Veronica R. Holley

Prospective Blinded Study of BRAFV600E Mutation Detection in Cell-Free DNA of Patients with Systemic Histiocytic Disorders

UNLABELLED Patients with Langerhans cell histiocytosis (LCH) and Erdheim-Chester disease (ECD) have a high frequency of BRAF(V600E) mutations and respond to RAF inhibitors. However, detection of mutations in tissue biopsies is particularly challenging in histiocytoses due to low tumor content and stromal contamination. We applied a droplet-digital PCR assay for quantitative detection of the BRAF(V600E) mutation in plasma and urine cell-free (cf) DNA and performed a prospective, blinded study in 30 patients with ECD/LCH. There was 100% concordance between tissue and urinary cfDNA genotype in treatment-naïve samples. cfDNA analysis facilitated identification of previously undescribed KRAS(G12S)-mutant ECD and dynamically tracked disease burden in patients treated with a variety of therapies. These results indicate that cfDNA BRAF(V600E) mutational analysis in plasma and urine provides a convenient and reliable method of detecting mutational status and can serve as a noninvasive biomarker to monitor response to therapy in LCH and ECD. SIGNIFICANCE Patients with BRAF(V600E)-mutant histiocytic disorders have remarkable responses to RAF inhibition, but mutation detection in tissue in these disorders is challenging. Here, we identify that analysis of plasma and urinary cfDNA provides a reliable method to detect the BRAF(V600E) mutation and monitor response to therapy in these disorders.

BRAF mutation testing with a rapid, fully integrated molecular diagnostics system

Fast and accurate diagnostic systems are needed for further implementation of precision therapy of BRAF-mutant and other cancers. The novel IdyllaTM BRAF Mutation Test has high sensitivity and shorter turnaround times compared to other methods. We used Idylla to detect BRAF V600 mutations in archived formalin-fixed paraffin-embedded (FFPE) tumor samples and compared these results with those obtained using the cobas 4800 BRAF V600 Mutation Test or MiSeq deep sequencing system and with those obtained by a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory employing polymerase chain reaction–based sequencing, mass spectrometric detection, or next-generation sequencing. In one set of 60 FFPE tumor samples (15 with BRAF mutations per Idylla), the Idylla and cobas results had an agreement of 97%. Idylla detected BRAF V600 mutations in two additional samples. The Idylla and MiSeq results had 100% concordance. In a separate set of 100 FFPE tumor samples (64 with BRAF mutation per Idylla), the Idylla and CLIA-certified laboratory results demonstrated an agreement of 96% even though the tests were not performed simultaneously and different FFPE blocks had to be used for 9 cases. The IdyllaTM BRAF Mutation Test produced results quickly (sample to results time was about 90 minutes with about 2 minutes of hands on time) and the closed nature of the cartridge eliminates the risk of PCR contamination. In conclusion, our observations demonstrate that the Idylla test is rapid and has high concordance with other routinely used but more complex BRAF mutation–detecting tests.

BRAF Mutation Testing in Cell-Free DNA from the Plasma of Patients with Advanced Cancers Using a Rapid, Automated Molecular Diagnostics System

