS1, RET, NTRK1) including 33 specific variants. The 3D workflow requires pre-amplification of gDNA, whereas RNA does not require any enzymatic treatment. After hybridization, the analytes (DNA/RNA) are pooled for simultaneous, single-lane, digital counting in total turnaround of 3 days. A total amount of 5ng DNA and 150ng RNA from two4-micron FFPE-sections was used for the assay without microdissection.

Result:
A total of 72 analyses (DNA/RNA) were performed with an evaluation pass of 97.2% (70/72 analyses yielded results) and 89% concordant results (64/72). Sensitivity of the technique was 92.1%. Among 41 SNVs interrogated in this study 34 were successfully detected (two not evaluable). Five new mutations were found involving NRAS, FBXW7, GNA11, FGFR2 and KRAS genes. Of those, only two were considered false positives as they were not confirmed by alternative sequencing and/or PCR. The remaining three were not assessable for test confirmation. For gene fusion analysis, 13 known positive samples were tested. All fusion transcripts were detected for ALK (n=5) RET (n=2) and NTRK1 (n=1). For ROS1 (n=5) there were 2 false negatives only detected by nCounter Elements target-specific assay.

Conclusion:
We have shown that the SNV detection chemistry can be successfully combined with fusion gene expression analysis by using the nCounter 3D™ single-workflow. The Nanostring nCounter Vantage 3D SNV:Fusions Lung Assay is highly efficient in detecting hotspot mutations as well as gene rearrangements. The assay is simple, features a brief hands-on time and requires low amounts of genomic material, supporting minimal use of precious samples.

Background:
Non-small cell lung cancer (NSCLC) is a heterogeneous disease with unique combinations of somatic molecular alterations in individual patients, as well as significant differences in populations across the world with regard to mutation spectra and mutation frequencies. Here we describe the mutational pattern and linked clinical parameters in a population-based Swedish NSCLC cohort.

Method:
The cohort consists of 354 patients treated surgically at the University Hospital in Uppsala between 2006 to 2010. DNA was extracted from either fresh frozen (n=200) or formalin fixed paraffin embedded (FFPE; n=154) tissues prior to library preparation with Haloplex capture probes and Illumina Hiseq sequencing. The gene panel covers all exons of 82 genes, that have been shown to harbor mutations relevant for NSCLC development, and utilizes a reduced target fragment length and two strand capture compatible with degraded FFPE samples.

Result:
In order to avoid a systematic technical bias between FFPE and fresh-frozen samples, we adapted the sequencing depth and the bioinformatic pipeline for variant calling to obtain uniform sequence coverage and mutational load across the two sample types. TP53 was the most frequently mutated gene in both adenocarcinoma (AdC; 47%) and squamous cell carcinoma (SqC; 85%). KEAP1 or NFE2L2 was mutated in 19% of AdC and 23% of SqC in a mutually exclusive fashion. In AdC, hotspot alterations in driver genes could be seen in KRAS (43%), EGFR (13%), ERBB2 (3%, exon 20 insertions), BRAF (2%) and MET (1%, exon 14 skipping). Mutations in STK11 were observed in 21% of AdC cases. In SqC, frequently mutated genes were MLL2 (26%), PIK3CA (20%), CDKN2A (15%) and DDR2 (4%). Survival analysis revealed a worse overall survival for AdC patients with a mutation in either TP53, STK11 or SMARCA4. In the KRAS-mutated group poor survival appeared to be linked to concomitant TP53 or STK11 mutations, and not to KRAS mutation as a single aberration. In SqC a worse overall survival could be observed for patients with MLL2 mutations. SqC patients with mutations in CSMD3 had trend for a better prognosis.

Conclusion:
Here we have evaluated the mutational status of a Swedish NSCLC cohort. Technical adaption allowed analysis across both FFPE and fresh-frozen samples. Overall, the high frequency of TP53 and KRAS mutations might be related to the large fraction of smokers. Poor prognosis was linked to mutations in TP53, STK11 or SMARCA4 in AdC and MLL2 mutations in SqC.

Background:
Pulmonary sarcomas (PSRC) are uncommon primary thoracic malignancies that are often clinically aggressive. Comprehensive genomic profiling (CGP) can identify biomarkers for both targeted and immunotherapy. We used CGP to analyze novel treatment options for patients with advanced PSRC.

Method:
CGP using hybridization-captured, adaptor ligation-based libraries for up to 406 genes plus select introns from 31 genes frequently rearranged in cancer was performed on 19 PSRC; 15 cases also underwent RNA sequencing for enhanced fusion detection in 265 of these genes. All classes of genomic alteration (GA) were assessed simultaneously: base substitutions, indels, rearrangements, and copy number changes. Clinically relevant GA (CRGA) are GA associated with drugs on the market or under evaluation in clinical trials. Tumor mutational burden (TMB) was determined on 1.1 Mb of sequenced DNA.

Result:
In this cohort were 10 sarcoma NOS, 5 pulmonary artery intimal sarcomas, 2 pleomorphic/MFH sarcomas, 1 primary IMT, and 1 primary SFT, including 1 stage I, 1 stage II, 9 stage III and 8 stage IV tumors. Patient median age was 52 years (range 33–81 years), with 7 female and 12 male patients. The mean GA per sarcoma was 5.7. Notable alterations not presently considered actionable affected TP53 (53%), CDKN2A (32%), CDKN2B (27%), and RB1 (21%). CRGA alterations were detected in PDGFRA, RICTOR, CDK4 and KIT (11% each), and EGFR, TSC2, ALK and BRAF (5% each); 9 (47%) PSRC featured ≥1 CRGA. An ALK fusion was detected in an IMT localized only to the lung and diagnosed as a primary lesion. Inactivation of SMARCA4 through mutation and loss of heterozygosity was found in 1 case. Mean TMB was 8.65 mutations/Mb (16% had TMB >10 mut/Mb, 11% had TMB >20 mut/Mb); cases with TMB >20 mut/Mb lacked characteristically targetable CRGA. All samples tested for MSI (n=7) were microsatellite stable. Assessment of therapeutic intervention and responses is ongoing.

Conclusion:
PSRC is an extremely rare primary lung malignancy characterized by a relatively high frequency of GA. CGP identified various potentially targetable alterations in this small series, particularly driver mutations or fusions in tyrosine kinases and cell cycle regulatory genes. Furthermore, characteristic genomic profiles can provide diagnostic insight, as for SMARCA loss or ALK fusions. This study also identified a significant number of PSRC with intermediate or high TMB, indicating potential immunotherapy options for these patients. Further study of CGP to help manage patient care and minimize suffering from this rare pulmonary malignancy appears warranted.

Background:
Less than half of lung adenocarcinoma (LUAD) patients harbour clinically actionable driver genes, emphasizing the need to explore alternative mechanisms of cancer gene deregulation. Long non-coding RNAs (lncRNAs) have emerged as important players in cell biology, and can be exploited by tumours to drive the hallmarks of cancer. Pseudogenes are DNA sequences that are defunct relatives of their functional protein-coding parent genes but retain high sequence homology. Interestingly, several lncRNAs expressed from pseudogene loci have been shown to regulate the protein-coding parent genes of these pseudogenes in trans due to sequence complementarity. We hypothesize that this phenomenon occurs more broadly than previously realized, and that these events provide an alternative mechanism of cancer gene deregulation in LUAD tumourigenesis that has clinical implications.

Method:
Illumina HiSeq reads were processed and aligned to the ENSEMBL annotation file in order to derive the most complete set of both protein-coding and non-coding genes. Two datasets were selected due to their paired nature, complete with both LUAD and non-malignant lung profiles (TCGA n=108, BCCA n=72). LncRNAs were filtered based on positional overlap within pseudogene loci, and a Wilcoxon sign-rank test was run to identify lncRNAs with significantly altered expression between paired tumour and normal tissues (FDR p<0.05). To identify lncRNAs that likely regulate protein-coding parent gene expression in trans, tumours were ranked by lncRNA expression, and protein-coding parent gene expression of top and bottom ranked tertiles was compared by Mann Whitney U-test (p<0.05). Survival analysis was performed using a Cox proportional hazard model.

Result:
Our analysis has identified 129 lncRNAs expressed from pseudogene loci that were significantly deregulated in LUAD in both datasets. Remarkably, many of these deregulated lncRNAs (i) were expressed from the loci of pseudogenes related to known cancer genes, (ii) had expression that significantly correlated with protein-coding parent gene expression, and (iii) protein-coding parent gene expression was significantly associated with survival. For example, RP11-182J1.1 is a lncRNA expressed from a pseudogene to EGLN1, a previously described cancer gene involved in regulation of tumour hypoxia. RP11-182J1.1 was underexpressed in LUAD and significantly positively correlated with EGLN1 expression. In addition, EGLN1 was significantly associated with patient survival (p=1.2e-08) emphasizing the clinical potential of these lncRNAs.

Conclusion:
This work uncovers evidence to suggest the lncRNA-pseudogene-protein-coding gene axis is a prominent mechanism of cancer gene regulation. Further characterization of this understudied gene regulatory mechanism could lead to novel therapies that silence oncogenes or reactivate tumour suppressor genes.

Background:
Identifying genomic alterations of actionable driver genes in non-small cell lung cancer (NSCLC) such as EGFR, ALK, ROS1 has been used as important evidences for firstline treatments. Patients with driver mutations received matched target drugs could have significantly longer progression free and overall survival.

Method:
FFPE tumor samples of 498 Chinese NSCLC patients including 279 males (56%) and 219 females (44%) with a median age of 60 were collected for next-generation sequencing (NGS)-based multi genes panel assay. Genomic alterations including single base substitution, short and long insertions/deletions, copy number variations, and gene rearrangement and fusions in selected genes were assessed.

Result:
Different histological subtypes of adenocarcinoma (417/498, 83.7%), squamous carcinoma (68/498, 13.7%), mixed carcinoma (6/498, 1.2%) and large cell carcinoma (7/498, 1.4%) were included in the Chinese NSCLC cohort. The top ranked genomic alterations in driver genes were EGFR (47.8%), KRAS (10.0%), ALK fusions (8.2%), PIK3CA (7.0%), HER2 (6.2 %), PTEN (3.6%), BRAF (2.6%), MET (3.6%), RET fusions (1.6%), and ROS1 fusions (0.8%), which counts up to 86.9% of the 498 patients with at least one driver mutation. In addition to common driver mutations, rare mutation types such as EGFR-KDD, EGFR-RAD51, AMOTL2-NTRK1 and KIF13A-RET were also detected by deep sequencing assay.

Conclusion:
Our study revealed the landscape of driver gene mutations in 498 Chinese NSCLC patients. Comparing to the largest public NSCLC cohort from Foundation Medicine, mostly Western populations (N=6823, PMID: 27151654), we identified similar frequencies of some driver genes, but more ALK fusions (8.2% vs 3.9%), EGFR mutations (47.8% vs 20.0%) as druggable target genes, and less KRAS mutations (10.0% vs 32.0%) consistent with reported results. Totally 78.7% of the Chinese patietns harbored at least one mutaiton in the 8 core driver genes including EGFR, ALK, BRAF, ERBB2, MET, ROS1, RET, or KRAS (vs. 71% in the FMI cohort). Our findings demonstrated that genomic profiling of driver genes in NSCLC showed significant differences among racial or ethnic groups, which indicated different treatment options between Eastern and Western populations.

Background:
The detection of circulating tumor cells and the corresponding expression of tumor genes has been utilized as a prognostication tool for assessing therapeutic response to various anti-cancer therapies including immune cell therapy, chemotherapy and natural products-based anti-cancer therapy at the Lung Center of the Philippines (LCP) since 2014. Recently, circulating tumor DNA (ctDNA) has emerged as a new promising test for noninvasive detection of several cancers including lung cancer. This study aims to evaluate the utility of ctDNA in prognostication of lung cancer vs. other cancer types.

Method:
Blood sample from cancer patients (7.5 ml) were extracted for DNA and RNA using the QIAamp Circulating Nucleic Acid Kit, Qiagen, and subjected to two tests, 1) detection of EGFR mutation using the therascreen EGFR Plasma RGQ PCR Kit, and 2) detection of tumor gene expression level using an in-house two gene panel (i.e. EGFR and ERBB2). Three groups were compared, group A (Lung cancer, 7 cases), group B (Breast cancer, 7 cases) and group C (one case of Ovarian, one case of Colon and one case of Mixed Epithelial). The protocol is approved by the Technical and Ethics Review Board of the hospital.

Result:
Data show that in groups B and C, no EGFR mutation was detected while in group A, two out of 7 cases showed Exon 19 Deletion. The in-house two gene panel showed group A to have two cases of upregulated ERBB2 (i.e., 5-9.5 folds), group B to have three cases of upregulated ERBB2 (i.e., 1.6-6.5 folds) and one upregulated EGFR (i.e., 15.7 folds) and group C to have one case of upregulated ERBB2 (i.e., 7.4). ctDNA with therascreen for EGFR mutation detection has less sensitivity (i.e.2/17) as compared to ctDNA with in-house two panel for tumor gene expression (i.e., 7/17), although specific to detect EGFR-associated lung cancer. The data also suggest a possible preponderance of copy number-driven cancer as compared to mutation-driven cancer in these set of patient samples.

Conclusion:
ctDNA-based tumor detection can be useful as a prognostication tool however it should be in combination with a compatible detection platform that may indicate copy-number driven or mutation-driven cancer.

Background:
Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a known tumor suppressor in non-small cell lung cancer (NSCLC). Because of the rarity of those mutations, associated clinical features and prognostic significance have not been thoroughly described so far. The aim of this study is to investigate mutations and prognosis of NSCLC harboring PTEN mutations.

Method:
A total of 402 patients with non-small-cell lung cancer were recruited between July 2012 and December 2014. The status of PTEN mutation and other genes were detected by the next generation sequencing.

Result:
PTEN gene mutation was detected in 1.99% (8/402) NSCLC patients, including A333fs*10 (2 patients), D252N (1 patient), P38S (1 patient), Q171E (1 patient), S59* (1 patient), S10R(1 patient) and Y225Ifs*18(1 patient), and median overall survival (OS) for these patients was 19.7 months. Among them, 7 patients with co-occurring mutations had a median OS of 23.3 months, and OS of the 1 patient without complex mutations was 14.6 months. No statistically significant difference was found between the two groups (P=0.35). Briefly, patients with (n=5) or without (n=3) co-occurring EGFR mutations had a median OS of 33.6 months and 16.0 months repectively (P=0.33); patients with (n=4) or without (n=4) co-occurring TP53 mutations had a median OS of 14.9 months and 33.5 months repectively (P=0.18); patients with (n=2) or without (n=6) co-occurring DNMT3A mutations had a median OS of 17.8 months and 24.8 months repectively (P=0.27).

Conclusion:
Our results demonstrated that decreased PTEN gene mutation correlated with poor overall survival in non-small-cell lung cancer patients. PTEN gene mutation may define a subset of patients with lung cancer appropriate for investigational therapeutic strategies.

Background:
PTPRD, encoding protein tyrosine phosphatases receptor type D, is located at chromosome 9p23-24.1, a loci frequently lost in many types of tumors. Recently, PTPRD has been proposed to function as a tumor suppressor gene. The aim of this study is to investigate mutations and prognosis of non-small-cell lung cancer(NSCLC) harboring PTPRD mutations.

Method:
A total of 962 patients with NSCLC were recruited between July 2012 and December 2014. The status of PTPRD mutation and other genes were detected by next generation sequencing.

Result:
PTPRD gene mutation was detected in 0.64% (6/962) NSCLC patients, including V693F (1 patient), V330L (1 patient), T1103A (2 patients), D388Y (1 patient) and R1692G plus G1213V (1 patient), and median overall survival (OS) for these patients was 31.4 months. Among them, all patients were PTPRD gene with co-occurring mutations. Briefly, patients with (n=2) or without (n=4) co-occurring EGFR mutations had a median OS of 41.0 months and 20.6 months repectively (P=0.06); patients with (n=4) or without (n=2) co-occurring TP53 mutations had a median OS of 27.6 months and 26.0 months repectively (P=0.79).

Conclusion:
PTPRD gene mutation coexist with other gene mutation in NSCLC. EGFR and TP53 gene accompanied may have less correlation with PTPRD mutation in NSCLC patients. Results of ongoing studies will provide more insight into effective treatment strategies for patients with PTPRD mutations.

Background:
Uncovering novel mechanisms of cancer-gene regulation may reveal new actionable targets to direct the treatment of patients who do not harbour targetable molecular drivers of lung cancer. Long non-coding RNAs (lncRNAs), are a class of transcripts that hold an emerging role in cell biology, particularly in gene regulation. These genes have since been implicated in cancer-associated phenotypes, and may represent attractive therapeutic intervention points; however, prediction of downstream regulatory targets of lncRNAs has been impeded due to their complex tertiary structure. Recently, a subset of lncRNAs has been shown to regulate the expression of neighbouring protein-coding genes in cis. Here we take a novel approach to identify lncRNAs deregulated in lung adenocarcinoma (LUAD) and examine their roles in the expression modulation of their cancer-associated protein-coding cis-partner genes.

Method:
RNA-sequencing was performed on 36 LUAD tumour samples with matched adjacent non-malignant tissue obtained via microdissection to 90% purity. Significantly deregulated lncRNAs and neighbouring protein-coding genes were identified by comparison of matched tumour and non-malignant normalized read counts (Wilcoxon Signed-Rank Test, FDR-BH<0.05). Fifty LUAD tumours with paired normal tissue from The Cancer Genome Atlas (TCGA) were used to validate these findings. Cox-Proportional Hazard analysis was performed on both datasets to assess survival associations of significantly deregulated lncRNAs.

Result:
Our approach revealed greater than 500 lncRNAs that were significantly deregulated between LUAD and matched normal tissues. Many of these lncRNAs have neighbouring protein-coding genes that also display deregulated expression patterns. Of particular interest are the protein-coding-target genes that have been previously implicated in cancer, including OIP5, which is involved in chromatin segregation, as well as HMGA1, which contributes to cell transformation and metastasis. In both of these cases, the neighbouring lncRNA is significantly underexpressed while the protein-coding gene is significantly overexpressed, suggesting a negative regulatory function of the lncRNA. Moreover, survival analyses revealed that patients with high expression of either OIP5 or HMGA1 had significantly shorter overall survival. Strikingly, patients with low expression of the lncRNA near OIP5 also displayed poorer overall survival, illustrating the clinical opportunity that these genes present.

Conclusion:
Our results highlight the landscape of lncRNA deregulation in LUAD and uncover a role of these non-coding transcripts in the cis-regulation of neighbouring protein-coding genes, many of which have been described in cancer and predict patient survival. Further characterization of this alternative lncRNA-mediated cancer-gene regulatory mechanism may reveal novel therapeutic targets that may improve treatment for LUAD patients without well defined molecular drivers.

Background:
The molecular analysis of non-small cell lung cancer (NSCLC) is limited by the availability of only small biopsies or cytological specimens that are procured for diagnostic purpose. The nuclease protection method provides the possibility to analyze minimal amount of formalin fixed paraffin embedded (FFPE) tissue without previous extraction steps. We tested this technique and compared it to the traditional methods RNA sequencing (RNAseq) and immunohistochemistry (IHC).

Method:
The nuclease protection method (HTGmolecular) in combination with next-generation sequencing was used to measure gene expression of 549 immune-oncology genes in FFPE samples from NSCLC-patients. Standardized minimal tissue amounts were used for 12 samples (4 tissue circles, 4µm thick, 1 mm in diameter, from a tissue microarray). Of these tissue sections also two corresponding original tumor biopsy were analyzed. RNA sequencing data was available from a corresponding fresh frozen tissue as well as IHC annotation of the immune markers FOXP3, CDH1, CD20, CD44, CD3, CD4, CD8 and PD-L1 on the analyzed tissue cores.

Result:
Of the 12 core preparations, 9 samples were successfully analyzed and fulfilled the quality criteria in the first run, the three others in a second re-analysis. The mRNA expression profiles of 12 samples measured with HTG on minute FFPE samples and RNAseq from fresh frozen tissue showed most often good correlations (r=0.41-0.87). HTG based mRNA data correlated with IHC expression for 5 of 8 genes (PD-L1 r=0.76, CD44 r=0.75, CDH1 r=0.61, CD8 r=0.60, CD4 r=0.54). RNAseq data showed good correlations with IHC for only 3 of 8 genes (CD44 r=0.91, PD-L1 r=0.86, CD8 r=0.67). Also, the HTG data of the two biopsies demonstrated very good correlations to the corresponding tissue cores and the RNAseq data (r>0.91). Finally, technical replicates of 10 of the minimal tissue core samples measured in different laboratories revealed relatively good concordance (r=0.71-0.94).

Conclusion:
The applied nuclease protection technique opens the possibility to multiplex and analyze the immune profile of 549 genes in minimal diagnostic biopsies with a high success rate. This is of great value for clinical use or in NSCLC clinical studies where the amount of tissue often is a limiting factor in companion diagnostics.

Background:
Pathologists may recognize the phenomenon of polyploidy in FISH, which may be misleading in interpretation of break apart fluorescence in-situ hybridization (FISH). The chance for single or split probe signals is likely to increase with the degree of polysomy. The aim of this study was to explore whether false positivity due to polyploidy occurs in practice.

Method:
A cohort of cases referred for study or patient care was collected from the archives. From the cases where the ALK and/or ROS1 in-situ hybridization test was repeated in our hospital the outcome of testing was compared. Additionally tumor DNA of an occasional case was tested by an orthogonal method (Ion Torrent Oncomine Focus Assay) for translocations.

Result:
Three cases with ALK FISH rearrangement elsewhere were diagnosed with polyploidy in the referral center. One case was reported with rearrangements in both the ALK and the ROS1 gene detected by FISH analysis. In the repeated FISH analysis the average number of co-localization signals in the tumor cell nuclei was 7.6 for ALK and 9.5 for ROS1 respectively (range 1 - 30). Moreover, the morphology of this case was a giant cell carcinoma, variant of pleomorphic carcinoma of the lung. Examination with an orthogonal method (Ion Torrent Oncomine Assay) revealed no translocations and the tumor cells were negative for ALK and ROS1 by immunohistochemistry proving the original report as false positive, supported by absence of response on crizotinib. In break apart FISH the 15% threshold for positivity was obtained in cells emphasizing that in cross sections of normal nuclei occasionally split signals or 3’ probe signals may be present even in diploid nuclei. In the range of 15-20% the chance of false positive FISH is >1%.[1] However, in polyploid tumors the higher number of probe signals within one nucleus comes with an increased chance of split or 3’ signals and a higher rate of false-positive results when maintaining a uniform threshold 15% irrespective of ploidy. Moreover, this may in case of ALK be an additional reason for discordancy with ALK immunohistochemistry, explaining the lack of response on targeted therapy in these patients.[2] 1. vLaffert Lung cancer. 2015;90:465 2. vdWekken. Clin Cancer Res.epub.

Conclusion:
In case of polysomy there is a increased chance of false positive in break apart FISH results. An addition technique should be used to confirm a positive FISH status in tumors with highly increased gene copy number due to polysomy.

Background:
Lymphocytes play important roles in cancer immunity. Tumor-infiltrate lymphocytes (TILs) are seen in non-small cell lung cancer (NSCLC) and generally classified according to their localization (epithelial area and stromal area). The distribution and the number of TILs are quite different. Cancer cells have an ability to evade from cancer immunity, and the several mechanisms of the ability have been reported; decreased expression of tumor antigen, inhibition of immune response, induction of immunosuppressive cells, and secretion of immunosuppressive cytokines. We hypothesized that the mechanisms of evasion from cancer immunity would influence TIL representation. In this study, we investigated the differences of TILs in histological differentiation, since we considered that histological difference could affect cancer immunity.

Method:
We retrospectively investigated surgical specimens between 2009 and 2015. Consecutive 20 cases with minimally invasive adenocarcinoma (MIA), lepidic adenocarcinoma (Ad lepidic), acinar or papillary adenocarcinoma (Ad aci/pap), solid adenocarcinoma (Ad solid) and squamous cell carcinoma (Sq) were selected (total 100 cases). We checked all fields of the tumors in the slice with maximum tumor-diameter microscopically at 100-fold magnification. TILs in the field were judged as positive when more than 10 lymphocytes flocking in tumor epithelial area or stromal area were observed. TILs of the tumors were assessed as the rate of the TIL positive fields in all, and separately evaluated in epithelial area and stromal area. Then, analysis of variance was used to assess the histological differences of TILs. Significant difference was considered as p-value was less than 0.05.

Result:
The average rates of TIL positive fields in epithelial area of MIA, Ad lepidic, Ad aci/pap, Ad solid and Sq were 11.2 ± 20.4%, 15.8 ± 20.4%, 26.9 ± 20.9%, 52.4 ± 30.0% and 27.8± 28.8%, respectively. The rate of Ad solid was significantly higher than those of MIA, Ad lepidic and Ad aci/pap, and the rate of Sq was also significantly higher than those of MIA and Ad lepidic. The average rates of TIL positive fields in stromal area of MIA, Ad lepidic, Ad aci/pap, Ad solid and Sq were 41.9 ± 26.1%, 51.2 ± 28.3%, 57.6 ± 23.2%, 67.7 ± 25.4% and 67.8 ± 30.0%, respectively. The rate of MIA was significantly lower than Ad solid and Sq.

Conclusion:
TILs were significantly different representation depending on the histology. Especially in adenocarcinoma, the TILs differed according to the grade of differentiation. These results might show that highly differentiated lung adenocarcinoma has low expression of tumor antigen.

Background:
2-deoxy-2-[fluorine-18] fluoro-D-glucose integrated with positron emission tomography (18F-FDG-PET) is clinically useful for the evaluation of cancer. The accumulation of 18F-FDG within tumor cells is implicated in the expression of glucose transporter 1 (GLUT1) and hypoxic inducible factor-1α (HIF-1α). Although anti-programmed death-1 (PD-1) antibody therapy is approved for non-small cell lung cancer (NSCLC), the predictive biomarkers remain unknown. It was recently reported that the expression of programmed death ligand 1 (PD-L1) was positively correlated with the expression of GLUT1 and HIF-1α. Based on these backgrounds, we investigated the relationship between the tumor immunity including PD-L1 expression and the degree of 18F-FDG uptake in surgically resected pulmonary squamous cell carcinoma (SQC).

Method:
One hundred and sixty-seven patients (153 men, 14 women) with SQC who underwent 18F-FDG PET were included in this study. Tumor sections were stained by immunohistochemistry for GLUT1, HIF-1α, PD-L1, CD4, CD8, and Foxp3. Relationships of clinicopathological and molecular biological features to the degree of FDG uptake and survival were analyzed.

Result:
The SUVmax of 18F-FDG was significantly correlated with the expression of PD-L1 (p=0.0224) and GLUT1 (p=0.0075). The PD-L1 expression was significantly correlated either with GLUT1 (p=0.0059), HIF-1α (p<0.0001) or CD8 (p=0.0006). Other pairs exhibiting significant correlation are as follows: GLUT1 and HIF-1α (p=0.0072), HIF1α and CD8 (p=0.0072), CD8 and Foxp3 (p<0.0001). Univariate analysis demonstrated that advanced stage (p=0.0027), elevated SUVmax (p=0.0233), and elevated PD-L1 expression (p=0.0157) were associated with unfavorable overall survival (OS). Multivariate analysis also revealed that advanced stage (p=0.0445), elevated SUVmax (p=0.0382), and elevated PD-L1 expression (p=0.0173) were independent prognostic factors predicting unfavorable OS.

Conclusion:
18F-FDG uptake was significantly correlated with the expression of PD-L1 and GLUT1 in SQC. 18F-FDG PET may reflect the immune response in the tumor microenvironment involved in the regulation of PD-L1 expression.

Background:
Elevated neutrophile-to-lymphocyte ratio (NLR) is a systemic inflammatory marker that has been associated with poor prognosis in NSCLC (Bar-Ad, 2016). There is, however, limited data of the effect of inflammatory markers on Nivolumab efficacy. We assessed whether there is an association between NLR and efficacy of Nivolumab in NSCLC. We also evaluated the value of neutrophil count percentage (NCP). Finally, to establish if the effect was predictive of Nivolumab or prognostic of a therapeutic effect, we studied also NLR and NCP in a cohort of chemotherapy-treated NSCLC.

Method:
Data from NSCLC patients treated with Nivolumab (N=40) in routine clinical practice in our Hospital between January 2015 and May 2017 were retrospectively collected. Population was dichotomized according to whether they had NLR≥5 or <5. Cut-off for NCP was established at 80% using the minimum p-value method. The association between NLR or NCP and progression free survival (PFS) and overall survival (OS) was analyzed by log-rank, Kaplan-Meier method and Cox proportional models. A cohort of chemotherapy-treated NSCLC patients (N=54) were also analyzed.

Result:
In Nivolumab cohort, median age was 67. Thirteen patients (32.5%) were NLR≥5 and five (12.5%) were NCP≥80%. In chemotherapy cohort, median age was 69. Thirty-one patients (57%) were NLR≥5 and ten (18.5%) were NCP≥80%. In Nivolumab cohort, PFS and OS were longer with NLR<5 (log-rank p<0.0001). This effect was also observed with NCP<80% (log-rank p<0.0001 -PFS-, p=0.01 -OS-). In chemotherapy-treated patients, a similar effect was observed. Complete data of median PFS and OS, and Cox proportional models is shown in table 1.

Treatment	Inflammatory marker	Median PFS (months)	Cox proportional models median PFS	Median OS (months)	Cox proportional models median OS
Nivolumab	NLR<5 ≥5	6 2	HR 6.7 CI 95% 2.9-15.3 p<0.000001	25 10.5	HR 4.4 CI 95% 1.9-9.2 p<0.0000001
Nivolumab	NCP<80% ≥80%	6 1.5	HR 0.09 CI 95% 0.02-0.34 p<0.0000001	21 9.5	HR 0.2 CI 95% 0.09-0.84 p=0.02
Chemotherapy	NLR<5 ≥5	15.5 6.5	HR 6.7 CI 95% 3.0-15.1 p<0.0000001	24 17	HR 8.9 CI 95% 3.6-21.9 p<0.0000001
Chemotherapy	NCP<80% ≥80%	4 2.5	HR 0.45 CI 95% 0.2-0.9 p=0.03	14 9.5	HR 0.35 CI 95% 0.16-0.75 p=0.007

Conclusion:
Systemic inflammation biomarker NLR, and to a lesser extent NCP are prognostic, but not predictive, factors of Nivolumab efficacy in NSCLC. NLR<5 and NCP<80% are associated with improved PFS and OS in NSCLC regardless of treatment evaluated.

Background:
Programmed cell death-1 ligand 1 (PD-L1), tumor infiltrating CD8-positive T lymphocytes (CD8+TILs), and cyclooxygenase-2 (Cox-2) has been used as a prognostic tool in lung adenocarcinoma.

Method:
We conducted a retrospective review of data from 170 patients who underwent pulmonary resection as the first treatment for clinical T1-2 N0 lung adenocarcinoma. We investigated the expressions of three biomarkers and EGFR mutation, and analyzed between expression levels and clinicopathological characteristics or prognosis. Then we classified tumors into four groups based on PD-L1 and CD8+TILs status, and evaluated the prognostic significance of Cox-2 expression according to tumor immune-microenvironment classification.

Result:
The high PD-L1 expression tumors showed a significantly larger number of CD8+TILs than low PD-L1 tumors, in contrast, the high Cox-2 expression tumors showed significantly fewer CD8+TILs than low Cox-2 tumors. A multivariate analysis showed that histological subtype, nodal metastasis, CD8+TILs count, and the PD-L1 expression were independent predictor of recurrence-free survival. Based on the classification of PD-L1 and CD8+TILs status, the prognosis in patients with low PD-L1 and high CD8+TILs was significantly better than other types. In patients with low PD-L1 and low CD8+TILs, the rate of EGFR mutation was significantly higher than other types and Cox-2 expression was a powerful predictor of prognosis.

