EGFR Testing Research Articles

Success and failure rates of tumor genotyping techniques in routine pathological samples with non-small-cell lung cancer

IntroductionIdentification of some somatic molecular alterations in non-small-cell lung cancer (NSCLC) has become evidence-based practice. The success and failure rate of using commercially available tumor genotyping techniques in routine day-to-day NSCLC pathology samples is not well described. We sought to evaluate the success and failure rate of EGFR mutation, KRAS mutation, and ALK FISH in a cohort of lung cancers subjected to routine clinical tumor genotype. MethodsClinicopathologic data, tumor genotype success and failure rates were retrospectively compiled and analyzed from 381 patient-tumor samples. ResultsFrom these 381 patients with lung cancer, the mean age was 65 years, 61.2% were women, 75.9% were white, 27.8% were never smokers, 73.8% had advanced NSCLC and 86.1% had adenocarcinoma histology. The tumor tissue was obtained from surgical specimens in 48.8%, core needle biopsies in 17.9%, and as cell blocks from aspirates or fluid in 33.3% of cases. Anatomic sites for tissue collection included lung (49.3%), lymph nodes (22.3%), pleura (11.8%), bone (6.0%), brain (6.0%), among others. The overall success rate for EGFR mutation analysis was 94.2%, for KRAS mutation 91.6% and for ALK FISH 91.6%. The highest failure rates were observed when the tissue was obtained from image-guided percutaneous transthoracic core-needle biopsies (31.8%, 27.3%, and 35.3% for EGFR, KRAS, and ALK tests, respectively) and bone specimens (23.1%, 15.4%, and 23.1%, respectively). In specimens obtained from bone, the failure rates were significantly higher for biopsies than resection specimens (40% vs. 0%, p=0.024 for EGFR) and for decalcified compared to non-decalcified samples (60% vs. 5.5%, p=0.021 for EGFR). ConclusionsTumor genotype techniques are feasible in most samples, outside small image-guided percutaneous transthoracic core-needle biopsies and bone samples from core biopsies with decalcification, and therefore expansion of routine tumor genotype into the care of patients with NSCLC may not require special tissue acquisition or manipulation.

29. Patterns of EGFR mutation testing pre-and post-establishment of an in-house molecular pathology service

<h3>Background</h3> Molecular testing of tumours is a rapidly growing area, with anatomical pathology laboratories being asked more frequently to use their material to perform molecular testing, either in-house or as a send-out test. In addition to the expense of the test itself, the retrieval and preparation of material for testing may be both time-consuming and labour-intensive, particularly if archives are not on-site or if a pathologist is required to re-examine the case to identify appropriate material. Limited information exists in the literature regarding adherence to guidelines, and without this information it is difficult to determine if the additional workload on laboratory personnel is justified. <h3>Aims</h3> To determine the timing, appropriateness and rates of EGFR mutation testing at the Austin and to identify any changes to these measures following establishment of an in-house service. <h3>Methods</h3> For EGFR mutation testing, specimen type, average request rates, turnaround times and test failure rates were determined for the 12 month period from January to December 2012. Following the establishment of in-house EGFR mutation testing, these rates were determined for the 3 month period of January to March 2013. <h3>Results</h3> EGFR tests were requested appropriately on all cases and were confined to cases of primary or metastatic lung adenocarcinoma only. Turnaround times improved significantly following introduction of in-house testing (average TAT time pre-introduc-tion, 39 days; average TAT post-introduction, 7 days). Request rates increased following introduction of in-house testing. Failure rates remained unchanged. <h3>Conclusions</h3> Although EGFR testing may have additional unaccounted-for costs for anatomical pathology laboratories, these costs are justified at the Austin by clinically-appropriate testing. Our data confirms increasing request rates although the additional convenience of in-house availability and more rapid TATs may also be contributing factors.

A Comparison of Methods for EGFR Mutation Testing in Non–Small Cell Lung Cancer

EGFR mutation testing of tumor samples is routinely performed to predict sensitivity to treatment with tyrosine kinase inhibitors for patients with non-small cell lung cancer. At least 9 different methodologies are employed in UK laboratories, and the aim of this study was to compare the sensitivity of different methods for the detection of EGFR mutations. Participating laboratories were sent coded samples with varying mutation loads (from 0% to 15%) to be tested for the p.Leu858Arg (p.L858R) missense mutation and c.2235_2249del exon 19 deletion. The p.L858R mutation and deletions within exon 19 of the EGFR gene account for ∼90% of mutation-positive cases. The 11 laboratories used their standard testing method(s) and submitted 15 sets of results for the p.L858R samples and 10 for the exon 19 deletion. The p.Leu858Arg (p.L858R) mutation was detected at levels between 1% and 7.5% by Sanger sequencing, pyrosequencing, real-time polymerase chain reaction (PCR), amplification refractory mutation system, and capillary electrophoresis single-strand conformation analysis. The c.2235_2249del mutation was detected at 1% to 5% by fragment size analysis, Sanger sequencing or real-time PCR. A mutation was detected in 24/25 (96%) of the samples tested which contained 5% mutated DNA. The 1% sensitivity claimed for commercial real-time PCR-targeted EGFR tests was achieved and our results show greater sensitivity for the Sanger sequencing and pyrosequencing screening methods compared to the 10% to 20% detection levels cited on clinical diagnostic reports. We conclude that multiple methodologies are suitable for the detection of acquired EGFR mutations.

