BRAF Hotspot Mutations Research Articles

Circulating tumor DNA (ctDNA) detection is associated with shorter progression-free survival in advanced melanoma patients

BRAF, NRAS and TERT mutations occur in more than 2/3 of melanomas. Its detection in patient’s blood, as circulating tumor DNA (ctDNA), represents a possibility for identification and monitoring of metastatic disease. We proposed to standardize a liquid biopsy platform to identify hotspot mutations in BRAF, NRAS and TERT in plasma samples from advanced melanoma patients and investigate whether it was associated to clinical outcome. Firstly, we performed digital polymerase chain reaction using tumor cell lines for validation and determination of limit of detection (LOD) of each assay and screened plasma samples from healthy individuals to determine the limit of blank (LOB). Then, we selected 19 stage III and IV patients and determined the somatic mutations status in tumor tissue and track them in patients’ plasma. We established a specific and sensitive methodology with a LOD ranging from 0.13 to 0.37%, and LOB ranging from of 0 to 5.201 copies/reaction. Somatic mutations occurred in 17/19 (89%) patients, of whom seven (41%) had ctDNA detectable their paired plasma. ctDNA detection was associated with shorter progression free survival (p = 0.01). In conclusion, our data support the use of ctDNA as prognosis biomarker, suggesting that patients with detectable levels have an unfavorable outcome.

The tumor genetics of acral melanoma: What should a dermatologist know?

Dermatologists stand at the gateway of individualization of classification, treatment, and outcomes of acral melanoma patients. The acral melanoma genetic landscape differs in vital ways from that of other cutaneous melanomas. These differences have important implications in understanding pathogenesis, treatment, and prognosis. The selection of molecularly targeted therapy must be adapted for acral melanoma. It is also critical to recognize that tumor development is far more complex than an isolated event, reliably treated by a medication acting on a single target. Tumors exhibit intratumor genetic heterogeneity, metastasis may have different genetic or epigenetic features than primary tumors, and tumor resistance may develop because of the activation of alternative genetic pathways. Microenvironmental, immune, and epigenetic events contribute and sustain tumors in complex ways. Treatment strategies with multiple targets are required to effectively disrupt the tumor ecosystem. This review attempts to translate the current molecular understanding of acral melanoma into digestible concepts relevant to the practice of dermatology. The focus is tumor genetics defining potentially treatable cancer pathways, contextualized within the relevant pathologic and molecular features.

Simultaneously Inhibiting BCL2 and MCL1 Is a Therapeutic Option for Patients with Advanced Melanoma.

There is an urgent need to develop treatments for patients with melanoma who are refractory to or ineligible for immune checkpoint blockade, including patients who lack BRAF-V600E/K mutations. This is often the case in patients diagnosed with rare melanoma subtypes such as mucosal and acral melanoma. Here, we analyzed data from the cutaneous melanoma The Cancer Genome Atlas Network (TCGA) transcriptomic and proteomic databases for differential expression of apoptosis molecules between melanomas with or without BRAF hotspot mutations. Our data indicated higher B-cell CLL/lymphoma 2 (BCL2) expression in melanoma without BRAF hotspot mutations, suggesting that BH3 mimetics, such as ABT-199 (venetoclax, a small molecule against BCL2), may be a potential therapeutic option for these patients. We explored the efficacy of combining two BH3 mimetics, ABT-199 and a myeloid cell leukemia sequence 1 (MCL1) inhibitor (S63845 or S64315/MIK665) in cutaneous, mucosal and acral melanomas, in vitro and in vivo. Our data indicate this combination induced cell death in a broad range of melanoma cell lines, including melanoma initiating cell populations, and was more potent in melanoma cells without BRAF-V600E/K mutations. Our knockdown/knockout experiments suggest that several pro-apoptotic BCL2 family members, BCL2-like 11 (apoptosis facilitator) (BIM), phorbol-12-myristate-13-acetate-induced protein 1 (NOXA) or BID, play a role in the combination-induced effects. Overall, our study supports the rationale for combining an MCL1 inhibitor with a BCL2 inhibitor as a therapeutic option in patients with advanced melanoma.

Abstract A66: Analytical validation of 7 droplet digital PCR assays detecting TERT, BRAF, and NRAS hotspot mutations in plasma-derived circulating tumor DNA (ctDNA)

