Targeting BRAF in cancers – from molecular diagnostics to personalized therapy

Detection of BRAF V600 Mutations in Metastatic Melanoma: Comparison of the Cobas 4800 and Sanger Sequencing Assays

Tumor heterogeneity and strategies to overcome kinase inhibitor resistance in cancer: lessons from melanoma

For patients with non-small cell lung cancer (NSCLC) to benefit from ALK inhibitors, sensitive and specific detection of ALK genomic rearrangements is needed. ALK break-apart fluorescence in situ hybridization (FISH) is the U.S. Food and Drug Administration approved and standard-of-care diagnostic assay, but identification of ALK rearrangements by other methods reported in NSCLC cases that tested negative for ALK rearrangements by FISH suggests a significant false-negative rate. We report here a large series of NSCLC cases assayed by hybrid-capture-based comprehensive genomic profiling (CGP) in the course of clinical care. Hybrid-capture-based CGP using next-generation sequencing was performed in the course of clinical care of 1,070 patients with advanced lung cancer. Each tumor sample was evaluated for all classes of genomic alterations, including base-pair substitutions, insertions/deletions, copy number alterations and rearrangements, as well as fusions/rearrangements. A total of 47 patients (4.4%) were found to harbor ALK rearrangements, of whom 41 had an EML4-ALK fusion, and 6 had other fusion partners, including 3 previously unreported rearrangement events: EIF2AK-ALK, PPM1B-ALK, and PRKAR1A-ALK. Of 41 patients harboring ALK rearrangements, 31 had prior FISH testing results available. Of these, 20 were ALK FISH positive, and 11 (35%) were ALK FISH negative. Of the latter 11 patients, 9 received crizotinib based on the CGP results, and 7 achieved a response with median duration of 17 months. Comprehensive genomic profiling detected canonical ALK rearrangements and ALK rearrangements with noncanonical fusion partners in a subset of patients with NSCLC with previously negative ALK FISH results. In this series, such patients had durable responses to ALK inhibitors, comparable to historical response rates for ALK FISH-positive cases. Comprehensive genomic profiling (CGP) that includes hybrid capture and specific baiting of intron 19 of ALK is a highly sensitive, alternative method for identification of drug-sensitive ALK fusions in patients with non-small cell lung cancer (NSCLC) who had previously tested negative using standard ALK fluorescence in situ hybridization (FISH) diagnostic assays. Given the proven benefit of treatment with crizotinib and second-generation ALK inhibitors in patients with ALK fusions, CGP should be considered in patients with NSCLC, including those who have tested negative for other alterations, including negative results using ALK FISH testing.

Molecular Biomarkers in the Personalized Treatment of Colorectal Cancer.

A diagnostic dilemma: Atypical melanocytic lesions arising in the setting of treatment with the BRAF inhibitor, vemurafenib

Detection of Mutant BRAF Alleles in the Plasma of Patients with Metastatic Melanoma

Anaplastic lymphoma kinase tyrosine kinase inhibitors (ALK-TKIs) are the standard treatment for advanced ALK-positive non-small cell lung cancer (NSCLC) allowing survivals up to 5 years. However, duration of responses is limited by the almost certain occurrence of drug resistance. Here, we report a case of a never smoker, 59-year-old female with metastatic ALK-positive adenocarcinoma, solid and signet ring patterns, who developed resistance to alectinib, a second-generation ALK-TKI, mediated by HER2 gene amplification. The patient received 22 months of crizotinib as first-line and subsequently 1-year of alectinib therapy. A study of resistance mechanism was performed with next generation sequencing (NGS) on tissue re-biopsy. A HER2-amplified emerging clone was identified by NGS in a liver metastasis and confirmed by fluorescent in situ hybridization (FISH) analysis. The resistant clone was detectable 2 months before disease progression in plasma cell-free DNA (cfDNA) using digital droplet PCR (ddPCR) copy number variation (CNV) assay and it was retrospectively traced in rare cells of the lung primary by FISH. To our best knowledge, this is first evidence of HER2 gene amplification as a resistance mechanism to ALK-TKI in a NSCLC. Future strategies against oncogene-addicted NSCLC might benefit of combined drug treatments, such as ALK and HER2 inhibition.

Prevalence and breakdown of non-small cell lung cancer BRAF driver mutations in a large UK cohort

The use of immunotherapy in treatment of non-small cell lung cancer (NSCLC) patients with the BRAF gene mutations is an area of active research and is an item of clinical trials. While BRAF mutations are relatively infrequent in NSCLC patients, comprising approximately 1-3% of cases, the V600E substitution stands out as the most prevalent subtype of BRAF mutations. The presence of this mutation in cancer cells qualifies the patients for first-line therapy with BRAF and MEK inhibitors. This study aims to evaluate the efficacy of immunotherapy in NSCLC patients with BRAF mutations. We presented a series of seven NSCLC cases with BRAF mutations, four of whom received immunotherapy or chemoimmunotherapy. We observed benefit from immunotherapy in all patients, but its duration depended on comorbidities and the presence of brain metastases. Utilization of the next generation sequencing (NGS) technique causes high detection frequency of BRAF mutations (4.7% of patients), although mutations other than V600E may predominate (4 out of 7 patients). In patients receiving immune checkpoint inhibitors (ICIs)-based therapy, the median progression-free survival (PFS) was 17 months from the start of immunotherapy, the overall objective response rate (ORR) was 50%, and disease control was achieved in all patients. Immunotherapy can benefit NSCLC patients with BRAF mutations, though its efficacy is affected by comorbidities and brain metastases. The use of NGS enhances mutation detection, highlighting the need for personalized treatment approaches in NSCLC management. The varying responses to treatments among the patients emphasize the complexity of NSCLC management and the necessity for a personalized approach.

Background: BRAF inhibitors have been approved in BRAF mutated malignant melanoma. However, in other BRAF mutated cancers, the effect of BRAF inhibition has been studied less extensively. BRAF mutated non-melanoma cancer patients (pts) were identified in a prospective genomic profiling study. The effect of targeted treatment was assessed by BRAF mutant allele fraction (AF) in circulating tumor DNA (ctDNA) and radiological response. Methods: Pts had tumor biopsies performed at enrolment and, if feasible, on or after treatment. Biopsies were subjected to whole exome sequencing, RNA sequencing and SNP array. Blood samples for ctDNA analysis were collected at baseline and longitudinally during treatment. Mutant BRAF fragments were quantified using droplet digital PCR (ddPCR) or by targeted next generation sequencing (NGS). Radiological response was evaluated according to RECIST 1.1. Results: Twenty-two BRAF mutated non-melanoma cancer pts (lung n=2, colorectal n=14, bile duct n=4, prostate n=1, pancreas n=1) were identified out of a total of 405 (5 %) pts enrolled in the Copenhagen Prospective Personalized Oncology (CoPPO) study. In all tumors we identified the BRAF V600E mutation except in one prostate cancer pt (BRAF K601E). Fifteen pts were treated with dabrafenib/trametinib (lung, bile duct), vemurafenib/panitumumab +/- irinotecan (colorectal) or vemurafenib (pancreas, prostate). Two pts were non-evaluable because one patient requested early termination and one has pending tumor evaluation. All pts had tumor reduction, except one patient with colon cancer. Six out of 13 (46%) pts achieved partial response (lung n=2, bile duct n=1, colorectal n= 3) and 7/13 (44%) had stable disease (prostate n=1, bile duct n=1, colorectal n= 5) as best response according to RECIST 1.1. Median progression-free survival was 20 weeks. Eighteen pts analysed by ddPCR or NGS at baseline had detectable mutant BRAF in the plasma obtained before initiation of therapy. Eight of the 18 pts had ctDNA assessed during treatment and all presented with a reduction in mutant BRAF AF corresponding to a reduction in tumor volume. Moreover, in 5/8 pts, time of best radiological response coincided with the lowest detectable BRAF mutant AF. Analysis of on- and post treatment biopsies and matched ctDNA samples are pending to identify putative mechanisms of resistance and will be reported at the meeting. Conclusion: Targeted therapy seems active in BRAF mutated non-melanoma cancer pts and treatment response is evaluable by monitoring mutant BRAF in ctDNA during therapy. Citation Format: Lise Barlebo Ahlborn, Ida Viller Tuxen, Olga Oestrup, Ane Yde Schmidt, Cecilia Brunhoff Håkansson, Finn Celius Nielsen, Ulrik Lassen, Christina Westmose Yde, Morten Mau-Sorensen. BRAF mutant allele fraction in circulating tumor DNA as marker of treatment response in BRAF mutated non malignant melanoma cancers identified in the Copenhagen Prospective Personalized Oncology study [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 5393. doi:10.1158/1538-7445.AM2017-5393

10568 Background: Effective targeted therapy sorely depends on comprehensive and precise genomic profiling. Besides, single nucleotide variations (SNVs) and InDels, gene fusions, as drivers and therapeutic targets of great importance, are not yet well characterized in Chinese patients. Methods: Formalin-fixed, paraffin-embedded (FFPE) tumor tissues from 1,384 pan-cancer patients were collected and sequenced using next-generation sequencing (NGS) targeting 40 cancer genes including SNV, fusion and assessing copy number variation (CNV) (AmoyDx HANDLE Classic Panel). qPCR and fluorescence in situ hybridization (FISH) were further applied to verify NGS detected fusion genes. Results: 1,384 patients were recruited including 890 lung cancers, 272 colorectal cancers, 174 gastric cancers, and 48 endometrial cancers. The prevalence of fusion genes (5.49%), including ALK-fusion (2.67%), ROS1-fusion (0.94%), RET-fusion (0.72%), NRG1-fusion (0.29%), and NTRK-fusion (0.07%), was nearly double the frequency of previously reported data from East Asians. Prevalence of fusion genes varied in different types of cancers. For instance, ALK (3.7%), ROS1 (1.5%), RET (1.0%) and NRG1 (0.2%) fusions were largely found in patients with non-small cell lung cancer (NSCLC), and were rarely detected in other cancer types. Further analysis of genomic alterations in fusion-positive patients revealed that, TP53 (21%) was the most frequently co-occurred mutated gene with ALK fusion, while RET fusions and ROS1 fusions were rarely accompanied by other mutated genes. In addition, two patients with RBPMS- NRG1 fusion were accompanied by BRAF and RB1 mutations, whereas no co-occurred mutation was found in patients with other partners of NRG1. Patients with inconsistent results of RNA-based NGS and FISH validation fusions are still being followed for treatment and survival, updated data will be available at the time of the presentation. Conclusions: Genomic alterations in real-world Chinese populations have specific characteristics, especially fusion genes. Further characterization of these variants is essential for clinics to guide appropriate targeted therapies.