Translating Genomics in Cancer Care

There is increasing enthusiasm for genomics and its promise in advancing personalized medicine. Genomic information has been used to personalize health care for decades, spanning the fields of cardiovascular disease, infectious disease, endocrinology, metabolic medicine, and hematology. However, oncology has often been the first test bed for the clinical translation of genomics for diagnostic, prognostic, and therapeutic applications. Notable hereditary cancer examples include testing for mutations in BRCA1 or BRCA2 in unaffected women to identify those at significantly elevated risk for developing breast and ovarian cancers, and screening patients with newly diagnosed colorectal cancer for mutations in 4 mismatch repair genes to reduce morbidity and mortality in their relatives. Somatic genomic testing is also increasingly used in oncology, with gene expression profiling of breast tumors and EGFR testing to predict treatment response representing commonly used examples. Health technology assessment provides a rigorous means to inform clinical and policy decision-making through systematic assessment of the evidentiary base, along with precepts of clinical effectiveness, cost-effectiveness, and consideration of risks and benefits for health care delivery and society. Although this evaluation is a fundamental step in the translation of any new therapeutic, procedure, or diagnostic test into clinical care, emerging developments may threaten this standard. These include "direct to consumer" genomic risk assessment services and the challenges posed by incidental results generated from next-generation sequencing (NGS) technologies. This article presents a review of the evidentiary standards and knowledge base supporting the translation of key cancer genomic technologies along the continuum of validity, utility, cost-effectiveness, health service impacts, and ethical and societal issues, and offers future research considerations to guide the responsible introduction of NGS technologies into health care. It concludes that significant evidentiary gaps remain in translating genomic technologies into routine clinical practice, particularly in efficacy, health outcomes, cost-effectiveness, and health services research. These caveats are especially germane in the context of NGS, wherein efforts are underway to translate NGS results despite their limited accuracy, lack of proven efficacy, and significant computational and counseling challenges. Further research across these domains is critical to inform the effective, efficient, and equitable translation of genomics into cancer care.

Treatment patterns and outcomes in patients with non-squamous advanced non-small cell lung cancer receiving second-line treatment in a community-based oncology network

ObjectivesThis retrospective study used the US Oncology iKnowMed™ database, billing claims, and chart reviews to report treatment patterns and outcomes in late-stage non-small cell lung cancer (NSCLC) in US community oncology practices. Materials and methodsEligibility criteria included non-squamous NSCLC, stage IIIB/IV at diagnosis, ECOG performance status (PS) <3, and initiation of 2nd-line therapy (defined as index date) between 1/1/2007 and 6/30/2011 with ≥1 year follow-up. Key outcomes were overall survival (OS), progression-free survival (PFS), time-to-progression (TTP), and time-to-hospitalization (post-index date). Kaplan–Meier and Cox proportional hazard models were used to characterize the distribution and predictors of outcomes. Results1168 patients were eligible for the study. The most frequent 2nd-line therapies were pemetrexed (54.4%), erlotinib-containing regimens (17.6%), and docetaxel (10.0%). Median OS and PFS were 7.5 (95% confidence interval [CI]: 6.6–8.4) and 4.1 (95% CI: 3.7–4.5) months, respectively; 57% of patients were hospitalized post-index date. EGFR testing rates were 2.3% before 2010, 15.2% in 2010, and 32.0% in 2011 (P<.001). Of EGFR-positive patients, 50.0% received erlotinib-containing regimens compared with 16.9% of EGFR-negative patients (P=0.001). An increased risk of shorter time-to-hospitalization, after controlling for other covariates, was associated with PS=1 (hazard ratio [HR]=1.51; P<.001) or PS=2 (HR=1.68; P=.001) compared with PS=0, pre-existing comorbid fatigue (HR=1.64; P=.003) compared with no comorbid fatigue, and progression (HR=1.92; P<.001), when it occurred, compared with no progression. Compared with other 2nd-line treatment, erlotinib-containing regimens prolonged adjusted TTP (HR=0.69; P=.015). ConclusionsThis retrospective observational study provides new insights into treatment patterns, biomarker testing, and outcomes in advanced NSCLC within the context of a large community oncology network. Outcomes of these community practice patients, although poor, were similar to those reported in 2nd-line clinical trials for relevant regimens. EGFR testing in community practice rose rapidly after 2010.

