Cecile Rose T. Vibat

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.

A highly sensitive and quantitative test platform for detection of NSCLC EGFR mutations in urine and plasma

Introduction: In approximately 60% of patients with NSCLC who are receiving EGFR tyrosine kinase inhibitors, resistance develops through the acquisition of EGFR T790M mutation. We aimed to demonstrate that a highly sensitive and quantitative next‐generation sequencing analysis of EGFR mutations from urine and plasma specimens is feasible. Methods: Short footprint mutation enrichment next‐generation sequencing assays were used to interrogate EGFR activating mutations and the T790M resistance mutation in urine or plasma specimens from patients enrolled in TIGER‐X (NCT01526928), a phase 1/2 clinical study of rociletinib in previously treated patients with EGFR mutant–positive advanced NSCLC. Results: Of 63 patients, 60 had evaluable tissue specimens. When the tissue result was used as a reference, the sensitivity of EGFR mutation detection in urine was 72% (34 of 47 specimens) for T790M, 75% (12 of 16) for L858R, and 67% (28 of 42) for exon 19 deletions. With specimens that met a recommended volume of 90 to 100 mL, the sensitivity was 93% (13 of 14 specimens) for T790M, 80% (four of five) for L858R, and 83% (10 of 12) for exon 19 deletions. A comparable sensitivity of EGFR mutation detection was observed in plasma: 93% (38 of 41 specimens) for T790M, 100% (17 of 17) for L858R, and 87% (34 of 39) for exon 19 deletions. Together, urine and plasma testing identified 12 additional T790M‐positive cases that were either undetectable or inadequate by tissue test. In nine patients monitored while receiving treatment with rociletinib, a rapid decrease in urine T790M levels was observed by day 21. Conclusions: DNA derived from NSCLC tumors can be detected with high sensitivity in urine and plasma, enabling diagnostic detection and monitoring of therapeutic response from these noninvasive “liquid biopsy” samples.

Mutation-enrichment next-generation sequencing for quantitative detection of KRAS mutations in urine cell-free DNA from patients with advanced cancers

Purpose: Tumor-derived cell-free DNA (cfDNA) from urine of patients with cancer offers noninvasive biological material for detection of cancer-related molecular abnormalities such as mutations in Exon 2 of KRAS. Experimental Design: A quantitative, mutation-enrichment next-generation sequencing test for detecting KRASG12/G13 mutations in urine cfDNA was developed, and results were compared with clinical testing of archival tumor tissue and plasma cfDNA from patients with advanced cancer. Results: With 90 to 110 mL of urine, the KRASG12/G13 cfDNA test had an analytical sensitivity of 0.002% to 0.006% mutant copies in wild-type background. In 71 patients, the concordance between urine cfDNA and tumor was 73% (sensitivity, 63%; specificity, 96%) for all patients and 89% (sensitivity, 80%; specificity, 100%) for patients with urine samples of 90 to 110 mL. Patients had significantly fewer KRASG12/G13 copies in urine cfDNA during systemic therapy than at baseline or disease progression (P = 0.002). Compared with no changes or increases in urine cfDNA KRASG12/G13 copies during therapy, decreases in these measures were associated with longer median time to treatment failure (P = 0.03). Conclusions: A quantitative, mutation-enrichment next-generation sequencing test for detecting KRASG12/G13 mutations in urine cfDNA had good concordance with testing of archival tumor tissue. Changes in mutated urine cfDNA were associated with time to treatment failure. Clin Cancer Res; 23(14); 3657–66. ©2017 AACR.

Performance and Diagnostic Accuracy of a Urine-Based Human Papillomavirus Assay in a Referral Population