Mesenchymal-epithelial transition (MET) gene amplification is a critical biomarker in non-small cell lung cancer (NSCLC), significantly influencing treatment decisions and prognostic evaluations. However, current detection methods such as fluorescence in situ hybridization (FISH) and next-generation sequencing (NGS) have limitations in speed, cost, and specificity, particularly when distinguishing between focal MET amplification and MET polysomy. This study introduces a novel digital PCR (dPCR) assay designed not only to detect MET amplification but also to differentiate between its focal and non-focal subtypes. The assay was evaluated against established FISH and targeted NGS panels using 55 NSCLC samples with known MET amplification statuses (26 positive and 29 negative) confirmed by FISH and NGS. Results The dPCR assay demonstrated high sensitivity (96.0%) and specificity (96.7%), achieving 100% concordance with FISH in differentiating focal MET amplification from MET polysomy. Additionally, the assay exhibited excellent precision, accuracy, and linearity (R2 = 1.00) in MET copy number quantification, surpassing NGS in diagnostic performance. Offering a robust, cost-effective, and efficient alternative to FISH, the dPCR assay significantly reduces the turnaround time (3 h versus 2 days) and provides a quantitative and objective method for MET amplification detection and subtype differentiation. This makes it suitable for clinical laboratories with limited molecular expertise. This study highlights the potential of the dPCR assay to complement existing molecular diagnostic techniques, delivering reliable and actionable results for MET-targeted therapy selection in NSCLC patients and thereby advancing precision oncology.

P2.16-08 Clinical Characteristics and Outcomes of Patients with BRAF Mutated Non-Small Cell Lung Cancer

Background: BRAF mutations occur in 2 to 3% of patients (pts) with non-small cell lung cancer (NSCLC). In these pts vemurafenib, a selective oral BRAF inhibitor is associated with a response rate (RR) of 42%, rising to 64% for combination treatment with dabrafenib and trametinib. Despite initial responses, most pts ultimately develop resistance to therapy. Mechanisms of resistance to BRAF inhibitors in NSCLC have only been reported in 2 pts (acquired KRAS G12D and primary resistance due to BRAF G469L) Objective: To assess the molecular mechanisms of resistance and to monitor disease response to treatment using liquid biopsies in NSCLC pts treated with BRAF inhibitors. Strategy: We performed a longitudinal genomic analysis of circulating-tumor DNA (ctDNA) in BRAF-mutated NSCLC pts treated in the AcSé vemurafenib program (NCT02304809) (n=44), or with the combination of dabrafenib and trametinib (n=6). We have collected 24 samples at baseline, 45 during follow-up and 9 at progressive disease (PD). ctDNA genotyping of 36 genes was performed using the Inivata InVisionFirst™ assay. Functional analyses of potentially resistant mutations and in vitro strategies to revert the resistant phenotype are ongoing. Results: Our preliminary analyses showed that BRAF mutations were detected at diagnosis in 16/24 pts, including 12 BRAF V600E mutations and 4 non-V600E mutations (i.e. G466V, G596R, G469A and K601E). 4/12 (34%) of BRAF V600E-mutated pts presented coexistent mutations, in FGFR2, CTNNB1, IDH1 or PI3KCA, whereas concomitant mutations in KRAS, NRAS or MYC were found in 3/4 (75%) of non-V600E cases. Analyses of response to treatment vs mutational profile will be presented. For the remaining 8/24 pts, TP53 mutations were found in 5 pts in absence of BRAF mutations, and no mutations were detected in 3 pts. Mechanisms of resistance were evaluated in 9 pts. One patient who progressed after 11 months on vemurafenib had MAP2K1 C121S and NFE2L2 p.31-32:GV/X mutations. In this patient, longitudinal ctDNA profiling revealed agreement between the %AF of BRAF and TP53 mutations and response to treatment, and detectable levels of the BRAF V600E and the MAP2K1 C121S mutations up to 6 months before the clinical confirmation of PD. Acquired PI3KCA H1047R and E545K mutations were seen in two pts, respectively, who progressed after 15 and 7 months of vemurafenib. Finally, a fourth patient who relapsed after 3 months on vemurafenib, presented a KRAS G12C mutation. All 4 cases also presented detectable levels of the BRAF V600E mutation at PD. In 3/8 pts, we detected the BRAF V600E mutation at PD but no other mutations; drivers of resistance may be present in genes outside this panel. ctDNA sequencing data on additional 7 pts at PD will be presented. Conclusion: Our results suggest that ctDNA genotyping might be an informative tool for monitoring disease response and resistance in NSCLC pts treated with BRAF-targeted therapies. Citation Format: Sandra Ortiz-Cuaran, Julien Mazières, Aurélie Swalduz, Washington René Chumbi Flores, Yohan Loriot, Virginie Westeel, Anne Pradines, Claire Tissot, Christelle Clement Duchene, Christine Raynaud, Xavier Quantin, Radj Gervais, Etienne Brain, Isabelle Monnet, Etienne Giroux Leprieur, Séverine Neymarc, Virginie Avrillon, Solène Marteau, Séverine Martinez, Gilles Clapisson, Nathalie Girerd-Chambaz, Celine Mahier, Nathalie Hoog-Labouret, Frank de Kievit, Karen Howarth, Emma Green, Clive Morris, Maurice Pérol, Jean-Yves Blay, Pierre Saintingy. Integrative analysis of resistance to BRAF-targeted therapies in lung adenocarcinomas [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 1841.

Background: Detection and monitoring of oncogenic mutations in cell-free urinary DNA opens the possibility of a new paradigm for a truly non-invasive method of individualized care for metastatic cancer patients, enabling the quantitation of mutational tumor load and respective concordance to therapeutic responsiveness followed by detection of emerging genomic alterations underlying acquired resistance. Methods: Cell-free DNA was isolated from single and/or multiple sequential urine samples from patients with advanced cancers and BRAF V600E, KRAS G12D or G12V mutations in the tumor tissue from a CLIA-certified laboratory, who progressed on systemic therapy. Assays for quantitative assessment of BRAF V600E, KRAS G12D and G12V mutations in cell-free urinary DNA were developed using droplet digital PCR methodology (RainDance, MA) with enrichment of mutation-containing DNA fragments by pre-amplification of BRAF and KRAS genes. Mutation sensitivity of at least 0.03% was achieved by spike-in experiments of input DNA from cell-lines containing BRAF and KRAS mutations. Healthy controls (N=6) yielded baseline signals that were ∼10-fold less than observed for 0.03% sensitivity. Results: Cell-free DNA was extracted from urine of 25 patients with diverse advanced cancers (colorectal cancer, n=8; melanoma, n=7; non-small cell lung cancer, n=6; papillary thyroid carcinoma, n=2; appendiceal carcinoma, n=1; and glioblastoma, n=1) with BRAF V600E (N=18), KRAS G12D (N=5) and KRAS G12V (N=2) in the tumor tissue. Of 18 patients with BRAF V600E mutations in the tumor, 17 (94%) had the same mutation in urinary cell-free DNA. In addition, all 5 (100%) patients with KRAS mutations (G12D, n=5; G12V, n=2) in the tumor tissue DNA had these same mutations in urinary cell-free DNA.A total of 5 patients with BRAF V600E mutations had longitudinal analysis of percentage of cell-free urinary DNA BRAF V600E mutation to wild-type in sequentially collected urine samples. Although the numbers are small the detected amount of BRAF mutant copies are in agreement with a clinical course. Conclusion: Our preliminary data suggest that detecting BRAF V600E, KRAS G12D, and G12V mutations in cell-free DNA from urine can offer a noninvasive alternative to mutation testing of tumor tissue with excellent concordance, and should be investigated further for testing and monitoring of mutation status in patients with cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B175. Citation Format: Filip Janku, Gerald S. Falchook, Sarina A. Piha-Paul, Aung Naing, Apostolia M. Tsimberidou, Veronica R. Holley, Daniel D. Karp, Ralph G. Zinner, Siqing Fu, Jennifer J. Wheler, David S. Hong, Funda Meric-Bernstam, Vanda M. Stepanek, Rayjalakshmi Luthra, Lorieta Leppin, Latifa Hassaine, Karena Kosco, Jason C. Poole, Mark G. Erlander. Detection and monitoring of BRAF and KRAS mutations in cell-free urinary DNA of metastatic cancer patients by droplet digital PCR. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B175.