Conclusion:
Clinical and pathological features in conjunction with tumor immune-microenvironment classification indicate that lung adenocarcinoma should be divided into different subgroups. The classification of PD-L1 and CD8+TILs status might predict the effect for immunocheckpoint inhibitors, EGFR tyrosine kinase inhibitors, and/or Cox-2 inhibitor.

Background:
In patients with resected non-small cell lung cancer, the relationships between PD-L1 expression and various clinicopathological characteristics have been examined. However, the association between cytological features and PD-L1 expression to the authors’ knowledge remains unknown. In the current study, the authors used small biopsy specimens to investigate whether morphologic feature correlated with PD-L1 expression in patients with advanced and inoperable lung adenocarcinoma.

Method:
Archival slides from ninety patients with lung adenocarcinoma who underwent small biopsy between October 2014 and May 2017 at Incheon St. Mary’s Hospital were reviewed. The small biopsy specimens were obtained from lung (52 cases), pleura (16 cases), lymph node (13 cases), brain (8 cases), and bone (1 cases). PD-L1 expression was detected by immunohistochemistry using the PD-L1 22C3 IHC assay. We examined the association of PD-L1 expression with pathological and molecular features (EGFR mutation, ALK and ROS-1 rearrangement) and was statistically correlated with histological characteristics.

Result:
PD-L1 expression in tumor cells was positive in 33 of 90 cases (36.7%). Higher PD-L1 expression (≥50%) was more frequent in marked nuclear pleomorphism (p<0.001), coarse chromatin pattern (p=0.006), predominant nucleoli (≥3μm)(p< 0.001), large nuclear diameter (>5 small lymphocytes) (p= 0.006), non-glandular feature (p<0.001) and atypical mitosis (p=0.034). There were no significant correlations between PD-L1 positivity and molecular features. In a multivariable logistic regression analysis, PD-L1 positivity was independently associated with prominent nucleoli (multivariable odds ratio, 6.688; 95% confidence interval [CI], 1.784–25.065; P = 0.005) and non-glandular feature (multivariable odds ratio, 4.539; 95% confidence interval [CI], 1.508–13.663; P = 0.007).

Conclusion:
Our study demonstrated that PD-L1 expression was associated with prominent nucleoli and non-glandular feature in lung adenocarcinoma, which might lead to a poor prognosis.

Background:
Bavituximab is a chimeric monoclonal antibody that targets the membrane phospholipid phosphatidylserine (PS) exposed on endothelial cells and cancer cells in solid tumors. Our previous studies showed that bavituximab enhances the activation of CD8+ TILs that correlates with increased cytokine production by lymphoid and myeloid cells in lung cancer with low PD-L1 expression suggesting that the interruption of the PD-1/PD-L1 axis by nivolumab may enhance the bavituximab effect in tumors.

Method:
Fresh tumor tissues obtained from consented patients with NSCLC, urothelial carcinoma or renal cell carcinoma at the time of surgical resection were utilized in a proprietary 3D ex vivo tumor miscrosphere assay, where 3D tumor microspheres were treated with bavituximab or nivolumab alone or in combination at 10 mg/ml for 36 hours. At the end of the treatment, a multiplex human cytokine assay was used to simultaneously analyze the differential secretion of cytokines, including human IFNg, in culture media as a surrogate of TIL activation. In addition, gene expression analysis of microspheres was performed using the NanoString PanCancer Immune Profiling panel which contains probes to quantitate 770 immune function genes.

Result:
Preliminary results indicate the combination treatment with bavituximab and nivolumab led to increased expression of genes involved in M1 polarization of tumor associated macrophages in a subpopulation of lung tumors that closely correlated with release of cytokines such as MIP1b (CCL4) which is a chemoattractant for natural killer cells, monocytes and a variety of other cells involved in tumor immune response.

Conclusion:
This lung patient derived ex-vivo approach indicates that bavituximab in combination with nivolumab may enhance immune response. This response likely involves M1 polarization of tumor associated macrophages and suggests potential clinical implications in the treatment of lung cancer.

Background:
Immunocheckpoint inhibitors targeting PD-1/PD-L1 axis have shown promising results in patients with non-small-cell lung cancer (NSCLC). It is well known that PD-L1 is induced by IFNg, however recent study shows that EGFR also affects PD-L1 expression in tumor cells.

Method:
We evaluated the expressions of PD-L1, EGFR, and HER2 in tumor tissues collected from patients with pStage IA–IIIA NSCLC using immunohistochemistry. Intensity scoring for staining was calculated with H-score (0-300). The relationships between their expression and clinicopathological characteristics were evaluated. For in vitro assay, the expression of PD-L1 was evaluated by flow cytometric analysis while cell signaling pathways were assessed with Phospho-Receptor Tyrosine Kinase (RTK) array in LC2/ad human lung adenocarcinoma cell line.

Result:
Of the total 91 tumors, 13 cases (14%) showed PD-L1 overexpression, 12 cases (13%) showed EGFR overexpression, and 35 cases (38%) showed HER2 overexpression in tumor cells. PD-L1 overexpression associated with poor clinical outcome and was positively correlated with EGFR expression while inversely correlated with HER2. To assess the regulation mechanism of PD-L1 expression via EGFR/HER2 signaling, LC2/ad cells were treated with EGF with or without inhibition of EGFR or HER2, after which PD-L1 expression was evaluated using flow cytometry. Consistent with previous reports, PD-L1 expression was clearly enhanced by EGF, and either EGFR-tyrsine kinase inhibitor Gefitinib and siRNA for EGFR blocked EGF-induced PD-L1 overexpression in LC2/ad cells. On the other hand, we found that siRNA for HER2 could not block EGF-induced PD-L1 overexpression. We compared EGF-induced signaling with IFNg-induced one by RTK array, and found EGF stimulation activated AKT, MAPK, and S6 ribosomal protein while IFN-g activated STAT1.

Conclusion:
PD-L1 overexpression is associated with poor prognosis and is positively correlated with EGFR expression but inversely correlated with HER2 expression in NSCLC. The expression mechanism of PD-L1 is different between EGFR and HER2 signaling in LC2/ad cell line. Additionally, both EGF and IFNg enhance PD-L1 expression but via different pathway in NSCLC cell line LC2/ad.

Background:
DNA damage-repair and autophagy may contribute to the efficacy of anticancer therapy. The gamma-H2AX is phosphorylated to repair DNA after the double stranded breakdown. Thus, this study was aimed to check the expression of gamma-H2AX and autophagy in cisplatin- and etoposide-treated lung cancer cell line.

Method:
A549 cells were cultured within Dulbecco’s Modified Essential Medium with 10% of fetal bovine serum. Cisplatin and etoposide were treated into the cells with time and dose dependent manners. MTT assay and light microscopy were performed to determine the LD50 of these drugs. Western blotting was performed to define the expression of proteins related to LC3A/B-I/II, NBR1 and DNA damage(gamma-HA2X).

Result:
Based on MTT assay, LC50’s were determined as the followings: LC50 of CDDP was 14.4 micromole/L and LC50 of etoposide was approximately 25 micromole/L. Chemoresistant cancer cell islets were defined the surviving cells under light microscopy with the treatment of cisplatin and etoposide. In 0-, 5-, 10- and 20-h time points, gamma HA2X showed increasing expressional pattern. This pattern was also similar in etoposide with the peak time point of 10 hour. LC3A/B-I/II and NBR1 expressions were increased by 25 micromole/L of CDDP and 100 micromole/L of etoposide whose patterns were also similar to gamma H2AX.

Conclusion:
Similar expressional patterns of DNA breakdowns (gamma H2AX) and autophagy(LC3A/B-I/II, NBR1) by cisplatin and etoposide may suggest the linkage between autophagy and DNA breakdown. Therefore, this tentative conclusion must be furthermore defined by the research for molecular networks in the chemoresistant lung cancer cells.

Background:
Programmed cell death-1 (PD-1) inhibitor is one of important medicines of immunotherapy for cancers. Programmed cell death ligand-1 (PD-L1) expression is a valuable predictor for selection of patients by PD-1 inhibitors therapy. However, the correlation of PD-L1 expression with clinicopathologic features, EGFR and ALK status, and prognosis of lung adenocarcinoma remain controversial.

Method:
421 lung adenocarcinoma patients with identified EGFR and ALK status in tumor tissues were enrolled. Using tissue microarrays, PD-L1 expression was evaluated using clone SP263 antibody by immunohistochemistry (IHC) on Ventana Benchmark automated staining system. The association of PD-L1 expression with clinicopathologic characteristics, EGFR and ALK status and prognosis of lung adenocarcinoma were analysed.

Result:
A total of 404 patients were available for evaluation. The positve incidence of PD-L1 expression was 22.5% (91/404) (using a cutoff of ≥ 25%). Multivariate Logistic regression analysis showed PD-L1 expression was associated with advanced stage (ORR 2.190, 95% CI 1.313-3.651, P = 0.003), solid predominant subtype (ORR 3.594, 95% CI 1.826-7.073, P < 0.001), and wild-type epidermal growth factor receptor (EGFR) (ORR 1.895, 95% CI 1.091-3.291, P = 0.023), while it was not associated with ALK rearrangement. In multivariate analysis for OS by Cox hazard proportion model, patients with early stage (HR 2.495, 95% CI 2.003-3.106, P < 0.001) and EGFR mutations (HR 1.635, 95% CI 1.310-2.040, P < 0.001) had a significantly longer survival, while PD-L1 expression was not associated with OS of patients with lung adenocarcinoma (HR 0.847, 95% CI 0.655-1.094, P = 0.203).

Conclusion:
Positive PD-L1 expression in lung adenocarcinoma tissues was significantly associated with tumor advanced stage, solid predominant subtype, and wild-type EGFR, however, PD-L1 expression may not be a predictor of OS for patients with lung adenocarcinoma.

Background:
INF-gamma secreted by CD8+ lymphocytes upregulates PD-L1 expression in cancer cells. We recently identified STAT3 and YAP1 as compensatory mechanisms of resistance to EGFR tyrosine kinase inhibition in EGFR mutant cells. STAT3 and YAP1 up-regulate CCL5 (Rantes) and CXCL5, respectively, with both chemokines attracting the myeloid-derived suppressor cell. STAT3 stimulates DNMT1 by repressing STAT1 and retinoic acid-inducible gene-I (RIG-I) expression. STAT1 and RIG-I are key mediators in INF-gamma signaling. We assume that alterations in the INF-gamma signaling pathway could be present in EGFR mutant NSCLC.

Method:
Total RNA from 53 EGFR mutant NSCLC patients was reversed transcribed and analyzed by qRT-PCR. STAT3, YAP1, RIG-I, STAT1, PD-L1, DNMT1 and CXCL5 mRNA were examined with specific primers/probes in triplicates. Progression-free survival (PFS) and overall survival (OS) were estimated.

Result:
Fifty-three EGFR mutant NSCLC patients treated with erlotinib were analyzed, 72% were female, 62% never-smoked, 70% had exon 19 deletion and 36% brain metastases. A positive correlation was found between RIG-1 and STAT1 (r=0.42, p=0.003). An anti-correlation trend was noted between STAT3 and PD-L1, YAP1 and PD-L1 and DNMT1 and STAT1. Median PFS was 22, 12.9 and 8.6 months for patients with high, intermediate and low PD-L1 mRNA, respectively (P=0.04). Median PFS was numerically longer for patients with low levels of DNMT1, RIG1 STAT1 and CXCL5, although the differences were not statistically significant. A similar trend was observed for OS.

Conclusion:
PD-L1 mRNA could be a prognostic marker in EGFR mutant NSCLC patients. Down-modulation of PD-L1 indicates alterations in pattern-recognition receptors (PRRs), like RIG-1 or downstream interferon signaling factors. The dysregualtion of the pathway is multifactorial, and the role of STAT3 and YAP1 hyperactivation merits further research. DNMT1 overexpression ablates STAT1. Since the cyclin-dependent kinase 4 (CDK4) interacts with DNMT1, therapies with CDK4 inhibitors can directly neutralize the main defects in the INF-gamma signaling pathway.

Background:
PD-L1 IHC test is an important biomarker for predicting the response of the immune checkpoint inhibitor against the PD1/PD-L1 axis. The FFPE tissue sample is an only validated specimen used in the clinical study, although it is sometimes difficult to obtain an enough tissue sample in advanced stage patients. Cytology specimen is an expected candidate. In this study, we evaluated the PD-L1 IHC expression on cytology cell block specimen (CB) and compared to the corresponding formalin-fixed-paraffin-embedded tumor tissue sample (FFPE-T).

Method:
Nine primary lung cancer patients who have both surgical resected FFPE-T and pleural effusion CB were recruited. CB was prepared as following; pleural fluid was centrifuged to collect the cell pellet, then fixed in formalin and embedded in paraffin. PD-L1 expression was evaluated using two clones (DAKO PharmDx kit, 22C3 and 28-8). Three pathologists (two certified, one path-trainee) and one cytotechnologist reviewed the slides independently. The proportion score of tumor cell (TPS) was evaluated and divided into 2-tier (positive, negative for 28-8) and 3-tier (no, low, high expression for 22C3) categories, according to the manufactural protocols. The correlation between CB and FFPE-T and the inter-observer agreement (kappa value) were calculated.

Result:
All samples were acceptable for PD-L1 evaluation. FFPE-T resulted in 2 positive, 7 negative (28-8); 3 low and 6 no expression (22C3), respectively. CB resulted in 5 positive, 2 negative (28-8); 3 low and 6 no expression (22C3), respectively. The TPS and tiered-category of CB did not correlate to those of FFPE-T, statistically. The concordant rate of tiered-category between FFPE-T and CB resulted in 4/9 (45.4%) for both clones. It can be explained by the heterogeneity of PD-L1 expression. The TPS and category judgment of two tests (28-8 and 22C3) within each observer were statistically correlated (R=0.588-0.951, p-value <0.001). The kappa value of the inter-observer agreement varied from 0.18 to 1.0, depending on the experience and education. Two certified pathologists reached moderate (kappa=0.59 for 28-8) to high (1.0 for 22C3) agreement on CB, but low (0.05 and 0.14) on FFPE-T. The kappa value between certified pathologist and path-trainee/ cytotechnologist was 0.6/ <0.01 for FFPE-T, and 0.18/0.57 for CB, respectively. These results seem to be influenced by the recognition of appropriate target tumor cells.

Conclusion:
Our study suggested that the properly processed cytology sample has a potential clinical utility for PD-L1 evaluation. The difficulty of target cell recognition on cytology specimen seems to be one of the critical issues of standardization.

Background:
Lung cancer is the leading cause of death among cancers. Despite improved surgical and novel radiation improvements, the overall prognosis remains poor. CD26/dipeptidyl peptidase 4 (DPP4) is a ubiquitously expressed transmembrane exopeptidase on the cell surfaces of many different cells including malignancies of breast, colon, and mesothelioma. Phase I data in mesothelioma with a specific antibody showed tolerability in mesothelioma patients.Our group found previously that the activity of CD26/DPP4 of lung adenocarcinoma (Adeno-CA) patients is four times higher than in normal tissue and the inhibition of CD26/DPP4 decreased the growth of lung tumors in experimental models. These data prompted us to analyze the expression of CD26/DPP4 in samples from lung cancer patients to unravel the role of CD26/DPP4 as a biomarker for lung cancer and a target for inhibition to reduce lung cancer burden. burden.

Method:
To identify CD26/DPP4 by immunohistochemistry (IHC), we tested four antibodies from Abcam, R/D systems, and Cell signaling technology on multi-organ tissue micro array (TMA) and human lung Adeno-CA cell lines (A549, H460, Gon8, Mai9) derived from advanced stage (IV) of human Adeno-CA. We selected the antibody from Cell signaling technology against CD26/DPP4. For the analysis of CD26/DPP4 by IHC in lung cancer samples, TMAs constructed from non-small cell lung cancer patients were used. The cohort consisted of 475 patients (Adeno-CA: 223; Squamous carcinoma: 252). The intensity of the staining was scored from 0 to 3 in a blinded manner. To quantify CD26/DPP4 in the supernatant of human lung Adeno-CA cell lines in vitro, ELISA was performed.

Result:
IHC scores revealed that Adeno-CA expresses significantly more CD26/DPP4 compared to squamous carcinoma (p<0.0001). Consistent with our previous findings, early stage cancer (IA) scores significantly higher than other stages IIB (p=0.0012), IIIA (p=0.0019), and IV (p=0.02) among Adeno-CA samples. We could not find CD26/DPP4 expression on human Adeno-CA cell lines by IHC, but the secretion of the protein in supernatant stays high (A549: 20pg/ml; H460: 161pg/ml; Gon8: 74pg/ml; Mai9: 648pg/ml).

Conclusion:
CD26/DPP4 expression was significantly higher at early stages of Adeno-CA samples when compared to advanced stages, supporting our previous findings. From the human cell line data, we suggest that advanced cancer secretes CD26/DPP4 more actively than early stage cancers. CD26/DPP4 seems to be a substantial target for inhibition of human Adeno-CA.

Background:
Despite undergoing curative-intent therapy, patients with early stage non-small cell lung cancer (NSCLC) have reduced survival rates of 43-73% due to the risk of distant metastasis(1-3). This underscores the acute need to identify new biomarkers for improved prognostication and to better understand the underlying biology that drives poor outcomes.

Method:
Tumor tissue from 189 patients with NSCLC who underwent curative intent surgery was stained for CD14 by IHC and qualitatively scored for low, moderate, or high expression. CD14 expression groups for all patients and stage I patients alone were analyzed for overall survival. In vitro studies were performed utilizing a coculture system of human lung cancer cell lines and freshly isolated CD14[+ ]cells.

Result:
We found that the level of CD14[+ ]cells within the tumor microenvironment (TME) was strongly associated with overall survival in all patients (p<0.001) and stage I patients alone (p=0.006). Patients with high CD14 TME staining had a median overall survival of 5.5 years versus 8.3 years and 10.7 years for moderate or low CD14 staining, respectively. The intensity of CD14 TME infiltration was associated with an advanced stage at time of diagnosis (p=0.049) and more positive lymph nodes found at surgery (p=0.012). The potential advantages of TME CD14[+ ]cells were investigated through a coculture system. Tumor growth kinetics were not altered with coculture. Lung cancer cells cocultured with CD14[+ ]cells recovered more quickly after exposure to chemotherapy. This improved recovery was observed only after a 48 hour coculture of tumor cells and CD14[+ ]cells. Figure 1



Conclusion:
CD14[+] cells are a prognostic marker within the TME of patients with resected lung adenocarcinoma. Our data suggests crosstalk with the CD14[+] cell infiltration results in a tumor survival benefit that drives poor patient outcomes.

Background:
The contribution of the tumour-immune microenvironment to tumour progression and patient outcome has become increasingly evident. Newly developed genomic tools have enabled the study of immune cell composition from bulk tumour data. However, such tools (e.g. CIBERSORT) do not provide the key spatial information that is crucial to understand tumour-immune cell interactions. To this end, we have developed a multispectral imaging platform that improves upon traditional analysis methods of cell segmentation and cell density calculations by further quantifying nearest-neighbour interactions (cell-cell spatial relationships). We apply this technology to investigate tumour-immune cell spatial relationships and their clinical significance to discover novel biological insights.

Method:
Whole tissue sections from 20 lung adenocarcinomas were stained for CD3, CD8, and CD79a and counterstained with haematoxylin. Multispectral images were acquired for five fields of view and analyzed to quantify cell types. Regions of Interest (ROIs) were then identified for the characterization of intra-tumoural and dense inflammatory regions. Image files including ROIs were analyzed in order to quantify cell-cell spatial relationships. Non-random patterns of immune cell distributions were identified using the Monte Carlo re-sampling method (500 iterations). Immune cell counts, densities, spatial relationships, and significant immune cell distributions were associated with clinical features by two-group comparison (Kruskal-Wallis p<0.001).​

Result:
Our analysis generated 234 image files for analysis, including ROIs. Each field of view contained an average of 16,400 cells. The densities of intra-tumoural CD3+CD8+ and CD3+ T cells were significantly lower in recurrent cases, agreeing with literature reports. Following Monte Carlo analysis, non-random cell-cell spatial proximities emerged that were not observed at a cell density level. For example, an increased proximity of CD3+ T cells and B cells was observed in never smokers, while a decreased proximity was observed in ever smokers.

Conclusion:
While immune cell densities are of clinical prognostic importance, their spatial organization within the tumour architecture is of functional importance (e.g. the inhibition of cytotoxic T cell activity by adjacent PD-L1 expressing cells). In addition to cell densities, our platform is capable of quantifying cell-cell spatial relationships, thereby providing further information for clinical associations and for the identification of novel prognostic interactions. This automated quantification could be used to complement visual diagnostics and improve prognostic interpretation of histology specimens.

Background:
Immunotherapy with immune checkpoint inhibitors have revolutionised the management of solid organ malignancy including melanoma and NSCLC. Direction has turned to the tumour immune microenvironment (TIM) to explore predictive biomarkers. The spatial arrangement of immune infiltrative cells has the potential to better explain the TIM. Vectra multispectral immunohistochemistry (IHC) allows accurate definition of the TIM and may help detect mechanisms of immune evasion.

Method:
Multispectral fluorescent immunohistochemistry with a panel including CKAE1/3, CD8, FOXP3 and PD-L1 (clone E1L3N, Cell Signalling Technology) was used to analyse the TIM in six patients (pts) with resected NSCLC (full face section from block). Respective tissue microarrays were collected in triplicate from each specimen and underwent conventional IHC scoring for PD-L1, tumour infiltrating lymphocytes (TILs), CD8, FOXP3 and scored (0,1,2,3). The spatial arrangement of lymphocytes relative to tumour cells, stroma and PD-L1 expression was examined.

Result:
All six pts had adenocarcinoma histology, with the following level of PD-L1 expression: low(0-5%;n=2), intermediate(5-50%;n=2) and high(>50%;n=2)Figure1. In PD-L1[hi] pts Vectra staining showed uniform staining of PD-L1 across the full face. CD8 lymphocytes were present mainly in tertiary lymphoid structures without evidence of clustering. In PD-L1[lo] pts, one had heterogenous staining of TILs with dense stromal clustering (3:1 ratio of stroma to intratumoural). Neither patient (PD-L1[lo]) demonstrated significant PD-L1 uptake on full section assessment. Of the PD-L1[int] pts, although heterogeneity in PD-L1 expression was evident across the full face, the majority of tumour rich areas stained positively and TILs were uniform in the stroma. FOXP3 had low expression across all 6 patients <1% almost uniformly in the stroma.Figure 1



Conclusion:
Although PD-L1 staining heterogeneity was limited in this small dataset, clear differences in immune-cell infiltrate were seen between full-face sections and limited cores. Multiplex Immunofluorescent IHC provides accurate quantification of immune infiltrates and spatial alterations within the TIM and may facilitate predictive biomarkers.

Background:
PD-L1 immunohistochemistry (IHC) testing is usually performed on core needle biopsy or surgical resection tissue blocks, and tumor proportion score (TPS) ≥50% is used to select patients to treat with Pembrolizumab immunotherapy. In this study, we evaluate the results using cytology cell block for PD-L1 IHC assay.

Method:
A total of 1423 consecutive cases of non-small cell lung cancer (NSCLC), including 368 cytology cell blocks, 813 small biopsies and 242 surgical resections, were included in the study. 151 cytology cell blocks had known fixation status, which were either directly fixed in formalin, or in alcohol first then refixed in formalin. IHC used Dako PD-L1 IHC 22C3 pharmDx. The TPS of ≥ 50% tumor cells was defined as positive. A total of 100 viable tumor cells were required for adequacy.

Result:
Of the cytology cell blocks, 93% of the specimens had sufficient numbers of tumor cells, and the rate was equivalent to the rate of small biopsies (93%). All resection specimens were shown to be adequate for testing. PD-L1 expression was positive in 42.1% of cytology cell blocks, statistically comparable to small biopsies (36.3%, P>0.05), but higher than in surgical resections (28.5%, p<0.05). The fixative methods did not affect the immunostaining, since the PD-L1 positive rate was of 41.9% in formalin only group, vs 40.4% in alcohol plus formalin fixed cell blocks (p>0.05). The PD-L1 positive rate appeared lower in cell blocks from bronchoalveolar lavage (BAL) (27%) as compared to fine needle aspiration (FNA, 42%) and pleural/pericardial fluid (45%), although the difference did not reach statistical significance (P>0.05).

Conclusion:
Our results demonstrate that PD-L1 IHC performs well with cytology cell blocks. The rate of positive PD-L1 was comparable between cytology blocks and small biopsies. As cytology cell blocks are commonly available from lung cancer patients, they can provide valuable resource for PD-L1 testing and can help to avoid rebiopsies. However additional correlation with clinical response will be helpful to further validate the cytology specimens.

Background:
Given the rapidly changing therapeutic landscape for non-small cell lung cancer (NSCLC), a thorough understanding of the tumor immune environment is critical to the appropriate selection of patients and testing of immuno-oncology agents. We established a project aimed at defining the comprehensive and integrated immunogenomic landscape in NSCLC, including immune, genomic and clinical data from 100 surgically resected lung cancers.

Method:
Tissue samples were collected at the time of surgery, and blood samples before and after surgery up to 1-year. Tissue samples were subjected to tumor infiltrating lymphocyte (TIL) isolation and expansion; generation of PDX models,WES and in-silico neoantigen prediction, RNA sequencing, RPPA, CyTOF, T cell receptor sequencing, and multiplex immunofluorescence evaluation of immune cells and markers. Blood samples were processed for cfDNA, microRNA, and cytokine profiling.

Result:
93 out of 131 enrolled patients with median age 67 years contributed samples to ICON; 44% males, 80% former smokers, 12% never smokers. Majority had adenocarcinoma (60%) and 26% SCC. 37 (40%) had stage I, 29 (31%) stage II, and 20 (22%) stage III disease; 14 patients received neoadjuvant chemotherapy. Median tumor size was 4.0cm and 79 (85%) underwent R0 and 9 (10%) R1 resection. TIL expansion was 68% successful. Phenotypic and functional analysis of TIL is ongoing. Preliminary analysis show suppression of intratumoral CD8[+] cells with low perforin levels and high CD8[+]PD1 levels as compared to normal tissue; however tumor CD8[+]CD28 co-stimulatory molecule expression was high. PDX success rate is 35%. IHC analyses show higher CD8[+]PD1, CD3, CD8[+]BTLA expression in SCC than in adenocarcinoma or normal tissue. WES and RNA sequencing show that median mutation burden is 9.3/MB in adenocarcinomas and 11.2/MB in SCC. Other immunogenomic analyses are in process.

Conclusion:
The ICON is an ambitious multi-team project designed to integrate multiple levels of tumor related data. Preliminary analysis demonstrates the project to be feasible, with a high rate of prospective sample acquisition. Tumor profiles from ICON will serve as a reference for upcoming neoadjuvant single and dual checkpoint immunotherapy trials. ICON Team: A. Weissferdt, A. Vaporciyan, A. Futreal, T. Karpinets, C. Yee, C. Haymaker, L. Federico, M.-A. Forget, G. Lizee, A. Talukder, J. Roszik, H. Tran, M. Vasquez, E. Prado, C. Behrens, E. Parra, J. Rodriguez-Canales, J. Fujimoto, L. Vence, J. Roth, I. Meraz, E. Roarty, L. Lacerda, L. Byers, S. Swisher, W. William Jr., P. Sharma, J. Allison, B. Fang, H. Wagner, E. Bogatenkova, I. Wistuba, J. Heymach and C. Bernatchez

Background:
PD-L1 expression level in lung cancer may be a predictive biomarker for the use of PD1/PD-L1 inhibitors. However, the reproducibility of PD-L1 staining using different antibodies and platforms is still a matter of debate. We investigated whether the PD-L1 expression in Non-small lung cancer is associated with specific clinical features or survival using four different antibodies.

Method:
PD-L1 status was assessed with IHC (AB clone SP142 and SP263 - Ventana, 22C3 and 28-8 - Dako) on archival FFPE surgical tumor specimens, arrayed on tissue microarrays (TMAs) with duplicate 1 mm cores from two institutions (Karolinska University Hospital and Nagasaki University Hospital). All patients (n = 682) underwent curative surgery between 1987 and 2015. The following cases were excluded from survival analysis (n = 89): R1 resection, early post-operative mortality, adjuvant chemo- or radiotherapy. PD-L1 staining was scored as positive if present in >1% of tumor cells, independently of staining intensity.

Result:
Patient and tumor characteristics were as follows. Median age (IQR): 68 years (27-89); gender: male/female 54%/46%; histology: squamous-cell carcinoma (SCC)/Non-squamous (N-Sq)-NSCLC/carcinoid 219 (32%)/394 (58%)/45(7%); p-stage: IA/IB/IIA/IIB/IIIA/IIIB 50%/26%/10%/10%/2%/0.2%. Median overall survival was 74 months. PD-L1 28-8 was positive in 11% of cases (SCC (56%)/N-Sq-NSCLC(40%), Pearson Chi-square p<0.0001). PD-L1 positivity (>50%) 22C3/SP263/SP142 was 10%/13%/3%. All carcinoids were negative for PD-L1. In PD-L1-SP263 positive cases, the staining intensity and distribution had a homogenous pattern between the 2 TMA cores. In NSCLC, PD-L1 positivity for each antibody was associated with tumour size (T1/T2-4; Fisher’s exact test, p<0.001) and grade of differentiation (G1, G2 and G3; p<0.0002). Statistically significant association between PD-L1 expression and OS was only observed using the clone SP263 (log-rank p=0.013).

Conclusion:
In this surgical series, the clone SP142 showed less PD-L1 expression in the tumour cells. PD-L1 expression was associated with tumour size, grading and only the clone SP263 showed association between its expression and survival ratio.

Background:
PD-L1 status in lung cancer may be a predictive biomarker for the use of PD1/PD-L1 inhibitors. Nonetheless, the reproducibility of PD-L1 staining using different antibodies and platforms is still a matter of debate. We investigated the performance of four different antibodies with two different platforms in two different institutions.

Method:
PD-L1 expression was assessed with IHC (AB clone SP142 and SP263 - Ventana, 22C3 and 28-8 - Dako) on archival FFPE surgical tumour specimens, arrayed on tissue microarrays (TMAs) with duplicated 1 mm cores from two institutions (Karolinska University Hospital and Nagasaki University Hospital). All patients (n = 682) underwent curative surgery between 1987 and 2015. PD-L1 staining was scored as positive if present in ³1% of tumor cells, independently of staining intensity. The slides were scanned and scored by seven experienced pathologists who estimated the expression of PD-L1 in the tumour and inflammatory cells. The statistical analyses were performed to compare the four different antibodies and the scoring of the pathologists on the tumour and inflammatory cells.

Result:
PD-L1 positivity ³1% of tumor cells using clones 28-8 / 22C3 / SP263 / SP142 was 32%/29%/33%/9%, respectively. All carcinoids were negative for PD-L1. SP142 showed a significantly lower mean score compared with other clones. PD-L1 positivity ³50% of tumour cells using clones 28-8 / 22C3 / SP263 / SP142 was 11%/10%/13%/3%, respectively. The pair comparison analysis in the tumour cells showed that the score from the highest agreement was between 28-8 and 22C3 (Kappa 0.75, CI95% 0.70-0.80) and the lowest concordance was between SP263 and SP142 (Kappa 0.21, CI95% 0.16-0.27). The evaluation of the concordance in the inflammatory cells and among the pathologists is ongoing.

Conclusion:
The present study shows a comparative analysis using four different antibodies. The clone SP142 shows significantly lower expression of PD-L1 in the tumour cells. The clones 28-8, 22C3 and SP263 showed an excellent concordance in the tumour cells with two different cut offs (>1% an >50%) showing a good reproducibility for the pathology assay. The highest agreement was between the clones 28-8 and 22C3. The lowest concordance was between the clones SP263 and SP142.