EGFR mutation in malignant pleural effusion of non-small cell lung cancer

EGFR testing has become the consensus before epidermal growth factor-tyrosine kinase inhibitorrs (EGFR-TKIs) treatment in non-small cell lung cancer(NSCLC) patients.Malignant pleural effusion is the common clinical manifestation in NSCLC patients,and EGFR testing by using different methods in pleural effusion cells and free nucleic acids has good prospect for predicting the efficacy of EGFR-TKIs. Key words: Carcinoma, non-small cell lung; Receptor, epidermal growth factor; Mutation; Malignant pleural effusion

Impact of the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology clinical practice guidelines for EGFR and ALK testing in lung cancer in Canada.

This paper summarizes the practical impact of the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology lung cancer biomarkers guidelines on the lung cancer approach in Canada, providing possible practical solutions for other similar health care systems in which scientific reality needs to be constantly balanced against economic reality.

EGFR activating mutations detected by different PCR techniques in Caucasian NSCLC patients with CNS metastases: short report

EGFR mutation testing has become an essential determination to decide treatment options for NSCLC. The mutation analysis is often conducted in samples with low percentage of tumour cells from primary tumour biopsies. There is very little evidence that samples from metastatic tissues are suitable for EGFR testing. We had evaluated the frequency of EGFR mutations with three highly sensitive PCR techniques in formalin-fixed, paraffin-embedded samples of 143 NSCLC patients with central nervous system (CNS) metastases. 32 corresponding primary tumours were also examined. We used PCR followed by DNA fragments length analysis (FLA), ASP–PCR and PNA–LNA PCR clamp techniques. We found 9 (6.29 %) EGFR gene mutations in CNS samples: 3 (2.1 %) in exon 19 and 6 (4.2 %) in exon 21. The full concordance between CNS metastases and primary tumour samples was observed. PCR followed by DNA–FLA and PNA–LNA PCR clamp were sensitive enough to detect exon 19 deletions. Two mutations in exon 21 were detected by ASP–PCR only, one L858R substitution was detected only by PNA–LNA PCR clamp. With respect to sensitivity, PCR followed by DNA–FLA achieved a level of detection of at least 10 % of mutated DNA for exon 19 deletion, as for ASP–PCR it was at least 5 % of mutated DNA for L858R substitution. Higher sensitivity of 1 % of mutated DNA was achieved by PNA–LNA PCR clamp technique for both mutations. The use of different methodological techniques authenticates the negative result of molecular tests.

Lung Adenocarcinoma Biomarker Incidence in Hispanic Versus Non-Hispanic White Patients

Lung cancer is the leading cause of cancer deaths in the United States and worldwide. Biomarker testing is critical to personalized therapy in lung adenocarcinoma and has been extensively investigated in non-Hispanic whites, Asians, and African Americans. However, little information addresses the underlying genetic changes in lung adenocarcinoma among Hispanic patients in the United States. To identify targetable biomarkers other than EGFR and EML4-ALK in Hispanic patients with lung adenocarcinoma. We tested DNA extracted from 85 lung adenocarcinoma specimens collected from 40 Hispanic and 43 non-Hispanic white patients for previously reported mutations in KRAS, MET, BRAF, mTOR, STAT3, JAK2, PIK3CA, AKT1 through AKT3, and PTEN with a custom Sequenom massARRAY assay (Sequenom, San Diego, California). Mutations in KRAS were identified in 11 cases (13%; 6 Hispanic [7%], 5 non-Hispanic white [6%]) and had no correlation with sex, age, or smoking history. Mutations in PIK3CA were identified in 2 of the 40 Hispanic patients (5%), including one patient (2.5%) with a concurrent KRAS mutation. The tumors were wild type for all other genes tested. Targetable biomarkers other than EGFR and EML4-ALK were identified in 7 of the 40 Hispanic patients (18%) and 5 of the 43 non-Hispanic white patients (12%), suggesting a similar mutational frequency. Our highly multiplexed genotyping assay detected actionable mutations in 14% (12 of 83) more patients than would have been identified by EGFR and EML4-ALK testing alone.

Awareness about EGFR testing and use of TKIs in advanced lung cancer: A questionnaire-based survey of medical oncologists from India.

e19116 Background: EGFR is an important driver mutation in lung cancer. The pattern of usage of EGFR mutation testing is not well defined in the Indian population. Methods: A total of 70 medical oncologists from India were interviewed to identify current practices in the management of advanced lung cancer. They were in clinical practice for 2 to 35 (median 9) years. Their affiliation included medical college (21), regional cancer center (6), corporate hospitals (29) and private practice (14). Their collective experience in conducting international clinical trials in lung cancer was significant (range 0 to 35 trials). Results: The number of new patients with lung cancer seen by them in a month was a median of 8 (range 1 to 20). EGFR testing on the lung cancer tissue was recommended in a median of 60 % (range 10 to 100 %) of paients and was actually carried out in a median of 30 % (range 0 to 90 %). Tissue samples were inadequate for EGFR testing in 30 % of cases (range 2 to 95 %). Method of EGFR testing used included IHC by 06, molecular by 52 and both by 12 of the oncologists. The time to obtain a report was 10 days (range 5 to 21 days). EGFR testing showed mutation/ overexpression in approximately 25 % of cases (range 0 to 70 %. In a large national tertiary cancer center with inhouse laboratory that did testing by PCR and sequencing, EGFR mutations were seen in 30 % of 481 samples. The EGFR mutations considered clinically important included del 19 by 56, L858R by 41 and T790M by 31 of the responding oncologists. The first line therapy used in EGFR mutation positive Lung cancer patients was platinum doublet chemotherapy by 7, gefitinib by 55 and erlotinib by 8 oncologists. The side effects of TKIs considered important were diarrhea by 64, mucositis by 47, rash by all 70, paronychea by 50, nausea vomiting by 52, fatigue by 51 and myelosuppression by 6. The common methods of managing these side effects included dose reduction by 22, drug interruption by 50, symptomatic treatment by 49 and counseling by 22. Conclusions: The awareness about EGFR mutation and use of TKIs among medical oncologist is increasing in India. However availability of sufficient tissue for molecular testing remains a problem.

The CAP-IASLC-AMP molecular testing guideline for the selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors.

11085 Background: The College of American Pathologists (CAP), the International Association for the Study of Lung Cancer (IASLC), and the Association for Molecular Pathology (AMP) jointly initiated an effort to establish evidence-based recommendations for the molecular analysis of lung cancers required to guide EGFR- and ALK-directed therapies, addressing which patients and samples should be tested, and when and how testing should be performed. Methods: Three co-chairs without relevant conflicts of interest were selected, one from each of the sponsoring societies: CAP (P.T.C.), IASLC (M.L.), and AMP (N.I.L.). Writing and advisory panels were formed from additional experts from these societies. Unbiased literature searches were performed to capture articles up to February 2012, yielding 1,533 articles whose abstracts were screened to identify 521 pertinent articles that were then reviewed in detail for relevance. Evidence was formally graded for each of the recommendations first formulated by the co-chairs and panel members at a public meeting. Each guideline section was assigned to at least two panelists. Successive drafts were circulated for comments to the writing panel, the advisory panel, the public (online posting), and the three professional societies. Results: We generated 37 guideline items addressing 14 areas of EGFR and ALK testing. The major, evidence-based recommendations are to test for EGFR mutations and ALK fusions in all patients with advanced stage adenocarcinoma, regardless of sex, race, or smoking history, and to prioritize EGFR and ALK testing over other molecular predictive tests. Recommendations and expert consensus opinions were generated for all other key aspects of EGFR and ALK testing in lung cancer related to oncology and pathology practice and technical issues in molecular testing. Conclusions: As scientific discoveries and clinical practice outpace the completion of randomized clinical trials, evidence-based guidelines developed by expert practitioners are vital for communicating emerging clinical standards and thereby improving patient outcomes.

Success and failure rates of tumor genotyping techniques in routine pathologic samples with non-small cell lung cancer.

8050 Background: Identification of somatic molecular alterrations in NSCLC has become evidence-based practice. The success and failure rate of using commercially-available tumor genotyping techniques in routine day-to-day NSCLC pathology samples is not well described. We sought to evaluate the success and failure rate of EGFR mutation, KRAS mutation and ALK FISH. Methods: Clinicopathologic data, tumor genotype success and failure rates were retrospectively compiled and analyzed from 381 patient-tumor samples sent for routine tumor genotype in clinical practice. Results: Mean age was 65, 61.2% women, 75.9% white, 27.8% never-smokers, 73.8% had advanced NSCLC and 86.1% adenocarcinoma histology. Tumor tissue was obtained from surgical biopsies in 48.8%, core biopsies in 17.9% and as cell blocks from aspirates/fluid in 33.3%. Anatomic sites for tissue collection included lung (49.3%), lymph nodes (22.3%), pleura (11.8%), bone (6.0%), brain (6.0%), among others. In the 207 tumors in which the three tests were ordered concurrently, the success rate for EGFR was 92.3%, for KRAS 91.8% and for ALK FISH 89.9%. The highest failure rates were observed when the tissue was obtained from core biopsies (30.8%, 20.5% and 30.8% for EGFR, KRAS and ALK tests, respectively) and bone specimens (23.1%, 15.4% and 23.1% for EGFR, KRAS and ALK tests, respectively). In specimens obtained from bone, the failure rate was significantly higher in non-surgical than surgical specimens (40% vs 0%, p=0.024 for EGFR) and decalcified than non-decalcified samples (60% vs 5.5%, p=0.021 for EGFR). Conclusions: The success rate of multiple tumor genomic analyses techniques for EGFR, KRAS and ALK gene abnormalities using routine lung cancer tissue samples was ~90%. The highest failure rates occurred in tumors obtained from core biopsies and in bone samples from core biopsies with decalcification; specimens that may need to be scrutinized before submission to molecular studies. Tumor genotype techniques are feasible in most other samples obtained with current tumor acquisition methods, and therefore expansion of routine tumor genotype into the care of patients with NSCLC may not require special tissue acquisition or manipulation.

Treatment patterns, biomarker testing, and outcomes in patients with nonsquamous non-small cell lung cancer (NSCLC) receiving second-line treatment in the US Oncology network.

e19139 Background: Non-small cell lung cancer (NSCLC) is the leading cause of cancer death in the US, with a 5-year survival rate of &lt; 20%. Treatment patterns and outcomes in late stage NSCLC in US community oncology practices are not well known. Methods: This retrospective study used the iKnowMed database, billing claims, and chart reviews. Eligibility criteria included non-squamous NSCLC, stage IIIB/IV at diagnosis, ECOG status &lt;3, and initiation of second-line therapy (defined as index date) between 1/1/2007-6/30/2011 with &gt; 1 year follow-up. Key outcomes were overall survival (OS), progression-free survival (PFS), time-to-progression (TTP), and time-to-hospitalization (from index date). Progression was defined as change in therapy and/or new metastatic lesion. Kaplan-Meier and Cox proportional hazard models were used to characterize the distribution and predictors of outcomes. Results: 1,168 patients were eligible for the study. The top three second-line therapies were pemetrexed (54.4%), erlotinib-containing regimens (17.6%), and docetaxel (10.0%). OS and PFS were 7.5 (95% CI: 6.6-8.4) and 4.1 (95% CI: 3.7-4.5) months, respectively; 57% of patients were hospitalized post-index date. EGFR testing rates were 2.3% pre-2010, 15.2% in 2010, and 32.0% in the studied 2011 period (p&lt;0.001). Of EGFR-positive patients, 50.0% received erlotinib-containing regimens compared to 16.9% of EGFR-negative patients (p=0.001). Baseline ECOG=1 (HR=1.49; p&lt;0.001) or ECOG=2 (HR=1.79; p&lt;0.001), compared to ECOG=0, existing co-morbid fatigue (HR=1.53; p=0.010) compared to no co-morbid fatigue, and progression (HR=1.78; p&lt;0.001), when it occurred, compared to no progression, increased risk of shorter time to hospitalization, adjusting for other factors. Compared to other second-line treatment, erlotinib-containing regimens prolonged adjusted TTP (HR=0.68; p=0.018). Conclusions: EGFR testing in community practice rose rapidly after 2010 suggesting improved personalized treatment given study evidence that EGFR inhibitors prolonged adjusted TTP. Also, ECOG ≥1 and progression, once it occurred, predicted shorter time-to-hospitalization.

Phase II, open-label study of erlotinib (ERL) in patients with NSCLC with activating mutations of the EGFR identified on tissue samples, plasma DNA, and CTC-trigger.

e19020 Background: Patients (pts) with advanced NSCLC and activating mutations of EGFR represent a biologically distinct disease that shows exquisite sensitivity to the reversible EGFR-TK inhibitors (TKIs) as ERL, demonstrated in phase III randomized studies. Unfortunately, tumor tissue needed for mutation analysis is in some cases insufficient for an accurate molecular characterization of the tumor before treatment and re-biopsies after treatment are challenging. Therefore it is crucial to develop new diagnostics techniques. Methods: Pts with histologically or cytologically confirmed stage IIIB/IV NSCLC, previously untreated with chemotherapy who present activating mutations of EGFR received ERL 150 mg/d until PD. The primary endpoint was 12-month PFS. Secondary endpoints included: ORR; OS; correlation between EGFR testing results obtained from basal tumor biopsies, from circulating tumor cells (CTC), from plasma DNA and by immunoistochemical detection; primary and secondary resistances and correlation to response and safety and tolerability. Results: Between 03-2011 and 11-2012, a total of 50 pts were enrolled from 7 centers. Median age was 63 years; 98% had adenocarcinoma, 54% had never smoked and 68% were female. Exon 19 deletions were more common (70%) than exon 21 (30%). EGFR screening methods applied at a site level were different: pyrosequencing (41%), Sanger (29%), SSCP (8%), Pyrosequencing and DNA melting (4%) and others (18%). Central evaluation confirmed the presence of an EGFR mutation in all cases. EGFR testing from CTC and from plasma DNA resulted feasible. EGFR mutations were identified by next generation sequencing in CTC preparations from 31 (84%) of 37 pts so far examined, corresponding to those present in matching tumor tissue. Treatment discontinuation was observed in 25 pts, mainly due to PD (30%) and death (10%). Results of plasma DNA and of immunoistochemical detection at baseline are ongoing as well as analysis on CTC and plasma DNA at progression. Conclusions: This trial confirms the feasibility of new diagnostics techniques and will help in improving understanding in the resistance mechanisms to EGFR TKIs. Clinical trial information: EudraCT Number: 2010-023892-24.

Clinical validation of mutational analysis of EGFR and KRAS in fine needle aspiration and small core needle biopsies using a real-time PCR method.

e19027 Background: About 75% of NSCLC is diagnosed at an advanced stage, thus analysis is often performed from small samples. There is a growing need for multiple biomarker testing when tissue is limited. We evaluated two real-time PCR based assays to assess EGFR and KRAS mutation status in limited tissue specimens and compared results to a lab validated method. Methods: 117 NSCLC patient samples (46 CNB, 71 FNA) were tested for EGFR and KRAS mutations using a single section. DNA was extracted directly from one stained smear in FNA samples (without pre-processing) and one 5-micron section in CNB using the cobas DNA Sample Preparation Kit. Sixty samples were also analyzed for both genes by direct sequencing using the same DNA. Eleven patients had matched CNB and FNA samples. Results: We tested 68 ADCs, 38 SqCCs, 8 NSCLC-NOS, 2 ADCs in situ non-mucinous, and 1 large cell carcinoma (Table). Median DNA concentrations for CNB and FNA were 14.17 ng/ul and 3.75 ng/ul. The failure rate for the cobas were 1.7% (EGFR), 6.8 % (KRAS) in FNA samples, and 0% for CNB. The failure rate for EGFR sequencing was 13.1% (FNA), 16.4% (CNB), and 0% for KRAS. The 11 patients with FNA and CNB showed concordant results between cobas and sequencing. One patient had two mutations; one (L782F) was detected only by sequencing. No discordant findings were seen other than invalid results comparing both systems. Conclusions: Assessment of mutations in limited tissue samples of CNB and FNA using a single section performed on the cobas EGFR and KRAS test is feasible and reliable. There was a lower invalid rate for EGFR testing on cobas test compared to Sanger sequencing. The cobas tests have rapid turnaround time to results, require limited input DNA, and are easier to use compared to sequencing. Still, it is mandatory that pathologists maintain careful quality control of the samples. [Table: see text]

Clinical impact of EGFR mutation fraction and tumor cellularity in EGFR mutation positive NSCLC.

e19077 Background: We investigated the impact of mutation fraction, tumour sample cellularity, and diagnostic specimen type on EGFR TKI response, time to treatment failure (TTF) and overall survival in advanced EGFR mutation positive NSCLC patients. Methods: From March 2010 to May 2012, EGFR testing in the province of Ontario (Canada) was conducted at a single centre, using fragment analysis for exon 19 deletion and Sau961 restriction enzyme digest for exon 21 mutation. Patients with EGFR mutation positive samples were identified, tumour cellularity, mutation fraction (percent tumour cells mutated) and clinical outcome data collected. Regression analysis was undertaken to assess the association between demographic variables, mutation fraction, tumour cellularity and sample type on clinical outcomes. Results: Of 173 patients identified to date, 153 received EGFR TKI and are included in this analysis, with median follow up of 10.6 months (range 0-44). Most are female (73%), never smokers (65%), have exon 19 deletions (55%; 45% exon21 L858R), and median age 64 years (range 34 to 95). Tumour specimens tested include resection (36%), cytology (32%), and core biopsy samples (32%). Median EGFR mutation fraction is 30% (range 1% to 96%); 18% had a low (&lt;10%) mutation fraction. Responses (any reduction) were seen in 66%, mixed response or stable disease in 28%, and progression in 7%. Median TTF of EGFR TKI therapy is 13.8 months. Median OS from TKI start is 24.1 months, with 1-, 2- and 3-year rates of 75%, 51% and 42%. In multivariable analysis, increasing mutation fraction was associated with EGFR TKI response (OR 2.5, p&lt;0.01), but not TTF or OS. Female sex was associated with TTF (HR 0.49, p=0.007), and testing of larger samples (lobectomy, wedge, excision) was associated with TTF and OS (HR 0.45, p=0.007; HR 0.26, p=0.0002). Exon 19 deletions are associated with a higher risk of death (HR 2.41, p=0.02) and a lower probability of response than L858R (OR 0.31, p=0.004). Conclusions: Pathologic features may be relevant to clinical outcomes in EGFR mutation positive NSCLC, including mutation fraction, sample cellularity, specimen tested and mutation type. Validation of the use of EGFR mutation fraction in clinical decision-making is ongoing.

Biomarker testing methods in breast, gastric, and lung cancers: A benchmarking survey of NCI cancer centers.

e22093 Background: Biomarkers are an integral part of cancer care. A variety of testing methods exist, allowing institutional selection of which technologies to use and in what order to run them. This descriptive study provides a benchmark on methods used in practice for biomarker testing in breast (BC), gastric/esophageal (GC), and non-small cell lung cancers (NSCLC). Methods: We conducted an IRB-approved web survey of the 58 NCI designated cancer centers (pathologists and oncologists) providing adult care in BC, GC, and NSCLC. We developed a survey instrument with 14 BC, 5 GC, and 10 NSCLC items asking about methods used, order of testing, turn around time (TAT) and if tests are internally developed (ID). Results: For BC, 32% (18/57) of sites run concurrent rather than reflex HER2 FISH and IHC testing, citing quality assurance reasons. Average TAT for concurrent vs reflex testing is 6.5 and 9.5 days. Rates of ID tests are 14% (8/57) for PgR, 12% (7/57) for ER and 7% (4/57) for HER2. For GC, 39% (21/54) of sites run concurrent HER2 FISH and IHC tests, citing reasons of quality assurance and evolving guidelines. TAT for concurrent vs reflex testing is 6.3 and 9.5 days. 19% (10/54) of sites use ID HER2 tests. All reporting sites use EGFR and ALK testing in NSCLC and 96% use KRAS. 24% (13/55) of sites run them concurrently with an average TAT of 7.6 days, while the average TAT for sites with sequential testing is 22.8 days. CAP recommends a 10 day TAT for NSCLC tests, and 92% (12/13) of sites with concurrent testing meet this guideline vs only 11% (4/38) of sites with sequential testing (p&lt;.0001). Rates of ID tests are 57% (30/53) for KRAS, 47% (26/55) for EGFR and 35% (19/55) for ALK, for which a kit is listed on the FDA drug label. There is a difference in the use of ID tests between BC and NSCLC, p&lt;.0148. Conclusions: Concurrent testing for BC and GC HER2, and for NSCLC markers is a common practice at NCI cancer centers; and it improves TAT. Concurrent NSCLC testing allows meeting CAP TAT guideline, but sequential testing does not. New markers (GC HER2, EGFR, KRAS and ALK) are commonly adopted, with a higher rate of internally developed tests than for BC HER2. [Table: see text]

Efficient detection of EGFR, KRAS, and BRAF mutations in cell-free DNA from pleural effusions.

e19151 Background: Detection of EGFR gene alterations is critical for predicting the response to tyrosine kinase inhibitors in patients with non-small-cell lung cancer. Analysis of other biomarkers might also be mandatory in the future, requiring more biological material. In routine practice, molecular testing is usually performed on tissues such as bronchial or transthoracic biopsies and surgical resections. We investigated the feasibility of using both cytology cell blocks and cell-free DNA from pleural effusions. Methods: Pleural effusion samples were centrifuged to form a pellet, fixed in formaldehyde, and processed as a cell block. DNA was extracted from 10-µm sections after paraffin removal using the Forensic kit and an iPrep system (Invitrogen). Cell-free DNA was extracted from the centrifugation supernatants using the QIAamp Circulating Nucleic Acid kit (Qiagen). Detection of molecular alterations were performed by allele-specific PCR (EGFR p.L858R mutation, BRAF p.V600E mutation), fragment size analysis (EGFR exon 19 deletions), and Sanger sequencing (KRAS mutations, codons 12 and 13). Results: Between January 2010 and December 2012, we tested 65 cell pellets (32 women and 33 men) and collected 34 paired supernatants. With the cell pellets, all the EGFR status were successfully determined (13 mutations; 20%), 9 mutations of KRAS on the 42 cases sequenced (17.6%), and 2 BRAF mutations on the 50 samples tested (4%) were found. We next investigated the potential use of the cell-free DNA from supernatants. Quantification by real-time PCR revealed significant amounts of DNA (mean = 306 ng extracted from 2 ml). EGFR, KRAS and BRAF tests were contributive for all these samples: 2 EGFR p.L858R mutations, 6 KRAS mutation (p.G12C, n=2; p.G12D, n=2; p.G13C, n=1 and p.G12F, n=1) and 1 BRAF p.V600E mutation were found. Most interestingly, the molecular status of the cell-free DNAs perfectly matched those obtained with the corresponding cell pellets. Conclusions: Pleural effusions provide high quality material for molecular testing and are suitable samples for routine practice. Cell-free DNA can also be successfully tested thus saving material for morphological / immunohistochemical / FISH analysis.

