Goran Cabrilo

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 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.

Multiplex KRASG12/G13 mutation testing of unamplified cell-free DNA from the plasma of patients with advanced cancers using droplet digital polymerase chain reaction

Background Cell-free DNA (cfDNA) from plasma offers easily obtainable material for KRAS mutation analysis. Novel, multiplex, and accurate diagnostic systems using small amounts of DNA are needed to further the use of plasma cfDNA testing in personalized therapy. Patients and methods Samples of 16 ng of unamplified plasma cfDNA from 121 patients with diverse progressing advanced cancers were tested with a KRASG12/G13 multiplex assay to detect the seven most common mutations in the hotspot of exon 2 using droplet digital polymerase chain reaction (ddPCR). The results were retrospectively compared to mutation analysis of archival primary or metastatic tumor tissue obtained at different points of clinical care. Results Eighty-eight patients (73%) had KRASG12/G13 mutations in archival tumor specimens collected on average 18.5 months before plasma analysis, and 78 patients (64%) had KRASG12/G13 mutations in plasma cfDNA samples. The two methods had initial overall agreement in 103 (85%) patients (kappa, 0.66; ddPCR sensitivity, 84%; ddPCR specificity, 88%). Of the 18 discordant cases, 12 (67%) were resolved by increasing the amount of cfDNA, using mutation-specific probes, or re-testing the tumor tissue, yielding overall agreement in 115 patients (95%; kappa 0.87; ddPCR sensitivity, 96%; ddPCR specificity, 94%). The presence of ≥ 6.2% of KRASG12/G13 cfDNA in the wild-type background was associated with shorter survival (P = 0.001). Conclusion(s) Multiplex detection of KRASG12/G13 mutations in a small amount of unamplified plasma cfDNA using ddPCR has good sensitivity and specificity and good concordance with conventional clinical mutation testing of archival specimens. A higher percentage of mutant KRASG12/G13 in cfDNA corresponded with shorter survival.

Development and Validation of an Ultradeep Next-Generation Sequencing Assay for Testing of Plasma Cell-Free DNA from Patients with Advanced Cancer

Purpose: Tumor-derived cell-free DNA (cfDNA) in plasma can be used for molecular testing and provide an attractive alternative to tumor tissue. Commonly used PCR-based technologies can test for limited number of alterations at the time. Therefore, novel ultrasensitive technologies capable of testing for a broad spectrum of molecular alterations are needed to further personalized cancer therapy. Experimental Design: We developed a highly sensitive ultradeep next-generation sequencing (NGS) assay using reagents from TruSeqNano library preparation and NexteraRapid Capture target enrichment kits to generate plasma cfDNA sequencing libraries for mutational analysis in 61 cancer-related genes using common bioinformatics tools. The results were retrospectively compared with molecular testing of archival primary or metastatic tumor tissue obtained at different points of clinical care. Results: In a study of 55 patients with advanced cancer, the ultradeep NGS assay detected 82% (complete detection) to 87% (complete and partial detection) of the aberrations identified in discordantly collected corresponding archival tumor tissue. Patients with a low variant allele frequency (VAF) of mutant cfDNA survived longer than those with a high VAF did (P = 0.018). In patients undergoing systemic therapy, radiological response was positively associated with changes in cfDNA VAF (P = 0.02), and compared with unchanged/increased mutant cfDNA VAF, decreased cfDNA VAF was associated with longer time to treatment failure (TTF; P = 0.03). Conclusions: Ultradeep NGS assay has good sensitivity compared with conventional clinical mutation testing of archival specimens. A high VAF in mutant cfDNA corresponded with shorter survival. Changes in VAF of mutated cfDNA were associated with TTF. Clin Cancer Res; 23(18); 5648–56. ©2017 AACR.

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 493: Quantity of KRAS mutations in cell-free DNA is associated with survival of patients with advanced cancers

Background: Cell-free (cf) DNA from plasma offers an easily obtainable material for KRAS mutation analysis for diagnostics and monitoring. There is emerging evidence that the percentage of mutant cfDNA in the wild-type background (mutant allele fraction, MAF) and/or absolute quantity of mutant cfDNA can be associated with survival of patients with advanced cancers. Methods: Plasma-derived cfDNA from patients with progressing advanced cancers was purified and 16 ng of DNA was tested with a KRAS multiplex assay to distinguish the wild-type allele from 7 of the most common mutations in the G12 and G13 hotspot of exon 2 using the QX200 Droplet Digital PCR™ platform (Bio-Rad). Results were compared to mutation analysis of archival primary or metastatic tumor tissue obtained at different points of clinical care from a CLIA-certified laboratory and clinical outcomes including survival. Results: Of the 117 patients (colorectal cancer, 71; non-small cell lung cancer, 12; melanoma, 10; pancreatic cancer, 5; ovarian cancer, 5; appendiceal cancer, 5; other cancers, 9), KRAS mutations were detected in 85 (73%) archival FFPE tumor samples and 85 (73%) plasma cfDNA samples. The two methods had overall agreement in 109 patients (93%; kappa, 0.83, standard error, 0.06; 95% confidence interval [CI], 0.71-0.94), sensitivity of 95% (95% CI, 0.88-0.99), specificity of 88% (95% CI, 0.71-0.96), even though median time from tissue to blood sampling was 18.5 months (1.1-134.4 months). A higher MAF (>7%) of KRAS in cfDNA as determined by 5% trimmed mean value was associated with shorter survival compared to lower ( Conclusions: A higher percentage of KRAS mutation in plasma cfDNA is an independent predictive factor for shorter survival in patients with advanced cancers. Citation Format: Kiran Madwani, Helen J. Huang, Dawne N. Shelton, Siqing Fu, Apostolia M. Tsimberidou, Sarina A. Piha-Paul, Aung Naing, David S. Hong, Daniel D. Karp, Debra L. Andrews, Goran Cabrilo, E. Scott Kopetz, Rajyalakshmi Luthra, Bryan K. Kee, Cathy Eng, Van K. Morris, George A. Karlin-Neumann, Funda Meric-Bernstam, Filip Janku. Quantity of KRAS mutations in cell-free DNA is associated with survival of patients with advanced cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 493.