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S. Toyooka
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MO16 - Prognostic and Predictive Biomarkers IV (ID 97)
- Event: WCLC 2013
- Type: Mini Oral Abstract Session
- Track: Medical Oncology
- Presentations: 10
- Moderators:S. Toyooka, J.C. Yang
- Coordinates: 10/29/2013, 16:15 - 17:45, Parkside Auditorium, Level 1
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MO16.01 - Different Micro-RNA expression in lung adenocarcinoma with molecular driver events (ID 2316)
16:15 - 16:20 | Author(s): L. Landi, P. Gasparini, C. Tibaldi, S. Carasi, L. Cascione, G. Alì, A. D'Incecco, G. Minuti, J. Salvini, A. Chella, G. Fontanini, C.M. Croce, F. Cappuzzo
- Abstract
- Presentation
Background
Oncogenic driver alterations identify several types of lung adenocarcinoma with different prognosis and sensitivity to targeted agents. MicroRNAs (miRNAs) are a new class of non-coding RNAs involved in gene expression regulation. How miRNAs are dysregulated in lung cancer with ALK translocation, EGFR or KRAS mutation is largely unknown. In the present analysis we aimed to investigate miRNAs expression according to a specific molecular driver and to correlate miRNAs deregulation with patient outcome.Methods
The study was conducted in a cohort of 67 lung adenocarcinoma patients (pts) including 17 ALK+ tumors, 11 ALK-/EGFR mutation+, 15 ALK-/KRAS mutation+, 24 ALK-/EGFR and KRAS wild-type and defined as triple negative cases. Matched normal lung tissues from 18 cases representative of the entire cohort were also included onto the analysis. RNA was isolated from formalin-fixed paraffin-embedded tissue (FFPE), using the Recover ALL kit (Ambion). NanoString nCounter system platform was used to generate the miRNA profile. We used Limma to test for differential expression analysis of data. Among the miRNAs evaluated, the miR-515 family expression between tissues was validated by RT-qPCRs, analyzed using the parametric t-test (unpaired, 2-tailed for validation).Results
miRNA expression profile clusters distinctly ALK+ pts from ALK- and normal lung tissue. Within the ALK- group we found specific miRNAs subsets able to sub-stratify KRAS versus EGFR careers clustering sharply triple negative versus EGFR mutation+ and triple negative versus KRAS mutation+. miRNAs belonging to the miR-515 family seems to be the most deregulated in the ALK+ versus ALK-. Although their expression is stably high in normal tissues and ALK+ class, they are highly downregulated in KRAS mutated versus EGFR mutated and versus triple negative (p-value <0.001 for all comparisons).Conclusion
miRNAs profile significantly differs in lung cancer pts with ALK translocation, EGFR mutations and KRAS mutations. Putative targets of deregulated miRNAs are under investigation to better define differences in driver-dependent pathway activation.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO16.02 - Tumor and Stroma Treg markers in resectable NSCLC (ID 2753)
16:20 - 16:25 | Author(s): M. Usó, J.J. Pérez Marcos, E. Jantus Lewintre, R. Sirera, R. Lucas, C. Hernando, C. Camps
- Abstract
- Presentation
Background
Immunosuppressive regulatory T lymphocytes (Tregs) have been proved to play a critical role in immune tolerance to tumor. In this study we have analyzed several markers related to Tregs, in both tumor and stroma areas in patients with resectable NSCLC.Methods
Tumor FFPE samples from 135 early-stage NSCLC patients were used in this retrospective study. The most representative areas of tumor cells and tumor stroma of each sample were carefully micro-dissected. RTqPCR using hydrolysis probes was performed to determine the expression of Treg markers such as: CD127, CD25, FOXP3, CTLA-4, IL-10, TGFB-1, LAG-3, GITR and TNF-a as well as CD4 and CD8. Relative gene expression was assessed using GAPDH and CDKN1B as endogenous controls and results were normalized against a human cDNA as a reference. FOXP3 protein expression was assessed by immunohistochemistry, in 80 of the 135 patients included in this study. The absolute number of FOXP3-positive lymphocytes was determined in both tumor and stroma areas by averaging the cell counts in 10 fields (400X). All statistical analyses were considered significant at p< 0.05.Results
Gene expression analyses revealed an over-expression of CD25 (5.40X and 7.95X, respectively) and down-expression of CD127 (0.28X and 0.37X, respectively) in both, tumor and stroma. There was a tendency toward higher expression of FOXP3 (1.67X and 2.01X, respectively) and CTLA-4 (1.92X and 1.76X, respectively) as well. Paired Wilcoxon test showed significant gene expression differences between tumor and stroma in FOXP3 (p=0.006), CD25 (p<0.0001), CD4 (p<0.0001), CD8 (p=0.028), IL-10 (p<0.0001) and TGFB-1 (p<0.0001). Survival analyses revealed that patients with a “Treg profile” (↑CD25/↓CD127) had a reduced overall survival (OS), whilst those patients with higher levels of the ratio FOXP3 stroma/tumor had worse time to progression (TTP) (Table 1). Spearman test revealed a significant association between stromal FOXP3 expression levels and the number of FOXP3-positive lymphocytes (by IHC) in stroma, p=0.006. Moreover, chi-square test showed that patients with squamous cell carcinoma histology presented a higher number of FOXP3-positive lymphocytes than those patients with adenocarcinoma, p= 0.035. Table 1: OS for “Treg profile” and TTP for Ratio FOXP3 Stroma/TumorOS Median (months) 95% CI p Others 74.33 65.96 - 82.69 0.003 "Treg profile" 29.90 4.91 - 6.54 TTP Median (months) 95% CI p ↓ Ratio FOXP3 Stroma/Tumor NR -- 0.040 ↑ Ratio FOXP3 Stroma/Tumor 32.50 16.25- 48.74 Conclusion