Background:
Side effects of immune checkpoint therapy are milder than with standard chemotherapy. Pulmonary toxicity has been described in 5% of patients, most of which are low grade pneumonitis with varied radiologic and pathologic appearances. Here we report lung pathology findings in 7 autopsy cases of patients who died while on immune checkpoint therapy.

Method:
Patient characteristics are shown in table 1. We evaluated tumor burden, the presence of tumor infiltrating lymphocytes (TIL), and type of lung injury. TIL were quantitated using a 4 tier system (0 -3+ = none, mild, moderate, extensive)

Table1. Summary of demographic and clinical information
Case	Age	Sex	Immune Tx	Pneumonitis	Chest Imaging
1	38	WM	3 mo	Yes	GGO, multifocal
2	65	WM	9 mo	No	Bilateral pleural effusion
3	54	WF	single dose	Yes	Bilateral reticular opacities
4	30	BM	4 mo	No	GGO, patchy
5	59	WM	7 mo	No	Pulmonaryemboli and tumor
6	72	WM	25 mo	Yes	GGO, bilateral, diffuse
7	68	WM	5 mo	Yes	GGO, bilateral, diffuse

Result:
Residual viable tumor with little or no therapy effects was present in all 7 cases. The 4 cases where pneumonitis was diagnosed clinically had at least focal DAD. The majority of tumors had at least scattered TIL present, while one tumor had a large number TIL.

Table 2. Summary of autopsy findings
Case	Tumor	TIL	LVI	DAD	Pneumonia	Tumor necrosis
1	ACA in 5 lobes	1+	Yes*	Focal	No	<5%
2	MM in LLL, LUL	3+	No	Focal RLL	No	None
3	ACA in 4 lobes	1+	Yes	LUL, RUL, LLL	LLL, RLL, LUL	None
4	MM in 4 lobes	1+	Yes	No	No	None
5	MM in RUL	1+	No	No	No	None
6	SCC in RLL, LL, LUL	2+	Yes	RUL, RML, RLL	No	10% and fibrosis
7	SCC in 5 lobes	1+	Yes	LUL, LLL	No	20-30%

ACA: adenocarcinoma; MM: Malignant melanoma; SCC: squamous cell carcinoma; *extensive LVI

Conclusion:
The majority of patients with clinical diagnosis of pneumonitis had DAD at autopsy and variable amount of viable tumor in the lungs and at least a few tumor infiltrating lymphocytes. Additional studies are pending to further characterize the phenotype of the TIL and to determine PD1 and PDL1/PDL2 expression on the tumor cells.

Background:
Pembrolizumab therapy for non-small cell lung cancer requires patient selection using PD-L1 immunohistochemistry (IHC). FDA approval was based on staining of resections or core biopsies. There is only limited data on PD-L1 expression in fine needle aspiration (FNA) specimens including EBUS of mediastinal lymph nodes.

Method:
Immunohistochemistry (IHC) was performed on an automated platform (Ventana Benchmark Ultra) using clone 22C3 (Dako) and the Optiview detection system (Ventana Medical Systems) on formalin fixed paraffin embedded FNA cell blocks (n:49), cell blocks from aspirated fluids (n:14), core biopsies (n:21) and 4 transbronchial biopsies. The cohort included of 18 squamous cell carcinomas, 56 adenocarcinomas (ACA), 2 adenosquamous carcinoma, 1 NUT-1 carcinoma, 7 NSCLC and 2 melanomas (age: 24-89, mean: 66, median: 69; 43 female and 45 male). Membranous PD-L1 staining of any intensity and extent was recorded in at least 100 tumor cells (tumor proportion score). The tumors were grouped as: no staining (< 1%), low expression (1-49%) and high expression (50% or more).

Result:
Twenty (23.3%) tumors showed no staining, 26 (30.2%) low expression and 40 (46.5%) high expression. Of the 56 adenocarcinomas 21 (37.5%) showed high expression, while of the 18 squamous cell carcinomas 7 (39%) showed high PD-L1 expression. The other tumors with high expression included one adenosqaumous carcinoma and 11 poorly differentiated carcinomas. One EBUS biopsy also had PDL1 assessed on a transbronchial biopsy of the primary tumor showing similar staining (30% versus 20%).

Conclusion:
PD-L1 immunohistochemistry appears to be feasible using formalin fixed cell blocks of fine needle aspirates and aspirated fluid specimens containing adequate number of viable tumor cells. High PD-L1 expression was seen in approximately half the tumors, which is greater than has been observed in resections/core biopsies, this finding merits further study. High expression of PD-L1 was seen in both squamous cell carcinoma and adenocarcinomas.

Background:
Understanding of the “profile” of PD-L1 expression and its interplay with immune cells will provide important insights into lung cancer pathogenesis, and immunotherapeutic strategies targeting this important immune checkpoint protein. The aim was to investigate the correlation between multiplex immunofluorescence (mIF) expression of PD-L1, density and nature of tumor infiltrating immune cells in non-small cell lung carcinomas (NSCLC), and correlate those profiles with clinical and pathological variables including patient outcome.

Method:
We studied 194 stage II/III patients that underwent pulmonary resection, including 98 adenocarcinoma (ADC), 59 squamous cell carcinoma (SqCC), 15 large cells carcinomas (LCC) and 22 neuroendocrine carcinomas (NEC), primary tumors. Formalin-fixed and paraffin embedded (FFPE) tissue microarrays were constructed with five 1.5 mm cores representative of histologic patterns found in each tumor. mIF was performed using the Opal 7-color fIHC Kit™, scanning in the Vectra™ multispectral microscope and analyzed using the inForm™ software (Perkin Elmer, Waltham, MA). The markers studied were grouped in two 6-antibody panels: Panel 1, AE1/AE3 pancytokeratins, PD-L1 (clone E1L3N), PD-1, CD3, CD8 and CD68; and Panel 2, AE1/AE3, Granzyme B, CD45RO and CD57, FOXP3, and CD20. General linear model was used to evaluate the interaction among primary vs metastatic tumors, histologic type and TAICs and Cox's proportional hazard model for overall survival (OS).

Result:
Fifty-eight % out of 164 tumors were positive for PDL-1+ expression (5% cut-off) in malignant cells (EA1/EA3+). Significant higher levels of PD-L1+ expression were detected in NEC compared with other histologies (ADC, SqCC and LCC) (P=0.006). In the same way, we observed higher densities of cytotoxic T lymphocytes (CD3+CD8+) in NEC when compared with the lowest expression in SqCC (P=0.02). Large cell carcinomas presented high levels of memory/regulatory T cells (CD3+FOXP3+CD45RO+) compared with other histologic types but the difference didn´t achieve statistical significance. No difference was found for CD3+PD-L1+, CD68+PD-L1+, natural killer T lymphocytes (CD3+CD57+) and B lymphocytes (CD20+) among the histologic types. Difference between primary and metastatic tumors was found only for naive/memory T lymphocytes (CD3+ CD45RO+) (P=0.04). High CD3+FOXP3+CD45RO+ and CD3+PDL1+ expression were independent favorable prognostic factor for DFS and OS adjusted by smoking, primary vs metastatic, and histologic type [HR 2.68, 95% (CI 1.37–5.24), P=0.004; HR 2.11 (CI 1.07-4.18, P=0.03].

Conclusion:
High abundance of CD3+PD-L1+ cells and memory/regulatory T cells CD3+FOXP3+CD54RO are favorable prognostic factors for resected NSCLC, highlighting the importance of comprehensive assessment of both tumor and immune cells. Supported by CNPq P246042/2012-5 e CNPq 301411/2016-6; FAPESP 2013/10113-7.

Background:
The FDA has approved different companion and complementary PD-L1 assays for selection of patients for different PD-L1 inhibitors. As a result, laboratories intending to implement FDA approved assays face significant operating and capital expenses. Several studies have demonstrated technical concordance between different PD-L1 assays, but their clinical validity in terms of the response to treatment has not entirely been explored. The aim of our study is to prospectively assess the staining performance of laboratory developed tests (LDT) for Ventana SP263 (nivolumab) and Dako 22C3 (pembrolizumab) clones on formalin fixed paraffin embedded samples, and to investigate the association between PD-L1 assays and response to PD-L1 inhibitors.

Method:
189 sequential lung tumor samples from patients with advanced NSCLC were prospectively stained with Ventana SP263 and Dako 22C3 clones. Both antibodies were optimized for use on the automated Ventana BenchMark ULTRA platform, and validated against corresponding FDA approved assays. Scoring algorithms for staining of the tumor cells approved by matched FDA assays were applied to all samples. Best overall response (BOR) for 43 patients treated with either nivolumab or pembrolizumab were assessed using RECIST v.1.1 and correlated with PD-L1 expression with SP263 and 22C3 using cut off points of 1%, 5% and 10%, 1-49% and 50%.

Result:
Ventana SP263 and Dako 22C3 LDTs showed good agreement with a concordance correlation coefficient of 0.86 (95% CI 0.82-0.90). Comparing the assays using the cutoffs of 1%, 5% or 10% for SP263 and the two cutoffs of 1% and 50% for 22C3 showed an association between the two assays as well (p<0.0001). There were no differences in BOR between pembrolizumab and nivolumab (p=0.12). For SP263, BOR was associated with a cut off point of 10% (Fisher’s p=0.030); while the 1% (Fisher’s p=0.09)and 5 % (Fisher’s p=0.054) cut off points were not associated with response. In contrast, for 22C3, BOR was associated with a cut off point of 1% (Fisher’s exact test p-value = 0.006), 5% (p 0.006) and 10% (0.006)

Conclusion:
Similar to FDA approved assays, Ventana SP263 and Dako 22C3 LDT, if properly validated, demonstrate good concordance, but are not interchangeable. Dako 22C3 is more sensitive assay and its expression shows better association with therapeutic response. Larger studies evaluating associations of alternative staining assays and a response to specific therapy are needed.

Background:
Tumour suppressor gene TP53 mutation is common in human cancers, especially playing an important role in lung cancer tumourgenesis. Some clinical studies have shown that TP53 alterations in non-small cell lung carcinoma (NSCLC) carry a worse prognosis and may relatively more resistant to chemotherapy and radiation. We conducted this study to evaluate the impact of TP53 assessed by limited targeted profiling, correlating with PD-L1 tumour expression and clinicopathological variables in resected NSCLC.

Method:
NSCLC patients who underwent curative resection between 1998 and 2006 at our institution were included. PD-L1 status was assessed using Ventana SP142 antibody on archival FFPE surgical tumour specimens, arrayed on tissue microarrays (TMAs) with triplicate 0.6 mm cores. PD-L1 was scored as positive if membranous staining was present in >1% of tumour cells aggregated across the replicate cores to address heterogeneity. In collaboration with the Lung Cancer Genomics Ireland Study, a targeted panel of 49 genes was assessed by Sequenom MassArray including TP53 and genes in MAPK and PI3K pathways. Clinicopathological data was obtained from hospital electronic database.

Result:
Seventy-two patients were included, of which 40 (58.0%) were males, with a median age of 66.0 years (range: 51.0 – 82.6). 54.2%, n=39 with adenocarcinoma histological subtypes, 45.8%, n=33 were ex-smoker and 42.9%, n=30 had Stage IB disease. Most patients had T2 stage (71.4%, n=50), N0 nodal disease (55.2%, n=37) and grade 2 differentiation (65.7%, n=46). Presence of TP53 mutation was identified in 22 patients (30.5%). Five patients had co-presence of TP53 mutation and PD-L1 positivity. There was no correlation between PD-L1 positivity with TP53 status, KRAS, PTPN11, PHLPP2, PIK3CA, MET and PIK3R1. The median disease-free survival in TP53 mutation with PD-L1 positivity was not reached. In univariate/unadjusted analysis, co-presence of TP53 mutation and PD-L1 positivity appear to have superior disease-free survival over TP53 wild-type and PD-L1 negativity, HR 0.17 (95%CI 0.01-0.78, p=0.018). A trend was seen with overall survival but not statistically significant (TP53 mutant, PD-L1 positive vs TP53 wild-type, PD-L1 negative: NR vs 23.1 months, HR 0.34 (95% CI: 0.0.5-1.11, p=0.079). Independent PD-L1 positivity appears to be associated with better prognosis: DFS HR 0.36 (95% CI 0.11-0.90, p=0.0272) and OS HR 0.47 (95% CI 0.19-0.98, p=0.0427).

Conclusion:
In our cohort, co-presence of TP53 mutation and PD-L1 expression was not associated with poorer survival among resected NSCLC patients. Independently, PD-L1 expression was associated with better survival, a finding which warrants further investigations as potential biomarker.

Background:
The death rate from lung cancer in Uzbekistan during 1990 to 2000 increased from 2.1 to 3.1 per 100,000 and in 2016, it reached 3.8 per 100,000 with steady rising. The most prevalent type of lung neoplasm in Uzbekistan is squamous cell carcinoma, whereas in other countries, adenocarcinoma of the lung is considered to be more frequently diagnosed than squamous cell carcinoma. Important key factors in cancer evaluation are diagnosis and prognosis. The aim of this study is directed to determine biological characterization of lung cancer by means of immunohistochemistry, which would be a crucial step towards to therapy.

Method:
Study consisted 564 patients (mean age 58; range, 34-82 years) with primary lung cancer registered at National Cancer Register in the period from 2000 to 2010. All patients were clinically diagnosed, resected, reviewed by a pathologist and followed for at least 72 months. Two different types of oncoproteins were chosen for this analysis: p53, a tumor suppressor gene; and VEGF, vascular endothelial growth factor for detecting the prognostic value of VEGF and its possible association with p53-gene mutation. Overall cancer-specific survival and cancer-free survival or the risk of recurrence was defined from the date ofoperation to the date of recurrence or last follow-up.

Result:
Among the 564 patients, immunoreactivity for VEGF was negative, weakly, moderately and strongly positive in 96 (17%), 141 (25%), 225 (40%) and 102 (18%) cases, respectively. Abnormalities in p53 expression were found in 220 (39%) of 564 carcinomas. A strong, statistically significant association was found between the presence of a p53 gene mutation and expression of VEGF (P<0.001). Tumors with high p53 expression showed significantly higher VEGF, also high expression of p53 correlated with mediastinal and lymph node metastasis. Survival and post-operative relapse time were shorter in patients with high p53 expression. Median follow-up was 34 months (1-72 months). The median time to recurrence was 9 months; the recurrence rate was 310 of 564 (55%). With 5-year follow-up on all patients, actual 5-year survival was 58%.

Conclusion:
P53 overexpression was an indicator of poor prognosis; in addition, it is generally believed that tumors with p53 alterations are more resistant to cancer chemotherapeutic agents than those without p53 mutation. Our study showed that among 40% p53 positive cases have patients with 18% VEGF overexpression, which is the further assay for future studies, the next step will be to examine the capability of target therapy in this group of patients.

Background:
Among the factors influence the prognosis of patients, cancer metastasis or cancer stage when first diagnosed at hospital is the most important. This article aims at finding the significant gene networks related with lung cancer patients’ overall survival and cancer stage by analyzing big data of gene expression with the algorithm of weighted gene coexpression network analysis (WGCNA).

Method:
A dataset containing 188 lung cancer patients synthesized from TCGA were applied for WGCNA to find the most significantly related modules with overall survival and cancer stage. Then GO and KEGG analysis were performed for further analysis.

Result:
Figure 1 Figure 2 A co-expression network concerning overall survival or cancer stage was constructed respectively. And the significant core genes were determined. The related pathways were also identified.





Conclusion:
Not only have we testified the present classical points concerning cancer progression and patients survival, but also some new discoveries have been identified.The genes of TBL1XR1, ATP11B, DCUN1D1 and ABCC5 played significant roles in deteriorating lung cancer patients overall survival. WNT pathway was significantly related with the patients overall survival. The genes of DUUN1D1, MRPL47, NDUFB5, DNAJC19, PIK3CA, ACTL6Aand ZNF639 promote lung cancer progressing, and lung cancer stage progress was also related with signaling pathways regulating pluripotency of stem cell.

Background:
Patients with non-small cell lung cancer are at high risk of developing brain metastases. The mainstay treatment for patients with brain metastasis is surgical excision, whereas radiation is used for multiple brain metastases. Regardless of the treatment, brain metastasis is associated with poor prognosis and the diminished quality of life. Here, we established experimental brain metastasis model allowed interrogation of the brain-specific requirements for cancer cell metastasis and evaluated antitumor efficacy of bevacizumab (BEV), a humanized monoclonal antibody targeting VEGF.

Method:
To produce brain metastases model, we transfected secreted NanoLuc (Nluc) genetic reporter vectors into NCI-H1915 cell line, which was established from a brain metastasis derived from a primary human lung carcinoma and injected into internal carotid artery of SCID mice with external carotid clamped with microclamp. Mice whose Nluc activities in plasma (Relative Light Unit; RLU/5μl) were detected 16 days after inoculation of NCI-H1915 cells were randomly allocated to control and BEV treatment groups (n=9). HuIgG or BEV (5 mg/kg) was intraperitoneally administered once a week (Day 1, 8, 15). Antitumor activity was evaluated by measuring Nluc activity (RLU/whole brain) in supernatant of brain parenchyma homogenized with cell lysis buffer on Day 22. Statistical analysis was performed using the Wilcoxon test.

Result:
Large metastatic nodules were macroscopically observed in brain on Day 22 in 6/9 mice in the control group, whereas those were not observed in any mice treated with BEV. Nluc activity in brain parenchyma homogenate (RLU, mean ± SD) in the BEV group (3.12E+9 ± 3.04E+9) was significantly lower than that in the control group (1.88E+10 ± 2.03E+10, p<0.05). Meanwhile, the weight of parenchyma (mg, mean ± SD) on Day 22 of control and BEV, was 417 ± 27.5 and 398 ± 15.6, respectively and there was no significant difference between them (p>0.05).

Conclusion:
In the secNluc-transfected H1915 hematogeneous metastasis model, BEV showed remarkable activity in reducing Nluc activity in brain parenchyma as well as emergence of visible legion. These results suggested BEV has efficacy against established hematogenous lung cancer brain metastasis lesions in the xenograft model and it may be one of the treatments for brain metastases from lung cancer.

Background:
The increase in targeted therapies and associated companion diagnostics (CDx) has led to the need for efficient determination of therapeutic eligibility from a single assay. Comprehensive genomic profiling (CGP) provides a solution, but due to the complexity and number of assays available today, standardization of validation has become critically important. We present here the first NGS-based universal CDx platform developed and performed in compliance with FDA 21 CFR part 820. The assay interrogates 324 genes, and is anticipated initially to have eight CDx indications (Table 1). The versatile assay design will facilitate streamlined development of future CDx indications.

Method:
DNA extracted from FFPE tumor tissue underwent whole-genome shotgun library construction and hybridization-based capture, followed by sequencing using Illumina HiSeq 4000. Sequence data were processed using a proprietary analysis pipeline designed to detect base substitutions, indels, copy number alterations, genomic rearrangements, microsatellite instability (MSI), and tumor mutational burden (TMB).

Result:
Concordance with FDA-approved CDx are shown in Table 1. Clinical validity was established such that the concordance between CGP and approved CDx were statistically non-inferior to that of two runs of approved CDx. For analytical validity, limit of detection (LoD) was at allele frequency 4% for known substitutions and indels. LoD was 16% tumor content for copy number amplifications, 30% for homozygous deletions, 11% for genomic rearrangements, 12% for MSI, and estimated 20% for TMB. Positive percent agreement (PPA) with an orthogonal NGS platform was 95.8% in substitutions and indels. PPA with FoundationOne was 98.3% across all variant types. Within-assay reproducibility was measured with PPA 99.4%.

Companion diagnostic	Indicated use	PPA	Comparator CDx assay
EGFR exon 19 deletions and L858R	erlotinib, afatinib or gefitinib in NSCLC	98.1% (106/108)	cobas® EGFR Mutation Test v2
EGFR T790M	osimertinib in NSCLC	98.9% (87/88)	cobas® EGFR Mutation Test v2
ALK rearrangements	crizotinib in NSCLC	92.9% (78/84)	Ventana ALK (D5F3) CDx Assay Vysis ALK Break-Apart FISH
KRAS	cetuximab or panitumumab in CRC	100% (173/173)	therascreen® KRAS PCR
ERBB2 (HER2) Amplifications	trastuzumab, pertuzumab and ado-trastuzumab-emtansine in breast and gastric cancer	89.4% (101/113)	Dako HER2 FISH PharmDx®
BRAF V600E/K	vemurafenib, dabrafenib, trametinib in melanoma	99.4% (166/167)	cobas® BRAF V600 PCR
BRCA1/2	rucaparib in ovarian cancer	N/A	N/A: Novel CDx that was previously validated in PMA P160018

Conclusion:
Rapid expansion of targeted therapies and CDx has necessitated a new approach and urgency to defining performance standards. We developed a universal CDx assay and established a robust approach for demonstrating clinical and analytical validity to support and accelerate the use of CGP for routine clinical care.

Background:
The aim of the study was to assess the prognostic value of 18 proteins as markers of inflammation and fibrosis (Table 1) in surgically treated non-small cell lung cancer patients and how the chronic obstructive pulmonary disease (COPD) as co-morbidity affected the prognostic significance.

Method:
Blood samples were collected from 207 lung cancer patients with an early-stage disease before surgery. 55,6% of the lung cancer group had COPD and the majority of these (86%) had moderate COPD (GOLD 2). We grouped the lung cancer patients with moderate, severe and very severe COPD (GOLD 2,3 and 4) in one group, and the lung cancer patients with mild (GOLD 1) or no COPD in another group. Serum levels of 18 proteins were measured by enzyme immunoassays. The proteins were selected because most were previously known to be associated with lung cancer and its prognosis, some with COPD.

Result:
Higher soluble tumour necrosis factor (TNF) receptor type 1 (sTNFR1) level was associated with better overall survival (OS) and progression-free survival (PFS) for lung cancer patients with COPD (OS p=0.006 and PFS p=0.030) and without COPD (OS p=0.040). High level of C-reactive protein (CRP) was significantly associated with poor overall survival regardless of COPD status. Higher osteoprotegerin (OPG) level was significantly associated with better PFS (p=0.014) and OS (p=0.027) in patients with lung cancer and COPD. In lung cancer patients with COPD, only CRP was significantly increased compared to patients without COPD of those three proteins.

Conclusion:
Whereas high levels of sTNFR1 and OPG, members of the TNF receptor superfamily, were associated with better prognosis, high level of CRP was associated with worse prognosis. This illustrates the complex role of inflammation and TNF-related molecules, particularly in the prognosis of non-small cell lung cancer, at least partly influenced by COPD as co-morbidity.

Background:
Thymidylate synthase (TS) is a nucleotide metabolism enzyme and a chemotherapeutic target. TS overexpression is associated with worst prognosis in NSCLC and is important for the pathological progression of tumors. We investigated the role of TS in epithelial-to-mesenchymal transition (EMT), a developmental process that allows the cancer cells to acquire features of aggressiveness like motility and chemoresistance.

Method:
Immunohistochemistry (IHC) on TS and EMT markers was performed in tissues from 61 NSCLC patients. EMT and cancer stemness markers were quantified in cultured NSCLC cell lines by western blotting. Migration assays were conducted using an automated wound-healing real-time quantification system. Spheres-forming assays were performed by growing cells in low-adherence plates. In addition, cells were stably infected with a lentiviral reporter plasmid in which the expression of m-Cherry fluorescent protein was driven by the full-length promoter sequence of the TS gene (TYMS).

Result:
A significant association between TS mRNA expression and the markers of EMT was found (p<0.01). This was confirmed at the protein level in cultured cell lines, and TS was found up-regulated following EMT induction by TGF-Beta. In addition, a strong association between TS and the powerful EMT driver ZEB1 was found by western blotting and validated by IHC in tissue specimens from NSCLC patients (p=0.02). Importantly, TS showed to regulate EMT, as a shRNA-mediated knockdown of TS could reduce in vitro the levels of ZEB1 and of other EMT markers, suppress the cells’ migratory and spheres-forming abilities and the chemoresistance. Furthermore, following infection with the fluorescent promoter reporter, we could FACS-sort two populations with distinct mesenchymal and epithelial-like phenotypes (TS-prom[high] and TS-prom[low], respectively) from NSCLC cells. Sorted cells, in fact, displayed differential expression of EMT markers (E-Cadherin, Vimentin and ZEB1), migratory ability and spheroids-forming properties (all p<0.001), while no differences were observed using a GAPDH promoter reporter as control.

Conclusion:
All together, these data indicated an unprecedented role for TS in governing cancer differentiation and aggressiveness. The lentiviral promoter reporter may lead to the identification of the regulatory elements and the transcription factors linking TS to EMT. With regards to possible translational implications, the cancer EMT status could be tested as a predictor of the response to TS-inhibiting drugs in NSCLC, and eventually used as a stratification criterion. In the longer run, these data could inspire the development of conceptually novel anti-TS agents that better suppress tumor's growth and aggressiveness.

Background:
Receptor tyrosine kinases (RTKs) such as epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (MET) are the best-known therapeutic targets in lung adenocarcinoma. Previously, we have revealed that stratifin (SFN, 14-3-3 sigma) acts as a novel oncogene, accelerating tumor initiation and progression of lung adenocarcinoma and interacts with ubiquitin-specific protease 8 (USP8) (IJC 2011, Mol Cancer 2015). USP8 is one of the deubiquitination enzymes that stabilize specific protein substrates by removing ubiquitin from the proteins, and is known to target receptor tyrosine kinases (RTKs). In this study, we investigated the molecular mechanism underlying the binding of SFN to USP8 in lung adenocarcinoma cells, as the role of this interaction in RTK stabilization was considered a promising avenue for identifying a useful therapeutic target for lung adenocarcinoma.

Method:
Expressions of USP8 and SFN in human lung adenocarcinoma tissues (n=193) were examined by immunohistochemistry and statistically analyzed with clinicopathological features of patients. Functional analysis of USP8 and SFN such as cellular proliferation assay, apoptosis assay, and wound healing assay was examined after siRNA-USP8 or SFN transfection. Regulation mechanism of USP8 and SFN on RTKs stabilization was demonstrated using co-immunoprecipitation, western blot analysis, and immunofluorescence.

Result:
USP8 specifically bound to SFN in lung adenocarcinoma cells. Both USP8 and SFN showed higher expression in human lung adenocarcinoma than in normal lung tissue, and their expression was mutually correlated. Expression of SFN, but not that of USP8, was significantly associated with histological subtype, pathological stage, and patient’s prognosis. In vitro, USP8 binds SFN at the early- and late-endosome in immortalized adenocarcinoma in situ (AIS) cells. Moreover, USP8 or SFN knockdown led to down-regulation of tumor cell proliferation, RTK expression, and expression of downstream factors including AKT and STAT3, as well as accumulation of ubiquitinated RTKs leading to lysosomal degradation. Additionally, transfection with mutant USP8 and mutant SFN, which are unable to interact each other, reduced the expression of RTKs and their downstream factors, indicating that interaction with SFN is important for USP8-mediated stabilization of RTKs via deubiquitination.

Conclusion:
RTKs are regulated by ubiquitin-lysosome system, and aberrant stabilization of RTKs contributes to the proliferative activity of many human cancers, including NSCLC. Here, we demonstrate SFN induces aberrant activation of USP8 and subsequently protects RTKs from lysosomal degradation, resulting in hyperactivation of these signaling pathways. SFN may be central to the development of a useful therapeutic strategy for both early and advanced lung adenocarcinomas.

Background:
We conducted this study to identify genetic variants in cancer-related pathway genes which can predict prognosis of NSCLC patients after surgery, using a comprehensive list of regulatory single nucleotide polymorphisms (SNPs) prioritized by RegulomeDB.

Method:
A total of 509 potentially functional SNPs in cancer-related pathway genes selected from RegulomeDB were evaluated. These SNPs were analyzed in a discovery set (n=354), and a replication study was performed in an independent set (n=772). The association of the SNPs with overall survival (OS) and disease-free survival (DFS) were analyzed.

Result:
In the discovery set, 76 SNPs were significantly associated with OS or DFS. Among the 76 SNPs, the association was consistently observed for 5 SNPs (ERCC1 rs2298881C>A, BRCA2 rs3092989G>A, NELFE rs440454C>T, PPP2R4 rs2541164G>A, and LTBP4 rs3786527G>A) in the validation set. In combined analysis, ERCC1 rs2298881C>A, BRCA2 rs3092989, NELFE rs440454C>T, and PPP2R4 rs2541164G>A were significantly associated with OS and DFS (adjusted HR aHR for OS = 1.46, 0.62, 078, and 0.76, respectively; P = 0.003, 0.002, 0.007, and 0.003 respectively; and aHR for DFS = 1.27, 0.69, 0.86, and 0.82, respectively; P = 0.02, 0.002, 0.03, and 0.008, respectively). The LTBP4 rs3786527G>A was significantly associated with better OS (aHR = 0.75; P = 0.003).

Conclusion:
Our results suggest that five SNPs in the cancer-related pathway genes may be useful for the prediction of the prognosis in patients with surgically resected NSCLC.

Background:
Protein glycosylation is a post-translational modification that is altered in cancer. However, it is yet unclear if there is a correlation between different glycosylation patterns of N-glycan and clinicopathological features. Therefore, we aimed to investigate differing glycosylation in advanced non-small cell lung cancer tissues by lectin expression assays and N-acetylglucosaminyltransferase gene expression analysis.

Method:
Formalin-fixed and paraffin-embedded biopsy specimens were obtained from 62 patients with lung adenocarcinoma (ADC) (2009-2011) who were diagnosed at Nihon University Itabashi Hospital. Tumor cells were excised and collected by laser microdissection, and each solubilized glycoprotein and mRNA was extracted. Expression levels of 44 lectins were analyzed by lectin array using a lectin chip. mRNA expression levels of mannosyl (beta-1,4-)-glycoprotein beta-1,4-N-acetylglucosaminyltransferase (MGAT3), mannosyl (alpha-1,3-)-glycoprotein beta-1,4-N-acetylglucosaminyltransferase, isozyme A (MGAT4a), mannosyl (alpha-1,6-)-glycoprotein beta-1,6-N-acetyl-glucosaminyltransferase (MGAT5), fucosyltransferase (FUT)7, and FUT8 were analyzed by qRT-PCR.

Result:
Expression levels of Datura stramonium lectin (DSA), Solanum tuberosum lectin (STL), and Aspergillus oryzae lectin (AOL) were significantly higher in drug-resistant ADCs than drug-sensitive ADCs (P = 0.036, 0.0005, and 0.035, respectively). qRT-PCR showed that overexpression of MGAT4a and MGAT5 was detected in 7/62 (11.3%) and 3/62 (4.8%) of ADCs, respectively. The prognosis of patients with MGAT4a and/or MGAT5 positive ADC was worse than those with negative ADC (P = 0.018, log-rank).

Conclusion:
The results of the study suggested different glycosylation of N-glycan in lung ADC tissues. MGAT4a and/or MGAT5 overexpression, in particular, may correlate with a poorer response to standard chemotherapy and poorer outcome in advanced lung ADCs.

Background:
Arginine depletion induced by PEGylated arginase 1 (BCT-100, PEG-BCT-100 or rhArg1peg5000) has shown promising anticancer effects among arginine auxotrophic cancers that are deficient in argininosuccinate synthetase (ASS1) and ornithine transcarbamylase (OTC). High endogenous arginase 2 (ARG2) was previously found in human lung cancers. Although high ARG2 does not induce immunosuppression nor affect disease progression, it may potentially affect the efficacy of PEGylated arginase 1 treatment. ARG2 was highly expressed in H520 squamous cell lung carcinoma (lung SCC) xenograft while undetectable in SK-MES-1 lung SCC xenograft. We postulated that high endogenous ARG2 expression might hamper anticancer effect of PEGylated arginase 1 in lung SCC.