Abstract Introduction: Serial monitoring of sensitive and specific blood-based biomarkers of disease burden in cancer patients could enable clinicians to adjust treatments at the earliest signs of disease progression when the likelihood of improved outcomes is probably greatest. Such assays require preanalytical, analytical, and clinical validation prior to use. We conducted analytical validation studies of 7 droplet digital PCR (ddPCR) assays to determine their performance characteristics in detecting mutant TERT, BRAF, and NRAS ctDNA. Methods: Probe-based ddPCR assays (Bio-Rad) detecting BRAF V600E, K, NRAS Q61K, R, L, and TERT C228T, C250T were studied. Preanalytical validation experiments using plasma collected in EDTA tubes investigated processing variables including plasma volumes, centrifugation speeds, and DNA extraction efficiencies, and evaluated the necessity of internal controls using spike-in experiments. Analytical validation studies assessed accuracy using plasma from healthy donors spiked with known amounts of mutant nucleosomal DNAs from control cell lines. Potentially interfering substances were evaluated using the AcroMetrix inhibition panel (Thermo Scientific). Precision/reproducibility was assessed using combinations of multiple reagent lots and operators. Assay specificity was determined using templates with different mutations to check cross-reactivity. Limits of Blank (LoB) and Limits of Detection (LoD) were calculated for each assay using 8 replicate wells of healthy donor plasma or mutant cell line DNA, respectively. Three-phase inter-laboratory harmonization was conducted using: 1) extracted DNA, 2) plasmas spiked with control DNAs, and 3) melanoma patient samples shipped in PAXgene Blood ccfDNA tubes (Qiagen). Results: Preanalytical validation yielded a double centrifugation protocol to reduce contaminating large DNA. Assays were highly accurate and precise with 80-90% recovery of spiked-in mutant templates and coefficients of variation (CVs) <10%, respectively. BRAF and NRAS mutation-specific assays also detected alternative specific mutations in their respective targets that were easily discernible from the perfect-match mutation as discrete clusters in the ddPCR output software. The LoBs ranged from 0.24 to 1.68 copies/mL and 0.016% to 0.058% fractional abundance; LoDs ranged from 0.01% to 0.05%. Inter-laboratory harmonization demonstrated CVs ranging from 1.5% to 15%. Conclusion: This ddPCR-based plasma analysis platform demonstrates low background levels in normal plasma, yielding high sensitivities to detect small amounts of ctDNAs with these mutations. The assays also demonstrate highly reproducible results between laboratories. These assays could be integrated into biomarker-driven clinical trials to evaluate their potential to inform more personalized clinical decision-making. Citation Format: Mahrukh M. Syeda, Broderick Corless, Iman Osman, Cindy Spittle, George Karlin-Neumann, David Polsky. Analytical validation of 7 droplet digital PCR assays detecting TERT, BRAF, and NRAS hotspot mutations in plasma-derived circulating tumor DNA (ctDNA) [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A66.

Successful mutational profiling of single circulating epithelial tumor cells (CETCs) from patients with solid cancers for guiding therapy.

e15527 Background: Circulating epithelial tumor cells (CETCs) are an important link between primary tumors and metastases and provide a readily accessible source of tumor material from patients with solid cancer. Therefore, they are representative for primary and metastatic lesions to evaluate treatment response in real-time and to guide therapeutic modifications. Beyond enumeration, information on mutations is extremely important in the clinical setting to monitor the development of the disease over time and to guide drug selection. Isolating single CETCs with high recovery and purity from the high background of blood cells is an indispensable prerequisite to investigate cell heterogeneity and provides a tool for a personalized medicine approach. Methods: Blood from patients with non-small cell lung cancer, colon cancer and malignant melanoma was analysed for CETCs using the maintrac approach. Between 8-20 EpCAM positive cells from each patient were isolated individually using a semiautomated capillary approach and deposited one by one into micro cups. For mutational profiling, EGFR, KRAS and BRAF hotspot mutations were analysed in CETCs after whole genome amplification. Results: DNA could be amplified from all individually isolated cells. EGFR mutations were detected in 17% of isolated tumor cells from patients with non-small cell lung cancer; KRAS mutations were detectable in 28% of cells from patients with colon cancer and BRAF mutations in 50% of evaluable cells. A high heterogeneity in mutation status was observed among CETCs within a patient. Conclusions: Individually isolating CETCs from the peripheral blood from patients with non-small cell lung cancer, colon cancer and melanoma allows not only to detect driver mutations but also to determine genotypic heterogeneity between cells and changes over time. Mutations in the CETCs resemble those detected in primary tumors present only in part of the cells. They can be used to monitor the development of disease and its status over time, particularly for its impact in guiding drug selection.

Performance of cell-free tumor DNA testing for 101 clinical laboratories on College of American Pathologists proficiency tests.

e13681 Background: Cell-free tumor DNA or circulating tumor DNA tests are increasingly used in clinical care to detect somatic mutations from solid tumors. However, data on laboratory performance characteristics using standardized samples is limited. Methods: Well-characterized reference materials were used for the College of American Pathologists (CAP) cell-free tumor DNA proficiency testing surveys, which consisted of stabilized DNA fragmented to simulate cell-free DNA in a synthetic plasma matrix. For the 2018A, 2018B, 2019A and 2019B surveys, laboratories tested for hotspot mutations (single and dinucleotide sequence changes) in EGFR, BRAF, KRAS, NRAS, and IDH1 at variant allele fractions ranging from 0.1% - 1.0%. As per CAP proficiency testing standards, results were scored according to the known mutation(s) engineered at designated variant allele fractions in each PT sample. Nine laboratories were excluded from analysis because they provided incomplete results. Statistical significance was calculated using a multivariate logistic regression model. Results: In 2018 and 2019, 101 laboratories submitted survey results for at least one proficiency testing mailing. There were 5088 total proficiency testing responses for EGFR, BRAF, KRAS, NRAS, and IDH1 mutations across 12 different samples. For the 3585 responses submitted for BRAF, KRAS, NRAS, and IDH1, sensitivity ranged from 94.6 – 100%, while specificity exceeded 99%. There were no significant differences in performance between analytical methodologies for BRAF, KRAS, NRAS, and IDH1 mutations. Performance characteristics for EGFR mutations among the 1503 responses showed a combined sensitivity of 87.1% and specificity of 98.7%. For laboratories detecting mutations in EGFR, next-generation sequencing methods exhibited a sensitivity (true positivity) of 95.7% while the sensitivity of non-NGS methods was lower at 81.7% ( P= 0.02). Conclusions: These findings demonstrate high sensitivity and specificity for clinical laboratories performing cell-free tumor DNA tests. For EGFR mutations, NGS outperformed non-NGS methods. These data suggest excellent overall agreement among laboratories performing clinical cell-free tumor DNA tests. Further investigation across variant allele fractions and additional variant types is warranted.