Gene expression of Treg markers in tumor microenvironment seem to play an important prognostic role in early-stage NSCLC patients. Furthermore, preliminary IHC analysis indicated a correlation between mRNA and protein levels for FOXP3 in NSCLC patients. Supported in part, by grants PS09/01149, RD06/0020/1024 and RD12/0036/0025 from Red Temática de Investigación Cooperativa en Cáncer, Instituto de Salud Carlos III (ISCIII).Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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- Abstract
- Presentation
Background
Estrogen receptor pathway has been reported to be interacted with epidermal growth factor receptor (EGFR) signal pathway. This study focused on the impact of intracellular ERβ localization (cytoplasmic or nuclear) on the efficacy of EGFR-TKI.Methods
Tumor tissue specimens from 149 stage IV NSCLC patients treated with EGFR-TKI were analyzed using immunohistochemistry (IHC) for ER expression (ERαorβ) and their associations with clinicopathological variables and clinical outcomes. Significance of cyto-ERβ expression was further examined in NSCLC cell lines.Results
The expression of ERα and ERβ was detected in 15% and 28.9% of the patients, respectively. Cyto-ERβ positive cases showed shortened progression free survival (PFS) compared with cyto- ERβ negative ones (3.1 months vs. 7.3 months, p=0.061). In the subgroup with concurrent EGFR mutation, the differences of PFS were enlarged with significant statistics (4.7 months vs. 10.9 months, p=0.042). COX’s proportional hazard model showed that female, EGFR mutation and c- ERβ negative expression were independent predictive factors for PFS. PC-9 cells present ERβ in cytoplasma as well as nucleus. Estrodial (E2) induced PC-9 cells moderately resistant to erlotinib with a 3-fold increase of IC50, and the resistance can be reversed by ER blocker (fulvestrant) or siRNA directed to ESR2. The function of E2 was accomplished by nongenomic activation (MAPK phosphorylation) caused by E2 via cyto- ERβ. Combination therapy with erlotinib and fulvestrant turned out to be far more effective than either treatment alone in PC-9 cells. Furthermore, 2 patients harboring both EGFR mutation and cyto-ERβ expression underwent PD of EGFR-TKIs, and re-obtained disease control after receiving combined EGFR-TKIs with fulvestrant.Conclusion
Cyto-ERβ expression may predict relatively poor efficacy to EGFR-TKI compared with non- cyto-ERβ expression in NSCLC patients harboring EGFR mutation.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO16.04 - Analysis of HER2 amplification in non-small cell lung cancers (NSCLCs) with acquired resistance (AR) to Epidermal Growth Factor Receptor (EGFR) tyrosine kinase inhibitors (TKIs) (ID 2951)
16:30 - 16:35 | Author(s): K. Politi, A. Wurtz, Z. Walther, G. Cai, V. Pirazzoli, M.A. Melnick, L. Reynolds, J. Boyer, A. Chiang, D. Morgensztern, S. Goldberg, R. Herbst, T. Lynch, S.N. Gettinger
- Abstract
- Presentation
Background
Recent studies have demonstrated the feasibility of rebiopsy in patients (pts) with EGFR mutant NSCLC at the time of AR to the EGFR tyrosine kinase inhibitors (TKIs) erlotinib or gefitinib, and provide estimates of the prevalence of well described mechanisms of AR including the EGFR T790M mutation, MET amplification and small cell lung cancer (SCLC) transformation. HER2 amplification has also been described in cases of AR to EGFR TKIs, however, its exact frequency is still unclear. Moreover, comprehensive analysis of paired pre- and post-treatment samples to establish whether HER2 amplification is acquired during treatment with TKIs have not been performed. This prompted us to further investigate HER2 amplification in EGFR mutant NSCLC cases.Methods
Pts with metastatic or recurrent NSCLC who developed AR while on a molecularly targeted agent were enrolled on an IRB approved repeat biopsy protocol. Tumor biopsies were obtained at the time of AR, and histopathological and molecular analyses of the tumors were performed. Known mechanisms of AR to EGFR TKIs were analyzed (T790M mutation, MET amplification and SCLC transformation) as well as amplification of HER2. The presence of T790M was assessed either by Taqman or pyro-sequencing (unless T790M status was available from an outside institution). HER2 and MET amplification were determined using fluorescence in situ hybridization (FISH).Results
41 pts with AR to EGFR TKIs (erlotinib or gefitinib) were enrolled at YCC between Jan 2012 and May 2013. Histological analysis of all specimens revealed transformation of adenocarcinoma to SCLC in 3 cases (7%). Depending on the availability of tissue, samples were prioritized for T790M analysis followed by MET and HER2 amplification. T790M was identified in 36% of pts; MET and HER2 amplification were found in 11% and 10% of samples respectively. In the two cases with HER2 amplification, analysis of the pre-treatment specimen revealed that amplification of this receptor tyrosine kinase preceded treatment with EGFR-TKIs, however, the amplification level was lower pre-treatment in both cases. Specifically the ratio of HER2 to CEP17 probes was 2.8 pre-treatment in both cases and increased to 4.3 and 8 following TKI treatment. HER2 amplification was mutually exclusive with the other tested mechanisms of resistance.Conclusion
T790M was the most commonly identified mechanism of AR to EGFR TKIs in the YCC cohort consistent with other studies. MET amplification, HER2 amplification and SCLC transformation were also observed. The observation that HER2 was amplified pre-treatment warrants further investigation of HER2 amplification in AR and pre-treatment specimens. Whole exome sequencing of specimens without known resistance mechanisms is ongoing.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO16.05 - DISCUSSANT (ID 3915)
16:35 - 16:50 | Author(s): T. John
- Abstract
- Presentation
Abstract not provided
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MO16.06 - Clinical, structural and biochemical characterization of EGFR exon 20 insertion mutations in lung cancer (ID 745)
16:50 - 16:55 | Author(s): D.B. Costa, H. Yasuda, E. Park, C. Yun, M.S. Huberman, G.R. Oxnard, L.V. Sequist, G. Riely, R. Soo, M.J. Eck, S.S. Kobayashi
- Abstract
- Presentation
Background
Epidermal growth factor receptor (EGFR) exon 20 insertion mutations account for ~10% of EGFR-mutated non-small-cell lung cancer (NSCLC), for the most part occur at the N-lobe of EGFR after its C-helix (after amino-acid M766) and have unsolved patterns of response to ATP-mimetic EGFR tyrosine kinase inhibitors (TKIs).Methods
To understand the patterns of resistance or response to EGFR TKIs of EGFR exon 20 insertion mutations, we decided to study representative mutations using in vitro systems, structural models and also NSCLCs with these specific EGFR mutations.Results
We selected three mutations located within the C-helix (A763_Y764insFQEA [identical to D761_E762insEAFQ], Y764_V765insHH and M766_A767insAI) and four mutations following the C-helix (A767_V769dupASV [identical to V769_D770insASV], D770_N771insNPG, D770_N771insSVD [identical to S768_D770dupSVD] and H773_V774insH [identical to P772_H773insH]) mutations. Our data indicates almost all EGFR exon 20 insertions are resistant to submicromolar concentrations of gefitinib or erlotinib; data that mirrors the lack of clinical response of NSCLCs with these mutations. The crystal structural and enzyme kinetic studies of a prototypical post C-helix EGFR TKI-resistant insertion, between residues D770_N771 (D770_N771insNPG), highlight that these mutations favor the active conformation (i.e., are activating), don’t alter EGFR’s ATP-binding pocket and are less sensitive than TKI-sensitive mutations. D770_N771insNPG is predicted to be 7.66 fold less sensitive than the TKI-sensitive EGFR-L858R. Unexpectedly, we identified the atypical EGFR-A763_Y764insFQEA as the only EGFR exon 20 insertion hypersensitive to EGFR TKIs using enzyme kinetic and cell line models. In patients with EGFR exon 20 mutated NSCLCs, the response rates to gefitinib or erlotinib were significantly higher for A763_Y764insFQEA (2/3; 66.6%) when compared to all other mutations within or following the C-helix (0/17, 0%; p=0.0158). The unorthodox homology model of A763_Y764insFQEA suggests a mechanism of activation (by shifting the register of the C-helix N-terminal) related to TKI-sensitive mutations (such as L858R or L861Q).Conclusion
Our findings not only explain the intricate interplay between different EGFR mutations and their response to EGFR TKIs, but also have clinical implications for the treatment of EGFR exon 20 insertion mutated NSCLCs. Therefore, based on our data and previously published reports the aforementioned mutations affecting amino acids V765 to V774 should be classified as non-sensitizing to the reversible EGFR TKIs gefitinib and erlotinib. Our models may usher the development of EGFR TKIs specific for EGFR exon 20 insertion mutations.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO16.07 - Higher frequency of genetic aberrations in KRAS- than in EGFR-mutated NSCLCs. A next-generation sequencing study on 96 samples. (ID 1094)
16:55 - 17:00 | Author(s): O.T. Brustugun, Å. Helland, A.R. Halvorsen, M. Lund-Iversen, O. Myklebost, E. Hovig, L. Meza-Zepeda
- Abstract
- Presentation
Background
Genetic subtyping is increasingly being clinically relevant in NSCLC, and the search for novel targetable driver mutations is warranted. We intended to study the frequency and types of a vast number of potential druggable genetic aberrations in a large cohort of non-small cell lung cancers of all major histological subtypes. Herein we report the first findings.Methods
Blood samples and tumor tissue was obtained from 96 operated early stage lung cancer patients admitted to Oslo University Hospital-Rikshospitalet in the period 2006-2011. Tissue was taken from the excised tumours, snap frozen in liquid nitrogen in the operation room, and stored at -80[o]C until DNA isolation. The tumor cell content in the specimens was found to be more than 70% in most samples. DNA was isolated from both tumor and corresponding blood sample according to standard procedures. High-throughput sequencing was performed using the SureSelect Human Kinome kit (Agilent Technologies), with capture probes that target 3.2 Mb of the human genome and include exons for all known kinases, select cancer-specific genes and their associated UTRs, in total 612 genes. The derived sequence reads were analyzed based on a pipeline including calling variations, somatic mutations, DNA copy number changes, indels and genomic rearrangements, as well as functional annotations.Results
Tissues from 48 females and 48 males were analyzed; 73 adenocarcinomas, 21 squamous cell carcinomas and 2 large cell carcinomas. 55 patients were in stage I, 27 in II and 14 in stage III. 13 patients were never-smokers. 25 samples harbored a KRAS-mutation and 10 an EGFR mutation. The number of mutated genes per sample varied from 1 to 81. The median number of mutated genes was 14 in the overall cohort, 15 in the EGFR wildtype/KRAS wildtype tumors, 17 in KRAS- mutated patients, 5 in the EGFR-mutated group and 6 in the never-smoking patients (of whom 4 patients were EGFR-mutated).Figure 1Conclusion
KRAS-mutated tumors contain the same amount of genetic aberrations as in wild-type tumors, whereas EGFR-mutated tumors show a much lower number of mutations per tumor. Never-smokers harbor a low number of mutations independent of EGFR-mutation status. Novel driver mutations are probably found in samples with low numbers of mutations.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO16.08 - Cytology samples (s) for EGFR, KRAS and ALK testing in Non-Small-Cell Lung Cancer (NSCLC) (ID 2439)
17:00 - 17:05 | Author(s): E. Carcereny, T. Moran, A. Estival, F. Andreo, M. Lletjos, E. Mijangos, J. Sanz, E. Castellà, L. Capdevila, M.D.L.L. Gil, L. Rodriguez, M. Hardy-Werbin, I. De Aguirre, R. Rosell
- Abstract
- Presentation
Background
Recent advances in targeted therapy in NSCLC have achieved impressive results in advanced disease. For molecular testing,cytology samples are not commonly used since is less likely to be adequate. At ICO Badalona- Germans Trias i Pujol Hospital we have used cytology specimens when biopsy was not available. We describe the general results when using cytology specimens in NSCLC to detect EGFR mutation, KRAS mutations and ALK translocations.Methods
From February 2007 to May 2012, 227 cytology samples from patients with NSCLC were collected at the Department of Pathology as cell block or fresh specimen over an apropiate slide. After that, tumor cells were(8-150) captured by laser microdissection. DNA sequencing for EGFR exons 18, 19, 20, 21, KRAS codons 12 and 13 was performed at Molecular Biology Laboratory( ICO-Badalona) and ALK translocation were analyzed at Pathology Department by FISHResults
EGFR mutations were tested in 227 samples.The overall output was 86.3% (not evaluable in 15 , insufficient tissue in 8, no tumor cells in 4, not done in 4). EGFR mutation was detected in 8.81% (20/227). KRAS mutation were tested in 41 samples with results in 33, 80.5% (2 not evaluable, insufficient tumor cells 3, no tumor 1 and not done 2 samples). KRAS mutation was positive 6 (14.6%). ALk translocation were tested in 9 p with results in 6 p ( 1 not evaluable and 2 insufficient tumor cells) Both cell-block and fresh specimen over an apropiate slide were used to perform molecular testing. The output for cell-block was 83.3%(124/148) and testing was not possible in 23(11 not evaluable, 6 insufficient tumor cells, 4 not tumor and 3 not done). The output for membrane was 91.1% (72/79) and was not possible in 7(4 were not evaluable, 2 insufficient tumor cells and not done in 1). 54.7% of samples were obtained from endobronquial ultrasound guided transbronquial needle aspiration of mediastinal adenopathies, 11.3% lung mass needle aspiration and 11.7% from pleural effusion.Conclusion
Our results support the potential use of cytology samples for molecular testing in NSCLC when biopsy specimens are not available. Both membrane preparations and cytology blocks have been used and are equally suitable for molecular testing.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO16.09 - Patterns of metastasis and survival in patients with PI3K-aberrant and FGFR1 amplified stage IV squamous cell lung cancers (SQCLCs) (ID 1666)
17:05 - 17:10 | Author(s): P.K. Paik, A.L. Moreira, L. Wang, N. Rekhtman, C.S. Sima, H. Won, M. Ladanyi, M.F. Berger, M.G. Kris
- Abstract
- Presentation
Background
The majority of actionable drivers in SQCLCs occur in the PI3K (30%) and FGFR1 (20%) pathways. The biologic behaviors and natural histories of these subtypes are not well characterized. Characterization of these data may help to elucidate the biologic relevance of these putative oncogenic events.Methods
As of October 2011, all patients with SQCLCs at MSK have undergone prospective, multiplex testing of their FFPE tumors for FGFR1 amplification (FISH, FGFR1:CEP8 ≥ 2.2), PIK3CA mutations (Sequenom and exon sequencing), PTEN loss (IHC, Cell Signaling), and PTEN mutations (exon sequencing), among others. The PI3K abberant group was defined as PIK3CA mutant, PTEN complete loss, or PTEN mutant. Patient characteristics, outcomes, and metastatic sites were identified. Survival probabilities were estimated using the Kaplan-Meier method. Group comparisons were performed with log-rank tests and Cox proportional hazards methods.Results
77 stage IV SQCLC patients were analyzed. Genotypes were: FGFR1 amplified (23%); PTEN loss (22%), PIK3CA mutant (8%), PTEN mutant (7%). Events were non-overlapping save for 2 cases with PTEN nonsense mutations and PTEN loss. The sole significant clinical difference (KPS, age, sex, lines of tx, smoking status) was sex (women in PI3K group 52% vs. in others 23%, p=0.02). Metastatic patterns for PI3K and FGFR1 vs. all others were:
Median OS for PI3K vs. all others: 9mo (95%CI:8-NR) vs. 16mo (95%CI:11-NR), p=0.004. Median OS for FGFR1 vs. all others: 20mo (95%CI:11-NR) vs. 10mo (95%CI:9-16), p=0.06. Multivariate analysis for risk of death: PI3K HR=3.3 (95%CI:1.5-7, p=0.003); FGFR1 HR=0.5 (95%CI:0.2-1.1, p=0.06); Age ≥65, HR=1.3 (95%CI:0.6-2.8, p=0.5); KPS≤70, HR=3.2 (95%CI:1.6-.6.4, p<0.001); Lines of therapy ≥ 2, HR=2.3 (95%CI=0.8-5.7, p=0.08), male gender, HR=0.7 (95%CI:0.3-1.4, p=0.3).Site PI3K p FGFR1 p Other Total Brain 6 (22%) 0.002 0 (0%) 0.6 0 (0%) 6 (7%) Pleura 5 (19%) 0.4 5 (28%) 0.7 9 (28%) 19 (25%) Liver 5 (19%) 0.4 1 (6%) 1 1 (3%) 7 (9%) Bone 8 (30%) 0.8 3 (17%) 0.7 10 (31%) 21 (27%) Lung 12 (44%) 0.8 10 (56%) 0.2 12 (38%) 34 (44%) Adrenal 3 (11%) 1 3 (17%) 1 4 (13%) 10 (13%) Pericardium 1 (4%) 1 1 (6%) 0.3 0 2 (3%) Conclusion
Patients with stage IV PI3K-aberrant SQCLCs have poorer survival compared to other patients with SQCLCs while patients with FGFR1 amplified SQCLCs have a trend towards better survival. Brain metastases in SQCLC are rare, and occurred exclusively in patients with PI3K-aberrant tumors. These data suggest that PI3K pathway activation confers a distinct biology, and that targeting this in SQCLC patients with brain metastases may be an effective therapeutic strategy. Whole exome and RNA-sequencing data from 8 resected SQCLC brain metastases (4 paired with lung primaries) will be presented.Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MO16.10 - DISCUSSANT (ID 3916)
17:10 - 17:25 | Author(s): J. Minna
- Abstract
- Presentation
Abstract not provided
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MS11 - Next Generation Technology for Detection and Treatment of Lung Cancer (ID 28)
- Event: WCLC 2013
- Type: Mini Symposia
- Track: Biology
- Presentations: 4
- Moderators:S. Toyooka, V.N. Tran
- Coordinates: 10/29/2013, 14:00 - 15:30, Bayside Auditorium B, Level 1
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MS11.1 - Next Generation Sequencing (ID 506)
14:05 - 14:25 | Author(s): R. Govindan
- Abstract
- Presentation
Abstract not provided
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MS11.2 - Validating Platforms for Routine Clinical Use (ID 507)
14:25 - 14:45 | Author(s): P. Waring
- Abstract
- Presentation
Abstract
In this presentation, we describe the process that our laboratory followed that led to successful accreditation, by the Australian National Association of Testing Authorities (NATA), for medical use of NGS in clinical practice. First, we evaluated three different amplicon –based MPS technologies in order to choose the platform of choice for clinical use. We compared the performance of two commercial somatic mutation panels (Life Technology’s AmpliSeq cancer panel and Illumina’s TruSeq amplicon panel) and a customized panel (Agilent’s HaloPlex). The panels shared 31 genes in common. The AmpliSeq panel was sequenced using the Ion Torrent platform and the TruSeq and HaloPlex panels were sequenced using the Illumina MiSeq platform. In-house bioinformatics and variant annotation and reporting pipeline were developed to allow data from all three panels to be compared. A training set of 28 FFPET samples with known missense or deletion mutations in EGFR, KRAS, BRAF, NRAS, PIK3CA and KIT were tested by all three panels. These samples, previously tested using NATA - accredited Sanger sequencing, SNaPshot and fragment analysis performed on an ABI3730, were used to empirically determine the parameters required for accurate mutation detection by MPS. Sample acceptance criteria included samples with at least 1mg of extractable DNA following macrodissection from tumour areas with at least 70% purity. Library quality was assessed by BioAnalyser and libraries were sequenced to a median depth of 2000x. The panels and platforms were compared for % aligned reads, % on - target reads, median and range of coverage, input DNA quantity and quality requirements, data quality and variability, cost, turn around time, ease of use, and accuracy of mutation detection. There was marked variation in the number and types of variants identified across the three panels. With minimum variant calling criteria of depth >50x, variant depth >20x, variant frequency >5% and base quality >15, we identified 18557 variants with AmpliSeq, 15064 variants with TruSeq and 3326 variants with Haloplex. 14229 of the TruSeq variants were SNPs (9319 were C>T), indicating DNA polymerase errors, while 12370 of the AmpliSeq variants were small indels (mostly in homopolymeric tracts) indicating errors in calling repetitive sequences. In total, 59 variants were identified by all three panels. The TruSeq and Ampliseq panels detected all 31 known somatic mutations, where as the HaloPex panel missed four mutations due to patchy on - target coverage. In panels with adequate coverage of regions of interest, the assay sensitivity was 100%. The TruSeq panel was chosen for clinical use, despite the requirement for higher DNA input (150ng compared to 10ng for AmpliSeq), primarily due to ease of use and less hands - on time by laboratory staff. We then performed reproducibility, repeatability, robustness and limit of detection experiments using the TruSeq panel. Initially, there was poor reproducibility of all variants, particularly SNVs, especially in samples with low input DNA (<50ng) or poor quality DNA (fragment size <250 bp). Most of the identified variants were random and present at low frequency, most being present at <1-2% allele frequency. These showed characteristics suggestive sequencing and polymerase errors, formalin – induced artifacts and misaligned repetitive sequences. To reduced the great excess of false positives, we restricted variant calling by establishing minimum allele frequencies, by eliminating unreported variants and by limiting alignment to clinically- relevant or actionable mutations. Variant reproducibility was increased to 38% by only calling SNVS >5% and indels >1% allele frequency that were contained within the COSMIC database. This was further increased to 92% by restricting variant calling to known clinically - relevant mutations listed on the www.mycancergenome web site. Reproducibility was increased further by strict adherence to sample and library quality control criteria (DNA amount 150ng DNA fragment size at least 250bp, minimum library concentration of 1nM, and minimum of 400,000 reads per sample) and by only calling mutations if present with allele frequency above 5% for FFPET samples and 1% for AML samples. Notabily, non - reproducible “mutations” in clinically relevant genes (eg KRAS G12A) were not infrequently encountered below these cut off values. A second independent test set of 82 FFPET samples with known missense and deletion mutations in EGFR, KRAS, BRAF, NRAS, PIK3CA, KIT and PDGFRA were analysed by the TruSeq panel. By strict adherence to the above criteria and restricting variant calling to clinically relevant mutations, 100% sensitivity and 100% specificity was achieved in the samples that met the criteria. In all, only 71% of the samples tested passed all quality control criteria. 12% of the samples failed the library preparation and were not processed. 17% of the samples passed the library QC criteria but failed the sample QC criteria. In each case the known mutation was identified. In conclusion, by strict adherence to sample and library QC and by restricting analysis to clinically-relevant mutations, the TruSeq amplicon cancer panel was able to detect common somatic missense and deletion mutations with an allele frequency >5% in FFPET samples with 100% specificity and sensitivity without the need for confirmation by an orthogonal method. However, confirmation by an orthogonal methods would be required for suspected mutations present at an allele frequency <5%, for mutations not known to be of clinical – relevance and for samples with low tumour purity, low DNA input or poor quality DNA. This study showed that deep sequencing of tumour tissue from FFPETs generated many low frequency artifacts due to sequencing, polymerase, formalin – induced chemical modifications and well as frequent mapping and variant calling errors. These artifacts and errors mostly occur at low allele frequency and can be difficult to distinguish from low frequency somatic mutations. Strict adherence to sample and library quality control criteria, allele frequency thresholds and clinically relevant mutations allows highly accurate mutation calling without the need for confirmation by an orthogonal method.Only Members that have purchased this event or have registered via an access code will be able to view this content. 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MS11.3 - The Role of Current Pathologic Techniques in the Next Gen World (ID 508)
14:45 - 15:05 | Author(s): I.I. Wistuba
- Abstract
- Presentation
Abstract
Over the past decade, significant progress has been made in the characterization of molecular and genetic abnormalities tumors from patients with non-small cell carcinoma (NSCLC) that are being used as molecular targets and predictive biomarkers to select patients for targeted therapy. Recent advances in expanding the available NSCLC targeted therapies require the analysis of a broad panel of molecular abnormalities in tumor specimens, including gene mutations, gene amplifications, gene fusions and protein expression by applying different methodologies to tumor tissue (biopsy) and cell (cytology) samples. The rapid development of technologies for large-scale sequencing (next-generation sequencing, NGS) has facilitated high-throughput molecular analysis holding various advantages over traditionally sequencing including the ability to fully sequence large numbers of genes in a single test and simultaneously detect deletions, insertions, copy number alterations, translocations, and exome-wide base substitutions (including known hot-spot mutations) in all known cancer-related genes [1,2]. Currently, NGS platforms, including whole genome, whole exome and targeted gene sequencing, represent emerging diagnostic methodologies for the detection of oncogenes fusions and mutations in tumor tissue specimens, including formalin-fixed and paraffin-embedded (FFPE) samples [3]. Technical challenges include sequencing samples of low quality and/or quality, reliable identification of structural and copy number variation, and assessment of intratumoral heterogeneity. In addition, the clinical use of the NGS sequencing data is not straightforward and there are several challenges related to data analysis, data storage and report generation [4]. There is growing consensus that tumor tissue specimens must represent the setting of the disease to be treated, and increasingly, more tissue samples are being obtained for molecular testing of advanced, metastatic and chemo-refractory NSCLC tumors (e.g., MD Anderson BATTLE Lung Cancer Program) [5]. However, the biopsy and cytology samples available for molecular testing in those metastatic refractory NSCLC tumors are likely to be more challenging samples for molecular testing, including NGS platforms. The role of the pathologist is becoming increasingly important to adequately integrate routine histopathology assessments and molecular testing, including NGS, with clinical pathology for the most accurate tumor diagnosis and subsequent selection of the most appropriate therapy. References: 1. Meyerson M, Gabriel S, Getz G: Advances in understanding cancer genomes through second-generation sequencing. Nat Rev Genet 11:685-96, 2010 2. Mwenifumbo JC, Marra MA: Cancer genome-sequencing study design. Nat Rev Genet 14:321-32, 2013 3. Ross JS, Cronin M: Whole cancer genome sequencing by next-generation methods. Am J Clin Pathol 136:527-39, 2011 4. Ulahannan D, Kovac MB, Mulholland PJ, et al: Technical and implementation issues in using next-generation sequencing of cancers in clinical practice. Br J Cancer 109:827-35, 2013 5. Kim ES, Herbst RS, Wistuba, II, et al: The BATTLE trial: personalizing therapy for lung cancer. Cancer Discov 1:44-53, 2011Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
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MS11.4 - Reporting and Interpreting Molecular Results (ID 509)
15:05 - 15:25 | Author(s): M.S. Tsao
- Abstract
- Presentation
Abstract
As molecular biomarkers are becoming routine in the clinical management of lung cancer patients, there is an increasing need to establish standards or guidelines for the reporting of molecular results. In the most ideal situation, reporting of tissue based molecular biomarker results should be integrated into the histopathology report of the tissue sample, to provide a more complete genotype-phenotype characterization of the tumor. This is particularly important for lung cancer as molecular profiling to date has clearly shown that many “driver” genomic aberrations are often closely associated with specific tumor histology. In fact, the current CAP/IASLC/AMP guideline on molecular testing in lung cancer recommends the use of histology (adenocarcinoma containing tumors) as a primary criterion to select lung cancer samples for EGFR and ALK testing. However, until reflex molecular testing becomes routine in pathology practice, molecular testing is often conducted at a laboratory that is separate from the one where the original tissue histopathological diagnosis was made. In such cases, it is important that the stand alone molecular report should also include some histopathological data that may be highly relevant to the interpretation of the results, or at the very least, refer to the relevant Pathology report. In the Pathology report, the data should include: (a) type of sample, whether it is paraffin embedded or fresh (e.g. fluid), (b) tumor diagnosis, subtypes and variants when applicable, (c) essential immunohistochemical markers that were assessed to support the diagnosis, (d) use of tissue processing solution or fixative that could adversely affect the quality of DNA for sequencing, e.g. acid and Bouin’s solution, (e) the approximate size of the tissue, (f) whether a tumor cell enrichment strategy was used, and (g) estimated tumor cellularity in the tissue area marked for isolation of DNA for testing. It is of utmost important that molecular reports are written in language that can be understood by the treating physicians and the pathologists, who are the end-users of the report. Typical laboratory reports should include patient identification codes, the date the sample was acquired (biopsy or resection) from the patient, the date the sample is received in the molecular testing laboratory, and the date the report is signed out. All this information provides not only important sample identification information, but also the real turnaround time of the reported results. Aside from a summary of the molecular results themselves, the report should include a concise but reasonable detailed methodological section, which also provides the performance features of the assay platform being used. It should specify the list of genes included in the assay, the type of aberrations that can be reliably detected, e.g. single nucleotide mutations, deletions, insertions, rearrangements, copy number changes, etc, and the sensitivity and specificity of the assay. The methodology section should also include the analytical software used for processing the data and identifying the genomic aberrations and the version of the normal reference sequence used for comparison with the sequence in question. If the methodology used is fairly new or represent emerging technology, such as next generation sequencing (NGS), then information about mutation verification technology or process may also be required (1). While molecular aberrations are integral to the complete pathological diagnosis of a tumor, in lung cancer their main clinical relevance is for their ability to predict patient response to a specific therapeutic agent, or for patient prognosis. In this context, especially if there are a number of genetic changes being reported (as example with NGS); it may be useful if the aberrations (often called variants) are classified into categories, which reflect their clinical utility. Although there is as yet no universally acceptable classification framework for reporting genomic aberrations identified by NGS platforms, broad categories that establish prognostic, biological or treatment relevance to the aberrations have been proposed or used. These variants have been classified into several “Levels” or “Tiers”, depending on the level of evidence for their predictiveness of response to specific drug. These levels have been derived from widely accepted classification schemes, such as those published by the American College of Medial Genetics (ACMG) for use in diseases such as Breast Cancer. The “actionable” aberrations are those demonstrating proven evidence for their association with high response rates to a specific drug or treatment strategy. The “potentially actionable” alterations are those with strong rationale but as yet proven clinical evidence for being associated high response rate to a specific drug. This group also include aberrations that have demonstrated evidence for response to a specific drug in one type of cancer, yet of unproven response pattern in a different tumor being studied. However, as NGS enables the discovery of a large number of genetic aberrations that typically occur in sporadic adult cancers, many aberrations fall into the category of “unknown therapeutic or biological significance”. While some of these could potentially be predictive markers of drugs that are already available for other reasons, most may not even be pharmacologically targetable. An important risk of conducting comprehensive genomic profiling in patient samples is the identification of “incidental” aberrations, which require clinical management that is not originally planned or anticipated (2). These aberrations could involve genes/mutations with known hereditary roles in cancer or non-cancer conditions, with potentially significant implication on patient and/or other family members. For these reasons, the ACMG recently convened a working group of experts to publish recommendations for reporting of incidental findings in clinical exome and genome sequencing. While these recommendations have been provided primarily as educational resources for medical geneticists and other health care providers (and are still quite controversial), the issues discussed should be considered when deciding upon the reporting strategy for profiling cancer samples using NGS technology. References: 1. Rehm HL, Bale SJ, et al. ACMG clinical laboratory standards for next-generation sequencing. Genet Med. 2013 Jul 25. doi: 10.1038/gim.2013.92. [Epub ahead of print] 2. Green RC, Berg JS, et al. ACMG recommendations for reporting of incidental findings in clinical exome and genome sequencing. Genet Med. 2013 Jul;15(7):565-74. doi: 10.1038/gim.2013.73. Epub 2013 Jun 20.Only Members that have purchased this event or have registered via an access code will be able to view this content. 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