Background: Human papillomavirus (HPV) testing from clinician-collected cervical and self-collected cervico-vaginal samples is more sensitive for detecting CIN2+/CIN3+ than cytology-based screening, stimulating interest in HPV testing from urine. The objective was to determine the performance of the Trovagene HPV test for the detection of CIN2+ from urine and PreservCyt cervical samples. Methods: Women referred for colposcopy at St Marys Hospital (London, United Kingdom), following abnormal cytology, were recruited to this diagnostic accuracy study by convenience sampling (September 2011 to April 2013). A total of 501 paired urine and cervical samples were collected. Primary outcomes were sensitivity for CIN2+/CIN3+ and specificity for <CIN2; secondary outcomes were comparisons with other HPV tests and agreement/kappa values between urine and cervical samples. Results: Trovagene HPV test sensitivity and specificity from PreservCyt were similar to well-established tests [sensitivity for CIN3+ (n = 145) 96.3% (95% confidence interval (CI), 89.6–99.2); CIN2+ (n = 81) 94.5% (95% CI, 89.4–97.6); specificity for <CIN2 25.3% (95% CI, 20.8–30.1)]. Sensitivity from urine was slightly, but not significantly, lower [CIN3+ 91.4% (95% CI, 83.0–96.5), P = 0.3; CIN2+ 88.3% (95% CI, 81.9–93.0), P = 0.06]. Specificity for <CIN2 was similar: 24.7% (95% CI, 20.3–29.5), P = 0.9. A total of 403 Trovagene cervical and 396 urine HPV tests were positive. Overall agreement between paired samples was 82.6% (95% CI, 79.3–86.0). Conclusions: Trovagene HPV tests performance on PreservCyt cervical samples was comparable with established HPV tests. Sensitivity in urine, although slightly lower, may nevertheless be adequate for self-sampling. This referral populations higher HPV positivity rate affects specificity, warranting further studies in a screening population. Impact: This may prove useful for women not attending for cervical screening. Cancer Epidemiol Biomarkers Prev; 26(7); 1053–9. ©2017 AACR.

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

High-Risk Human Papillomavirus Detection in Urine Samples from a Referral Population with Cervical Biopsy-Proven High-Grade Lesions

The aim of the study was to evaluate the performance of the HPV-HR test to detect high-risk human papillomavirus (HPV) in urine samples in comparison with a commercial molecular HPV test. Materials and Methods This is a prospective study, in which 350 patients diagnosed previously with cervical intraepithelial neoplasia (CIN) grade 2 or higher were enrolled. Urine and cervical specimens were collected. Urine was tested with the HPV-HR test and cervical specimens were tested with the Cobas. Results Of the 336 evaluable patients, there were 271 cases of CIN 2+, of which 202 were CIN 3+ and the remaining 65 patients were less than CIN 2. Positivity was 77.1% (95% confidence interval [CI] = 72.5–81.5) for the urine samples and 83.6% (95% CI = 79.6–87.6) for the cervical samples. Agreement between cervical and urine samples for HPV detection was 79.8% (&kgr; = 0.363; 95% CI = 0.243–0.484). Sensitivity for CIN 2+ was 83.4% (95% CI = 78.4–87.6) for urine and 90.8% (95% CI = 86.7–92.9) for cervical samples. The sensitivity for CIN 3+ was 85.6% (95% CI = 80.0–90.2) for urine and 92.6% (95% CI = 88.0–95.8) for cervical samples. Specificity for worse than CIN 2 was 50.8% (95% CI = 33.7–59.0) and 46.2% (95% CI = 33.7–59.0) for urine and cervical samples, respectively. Conclusions Although these results demonstrated slightly higher detection rates for HR-HPV and clinical sensitivity in cervical samples than in urine, when compared with histological diagnoses, urine sampling is a viable alternative to access women who do not participate in routine screening programs.

Sensitivity of TargetSelector in clinical experience in ctDNA profiling of NSCLC 2000 cases.

e23033Background: Targeted cancer therapy relies on identifying specific DNA mutations from a patient’s tumor. Tyrosine kinase inhibitors (TKIs) tend to be effective for non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) activating mutations, of which exon 19 deletions (Del19) and L858R are most common. Acquired resistance to TKI therapy is associated with a T790M mutation. Standard biomarker analyses may not reflect tumor heterogeneity; they entail tissue biopsies often with surgical complications. To address these limitations, Biocept developed a minimally invasive method to characterize cancer biomarkers in blood. Biocepts proprietary TargetSelector assays selectively amplify relevant mutations from circulating tumor DNA (ctDNA). Clinical validations demonstrated high concordances between molecular tests in blood vs tissue. As further validation, EGFR mutation detection frequencies were compared to US averages (mycancergenome.org). Here we analyze 2000 blood samples receiv...