Method:
The in vivo effect of PEGylated arginase 1 was studied using 2 lung SCC xenograft models (SK-MES-1 and H520). Protein expression, arginine concentration and apoptosis were investigated by Western blot, ELISA and TUNEL assay respectively.

Result:
PEGylated arginase 1 (60 mg/kg) suppressed tumor growth in SK-MES-1 but not in H520 xenograft. ASS1 was highly expressed in SK-MES-1 xenograft while expression of OTC remained low in both xenografts. Serum arginine level was decreased significantly by PEGylated arginase 1 in both xenograft models. On the other hand, intratumoral arginine level was reduced by PEGylated arginase 1 treatment in SK-MES-1 xenograft only. In H520 xenograft, intratumoral arginine level in control arm was already very low which could not be further lowered in PEGylated arginase 1 treatment arms. G1 arrest was indirectly evidenced by downregulation of cyclin A2, B1, D3, E1 and CDK4 with PEGylated arginase 1 in SK-MES-1 xenograft only. Moreover, suppression of proliferation factor Ki67 and activation of apoptosis were induced by PEGylated arginase 1 in SK-MES-1 xenograft only.

Conclusion:
PEGylated arginase 1 treatment was effective in lung SCC xenograft with low endogenous ARG2 expression. High endogenous ARG2 level may explain low intratumoral arginine level in lung SCC xenograft. ARG2 may serve as an additional predictive biomarker, other than ASS1 and OTC, in PEGylated arginase 1 treatment in lung SCC. Acknowledgment: This research was supported by Hong Kong Anti-Cancer Society, HKSAR.

Background:
Recent development of cancer panels based on target sequencing technology can detect genomic mutations which are useful to choose target agents for advanced stage lung cancer. We investigated if the result of panel-detected mutation patterns can predict prognosis of early stage lung cancer.

Method:
Among the 350 cases whose postoperative tissues were tested with cancer panel, we selected 338 cases excluding 9 recurrent tumors and 3 cases who received neo-adjuvant treatment. The mean age was 60.7+/-11.4 years (Male 197, female 141). Adenocarcinoma (293) was the most common followed by squamous cell (26), larger cell (5) and others (14). We classified the mutations patterns into three group; SN group (n=126) where a single nucleotide mutation (EGFR, KRAS, NRAS, CTNNB1) was detected, SV group (n=117) where a structural variation (SV) (indel, fusion, splicing) was present, and NO group (n=95) where no significant mutation was found. We investigated the association of mutation patterns with various clinical variables and their impact on the prognosis.

Result:
The mutation was rarely found in squamous cell cancer and the SVs were more frequently found in never-smoked patients. The 5 year overall survival was 73.8+/-3.1% and the 5 year disease-free survival was 46.3+/-3.2%. The disease-free survival of SV group was inferior to that of SN group (p=0.029). When we selected only adenocarcinoma patients and stratified them by clinical stage, the SV group showed poorer disease-free survival to that of SN group in clinical stage I (p=0.017). However, when stratified by pathologic stage, there was no difference. When we investigated discrepancies between clinical and pathologic stages, up-stage from clinical stage I to pathologic N2 was more frequently found in SV group. Figure 1



Conclusion:
Our observation suggests that if structural variations are detected in clinical stage I adenocarcinoma, more aggressive mediastinal lymph node evaluation is mandatory and a different treatment strategy may be investigated.

Background:
Epidermal Growth Factor-like repeats and Discoidin I-Like Domains 3 (EDIL3), is a secreted glycoprotein associated with endothelial cell surface and extracellular matrix. Overexpressed EDIL3 can contribute to carcinogenesis by promoting cancer vascularization and epithelial–mesenchymal transition. Therefore, EDIL3 might be a good candidate target for developing novel cancer anti-angiogenic therapy. Although the associations among EDIL3, mesenchymal phenotype, and angiogenesis have been observed in several malignancies, no study has examined the relationships among EDIL3, mesenchymal phenotype, and angiogenesis or the prognostic significance of EDIL3 expression in non-small cell lung carcinoma (NSCLC) patients. We examined the prognostic significance of EDIL3 expression and its correlations with mesenchymal phenotype and microvessel density in NSCLC.

Method:
A total of 268 NSCLC specimens were evaluated retrospectively by immunohistochemical staining for EDIL3, epithelial–mesenchymal transition (EMT) markers (e-cadherin, β-catenin, and vimentin), and CD31 to measure microvessel density. we performed real-time qRT PCR for EDIL3 expression

Result:
EDIL3, e-cadherin, β-catenin, and vimentin were expressed in 16%, 22.8%, 3.7%, and 10.1% of the specimens, respectively. The mRNA level of EDIL3 in tumor was correlated with the level of EDIL3 protein expression using immunohistochemistry. In lung adenocarcinoma patients, EDIL3 expression was significantly correlated with mesenchymal phenotype (low e-cadherin expression and high vimentin expression) and increased microvessel density (P < 0.001, P = 0.001, and P = 0.023, respectively). In lung squamous cell carcinoma patients, EDIL3 expression was significantly correlated with mesenchymal phenotype (low e-cadherin expression and high vimentin expression) (P = 0.021 and P = 0.002, respectively). In lung adenocarcinoma patients, EDIL3 was an independent prognostic factor for overall survival in a multivariate analysis (hazard ratio: 2.552, P = 0.004).

Conclusion:
EDIL3 is significantly correlated with mesenchymal phenotype, angiogenesis, and tumor progression in lung adenocarcinoma.

Background:
The development of robust, feasible and clinically useful molecular classifiers for early stage NSCLC patients to assess the risk of developing post-resection recurrence is an unmet medical need. Here we identified and validated the clinical utility of two different histotype-specific protein-based prognostic signatures to stratify the five-year risk of lung cancer recurrence or death in patients with either early lung adenocarcinoma (ADC) or early squamous cell carcinoma (SCC). The signatures are based on the immunohistochemical detection of three and five proteins, for ADC and SCC respectively

Method:
A total number of 562 lung cancer patients were included in this study (n=350 for ADC and n=212 for SSC). A training cohort was used to assess the value of the prognostic signatures based on immunohistochemical (IHC) detection (n=239 ADC and n=117 SSC). The prognostic signatures were developed by Cox regression analysis and were comprised of three and five proteins, respectively for ADC and SCC. Overfitting and optimism were quantified and calibrated by internal validation by applying shrinkage and bootstraping combination. The performance of the models was externally validated in a second cohort of 111 and 95 patients with stage I-II lung ADC and SCC, respectively.

Result:
The prognostic indexes (PIs) generated by the models were significant predictors of five-year outcome for disease-free survival: [P<0.001, HR=2.88 (95% CI, 1.77-4.69)] for ADC and [P<0.001; HR=2.97 (95% CI, 1.84-4.79)] for SCC; and overall survival: [P<0.001, HR=4.04 (95% CI, 2.30-7.10)] for ADC and [P=0.006; HR=1.86 (95% CI, 1.20-2.88)] for SCC, independently of other clinicopathological parameters. The prognostic ability of both PIs was externally validated in the second cohort of early stage lung cancer patients (P<0.05). The molecular classifiers added significant information to pathological stage. Combined models including both PIs and the pathological stage (CPIs) improved the risk stratification in both cases (P<0.001). Moreover, using the CPI value we were able to select the group of stage I-IIA patients who could obtain a benefit from platinum-based adjuvant chemotherapy treatment (P<0.05) in both histological subtypes.

Conclusion:
This study identifies and validates two protein-based prognostic signatures that accurately identify early lung cancer patients with high risk of recurrence or death. More importantly, the proposed models may be valuable tools to identify the subset of stage I-IIA patients for whom adjuvant chemotherapy could be beneficial.

Background:
Up to 30% stage I non-small cell lung cancer (NSCLC) patients undergone the tumor resection suffer from recurrence within 5 years. Applicable prognostic biomarkers are required to identify those with high risk of relapse after the surgery, for modified adjuvant therapy to potentially improve survival. This study aims to identify the microRNAs (miRNAs) that associate with recurrence and metastasis of stage I lung adenocarcinoma (ADC).

Method:
Two groups of stage I lung ADC patients were enrolled, with 40 cases for each. For the patients in the group RM, the tumor recurrence and/or metastasis occurred within 2 years after the surgical resection. For the patients in the group NRM, the tumor recurrence and/or metastasis had not reappeared for 5 years after the surgery. The tumor tissues were enriched from formalin-fixed and paraffin-embedded tissue sections, and the total RNAs were extracted. The Agilent human microRNA microarrays were used to generate miRNA profiles and explore the aberrantly expressed miRNAs in the samples investigated. The interesting differentially expressed miRNAs between the two groups were then validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in an independent cohort of 80 cases of stage I lung ADC, which was also divided into two groups, the RM and NRM.

Result:
The miRNA expression profiling revealed that a panel of miRNAs were differentially expressed between the group RM and group NRM. Cluster analysis showed that this panel of miRNAs could distinguish patients in the group RM from those in the group NRM. Moreover, according to target gene prediction and KEGG pathway analysis of these miRNAs, predicted target genes of 2 miRNAs (miR-3621 and miR-2467-3p) in the panel are involved in the non-small cell lung cancer pathways. The expression of these miRNAs were tested subsequently by qRT-PCR in the tumor samples of the independent cohort, using miR-191 as an internal reference. The preliminary data showed that among them, the mean relative expression fold change (2[-ΔCt]) of miR-2467-3p in the group RM was significantly lower than that in the NRM (p = 0.014).

Conclusion:
Our finding suggests that a panel of miRNAs may be regarded as candidate biomarkers for prognosis of early stage lung ADC. Among them miR-2467-3p was aberrantly down-regulated in the tumor tissues of patients in the group RM; accordingly, miR-2467-3p (and its predicted target genes) may play a role in recurrence and/or metastasis of stage I lung ADC after the surgical resection.

Background:
Squamous cell carcinoma (Sq) is second major histological subtype of lung cancer. Unlike in the case of adenocarcinoma (Ad), Sq has only few molecular target drug. MicroRNA (miR) is a major part of post-transcriptional regulators functioning as tumor suppressor genes or oncogenes. MiR will regulate target molecules related to carcinogenesis and malignancy in Sq.

Method:
Using The Cancer Genome Atlas dataset including copy number variation, RNA sequence, miR sequence, clinicopathological feature from 484 lung cancer cases, the correlation between genomic copy number and expression of miR was analyzed. 245 samples of Sq and 239 samples of Ad were included. The raw counts of each mature miR fragments with different precursor were merged and calculated from miR-seq isoform files by R project (http://www.r-project.org/) Segmented copy number variation datasets were processed with R package CNTools of Bioconductor project. Independent two-group Mann-Whitney U test was used to compare different expression between Sq and Ad. MiR expression according to copy number variation was analyzed using Pearson correlation coefficient r-score. To identify the miR target sites of mRNAs, targetscan-Perl scripts were used (http://www.targetscan.org/).

Result:
From 1,001 mature miR fragments, 34 miRs were identified as the candidates especially for Sq distinguished from Ad. Furthermore, four miRs were up-regulated in amplified regions and independently associated with poor prognosis in Sq. Moreover, those who had the tumor with high expression in three of four miR simultaneously showed worst prognosis. To explore miR-mRNA network, we also predicted the target genes for each miR. From 734 common target genes, three showed positive correlation with the expression of three miRs. Among them, the expression of 109 mRNAs inversely correlated with that of 3 miRs. From 109 mRNA, the expression of 24 mRNAs inversely correlated with that of all the 3 miRs and only 2 mRNA expression showed low levels in Sq compared with Ad or normal tissues.

Conclusion:
Three miRs up-regulated in Sq were associated with poor prognosis through the regulation of two common target genes.

Background:
MicroRNAs are a class of small non-coding RNAs that range in size from 19-25 nucleotides. They have been shown to regulate a number of processes within tumour biology, including metastasis, invasion and angiogenesis. More recently, miRNAs have been linked to chemoresistance in solid tumours, including lung cancer. Their role in cisplatin resistance has yet to be determined.

Method:
MicroRNA expression within a panel of age-matched parent (PT) and cisplatin resistant (CisR) NSCLC cell lines was profiled using the 7[th] generation miRCURY LNA arrays (Exiqon) and subsequently validated by qPCR. Significantly altered miRNAs within the CisR sublines were manipulated using antagomirs (Exiqon) and Pre-miRs (Ambion) and functional studies were carried out in the presence and absence of cisplatin. To examine the translational relevance of these miRNAs, their expression was examined in a cohort of chemo-naïve patient-matched normal and lung tumour tissue and serum from NSCLC patients of different histologies. A xenograft model of cisplatin resistance was carried out in which 1x10[3] H460 PT or CisR cells were injected into 5-7week old NOD/SCID mice. Tumour volume was measured over time and harvested once the tumour mass measured 500mm[3] and formalin-fixed and paraffin embedded (FFPE). Expression of the 5-miR signature was analysed within FFPE murine tumours and compared between PT and CisR tumours.

Result:
Profiling and subsequent validation revealed a 5-miR signature associated with our model of cisplatin resistance (miR-30a-3p, miR-30b-5p, miR-30c-5p, miR-34a-5p, miR-4286). Inhibition of the miR-30 family and miR-34a-5p reduced clonogenic survival of CisR cells when treated cisplatin. Expression of the miRNA signature was significantly altered in both adenocarcinoma (AD) and squamous cell carcinoma (SCC) relative to matched normal lung tissue and between SCC and AD tissue. miR-4286 was significantly up-regulated in SCC sera compared to normal control and AD sera. Similarly to the cell line expression of the miRNAs, the miR-30 family members and miR-34a-5p were up-regulated in the CisR xenograft FFPE tissue relative to PT.

Conclusion:
A novel miRNA signature associated with cisplatin resistance was identified in vitro, genetic manipulation of which altered clonogenic response to cisplatin. The 5-miR signature showed both diagnostic and prognostic biomarker potential across a number of diagnostically relevant biological media.

Background:
We have previously demonstrated the involvement of Ran Binding Protein 9 (RanBP9) in the DNA Damage Response (DDR) in Non Small Cell Lung Cancer (NSCLC) cells. Here, we investigate its role in response to DNA-damaging agents in vitro and as prognostic and predictive biomarker for NSCLC patients.

Method:
First, by IHC, we evaluated RanBP9 expression in tumor vs normal adjacent tissue (NAT). Then, we generated A549 RanBP9 WT and KO NSCLC cells using CRISPR/Cas9. We treated A549 RanBP9 WT and KO with cisplatin (CDDP) and PARP inhibitors. We assessed response to treatment by measuring cell toxicity, apoptosis and proliferation. Finally, we determined the expression of RanBP9 in cohort of NSCLC patients previously enrolled in the TAILOR trial.

Result:
In the present study, we report that significant overexpression of RanBP9 is a common event in lung cancer, as shown by an extensive immunohistochemical analysis of RanBP9 levels in 148 lung tumors of different histotypes and their normal adjacent tissue (p<0.02 - 0.001). RanBP9 expression was maintained/acquired in the nodal metastasis from 30 NSCLC patients, indicating its potential involvement in tumor aggressiveness. We also show that RanBP9 KO A549 NSCLC cell lines display a reduced DDR and higher levels of apoptosis upon cisplatin treatment both in vitro and in vivo. Accordingly, a retrospective analysis of 134 NSCLC patients revealed that higher levels of RanBP9 are associated with tumor stage (p<0.0001), and low response to platinum compounds as first-line treatment (PFS, HR~ (RanBP9 positive versus negative)~ 1.71, 95% CI 1.142 - 2.563, p = 0.0093; OS HR~ (RanBP9 + vs -) ~1.942, 95% CI 1.243-3.033, p=0.0036). Finally, we show that ablation of RanBP9 is associated with overactivation of Poly(ADP-ribose) Polymerase (PARP) and increases sensitivity to PARP inhibitors. Moreover, that use of PARP inhibitors enhances cisplatin anti-neoplastic efficacy in the absence of RanBP9.

Conclusion:
We identified RanBP9 as a novel predictive biomarker of response to genotoxic treatments in NSCLC patients. We also report that RanBP9 affects the response of NSCLC cells to PARP inhibitors in vitro. Our results open new avenues for the treatment of NSCLC patients based on their level of expression of RanBP9.

Background:
The vast majority of cancer driver genes in non-small cell lung cancer (NSCLC) are characterized by activating mutations or high gene copy number amplifications. To identify tumorigenic genes without any genomic aberrations remains difficult. Network analysis of gene expression provides the possibility to describe the relations of genes to each other and by that to estimate their importance.

Method:
To analyze gene networks of NSCLC we applied the PageRank algorithm that was established by Google primarily to order the importance of websites. Data from NSCLC cancer tissue (n=1002) and normal lung (n=110) were retrieved from the cancer genome atlas (TCGA) and the highest expressed genes (n=16000) were ranked according to their importance in normal lung tissue as well as in NSCLC tissue. Subsequently, the difference in rank between normal and cancer was analyzed. Four comparative categories were defined and were analyzed with respect to their cellular function (GO annotation) and survival. Additionally, organ specific (n=163), housekeeping (n=68) and lung cancer related genes (n=62) were compared in the networks.

Result:
Genes with the highest importance (top 100) in normal lung tissue were connected with cellular metabolic processes or membrane transport. In cancer, most genes (top 100) were related to cell cycle and mitosis, chromosomal localization and DNA processing. There was no overlap between the two lists. Organ specific genes increased in average in their rank (p<0.001) while housekeeping genes decreased (p<0.001). Notably, cancer related genes did not significantly change their relevance in the network. Among the genes (top 100) that increased rank from normal to cancer, many were related to antigen processing and presentation.

Conclusion:
The PageRank algorithm provides the possibility to unbiasedly evaluate the importance of genes in the gene expression network of cancer. Surprisingly, not traditional cancer related genes but several hitherto not recognized genes were identified to be of regulatory importance and may be target for therapy. Once again, our results indicate the significance of the immune response in changes related to cancer.

Background:
Lung cancer is the leading cause of cancer-associated mortality in the United States. LKB1 loss, for which there are no targeted therapies, occurs in 30% of lung adenocarcinomas. Our group has developed an RNA-based genetic signature for LKB1 loss and applied it to samples in the Cancer Genome Atlas (TCGA). Our studies have identified a novel role for LKB1 in regulation of DNA methylation and histone acetylation/methylation. While global demethylation has been noted in many cancers, LKB1-deficient lung tumors display a greater degree of demethylation compared to tumors that express functional LKB1.

Method:
RNA-Seq results the TCGA lung adenocarcinoma provisional dataset were analyzed using a genetic classifier for LKB1 loss; samples were separated into wildtype and loss cohorts. Approximately 420 of the samples classified were also characterized using Illumina 450k methylation arrays. We used this data to analyze differentially methylated CpGs. Motif analysis of common transcription factor binding sites near hypomethylated CpGs was accomplished using HOMER. To assess transcription factor localization and binding in vitro, we used a retroviral gene expression system to restore LKB1 function in previously deficient lung cancer cell lines and a CRISPR/Cas9 approach to knock out LKB1 in wildtype cell lines.

Result:
We observed that LKB1 loss is associated with widespread demethylation of CpG islands throughout the genomes of TCGA samples. Of approximately 138,000 differentially methylated probes, 131,000 are significantly hypomethylated in LKB1 loss (adj. p-value cutoff = .01). We observed that DNMT1, which maintains methylated CpG sites, is downregulated following LKB1 loss. HOMER motif analysis of common transcription factor binding sites in the top 5000 hypomethylated sites implicated several transcription factors that are associated with hypomethylated CpGs—most notably FOXA1/2/3, Nur77, CEBPB, and KLF5; the FOXA family and KLF family have been described as pioneering transcription factors in genomic demethylation. Fractionation and Western blot of A549 cells show that inducing LKB1 expression attenuates FOXA1 and FOXA3 chromatin binding as well as overall expression of FOXA1 and FOXA2.

Conclusion:
These results have broad implications for gene regulation in LKB1-loss lung tumors and a full understanding of these changes might uncover drug targets specific for these tumors. LKB1’s association with global demethylation suggests that therapeutics targeting methylation such as decitabine may have different effects on LKB1 loss and LKB1 WT tumors, a hypothesis which we are currently testing. We are also continuing to study FOXA1/2/3 and KLF5 to determine the mechanism by which LKB1 regulates its demethylation program.

Background:
Evidence suggests a striking significance of BRG1 in non-small cell lung cancer (NSCLC) development and prognosis including its ability to modulate NSCLC response to commonly used chemotherapy. The human SW1/SNF (switching defective/ sucrose non-fermenting) family of chromatin remodeling complexes are composed of multi-subunit proteins among which are the BRG1 and INI-1. Both BRG1 (SMARCA4) and INI-1 (SMARCB1, BAF47, SNF7) are implicated in different cancers. We aimed at identifying the frequency of the SW1/SNF protein components loss and the significance in NSCLC. Given that inactivating BRG1 mutations could coexist with TP53 alterations in NSCLC cell lines, we also included the tumor suppressor protein p53 in our analysis.

Method:
We analyzed the expression of the SW1/SNF subunits (BRG1 and INI-1) and p53 proteins from resected stage I – III NSCLC using the immunohistochemistry. Mouse monoclonal anti-p53 (DO-7, Dako Omnis) and anti-INI-1 (MRQ-27, Cell Marque) antibodies were used for p53 and INI-1 protein detection respectively. BRG1 was detected using the rabbit monoclonal anti-BRG1 antibody (EPNCIR111A, Abcam). Clinical data were obtained from the Glans-Look lung cancer database and patients diagnosed of NSCLC from 2003 to 2006 were included in the study.

Result:
A total of 150 cases were identified, {Adenocarcinoma n =82 (54.7%), Squamous cell carcinoma n = 50 (33.3%), Large cell carcinoma n = 7 (4.7%) and others n = 11 (7.3%: Adenosquamous, Bronchioalveolar and Giant cell carcinomas)}.The BRG1 protein was absent in 6% (9/150) of cases with the overall highest number (3.3%) seen in adenocarcinoma. Whereas, normal INI-1 protein was expressed in all samples. Within NSCLC subtypes, the rate of BRG1 loss was highest in the large cell carcinomas at 28.1% (2/7) and lowest in squamous cell subtype at 2% (1/50) while only 6.1% (5/82) of adenocarcinoma showed BRG1 loss. The frequency of aberrant p53 protein expression (including overexpression, cytoplasmic and complete expression loss) was at 63% (95/150) and 6 (2 large cell and 4 adenocarcinoma) of the 95 abnormal p53 cases (6.3%) had BRG1 loss.

Conclusion:
BRG1 loss seems to be a low occurrence in NSCLC. Although a high rate of BRG1 inactivating mutations were previously reported in NSCLC cell lines, the present result suggests that these mutations may still result in the expression of possibly an abnormal BRG1. BRG1 loss appears to coexist with p53 abnormality in NSCLC. Additional multivariate and outcome analysis will be presented.

Background:
Neuropilin-1 (NP1) is expressed by a wide variety of human tumour cell lines and diverse human neoplasms, and is implicated in mediating the effects of VEGF on the proliferation, survival and migration of cancer cells. It is extensively expressed in tumour vasculature, where NP1 over-expression is associated with tumour progression and poor clinical outcome. In this study, we examined the effects of targeting NP1 in NSCLC both in vitro and in vivo.

Method:
A panel of NSCLC cell lines (H460, H647, A549 and SKMES) were screened for NP1 at the mRNA and protein levels by RT-PCR and Western blotting, respectively. Cellular expression and localisation of NP1 was further examined by immunocytochemistry, while a panel of retrospective resected lung tumours and matched normal tissues were stained by immunohistochemistry. The effects of targeting NP1 on cell proliferation (BrdU ELISA), apoptosis (FACS, HCS) and downstream survival signalling pathways (Western Blot) were examined under normoxic and hypoxic (0.1% O~2~) cell growth using anti-NP1 neutralising antibodies. Cell survival was assessed in response to treatment of NSCLC cells with a range of chemotherapeutic agents in combination with NP1 neutralising antibodies. Using a human xenograft model, tumour growth studies were carried out in nude mice following subcutaneous injection of NP1 over-expressing cells relative to empty vector controls.

Result:
All lung cancer cell lines examined expressed NP1 with the exception of the H460 cell line. Immunocytochemistry analysis confirmed cellular expression and localisation of this receptor, particularly in the leading edges of migrating cells, suggesting a possible role in cell migration. In a small cohort of resected NSCLC patients, tumour expression of NP1 was high relative to their matched normal lung tissues in adenocarcinoma, squamous cell and large cell neuroendocrine carcinomas. Cell proliferation and apoptosis were significantly altered in NSCLC cells expressing NP1. While hypoxia induced the expression of NP1, treatment of cells with NP1 neutralising antibodies reduced hypoxia-mediated cell proliferation and decreased expression of PI3K and MAPK signalling pathways. In a preliminary study, treatment with NP1 neutralising antibodies sensitised NSCLC cells to the cytotoxic effects of chemotherapy. In vivo, H460 cells over-expressing NP1 significantly increased tumour growth in NOD/SCID mice relative to empty vector controls.

Conclusion:
These data suggest a role for the Neuropilin-1 receptor in promoting cell survival and tumour growth in NSCLC and may offer potential as a therapeutic biological strategy in lung cancer.

Background:
c-MET protein overexpression has been proposed as potential prognostic as well as predictive biomarker for targeted therapy in non-small cell lung cancer (NSCLC), albeit its role in the clinical setting has not been firmly established yet and no clear cut-off value exists for immunohistochemistry (IHC) score.

Method:
We designed a retrospective cohort study, consisting of 725 patients with surgically removed non-small cell lung cancer. IHC was conducted in tissue microarrays (TMA) from lung tumors and healthy tissue adjacent to the tumor, using a specific antibody against human c-MET (MET PharmDx). IHC staining was quantified using H-scores (range 0-300). Association between c-MET H-score and overall survival (OS) as well as progression-free survival (PFS) was explored.

Result:
c-MET H-score over 20 had a significant protective impact on OS in the multivariate analysis in the whole study population, both as continuous variable (p=0.014), as well as dichotomous variable with HR=0.79 (95%CI: 0.64-0.97, p-value = 0.022). The prognostic effect of c-MET H-score over 20 was stronger in patients who received adjuvant treatment with a HR=0.61 (95% CI: 0.40-0.93, p-value=0.022). In the subgroup of adenocarcinoma and squamous cell carcinoma patients with stage IIA-IIIB disease, the prognostic impact of c-MET was significant even in the univariate analysis (HR=0.60, 95% CI: 0.43-0.83, p-value=0.002).

Conclusion:
c-MET H score > 20 is a positive prognostic biomarker for OS in early stage NSCLC. This benefit seems to be strongly correlated to adjuvant chemotherapy, therefore rendering c-MET H-score > 20 a possible predictive biomarker for platinum-based adjuvant chemotherapy in early stage NSCLC.

Background:
Next generation sequencing has contributed to understanding the biology of NSCLC and to improve therapy selection. The prevalence of other oncogenic alterations beyond EGFR, ALK and KRAS in Argentina remains unknown. The aim of this study was to characterize the genomic lanscape of NSCLC tumors from 45 patients in our institution by NGS.

Method:
Prospective observational study. We included 45 patients, over 18 years old, with diagnosis of NSCLC and adequate tumor sample. DNA was purified from FFPET samples. Targeted NGS was done with Colon and Lung Cancer Research Panel v2 (Ion Torrent™ AmpliSeq™ technology) for 22 genes with Ion 520 chip, sequenced in Ion S5 Next Generation Sequencing Systems.

Result:
Forte five patients were included in the analysis, 19 female (42%) and 26 male (58%). Median age was 57 years old (range 34-89). Most patients had lung adenocarcinoma 43 (96%), 1 squamous (2%) and 1 adenosquamous histology (2%). A total of 28 patients (62%) had stage IV lung cancer, 18% stage III, 4% stage II and 16% stage I. From 43 evaluable samples, 65 mutations were detected: TP53 n=21, KRAS n=20, EGFR n=9, BRAF n=5, MET n=3, ERBB2 n=2, FGFR3 n=2, PI3K n=2, FGFR2 n=1. Of these, 10 are associated with clinical benefit with approved targeted therapies. Two samples had novel EGFR mutations and 2 had EGFR co-activating mutations (Del19 + L858R; and G719C + S768I). KRAS and TP53 co-mutations were present in 50% of KRAS mutant samples. We encountered 26 variants of unknown significance. In our population, 37 samples (86%) had EGFR Q787Q (c.2361G>A) polymorphism.Figure 1



Conclusion:
The distribution of oncogene mutations in patients with NSCLC in our institution in Argentina is similar to other western countries with the exception of higher KRAS mutant patients in this cohort. An ongoing larger trial will provide further information for our country and the region.

Background:
Programmed death 1 immune checkpoint inhibitor antibody has been effective in patients with PD-L1 positive advanced NSCLC. However, surgically resected specimens or core-needle biopsy samples were used to estimate drug potency in past clinical trials.The aim of this study is to prospectively investigate small sample reliability for NSCLC to determine the PD-L1 expression status.

Method:
We prospectively enrolled patients who underwent diagnostic biopsy by any procedures (under rigid bronchoscopy, flexible bronchoscopy, CT & US-guided core-needle, excisional method) from March 2017 to March 2018 (ongoing study). Pathologically confirmed non-small cell lung cancer (NSCLC), PD-L1 expression was evaluated in our institution using companion diagnostic PD-L1 immunohistochemistry：PD-L1 IHC 22C3 pharmDx (Daco) with autostainer Link 48, detecting a driver mutation in parallel. We evaluated: 1) the total number of tumor cells and sample size; 2) compared PD-L1 expression for each procedure using tumor proportion score: TPS (＜1%, 1～49%, 50%≦), 3) the concordance rate of PD-L1 expression status by biopsy and surgical materials; and 4) the efficacy of administration for PD-1 immune checkpoint inhibitor antibody.

Result:
During the first three months 44 cases of PD-L1 expression were evaluated. 33 cases were sampled by bronchoscopy (6 under rigid scope with BF 1T260, 17 TBBs using 1T260/P260F in 4/13 cases, 10 TBNAs), and 7 cases were CT-guided core-needle biopsy. The TPS (＜1%, 1～49%, 50%≦) was 8/6/10 for TBB, 3/4/2 for TBNA, and 4/0/3 for CT-guided. Four cases harboring EGFR mutation showed a lower PD-L1 expression (＜10%).

Conclusion:
Utilizing smaller samples to evaluate PD-L1 expression, and the frequency of TPS were comparable to past clinical trials using larger samples. Smaller samples might be an accurate alternative to assess PD-L1 expression. Current data will be updated at the conference.

Background:
Lung cancer is the most lethal malignancy in worldwide. We have previously compared genetic abnormality profiles in early-stage lung adenocarcinoma using array-comparative genomic hybridization (CGH) and found that Epithelial cell transforming sequence 2 (ECT2) amplification and overexpression a new prognostic marker in early-stage lung adenocarcinoma (Cancer Science, 2014). ECT2 is an oncogene that is overexpressed in several types of human cancer and has tumorigenic activity. ECT2 is localized in the nucleus of normal cells, and its function is associated with cytokinesis. In cancer cells, ECT2 exists in not only nucleus but also cytoplasm. However, cytoplasmic ECT2 is thought to promote tumor growth and invasion. In the present study, we aimed to explore the expression of cytoplasmic ECT2 and to assess its functional and prognostic significance in lung adenocarcinoma. First, we examined the subcellular localization of the ECT2 protein in lung adenocarcinoma cells. Subsequently, we investigated the biological significance of cytoplasmic ECT2 that mediated its phosphorylation state. Finally, we examined the clinicopathological attributes of cytoplasmic ECT2 in terms of patient outcome.