Accurate detection of KRAS, NRAS and BRAF mutations in metastatic colorectal cancers by bridged nucleic acid-clamp real-time PCR

BackgroundPatients with metastatic colorectal cancer can benefit from anti-EGFR therapy, such as cetuximab and panitumumab. However, colorectal cancers harboring constitutive activating mutations in KRAS, NRAS and BRAF genes are not responsive to anti-EGFR therapy. To select patients for appropriate treatment, genetic testing of these three genes is routinely performed.MethodsWe applied bridged nucleic acid-clamp real-time PCR (BNA-clamp PCR) to detect somatic hotspot mutations in KRAS, NRAS and BRAF. PCR products from BNA-clamp PCR were subsequently analyzed Sanger sequencing. We then compared results with those from the PCR–reverse sequence-specific oligonucleotide probe (PCR-rSSO) method, which has been used as in vitro diagnostic test in Japan. To validate the mutation status, we also performed next generation sequencing using all samples.ResultsIn 50 formalin-fixed paraffin-embedded tissues, KRAS mutations were detected at frequencies of 50% (25/50) and 52% (26/50) by PCR-rSSO and BNA-clamp PCR with Sanger sequencing, respectively, and NRAS mutations were detected at 12% (6/50) and 12% (6/50) by PCR-rSSO and BNA-clamp PCR with Sanger sequencing, respectively. The concordance rate for detection of KRAS and NRAS mutations between the two was 94% (47/50). However, there were three discordant results. We validated these three discordant and 47 concordant results by next generation sequencing. All mutations identified by BNA-clamp PCR with Sanger sequencing were also identified by next generation sequencing. BNA-clamp PCR detected BRAF mutations in 6% (3/50) of tumor samples.ConclusionsOur results indicate that BNA-clamp PCR with Sanger sequencing detects somatic mutations in KRAS, NRAS and BRAF with high accuracy.

Relevance of calcification and contrast enhancement pattern for molecular diagnosis and survival prediction of gliomas based on the 2016 World Health Organization Classification

ObjectivesThe significance of conventional neuroimaging features for predicting molecular diagnosis and patient survival based on the updated World Health Organization (WHO) classification remains uncertain. We assessed the relevance of neuroimaging features (ring enhancement [RE], non-ring enhancement [non-RE], overall gadolinium enhancement [GdE], and intratumoral calcification [IC]) for molecular diagnosis and survival in glioma patients. Patients and methodsWe evaluated 234 glioma patients according to the updated WHO classification. Isocitrate dehydrogenase (IDH), H3F3A, BRAF hotspot mutations, TERT promotor mutation, and chromosome 1p/19q co-deletion were examined. RE, non-RE, GdE, and IC were evaluated as significant neuroimaging findings. Kaplan-Meier analyses were performed to evaluate overall survival (OS) and the correlations of prognostic factors were evaluated by log-rank tests. Univariate and multivariate analyses were performed to detect prognostic factors for OS. ResultsA total of 207 patients were eligible. In 110 patients presenting RE, 102 (93%) were glioblastoma (GBM), IDH-wild type. In 97 patients without RE, presence of GdE or IC were not significantly different between IDH-mutant and -wild type tumors, whereas presence of GdE was a significant indicator of higher WHO grades. IC was the only significant finding for 1p/19q co-deleted tumors. TERT promoter mutation was observed in 7/17 patients with diffuse astrocytic glioma, IDH-wild type; recently-defined as “molecular GBM.” IC, RE, and GdE were observed with lower prevalence in molecular GBMs. While presence of RE, GdE, and absence of IC were significant factors of OS in overall cohort, presence of GdE was not significant in OS in cases without RE, and IDH-mutant tumors. IC was a significant predictor of favorable OS in cases without RE and IDH-wild type tumors. Multivariate analysis also validated these findings. ConclusionGdE alone is not a significant predictor of IDH mutation status, but the pattern of enhancement is a significant predictor with RE demonstrating high sensitivity and specificity for GBM, IDH-wild type. Predicting “molecular GBM” by conventional neuroimaging is difficult. Moreover, GdE is not a significant factor of survival analyzed with pattern of enhancement or molecular stratifications. IC is an important radiographic finding for predicting molecular diagnosis and survival in glioma patients.