Abstract 30: Quantitative monitoring of EGFR mutations in urinary circulating tumor DNA enables non-invasive pharmacodynamic assessment of anti-EGFR drug response

Background: Acquisition of the EGFR T790M resistance mutation is a hallmark of disease progression in patients with metastatic EGFR mutant lung adenocarcinoma treated with first generation anti-EGFR inhibitors. Utilizing a single copy sensitivity mutation detection platform and implementing daily collection of urine samples, we sought to demonstrate the feasibility of detecting EGFR mutations in urinary ctDNA and understand mechanisms of resistance to targeted therapies in patients with EGFR-mutated lung adenocarcinoma. Methods: In a biomarker study of 100 patients with EGFR-mutant metastatic lung adenocarcinoma (34 patients enrolled), urine was collected at either daily or monthly time points up to 4 months prior to radiologic detection of progression on erlotinib, and at multiple time points post-progression on next line therapy. Urinary ctDNA was extracted by a method that preferentially isolates short, fragmented ctDNA. Quantitative analysis of EGFR activating mutations and T790M resistance mutation was performed using blocker technology and PCR enrichment coupled with NGS detection (MiSeq). Urine was collected daily after the initiation of second line anti-EGFR therapy in 10 patients. Early pharmacodynamic events that occur within the first hours to days of anti-EGFR therapy were further studied by quantitating ctDNA for EGFR exon 19deletions, L858R, and T790M. Results: Interim analysis was conducted on 34 patients receiving first line anti-EGFR therapy with erlotinib; twenty-two of 34 patients demonstrated radiographic progression. Analysis of longitudinal samples revealed that the EGFR T790M mutation was detected in the urine specimens of 15 out of 22 (68%) patients on erlotinib. All 10 patients who were positive for T790M mutation by tissue were also positive by urine. Urine testing identified five additional T790M-positive patients who had a high clinical suspicion of T790M progressive disease. Three of these patients were tissue negative but both plasma and urine positive for T790M. EGFR T790M was detected up to 15 weeks prior to radiolographic detection of progression on first line erlotinib. Early peaks in ctDNA on days 1-4 correlated with tumor lysis. An observed sustained decrease in mutational levels after week 1 of therapy confirms the cytostatic effect of the tyrosine kinase inhibitor (TKI). The size of the initial peaks in ctDNA for EGFR exon 19deletions, L858R, and T790M correlated with CT radiographic response after two cycles of therapy. Conclusion: We demonstrate that the T790M mutation can be successfully detected in urinary ctDNA months before progression on anti-EGFR TKIs. Urinary ctDNA testing identifies additional patients who potentially are eligible for anti-T790M treatment. Initial results from 10 patients demonstrated that kinetic changes in EGFR ctDNA mutational load after drug adminstration can be used with pharmacokinetic data to better understand dyanmic changes in tumor biology, drug bioavailability, and assist in early drug development. The clinical utility of daily kinetic monitoring of ctDNA in urine after drug adminstration is being further validated in a larger study. Citation Format: Hatim Husain, Karena Kosco, Saege Hancock, Errin Samuelsz, Shiloh Guerrero, Brian Woodward, Cecile Rose Vibat, Vlada Melnikova, Mark Erlander, Scott Lippman, Razelle Kurzrock. Quantitative monitoring of EGFR mutations in urinary circulating tumor DNA enables non-invasive pharmacodynamic assessment of anti-EGFR drug response. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 30.

Abstract 3146: Circulating tumor DNA assay performance for detection and monitoring of KRAS mutations in urine from patients with advanced cancers