Method:
ECT2 expression was evaluated in an immortalized lung epithelial cells (PL16B) and eight lung adenocarcinoma cell lines Calu-3, A549, RERF-LC-KJ, NCI-H1650, PC-9, NCI-H23, NCI-H1975, and HCC827 using Immunoblotting, RT-PCR, Immunofluorescence, and Immunohistochemistry. In order to assess the clinicopathologic characteristics of cytoplasmic ECT2, we examined 50 cases of surgical specimens lung adenocarcinoma by immunohistochemistry. Twenty fresh scraping samples of lung adenocarcinoma were also used to evaluate the expression of Phosho-ECT2 (T790). The Kaplan–Meier method and Cox regression analyses represent the prognostic significance of cytoplasmic ECT2 in lung adenocarcinoma.

Result:
We found that ECT2 expressed in eight lung adenocarcinoma at variable degree levels. In PL16B cells, ECT2 was localized in the nucleus, whereas in lung adenocarcinoma cell lines ECT2 distributed in both the cytosol and the nucleus. Importantly, overexpression of ECT2 leads to aberrant cytoplasmic localization in lung adenocarcinoma cells. We also found that cytoplasmic ECT2 was phosphorylated and accumulated at the cell membrane in lung adenocarcinoma cell lines and surgical specimens. The phosphorylated form of ECT2 was reported to correlate with malignant attributes of lung adenocarcinoma and our clinical analysis showed that cytoplasmic ECT2 expression was significantly associated with poor outcome (OS; P=0.002, DFS; P =0.001), and was an independent prognostic factor in lung adenocarcinoma.

Conclusion:
We demonstrate that aberrant localization of ECT2 to the cytoplasm is a specific feature of lung adenocarcinoma, and provide a new potential prognostic biomarker in lung adenocarcinoma.

Background:
Programmed death-ligand 1(PD-L1) expression in Non-small Cell Lung Carcinoma (NSCLC) is tissue based predictive marker. However, the differential expression of PD-L1 in tumor microenvironment between synchronous primary tumor in lung and metastatic side, especially brain metastasis remains unclear. This study addresses whether there is concordance of PD-L1 status and tumor infiltrating lymphocytes (TIL) together with intensity of CD8 lymphocytes, between the synchronous primary tumor and brain metastasis.

Method:
PD-L1 expression was evaluated immunohistochemically in primary lung tumor and synchronous brain metastasis by using the Dako PD-L1 IHC 22C3 pharmDx kit (SK006) . TIL were scored via CD3 and CD8 positivity immunohistochemically. PD-L1 expression was also compared with TIL proportion and intensity of CD8 lymphocytes between paired tumor tissues. All brain metastatic tissues had been removed before any treatment therefore;the study allowed the comparison of lung carcinoma and their native brain metastasis (BM).PD-L1 scoring was categorized based on the proportion of membranous immuno-positive tumor cells using cutoff values 0-1%, 1-49% and 50%. CD3 and CD8 positivity in TIL was evaluated semi quantitatively. Spearman rank correlation test was used for statistical analysis.

Result:
Primary tumor tissues and synchronous brain metastases of 24 NSCLC cases were included the study. Histopathological suptype of the cases were17/24 (70.83 % ) adenocarcinomas and 2/24 squamous cell carcinoma (8.3%), 1/24 adeno-squamous Ca.(4.16%) and 4/24 large cell carcinomas and pleomorphic carcinomas (16.6%). Tumor subtypes were the same in both primary tumor and BM specimen. In primary tumors, PD-L1 positivity was observed in 25% (6/24), 37.5% (9/24) and 37.5% (9/24) using cutoff values 0-1% , 1-49% and 50% respectively. PD-L1expresion score and pattern showed significant correlation in paired BM (Spearman rho= .731,p ˂0.001). However the PD-L1 expression on TIL cells and proportion of TIL infiltration was not demonstrated same concordance (Spearman rho= .548, p=0.006). When CD8 lyhmpocyte intensity among the TIL cells was compared between primary tumor and BM, only 54.16% (13/24) of the pair tumors were compatible.

Conclusion:
This study demonstrates that the concordance of PD-L1 expression between primary NSCLC and BM is very high. Thus, evaluation of PD-L1 in either primary lung tumors or brain metastasis would be useful for choosing anti PD-1/PD-L1 immunotherapy in patient with NSCLC with BM. Due to the low compatibility of CD8 intensity in TIL cells, may not be sufficient enough as a therapeutic target to use for both primary and brain metastases. Further research on this subject is required to gain deeper insigth in to the mecanism/biology of CD8 lymhpocites intensity in TIL cells and PD-L1 expression in NSCLC.

Background:
Substantial advances have been made in the understanding of the biology of NSCLC in relation to the characterization of molecular abnormalities such as activations of oncogenes by mutations, translocations and amplifications, which are being used as molecular targets and predictive biomarkers. Molecular analysis of NSCLC, adeno carcinoma (AC) is now the standard of care for therapy selection.

Method:
We determined the frequency of molecular alterations in EGFR and gene fusion ALK in our Caucasian and Hispanic populations to decide the adequate treatment . 123 small biopsies and resection specimens of patients with NSCLC (AC) in different institutions of Cordoba were studied during a period (2014 2017). In addition to histopathology type, we analyzed immunohistochemistry (IHC) characteristics and molecular profiles and several clinical variables were studied as well. Different tests were used to detect alterations of EGFR and fusion gene EML4-ALK expression, with the aim to identify our own profile and decide the adequate therapeutical option. EGFR mutation was studied by therascreen kit, PCR, in order to detect genetic alterations in exons 18, 19, 20 and 21. ALK translocations were analyzed by FISH (Vysis- Break Apart, Abbott) and IHC (clon D5F3, ventana, Roche). We correlated the molecular profile with different clinical variables (age, gender, and tobacco habits). The statistical method used was the multiple regression logistic model.

Result:
78 men and 45 women out of 123 samples were tested for EGFR expression. Eleven men and sixteen women expressed EGFR positive. Activating kinase-domain mutations in EGFR were identified in 27 pts (21, 95 %): exon 19 deletion = 17, L858R = 6, exon 20 insertion = 2, other = 2. EGFR alterations were associated with gender (p=0.044), women showed more alterations of the genes. Age and smoking habit of patients did not show significant association (p=0.757 and p=0.547, respectively). We used the multiple regression logistic model to correlate EGFR expression to age, gender, tobacco habits. We identified 4 pts (5%) with fusion gene EML4-ALK. ALK alterations were not related to gender (p=0.449), age (p=0.837) and smoking habit (p=0.452).

Conclusion:
Our results showed a comparable frequency in EGFR mutations and gene fusion ALK in western population, in relation to the data published. These results allow a proper diagnosis to provide pts with the most adequate therapy.

Background:
Some patients with advanced non-small cell lung cancer (NSCLC) show prolonged disease stabilization on treatment with an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), gefitinib, erlotinib and afatinib provide remarkable response rates and survival in NSCLC patients harboring epidermal growth factor receptor-activating mutations, and are therefore standard first-line treatment in these patients, given the long-term exposure of such patients to EGFR-TKIs, from those observed in patients, to evaluate the efficacy and antitumor activity and toxicity, this study aimed to compared reasons severe adverse events (AEs) and survivors for NSCLC patients treated with these three EGFR-TKIs.

Method:
We conducted this retrospective study at a single teaching hospital in southern Taiwan from January 2014 to December 2015, the patients with advanced or metastatic EGFR mutation-positive NSCLC who received gefitinib, erlotinib, or afatinib as first-line treatment, used the Kaplan-Meier method to estimate survival, the data was analyzed by using SPSS 20.0 software.

Result:
Of the 192 patients diagnosed with stage IIIB and IV NSCLC(Non-squamous) in the study period, the analysis 88 (45.8%) had EGFR-activating mutations, of the 37 males, 51 females, median age was 63 years (range, 29-94 years). Sixty-two patients (70%) never smoked. 84 (95%) had adenocarcinoma,and received first-line therapy with gefitinib (n = 55, 63%), erlotinib (n = 10, 11%), and afatinib (n = 23, 26%).Rash and diarrhea were the most common drug-related toxicities, encountered in 68.2% and 53.4% of patients, The overall response and disease control rates were 58 and 80 %(Table 1), respectively, and the median progression-free survival and overall survival were 16 and 23 months, respectively. Figure 1



Conclusion:
First-line target therapy a preferred standard treatment in advanced NSCLC harboring sensitive EGFR mutations. treatment with TKI was feasible and lead to prolonged overall survival.

Background:
It is unclear whether there is a difference in the efficacy of treatment by EGFR-TKI between patients with postoperative recurrent non-small cell lung cancer (NSCLC) and those with stage IV NSCLC harboring EGFR mutations.

Method:
The records of NSCLC patients harboring EGFR mutations who were treated with gefitinib or erlotinib were retrospectively reviewed, and the treatment outcomes were evaluated. Moreover, we performed an immunohistochemical analysis of PD-L1 expression in tumor lesions of the postoperative recurrence group.

Result:
In 205 patients, both the progression-free survival time (9.4 versus 16.9 months) and the median survival time (24.7 versus 37.4 months) were significantly longer in the postoperative group than stage IV group. Additionally, multivariate analysis identified that postoperative recurrence was as an independent predictor of the PFS and OS, as well as performance status and smoking status. The PFS durations were 15.7 and 16.6 months for the high PD-L1 expression and low PD-L1 expression groups, respectively, and a significant difference was not observed (P = 0.73).

Conclusion:
The findings of this study provide a valuable rationale for considering postoperative recurrence as a predictive factor for favorable PFS and overall survival in patients with NSCLC harboring activating EGFR mutations receiving EGFR-TKI.

Background:
Tyrosine kinase inhibitor such as Gefitinib rather than conventional chemotherapy is the standard of care for advanced lung adenocarcinoma (LA) with mutant epidermal growth factor receptor (mEGFR). However, its cost-effectiveness is less clear. The aim of our study is to compare the cost and effectiveness of 1[st] line gefitinib vs platinum-based chemotherapy for clinical stage IV LA via this population-based propensity score (PS) matched analysis.

Method:
We identified eligible patients diagnosed within 2011 through a comprehensive population-based database containing cancer and death registries, and reimbursement data in Taiwan. The primary duration of interest (DOI) was two years within diagnosis. Effectiveness was measured as survival whereas direct medical cost was measured from the health care sector’s perspective. In supplementary analysis (SA), we estimated the cost-effectiveness under potential unmeasured confounder(s) and the cost-effectiveness if the DOI was three years.

Result:
Our study population constituted 240 patients [Table 1]. Within 2 years, gefitinib was both more effective [mean overall survival 1.48 vs 1.47 life-year] and cost-saving [mean 78770 vs 82684, 2015 US dollars] when compared to chemotherapy. In SA, our results was sensitive to potential unmeasured confounder(s) but remained cost-effective when DOI was 3 years.

Table 1. Patient characteristics of the propensity-score matched final study population

		Gefitinib		Platinum-based chemotherapy		standardized difference (rounded)
		number	(%)	number	(%)
age	<65	76	(63.33)	75	(62.5)	0.017
	>=65	44	(36.67)	45	(37.5)
gender	female	60	(50)	60	(50)	0
	male	60	(50)	60	(50)
residency	non-north	55	(45.83)	56	(46.67)	0.017
	north	65	(54.17)	64	(53.33)
comorbidity	without	62	(51.67)	64	(53.33)	0.033
	with	58	(48.33)	56	(46.67)
smoking history	no	79	(65.83)	79	(65.83)	0
	yes	41	(34.17)	41	(34.17)
performance status	0-1	113	(94.17)	113	(94.17)	0
	2	7	(5.83)	7	(5.83)

Conclusion:
1[st] line gefitinib was cost-effective for clinically stage IV LA with mEGFR from health care sector’s perspective when compared to platinum-based chemotherapy.

Background:
Approximately one-third of non-small-cell lung cancer (NSCLC) patients harboring EGFR mutations develop central nervous system (CNS) metastases as the progressive pattern during EGFR-TKIs treatment. We design this study to evaluate the feasibility and efficacy of erlotinib as the subsequent therapy when intracranial tumor progression occurs during gefitinib or icotinib treatment.

Method:
The study reviewed the clinical data of patients with advanced NSCLC who received erlotinib treatment after CNS progression of gefitinib or icotinib in Cancer Hospital Chinese Academy of Medical Sciences from June 2012 to July 2016.

Result:
In 29 eligible patients, the objective response and disease control rates of erlotinib for intracranial lesions (ICLs) were 10.3% and 86.2%, and for extracranial lesions (ECLs) were 0% and 93.1%, respectively. The median progression-free survival (PFS) of patients treated with erlotinib was 6.28 months (HR 1.729, 95% CI 2.887–9.663) and the median overall survival (OS) was 11.73 months (HR 2.394, 95% CI 7.036–16.422). Synchronous ICLs and ECLs progression in initial EGFR-TKIs treatment (HR 2.467, 95% CI 1.029-5.915, P=0.043) was found to be an independent adverse prognostic factor for PFS of erlotinib. The patients who received ≥ 2 lines of treatment before erlotinib had a poorer PFS (HR 3.340, 95% CI 1.369-8.152, P=0.008) and OS (HR 2.563, 95% CI 1.025-6.412, P=0.044). The median intracranial progression-free survival (iPFS) for initial EGFR-TKIs was 14.22 months (range, 0.56-35.12 months) and showed no significant difference in PFS and OS of erlotinib re-treatment in subgroup analyses. As for safety profile, the most common adverse events of erlotinib were hematological (44.8%), gastrointestinal reaction (20.7%) and rash (37.9%) in grade 1/2.

Conclusion:
Erlotinib can be used when intracranial tumor progression occurs during gefitinib or icotinib treatment. The progressive pattern of initial EGFR-TKIs and multiple prior treatments may influence the survival of patients with erlotinib re-treatment. Prospective studies are needed to confirm these results.

Background:
Maintenance therapy with full-dose erlotinib for patients with advanced non-small cell lung cancer (NSCLC) has demonstrated a significant overall survival (OS) benefit. However, 150 mg/day of erlotinib seems too toxic as maintenance therapy. This study aimed to evaluate the efficacy and safety of low-dose erlotinib (25 mg/day) as maintenance treatment after platinum doublet chemotherapy in NSCLC harboring epidermal growth factor receptor (EGFR) mutation.

Method:
Activated EGFR-mutation-positive NSCLC patients who did not progress after first-line platinum-doublet chemotherapy, ≥20 and ≤85 years old, with performance status (PS) 0–3 were included in this study. Low-dose erlotinib (25 mg/day) was administered until disease progression. The primary endpoint was overall response rate (ORR). Secondary endpoints included progression-free survival (PFS), OS, and safety. The required sample size was 40 patients.

Result:
The study was stopped early, after achieving only 28% of planned enrollment, due to poor accrual. Between April 2011 and May 2014, 11 patients (male/female, 5/6; median age, 72 years; PS 0/1, 8/3; stage IV/relapse after surgery, 9/2; exon 19 deletions/L858R, 7/4) were enrolled and accessible in this study. Partial response (PR) was observed in 6 patients (56%). Median PFS was 14.9 months [95% confidence interval (CI), 2.7–27.1 months] and median OS was 40.6 months [95% CI, 24.7-56.5 months] (Figure 1). Toxicities were generally mild. Only one patient developed grade 3 aspartate aminotransferase (AST)/alanine aminotransferase (ALT) elevation. Eight patients developed grade 1 skin rash. No treatment-related deaths were observed. Ten patients progressed, and recurrences included brain metastases (n=3), pulmonary metastasis (n=3), local recurrence (n=2), local recurrence plus brain metastasis (n=1), and bone metastasis (n=1). Figure 1



Conclusion:
The study was stopped early due to poor accrual. However, our study suggests that maintenance therapy with low-dose erlotinib might be useful and tolerable in selected NSCLC patients harboring EGFR mutation.

Background:
Lung cancer is the fourth most common cancer in Brazil with 28,220 new cases in 2017. It is the main cause of cancer-related deaths with 23,393 deaths in 2013. In the 2000s, better understanding of molecular pathways led to the development of targeted treatments. The introduction of EGFR tyrosine kinase inhibitors (TKI) led to significant improvements in Response Rate and Progression-Free Survival for patients with activating mutations. Nevertheless, this treatment is not available in the Brazilian Public Health System based upon its costs andthe absence of Overall Survival gain in randomized clinical trials. The aim of this study was to assess the potential number of life-years lost and the cost associated with lack of treatment.

Method:
We estimated the number of eligible cases for treatment using epidemiological data from the National Cancer Institute (INCA) plus the national database on the frequency of EGFR gene mutations since July 2010 (gefitinib approval in Brazil). We based the differences in survival between patients treated with EGFR TKIs and chemotherapy using the curves of The Lung Cancer Mutation Consortium. The costs of TKI treatment were based on the national reference ($1,200 monthly) and was compared with the amount reimbursed by the Brazilian Public Health System for chemotherapy ($350 monthly).

Result:
The number of eligible cases for EGFR TKIs in the Brazilian Public Health System is around 2,224 patients each year. Since gefitinib approval, the estimated number of years of life lost due to the lack of access to EGFR TKIs was 2,668 annually. Considering only drug acquisition costs, we need nearly 150 million dollars to incorporate TKIs into the public health care system. The cost per incremental life-year gained over chemotherapy was 585 dollars. Although our analysis does not consider quality-of-life, the cost of one life-year gain is lower than three times the Brazilian GDP per capita (approximately 35,000 dollars).

Conclusion:
The lack of access to EGFR TKIs cost more than 18,676 years of live in Brazil in the past 7 years. Treatment would also be cost-effective.

Background:
Skin rash is the most common adverse event induced by epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). The efficacy of tetracycline for EGFR-TKI-related skin rash has been reported. However, a skin rash is often observed despite the use of tetracycline. Some studies have reported that skin rash is caused by a type of inflammation. Hence, there is a possibility that loxoprofen, a non-steroidal anti-inflammatory drug, can prevent these skin rashes.

Method:
We conducted a single-center, retrospective study to investigate the efficacy of loxoprofen for EGFR-TKI-related skin rash. The patients had non-small cell lung cancer and received EGFR-TKIs at the Chemotherapy Research Institute, Kaken Hospital from October 2011 to March 2017. We divided them into two groups: those who received EGFR-TKIs along with loxoprofen (loxoprofen (+) group; n = 12) and without loxoprofen (loxoprofen (−) group; n = 37), and investigated the incidence of EGFR-TKI-related skin rash.

Result:
There was no significant difference in the baseline characteristics between the groups. Grade 1 and 2 EGFR-TKI-related skin rash were more common in the loxoprofen (−) group than in the loxoprofen (+) group (grade 1; 90% versus 60%, P = 0.007, grade 2; 50% versus 0% P = 0.043, log-rank analysis). Multiple regression analysis indicated that the use of loxoprofen was a predictive factor that reduced the incidence of grade 1 skin rash (P = 0.0046). Figure 1



Conclusion:
Our study showed that loxoprofen combined with EGFR-TKIs could prevent skin rash, decreasing the risk by more than 65%. Our results suggest that loxoprofen can prevent and treat EGFR-TKI-related skin rash. Thus, we conclude that loxoprofen could be a new treatment option for EGFR-TKI-related skin rash.

Background:
Erlotinib (ERL) is modestly active to non-small cell lung cancer (NSCLC) with wild type epidermal growth factor receptor (EGFR). We hypothesized that an intermittent delivery of erlotinib and docetaxel (DOC) would increase efficacy.

Method:
This was a multi-center, single-arm phase I/II study in patients with wild type EGFR NSCLC who failed one prior chemotherapy. The phase I was designed a standard 3+3 dose escalation design to determine feasibility, the maximum tolerated dose (MTD) and phase II recommend dose (RD) of ERL on days 2 to 16, in combination with a fixed dose of 60mg/m[2] DOC on day 1. The phase II primary endpoint was objective response rate (ORR) by independent review committee. This study required 41 patients with expected ORR of 30% and threshold ORR of 10% (one-sided α= 0.025; β=0.1). The target number was 45 patients assuming the loss of follow-up cases. All eligible patients had ECOG performance status of 0/1 and adequate organ functions.

Result:
Between Mar 2009 and Dec 2010, 12 patients were enrolled in the phase I, and between May 2011 and Feb 2015, 46 patients in the phase II. Five patients were excluded from per protocol set, because of deviation of entry criteria. Planned dose escalation was completed without reaching a MTD. The RD was determined as 150 mg/dose of ERL. In the phase II, the ORR was 17.1% (95%CI, 7.2-32.1). The median progression free survival and median overall survival were 3.48 months (95%CI, 3.06-4.50) and 11.27 months (95%CI, 8.61-16.56), respectively. Gender, smoking status, or concomitant drugs which influence the ERL metabolism had no significant differences in ORR, or disease control rate. All 46 patients were evaluable for toxicity. The grade 3 non-hematological toxicities included 9 (19.6%) febrile neutropenia, 7 (15.2%) appetite loss, 3 (6.5%) oral mucositis and 3 (6.5%) infections. The grade 4 hematological toxicities were 31 (67.4%) neutropenia. Two treatment related deaths were observed; interstitial lung disease, and pleural infection.

Conclusion:
Intermittent dosing of ERL plus DOC is clinically feasible, but has no statistically significant improvement of ORR, in patients with recurrent NSCLC with wild type EGFR.

Background:
It is important to clarification of mechanisms of acquired resistance for epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) to determine the next treatment. EGFR T790M and hepatocyte growth factor (HGF) over expression has been observed in 50 to 60% of lung cancer patients who acquired resistance to the first generation EGFR-TKIs. However mechanisms of acquired resistance for the second generation EGFR-TKI, afatinib have not be clear.

Method:
We analyzed plasma T790M and HGF in twenty lung adenocarcimona patients treated with afatinib. Seven patients treated with afatinib as the first EGFR-TKI treatment and thirteen patients treated as EGFR-TKI re-challenge after acquired resistance for first generation EGFR-TKI. EGFR mutations in plasma DNA were detected using the mutation-biased PCR and quenched probe system. HGF level in plasma was measured by enzyme-linked immunosorbent assay.

Result:
In patients treated with afatinib as the first line treatment, no patients detected plasma T790M before afatinib treatment, but one of seven patients detected plasma T790M at the time of acquired resistance. Five of seven patients detected elevation of plasma HGF level when they had progressive disease. In patients treated with afatinib as EGFR-TKI re-challenge, ten of thirteen patients detected plasma T790M before afatinib treatment and nine of them had also T790M positive at the time of acquired resistance for afatinib. Two of three patients who had not detected plasma T790M before afatinib treatment become plasma T790M positive at the time of acquired resistance. Six patients who detected T790M treated with osimertiib and five of them demonstrated partial response (PR).

Conclusion:
T790M might be also major mechanism of acquired resistance in afatinib. Elevation of plasma HGF level might be detected more high frequency at the time of acquired resistance for afatinib than first generation EGFR-TKIs.

Background:
North East Japan Study Group (NEJ) 005/ Tokyo Cooperative Oncology Group (TCOG) 0902 study has demonstrated that first-line concurrent (C) and sequential alternating (S) combination therapies of EGFR tyrosine kinase inhibitor (gefitinib) plus platinum-based doublet chemotherapy (carboplatin/pemetrexed) offer promising efficacy with predictable toxicities for patients with EGFR-mutant NSCLC (ASCO2014, Ann Oncol 2015). However, overall survival (OS) data were insufficient because of the lack of death events in the primary report.

Method:
Progression-free survival (PFS) and OS were re-evaluated at the final data cutoff point (March 2017) for the entire population (N = 80).

Result:
At the median follow-up time of 35.6 months, 88.8% of patients had progressive disease and 77.5% of patients had died. Median PFS was 17.5 months for the C regimen and 15.3 months for the S regimen (p = 0.13). Median OS time was 41.9 with the C regimen and 30.7 months with the S regimen (p = 0.036). Updated response rates were similar in both groups (90.2% and 82.1%, respectively; p = 0.34). Patients who had common mutations showed no significant differences in PFS according to type of mutation. Patients with Del19 displayed relatively better OS (median: 45.3 and 33.3 months for C and S regimens) than those with L858R (31.4 and 28.9 months). No severe adverse events including interstitial lung disease have occurred during the follow-up period since the primary report. In an exploratory analysis, there was no significant difference in post progression survival and overall survival between patients with progression of target or non-target lesions and those progressed with new lesions.

Conclusion:
This updated analysis has confirmed that PFS is improved with first-line combination therapies compared to that with gefitinib monotherapy, and the C regimen in particular offers an overall survival benefit of 42 months in the EGFR-mutated setting. Our on-going NEJ009 study will clarify whether this combinational strategy can be incorporated into routine clinical practice.

Background:
T790M mutation test by the approved in vitro diagnostic agent (cobas v2.0) is essential for the use of osimertinib and opportunities for re-biopsy are increasing.

Method:
Success rate and T790M mutation detection rate were retrospectively investigated in 22 cases of 18 patients who took resistance to the 1st and 2nd generation EGFR-TKI at the Saga University Medical School Hospital and underwent re-biopsy. T790M with plasma DNA was examined by MBP-QP, a highly sensitive detection system developed in our laboratory, and cobas v2.0.

Result:
All patients were adenocarcinoma and the mutation status was 8 patients of del 19 and 10 patients of L858R. Re-biopsy sites were 7 bones, 4 primary sites, 3 cases of liver, pleura, lymph nodes, and 2 other sites, respectively, and surgical excision was 31.8%, median time to biopsy from informed consent was 10.5 days (range:1-39). The success rate was 95.5% and the T790M detection rate by cobas v2.0 was 52.3% for all cases, and there was no significant difference between 55.6% for del 19 and 50.0% for L858R. The plasma T790M detection rate using MBP-QP collected at the same time was 65.0% for all cases and there was no significant difference between 55.6% for del 19 and 72.7% for L858R. We experienced one case of atypical angina as a serious complication. Case presentation: ≪No.1≫ First biopsy as re-biopsy (bone): cobas method was negative and MBP-QP method was positive and could not be administered with osimertinib. Second biopsy as re-biopsy (liver): both cobas method and MBP-QP method were positive and could be administered with osimertinib and had a remarkable response. ≪No.2≫ Axillary lymph node biopsies were performed twice, but cobas method was negative and MBP-QP method was positive, and osimertinib could not be administered. Thereafter, hepatic metastasis was biopsied, but both cobas method and MBP-QP method were negative. When plasma specimens by cobas method were tested after insurance approval, plasma T790M was positive, and osimertinib could be finally administered. Although it was effective for axillary lymph node metastases, it had no effect on liver metastases.

Conclusion:
The sensitivity of the test method and the tumor heterogeneity between individuals may influence the detection of T790M mutation. In order to reliably administer osimertinib to patients with indication, it is desirable to establish an appropriate time and site for re-biopsy, and establish examination methods.

Background:
A significant subgroup of non-small cell lung cancers harbor epidermal growth factor receptor (EGFR) mutations. Third-generation EGFR tyrosine kinase inhibitors (EGFR-TKIs) were developed to overcome EGFR T790M-mediated resistance to first- and second-generation EGFR-TKIs. Third-generation EGFR-TKIs, such as osimertinib and nazartinib, are effective for patients with EGFR T790M mutation. However, there are no direct comparison data to guide the selection of a third-generation EGFR-TKI for patients with different EGFR mutations. We previously established an in vitro model to estimate the therapeutic windows of EGFR-TKIs by comparing their relative efficacies against cells expressing mutant or wild type EGFRs. The present study used this approach to characterize the efficacies of third-generation EGFR-TKIs and compare them with those of other EGFR-TKIs such as erlotinib and afatinib.

Method:
We evaluated the drug sensitivity and EGFR downstream signals of human lung cancer cell lines (PC9, H3255, H1975, PC9ER, BID007) and Ba/F3 cells harboring clinically relevant EGFR mutants for first (erlotinib), second (afatinib) and third (osimertinib and nazartinib) generation EGFR-TKIs with MTS assay and western blotting. An in vitro model of mutation specificity was created by calculating the ratio of IC50 values between mutated and wild type EGFR.

Result:
Among various mutation types, sensitivities to EGFR-TKI were different. For classic EGFR mutations, exon 19 deletion and L858R, with or without T790M osimertinib showed lower IC50 values and wider therapeutic windows than nazartinib. Afatinib, osimertinib and nazartinib inhibited the phosphorylation of EGFR, AKT, and ERK for human cell lines and Ba/F3 cells harboring these EGFR mutations. For uncommon EGFR mutations, G719S or L861Q, afatinib showed lowest IC50 values. For G719S+T790M or L861Q+T790M, the IC50 values of osimertinib and nazartinib were around 100 nM, 10 to 100 fold higher than those of classic+T790M mutations. On the other hand, osimertinib and nazartinib showed similar efficacies in cells expressing EGFR exon 20 insertions. For C797S mutations, no EGFR-TKIs demonstrated efficacy.

Conclusion:
The present study provides fundamental osimertinib and nazartinib sensitivity/resistance data in cells expressing a range of EGFR mutations, including uncommon EGFR mutations. The findings highlight the diverse mutation selective sensitivity pattern of EGFR-TKIs. These data may help to inform the selection of EGFR-TKIs for non-small cell lung cancer patients harboring EGFR mutations.

Background:
Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) is the standard therapy for advanced lung adenocarcinomas with common EGFR mutations. However, the efficacy of EGFR-TKIs in patients with these uncommon EGFR mutations (other than exon 19 deletions or exon 21 L858R mutation) remains undetermined.

Method:
Seven hundred and fifty-five non-small cell lung cancer (NSCLC) patients with EGFR mutation analyses for TKI therapy were identified between October 2010 and December 2015 in East of China. And 66 patients bearing uncommon EGFR mutations were included to collect data from TKI response and prognosis.

Result:
Rare sensitive mutations (G719X, L861Q, S768I), primary resistant mutation (Ex20 ins), and complex mutations (G719X + L861Q, G719X+S768I, 19 del+T790M, 19 del+L858R, L858R+S768I, and L858R+T790M) of EGFR were identified in 37 (56.1%), 9 (13.6%), and 20 (33.3%) patients, respectively. TKI treatment in patients harboring uncommon EGFR mutations exhibited a tumor response rate of 28.8% and a median progression-free survival (PFS) of 4.8 months. Importantly, patients with complex EGFR mutations had significantly longer PFS when compared with the remaining sensitizing rare mutations or Ex20 ins (8.6 versus 4.1 versus 3.1 months; p=0.041). Furthermore, complex EGFR mutations were independent predictors of increased overall survival in NSCLC patients (Hazard Ratios=0.31; 95% confidence intervals: 0.11-0.90; p=0.031). Among them, patients harboring Del-19 compound L858R mutations showed a tendency to have higher response rate and improved median PFS than those regarding patients with other complex mutations patterns (66.7% verse14.3%, p=0.021; 10.1 verse 8.6 months, p=0.232).

Conclusion:
Personalized treatment should be evolving in different types of uncommon EGFR mutations. And complex mutations of EGFR may benefit more from EGFR-TKIs than other uncommon mutations in Chinese NSCLC patients.

Background:
The efficacy and safety of afatinib therapy among elderly (aged 75 or older) populations diagnosed with EGFR-mutated non-small cell lung cancer (NSCLC) has not been evaluated yet. This phase I trial was conducted to determine the maximum tolerated dose (MTD), recommended phase II dose of afatinib in elderly patients with advanced NSCLC harboring EGFR mutations.

Method:
The study used a standard 3 + 3 dose escalation design. Patients aged 75 years or older advanced NSCLC harboring EGFR mutations were enrolled. Patients were required to have an Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 0-1 and adequate organ function. The doses of afatinib, which was given once daily, were planned as follows: level 1, 20 mg/body; level 2, 30 mg/body; level 3, 40 mg/body. Dose-limiting toxicity (DLT) was defined as grade 4 hematologic or grade 3 non-hematologic toxicity. DLT was evaluated during day1-28.