Small cell lung cancer (SCLC) among patients who are never smokers.

7593 Background: Although most patients (pts) with SCLC are current or former smokers, SCLC has been reported in pts who are never smokers, most recently in pts with EGFR-mutant lung cancers who develop acquired resistance (AR) to EGFR tyrosine kinase inhibitors (TKIs). We describe clinical, pathologic, and molecular characteristics of never-smoking pts with SCLC at diagnosis and in the AR setting. Methods: We identified cases through systematic review of pts seen at MSKCC from 2005 – 2012. Smoking history was obtained prospectively. SCLC diagnosis was confirmed by expert pathology review. We collected age, sex, stage, treatment, and survival data. EGFR, KRAS, PIK3CA, and ALK testing and next generation sequencing of 279 cancer genes was performed on available samples. Results: 2.2% (23/1040, 95% CI 1.5 to 3.3%) of pts with SCLC seen at MSKCC were never smokers: 61% women, median 64 years, 74% extensive stage, and 22% with brain metastases at diagnosis. 83% (19/23) had de novo SCLC, whereas only 17% had SCLC as AR to EGFR TKI after treatment for EGFR-mutant lung cancers, all of whom had persistent EGFR mutation confirmed at resistance. Median survival from SCLC diagnosis is 23 months (95%CI: 11-26) for all pts and 23 months (95% CI: 8–27) for the 19 pts with de novo SCLC. Pathologic review demonstrated 19 cases of pure SCLC and 4 mixed histology cases with SCLC and other histologies. Treatment history was available for 15/19 pts with de novo SCLC: 53% etoposide-platinum sensitive. ALK rearrangement and KRAS mutations were identified in 0/5 and 0/10, respectively. One pt with de novo mixed SCLC and adenocarcinoma had an EGFR mutation and another pt with de novo pure SCLC had EGFR and PIK3CA mutations. Mutations were identified in p53 and Rb1 with amplification in TERT in 1 sample to date tested with next generation sequencing. Conclusions: 2% of pts with SCLC are never smokers. While transformation to SCLC can occur in the setting of AR to EGFR TKI, de novo SCLC occurs in the majority of our never smokers with this disease. EGFR mutations uniformly exist in SCLC in the AR setting. EGFR mutations were rare, and we found no KRAS mutations or ALK rearrangements. Comprehensive, multiplexed genotyping can aid in providing optimal care and facilitate research in this unique population.

Epidermal growth factor receptor mutation (EGFRMUT) in non-small cell lung cancer (NSCLC) in the Middle East.

e19035 Background: EGFR mutation is an important therapeutic marker for the management of NSCLC with known ethnic variations. Our study aims at determining the prevalence of the mutation in the Middle East and its correlation with demographic and clinical features. Methods: A multisite retrospective study was conducted including institutions from Saudi Arabia, UAE, Qatar and Egypt. All consecutive patients (pts) with NSCLC tested for EGFRMUT were eligible. Data were obtained on demographic and disease characteristics, and EGFR test results. Institutional Review Board approval was obtained from all participating sites. Results: Data on 201 patients were obtained. Median age was 61 years (51 - 69), 140 (69.65%) were males and 174 pts were Arab (86.57%) Tumor histology subtypes were adenocarcinoma in 175 patients (87.07%), squamous cell carcinoma in 15 pts (7.46%) and others in 11 pts (5.48%). EGFRMUT was detected in 66 pts, with (36.50%) prevalence in adenocarcinoma. No squamous cell carcinoma harbored EGFRMUT. The most frequent abnormality detected was in exon 19 in 39 pts (57.36%), abnormalities in exon 21 in 22 pts (32.35%) and in exon 18 in 7 pts (10.29%). Two pts had dual mutations on exons 19 and 21. Univariate and multivariate analyses revealed significant predictors for having EGFRMUT to be: female gender (42.42 % vs. 27.7% in males, p= 0.0096), non-smoking status (81.82% vs. 16.21% in smokers, p=0.0001) and adenocarcinoma subtypes (36.25 % vs. 15.38% in others, p=0.035). There was no difference between Arabs and non-Arabs. Conclusions: This is the first study from our region to report the prevalence of EGFRMUT in ME population which seems to be similar to Far East population with similar clinical predictors. Studies about the impact of targeted therapy in our patient’s population are underway.