625P - Clinical relevance of circulating tumour (ct)DNA genotyping for first-line cetuximab-based treatment monitoring in metastatic colorectal cancer (mCRC): A prospective multicentric study

BackgroundGenotyping of ctDNA characterizes the molecular profile and monitors tumor molecular dynamics, but the clinical applicability of next generation sequencing (NGS) DNA sequencing has not been assessed in a large prospective cohort of mCRC patients (pts). MethodsmCRC pts with RAS wt tumors treated in first line with chemotherapy (CT) + cetuximab in 16 Spanish hospitals were included. Plasma samples were collected baseline (BL) and every 2 cycles until progression. ctDNA was isolated and sequenced with NGS platform Oncomine Colon cfDNA Assay which identifies hotspot mutations (mut) in AKT1, BRAF, CTNNB1, EGFR, ERBB2, FBXW7, GNAS, KRAS, MAP2K1, NRAS, PIK3CA, SMAD4, TP53 and APC. RAS ctDNA mut were also assessed by dPCR (BEAMing) at BL. Detection of any mut in plasma was considered as ctDNA+. Mut in BRAF/RAS/MAP2K1/PIK3CA were considered as anti-EGFR resistance. Results101 pts were enrolled (65.3% male; median age 64.5y; 77.6% left colon). RAS mut were detected in 11 pts BL by BEAMing. After quality assessment, 84 samples were sequenced by NGS BL and at least one mut was detected in 65 samples (77.3%; median 1, range 0-5) and 10 RAS mut were detected. Plasma samples from 47 pts were also analyzed after 4 cycles of treatment (C4) and mut were detected in 30 pts (63.8%; median 1, range 0-3). Median mutant allele fraction for paired samples was 24.9 at BL and 0.56 at C4 (p=0.0045). Treatment clinical benefit (CB: PR, CR and SD≥16weeks) was observed in 72 out of 85 pts. BL ctDNA+ or C4 ctDNA change were not associated with CB (p=0.722). Assessment of RAS BL mut was not associated with CB (p=0.243 by NGS and p=0.712 by BEAMing), while the absence of RAS mut at C4 predicted treatment CB (93.2% vs. 6.8% RAS mut p=0.02). Median PFS for all pts was 10.13 month. ctDNA+ at BL or C4 were not correlated with PFS (HR 0.8; p=0.73). No differences in PFS were found in ctDNA RAS mut vs RAS wt BL (BEAMing+ HR=1.2 p=0.79 and NGS+ HR=2.6 p=0.052). Among all anti-EGFR resistant mut, only ctDNA RAS mut were detected at C4 (N=4). Persistence or emergence of RAS mut at C4 were strongly associated with PFS (12.9m vs. 4.2. HR 8.8 p=0.0003). ConclusionsEarly RAS ctDNA dynamics predicts benefit to first line CT+cetuximab in mCRC pts. Legal entity responsible for the studyGrupo de Tratamiento de los Tumores Digestivos (TTD), Spain. FundingMerck-Serono. DisclosureJ. Vidal Barrull: Honoraria (self), Advisory / Consultancy: Merck-Serono; Advisory / Consultancy: Amgen; Honoraria (self): Sysmex-Inostics. E. Elez Fernández: Honoraria (self), Advisory / Consultancy: Amgen; Honoraria (self), Advisory / Consultancy, Research grant / Funding (self): Merck-Serono; Honoraria (self), Advisory / Consultancy: Roche; Honoraria (self), Advisory / Consultancy: Sanofi. F. Losa: Honoraria (self): Amgen; Honoraria (self): Merck-Serono. R. Salazar: Honoraria (self), Advisory / Consultancy: Amgen; Honoraria (self), Advisory / Consultancy: Merck-Serono; Advisory / Consultancy: Guardant-Helth; Honoraria (self), Advisory / Consultancy: Roche; Honoraria (self), Advisory / Consultancy: Ferrer; Advisory / Consultancy: Pfizer; Honoraria (self), Advisory / Consultancy: Novartis; Advisory / Consultancy: Lilly; Honoraria (self), Advisory / Consultancy: MSD. J. Tabernero: Honoraria (self), Advisory / Consultancy: Array Biopharma; Honoraria (self), Advisory / Consultancy: Merck-Serono; Honoraria (self), Advisory / Consultancy: Molecular Partners; Honoraria (self), Advisory / Consultancy: Sanofi; Honoraria (self), Advisory / Consultancy: Roche; Honoraria (self), Advisory / Consultancy: Novartis; Honoraria (institution), Advisory / Consultancy: MSD; Honoraria (self), Advisory / Consultancy: Foundation Medicine; Honoraria (self), Advisory / Consultancy: Halio DX SAS. E. Aranda Aguilar: Honoraria (self): Celgene; Honoraria (self): Merck-Serono; Honoraria (self): Roche; Honoraria (self): Sanofi. B. Bellosillo: Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: Merck-Serono; Advisory / Consultancy: Roche; Honoraria (self), Advisory / Consultancy: Biocartis; Advisory / Consultancy: Novartis; Advisory / Consultancy: Thermofisher. C. Montagut Viladot: Honoraria (self), Advisory / Consultancy: Amgen; Honoraria (self), Advisory / Consultancy, Research grant / Funding (self): Merck-Serono; Advisory / Consultancy: Sysmex-Inostics; Advisory / Consultancy: Sanofi; Honoraria (self), Advisory / Consultancy: Roche; Advisory / Consultancy: Bayer; Honoraria (self), Advisory / Consultancy: Biocartis; Honoraria (self), Advisory / Consultancy: BMS; Honoraria (self), Advisory / Consultancy: Guardant-Helth; Advisory / Consultancy: Symphogen. All other authors have declared no conflicts of interest.