Result:
From February 2015 to October 2016, 15 patients were enrolled from 3 participating institutions. Patient characteristics were: male/female 3/12; median age 79 (range 75-87); Performance status 0/1 2/13. Six patients have been treated at level 1, 3 and three patients have been treated at level 2, respectively. At level 1, 1 of 6 patients showed DLTs. The patient grade 3 rush, grade 3 anorexia, and grade 3 infection was observed. At level 2, none of 3 patients experienced a DLT. At level 3, 2 to 6 patients were observed DLTs. One patient developed grade 3 diarrhea, another patient developed grade 3 diarrhea and grade 3 anorexia. Most frequent adverse events (AEs, any grade) were diarrhea, paronychia, rush, and nausea. Most patients at level 3 required dose reduction in three months. No treatment-related deaths were observed at either level. An objective response was 73.3%.

Conclusion:
We considered level 3 was the MTD and recommended phase II dose level was 3. Clinical trial information: UMIN000016441.

Background:
Central nervous system (CNS) is a common site of metastases of non-small cell lung cancer (NSCLC). The prognosis for patients with brain metastases and/or leptomeningeal metastases is extremely poor. NSCLC harboring epidermal growth factor receptor (EGFR) mutation generally shows good response to tyrosine kinase inhibitors (TKI). However, the efficacy of EGFR-TKI in patients with CNS metastases is unclear. And the data on the occurrence of leptomeningeal metastases in the patients with brain metastases after use or EGFR-TKI remain limited.

Method:
We retrospectively evaluated clinical outcome and background of EGFR mutant NSCLC patients with CNS metastases who received EGFR-TKI for the first line drug therapy between January 2008 and December 2014 in the facilities belong to Niigata lung cancer treatment group.

Result:
A total of 104 eligible patients were enrolled. The response rate was 62%. The median time to treatment failure was 7.8 months. The median survival time (MST) was 24.0 months. The response rate of CNS was 37%. The median CNS-progression free survival (PFS) was 13.2 months. There was no statistical significant difference in TTF, overall survival (OS) and CNS-PFS between patients with exon 19 deletion and those with exon 21 L858R point mutation (mTTF 8.3 vs. 7.8 months, MST 26.1 vs. 24.9 months, mCNS-PFS 14.4 vs. 12.4 months) or between patients treated by Gefitinib and those treated by Erlotinib (mTTF 8.4 vs. 6.3 months, MST 26.0 vs. 20.2 months, mCNS-PFS 13.8 vs. 13.2months). Brain radiotherapy prior to EGFR-TKI prolonged TTF (11.2 vs. 6.8 months) and tended to prolong CNS-PFS (15.6 vs. 11.1 months), but was not significantly associated with OS (MST 26.1 vs. 24.0 months). There was no significant difference in treatment outcome between patients who received stereotactic irradiation and those who received whole brain irradiation as brain radiotherapy prior to EGFR-TKI. Leptomeningeal metastases (LM) were primarily found in 8 of 104 patients (8%), and those occurred subsequently during the clinical course in 19 patients (18%). Median time to occurrence of LM in the patients who had LM subsequently was 14.5 months. There was no significant difference in OS between patients who had LM subsequently and those without LM during the course (MST 28.1 vs. 24.9 months). MST from diagnosis of subsequent LM was 3.7 months.

Conclusion:
EGFR-TKI showed favorable effect for EGFR mutant NSCLC patients with CNS metastases. A longer TTF and CNS-PFS were observed with prior brain radiotherapy. Prognosis after occurrence of LM was poor.

Background:
Osimertinib, a third-generation EGFR tyrosine kinase inhibitor targeting EGFR T790M acquired resistance mutation, has demonstrated strong clinical activity. Therefore, we have to do a second biopsy to detect T790M when the tumor has progressed. On the other hand, liquid based assays are expected as minimally invasive methods for detecting resistance mutations. Recently, liquid-biopsy for detecting EGFR T790M mutation has been approved by the Japanese ministry of health, labor and welfare. The aim of this study is to assess the clinical utility of liquid biopsy for detecting EGFR T790M mutation.

Method:
Seventeen consecutive patients who underwent cobas® EGFR Mutation Test ver.2 for liquid biopsy from January to April 2017 were enrolled. Patient information and examination results were collected from electronic medical records and analyzed retrospectively. Concordance between liquid biopsy and tissue or cytological specimen was assessed in patients who underwent re-biopsy at the same time as liquid biopsy.

Result:
Median age: 70 (51-82); Women: 10 (58.8%); Never smoker: 12 (70.6%); Adenocarcinoma: 17 (100%); Stage IV: 16 (94.1%); Primary EGFR mutation: exon19 deletion = 11 (64.7%), exon21 L858R = 5 (29.4%), exon18+20 mutations = 1 (5.9%). Two patients (11.8%) were positive for T790M mutation in plasma. Tissue biopsy or cytology was done in 8 cases at the same time as liquid biopsy. Only one patient of four patients who were positive for T790M in tissue or pleural effusion was positive for T790M in plasma (sensitivity: 25%). One patient of two patients who were positive for T790M in plasma was negative for T790M in spinal fluid.

Conclusion:
There is no doubt that liquid biopsy is a minimally invasive and very convenient method. However, at least currently, liquid biopsy cannot replace tissue biopsy in clinical setting because of its sensitivity. In order to deliver the appropriate medication to the patient, it is necessary to selectively use well the tissue biopsy and liquid biopsy.

Background:
As LUX-Lung8 has shown significant improvements in the progression-free survival and the overall survival with afatinib when compared with erlotinib, afatinib could be an additional option for the second-line treatment of patients with squamous cell lung cancer. The selection process of patients on the basis of EGFR mutations would likely result in a population with a greater sensitivity to afatinib. However squamous cell lung cancer is not routinely examined for EGFR mutation status because of the low frequency of positive results and the poor clinical response of squamous cell lung cancer with EGFR sensitive mutations to gefitinib, compared to that of adenocarcinoma. We reviewed clinical features of squamous cell lung cancer with EGFR mutations at our hospital and their responses to EGFR-tyrosine kinase inhibitors.

Method:
The medical records of this retrospective review were taken from patients with histological or cytological proven squamous cell lung cancer from April 2008 to May 2017 at NTT Medical Center Tokyo. The patients were tested for EGFR mutation status with PNA-LNA clamp method. The electronic medical records were reviewed to obtain clinical and demographic data, including gender, age, smoking history, clinical stage of lung cancer, treatment, and survival.　Based on the chest CT findings, the patients were categorized into three groups according to the underlying pulmonary disease; normal lungs, emphysematous lungs and fibrotic lungs. The differences among the categorized groups were then compared.

Result:
Of 441 patients with squamous cell lung cancer, 23 patients (5.2%) were tested EGFR mutation status. Of 23 patients, we identified 5 (21.7%) patients with an EGFR mutation (Exon 19 deletion 3, L858R 2). All patients with EGFR mutation were female and never-smokers. EGFR mutations were identified in 4 (44.4%) of 9 patients with normal lungs, 1(8.3%) of 12 with emphysema, and 0 (0%) of 2 with pulmonary fibrosis. Of the 5 patients with EGFR mutation, 2 patients received gefitinib and 2 patients received afatinib. Although the two patients treated with gefitinib did not respond to treatment (SD 1, PD 1), the two patients treated with afatinib responded well (PR 2).

Conclusion:
Squamous cell carcinoma patients with sensitive EGFR mutations had a prognosis comparable to patients with adenocarcinoma. Selecting an afatinib treatment for patients on the basis of the underlying pulmonary diseases(normal lungs) and the smoking status (never smoker) for the EGFR mutation test would likely result in a population with a greater sensitivity to afatinib.

Background:
The identification of specific molecular drivers of pathogenesis is now crucial for treatment with targeted therapies in NSCLC. It is reported that ALK-rearrangement shows an intratumor heterogeneity in mixed adenocarcinomas and adenosquamous carcinomas, while EGFR-mutation is generally homogeneously expressed. Because of this intratumor heterogeneity, there is concern about underestimation of molecular markers in biopsy. Thus, we compared the diagnostic yields of fine-needle aspiration (FNA), core-needle biopsy (CNB), and surgical resection sample for EGFR-mutation and ALK-rearrangement.

Method:
This retrospective study was approved by institutional review boards, and informed consent was waived. Diagnostic yields of EGFR-mutation pyrosequencing tests and ALK-rearrangement FISH studies performed during a 6 year period (Jan 2010 - Dec 2016) were investigated stratified by the 3 tissue sampling methods: FNA, CNB and surgical resection. The diagnostic yields of positive results of EGFR-mutation and ALK-rearrangement tests were compared according to the tissue sampling methods.

Result:
Among the 1617 EGFR-mutation pyrosequencing tests, the number of surgical resection sample, CNB, and FNA was 996, 262, and 359, respectively, and the positive results were 41.7% (95% confidence interval 38.6–44.8), 40.8% (35.1–46.9), 38.2% (33.3–43.2), respectively. On the other hand, in a total of 221 ALK-rearrangement FISH studies, positive results of surgical resection sample (n = 60), CNB (n = 87), FNA (n = 74) were 45% (95% confidence interval 33.1–57.5), 44.8% (34.8–55.2), 31.1% (21.7–42.3), respectively.

Conclusion:
Diagnostic yields of FNA, CNB and surgical resection samples were not significantly different in EGFR-mutation pyrosequencing tests, and this probably reflects known intratumor homogeneity of EGFR-mutation. In the ALK-rearrangement FISH study, the diagnostic yield of FNA was lower than that of surgical resection or CNB samples. This difference is presumably due to intratumor heterogeneity, and caution should be made as it may cause underestimation of ALK-rearrangement in biopsy samples.

Background:
This study points out an issue of PCR-based methods to detect exon 19 deletions. PCR methods are used for clinical examination, because they are useful, rapid and cost-effective to detect EGFR mutations. Exon 19 deletions are most important among EGFR mutations to dictate EGFR tyrosine kinase inhibitors (EGFR-TKIs) therapy in non-small cell lung cancer (NSCLC), and have various species. The sensitive PCR methods select major exon 19 deletions, but cannot detect unusual variants. We investigated the clinical significance of minority exon 19 deletions.

Method:
A series of 73 NSCLC patients, which were treated with EGFR-TKI for recurrent disease after they had undergone surgery from 1992 to 2004. In all cases, Microdissenction and direct sequence were performed. Scorpion Amplification Refractory Mutation System (ARMS) and cobas version 2.0, as sensitive PCR methods, were performed in 47 patients along with sizing capillary electrophoresis for the exhaustive detection of exon 19 deletions.

Result:
EGFR mutations were detected from 35 patients (47.9%), which were one exon 18 mutation, 23 exon 19 deletions, and 11 exon 21 mutations in all 73 cases. The catalog of somatic mutation in cancer (COSMIC) database included 174 different exon 19 deletions in 4190 submitted cases at December 2014. E746_A750 deletion was most common deletion of exon 19, accounting for 70% of the total exon 19 deletions (2931/4190). Predicted frequency of exon 19 tested by Scorpion-ARMS was 81.6% (3419/4190), and that of cobas version 2.0 had 82.5% (3457/4190) in the detection of various exon 19 deletions. In clinical samples of this study, Scorpion-ARMS and cobas version 2.0 failed to detect four exon 19 deletions, in two squamous cell carcinomas (EGFR-TKI effects were stable disease and no assessable patient) and two papillary adenocarcinomas (EGFR-TKI effects were stable disease and no assessable patient). One of papillary adenocarcinoma had minority deletion ‘T751_I759 deletion and insertion S’, which had long stable disease for 43.4 months in EGFR-TKI therapy. Sizing capillary electrophoresis was able to detect this deletion.

Conclusion:
This study suggests sizing capillary electrophoresis is necessary for the exhaustive detection of exon 19 deletions, and may identify tumors responsive to EGFR-TKIs therapy, especially those with unusual deletions.

Background:
Although targeted therapy and immuno-oncology have shifted paradigm in treating lung cancer, platinum-based combination is still the standard of care for advanced lung cancer patients, especially for those without epidermal growth factor receptor (EGFR) mutation or anaplastic lymphoma kinase translocation. Based on the JMDB study conducted by Scagliotti et al., pemetrexed/platinum combination is a preferred treatment for nonsquamous non-small cell lung cancer (NSCLC). However, which chemotherapeutic regimen is better for patients with EGFR-negative nonsquamous NSCLC remains unclear. Thus, the object of this study was to compare pemetrexed-based chemotherapy to non-pemetrexed-based chemotherapy for EGFR-negative advanced nonsquamous NSCLC.

Method:
A total of 183 patients with EGFR-negative nonsquamous NSCLC who were treated with platinum-based doublet between January 2009 and December 2016 were retrospectively enrolled for this study. Progression-free survival (PFS) and overall survival (OS) of different treatment regimens were analyzed and compared.

Result:
Drugs combined with platinum as first-line chemotherapy were as follows: pemetrexed (n = 97, 53%), gemcitabine (n = 52, 28%), paclitaxel (n = 24, 13%), and docetaxel (n = 10, 6%). Among 97 patients in the pemetrexed group, 50 (52%) received continuation maintenance chemotherapy with pemetrexed after 4 cycles of combination. In the non-pemetrexed group, 7 (8%) patients received switch maintenance chemotherapy with erlotinib (n = 5) or pemetrexed (n = 2). Median PFS and OS of all patients were 5.1 months (95% confidence interval [CI]: 3.8-5.9 months) and 15.3 months (95% CI: 8.9-18.4 months), respectively. Median PFS was significantly higher in the pemetrexed group compared to that in the non-pemetrexed group (7.2 vs 4.1 months, p < 0.05). In addition, median OS showed an increasing tendency in the pemetrexed group compared to that in the non-pemetrexed group but it was not statistically significant (19.5 vs 14.3 months, p = 0.06). Multivariate analyses showed that the use of pemetrexed-based regimen was independently associated with better PFS, but not with better OS.

Conclusion:
Although sample size was relatively small, our data suggest that pemetrexed-based treatment was more beneficial compared to non-pemetrexed based treatment for EGFR-negative advanced nonsquamous NSCLC. Further large-scale studies are needed to confirm our findings.

Background:
Despite the high response rate in patients with EGFR-mutation positive NSCLC, treatment with EGFR-TKIs is not curative and eventually there is disease progression. In patients with acquired resistance to 1[st] generation EGFR-TKIs, previous studies have demonstrated that afatinib had some clinical activity. We previously reported that the combination of gefitinib, pemetrexed and carboplatin showed promising antitumor efficacies in EGFR-mutated lung cancer patients. In this phase I trial, we assessed the safety and efficacy of afatinib combined with pemetrexed and carboplatin in NSCLC patients who acquired resistance.

Method:
Patients with EGFR-mutation positive metastatic NSCLC, who had received 1[st] line gefitinib or erlotinib and developed disease progression were eligible. Patients received pemetrexed 500 mg/m[2] and carboplatin AUC=5 on day 1 in all cohorts, and afatinib at doses of 20, 30 and 40 mg/body from day 8 to 18 of 21-day cycle. DLT was assessed after the first cycle, and doses were escalated in cohorts of 3 to 6 patients.

Result:
Eleven patients were enrolled to this trial and 9 patients were evaluable for safety and efficacy. At an afatinib dose of 30mg/body, 3 patients experienced DLT (grade 3 diarrhea, grade 3 hypokalemia, grade 4 thrombocytopenia, grade 3 amylase elevation and grade 3 gamma-glutamyl transferase). The overall response rate was 20% (95% C.I. 5.7 to 51) and median progression free survival was 16.2 months (95% C.I. 4.7 to not reached).

Conclusion:
The MTD of afatinib is 20mg/body in combination with pemetrexed 500 mg/m[2] and carboplatin AUC=5 on day 1 every 21 days. This combination demonstrated activity in EGFR mutation positive NSCLC with acquired resistance to 1[st] line EGFR-TKIs.

Background:
Anti-angiogenic agents have been reported to have clinical activity EGFR mutant NSCLC with/without EGFR Tyrosine kinase inhibitor (TKI). We reported clinical outcomes of nintedanib plus docetaxel in refractory NSCLC according to EGFR mutation status during a Korean nintedanib NPU program.

Method:
Patients with NSCLC were eligible if they failed at least one prior systemic treatment. Docetaxel was administered either 75mg/m[2] or 37.5mg/m[2] on D1, D8 q every 3weeks plus nintedanib 200mg orally twice daily. Nintedanib treatment was continued until disease progression or unacceptable toxicity after 4-6 cycles of combination therapy.

Result:
62 patients were enrolled. 28 patients with activating EGFR mutation (17 in exon19 deletion, 8 exon21 L858R/L861Q, 1 exon 18 G719X, 1 in exon20 duplication, 1 in exon19 deletion and exon20 T790M) progressed after EGFR-TKI, 25/28 patient also progressed after platinum doublet chemotherapy were enrolled. Only for 2 patients EGFR mutation status were unknown. Patients were heavily pretreated, with 38.7% patients receiving nintedanib plus docetaxel as ≥ 4[th] line therapy. 5 patients had received bevacizumab. For patients with response assessment reported objective response rate was 30.6% and median PFS and OS were 3.9 months (95% CI 3.4-4.4) and 11.7months (95% CI 5.2-18.1) respectively in overall patients. Based on EGFR mutation status, objective response rate was 52.1% vs 24.1% (EGFR mut(+) vs EGFR mut (-), p=0.47) and median PFS was 5.9 vs 3.6 months (EGFR mt(+) vs EGFR mt(-), p=0.031). No treatment related death was reported. Common grade 3/4 adverse event were neutropenia (33.8 %), and reversible elevated liver enzyme(9.7%). 10 patients in 150mg twice daily and 2 patients with 100mg twice daily administered grade 3 adverse events. Figure 1



Conclusion:
Nintedanib plus docetaxel was well-tolerated and had clinical activity in refractory NSCLC. Specifically, EGFR TKI resistant EGFR mutant NSCLC may be a good candidate for nintedanib plus docetaxel.

Background:
From October 2013 there is a possibility to treat patients with NSCLC and with activated epidermal growth factor receptor (EGFR) mutations with three TKI (afatinib, erlotinib, gefitinib) in the Czech Republic. We have tried to find differences among patient groups treated with single TKI in 1st line of treatment.

Method:
Only patients with EGFR mutations and in which 1st line treatment started in October 2013 and later were included in analysis. With respect to defined inclusion criteria. We analysed 287 patients (gefitinib - 138 patients, afatinib - 102 patients, erlotinib - 40 patients.). Descriptive statistics and frequency tables were used to characterize the sample data set. Statistical significance of differences among three TKI inhibitors subgroups was assessed using the Fisher’s exact test or Kruskal-Wallis test for continuous variables. Overall survival (OS) was defined as the time from 1st line TKI inhibitor treatment initiation to the date of death due to any cause. Progression-free survival (PFS) was defined as the time from 1st line TKI inhibitor treatment initiation to the date of first documented progression or death due to any cause. OS and PFS were estimated using Kaplan-Meier method and all point estimates include 95% confidence intervals (95% CI). All statistical tests were performed at a significance level of α=0.05.

Result:
Between these three groups of patients there was no statistically significant difference in sex (p=0.972), in age (p=0.031), in smoking habits (p=0.877), in type of EGFR mutation (p=0.437), in adenocarcinoma proportion (p=0.07). Between these three groups, there was statistically significant difference according to performance status (< 0.001), patients treated with afatinib have better PS in common. Between these three groups, there was no statistically significant difference according to disease control (CR+PR+SD) (p=0.626) and in response to treatment (CR + PR) (p = 0.791). There was no statistically significant difference in PFS survival (p=0,015) and in overall survival ((p=0.046). There was no statistically significant difference in the occurrence of adverse events (p=0.002).

Conclusion:
We have not found any important difference in basic characteristic of patients treated in 1st line treatment with TKI (sex, age, smoking, histology and type of EGFR mutations). We have not found any important difference in response to treatment, in disease control, PFS, in overall survival and in occurrence of adverse events. We found a significant difference in PS segmentation at treatment initiation – patients treated with afatinib have better PS than others.

Background:
Administering the best treatment after acquiring resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) requires the knowledge of the resistance status. In this trial, the treatment efficacy of osimertinib (AZD9291) was assessed in patients with non-small-cell lung carcinoma (NSCLC) harboring T790M resistance mutation, which was detected in the circulating tumor DNA (CtDNA) without re-biopsy of the tumor tissue.

Method:
To prove 60% response rate of osimertinib compared to 30% as null hypothesis, and considering 10% drop out rate, 19 subjects were recruited. To extract CtDNA, 15 mL of peripheral blood was withdrawn and centrifuged immediately before storage. Cobas® v2 RUO (Roche diagnostics) and PANA mutyper® (Pangene, Korea) were used to detect the EGFR mutations from CtDNA. Osimertinib was prescribed as an 80 mg tablet once in a day irrespective of the food intake.

Result:
Eighty patients with acquired resistance to prior EGFR TKIs were screened for T790M resistance mutation, and the CtDNA of 21 subjects (26.3%) showed T790M mutation. T790M mutation was detected by both PANA mutyper® and Cobas® in 13 cases, T790M was detected only by PANA mutyper® in 4 cases, and only by Cobas® in 4 cases. Nineteen subjects (age: 64.4 ± 11.6 years old, 14 women, 5 men) were enrolled in this prospective single arm trial from September 2016 to April 2017. Prior EGFR TKIs were afatinib (n=3), erlotinib (n=4), gefitinib (n=10), erlotinib and afatinib (n=1), and gefitinib and afatinib (n=1). Twelve subjects had exon 19 deletion of EGFR gene, 4 had L858R point mutation, one showed exon 19 deletion and L858R, 1 had G719X, and 1 case showed no activating mutation. The response to osimertinib was evaluable in 15 subjects; 4 subjects dropped out from this trial before response evaluation. Among the 15 subjects (efficacy analysis set), partial remission was observed in 10 cases (66.7%).

Conclusion:
Assuming 40% of screened patients are harboring T790M mutation, sensitivity of CtDNA testing is 65% using both tests, and 53% with either test. Toxicity and survival analyses will be followed. (ClinicalTrials.gov Identifier: NCT02769286)

Background:
In patients with EGFR-mutant non-small-cell lung cancer (NSCLC), progression inevitably occurs after 9 to 14 months of treatment with EGFR tyrosine kinase inhibitors (TKIs). EGFR T790M mutations have been identified as the most common mechanism of acquired resistance. Analyses assessing the frequency of acquired T790M mutations have mainly been conducted in patients receiving the first-generation EGFR TKIs erlotinib and gefitinib, however limited data is available on the prevalence of this mutation after failure of the ErbB family blocker afatinib. This retrospective analysis aimed at determining the prevalence of EGFR T790M mutation in patients who had benefitted from afatinib therapy, but ultimately developed progression. Another objective was the assessment of response to the subsequent treatment with the third-generation EGFR TKI osimertinib, which is the treatment of choice for patients who have developed T790M mutations.

Method:
The analysis included consecutive patients with stage IV adenocarcinoma of the lung and sensitizing EGFR mutations who had progressed on first-, second- or third-line afatinib treatment after experiencing at least 3 months of disease stabilization. Mutation status was assessed using liquid biopsy or both liquid biopsy and tissue re-biopsy. Patients with confirmed T790M mutation received osimertinib.

Result:
T790M mutations were found in 27 of 48 patients, corresponding to a prevalence rate of 56.3%. The concordance rate between liquid biopsy and re-biopsy was 80%. In the total cohort, the objective response rate (ORR) obtained with afatinib was 89.6%, and in the patients who developed T790M mutation, 92.6%. Complete responses (CR) occurred in 25.0% and 37.0%, respectively, and partial responses (PR) in 64.6% and 55.6%, respectively. In the patients who received osimertinib after the discovery of T790M mutations, ORR was 81.5%, with CR and PR rates of 22.2% and 59.3%, respectively.

Conclusion:
The prevalence of acquired T790M mutations as assessed in this cohort was consistent with the mutation rates reported for patients who progressed on first-generation EGFR TKI treatment. T790M mutation appears to be the main mechanisms of acquired resistance to afatinib. The development of this mutation was not affected by any baseline characteristics. These real-world data confirm that for patients with advanced, EGFR-positive NSCLC who progressed on afatinib and developed T790M mutations, osimertinib therapy elicited excellent response rates, with a substantial proportion of patients achieving complete remissions.

Background:
Acquired Resistance to EGFR-TKI is inevitable to occur to most of NSCLC treated by first generation TKI such as Gefitinib or Erlotinib. There have been many secondary resistance mutations discovered to date including, L747S, D761Y, T790M and T854A . But in our series, the most common secondary resistance mutations was T790M type and was studied in details as to the occurrence, the treatment and the response to the treatment.

Method:
42 patients from our own series of advanced Non-small cell lung cancer(NSCLC) , were identified as having EGFR mutations for which TKI was used for the treatment initially. Resistance to first generation TKI was found due to Mutation T790M in exon 20 of EGFR gene . A total of 21 patients 50%(from 42) harbored T790M mutation. Interestingly enough, only 5 cases from classical mutated exon19 EGFR ( 5/22) , 6 cases from classical mutated exon 21 L858R (6/7) and 10 cases from non –classical mutated exon 19 and 21(10/13). The treatment of those who harbored T790M was uniformly given: 1. Multi-targeted method , Bevacizumab and Afatinib second generation TKI 2.Osimertinib, third generation TKI to all patients when failed Afatinib.

Result:
At one year time, all 21 patients survived the treatment. Beyond one year with closed follow-up, the results showed, 1. All 5 cases from classical mutated exon 19 and 6 cases from mutated exon 21 survived the disease and continued the treatment with Osimertinib. 2. 1 case from non-classical mutated exon 19 and 21 survived The overall success rate was 5+6+1 = 12/ 21 = 57% All 9 cases who failed the treatment showed progression of cancer .

Conclusion:
Patients who harboured T790M mutation from classical mutated exon 19 and exon 21 EGFR continued to respond to the treatment very well clinically. Obviously, Osimertinib was specific for T790M mutation and no further acquired resistance mutation in their cancer cells. But for those who failed Osimertinib treatment they must have additional acquired resistance mutation along with T790M. Future Plan : Consider Immune Checkpoint Treatment

Background:
Toxicity analysis in randomized studies is classically reported as maximum grade toxicity occurring during the course of treatment. Unfortunately, the duration of toxicity is neglected. Patients receiving targeted therapy like gefitinib frequently have low grade continuous toxicities. Longitudinal toxicity analysis may be a more appropriate method of quantifying and comparing toxicity.

Method:
EGFR mutated NSCLC patients treated with gefitinib or pemetrexed-carboplatin as a part of randomized study were selected for this analysis. Patients were evaluated on the 7th day after the start of therapy and then on days 15, 21, 42, 63, 84,105 and then subsequently every 2 months till progression. Toxicities in accordance with CTCAE version 4.02 occurring during each visit were documented at each visit. Three toxicities were selected for this analysis and these were diarrhea, skin rash and fatigue. R software was used for analysis. Maximum grade toxicity and longitudinal time -toxicity analysis was performed. Toxicities were compared between the 2 arms using both methods.

Result:
Among the 290 patients, half each were randomized to gefitinib and pemetrexed-carboplatin arm respectively. Any grade diarrhea was seen in 22 % of patients receiving gefitinib as opposed to 12% receiving pemetrexed-platinum (p-0.12%). Similar higher proportion of toxicity was seen in gefitinib arm for skin rash (33% versus 13%, p-0.00).The proportion of fatigue in gefitinib and pemetrexed -platinum arm were similar (6% versus 9%, p-0.59). The longitudinal time-toxicity analysis revealed that both rash and diarrhea were higher and more sustained in gefitinib arm. The difference area under the curve for rash and diarrhea were 10 units (p-0.192) and 21.89 units (p-0.09) respectively. The fatigue was similar in both arms (p-0.779)

Conclusion:
Longitudinal time -toxicity analysis provides information which is clinically relevant and might impact patients quality of life and hence should be performed in patients receiving targeted therapies.

Background:
AST2818 (Alflutinib) was designed to inhibit EGFR active mutations as well as the T790M acquired resistant mutation. The purpose of this study was to determine the safety and antitumor activity of AST2818 in EGFR T790M positive non-small cell lung cancer (NSCLC) patients after the first-generation EGFR-TKIs treatment failure.

Method:
Patients with histologically diagnosed, EGFR T790M mutant stage IV NSCLC were considered eligible, and they should have documented disease progression on EGFR-TKIs. In a 3+3 dose-escalation design, AST2818 was orally administered every day on a 21-day cycle at doses ranging from 20mg to 240 mg (NCT02973763). AST2818 was then explored in a dose-expansion cohort at doses ranging from 40 to 240 mg every day. Plasma samples were collected to evaluate pharmacokinetics of AST2818. EGFR T790M mutation in tissue samples was detected by amplification refractory mutation system. The primary endpoint was to determine dose limiting toxicity and objective response rate (ORR). Adverse events (AEs) were evaluated by CTCAE 4.03, and efficacy was assessed per RECIST v1.1 every 6 weeks.

Result:
From December 27, 2016 to August 21, 2017, 17 patients received at least one dose of AST2818 across four cohorts (20mg, 40mg, 80mg and 160 mg QD). Maximum tolerated dose has not been reached. The most common treatment-related AEs were grade 1 proteinuria (25%, 3/12). Other AEs included fatigue and prolonged Q-T interval, etc, all less than 10% and grade 1 or 2. The first 12 patients had been evaluated with an ORR of 58.3% (7/12) and a disease control rate of 91.7% (11/12). Profound and sustained tumor regression had already been observed at 20mg cohort. AST2818 plasma exposure, measured as Cmax and AUC 0-24h showed a dose-proportional increase. Figure 1



Conclusion:
AST2818 was well tolerated and had promising clinical activity with durable disease control in EGFR T790M mutant NSCLC after first-generation EGFR-TKIs treatment failure.

Background:
Lung cancer remains a leading cause of mortality worldwide. Evolution in molecular biology has expanded and the identification of somatic mutations in the epidermal growth factor receptor (EGFR) as a clinically relevant oncogene also selected a subgroup of patients. Most commonly described as activating mutations, both deletion of exon 19 and L858R point mutation in exon 21 account for nearly 90% of all EGFR mutated patients. These patients usually benefit from tyrosine kinase inhibitors (TKIs). On the other hand, some patients harboring specific EGFR mutations – such as exon 20 insertions – do not benefit from the same strategy.

Method:
We describe a case report of a patient with advanced lung cancer. A female, 39-year-old patient was first diagnosed in late 2015 when she presented with dyspnea and lower back pain. Biopsy of the primary lung mass as well as a bone lytic lesion both revealed an adenocarcinoma. PET-CT showed extensive lymphangitic carcinomatosis, bone and cervical lymph node involvement. She underwent 5 cycles of cisplatin-pemetrexed with a good radiologic partial response and very good clinical response. During chemotherapy, the first molecular test (Cobas Z 480 Roche) became available showing an insertion of exon 20 in the EGFR gene.

Result:
Patient then requested an alternative treatment given that she was not inclined to accept maintenance with chemotherapy. We then proposed a short trial with afatinib, even though the chances of response were very low. She was started on afatinib PO 40mg/day on March 14[th,] 2016. Three weeks later the patient developed a grade 3 diarrhea and the drug was withheld until her symptoms resolved. She resumed afatinib PO at 30mg/day on April 20[th,] 2016. Her first radiologic evaluation two months later showed stable disease and she was kept on treatment and reported to feel healthier. Her second evaluation on November 2016 then showed a partial response, mainly in the bone and she was continued on oral TKI. New imaging studies on March 2017 revealed a progression of her disease only 12 months later.

Conclusion:
There is a lack of data addressing patients harboring rare and unique EGFR gene mutations. Most exon 20 insertions identified in patient samples have not been tested against reversible EGFR TKIs. Extrapolations from the few tested mutations might not apply for other exon 20 mutations. It is imperative that patient-derived cell lines of common EGFR exon 20 insertion mutations are developed to enhance our preclinical understanding on these tumors.

