Common EGFR Research Articles

Abstract LB-020: Characterization of a novel BRAF in-frame deletion reveals a distinct mutational activating mechanism for oncogenic kinases

Abstract Kinase domain mutations are frequent drivers of many different types of cancer. While extensive studies of kinases such as BRAF and EGFR have provided insight into the mechanistic basis for many oncogenic point mutations such as the commonly occurring BRAFV600E and EGFRL858R mutations, the activation mechanism of short, in-frame deletions (such as the common EGFR exon19 deletion in non-small cell lung cancer) remains to be fully understood. In this work, we have discovered the activation mechanism for novel in-frame deletions in BRAF that have been reported in a small subset of thyroid cancers (TCGA Thyroid data set). These BRAF deletions structurally align with the EGFR exon19 deletion (most common being ΔELREA) and a similar deletion recently identified in HER2-positive breast tumor samples (ΔLRENT). These deletions target the β3-αC loop within the N-lobe of the kinase domain, which provides essential flexibility to the αC-Helix allowing the kinase to toggle between inactive and active conformations. Using molecular modeling studies, we demonstrate that all of these deletions genetically compromise this flexibility and constitutively “snap” the αC-Helix in the active conformation. Further characterization of the BRAF deletions shows that similar to BRAFV600E mutations, the β3-αC deletions are kinase-activating, CRAF-independent, and confer dimer-independent activity. Interestingly, cells with this deletion are sensitive to BRAF kinase inhibitors that bind to the active conformation (such as GDC-0879), but are innately resistant to the BRAF kinase inhibitor vemurafenib (which binds BRAF in the αC-Helix inactive, outward-shifted conformation). Similarly, modeling predicts all known β3-αC deletions would confer innate resistance to αC-Helix outward-shifting drugs such as vemurafenib or lapatinib. Further, we speculate that a β3-αC deletion could confer acquired resistance in vemurafenib-treated or lapatanib-treated patients. While many of the more common oncogenic point mutations have been suggested to function in part by destabilizing the inactive conformation and hence promoting the active conformation, it is becoming increasingly clear that oncogenic events that alter the αC-Helix region to directly promote the active conformation provide an alternative mechanism of kinase activation in cancer. Taken together our work underscores the importance of conformation specific kinase inhibitors to target mutationally activated kinases in cancer. Citation Format: Scott Foster, Aysegul Ozen, Dan Whalen, Kyung Song, Georgia Hatzivassiliou, Sarah Hymowitz, Nick Skelton, Shiva Malek. Characterization of a novel BRAF in-frame deletion reveals a distinct mutational activating mechanism for oncogenic kinases. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-020. doi:10.1158/1538-7445.AM2015-LB-020

Abstract 563: Evaluation of EGFR mutation analysis performed by Sanger sequencing versus real-time PCR (EGFR RGQ PCR Kit)

Abstract EGFR mutation analysis of exons 18, 19 and 21 is a standard analysis in routine molecular pathology for prediction of response of EGFR-tyrosine kinase inhibitors (EGFR-TKI) in non-small cell lung cancer (NSCLC). The aim of this study was to evaluate the mutation detection using two widely used mutation analysis systems. For this purpose we analyzed exons 18, 19 and 21 of EGFR in 718 NSCLC with both nested PCR followed by direct Sanger sequencing and the EGFR RGQ PCR Kit (Qiagen). 694/718 samples were amplifiable and 676 samples allowed interpretable results by both methods. We found mutations in 74/676 (10,1%) samples by Sanger sequencing and in 91/676 (13,0%) using the RGQ Kit. There were 10/676 (1,5%) cases showing mutations only by Sanger Sequencing but not by the RGQ Kit. These mutations comprised four common EGFR-TKI sensitivity mutations in Exon21 (p.L858R (n = 2) and p.L861Q (n = 2)) as well as a complex deletion-insertion in exon 19 (n = 1). The other 5 mutations detected only by Sanger sequencing were uncommon and very rare mutations located in exon 18 and 21 which cannot be detected by the RGQ Kit due to its primer design. Vice versa, we found mutations in 21/676 samples using the RGQ Kit but not with Sanger sequencing. 20 of these mutations were common EGFR-TKI sensitivity mutations in exon 19 (deletions or complex delins, n = 15) and in exon 21 (p.L858R, n = 5) as well as a “p.G719X” mutation (n = 1) in exon 18. According to these data which includes mutations only in exons 18, 19 and 21 but not in exon 20 the mutation detection rate is higher using the RGQ-Kit than using the Sanger sequencing method (13,0% vs. 10,1%) especially in detecting common EGFR mutations associated with EGFR-TKI sensitivity. However the RGQ Kit fails in detecting uncommon and rare EGFR mutations for that this Kit is not designed and may produce non-informative results in some cases when ΔCT values are out of valid ranges whereas nested PCR followed by Sanger sequencing may still work. Citation Format: Wolfgang Dietmaier, Irene Schardt, Petra Rümmele, Eva Geissinger, Arndt Hartmann, Robert Stöhr. Evaluation of EGFR mutation analysis performed by Sanger sequencing versus real-time PCR (EGFR RGQ PCR Kit). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 563. doi:10.1158/1538-7445.AM2015-563

EGFR mutation pattern in African American population in a community-based academic center.

8069 Background: The two most common EGFR mutations, L858R in exon 21 and deletions in exon19, represent around 90% of all EGFR mutations in NSCLC and convey sensitivity to EGFR TKIs. Little is kno...

Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Treatment Response in Advanced Lung Adenocarcinomas with G719X/L861Q/S768I Mutations

Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) is the standard therapy for advanced lung adenocarcinomas with common EGFR mutations. Preclinical studies have suggested that uncommon G719X, L861Q, and S768I mutations are also sensitive to EGFR-TKIs. However, the efficacy of EGFR-TKIs in patients with these uncommon mutations remains unclear. A nationwide survey was performed to collect data from gefitinib and erlotinib treatment outcomes of patients with stage IIIB/IV lung adenocarcinoma bearing EGFR G719X/L861Q/S768I mutations. The results were compared with those regarding patients with exon 19 deletions or L858R mutations. One hundred and sixty-one patients with uncommon EGFR mutations were enrolled from 18 institutes throughout Taiwan. Mutations of G719X, L861Q, S768I, G719X + L861Q, and G719X + S768I were observed in 78, 57, 7, 9, and 10 patients, respectively. After receiving EGFR-TKI treatment, patients with uncommon mutations exhibited a significantly inferior tumor response rate (41.6% vs. 66.5%; p < 0.001) and progression-free survival (median, 7.7 vs. 11.4 months; p < 0.001) than patients with common mutations. Among the patients who used EGFR-TKIs as first-line treatment, there was a significant difference in overall survival between these two groups of patients (median, 24.0 vs. 29.7 months; p = 0.005). Gefitinib and erlotinib are active in patients with G719X/L861Q/S768I mutations; however, less effective than in those with common mutations.

The impact of common and rare EGFR mutations in response to EGFR tyrosine kinase inhibitors and platinum-based chemotherapy in patients with non-small cell lung cancer.

In non-small cell lung cancer (NSCLC), the association between common EGFR mutations (Del EX19/L858R) with EGFR tyrosine kinase inhibitors (EGFR-TKIs) has been well established. However, this has not been investigated for rare EGFR mutations or their impact on treatment response and outcome to EGFR TKIs (primary objective) and chemotherapy (secondary objective). In an observational prospective cohort, we analyzed 188 NSCLC patients from Mexico, Colombia and Costa Rica with EGFR mutations. As a first line of treatment, 66.5% received platinum-based chemotherapy. All patients received TKIs in first-line treatment or after progression to chemotherapy. The clinical-pathological characteristics as well as the f of common and rare EGFR mutations associated with treatment response were analyzed. Of all patients, 79.5% had common and 20.5% had rare EGFR mutations. Lepidic and acinar adenocarcinomas were associated with common EGFR mutations (p=0.010). Patients with common EGFR mutations had higher response rates to EGFR-TKIs than those who had rare EGFR mutations (63.8 vs 32.4%, p<0.001). Women had increased progression-free survival (PFS) to EGFR-TKIs than men (16.4 vs 9.5 months, p=0.02). The median PFS and overall survival (OS) were better in patients with common EGFR mutations (15.5 vs 3.9 months, p<0.001; and 37.3 vs 17.4 months, p<0.001) respectively. Our findings suggested that only patients with rare EGFR mutations could receive platinum-based chemotherapy as a first-line treatment, due to their low response rates and short PFS in response to EGFR-TKIs. Consequently, EGFR-TKIs could be reserved as a second- or third-line treatment. In patients with EGFR mutations, women have better PFS to EGFR-TKIs than men, and rare EGFR mutations are more frequent in high grade adenocarcinomas than in low grade tumors.

The Importance of Brain Metastasis in EGFR Mutation Positive NSCLC Patients.

Introduction. Brain metastasis is a poor prognostic marker in lung cancer. However it is not known whether amongst patients with EGFR mutation those with brain metastases have a worse outcome. Methods. We compared the survival outcomes between EGFR mutation positive patients with and without brain metastases. In this retrospective analysis of prospective database of all metastatic lung cancer patients at our centre between July 2009 and December 2012, patients were treated with either combination chemotherapy or oral TKI. All patients with brain metastases received whole brain radiation. Kaplan Meier method was used for survival analysis and compared using log rank test. Results. 101 patients with EGFR mutated, metastatic lung cancer were studied. Fourteen had brain metastases and 87 did not. The common EGFR mutations were exon 19 deletion (61.3%) and exon 21 L858R mutation (28.7%). Overall response was 64% in extracranial metastasis group as compared to 50% in brain metastasis group. There was a significant worsening of median OS in the patients with brain metastases (11.6 months) compared with only extracranial metastases (18.7 months), P = 0.029. Conclusion. Amongst patients with EGFR mutant NSCLC, the presence of brain metastases leads to a worse outcome as compared to patients with extracranial metastases alone.

Cardiac Safety of Afatinib: Review of Clinical Trial Data: Metastatic Non-Small Cell Lung Cancer

Afatinib, an oral irreversible ErbB family blocker that inhibits EGFR, HER2, and HER4, is FDA-approved for treating metastatic non-small cell lung cancer (NSCLC) with common EGFR mutations (DEL19/L858R). Trial LUX-Lung 3 [LL3] showed a 13.6 month progression-free survival benefit for afatinib vs 6.9 months with cisplatin/pemetrexed in this population (P=.001). As anti-HER2 therapies have been associated with cardiac dysfunction, we assess cardiac events from 2 phase 3 trials of afatinib and a pooled analysis of additional trials.

Overall Survival (OS) With Afatinib Versus Chemotherapy in Patients (Pts) With Advanced Non-Small Cell Lung Cancer (NSCLC) Harboring Common (Del19/L858R) Epidermal Growth Factor Receptor Mutations (EGFR Mut): Results of LUX-Lung 3 (LL3) and LUX-Lung 6 (LL6): Metastatic Non-Small Cell Lung Cancer

Two trials LL3 (global) and LL6 (Asia) previously demonstrated significant improvements in the primary endpoint of progression-free survival with first-line afatinib (A) vs standard chemotherapy (CT) in pts with EGFR mut-positive NSCLC. In pts with common EGFR mut (Del19/L858R), the HR was 0.47 vs pemetrexed/cisplatin (LL3) and 0.25 vs gemcitabine/cisplatin (LL6). Here, we present the mature OS analysis in these pts from both trials and OS outcomes in non-Asian pts.

187P - Common and Rare Egfr Mutations in Non-Small Cell Lung Cancer

ABSTRACT Background: In non-small cell lung cancer (NSCLC), the association between common EGFR mutations (Del EX19/ L858R) with EGFR tyrosine kinase inhibitors (EGFR-TKIs) has been well established. Not so for rare EGFR mutations, not even their impact on treatment response to platinum-based chemotherapy. Methods: We analyzed 188 NSCLC patients’ harbouring EGFR mutations from Mexico, Colombia and Costa Rica. Patients were treated according to international guidelines for the treatment of lung cancer. As first line treatment, 64.1% receive platinum-based chemotherapy and 30.8% receive EGFR-TKIs, after chemotherapy progression. Clinical-pathological characteristics and presence of common and rare EGFR mutations regarding treatment response were analysed. Protocol is registered with ClinicalTrials.gov, number NCT01023828. Results: Of all patients, 79.5% had common and 20.5% had rare EGFR mutations. Lepidic and acinar adenocarcinoma were associated with common EGFR mutation (p = 0.010). Patients who harboured common EGFR mutations had higher response rates to EGFR-TKIs versus those who had rare EGFR mutations (63.8% cf 32.4%, p Conclusions: Our findings suggested that patients who harboured rare EGFR mutations only could receive platinum-based chemotherapy as first-line treatment and EGFR-TKIs could be reserved as second- or third-line treatment. Disclosure: All authors have declared no conflicts of interest.

Common and Rare Egfr Mutations (Egfrm) in the Radiant Trial

Common and Rare Egfr Mutations (Egfrm) in the Radiant Trial

Updated Analysis of Response and Patient-Reported Outcomes (Pro) in Two Large Open-Label, Phase III Studies (Lux-Lung 3 [Ll3] and Lux-Lung 6 [Ll6]) of Afatinib (A) Versus Chemotherapy (Ct) in Patients (Pts) with Advanced Nsclc Harboring Egfr Mutations (Mut)

ABSTRACT Aim: A, an oral, irreversible ErbB family blocker of EGFR, HER2, ErbB3 and ErbB4 signalling, improved progression-free survival (PFS), showed higher objective response rates (ORR), faster time to response (TTR) and favourable trends in PROs versus cisplatin/pemetrexed (LL3; 345 pts recruited globally) and cisplatin/gemcitabine (LL6; 364 Asian pts) in treatment-naive pts with stage IIIB/IV EGFR mut positive NSCLC. We present an updated analysis of PFS, response and PRO data. Methods: Pts were randomized 2:1 to 40 mg A or up to 6 cycles of CT. The primary endpoint was PFS; secondary endpoints included ORR and PROs. PROs were assessed by EORTC QLQ-C30 and QLQ-LC13 questionnaires from randomization until disease progression. Updated PFS, response and PRO analyses were completed at the time of primary OS analysis, during which 21 (LL3) and 23 (LL6) pts were still on A treatment. Results: Updated PFS analysis confirmed superiority of A in both trials. ORR, TTR and PRO analyses were consistent with earlier findings. ORR was 57% [A] vs 23% [CT] in LL3; 68% [A] vs 23% [CT] in LL6. Of those with OR, pts treated with A responded faster than those treated with CT, with response reported at the time of first imaging (week 6) in 72% vs 58% in LL3 and 73% vs 57% in LL6. Worsening of cough and dyspnea was significantly delayed with A vs CT. A greater magnitude of effect on PROs with A was seen in pts with common EGFR mut. Median time to deterioration (months) in symptoms of cough, dyspnea and pain LL3 LL6 A C/P HR; p-value A G/C HR; p-value All randomised Cough 27.0 8.0 0.59; 0.006 31.1 10.3 0.46; Dyspnea 10.4 2.9 0.68; 0.013 7.7 1.7 0.53; Pain 4.2 3.1 0.83; 0.188 6.9 3.4 0.70; 0.022 Common mutations Cough Not estimable 10.2 0.51; 0.001 31.1 Not estimable 0.43; Dyspnea 14.5 2.7 0.54; 8.3 1.7 0.53; Pain 4.8 3.1 0.74; 0.052 6.4 2.7 0.67; 0.016 Conclusions: In addition to significant delay in disease progression, A induces a superior and faster response in pts with EGFR mut NSCLC and leads to long-lasting control and delay in worsening of lung cancer symptoms vs CT. Disclosure: L.V. Sequist: Uncompensated Advisory boards for: Boehringer Ingelheim, AstraZeneca, Merrimack, Novartis, Taiho; J. Feng: Corporate sponsored research for Boehringer Ingelheim; S. Lu: Advisory boards for: AstraZeneca and Boehringer Ingelheim; M. Schuler: Advisory boards for AstraZeneca, Boehringer Ingelheim, Novartis, Pfizer Corporate sponsored research for Boehringer Ingelheim, Novartis Other substantive relationships with University Duisburg-Essen (Patents); T. Mok: Advisory boards for AZ, Roche, Eli Lilly, Merck Serono, Eisai, BMS, AVEO, Pfizer, Taiho, BI, Novartis, GSK Biologicals, Clovis Oncology, Amgen, Janssen, BioMarin Pharmaceutical Board of directors on IASLC Corporate sponsored research for AZ; N. Yamamoto: Other substantive relationships with TAIHO, ONO, CHUGAI, Pfizer, Eli Lilly, AstraZeneca, Boehringer Ingelheim; K.J. O'Byrne: Advisory Board, Travel expenses and Honoraria for: Boehringer Ingelheim; V. Hirsh: Advisory board for: Boehringer Ingelheim; C. Zhou: Advisory boards for Boehringer Ingelheim, F. Hoffmann-La Roche, Eli Lilly; D. Massey: Employee of Boehringer Ingelheim Ltd; J. Lungershausen: Employee of Boehringer Ingelheim GmbH; J. Yang: Advisory boards for Astrazeneca, Boehringer Ingelheim, Roche/Genentech, Merck Serono, Pfizer, Clovis Oncology, Novartis, Eli Lilly, Takeda, Innopharma, Bayer Corporate sponsored research for Boehringer Ingelheim. All other authors have declared no conflicts of interest.

Detection of Common and Less Frequent EGFR Mutations in Cytological Samples of Lung Cancer

Objective: Lung cancer represents the leading cause of cancer death. EGFR mutations, detected in 10-40% of lung adenocarcinomas, are an essential key to therapeutic management. EGFR-activated mutations comprise mainly deletions in exon 19 and point mutations in exon 21. Although histology is the traditional method of detection, we investigated the role of cytology in EGFR mutations. Study Design: A total of 774 lung cancers were studied for EGFR mutations (676 histological and 98 cytological samples), including 424 adenocarcinomas, 326 non-small cell lung carcinomas not otherwise specified, and 24 squamous cell carcinomas. Results: We had a total of 164 (21.2%) cases of mutations. Common mutations were short in-frame deletions in exon 19 (53.7%) and single-nucleotide substitutions in exon 21 (34.1%); less frequent mutations included single-nucleotide substitutions in exon 18 (3.7%) and in-frame insertions/deletions in exon 20 (8.5%). Histologically, EGFR mutations in exons 19 and 21 occurred in 19.4% and in exons 18 and 20 in 2.2%, while the rates cytologically were 13.3% for exons 19 and 21 and 5.1% for exons 18 and 20. Conclusions: The sensitivity for the detection of EGFR mutations in cytological samples overlaps histology, so the use of cytological material constitutes an adequate approach for treatment selection in patients with locally advanced or metastatic lung cancer.

Overall survival (OS) in patients (pts) with advanced non-small cell lung cancer (NSCLC) harboring common (Del19/L858R) epidermal growth factor receptor mutations (EGFR mut): Pooled analysis of two large open-label phase III studies (LUX-Lung 3 [LL3] and LUX-Lung 6 [LL6]) comparing afatinib with chemotherapy (CT).

8004^ Background: Afatinib (A) is an oral, irreversible ErbB family blocker of EGFR, HER2, ErbB3 and ErbB4 signalling. LL3 compared A with cisplatin/pemetrexed in 345 pts recruited globally and LL6 compared A with gemcitabine/cisplatin in 364 Asian pts. The primary analysis (2012) showed improved progression-free survival (PFS) with A versus CT in the overall EGFR mut positive population (HR=0.58 [LL3], HR=0.28 [LL6]) and pts with common (Del19/L858R) EGFR mut (HR=0.47 [LL3], HR=0.25 [LL6]). The FDA has approved A for the first-line treatment of pts with advanced NSCLC harboring common EGFR mut. Here we present a pooled analysis of mature OS data among such pts. Methods: Treatment-naive pts with EGFR mut stage IIIB/IV NSCLC were randomized 2:1 to 40 mg A or up to 6 cycles of standard CT and stratified by EGFR mut and race (LL3). The primary endpoint was PFS, with OS as a key secondary endpoint. Adverse events were also recorded. Results: The pooled analysis included 631/709 pts randomized into LL3 and LL6...

Cardiac safety of afatinib: Review of clinical trial data.

e19037 Background: Afatinib, an oral irreversible ErbB family blocker, is approved for treating non-small cell lung cancer (NSCLC) with common EGFR mutations (DEL19/L858R). As anti-HER2 therapies h...

The application of real-time PCR technique to detect rare cell clones with primary T790M Substitution of EGFR gene in metastases of non-small cell lung cancer to central nervous system in chemotherapy naive patients.

The time-limited efficacy of reversible EGFR-TKIs in patients with advanced non-small cell lung cancer (NSCLC) with EGFR gene activating mutations is associated with development of treatment resistance after some period of therapy. This resistance predominantly results from secondary mutations located in EGFR gene, especially T790M substitution. There is limited information available concerning the prevalence of primary T790M mutations in patients with metastatic NSCLC tumors before treatment with EGFR-TKIs. The aim of work was to assess the prevalence of de novo T790M mutations in EGFR gene in tissue samples from NSCLC metastatases in central nervous system (CNS) in both chemotherapy and EGFR-TKI naive NSCLC patients. We analyzed DNA samples isolated from paraffin-embedded tissue from CNS metastases for T790M mutations using real-time PCR and TaqMan probe against the T790M mutant sequence. The tissue samples were taken during palliative neurosurgery in 143 NSCLC patients. Amplification of the T790M-specific sequence was detected in 25 patients (17.5 %). The quantity of mutated DNA was less than 1 % in all samples with amplification, and in vast majority (20 patients, 14 % of all samples) it was even less that 0.1 %. In 5 patients (3.5 %) quantity of mutated DNA ranged from 0.1 to 1 % and true positive results of T790M mutation presence in these patients were most possible. Amplification of this sequence was not concurrent with common EGFR mutations and was not associated with sex, smoking status and pathological type of cancer. There is a possibility to detect the primary T790M mutation in brain metastases of NSCLC in EGFR-TKIs naïve patients.

The Relationship Between Common EGFR, BRAF, KRAS Mutations and Prognosis in Advanced Stage Non-Small Cell Lung Cancer with Response to the Treatment in Turkey.

The aim of the study was to evaluate EGFR (exon 19 deletion, exon 21 L858R point mutation), KRAS and braf mutation rates besides their relationship with survival and response to the treatment in non small cell lung cancer (NSCLC). We evaluated 513 NSCLC patients followed-up between January 2004 and November 2009 according to our registration data, retrospectively. Only 42 advanced stage NSCLC patients had enough tumor tissue material in paraffin blocks for all mutation analysis. The patients were evaluated retrospectively for clinicopathological features, EGFR, KRAS and BRAF mutations, erlotinib treatment, time to progression (TTP) and overall survival (OS). Mutation rates were as 7.14% (two patients) for EGFR exon 19 deletion; 4.76% (one patient)for KRAS codon 61 deletion and 2.38% for BRAF V600E mutation. They had neither EGFR exon 21 point mutation nor different mutations together. Median follow-up was 26 months (5-83) for all patients. It was 43 months (23-83) for the patients who had erlotinib and 23 months (5-61) for those who did not. Ten (23.8%) patients had erlotinib. There was significant survival difference between the patients taking erlotinib and the others (28 ± 3 months versus 15 ± 4 months, p= 0.05). TTP and OS were longer in the patients who had mutations, however the difference was not significant (p= 0.119 and p= 0.06). To our knowledge, this is the first study evaluating EGFR, KRAS and BRAF mutations in advanced stage NSCLC in Turkey .

12 VEGF autocrine survival signalling is mediated via neuropilin 1 receptor in NSCLC cells

Methods: Data on all tested patients was prospectively collected from the inauguration of the EGFR genetic service. There were three specific aims in the study. First, did the outcomes in an off-trial population with an often poor socio-economic status match those reported in a clinical trial population? Second, did the Scottish prevalence of common EGFR mutations reflect what has been described internationally? Finally, were there any interesting EGFR genetic variants and what was their potential response to erlotinib? Results: Between July 2012 and June 2013, 49 EGFR mutations were detected from 488 patients tested (10.04%) who had a new diagnosis of first line metastatic non-squamous NSCLC. Of those mutated cancers, 34 had a tyrosine kinase inhibitor (TKI) sensitising variant, 4 patients had a TKI resistance variant, 4 had both a TKI sensitising and resistance mutation, and 7 had mutations of uncharacterised clinical significance. One patient with a c.2543C>T; p.Pro848Leu mutation demonstrated a clinical response after two months of erlotinib treatment, a genetic variant with no prior description of benefit from EGFR inhibition. Conclusions: Interesting genetic EGFR variants have been identified in the west of Scotland NSCLC population, one of which has demonstrated a previously uncharacterised clinical response to erlotinib. To date, prevalence of EGFR mutation and response to TKI treatment in the west of Scotland has reflected that reported in an international trial population.