Cell-free (cf) DNA from plasma offers an easily obtainable material for BRAF mutation analysis for diagnostics and response monitoring. In this study, plasma-derived cfDNA samples from patients with progressing advanced cancers or malignant histiocytosis with known BRAFV600 status from formalin-fixed paraffin-embedded (FFPE) tumors were tested using a prototype version of the Idylla BRAF Mutation Test, a fully integrated real-time PCR-based test with turnaround time about 90 minutes. Of 160 patients, BRAFV600 mutations were detected in 62 (39%) archival FFPE tumor samples and 47 (29%) plasma cfDNA samples. The two methods had overall agreement in 141 patients [88%; κ, 0.74; SE, 0.06; 95% confidence interval (CI), 0.63–0.85]. Idylla had a sensitivity of 73% (95% CI, 0.60–0.83) and specificity of 98% (95% CI, 0.93–1.00). A higher percentage, but not concentration, of BRAFV600 cfDNA in the wild-type background (>2% vs. ≤ 2%) was associated with shorter overall survival (OS; P = 0.005) and in patients with BRAF mutations in the tissue, who were receiving BRAF/MEK inhibitors, shorter time to treatment failure (TTF; P = 0.001). Longitudinal monitoring demonstrated that decreasing levels of BRAFV600 cfDNA were associated with longer TTF (P = 0.045). In conclusion, testing for BRAFV600 mutations in plasma cfDNA using the Idylla BRAF Mutation Test has acceptable concordance with standard testing of tumor tissue. A higher percentage of mutant BRAFV600 in cfDNA corresponded with shorter OS and in patients receiving BRAF/MEK inhibitors also with shorter TTF. Mol Cancer Ther; 15(6); 1397–404. ©2016 AACR.

Abstract 2689: Overcoming BRAF/MEK resistance using vemurafenib with crizotinib or sorafenib in patients with BRAF-mutant advanced cancers: phase I study

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: BRAF inhibitors are effective in advanced melanoma and other cancers with BRAF V600mutations; however, patients ultimately develop therapeutic resistance through activation of alternative signaling pathways such as HGF/MET, PDGFR and CRAF. We hypothesized that combining the BRAF inhibitor vemurafenib and MET inhibitor crizotinib or PDGFR/CRAF inhibitor sorafenib can increase efficacy by overcoming intrinsic and acquired resistance. Methods: We designed a phase I study (3+3 design) to determine the safety of vemurafenib (240-960 mg PO BID q 28 days) with crizotinib (250 mg PO daily or BID q 28 days) in arm A or sorafenib (200 mg PO daily to 400mg PO BID q 28 days) in Arm B in patients with BRAF-mutant advanced cancers. Endpoints included maximum tolerated dose (MTD), dose limiting toxicities (DLT), safety, response (RECIST 1.1) and plasma cell-free DNA mutation analysis. Results: To date, 29 patients (Arm A, n = 11, vemurafenib 240-960mg PO BID with crizotinib 250mg PO daily; Arm B, n = 18, vemurafenib 240-720mg PO BID with sorafenib 200 mg PO BID to 400/200 mg PO), median age of 53 (33-76) years; median number of 3 (1-5) prior therapies including 22 (76%) patients with prior BRAF or MEK inhibitors were treated. Patients (melanoma 17/29, 59%; papillary thyroid cancer 4/29, 14%; colorectal cancer 3/29, 10%; lung adenocarcinoma 2/29, 7%; other 3/29, 10%) had BRAF V600E (n = 24), V600K (n = 3) or other BRAF mutations (n = 2). The MTDs have not been reached and no DLTs have been observed. Significant drug related toxicities included grade (G) 3 thrombocytopenia (n = 1) in arm A and G3 hypertension (n = 1), G3 headache (n = 1), G3 diarrhea (n = 1) in Arm B. In Arm A, 3 of 11 (27%) patients (melanoma refractory to BRAF monotherapy, -40% for 7.9 months; melanoma refractory to BRAF monotherapy, -36% for 6.0+ months and lung adenocarcinoma, -50% for 13.9 months) attained a partial response (PR) and 1 (9%) patient (melanoma refractory to MEK inhibitor, +3% for 12.5 months) with stable disease (SD) > 6 months. In Arm B, 3 of 18 (17%) patients (melanoma, -46% for 7.6 months; lung adenocarcinoma, -61% for 7.3 months and ovarian cancer refractory to MEK inhibitor, -50% for 18.7 months) attained a PR and 3 (17%) patients (papillary thyroid refractory to MEK inhibitor, -10% for 28.4 months; papillary thyroid cancer refractory to BRAF inhibitor, -6% for 7.3+ months and melanoma refractory to BRAF monotherapy -8% for 7.5 months) attained SD > 6 months. In patients with longitudinal assessment of plasma cfDNA changes in the amount of BRAF mutant DNA corresponded with clinical course (data will be presented). Conclusions: Preliminary data suggest that vemurafenib in combination with crizotinib or sorafenib are well tolerated with encouraging activity in patients previously treated with BRAF or MEK inhibitors. Citation Format: Shumei Kato, Aung Naing, Gerald Falchook, Veronica R. Holley, Vivianne M. Velez-Bravo, Sapna Patel, Ralph G. Zinner, Sarina A. Piha-Paul, Apostolia M. Tsimberidou, David S. Hong, Razelle Kurzrock, Funda Meric-Bernstam, Filip Janku. Overcoming BRAF/MEK resistance using vemurafenib with crizotinib or sorafenib in patients with BRAF-mutant advanced cancers: phase I study. [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 2689. doi:10.1158/1538-7445.AM2015-2689

Abstract 5607: BRAF and KRAS mutation testing in plasma cell-free DNA with ICE COLD-PCR in patients with advanced cancers

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Cell-free (cf) DNA in the plasma of cancer patients offers an easily obtainable, low-risk, inexpensive, and repeatedly available source of biologic material for mutation analysis and monitoring of molecular changes throughout cancer therapy. Methods: DNA in plasma from patients with advanced cancers who progressed on systemic therapy was tested for BRAF V600 and KRAS G12 and G13 mutations using the ICE COLD-PCR platform. ICE COLD-PCR, “Improved and Complete Enrichment COamplification at Lower Denaturation,” selectively amplifies mutant DNA by exploiting differences in denaturation temperatures between mutant DNA duplexes and normal “wild-type” DNA duplexes. KRAS Exon 2 and BRAF Exon 15 ICE COLD-PCR was performed on plasma samples. Amplicons were analyzed using Sanger sequencing and results were compared to the mutation status of archival primary or metastatic tumor tissue as determined in a CLIA-certified laboratory during routine clinical care. Results: Plasma samples from 77 patients with advanced cancers and known tumor tissue BRAF and/or KRAS mutation status (colorectal cancer, n=38; melanoma, n=17; non-small cell lung cancer, n=7; other cancers, n=15) were obtained before treatment and, if possible, sequentially during therapy and tested for BRAF (42), KRAS (34) or BRAF and KRAS (1) mutations in cfDNA. BRAF mutations were detected in 93% (40/43) of archival tumor samples compared to 70% (30/43) of plasma cfDNA samples (agreement 77%). In addition, 20 patients treated with systemic therapy had serial plasma samples collected and the change in relative abundance of BRAF-mutant compared to wild-type cfDNA corresponded with the clinical course of 15 patients and was discrepant for 1 patient; in 5 patients no BRAF mutated cfDNA was detected at any time point. KRAS mutations were detected in 83% (29/35) of archival tumor samples compared to 74% (26/35) of plasma cfDNA samples (agreement 80%). In addition, 12 patients treated with systemic therapy had serial plasma collected and the change in relative abundance of KRAS-mutant compared to wild-type cfDNA corresponded with clinical course in 10 patients; in 2 patients no KRAS mutated cfDNA was detected at any time point. Conclusions: Detection of BRAF and KRAS mutations in cfDNA can provide a fast and noninvasive alternative to mutation testing in tumor tissue with a potential to be used for monitoring response to cancer therapy. Citation Format: Filip Janku, Ben Legendre, Katherine Richardson, Gerald S. Falchook, Aung Naing, Veronica R. Holley, Siqing Fu, David S. Hong, Sarina A. Piha-Paul, Jennifer J. Wheler, Ralph G. Zinner, Vivek Subbiah, Apostolia M. Tsimberidou, Daniel D. Karp, Vanda M. Stepanek, Goran Cabrilo, Rajyalakshmi Luthra, Funda Meric-Bernstam, Agop Y. Bedikian, Bryan K. Kee, Cathy Eng, Michael J. Overman, Kevin B. Kim, Amy Kruempel, Jaclyn Pope, Courtney Cubrich, Grant Wu, Marcia Lewis, Razelle Kurzrock. BRAF and KRAS mutation testing in plasma cell-free DNA with ICE COLD-PCR in patients with advanced cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5607. doi:10.1158/1538-7445.AM2014-5607

Abstract 5584: BRAF mutation testing in cell-free DNA from plasma of patients with advanced cancers using a novel, rapid, automated molecular diagnostics prototype platform (IdyllaTM)

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Cell-free (cf) DNA from the plasma of cancer patients offers an easily obtainable, low-risk, inexpensive and repeatedly applicable source of biologic material for mutation analysis of druggable targets and monitoring molecular changes in tumor(s) during and after therapeutic interventions. Novel, fast, and accurate diagnostic systems are needed for further development of plasma cfDNA testing in personalized therapy. Methods: cfDNA from plasma samples of patients with advanced cancers who progressed on systemic therapy was purified and 100 ng was used for testing for V600 BRAF mutations using the prototype molecular diagnostics (IdyllaTM) fully integrated real-time PCR-based prototype platform (Biocartis, Mechelen, Belgium) with a quick turnaround time (< 90 minutes). The IdyllaTM platform and the BRAF V600 mutation prototype assay were used for research purposes only. Results were compared to mutation analysis of archival primary or metastatic tumor tissue from a CLIA-certified laboratory if available. Results: cfDNA was extracted from plasma samples of 81 patients with advanced cancers (colorectal, n=37; melanoma, n=16; non-small cell lung, n=10; breast, n=4, Erdheim-Chester histiocytosis, n=3; appendiceal, n=3; other cancers, n=8). BRAF mutations were detected in 31% (25/81) of plasma samples and in 42% (30/72) of available archival tumor samples, resulting in concordance for patients who had plasma and tissue tested (N=72) in 90% (65/72) of cases (kappa=0.80, 95% confidence interval 0.65- 0.94). In 6 of 7 discrepant cases identical plasma cfDNA samples were tested using an alternative cfDNA BRAF mutation PCR-based method (BEAMing, Sysmex Inostics, Baltimore, MD), which yielded 100% agreement. Longitudinally collected plasma samples were available in 3 patients (appendiceal, melanoma and papillary thyroid cancer) treated with BRAF targeting combinations and changes in the amount of BRAF-mutant cfDNA corresponded with changes in tumor markers or subsequent responses to therapy visualized via imaging. Conclusions: Detecting V600 BRAF mutations in cfDNA from plasma using the IdyllaTM platform is a fast and noninvasive alternative to mutation testing of tumor tissue with an acceptable level of concordance and sensitivity, and should be investigated further for testing and monitoring of BRAF mutation status in patients with cancer. Citation Format: Helen J. Huang, Bart Claes, Gerald S. Falchook, Veronica R. Holley, Aung Naing, Sarina A. Piha-Paul, Apostolia M. Tsimberidou, Ralph G. Zinner, Daniel D. Karp, Siqing Fu, Vivek Subbiah, David S. Hong, Jennifer J. Wheler, Vanda M. Stepanek, Goran Cabrilo, Rajyalakshmi Luthra, Agop Y. Bedikian, Bryan K. Kee, Cathy Eng, Michael J. Overman, Kevin B. Kim, Tania Ivens, Erwin Sablon, Geert Maertens, Razelle Kurzrock, Funda Meric-Bernstam, Filip Janku. BRAF mutation testing in cell-free DNA from plasma of patients with advanced cancers using a novel, rapid, automated molecular diagnostics prototype platform (IdyllaTM). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5584. doi:10.1158/1538-7445.AM2014-5584

Abstract B177: Non-invasive tumor mutation detection of cell-free urinary DNA using massively parallel deep DNA sequencing.