Diffuse leptomeningeal enhancement in a patient with rapidly progressive dementia: Answer

Diffuse leptomeningeal enhancement in a patient with rapidly progressive dementia: Answer

Whole transcriptome analysis reveals correlation of long noncoding RNA ZEB1-AS1 with invasive profile in melanoma

Melanoma is the deadliest form of skin cancer, and little is known about the impact of deregulated expression of long noncoding RNAs (lncRNAs) in the progression of this cancer. In this study, we explored RNA-Seq data to search for lncRNAs associated with melanoma progression. We found distinct lncRNA gene expression patterns across melanocytes, primary and metastatic melanoma cells. Also, we observed upregulation of the lncRNA ZEB1-AS1 (ZEB1 antisense RNA 1) in melanoma cell lines. Data analysis from The Cancer Genome Atlas (TCGA) confirmed higher ZEB1-AS1 expression in metastatic melanoma and its association with hotspot mutations in BRAF (B-Raf proto-oncogene, serine/threonine kinase) gene and RAS family genes. In addition, a positive correlation between ZEB1-AS1 and ZEB1 (zinc finger E-box binding homeobox 1) gene expression was verified in primary and metastatic melanomas. Using gene expression signatures indicative of invasive or proliferative phenotypes, we found an association between ZEB1-AS1 upregulation and a transcriptional profile for invasiveness. Enrichment analysis of correlated genes demonstrated cancer genes and pathways associated with ZEB1-AS1. We suggest that the lncRNA ZEB1-AS1 could function by activating ZEB1 gene expression, thereby influencing invasiveness and phenotype switching in melanoma, an epithelial-to-mesenchymal transition (EMT)-like process, which the ZEB1 gene has an essential role.

Evaluation of KRAS, NRAS and BRAF hotspot mutations detection for patients with metastatic colorectal cancer using direct DNA pipetting in a fully-automated platform and Next-Generation Sequencing for laboratory workflow optimisation.

BackgroundAssessment of KRAS, NRAS (RAS) and BRAF mutations is a standard in the management of patients with metastatic colorectal cancer (mCRC). Mutations could be assessed using next-generation sequencing (NGS) or real-time PCR-based assays. Times to results are 1 to 2 weeks for NGS and 1 to 3 days for real-time PCR-based assays. Using NGS can delay first-line treatment commencement and using PCR-based assays is limited by the number of possible analysed targets. The Idylla system is a real-time PCR cartridge-based assay, able to analyse hotspots mutations using one section of FFPE tumour tissue sample. To combine short delays and analysis of a large gene-panel, we propose here a laboratory workflow combining the Idylla system and NGS and compatible with FFPE samples with low tissue quantity. In this study we evaluated and validated the Idylla system for the analysis of RAS and BRAF mutations by pipetting directly DNA in the cartridge instead of FFPE section as recommended by the manufacturer.Materials and methodsDNA extracted from 29 FFPE samples from mCRC patients with NGS-characterized RAS and BRAF mutations were tested with the Idylla KRAS and the Idylla NRAS-BRAF mutation tests to assess sensitivity, specificity, reproducibility and limit of detection of each test.ResultsA 100% concordance was found between NGS and Idylla results for the determination of KRAS (12/12), NRAS (12/12) and BRAF (11/11) mutations with a sensitivity and a specificity of 100%. The system showed a good reproducibility with CV inferior to 3%. LOD was reached with 2.5 ng of DNA for KRAS and NRAS mutations and 5 ng of DNA for BRAF mutations.ConclusionsThe analysis of RAS and BRAF mutations using DNA pipetted directly in the cartridge of the Idylla system showed a good sensitivity, specificity, reproducibility and LOD, and can be integrated in a laboratory workflow for samples with few tissue without compromising a further complete tumour characterization using NGS.

Abstract 2521: Molecular profiling of T790M-negative NSCLC patients progressing on EGFR-TKI enrolled in the CL1-49076-003 trial with a MET/AXL/FGFR inhibitor in combination with gefitinib

Abstract Background Non-small cell lung cancer (NSCLC) tumors with mutations in the EGF receptor (EGFR) relapse to therapy with EGFR tyrosine kinase inhibitors (EGFR TKIs) due to a variety of mechanisms, such as emergence resistance mutations, dysregulation of AXL, MET, HER2 or FGFR1 receptors or histological transformation. The CL1-49076-003 trial of the MET/AXL/FGFR inhibitor S49076 in combination with gefitinib enrolled T790M-negative patients in progression to first-line EGF TKIs and showing dysregulation of MET and/or AXL. Molecular screening was performed in 47 patients, 23 of them met the molecular eligibility criteria and 14 were enrolled in the trial. Baseline biopsies of the 14 patients were submitted to molecular profiling. Methods Ten patients had enough material available for Next Generation Sequencing (NGS), that was performed using the GeneRead QIAact Lung UMI Panel (Qiagen, Hilden, FRG). The panel included mutation detection in 16 genes, copy number variations in 5 and the MET exon 14 splicing variant. Amplifications detected by NGS were confirmed by FISH. Of the 4 patients with insufficient material for NGS, 3 were submitted to FISH for HER2 and MET and quantitative PCR for BRAF and PIK3CA hotspot mutations. Finally, of the remaining patient only FISH for MET could be performed. Results Copy number gains were the most prevalent alterations in patients progressing to EGFR TKIs. Four of 14 evaluable patients (29%) showed MET amplification, 4/13 (31%) HER2 amplification and 3/10 (30%) EGFR copy number gains. FGFR1 amplifications were absent in the patient cohort. The baseline sensitizing mutation was confirmed in all cases. Two patients showed additional mutations that could be related to resistance, namely a p.G724S mutation in EGFR and a p.N784fs*2 mutation in MET. Remarkably, these two patients did not present gene amplifications. No other mutations were detected in the rest of genes analyzed, including BRAF, PIK3CA, KRAS, NRAS or ERBB2. Among the 4 patients with more than 6 months of progression free survival, 2 had MET amplifications. The patient with the p.G724S experienced rapid progression of target lesions. Conclusion Next Generation Sequencing can be used to determine mechanisms of resistance to EGFR TKIs at progression, and can give useful clinical information in order to select therapies for second line treatment Citation Format: Monica Garzon Ibanez, Nuria Jordana Ariza, María González Cao, Ruth Román Lladó, Alejandro Martínez Bueno, Lidia Alonso Landeira, María de los Llanos Gil, Miguel Ángel Molina Vila, Niki Karachaliou, Veronika Smutna, Valerie Cattan, Rafael Rosell, Santiago Viteri. Molecular profiling of T790M-negative NSCLC patients progressing on EGFR-TKI enrolled in the CL1-49076-003 trial with a MET/AXL/FGFR inhibitor in combination with gefitinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2521.

Mutational Signatures in Mandibular Ameloblastoma Correlate with Smoking

Ameloblastoma is a rare tumor of odontogenic epithelium, the low incidence rate of which precludes statistical determination of its molecular characterizations. Despite recent genomic and transcriptomic profiling, the etiology of ameloblastomas remains poorly understood. Risk factors of ameloblastoma development are also largely unknown. Whole exome sequencing was performed on 11 mandibular ameloblastoma samples. We identified 2 convergent mutational signatures in ameloblastoma: 1) a signature found in multiple types of lung cancers with probable etiology of tobacco carcinogens (COSMIC signature 4) and 2) a signature present in gingivobuccal oral squamous cell carcinoma and correlated with tobacco-chewing habits (COSMIC signature 29). These mutational signatures highlight tobacco usage or related mutagens as one possible risk factor of ameloblastoma, since the association of BRAF mutations and smoking was demonstrated in multiple studies. In addition to BRAF hotspot mutations (V600E), we observed clear inter- and intratumor heterogeneities. Interestingly, prior to BRAF mutation, important genes regulating odontogenesis mutated (e.g., corepressor BCOR), possibly playing important roles in tumorigenesis. Furthermore, recurrent mutations in the CDC73 gene, the germline mutations of which predispose patients to the development of jaw tumors, were found in 2 patients, which may lead to recurrence if not targeted by therapeutic drugs. Our unbiased profiling of coding regions of ameloblastoma genomes provides insights to the possible etiology of mandibular ameloblastoma and highlights potential disease risk factors for screening and prevention, especially for Asian patients. Because of the limited sample size and incomplete habitual, dietary, and occupational data, a causal link between tobacco usage and ameloblastoma still requires further investigations.

Abstract 2621: Genomic determinants of progression and response to therapy in prospectively characterized glioma patients