Common and Rare EGFR and KRAS Mutations in a Dutch Non-Small-Cell Lung Cancer Population and Their Clinical Outcome

IntroductionIn randomly assigned studies with EGFR TKI only a minor proportion of patients with NSCLC have genetically profiled biopsies. Guidelines provide evidence to perform EGFR and KRAS mutation analysis in non-squamous NSCLC. We explored tumor biopsy quality offered for mutation testing, different mutations distribution, and outcome with EGFR TKI.Patient and MethodsClinical data from 8 regional hospitals were studied for patient and tumor characteristics, treatment and overall survival. Biopsies sent to the central laboratory were evaluated for DNA quality and subsequently analyzed for mutations in exons 18–21 of EGFR and exon 2 of KRAS by bidirectional sequence analysis.ResultsTumors from 442 subsequent patients were analyzed. For 74 patients (17%) tumors were unsuitable for mutation analysis. Thirty-eight patients (10.9%) had EGFR mutations with 79% known activating mutations. One hundred eight patients (30%) had functional KRAS mutations. The mutation spectrum was comparable to the Cosmic database. Following treatment in the first or second line with EGFR TKI median overall survival for patients with EGFR (n = 14), KRAS (n = 14) mutations and wild type EGFR/KRAS (n = 31) was not reached, 20 and 9 months, respectively.ConclusionOne out of every 6 tumor samples was inadequate for mutation analysis. Patients with EGFR activating mutations treated with EGFR-TKI have the longest survival.

Abstract 4227: Simple identification of specific mutations using allele-specific PCR primers and micro capillary electrophoresis (WAVE MCE).

Abstract Transgenomic, Inc. has developed a size-based analysis for the detection of all EGFR Exon 19 deletions (including complex mutation) greater than 6 base pairs (ACE™ Kit EGFR Exon 19). Currently there are over 70 known EGFR Exon 19 deletions in the region important for response of non-small cell lung cancer (NSCLC) to thymidine kinase inhibitors (TKIs). The majority of these deletions are rare, but the patients may benefit from TKI treatment if the deletion is present. In most current testing paradigms, a NSCLC patient with one of these rare mutations would be considered EGFR mutation negative and not receive TKI therapy. We have designed an easy, size-based PCR assay to detect any of the deletions present in any sample after a single PCR reaction. The assay is based on the separation of PCR fragments of different sizes using microcapillary electrophyoresis (MCE). Transgenomic's WAVE® MCE platform can discriminate the deletions from the wild-type sequence in EGFR Exon 19, however the exact nature of the mutation cannot be determined. If the exact nature of the mutation is required, the PCR fragment can be sequenced. To confirm that the platform can detect common and rare mutations, plasmids containing various EGFR Exon 19 deletions were synthesized, mixed with EGFR wild-type plasmids and analyzed on the WAVE® MCE platform. The Limit of detection for the plasmid mixing experiments is between 0.5 and 1% for all plasmid constructs tested. These plasmids include 6, 9, 15, 21, 24, and 25 bp deletions . As all samples tested have some wild-type sequence present, 6 base pair deletions cannot be resolved from the wild-type fragment. Several of the deletion constructs contain those not detected by any allele-specific kit, however patients with these deletions, as determined by Sanger sequence analysis, have benefitted from TKI therapy. In addition cell line DNA and DNA isolated from FFPE sections have been analyzed for the detection of EGFR Exon 19 deletions and similar limits of detection (0.5-1.0%) were seen. This simple, size-based analysis for EGFR Exon deletions will result in NSCLC patients with both common and rare EGFR Exon 19 deletions being able to receive the treatment option best suited for their molecular profile. Citation Format: Katherine A. Richardson, Grant Wu, Rui Lin, Yanggu Shi, Phil Eastlake, Benjamin Legendre. Simple identification of specific mutations using allele-specific PCR primers and micro capillary electrophoresis (WAVE MCE). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4227. doi:10.1158/1538-7445.AM2013-4227

Molecular diagnostic alterations in squamous cell carcinoma of the head and neck and potential diagnostic applications

Head and neck squamous cell carcinoma (HNSCC) is a common malignancy that continues to be difficult to treat and cure. In many organ systems and tumor types, there have been significant advances in the understanding of the molecular basis for tumorigenesis, disease progression and genetic implications for therapeutics. Although tumorigenesis pathways and the molecular etiologies of HNSCC have been extensively studied, there are still very few diagnostic clinical applications used in practice today. This review discusses current clinically applicable molecular markers, including viral detection of Epstein-Barr virus and human papillomavirus, and molecular targets that are used in diagnosis and management of HNSCC. The common oncogenes EGFR, RAS, CCND1, BRAF, and PIK3CA and tumor suppressor genes p53, CDKN2A and NOTCH are discussed for their associations with HNSCC. Discussion of markers with potential future applications is also included, with a focus on molecular alterations associated with targeted therapy resistance.