Background: Previous studies have demonstrated the ability to detect tumor DNA mutations by PCR methodologies within cell-free urinary DNA in metastatic cancer patients. This has opened up the possibility to use massively parallel deep sequencing approaches for more global profiling of tumor mutations using cell free urinary DNA from these patients. Methods: In order to achieve clinical utility the sequencing of cell-free urinary DNA must overcome two technical hurdles: 1) the need for extremely high sensitivity, between 0.01-0.05% and 2) an ultra-short DNA footprint (∼30 bp). Here we report the development of a method using cell free DNA extracted from urine that enriches for extremely low levels of mutated tumor DNA and thereby provides a high detection sensitivity. Detection of the KRAS gene mutations were used as a model system; the developed assay utilizes a 31bp footprint, contains a pre-amplification step that specifically enriches mutated DNA fragments and detects 8 KRAS mutations at the codon 12 and 13 sites. Spiking experiments with DNA derived from cell lines harboring the KRAS G12D mutation were completed to determine the limits of sensitivity of our method. Results: Mutant DNA input at amounts of 0.2%, 0.05%, 0.01% and 0.0% of the total DNA returned observed detection levels of 18.25%, 4.45%, 1.84% and 0.54% respectively as a percentage of the total sequence reads with the detection of mutant DNA of 0.01% within the theoretical limit of the assay. To test our assay, we sequenced 30 ng of extracted urinary DNA from a stage IV colorectal carcinoma patient with a known mutation at the G12D site. Enrichment of the mutation presented at a level of 13.06% of our total sequence, corresponding to an input amount of approximately 0.14% mutational load in the patients urine. Ongoing analysis of a larger number of patients will be presented to further evaluate the clinical sensitivity and specificity of KRAS mutations. Conclusion: Further development of massively parallel DNA sequencing to detect mutations from cell-free urinary DNA has the potential to monitor metastatic patients for response, non-response and the emergence of resistance mechanisms to molecularly targeted therapies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B177. Citation Format: Jason C. Poole, Filip Janku, Veronica R. Holley, Jennifer J. Wheler, Funda Meric-Bernstam, Rayjalakshmi Luthra, Lorieta Leppin, Lafifa Hassaine, Karena Kosco, Mark G. Erlander. Non-invasive tumor mutation detection of cell-free urinary DNA using massively parallel deep DNA sequencing. [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 B177.

Abstract B26: Actionable mutations in cell-free DNA in plasma of patients with advanced cancers referred for experimental targeted therapies.

Background: Actionable mutations confer a survival advantage to cancer cells and can provide therapeutic targets. Cell-free (cf) DNA in the plasma of cancer patients offers an easily obtainable, low-risk, and inexpensive source of biologic material for mutation analysis. Methods: cfDNA in plasma samples from patients with advanced cancers who progressed on systemic therapy was tested for 21 mutations in BRAF (codon 600), EGFR (exons 19-21), KRAS (codons 12, 13), and PIK3CA (exons 9, 20) using the BEAMing method. Results were compared to mutation analysis results from archival primary or metastatic tumor tissue from a CLIA-certified laboratory. Results: cfDNA was extracted from 155 patients with advanced cancers. BRAF mutations were detected in 27% (33/122) plasma samples and in 30% (37/122) of archival tumor samples resulting in agreement in 88.5% cases (kappa=0.72, 95% confidence interval [CI] 0.58- 0.86). EGFR mutations were detected in 8% (6/77) plasma samples and in 6% (5/77) tumor samples resulting in agreement in 98.7% cases (kappa=0.90, 95% CI 0.71- 1.09). KRAS mutations were detected in 50% (55/110) of plasma samples and in 53% (58/110) of tumor samples, resulting in agreement in 82.7% cases (kappa=0.66, 95% CI 0.51- 0.80). PIK3CA mutations were detected in 20% (19/94) of plasma samples and in 14% (13/94) of tumor samples, resulting in concordance in 91.5% cases (kappa=0.70, 95% CI 0.51- 0.89). Of interest, 2 of 9 patients with BRAF mutations (both melanoma) in the tumor, but not in cfDNA had plasma collection at the time of progression on prior BRAF targeting therapy. Also, a patient with V600E BRAF-mutated melanoma, who previously progressed on a MEK inhibitor, displayed a G12R KRAS mutation in cfDNA and a patient with V600K BRAF-mutated melanoma with additional E542K and H1047R PIK3CA mutations in cfDNA was ultimately refractory to BRAF inhibitor treatment. Also, a V600K BRAF mutation was found in the cfDNA of a patient with colorectal cancer and a G12V KRAS mutation. In addition, 2 patients with EGFR-mutated lung adenocarcinoma (exon 19 and L858R), who progressed on erlotinib, had T790M EGFR (n=2) and E545K PIK3CA (n=1) mutations in cfDNA, plausibly explaining secondary resistance to erlotinib. A patient with Erdheim-Chester disease and a V600E BRAF mutation also had a T790M EGFR mutation in cfDNA. Finally, a patient with H1047R PIK3CA mutant ovarian cancer showed an additional G12C KRAS mutation in cfDNA and experienced a partial response followed by early progression on a PI3K inhibitor. Patient characteristics, mutation types and detailed discrepancy analysis will be presented. Conclusions: Detecting actionable mutations in cfDNA is a noninvasive alternative to mutation testing in tumor tissue with an acceptable level of concordance, and should be investigated further for selecting appropriate targeted therapies and monitoring mutation profiles. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B26. Citation Format: Filip Janku, Philipp Angenendt, Aung Naing, Gerald S. Falchook, Apostolia M. Tsimberidou, Veronica R. Holley, Siqing Fu, David S. Hong, Jennifer J. Wheler, Sarina A. Piha-Paul, Ralph G. Zinner, Daniel D. Karp, Vivek Subbiah, Kevin B. Kim, Funda Meric-Bernstam, Frank Diehl, Razelle Kurzrock. Actionable mutations in cell-free DNA in plasma of patients with advanced cancers referred for experimental targeted therapies. [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 B26.

Abstract LB-170: Droplet digital PCR detection and longitudinal monitoring of BRAF mutations in cell-free urinary DNA of patients with metastatic cancers or Erdheim-Chester disease

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, which would enable 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 or Erdheim-Chester disease and BRAF V600E mutation in the tumor tissue from a CLIA-certified laboratory, who progressed on systemic therapy. Assays for quantitative assessment of BRAF V600E mutation in cell-free urinary DNA were developed using digital droplet PCR methodology (RainDance, MA) with enrichment of mutation-containing DNA fragments by pre-amplification of the BRAF gene. Detection limits were established as wild-type ( 0.107%). Results: Cell-free DNA was extracted from urine of 33 patients with diverse advanced cancers (melanoma, n=11; colorectal cancer, n=8; non-small cell lung cancer, n=4; papillary thyroid carcinoma, n=4; other, n=4) or Erdheim-Chester disease (n=1) with BRAF V600E mutation in the tumor tissue. Of these 33 patients, 25 (76%) had the same mutation in urinary cell-free DNA (mutant, n=14; low-mutant, n=11). In addition, 17 patients had longitudinal analysis of percentage of BRAF V600E mutation to wild-type in sequentially collected urine samples and the dynamics of BRAF V600E mutation in the urine correlated with response to therapy in 13 (76%) of them. Conclusion: Our data suggest that detecting BRAF V600E mutation in cell-free DNA from urine can offer a noninvasive alternative to mutation testing of tumor tissue with acceptable concordance and should be investigated further for testing and monitoring of mutation status in patients with cancer and Erdheim-Chester disease. Citation Format: Filip Janku, Gerald S. Falchook, Sarina A. Piha-Paul, Aung Naing, Veronica R. Holley, David S. Hong, Vivek Subbiah, Daniel D. Karp, Ralph G. Zinner, Siqing Fu, Jennifer J. Wheler, Funda Meric-Bernstam, Vanda M. Stepanek, Rajyalakshmi Luthra, Lorieta Leppin, Latifa Hassaine, Karena Kosco, Jason C. Poole, Cecile Rose T. Vibat, Mark G. Erlander. Droplet digital PCR detection and longitudinal monitoring of BRAF mutations in cell-free urinary DNA of patients with metastatic cancers or Erdheim-Chester disease. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-170. doi:10.1158/1538-7445.AM2014-LB-170

Abstract C198: BRAF mutation testing of archival tumor samples with a novel, rapid, fully-automated molecular diagnostics prototype platform.

Background: Novel, fast, and accurate diagnostic systems are needed for further implementation of personalized therapy. Mutations in the BRAF gene can provide actionable targets for cancer therapy in melanoma and other tumor types. Methods: The molecular diagnostics (MDx) prototype platform (Biocartis, Mechelen, Belgium) is a fully integrated real-time PCR-based system with high sensitivity (1%) and quick turnaround time (< 90 minutes), which requires no sample preparation and <2 min hands-on time. Archival formalin-fixed paraffin-embedded tumor samples (1x to 5x of 10 µm shavings) from patients (pts) with advanced cancers previously tested for V600 BRAF mutations in a CLIA-certified Molecular Diagnostic Laboratory (PCR-based sequencing, Sequenom MassARRAY) were tested for BRAF V600 mutations using the MDx prototype platform. MDx prototype platform and the BRAF V600 mutation prototype assay were used for research purposes only. Concordance between different methods and treatment outcomes with BRAF/MEK inhibitors were analyzed. Results: Seventy-nine pts (melanoma, n=34; colorectal, n=20; papillary thyroid, n=6; ovarian, n=4; other cancers, n=15) with available tissue and CLIA laboratory BRAF results were identified (BRAF V600 mutation, n=49; wild-type BRAF, n=30). Of the 70 pts for whom the same tissue block was used for MDx and CLIA, BRAF results (V600 mutation yes/no) were concordant in 67 of them (96%; kappa 0.91; 95% CI 0.81-1.01; discrepancies: V600E mutation from CLIA only in prostate cancer, V600E mutation from CLIA only in melanoma, V600E mutation from MDx only in colon cancer). In addition, BRAF results by MDx were discrepant with CLIA in one case (melanoma) in the mutation subtype (V600K vs. V600E). In all 79 pts (including 9 patients with different blocks tested) MDx and CLIA BRAF results (V600 mutation yes/no) were concordant in 75 (95%; kappa 0.89; 95% CI 0.79-1.00) of them (additional discrepancy: V600K mutation from CLIA only in colon cancer). Of 49 pts with BRAF mutations detected by MDx, 30 were treated on protocols (on the basis of the CLIA lab results) with BRAF/MEK inhibitors and 8 (27%) had a partial response. Of interest, 2 of 3 pts with BRAF mutations from CLIA, but not MDx, received a BRAF/MEK inhibitor and did not respond. Detailed patient characteristics, mutation types and discrepancy analysis will be presented. Conclusions: The BRAF V600 mutation MDx prototype platform is a fast (turn-around time about 1.5 hours) and simple (<2 minutes hands-on time) test for determining BRAF mutation status, having 96% concordance with CLIA laboratory results in identical tissue blocks. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C198. Citation Format: Helen J. Huang, Bart Claes, Gerald S. Falchook, Benoit Devogelaere, Aung Naing, Siqing Fu, Sarina Piha-Paul, David S. Hong, Veronica R. Holley, Apostolia M. Tsimberidou, Vanda M. Stepanek, Kevin B. Kim, Laura S. Angelo, Vivek Subbiah, Jennifer J. Wheler, Ralph G. Zinner, Daniel D. Karp, Rajyalakshmi Luthra, Erwin Sablon, Geert Maertens, Funda Meric-Bernstam, Razelle Kurzrock, Stefan Scherer, Filip Janku. BRAF mutation testing of archival tumor samples with a novel, rapid, fully-automated molecular diagnostics prototype platform. [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 C198.