Abstract While the genomic landscape of low- and high-grade gliomas has been characterized and incorporated into further disease classification, its relationship to disease progression and treatment response remains poorly understood. To address this question, we integrated prospective clinical sequencing of 1,007 primary and recurrent tumors from 924 glioma patients with clinical and treatment phenotypes. Genomic alterations in effectors of cell cycle progression were a biomarker of aggressive disease in 1p19q intact IDH-mutant disease, arising preferentially in enhancing and recurrent tumors. When present at recurrence, the lesions were associated with rapid disease progression. These alterations were also present in all astrocytic tumors that ultimately relapsed with alkylating therapy-associated hypermutation. Analysis of matched pre-treatment and post-progression tumors indicated that cell cycle alterations preceded the emergence of hypermutation. In total, 391 patients (42%) harbored a potentially therapeutically actionable lesion in their tumor of which 73 patients (19%) received a diversity of targeted therapies. Both the type of mutation and its clonality were associated with therapeutic sensitivity in this cohort. For instance, RAF dimer-dependent BRAF hotspot mutations arose predominantly in IDH-wildtype glioblastomas, were subclonal, and affected patients did not respond to MAPK-directed therapy. In contrast, BRAF V600 hotspot mutations arose clonally in histologically distinct gliomas, both low- and high-grade, and responded to MAPK-directed therapy (either RAF, MEK or ERK inhibition). Collectively, these data reveal previously unrecognized genomic determinants of disease progression and treatment response in diverse types of glioma and serve as a rationale for utilizing genomic information in clinical care of patients with glioma. Citation Format: Philip Jonsson, Andrew L. Lin, Shahiba L. Ogilvie, Shweta S. Chavan, Andrew T. McKeown, Natalie M. DiStefano, Marc Rosenblum, Lisa M. DeAngelis, Ingo K. Mellinghoff, Barry S. Taylor. Genomic determinants of progression and response to therapy in prospectively characterized glioma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2621.

Clinical utility of circulating DNA analysis for rapid detection of actionable mutations to select metastatic colorectal patients for anti-EGFR treatment

While tumor-tissue remains the 'gold standard' for genetic analysis in cancer patients, it is challenged with the advent of circulating cell-free tumor DNA (ctDNA) analysis from blood samples. Here, we broaden our previous study on the clinical validation of plasma DNA in metastatic colorectal cancer patients, by evaluating its clinical utility under standard management care. Concordance and data turnaround-time of ctDNA when compared with tumor-tissue analysis were studied in a real-time blinded prospective multicenter clinical study (n = 140 metastatic colorectal patients). Results are presented according to STARD criteria and were discussed in regard with clinical outcomes of patients. Much more mutations were found by ctDNA analysis: 59%, 11.8% and 14.4% of the patients were found KRAS, NRAS and BRAF mutant by ctDNA analysis instead of 44%, 8.8% and 7.2% by tumor-tissue analysis. Median tumor-tissue data turnaround-time was 16 days while 2 days for ctDNA analysis. Discordant samples analysis revealed that use of biopsy, long delay between tumor-tissue and blood collection and resection of the tumor at time of blood draw, tumor site, or type of tissue analyzed seem to affect concordance. Altogether, the clinical data with respect to the anti-epidermal growth factor receptor response (RAS status) and the prognosis (BRAF status) of those discordant patients do not appear contradictory to the mutational status as determined by plasma analysis. Lastly, we present the first distribution profile of the RAS and BRAF hotspot mutations as determined by ctDNA analysis (n = 119), revealing a high proportion of patients with multiple mutations (45% of the population and up to 5 mutations) and only 24% of WT scored patients for both genes. Mutation profile as determined from ctDNA analysis with using various detection thresholds highlights the importance of the test sensitivity. Our study showed that ctDNA could replace tumor-tissue analysis, and also clinical utility of ctDNA analysis by considerably reducing data turnaround time.

Abstract 1561: Gene expression analysis identifies heterogeneity in cutaneous melanoma subjects with disruptive MC1R alleles and BRAF hotspot mutations

Abstract Background: Previous epidemiological and population sequencing studies have established that subjects with melanocortin 1 receptor (MC1R) germline mutations, associated with red hair and light skin phenotypes, have an increased risk of melanoma. However, several conflicting reports about the role of BRAF somatic mutations in MC1R subjects exist in the literature. We hypothesise that this conflict is due to biological process driven heterogeneity within MC1R subjects carrying disruptive alleles. To understand the heterogeneity, we analysed the TCGA cohort at the gene expression level using mRNA sequencing data. Method: From the previously published studies, we identified 68 cutaneous melanoma subjects who were of white ethinicity, had at least one disruptive allele of MC1R, and had BRAF mutations. Associated clinical and mRNA sequencing data were downloaded from the Firebrowse website. An unbiased hierarchical clustering analysis was performed using the preprocessed mRNA data containing 14669 gene expressions. The resultant clusters were then characterised using available clinical variables. A differential expression analysis was performed to identify the genomic signatures of these clusters. The resultant p-values were corrected for false discovery rate (FDR) using the Benjamini-Hochberg approach. Genes with corrected p-values less than 0.01 were further analysed using gene set enrichment analysis (GSEA) preranked lists. Results: An unbiased clustering identified two clear and separable clusters with 40 and 28 subjects respectively. The UV-signature (dipyrimidine C>T load) was correlated with these clusters (p-value = 0.047). However, no significant differences in age at the time of diagnosis, overall survival, and total mutation load between clusters were observed. At the genomic level, 2588 genes were significantly differentially expressed between two clusters (FDR corrected p- value less than 0.05). The top 10 differentially expressed genes included ATF2, MSH6, SP3, MAP3K2, and VEGFB. Further GSEA revealed gene sets playing roles in several pathways and biological processes, including oxidative phosphorylation, UV response in keratinocytes, DNA repair, cell cycle, and cellular response to stress. Conclusion: Cutaneous melanoma subjects with disruptive MC1R alleles and BRAF hotspot mutations have heterogeneous gene expression profiles with several key oncogenes differentially expressed. This suggests potential roles of other pathways, either independently or in cooperation with BRAF mutations, in melanomagenesis in MC1R bearing patients. Citation Format: Piyushkumar A. Mundra, Candelaria Bracalente, Lucas Trucco, Nathalie Dhomen, Richard Marais. Gene expression analysis identifies heterogeneity in cutaneous melanoma subjects with disruptive MC1R alleles and BRAF hotspot mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1561. doi:10.1158/1538-7445.AM2017-1561

Identification of somatic TERT promoter mutations in familial nonmedullary thyroid carcinomas.

The genes causing familial nonmedullary thyroid carcinoma (FNMTC) identified to date are only involved in a small fraction of the families. Recently, somatic mutations in TERT promoter region and in EIF1AX gene were reported in thyroid tumours of undefined familial status. The aim of this study was to investigate the role of TERT and EIF1AX mutations in familial thyroid tumours. The promoter region of TERT was sequenced in leucocyte DNA of the probands from 75 FNMTC families. In thyroid tumours from 54 familial cases, we assessed somatic TERT promoter, RAS and BRAF hotspot mutations, and the whole EIF1AX gene. No potentially pathogenic germline variants were identified in TERT in the 75 FNMTC families' probands. In the 54 carcinomas, we identified five cases (9%) with hotspot somatic TERT promoter mutations. BRAF mutations were found in 41% of the tumours. All TERT-positive samples were also positive for BRAF p.Val600Glu, and this co-occurrence was found to be statistically significant (P=.008). RAS mutations were detected in four tumours wild-type for TERT (7%). Evaluation of tumour mutation data together with the patients' clinicopathological features revealed a significant correlation between TERT plus BRAF mutations and advanced tumour stage (T4) (P=.020). No mutations were identified in EIF1AX. The results of this study suggest that TERT promoter and EIF1AX mutations are not frequently involved in FNMTC aetiology. However, we show for the first time that TERT alterations are associated with familial thyroid tumour progression. Our data also suggest that TERT mutations are more often found in concomitance with BRAF mutations in advanced stages of FNMTC.

HGG-15. MAPK PATHWAY ACTIVATION IN K27M MUTATED GLIOMAS – ASSOCIATION WITH CLINICAL PARAMETERS AND SURVIVAL

AbstractK27M midline gliomas are defined by point mutations within the chromatin modifiers H3 and are represented as a new separate entity in the new version of the WHO classification for tumors of the central nervous system. These tumors arise in midline structures of the brain, the brain stem and the spinal cord. They occur in children and young adults and are usually associated with a bad clinical outcome. We and others previously reported Mitogen-activated protein kinase (MAPK) pathway activation via BRAF or FGFR1 hotspot mutations in single cases of K27M gliomas. In contrast to K27M mutations, MAPK activation is strongly associated with pediatric low-grade astrocytomas, that show a favorable survival. In the context of K27M glioma, the clinical significance of co-occuring BRAF or FGFR1 mutations remains unclear. We screened a cohort of patients with K27M mutated gliomas for BRAF or FGFR1 mutations. So far, we detected three cases with FGFR1 mutations (K656E and N546K) and two cases with BRAF V600E mutations. These cases showed a favorable survival in comparison to other K27M cases in our series and in comparison to previously reported patient cohorts of K27M glioma. Our results shed light on the frequency and clinical impact of MAPK pathway activation in K27M mutated gliomas with implications for targeted therapies and prognosis of patients.

Mutational profiling of acral melanomas in Korean populations.

The proportion of acral melanoma (AM) is much higher in Asian populations than in Caucasian populations. Although mutational profiles associated with AM have been discovered in Caucasian populations, knowledge of its genetic alterations in Asian populations is limited. To describe the molecular nature of AM in Korean patients, we performed mutational profiling of AM and matched normal tissues in patients. Fifty-one formalin-fixed paraffin-embedded AM samples and 32 matched pairs from patients' saliva DNA were analysed by next-generation sequencing. Only mutations confirmed via digital droplet PCR or in BRAF, KIT and NRAS, the most frequently altered cancer genes in cutaneous melanoma, were considered as positive. The relationship between mutational status and clinicopathological features were examined. Of the 47 AM patients screened, alteration of BRAF, NRAS and KIT genes was observed in 6.4%, 4.3% and 8.5%, respectively. We also tested matched normal tissues of patients to identify tumor-specific mutations. Examination of the mutational profile in a cohort of 28 primary melanomas and matched normal controls found BRAF mutations in two cases (7.1%), KIT mutations in three cases (10.7%) and CTNNB1 mutations in one case (3.6%). The BRAF, NRAS and KIT mutation status did not correlate with clinicopathological characteristics. Our results show that KIT, NRAS and BRAF hotspot mutations occur at a low frequency in Korean populations. We also observed a case with the CTNNB1 mutation, which raises the possibility that other pathways are associated with AM development.