Introduction: Non-invasive urinary ctDNA-based liquid biopsy approach can be used to detect and track cancer driver mutations for rapid diagnosis and disease monitoring. Using highly sensitivity ctDNA mutation detection platform, we examined the detection of KRAS G12/13 mutations in urine obtained from advanced cancer patients, assessed urine sample requirements, and compared the results with matched tumor tissue in patients with advanced cancers. Methods: 41 patients with advanced solid cancer with KRAS mutations on archival tumor from CLIA laboratory testing were prospectively enrolled with informed consent (colorectal cancer, n = 29; non-small cell lung cancer, n = 6; pancreatic cancer, n = 2; ovarian cancer, n = 2; other, n = 2). Urine was collected before and during experimental therapies. Urinary DNA was isolated using a method that enriches for highly fragmented, systemically derived cell-free DNA. KRAS G12/13 analysis was performed using mutation enrichment PCR coupled with next generation sequencing (MiSeq). Analytical sensitivity of the KRAS G12/13 assay is 0.006% mutant alleles in the background of 60 ng wild-type (wt) DNA and 0.002% mutant alleles in 360 ng wt DNA. Clinical data was collected retrospectively from the electronic medical record. Results: For 41 patients enrolled on a study, urine volumes in pretreatment samples ranged from 13 to 120 mL (median, 55 mL). Urinary DNA yields were 151 to 23059 ng (median, 1039 ng). Using tissue as the reference, the positive percent agreement (PPA) between urine and tumor KRAS G12/13 test results was 54% (22/41) for urine samples with all volumes (13-120 mL) and any DNA input amount (2-360 ng) and 92% (12/13) for urine samples with volumes ≥50 mL and DNA input amount ≥60 ng. For metastatic CRC patient cohort, the PPA between urine and tumor KRAS G12/13 test result was 60% (18/30) for urine samples with all volumes and any DNA input amount (20-120 mL, 2-360 ng) and 100% (10/10) for urine samples with volumes ≥50 mL and DNA input amount ≥60 ng. Feasibility of longitudinal monitoring KRAS G12/13 mutational burden in urine of patients treated with experimental therapies was demonstrated. Conclusion: KRAS G12/13 mutational status can be assess in urinary DNA with highest PPA amongst patients with urine volume ≥50 mL and DNA input amount ≥60 ng (92%). KRAS mutation detection from urine should be considered as a viable approach, particularly when tumor tissue is not available. Citation Format: Takeo Fujii, Cecile Rose T. Vibat, Daniel D. Karp, Sarina A. Piha-Paul, Vivek Subbiah, Apostolia M. Tsimberidou, Siquing Fu, David S. Hong, Helen J. Huang, Kiran Madwani, Debra L. Andrews, Saege Hancock, Aung Naing, Rajyalakshmi Luthra, Bryan K. Kee, Scott Kopetz, Mark G. Erlander, Vlada Melnikova, Funda Meric-Bernstam, Filip Janku. Circulating tumor DNA assay performance for detection and monitoring of KRAS mutations in urine from 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 3146.

Abstract A31: Assessment of EGFR mutations in matched urine, plasma and tumor tissue in NSCLC patients treated with rociletinib (CO-1686)

Background: The acquisition of suitable tumor tissue is a challenge for a significant fraction of late-stage NSCLC patients who require EGFR testing to inform choice of therapy. An alternative for these patients could be the assessment of EGFR mutations in circulating tumor DNA (ctDNA). In this study, we examined the detection of EGFR T790M mutation in ctDNA from urine, assessed urine sample requirements, and compared the results with contemporaneously matched tumor tissue and plasma in TIGER-X (NCT01526928), a Phase 1/2 clinical study of rociletinib in previously treated mutant EGFR patients with advanced NSCLC. Rociletinib is an oral, potent, small-molecule irreversible tyrosine kinase inhibitor that selectively targets mutant forms of EGFR, including T790M, L858R and Del(19), while sparing wild-type EGFR. Methods: 63 Stage IIIB/IV NSCLC patients enrolled in either Phase 1 or 2 components of TIGER-X and representing all therapeutic dose groups consented to optional urine collection. Maximum sample volumes were 100 mL for urine and 2 mL for plasma. To maximize assay sensitivity in urine, samples containing the recommended sample volume of ≥90 mL (≥ 90% of maximum in this study) were evaluated; all samples received were processed to assess this recommendation. Urinary and plasma ctDNA were tested for mutations by the same EGFR assays using a sensitive and quantitative short footprint assay method that employs a mutation enrichment step followed by next generation sequencing. Results: Urine volumes ranged from 8-100 mL with a median DNA yield of 313 ng (N = 63). The median DNA yield was 299 ng for urine specimens with volume Conclusions: The analysis of ctDNA from urine identified a similar proportion of T790M+ patients as tissue-based testing with highest PPA amongst patients with urine volumes ≥90 mL. Discordant samples between urine and tissue that were not identified by the tumor test may be explained by tumor heterogeneity and/or inadequate biopsy. EGFR mutation detection from urine increases with urine volume and DNA yields and should be considered as a viable approach, particularly when tumor tissue is not available. Lastly, monitoring urine ctDNA T790M mutations longitudinally with baseline and post-therapy sampling could be clinically useful to determine benefit from therapy. