Jason C. Poole

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.

Abstract 5238: Methodology for single copy detection and quantitative monitoring of clinically actionable circulating tumor DNA mutations in urine from cancer patients

Background: Non-invasive detection and monitoring of circulating tumor DNA (ctDNA) mutations for personalized treatment of cancer patients can be realized by combining the practical advantages of urine as a ctDNA sample source with high throughput of next-generation sequencing (NGS). Methods: Our platform couples an extraction process capable of isolating ctDNA from the entire void volume of a urine sample (∼100ml) with an ultra-sensitive NGS-integrated mutation enrichment method with single copy detection sensitivity. Assays have been developed and validated to interrogate clinically actionable mutations/deletions in the KRAS, BRAF and EGFR (Exons 19, 20, 21) oncogenes in both urine and plasma samples. For mutation detection with high sensitivity, a novel allele-specific competitive cycling (ASCC) method was used prior to NGS to amplify ultra-short target DNA (31-45 bp) using kinetically-favorable binding conditions for a wild type (WT) blocking oligonucleotide. Enriched amplicons were sequenced and a proprietary algorithm was used to quantify the mutant ctDNA input level in analytical and clinical samples. Results: An extraction method optimized for enrichment of fragmented urine ctDNA enabled isolation of large amounts of DNA (mean ∼2 μg) from advanced stage patients with different cancer types. Analytical performance characterization demonstrated sensitivity of 0.0047 - 0.01% mutant copies in WT/mutant DNA blends. To show that our assays have a true single copy detection threshold, we used DNA blends with defined mutant spike-in levels of 2 - 60 copies distributed over 20 wells (to obtain 0 - 3 mutant copies/well). The observed distribution of positive and negative hits matched the theoretical hit rate of an ideal Poisson distribution for these replicates, confirming single copy sensitivity of our assays. Using the KRAS ASCC assay, 1 to 17,555 mutant fragments were detected per 1 mL of urine collected from KRAS tissue biopsy-positive advanced cancer patients. Analysis of serial patient-matched urine and plasma longitudinal samples from KRAS, BRAF, or EGFR tissue positive patients demonstrated a high level of concordance between urine and plasma samples and feasibility of monitoring mutation load in a variety of clinical settings, including monitoring post-surgery and responsiveness to targeted therapy or chemotherapy. Conclusion: The analytical characterization and clinical feasibility studies demonstrate that this methodology can successfully detect and quantitate mutational load in urinary ctDNA, thus enabling for the dynamic monitoring of therapy response, drug resistance, and minimal residual disease in cancer patients from a truly non-invasive sample. Citation Format: Karena Kosco, Jason C. Poole, Saege Hancock, Errin Samuelsz, Timothy T. Lu, Erin Clark, Latifa Hassaine, Shiloh Guerrero, Cecile Rose T. Vibat, Vlada Melnikova, Mark G. Erlander. Methodology for single copy detection and quantitative monitoring of clinically actionable circulating tumor DNA mutations in urine from cancer patients. [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 5238. doi:10.1158/1538-7445.AM2015-5238

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 5240: Comparative levels of KRAS mutations circulating tumor DNA for association with overall survival in patients with non-resectable pancreatic cancer

Background: The median overall survival (OS) time of patients with non-resectable pancreatic cancer varies widely. Diagnostic tools are presently lacking to predict patient outcome at diagnosis. The vast majority of pancreatic tumors harbor KRAS mutations. In this study, we evaluated whether quantitative baseline and longitudinal monitoring of KRAS mutations in plasma circulating tumor DNA (ctDNA) may be used to stratify patients for predicting outcome. Methods: The Danish BIOPAC study prospectively collected plasma from patients with non-resectable pancreatic cancer undergoing treatment with gemcitabine or FOLFIRINOX. Archival (3-5 years) plasma specimens were collected from 113 patients pre-treatment (baseline),on chemotherapy, as well as at multiple additional time intervals for up to 977 days from baseline. Interim analysis of ctDNA KRAS was conducted (after 105 deaths). Levels of ctDNA KRAS mutations were assessed in 35 patients with long OS (median 473 days; range 360-1134), 33 patients with medium OS (median 227 days; range 155-349) and 37 patients with short OS (median 94 days; range 21-146). PCR enrichment of KRAS G12A/C/D/R/S/V, and G13D mutations was performed, followed by massively parallel deep sequencing and quantification with standardization of reporting number of copies detected per 105 genome equivalents (GE). Results: In a prospective-retrospective biomarker study of 113 patients, interim analysis of ctDNA KRAS was conducted (after 105 deaths). 92 of 105 patients had evaluable baseline plasma samples. Number of mutant KRAS copies was higher in patients with short OS (median 661; range 0-190,490 copies/105 GE) versus with median OS (median 103; range 0 to 275,918 copies/105 GE) versus with long OS (median, 15; range, 0-1,369 copies/105 GE). Longitudinally, KRAS mutation levels remained mostly low with long OS (last time point median 9; range 0-70,451 copies/105 GE) vs. medium OS (median 155; range 0-314,103 copies/105 GE) or short OS where levels increased or remained high (median 803; range 0-138,508 copies/105 GE). As this dramatic difference in systemic KRAS levels may reflect distinct tumor phenotypes, the underlying tumor biology was further investigated by interrogating additional cancer mutational hotspots (using massively parallel deep sequencing) in plasma ctDNA of patients stratified by systemic KRAS and the OS. Conclusion: Shorter OS in patients with non-resectable pancreatic cancer tended to associate with high levels of ctDNA KRAS mutations at diagnosis and with post-treatment elevation of KRAS mutations. ctDNA KRAS mutation levels in patients with non-resectable pancreatic cancer observed at diagnosis or on treatment may predict patient outcome and could reflect distinct underlying tumor biology. Citation Format: Julia S. Johansen, Cecile Rose T. Vibat, Saege Hancock, Latifa Hassaine, Errin Samuelsz, Inna Chen, Eric A. Collisson, Dan Calatayud, Benny V. Jensen, Jane Preuss Hasselby, Timothy T. Lu, Jason C. Poole, Vlada Melnikova, Mark G. Erlander. Comparative levels of KRAS mutations circulating tumor DNA for association with overall survival in patients with non-resectable pancreatic cancer. [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 5240. doi:10.1158/1538-7445.AM2015-5240

Abstract 5237: Monitoring minimal residual disease by urinary or plasma circulating tumor DNA of KRAS mutation burden in colorectal cancer patients with resectable liver metastases

Background: Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the third leading cause of cancer deaths. Over half of patients with CRC will develop liver metastases. Surgical resection, in combination with systemic therapies, greatly improves long-term outcomes, and around 40% of patients with resected liver limited disease are alive 5 years after diagnosis. While tumor staging and radicality of surgery are commonly used for prognostic assessment, better non-invasive markers are needed for monitoring chemo-responsiveness, following minimal residual disease (MRD), and guiding complex treatment decisions in these patients. This study evaluated the utility of quantitating KRAS mutation burden in urinary and plasma ctDNA as a means of monitoring MRD in surgical CRC patients with liver limited metastases. Methods: We developed a novel, NGS-based method for enrichment and quantitative detection of KRAS mutations in urinary and plasma ctDNA. A blinded retrospective study was conducted on 20 Stage I-IV CRC patients, 15 of whom had undergone curative or palliative intent surgical resection of primary tumor or liver metastases in combination with various systemic therapies. Results: Archived, matched urine and plasma samples were collected from 20 patients with KRAS positive primary tumor. For operable patients, specimens were collected prior to surgery, during and immediately after surgery, plus additional time points post-surgery. A total of 193 plasma and urine samples archived for 3-5 years were tested. All 101 of 101 plasma samples (100%) and 79 of 92 urine samples (86%) had sufficient DNA and were deemed evaluable. In a blinded analysis, a correct KRAS mutation that correlated with KRAS mutation in tissue was identified in 95% of evaluable baseline plasmas (19 of 20) and 92% of evaluable baseline urines (11 of 12). In one patient, a KRAS mutation distinct from that identified in the tumor biopsy was detected consistently across all serial ctDNA samples. Overall, we observed a clear correlation and highly comparable fold change between plasma and urinary ctDNA KRAS levels on treatment. Significantly, in all patients with curative intent surgery, ctDNA KRAS levels were undetectable in urine or plasma after surgery. In contrast, in 8 of 10 patients with incomplete, palliative surgery, the ctDNA KRAS signal remained detectable or increased after surgery. Further correlation between ctDNA KRAS and clinical outcomes will be discussed. Conclusion: We demonstrate for the first time that quantitative changes of mutational KRAS burden in plasma and urinary ctDNA are highly correlated. We further demonstrate clinical applicability of urinary ctDNA KRAS analysis for monitoring quantitatively, with single molecule sensitivity, the MRD post-surgery for CRC patients with liver resectable metastases. Supported by Grant no. 13660. Citation Format: Vlada Melnikova, Jason C. Poole, Cecile Rose T. Vibat, Lucie Benesova, Barbora Belsanova, Saege Hancock, Latifa Hassaine, Errin Samuelsz, Timothy T. Lu, Mark G. Erlander, Marek Minarik. Monitoring minimal residual disease by urinary or plasma circulating tumor DNA of KRAS mutation burden in colorectal cancer patients with resectable liver metastases. [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 5237. doi:10.1158/1538-7445.AM2015-5237

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 B175: Detection and monitoring of BRAF and KRAS mutations in cell-free urinary DNA of metastatic cancer patients by droplet digital PCR.

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.