Background:
EGFR-mutated lung cancer patients are mostly found in never smoker but at least one third of the patients are ever smokers. Clinical outcomes are known to be variable according to the smoking history among EGFR-mutated lung adenocarcinoma patients when treated with EGFR-TKIs. The aim of this study is to investigate whether cumulative smoking dose affects the clinical outcomes such as progression-free survival (PFS) and overall survival (OS) in EGFR-mutated lung adenocarcinoma patients treated with EGFR-TKIs

Method:
We retrospectively analyzed 142 advanced or recurrent lung adenocarcinoma patients who harbored activating EGFR mutations (exon19 deletion or exon 21 L858R) and had received gefitinib, erlotinib or afatinib. Detailed smoking histories and smoking dose were obtained from all patients. The patients were classified into 4 groups by cumulative smoking dose (never smoker, ≤10 pack-years (PYs), 11-30 PYs and > 30 PYs). PFS and OS were analyzed according to smoking subgroups by Kaplan- Meier curves.

Result:
Among 142 EGFR-mutated patients, 91(64.1%) were never-smokers, 12(8.5%) were minimal smokers with ≤10 PYs, 22(15.5%) were moderate smokers with 11-30 PYs, and 17(12%) were heavy smokers with more than 30 PYs. Cumulative smoking dose was inversely associated with median PFS in dose-dependent manner with statistical significance. (11.8 months, 10.9months, 7.4 months, 3.9 months. p < 0.05). Kaplan-Meier curves of OS showed statistically significant negative association between cumulative smoking dose and median OS. (33.6months, 26.3months, 20 months, 8.9months: p<0.001) However, minimal smoker group less than 10 PYs showed very similar clinical outcomes of PFS and OS with never smoker group. In the multivariate analysis adjusted for age, sex, performance status, stage, and time point of EGFR-TKI treatment, cumulative smoking dose was an independent predictive factor for the disease progression (hazard ratio, 3.29; 95% confidence interval(CI), 1.68-6.45 p <0.001) and short OS(HR 4.3, 95% CI 2.1-8.7 p<0.001) to EGFR-TKIs.

Conclusion:
Cumulative smoking dose inversely affects the duration of response and survival to EGFR-TKIs in EGFR-mutated lung adenocarcinoma patients. Profiling of smoking-related gene signatures might be valuable for therapeutic decision besides EGFR mutation test in lung adenocarcinoma patients.

Background:
Combinations of gefitinib and radiotherapy have been observed to have synergistic and anti-proliferative effects on lung cancer in vitro, but clinical studies were limited. In clinical setting, patients who presented with respiratory difficulties like SVCS, radiotherapy should be given immediately to address the emergency while waiting for the results of EGFR mutation test.

Method:
We did a preliminary study to evaluate the efficacy of gefitinib and radiotherapy combination in lung adenocarcinoma in Persahabatan National Respiratory Refferal Hospital Jakarta Indonesia. Subjects were consecutively recruited between January 2013 to December 2016

Result:
Thirty one lung adenocarcinoma with EGFR mutations were enrolled. Most of them were male (51.61%) with median age of 54.5 years old (range 38-70 years old). Epidermal Growth Factor Reseptor (EGFR) mutation characteristics were on exon 21 L858R (61.30%); exon 21 L861Q (16.12%) and exon 19 deletion (22.58%). Radiotherapy were given at doses between 30-60 Gy. Among these subjects, Progression Free Survival (PFS) were 185 days (CI95%; 123.69-246.30), 1 year survival rate (1-ysr) was 45.2%. and overall survival (OS) are 300 days (CI95%;130.94-469.06). There were no grade 3/4 hematological and non hematological toxicities recorded. The most frequent Grade 1 and 2 non hematological toxicities were skin rash, diarrhea, paranochia and monoliasis and might be related to TKI.

Conclusion:
The combination of TKI with radiation may be considered in subjects with respiratory distress.

Background:
Advanced lung adenocarcinoma with epidermal growth factor receptor (EGFR) gene mutation in exon 18 to 21 is sensitive to EGFR tyrosine kinase inhibitors (TKI) such as gefitinib, erlotinib, and afatinib. However acquired resistance to EGFR-TKI develops after initial treatment, primarily due to T790M mutation. In March 2016, the Japanese Ministry of Health, Labour and Welfare approved osimertinib for the patients with advanced non-small cell lung cancer harboring a T790M mutation. In the same year, patients with a T790M secondary mutation were reviewed at Hiratsuka Kyosai Hospital for a period of 9 months. All patients were previously treated with an EGFR-TKI (with or without or prior/subsequent chemotherapy).

Method:
Data were obtained retrospectively by analysis of the medical records of patients who underwent molecular diagnostic testing for T790M mutation. All patients had stage IV adenocarcinoma.

Result:
A total of 8 patients with T790M mutation were identified. Molecular testing was performed using re-biopsy specimens of the primary tumor, metastatic lymph node, or circulating DNA in the plasma of patients. Seven out of 8 patients received osimertinib 80mg once daily. All patients treated with osimertinib received other treatments previously, including EGFR-TKI and standard chemotherapy. The overall response rate (ORR) was 87%. Most common adverse events included diarrhea (28.6%), rash (14.2%), nausea (14.2%). Adverse events of Grade 3 to 4 severity were not reported.

Conclusion:
These findings suggest that third-line or later osimertinib for advanced lung adenocarcinoma with T790M results in high ORR and managed tolerability. The use of molecular testing may improve treatment outcome in these patients.

Background:
Non-small cell lung cancer (NSCLC) patients with activating epidermal growth factor receptor (EGFR) mutations are treated with EGFR-tyrosine kinase inhibitors (TKIs). However, most patients acquire resistance to EGFR-TKIs and receive subsequent treatments. To determine the optimal treatment for patients with TKI-resistance, we retrospectively examined the outcomes in advanced or recurrent NSCLC patients and analyzed the efficacy of the prevalent treatment options for those with TKI-resistance, using propensity score modeling.

Method:
EGFR-mutated NSCLC patients who acquired resistance to EGFR-TKIs during their first-line EGFR-TKI therapy were assigned to the TKI-resistant group based on the response of progressive disease (PD) according to the Response Evaluation Criteria in Solid Tumors. Patients with wild-type (WT) EGFR were assigned to the EGFR-WT group. By multivariate analysis of the two groups, a propensity score for chemotherapy use was calculated for each patient using logistic regression model. TKI treatment-free survival (TFS) was defined as "the overall survival (OS) - total progression-free survival (PFS) of every EGFR-TKI therapy".

Result:
A total of 415 patients with NSCLC were screened for EGFR mutations in the National Center for Global Health and Medicine, from April 2007 through March 2012. Of these, 158 (39%) patients harbored EGFR mutations, and 101 of these patients with activating EGFR mutations developed TKI-resistance. Seventy-five patients with EGFR-mutations who acquired TKI-resistance received a second-line chemotherapy or other EGFR-TKIs. Fifty-seven patients (75%) in the TKI-resistant group received ≥2 lines of EGFR-TKI treatments (beyond PD). Of the 252 EGFR-WT patients, 139 patients who received first-line chemotherapy or EGFR-TKIs formed the EGFR-WT group. OS was significantly longer in the TKI-resistant group compared to the EGFR-WT group (median, 43.8 vs 14.8 months, p<0.001). TFS did not significantly differ between the two groups (median, 16.6 vs 14.4 months, p=0.83). TKI-resistant patients receiving three or two lines of EGFR-TKIs had a better total PFS than those receiving a single line of EGFR-TKI (median, 28.2 vs 21.1 vs 9.0 month, p<0.001). In the propensity score-adjusted multivariate analysis, TFS was significantly associated with the post-operative recurrence (hazard ratio [HR] 0.40, p<0.000) and the use of chemotherapy (HR 0.32, p=0.005). Total PFS of EGFR-TKIs significantly correlated with the post-operative recurrence (HR 0.27, p=0.02) and sequential use of other EGFR-TKIs (HR 0.25, p=0.03).

Conclusion:
The use of chemotherapy prolonged the TFS in TKI-resistant NSCLC patients to the same extent as that seen in EGFR-WT patients. In TKI-resistant patients with EGFR mutations, sequential use of different EGFR-TKIs improved the total PFS of EGFR-TKIs.

Background:
Although previous studies demonstrated that the PFS of geftinib, erlotinib and afatinib as first line treatment is superior to chemotherapy in advanced NSCLC patients harboring activating EGFR mutation, the efficacy of icotinib therapy as first line therapy has not yet been elucidated.

Method:
Patients with IIIB/IV NSCLC and a confirmed activating mutation of EGFR (exon 19 deletion or exon 21 L858R substitution) received oral icotinib (125 mg, three times per day) until disease progression or unacceptable toxic effects. The primary outcome was overall survival (OS), and the second endpoint were progression-free survival (PFS), objective response rate(ORR), disease control rate(DCR).

Result:
1615 patients with advanced NSCLC receiving icotinib from Oct. 2012 to Apr. 2016 were screened in four cancer centers in Qingdao city, P R.China, and 79 patients with intact follow-up data received icotinib in first-line setting and were enrolled.Of them, the number of women and men were 51 (64.56/%) and 28 (35.44/%) respectively. 27 (34.18%)patients were with EGFR exon 19 deletion, and 52(65.82%) patients with exon 21 L858R mutation. The median follow-up period at the time of analysis was 24.50 months. The objective response rate(ORR) was 45.57% (36/79) , and the disease control rate(DCR) reached 89.87%(71/79). One (1.27%) patient had CR, and 35(44.30%) patients had PR, 35(44.30%) patients had SD, 8(10.13%) patients with PD. The median PFS and OS were 13.61 months and 31.11 months respectively of overall population. The median PFS was 12.30 months in EGFR exon 19 mutation subgroup and 14.00 months in exon 21 mutation subgroup respectively(p=0.441). The exon 19 mutation group had a significantly longer median overall survival than exon 21 mutation group (34.72 months, vs. 28.66 months,log-rank p=0.006, hazard ratio, 0.31 95% CI, 0.13 to 0.71). Meanwhile, there is significant difference of overall survival during different ECOG PS subgroups(ECOG PS 0-1 versus ECOG PS 2-3,32.59 months versus 20.59 months,p=0.002,HR 3.09,95%CI, 1.46 to 6.52).

Conclusion:
The study illustrate that icotinib is effective as first-line treatment for patients with advanced NSCLC and sensitive EGFR mutation. The patients harboring EGFR exon 19 deletion mutation yield similar PFS, but longer OS compared to those harboring exon 21 L858R mutation.

Background:
Osimertinib, an irreversible EGFR-TKI with activity also against the resistance mutation T790M, has a high brain permeability surmising intracerebral efficacy in T790M-negative cases. We assessed the efficacy of osimertinib in T790M-positive and –negative patients.

Method:
The TREM-study is an investigator initiated phase 2, single-arm, multi-center clinical trial conducted in five Northern European countries. Patients with advanced EGFR-mutated NSCLC with progression after at least one EGFR-TKI were assigned to treatment with osimertinib 80 mg daily until radiological progression or death. Both T790M-positive and –negative patients were enrolled, as well as patients with stable and asymptomatic brain metastases. The primary endpoint was objective response rate (ORR) according to RECIST 1.1. Secondary endpoints were progression free survival (PFS), duration of response (DoR), disease control rate (DCR) and overall survival (OS). We here present data from a subset of patients with brain metastases at study entry.

Result:
Of 147 included patients, 34 presented with CNS-metastases at inclusion. This subset of patients had poorer performance status at baseline than the full study cohort (31 % with ECOG 2 in the CNS-subgroup vs 17 % in the full study cohort) and the median age was lower (61.5 vs 65 years), otherwise similar to the full cohort in terms of baseline characteristics. 69 % (20/29) were T790M-positive and 31 % (9/29) negative. 5 patients had unknown T790M-status. 28 patients were evaluable for response. ORR was 39 % (11/28) and DCR 75 % (21/28). For T790M-positive patients ORR was 53 % (9/17) and DCR 88 % (15/17), in T790M-negatives 13 % (1/8) and 38 % (3/8) respectively. Median DoR in T790M-positive patients was 14.7 months (95 % CI 6.4-22.9) and 5.5 months in one T790M-negative patient. Two patients had ongoing responses after 15.9 and 17.5 months at data cutoff. Median PFS in the CNS-subgroup was 7.2 months (95 % CI 4.1-10.3 months) vs 9.7 months (6.3-13.1) in patients without CNS metastases, p=0.300, regardless of T790M-status. In the CNS-subgroup PFS in the T790M-positive patients was 10.1 months (7.9-12.3) vs 2.0 months (0.9-3.2) in the T790M-negative patients, p<0.001. Of 18 patients who had progressed at cutoff, 7 had CNS as site of progression (4 T790M-negative, 2 unknown and only one T790M-positive).

Conclusion:
Although a limited number of patients in this subgroup analysis, our results show that osimertinib has similar efficacy in patients with CNS disease as without, whereas the benefit in T790M-negative patients may be substantially lower.

Background:
Osimertinib is a third generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), which showed its efficacy for T790M resistant mutation in patients with advanced and recurrent non small cell lung cancer (NSCLC). The efficacy and toxicity of osimertinib comparing to previous EGFR-TKIs are not fully elucidated. Since every patient receiving osimertinib has received previous EGFR-TKI therapy, we compared the efficacy and toxicity of those agents in the same patients.

Method:
We retrospectively reviewed medical records of patients with T790M mutation positive advanced and recurrent NSCLC, who had disease progression after previous EGFR-TKI, the standard first line therapy, and started osimertinib between April 2016 and March 2017 at National Caner Center Hospital. Progression free survival (PFS) of osimertinib, and 1st line EGFR-TKI PFS of the same patients were calculated by Kaplan-Meier method. Objective response rate (ORR) was assessed according to RECIST version 1,1. Adverse events (AEs) were also reviewed to evaluate the difference of safety profiles between osimertinib and previous EGFR-TKIs.

Result:
A total of 46 patients with T790M positive NSCLC received osimertinib after the failure of first line EGFR-TKI treatment. At May 2017, the median follow-up time since the start of osimertinib was 7.8months. The median age was 65 (range 36-82), the median number of treatment received before osimertinib was 3 (range 1-9), and the median wash out time of 1st line EGFR-TKI till the start of osimertinib was 14.0 months. The median PFS of osimertinib is not reached. The median PFS of first line EGFR-TKI was 15.2 months. ORR of osimertinib and first line EGFR-TKI was 56.0% and 65.2%, respectively. The most frequent AEs of any grade of osimertinib were rash, dry skin, paronychia, and diarrhea (39.4%, 35.8%, 32.1%, and 30.2%, respectively). Rash, paronychia, and diarrhea over grade 2 was 6.5%, 6.5%, and 0% with osimertinib, compared to 0%, 12.5%, and 4.1% with gefitinib, and 41.7%, 8.3%, and 0% with erlotinib. The incidence of pneumonitis with osimertinib treatment was 10.9% (5 cases) in any grade, and 6.5% (3 cases) in grade 3 to 4, though 2 of them (1 case in grade 1 and 1 in grade 3) had received nivolumab as the prior chemotherapy. Except for pneumonitis, there was no AE leading to permanent discontinuation related to osimertinib.

Conclusion:
Osimertinib showed the efficacy and feasibility even in practical use. Adverse effect of osimertinib was generally better tolerated than previous EGFR-TKIs, except for pneumonitis.

Background:
The resistance mutation T790M emerges in around 60 % of EGFR-TKI treated NSCLC patients. Osimertinib is approved only in T790M-positive patients. We assessed the efficacy of osimertinib in both T790M-positive and -negative patients and here present results from T790M-negative patients.

Method:
In this investigator initiated, multicenter, single-arm, phase 2 clinical trial conducted in five Northern European countries, patients with progression on at least one previous EGFR-TKI and with measurable disease by RECIST 1.1 were assigned to treatment with 80 mg of osimertinib daily until radiological progression or death. Rebiopsy for assessment of mutational status was done after inclusion. Plasma samples were collected for translational research purposes (not analyzed yet). The primary endpoint was objective response rate (ORR) according to RECIST 1.1. Secondary endpoints were progression free survival (PFS), duration of response (DoR), disease control rate (DCR) and overall survival (OS).

Result:
T790M-status was assessable in rebiopsies from 120 of 147 included patients. Of these, 42 patients (35 %) were T790M-negative in tissue. 55 % (23/42) of the T790M-negative patients had exon 19 deletion at diagnosis as opposed to 71 % of the T790M-positives, other baseline factors were similar between the two groups. ORR in the T790M-negative group was 19 % (7/36) including one patient with complete response. In the T790M-positive group, ORR was 52 % (37/71), no complete responses. DCR was 64 % (23/36) and 87 % (62/71), respectively. All responses were confirmed. Median DoR was 11.0 months (95 % CI 3.0-19.1) in the negatives and 12.0 months (8.7-15.2) in the positives, p = 0.887. In the T790M-negative group, median PFS was 5.5 months (2.6-8.3) vs 10.8 months (8.2-13.4) in the T790M-positive group, p = 0.009. Subgroup analyses were performed in the T790M-negative group and there was significant higher median PFS in patients without CNS-metastases (5.6 vs 1.6 months, p = 0.007), in patients with duration of previous TKI-treatment over median (15 months) vs under (9.7 vs 3.5 months, p = 0.044) and in patients with one previous line of TKI vs two or more lines (7.3 vs 2.0 months, p = 0.007).

Conclusion:
T790M-negative patients who respond have similar DoR as T790M-positive patients. T790M-negative patients without CNS-metastases and with durable response on first EGFR-TKI could benefit from osimertinib.

Background:
The aim of the study was to demonstrate whether early variations in the levels of serum 4 tumor markers (TMs), carcinoembryonic antigen [CEA], cancer antigen [CA]125, CA19-9, and CA15-3), after TKI target therapy were associated with treatment response and progression-free survival (PFS) in patients with lung adenocarcinoma.

Method:
Patients with stage IIIB-IV lung adenocarcinoma taking epidermal growth factor receptor (EGFR) TKIs or anaplastic lymphoma kinase (ALK) inhibitors were enrolled prospectively from June 2012 to February 2015. According to the variations of percentage of change in 4-TM levels (4-TM~pc~), we divided patients into ascending (increases in 4-TM~pc~ over the 7[th]- 14[th ]day) and descending (decreases in 4-TM~pc~ over the 7[th]- 14[th ]day) groups.

Result:
In all, 184 patients were enrolled, and 89% had at least one of the pre-treatment evaluable TMs and were further analyzed. A good response to the TKI target therapy was accurately predicted in the descending group, as determined using receiver operating characteristic curve analysis (an area under the curve, 0.83). The kappa value between type of 4-TM~pc~ and measurable radiographic lesions was 0.762. Multivariate Cox hazards model analyses demonstrated that the type of 4-TM~pc~ and mutation status were the strongest predictors of PFS (descending versus ascending, hazard ratios [HR] 0.30, 95% confidence interval [CI], 0.19–0.47; sensitive mutation versus wide type, HR 0.30, 95% CI, 0.19–0.48).

Conclusion:
Type of 4-TM~pc~ 14 days after TKI target therapy is associated with an image response and PFS, without regarding mutation status, in patients with advanced lung adenocarcinoma.

Background:
EGFR is one of the most commonly mutated proto-oncogenes in lung cancer. The frequency of these mutations varies from approximately 10% of lung adenocarcinomas in North American and European populations to 50% in Asia. The leucine to arginine substitution at position 858 (L858R) in exon 21 and short in-frame deletions in exon 19 are the most common sensitizing mutations, comprising approximately 90% of cases. Approximately 50% of EGFR TKI resistance is due to a second site mutation, the T790M mutation occurring within exon 20.

Method:
To describe the "real life" experience in our country regarding the compassionate use of Osimertinib in patients with diagnosis of lung cancer with EGFR mutation and progression to tyrosine kinase inhibitors with detectable T790M mutation. We also include patients with de novo T790M mutation. We evaluated demographic data, the diagnostic method of resistance, sites of disease progression, toxicities and treatment efficacy.

Result:
We analyzed 17 patients from 10 different centers in Argentina in the period between January 2016 and May 2017. Median age: 61 years old, female 76%, PS: 0-1: 86%, non-smokers: 59%, histology: 15 adenocarcinoma and 2 undifferentiated, 14/17 with stage IV at diagnosis. EGFR mutations: Exon 19: 9 patients, 3 patients with evidence of T790M de novo, the remainder between exons 21 and 18. At the time of diagnosis, 13 patients start treatment with tyrosine kinase inhibitors, and the median time of response was 17 months. The most frequent site of progression was lung/pleura (85%) and liver (21%). Re-biopsy was performed in 13/17 patients. The major difficulty for successful biopsy was the site or the complexity of the procedure to be performed. A liquid biopsy was performed in 6 of 17 patients, 4 were positive. ORR with Osimertinib was 68%. The most common side effects were diarrhea: 31% (only 1 patient with grade 3), rash 18%. One patient had liver toxicity (grade 3 hepatitis).

Conclusion:
Osimertinib showed better adhesion and tolerance profile than tyrosine kinase inhibitors. Osimertinib offers an excellent toxicity profile with overall response rates similar to those described in publications.

Background:
Lung cancer remains the leading cause of cancer-related death worldwide. Though most patients with EGFR activating mutations are sensitive to EGFR tyrosine kinase inhibitors (TKIs), tumors will inevitably acquire resistance to first generation EGFR TKIs. EGFR T790M mutation accounts for about 50% of these resistance cases. Droplet digital PCR (ddPCR) and cobas enables quantification of specific mutated ctDNA. In this study, these methods were used to detect and evaluate the ctDNA response after Osimertinib treatment.

Method:
In the screening period of ASTRIS (D5160C00022) study in single center, tumor and blood samples from 69 stage ⅢB-Ⅳ NSCLC patients defined as acquired resistance to first generation EGFR TKIs (Gefitinib, Elortinib or Ecotinib) were collected. The cobas® Mutation Test v2 kit was used to detect EGFR mutations in FFPE or plasma samples. Plasma T790M mutation of both Osimertinib naïve and treated patients were quantified by Droplet digital PCR (ddPCR).

Result:
The T790M mutation rate of FFPE tissue cobas, plasma cobas and plasma ddPCR testing were 54.5%, 21.3% and 30.4% respectively. Taking the FFPE tissue cobas testing as gold standard, the sensitivity and specificity of plasma ddPCR to detect T790M mutation was 66.7% and 63.6%. Taking all testing methods into account, the T790M positive rate was 52.2%. In all of the plasma cobas positive cases, T790M mutation was detected by ddPCR. In 10 tumor biopsy cobas negative cases, 3 were positive defined by ddPCR. In 23 patients received Osimertinib treatment, the OOR was 60.9%, quantification of T790M after 6 weeks of treatment decreased to very low level, no association was observed between response status and T790M mutation decrease.

Conclusion:
ddPCR is more sensitive in plama ctDNA testing and should be performed even tumor tissue biopsy yielded negative results in certain cases. Osimertinib significantly decreased plasma T790M level, but not associated with clinical response.

Background:
Avitinib is an oral, potent, irreversible epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor selective for T790M resistance mutations. We report the safety, the intracranial/extracranial efficacy, and the blood brain barrier (BBB) penetration rate of Avitinib in non-small cell lung cancer(NSCLC) patients with EGFR T790m mutation. The data come from Peking Union Medical College hospital-a single center of the Phase 1, open-label, multicenter study (NCT02330367).

Method:
NSCLC Patients with acquired EGFR T790m (+) were enrolled. Patients were orally administered with dose escalating from 150 mg to 300 mg twice daily for 28-continuous-day cycles until disease progression. Blood (2mL) and cerebrospinal fluid (CSF) samples (2ml) were collected for concentration analysis on day 29 in available patients with brain metastases (BM). Tumor response was assessed on day 29 and then every 8 weeks.

Result:
Sixteen patients were included. Nine patients had asymptomatic BM, and all the nine patients had more than 3 BM lesions. The most frequent adverse events were the elevated hepatic transaminases (10/16, 62.5%) and diarrhea (5/16, 31.3%), Most were mild and reversible. 9 Patients (56.3%) achieved Partial Response (PR), 6 (37.5%) achieved Stable Disease (SD). Median Progress Free Survival (PFS) was 247 days (95%CI: 154.8-339.2), and the Median Overall Survival (OS) was 536 days (95%CI: 363.6-708.4). Of the 7 evaluable BM patients, the median intracranial PFS was 142 days (95% CI 31.1-252.9), with two patients progressed first in intracranial disease, while five patients had concurrent intracranial and extracranial progression after avitinib treatment. The cytologic analysis of CSF showed one meningeal metastases who accepted intrathecal injection with methotrexate and dexamethasone later. The blood and CSF analysis of 5 BM patients showed the BBB penetration rate were 0.046%-0.146% (Table).

patients	CSF Con. (ng/ml)	Plasma Con. (ng/ml)	Per.%	Intracranial PFS (days)	Extracranial PFS (days)
1	0.106	231	0.046	566	566
2	0.425	291	0.146	60	177
3	0.487	631	0.077	142	142
4	4.05	2940	0.138	138	138
5	1.72	1890	0.091	28	28
6	24	227	10.6[1]	218	218
7				308	350
8				550[2]	252

[1]Her BBB was broken by postocular metasatses. [2]He accepted brain radiotherapy before avitinib, and he also accepted chemotherapy with pemetrexed plus carboplatin plus endostatin after progression from avitinib. He was excluded from the analysis of intracranial PFS.

Conclusion:
Avitinib is well tolerated and efficacious in EGFR T790m(+) NSCLC patients. Its concentration in CSF is low, and the penetrability of BBB is weak. The median intracranial PFS for asymptomatic BM is relative short comparing to extracranial disease. Further studies are proceeding.

Background:
The aim of this registry was to collect demographic, diagnostic, treatment, and outcome information about Turkish patients with non-small cell lung cancer (NSCLC).

Method:
It was designed as a multicenter, cross-sectional, non-interventional study conducted on new and previously diagnosed lung cancer cases applied to 28 medical oncology outpatient clinics between 2012 and 2015. A total of 3790 male (85,5%) and female (14,5%) patients >18 years were included.

Result:
Mean age of patients was 60,4 (SD:±9,2) years. 79,4% of patients had smoking history and 19,6% of patients had familial cancer history. 63,5 % of patients were at stage IIIB and IV. 47,1% of total patients had adenocarcinoma and 42,2% had squamous cell carcinoma. In patients with no smoking history, adenocarcinoma histology was more dominant; 58,7% adenocarcinoma vs 30,6% squamous cell carcinoma. EGFR testing rate increased throughout the years but dependent to late market access of TKIs for EGFR mutation positive patients the testing rate for total patients is only 14,4%. EGFR mutation positivity rate in tested patients is 21,75% and 58,5% of these patients had exon 19 deletion. Overall survival of stage 4 patients was 15.9 months. For patients tested positive for EGFR mutation, erlotinib significantly improved the overall survival to 34,5 months (erlotinib users) vs 30,2 months (non-users) (p=0,043).

Conclusion:
In this multicenter, cross-sectional, non-interventional study we had an overall picture of Turkish NSCLC patients. 63,5% of NSCLC patients were at stage IIIB-IV and EGFR mutation positivity rate was 21,75%. For EGFR mutated patients, erlotinib is an effective treatment option and significantly improves overall survival.

Background:
In this study (NCT02206763), momelotinib, an inhibitor of Janus kinases 1 and 2, was administered in combination with erlotinib, a tyrosine kinase inhibitor (TKI) in patients with TKI-naïve epidermal growth factor receptor (EGFR)-mutated metastatic non-small cell lung cancer (NSCLC), to determine the maximum tolerated dose and safety of momelotinib in combination with erlotinib. As previously reported, dose limiting toxicities (DLTs) of grade 3 diarrhea (n=1) and grade 4 neutropenia (n=1) without fever were seen at dose level (DL) 2B and trial enrollment was halted. Here, we report the final results.

Method:
Patients received oral erlotinib 150 mg QD (including 11-31 day run-in). Momelotinib was administered orally in a standard 3+3 dose-escalation design: DL1, momelotinib 100 mg QD; DL2A, 200 mg QD; and DL2B, 100 mg BID. DLTs were evaluated in the first 28 days. Plasma samples were collected for PK/PD analyses.

Result:
Eleven patients enrolled: 3 in DL1, 3 in DL2A, and 5 in DL2B. The median duration of exposure to momelotinib was 40 weeks (range 2.4-63.1) and median number of cycles was 10 (range 0.6-15.8). Treatment was discontinued for progressive disease (n=7), adverse event (n=3), and patient decision (n=1). The objective response rate was 54.5% (90% CI: 27.1%–80.0%) and all responses (n=6) were partial responses; 4 patients had stable disease and 1 patient had progressive disease. The median duration of response was 7.1 (90% CI: 4.4–9.6) months. The median progression-free survival was 9.2 (90% CI: 6.2–12.4) months. The estimated median overall survival was not reached. The most common treatment-emergent adverse events (TEAEs) were decreased appetite, dry skin, and fatigue (7 patients each) and diarrhea (6 patients). In addition to the patient with grade 4 neutropenia (DLT), decreased neutrophil count was recorded in 4 additional patients (grade 1-2 [n=3], grade 3 [n=1]); median time to first neutrophil abnormality was 0.5 (range 0.5–3.7) months. Momelotinib-related TEAEs of interest (one patient each) included grade 1 sensory peripheral neuropathy, grade 1 paresthesia, and reactivation of hepatitis B. There was one momelotinib-related serious adverse event, grade 3 pneumonitis. There was no PK interaction between momelotinib and erlotinib.

Conclusion:
The combination of momelotinib and erlotinib had more toxicity than expected at DL2B. Neutropenia was common. Although the small number of patients in this phase 1 study limits our ability to make a definitive conclusion regarding efficacy, the response rate and progression-free survival was similar to previous reports with erlotinib alone.

Background:
Activating mutations of the epidermal growth factor receptor (EGFR) are key drivers of non-small cell lung cancer (NSCLC) in approximately 10-15% of Western patients and 30-35% of Asian patients. Patients with common activating mutations (L858R and del19) typically have objective tumour response rates (OTRR) of 56-74% and median progression free survival (PFS) of 9-13 months with first-generation EGFR tyrosine kinase inhibitors (TKI) such as erlotinib or gefitinib. The most common mechanism of acquired resistance to first generation EGFR TKI is the T790M mutation (50-60% of cases). Osimertinib is an irreversible EGFR TKI effective against the T790M resistance mutation with OTRR of 51-71% in T790M positive disease. However, acquired resistance invariably develops, and median PFS is approximately 10 months. Mechanisms of resistance include C797S mutations and loss of T790M. Novel strategies that prevent or delay resistance to osimertinib are likely to enhance its durability of response and clinical benefit in EGFR-T790M positive NSCLC.

Method:
DESIGN: Open-label, single arm, multi-centre, phase 2 trial with safety run in. ELIGIBILITY: Adults with advanced, EGFR mutated NSCLC, acquired resistance to first or second generation EGFR TKIs, and mutation of T790M. ENDPOINTS: PFS rate at 12 months, feasibility of alternating osimertinib and gefitinib, time to progression and PFS time, objective tumour response rate, overall survival and adverse events. Tertiary correlative objectives include changes in plasma cfDNA levels for activating EGFR mutations and T790M over time, their relationships with OTRR and the mechanism of resistance. TREATMENT: Osimertinib 80mg daily for 8 weeks (2 cycles), then gefitinib 250mg daily for 4 weeks alternating with osimertinib 80mg daily for 4 weeks (i.e. alternating 4 weekly cycles of each drug) until disease progression or prohibitive toxicity. STATISTICS: A total of 45 participants provides 90% power, with a 1-sided type 1 error rate of 10%, to distinguish the observed proportion alive and progression free at 12 months from true rates of 45% (not worthy of pursuit) and 65% (worthy of pursuit) using a Simon, 2-stage, minimax design allowing for 4 ineligible or inevaluable participants. ASSESSMENT: CT scans 8-weekly until disease progression. Plasma collections at baseline and at the start of each 4-week cycle. OSCILLATE is an investigator-initiated cooperative-group trial led by ALTG, in collaboration with NHMRC Clinical Trials Centre, University of Sydney, with support from Astra-Zeneca.

Result:
Section not applicable

Conclusion:
Section not applicable

Background:
Liquid biopsy has been approved as an optional method to detect clinically relevant EGFR mutations in NSCLC. WJOG8114LTR is a prospective, multi-institutional study of liquid biopsy in EGFR mutated NSCLC patients. Previously, we reported that complete molecular response at 4 weeks could be an early surrogate marker of durable efficacy. Here, we report updated results.

Method:
Chemotherapy naïve, advanced NSCLC patients with EGFR-sensitizing mutation received afatinib monotherapy (40 mg/body) until progressive disease (PD) or unacceptable toxicity. Plasma DNA was obtained from patients at baseline, weeks 2, 4, 8, 12, 24, 48, and at PD. Three types of clinically relevant EGFR mutations (exon 19 deletion, exon 20 T790M and exon 21 L858R) will be analyzed using plasma DNA with multiplexed, pico-droplet digital PCR assay (RainDrop® system, RainDance Technologies, Billerica, MA). Complete molecular response (CMR) was defined as mutant allele event/frequency of exon 19 deletion or exon 21 L858R below the cutoff for the positivity by digital PCR in plasma. This study was registered at UMIN (ID: 000015847).

Result:
Fifty-seven patients were registered in the study. Efficacy of afatinib was comparable to previous reports (overall response rate: 78.6%, and median progression-free survival (mPFS): 14.2 months). At baseline, 62.5% of patients (35/56) were positive for EGFR mutation in plasma. Among those, CMR rate at 2, 4, 8, 12, 24 weeks was 60.6%, 87.5%, 93.8%, 87.1%, and 83.3%, respectively. About 40% of patients who achieved CMR at any time point maintain CMR at 48 weeks and had durable progression-free survival (more than 400 days). At the time of analysis, 17 patients experienced disease progression, and 14 plasma samples were collected. Of those, 8 (57.1%) were positive for mutation in plasma. In five patients, plasma progression was observed prior to radiological progression. Exon 20 T790M was detected in five patients (detection rate: 62.5%).

Conclusion:
Among EGFR mutated NSCLC patients, liquid biopsy was a useful method to predict durable efficacy and progression. Applicability of liquid biopsy should be explored in further study.

Background:
EGFR mutation is a strong predictor of the response to EGFR-TKI, but 10-30% of EGFR-TKI naive patients do not respond to the first line EGFR TKI therapy. Inflammation plays an important role in the initiation, progression, invasion and metastasis of cancer. Recentry study has demonstrated that hematological markers of inflammation such as LMR (lymphocyte to monocyte ratio), RDW (red cell distribution width), and MPV (mean platelet volume) are valuable biomarker in various types of human cancers. The purpose of the present study is to analyze whether hematological markers of inflammation is a prognostic factor in EGFR mutant NSCLC treated with EGFR TKI.

Method:
We retrospectively analyzed 75 advanced or recurrent NSCLC patients with common EGFR mutation treated with EGFR-TKI between 2008 to 2017 at the University of Tokyo hospital. Patients with de novo T790M mutaion, systemic corticosteroids or active infection were excluded from the analysis. We analyzed whether the hematological markers of inflammation before the TKI therapy impact the PFS of TKI therapy. The following variables were included: LMR, RDW, MPV, EGFR mutation subtype (Exon19 deletion of L858R) , ECOG performance status, age and gender. The PFS was estimated by the Kaplan-Meier method and were compared by the log-rank test. Prognostic factors for PFS were assessed by Cox’s proportional hazards regression model. Statiscital analysis was performed using the survival package in the R software.

Result:
Low LMR and high MPV were associated with shoter PFS (log-rank test, p=0.000057 and p=0.03 respectively), but RDW was not associated with PFS. In the multivariate analysis, low LMR (hazard ratio (HR) 2.9; p=0.00015), high MPV (HR 1.7; p=0.039), and poor PS (HR 2.0; p=0.046) were independent risk factors for shoter PFS.

Conclusion:
Low LMR and high MPV are independent risk factors for shorter PFS in patients treated with EGFR-TKI.

Background:
ADJUVANT (CTONG 1104) is the first randomized trial shows significantly prolonged disease-free survival (DFS) in completely resected stage II-IIIA (N1-N2) epidermal growth factor receptor (EGFR)-mutation positive non-small-cell lung cancer (NSCLC) through adjuvant gefitinib compare with vinorelbine plus cisplatin (VP). Further we aim to analyze the treatment failure pattern in ADJUVANT study.

Method:
In the ADJUVANT trial, a total of 222 patients with completely resected stage N1–N2 EGFR-mutation positive NSCLC were randomized 1:1 into gefitinib group (250mg, QD, 24 months ) or vinorelbine (25mg/m[2] Day 1/Day 8) plus cisplatin (75mg/m[2] Day 1) group (every 3 weeks for 4 cycles) respectively. Any recurrence or metastases occurred during the follow-up period was defined as treatment failure. Recurrent pattern in both group were analyzed with follow-up data (until Mar 9[th] 2017) integrated.

Result:
At the Data cut-off date for the primary analysis of DFS, 124 progression events (55.9% maturity overall) had occurred; 114 patients had disease recurrence，10 patients died before disease recurrence. Analysed recurrent pattern include lung, brain, liver, bone adrenal gland, pleura, pericardium, spleen and regional lymph nodes metastasis. Even no significant differences were found, highest proportion of patients in both group(18.9% for VP and 26.1% for gefitinib, p=0.199) surfer brain metastasis with lung metastases being the second common recurrent site. Time to brain metastases showed no significantly difference between the two groups (not reach vs 40.8m, p>0.05). Among the 29 brain metastases patients with gefitinib, the brain metastases occurred in 17 patients during the gefitinib treament, and 12 patients relapse after the gefitnib termination. Figure 1



Conclusion:
Compared with other site metastases, lung, brain and regional lymph nodes metastases account for major proportion of recurrence in ADJUVANT study. (NCT01405079)

Background:
Although patients with non-small cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) mutations respond well to EGFR tyrosine kinase inhibitors (TKIs), most progress after approximately 1 year. At the time of progression, the EGFR T790M mutation is detected in more than half of these tumors. NSCLC harboring the T790M mutation shows a high response rate to osimertinib. However, the heterogeneous nature of NSCLC may limit the efficacy of osimertinib to those lesions with the T790M mutation, and a subset of patients may show a mixed response (MR), whereby some lesions shrink while others progress at the first evaluation. Previous study showed that about 20% of NSCLC patients exhibit MR to systemic therapies including EGFR-TKI. However, little is known about characteristics of MR to osimertinib.

Method:
To determine the frequency of a MR, we retrospectively reviewed patients treated with osimertinib for NSCLC harboring the EGFR T790M mutation at the National Cancer Center Hospital.

Result:
Between April and December 2016, 45 patients (median age 65 [36‒82] years, 19 [42%] males) who had NSCLC with the T790M mutation received osimertinib. The median numbers of previous chemotherapies and EGFR-TKI therapies received by the patients were 1 and 2, respectively. All measurable tumor lesions showed a response to therapy in 35 (77%) patients and progression in three (7%) patients. A MR was seen in seven (16%) patients. Of these seven patients, the re-biopsy specimens in which T790M was detected were derived from the primary lesion in six and a metastatic lymph node in one patient. Two types of MRs were seen among these seven patients: (1) the tumor including the re-biopsy site responded, while the other lesions progressed (five patients), and (2) the tumor including the re-biopsy site progressed (two patients). The most frequent progressive sites were liver and lung metastasis (four patients, respectively). Three patients continued to receive osimertinib after the MR, one of whom underwent drug-eluting bead transarterial chemoembolization (DEB-TACE) for progressive liver metastasis and achieved disease control on osimertinib for an additional 4 months after the MR.

Conclusion:
A MR to osimertinib was seen in 16% of patients with NSCLC harboring the EGFR T790M mutation. This suggests that resistance mechanism of 1st line EGFR-TKI may differ by the site in same patient. Since benefit of osimertinib is mainly seen in T790M mutation, addition of local therapy may be beneficial for patients who develop a MR to osimertinib.

Background:
Adenoid cystic carcinoma (ACC) is an rare form of malignant neoplasm that arises from secretory glands in salivary glands of the head and neck but cases primary from respiratory tract is rare. Epidermal Growth Factor Receptor activating mutation is common in lung adenocarcinoma in Asian and responded well with Tyrosine Kinase Inhibitor.

Method:
We present the case of a 48 year old female complaining chest pain and shortness of breath.

Result:
The patient was referred to the hospital due to chest pain and shortness of breath since 4 months. Initial CXR showed pulmonary mass and chest CT scan showed giant pulmonary mass but no nodes enlargement. Lobectomy were performed and histopathology evaluation showed adenoid cystic carcinoma. The patient underwent radiotherapy chemotherapy. Four months later the symptoms increased and tumor grow. EGFR mutation were done and positive for activating mutation (exon 19 deletion) and Gefitinib were started. The patient was stable for 1 year with gefitinib with minor side effects.

Conclusion:
EGFR mutation testing should be considered in a rare pulmonary mass such as adenoid cystic carcinoma.

Background:
Few uncommon EGFR mutations existed in NSCLC patients, such as G719X mutation on 18 exon. The best treatment option for G719X mutation is unclear, and it is usually excluded from clinical trials using EGFR TKI therapy. Here we studied the clinical data of patients harboring G719X mutation in real world and their sensitivity to EGFR TKIs.

Method:
Between January 2011 and December 2016, we retrospectively collected the clinical data of stage IIIB/IV NSCLC patients harboring G719X mutation at Peking Union Medical College Hospital.

Result:
A total of 830 NSCLC patients were found to harbor common sensitive EGFR mutations ( 417 patients harbored 19 exon deletion，413 patients harbored 21 L858 mutation, respectively), while 27 (27/857, 3.15%) patients harbored G719X mutation on 18 Exon, using amplification refractory mutation system (ARMs). 19 (19/27, 70.4%) patients with G719X mutation were treated with EGFR TKIs, 11 (57.9%) with Gefitinib, 5 (26.3%) with Icotinib, and 3 (15.8%) with Erlotinib, respectively. The median age was 58.3 years ( range from 30 to 79 years). There were 11(57.9%) females, and 6 (31.6%) patients with history of heavy smoking. 3 (15.8%) patients had baseline central nervous systemic metastasis. 11(57.9%) patients had unique G719x mutation, while 8 patient had compound mutations (5 patients had G719+20s768I, 2 patients had G719+L861Q, and 1 patient had G719+19del). 9 (47.4%) patients gained PR, 7 (36.8%) patients gained SD, and 3 (15.8%) achieved PD, the ORR was 47.4%, and the DCR was 84.2%. The median PFS was 8.8 months (95% CI: 0.932-16.67). The median PFS of first-line TKI therapy was longer than second-line TKI therapy (10.8months vs. 4.0months respectively), but it didn’t got statistical significance (p=0.226). The median OS was 15.3 months (95%CI: 12.3-18.3), with 6 patients still alive. There were no intolerant adverse effect associating with EGFR TKIs.

Conclusion:
These results suggest that EGFR TKI therapy is effective in patients with G719X mutations. EGFR TKI could be a treatment choice better than chemotherapy for patients harboring G719X mutation.

Background:
Patients with advanced stage of non small cell lung cancer (NSCLC) have been historically treated with a limited number of cycles of first-line chemotherapy, with platinum-doublets as the most commonly used regimen, after which, those with tumour response or stable disease are observed for evidence of disease progression; progression is followed by second-line therapy in proper patients. In patients with advanced NSCLC harboring EGFR mutations, the use of EGFR TKIs in first-line treatment has shown a large PFS benefit with almost no toxicity when compared with chemotherapy. For patients with lung adenocarcinoma and activating EGFR mutations who received EGFR TKIs median overall survival (OS) ranges between 24 and 30 months contrasts with the plateau of 10 months reached with first line platinum-based chemotherapy in populations not selected by molecular profiling.

Method:
We analyze all patients attended in our hospital with NSCLC, who had received EGFR-TKI since 2010 to 2015 when hoarbouring EGFR mutation. For descriptive purposes, continuous variables were summarized as arithmetic means, medians and standard deviations, and categorical variables were reported as proportions with 95% confidence intervals (95% CI). PFS and OS were measured from the day of EGFR TKI treatment to the date of progression or death, respectively, and analyzed with the Kaplan-Meier technique.

Result:
The amount of patients were 46. Six types of EGFR gene mutations were found: delection in exon 19, exon 18 (G719), exon 20 (T790 and S768I ) and in exon 21 (L861Q and L858R). Two patients were combinations of G719 and L861Q, the third was a combination of G719 plus S768I, and the fourth was a combination of delection in 19 exon and T790 mutation. 13 patients (24.5%) could recieve second and furthers lines of therapy after EGFR ITKs, including: chemotherapy, immunotherapy and second generation EGFR TKIs. PFS was 9,98 months [4,7-15,25; IC95%.] OS was 23,45 months [11,26-35,6; IC 95%]

Conclusion:
The outcome of this study is to describe patient population receiving EGFR-TKIs. As expected most of them harbored the common sensitizing EGFR mutations L858R and delection in exon 19;both were higher in women and specially L858R mutation in non smokers. Nowadays treatment of patients with advanced NSCLC should be individualized, based on the molecular and histological features of their tumour. When harbouring an activating EGFR mutation, first-line treatment should be with an EGFR TKI.

Background:
EGFR tyrosine kinase inhibitors (EGFR-TKIs) improve progression-free survival (PFS) in patients with EGFR-mutant NSCLC, but disease progression limits efficacy. Retrospective studies show a survival benefit to local ablative therapy (LAT) in patients with oligoprogressive disease (progression at a limited number of anatomic sites).

Method:
This is a prospective study of LAT in patients with oligoprogressive EGFR-mutant NSCLC. Patients with no prior EGFR-TKI therapy (cohort 1) or progression after 1[st]/2[nd] generation EGFR-TKIs with acquired T790M mutation (cohort 2) receive osimertinib. Upon progression, eligible patients with <= 5 progressing sites undergo LAT and resume osimertinib until disease progression. Patients previously treated with osimertinib qualifying for LAT upon disease progression are also eligible for treatment (cohort 3). Primary endpoint: evaluation of safety and efficacy of reinitiation of osimertinib after LAT (assessed by PFS2). Additional goals are assessment of mechanisms of resistance to osimertinib by multi-omics analyses of tumor, blood, and saliva.

Result:
Between 04/2016 and 06/2017, 18 patients were enrolled (cohort 1: 11, cohort 2: 4, cohort 3: 3). Median age was 57 (range 36-71). The most common adverse events (AEs) on osimertinib treatment were rash, diarrhea, thrombocytopenia, and alanine transaminase elevation with most of the AEs being grade 1 or 2. Among evaluable patients, confirmed objective response rates prior to LAT in cohorts 1 and 2 were 66.7% (6/9) and 75% (3/4), respectively, with 11.3 months median PFS (95% CI: 3.4-11.3 months) in cohort 1 and 8.9 months in cohort 2 (95% CI not defined due to the small number of patients). To date, 7 patients had progressive disease, 6 of which had oligoprogression and subsequently underwent LAT (cohort 1: 2; cohort 2: 1; cohort 3: 3). Three patients were treated with combination of surgery and radiotherapy (RT), 2 patients with surgery, and 1 patient with RT. Whole exome sequencing (WES) and RNA-seq were performed on tumor tissues obtained pre-treatment and upon progression on osimertinib. MET amplification, transformation to small cell lung cancer, and EGFR C797S mutation were identified as mechanisms of resistance to osimertinib. One patient with MET amplification was referred for a clinical trial of osimertinib and savolitinib, a MET inhibitor, upon second progression on osimertinib re-challenge, and had a response to treatment.

Conclusion:
Patients with EGFR-mutant NSCLC and oligoprogression after EGFR-TKI therapy can be safely treated with LAT. In select patients, this approach could potentially maximize duration of EGFR-TKI treatment and prevent premature switching to other systemic therapies.

Background:
Detection of targetable driver mutations in NSCLC may depend on the method employed. We carried out an audit to determine whether the EGFR mutation (EGFR-M) and ALK rearrangement (ALK-R) detection rate is dependent upon on the testing method used. We also sought to assess if EGFR-M and ALK-R was associated with baseline demographic characteristics.

Method:
Retrospective analysis of NSCLC patients who underwent testing for EGFR-M and ALK-R from January 2012 till May 2017. Methods used for EGFR-M were Real time ARMS PCR and gene sequencing while Break Apart FISH and D5F3 immunohistochemistry(IHC) were used for ALK-R testing.

Result:
Of the 599 patients tested for EGFR-M, 541 (90.3%) had interpretable results with an overall prevalence of 21.4%(n=116). Real time ARMS-PCR and gene sequencing yielded 95.9% and 81.9% interpretable results respectively. ALK-R testing was done in 462 patients of whom 431 (93.3%) had interpretable results, of which 8.6%(n=37) were positive. D5F3 IHC and Break Apart FISH yielded 94.7% and 82% interpretable results respectively. Mean age was 59.2 and 54.0 years respectively for EGFR-M and ALK-R patients with 54.3% and 45.1% being females. Mutations in exon 19 were the most common (n=81, 69.8%) followed by exon 21 L858R (n=30, 25.9%). 87/116 (75%) and 19/37 (51.4%) of EGFR-M and ALK-R patients received EGFR-TKIs and crizotinib respectively. Table shows differences in prevalence of EGFR-M and ALK-R prevalence in relation to gender, smoking status, histology and testing method used.

Table 1 Smoking, gender and histologic profile of the patients tested for EGFR mutations and ALK rearrangements

	EGFR-M positive (n=116)	ALK-R positive (=37)
Overall	21.4%	8.6%
Adenocarcinoma only	23.8%	9.5%
Females vs. males	37.1% vs. 14.3%	12.5% vs. 6.8%
Non-smokers vs. smokers	34.6% vs. 11.9%	11.6% vs. 6.5%
Female non smokers	39.9%	12.4%
Male non-smokers	26.1%	10.5%
Male smokers	11.1%	6.3%
Females with adenocarcinoma	40.7%	13.3%
Method used for testing	23.1% Real time ARMS PCR vs. 17.8% gene sequencing	9.0% D5F3 IHC vs. 4.9% Break Apart FISH

Conclusion:
Real time ARMS-PCR and D5F3 IHC are more sensitive methods for detecting EGFR-M and ALK-R respectively. Prevalence of a targetable driver in North Indian NSCLC patients ranges from 52.3% amongst female non-smokers to 17.4% of male smokers which are very encouraging results from both the patients and the treating oncologists perspectives. Higher percentage of EGFR-M patients receive targeted therapy as compared to ALK-R.

Background:
There are few effective strategies for C-MET positive advance non-small-cell lung cancer(NSCLC) patients with epidermal growth factor receptor(EGFR) inhibitor resistance.The efficacy of PD-1 blockade immunotherapy and even the status of PD-L1 expression in such population is unclear.

Method:
Patients diagnosed as advanced NSCLC synchronously tested for EGFR status, expression of PD-L1 and C-MET at the Guangdong Lung Cancer Institute (GLCI) from 2015 to 2017 were collected.PD-L1 expression on tumor cells and immune cell was evaluated using a three-tiered grading system. C-MET positive was define as immunohistochemistry staining (2+/3+) in ≥ 50% of tumor cells. A chi-squared test was used to assess the relationships between C-MET positive and PD-L1 expression.

Result:
A total of 487 eligible cases were selected including 166 EGFR mutant and 321 wild type patients.In the general population(n=487),the difference of PD-L1 expression were observed between C-MET positive group and C-MET negative group (65.3% vs 31.7%, P=0.001),which was in accordance with the result from the Cancer Genome Atlas (TCGA) dataset (n=512,P<0.001).Furthermore,among the EGFR mutant patients (n=166), PD-L1 expression was showed in 58.1% of C-MET positive group and 28.5% of C-MET negative group,P value <0.001. Subsequently,T790M negative was identified in 55%(47/86) of EGFR TKI resistant patients (n=86).In this subgroup,a significant increase of PD-L1 expression was demonstrated in C-MET positive group compared to C-MET negative group(66.7% vs 34.6%,P=0.027).Finally, clinical efficacy of immunotherapy was further confirmed in 2 C-MET positive advanced lung adenocarcinoma patients with remarkable response to PD-1 blockade immunotherapy who had disease progression after C-MET inhibitors.Figure 1



Conclusion:
C-MET positive maybe associated with high PD-L1 expression in advanced NSCLC providing therapeutic insight into targeting the PD-1/PD-L1 pathway in EGFR inhibitor-resistant NSCLC with C-MET positive and T790M negative.

Background:
Afatinib, an irreversible ErbB-family blocker, has shown activity for patients with advanced non-small-cell lung cancer (NSCLC) after failure of gefitinib or/and erlotinib in LUX-LUNG1 trial; however, efficacy data of EGFR-TKI re-challenge with afatinib is insufficient.

Method:
We retrospectively reviewed medical record of the patients with advanced NSCLC harboring EGFR mutation whose disease progressed after the treatment with gefitinib or erlotinib and received EGFR-TKI re-challenge at Kyoto University Hospital between Apr 2008 and Mar 2017, and compared the efficacy of afatinib after the failure of gefitinib or erlotinib and erlotinib after the failure of gefitinib.

Result:
Sixty-two patients were identified, including 13 patients with afatinib and 49 patients with erlotinib. Patient characteristics, such as age, sex, ECOG-PS and smoking status, were not significantly different between the treatment groups. In the afatinib group, 8 patients had received erlotinib as their initial EGFR-TKI and 5 patients had received gefitinib. In the erlotinib group, all patients had received gefitinib as their initial EGFR-TKI. EGFR T790M mutation status was unknown in all patients when EGFR-TKI was re-administrated. Overall response rate (ORR) was 12.7% and disease control rate (DCR) was 54.5% in the entire population. ORR in the afatinib group and the erlotinib group were 7.7% and 14.3%, respectively (p=0.513), and DCR in the afatinib group and the erlotinib group were 61.5% and 52.4%, respectively (p=0.561). Median progression-free survival was 2.4 months in the entire population (95% confidence interval [CI], 1.8-3.3), and 3.9 months in the afatinib group and 2.3 months in the erlotinib group, respectively (hazard ratio [HR] = 0.698 (95% CI, 0.351-1.285), p=0.258).

Conclusion:
Re-challenge with EGFR-TKI demonstrated clinical benefit as previously reported. No significant difference was observed between the efficacy of afatinib after the failure of gefitinib or erlotinib and that of erlotinib after the failure of gefitinib.

Background:
Primary EGFR dual mutations comprising T790M and exon 19 or 21 mutation (SM, sensitizing mutations) are rare in the Caucasian population, and there are only limited data about the treatment results with EGFR-TKIs in this setting. In 2016 we published results of the Slovakian retrospective study, in which six cases of the primary EGFR dual mutations T790M and SM were found among 3883 patients with NSCLC tested for EGFR mutations. Here we present the treatment results with updated PFS and OS data.

Method:
In this retrospective multicentre study the databases of the molecular/genetic diagnostic centres were searched for patients with primary dual EGFR mutations T790M and SM. Data about treatment results in these patients were obtained from the databases of the participating institutions and patient files. Kaplan-Meier survival analyses were done with MedCalc software, v. 17.5.

Result:
Patients’ characteristics and the treatment results are in the Table. PS was improved after two months of treatment in patients with initial PS over 1, and remained unchanged in those with PS 1. There were no unexpected AEs. Median PFS was 16 months, 95%CI: 12 - 23 months, median OS: 26 months, 95%CI: 18 - 26+ months. Table: Characteristics of patients with primary dual EGFR mutations T790M and SM (sensitizing mutation), and results of treatment with EGFR-TKIs

Gender (M/W)	Age (yrs)	Smoking status	PS	NSCLC histology	Stage	T790M + SM	Treatment line/TKI	Response	PFS (mo)	OS (mo)
W	57	Never	3	AC	IV	Del 19	1st/afatinib	PR	12	28+
W	64	Never	1	AC	IV	Del 19	2nd/erlotinib	SD	16	18
W	71	Never	1	AC	IV	Del 19	1st/afatinib	SD	10	17+
W	72	Ex	2	AC	IV	Del 19	1st/gefitinib	SD	54+	54+
W	72	Never	1	NOS	IV	Del 19	1st/erlotinib	PR	20	26
W	75	Never	1	AC	IV	Del 19	1st/afatinib	SD	24	25

Conclusion:
Results in our group of patients with primary dual EGFR mutations T790M and SM treated with first or second generation ERGFR TKIs are comparable with results seen in NSCLC patients with the SM only. The quantitative analysis of these mutations using either the recent tumour tissue or the blood sample is available at present. It might be useful in decision making about the use of the first – second, or the third generation EGFR-TKI, based on the prevailing mutation.

Background:
Clinical features of epidermal growth factor receptor (EGFR) mutations: L858R, deletions in exon 19, T790M, G719X, and L861Q in non-small-cell lung cancer (NSCLC) are well-known. The clinical significance of other uncommon EGFR mutations, such as A763_Y764insFQEA, is not well understood. This study is aimed to improve the understanding of A763_Y764insFQEA, and the clinical response to icotinib of NSCLC patients with such an uncommon mutation.

Method:
Six cases of EGFR exon 20 A763_Y764insFQEA mutation and twelve cases of EGFR exon 20 other insert mutation NSCLC patients were retrospectively analyzed until the progress of the disease or the emergence of the side effects and clinical efficacy was observed after months of followed-up.

Result:
Six cases with EGFR exon 20 A763_Y764insFQEA and twelve cases of EGFR exon 20 other insert mutation NSCLC patients mutation manifested the median PFS (9.0months vs 1.2months, P<0.001). Clinical efficacy of icotinib with advanced NSCLC harboring EGFR exon 20 mutations between A763_Y764insFQEA and other insert mutation (ORR:33.33%, DCR: 100% vs ORR: 0, DCR: 16.16%).

Conclusion:
EGFR exon 20 A763_Y764insFQEA mutation of clinical benefit from icotinib is remarkable, and it close to the common mutation of clinical benefit. It illustrates the value of in-depth molecular testing with NGS of EGFR wild type NSCLC patients.

Background:
Rociletinib, an oral, irreversible tyrosine kinase inhibitor (TKI), selectively targets activating mutations in EGFR and the acquired resistance mutation T790M and demonstrated antitumor activity in the phase 1/2 TIGER-X study (NCT01526928). Initial results are reported from the TIGER-3 study (NCT02322281) of rociletinib vs chemotherapy in EGFR TKI-resistant patients with EGFR-mutated NSCLC.

Method:
Eligibility criteria included: metastatic or unresectable, locally advanced, EGFR-mutated NSCLC; radiological progression on most recent TKI therapy; ≥1 line of platinum doublet chemotherapy. Patients were not selected based on T790M status. Patients (N=900) were to be randomized (1:1:1) to rociletinib 500 or 625 mg BID or investigator’s choice of chemotherapy (pemetrexed, gemcitabine, docetaxel, or paclitaxel). The primary endpoint was investigator-assessed progression-free survival (PFS) (RECIST v1.1); objective response rate (ORR) was a secondary endpoint. The rociletinib dosing groups were combined and compared with chemotherapy in a step-down procedure (patients with a centrally confirmed T790M mutation, followed by all randomized patients).

Result:
TIGER-3 enrollment was halted upon discontinuation of rociletinib development in NSCLC in 2016; therefore, target enrollment was not achieved. TIGER-3 enrolled 75 patients in the rociletinib groups [500 mg BID, n=53; 625 mg BID, n=22] and 74 in the chemotherapy group. Median age was 62 years, 69.1% had ECOG Performance Status of 1, 39.6% were Asian, 58.4% were female, and median number of prior therapies was 3. PFS and ORR data are presented in the Table. The most common adverse events (all grade; grade ≥3) in the rociletinib group were diarrhea (62.7%; 2.7%), hyperglycemia (58.7%; 24.0%), nausea (37.3%; 4.0%), fatigue (37.3%; 8.0%), and decreased appetite (37.3%; 0%) and in the chemotherapy group were nausea (27.4%; 5.5%), anemia (24.7%; 2.7%), and fatigue (24.7%; 9.6%).

Outcome	Centrally Confirmed T790MPositive		Centrally Confirmed T790MNegative		Intent-to-Treat Population*
	Rociletinib[†] (n=25)	Chemotherapy (n=20)	Rociletinib[†] (n=36)	Chemotherapy (n=41)	Rociletinib[†] (n=75)	Chemotherapy (n=73)
Median PFS, mo (95% CI)	6.8 (3.7–12.2)	2.7 (1.3–7.0)	4.1 (2.5–4.6)	1.4 (1.3–2.7)	4.1 (2.8–5.5)	2.5 (1.4–2.9)
HR (95% CI)	0.570 (0.285–1.140); P=0.105		0.532 (0.322–0.878); P=0.011		0.609 (0.423–0.875); P=0.006
Confirmed ORR, n (%) [95% CI]	9 (36.0) [18.0%–57.5%]	3 (15.0) [3.2%–37.9%]	3 (8.3) [1.8%–22.5%]	2 (4.9) [0.6%–16.5%]	13 (17.3) [9.6%–27.8%]	6 (8.2) [3.1%–17.0%]
*Includes patients with undetermined T790M mutation status. [†]Rociletinib 500 mg BID and 625 mg BID dose groups were pooled for the analysis. CI, confidence interval; HR, hazard ratio.

Conclusion:
Incomplete enrollment precluded hypothesis testing. However, the data show a trend toward longer PFS and higher ORR with rociletinib.

Abstract not provided

Background:
Immunotherapy and DNA damage repair inhibitors have the potential to play a role in the treatment of patients with extensive-disease small cell lung cancer (ED-SCLC), due to high mutation load and genomic instability in this disease setting. Durvalumab, a selective, high-affinity, engineered human IgG1 mAb blocks PD-L1 binding to PD-1 and CD80. Tremelimumab is a selective human IgG2 mAb targeting CTLA-4. Durvalumab plus tremelimumab demonstrated clinical activity and manageable tolerability in a Phase 1b study in NSCLC (NCT02000947). AZD1775, a small-molecule inhibitor of DNA damage checkpoint kinase WEE1, potentiates genotoxic chemotherapies. AZD1775 plus carboplatin showed antitumor activity and acceptable safety in a Phase 2 study in platinum-refractory p53-mutated ovarian cancer (NCT01164995).

Method:
BALTIC (NCT02937818) is a Phase 2, open-label, multicenter, multi-arm, exploratory, signal-searching study to assess the preliminary activity of novel treatment combinations in patients with platinum-refractory/resistant ED-SCLC. Inclusion criteria include disease progression during, or within 90 days of completing, first-line platinum-based chemotherapy; WHO/ECOG performance status of 0/1; and life expectancy ≥8 weeks. Each study arm is independent and will open sequentially to enroll up to 20 patients. The study will open initially with two arms. Patients will receive durvalumab 1500 mg + tremelimumab 75 mg i.v. q4w for 4 doses, followed by durvalumab monotherapy 1500 mg i.v. q4w (Arm A); and oral AZD1775 225 mg bid for 2.5 days from Day 1 + carboplatin AUC 5 on Day 1 i.v. q3w (Arm B). Treatment will continue until confirmed disease progression or discontinuation. Further arms will be added to assess other combinations once tolerable dosing regimens have been established. The primary endpoint is investigator-assessed ORR (RECIST v1.1). Secondary endpoints include duration of response, disease control rate, time-to-response, PFS, OS, pharmacokinetics, safety and tolerability. Gene expression levels and biomarkers will also be explored. Recruitment is ongoing.Figure 1



Result:
Section not applicable

Conclusion:
Section not applicable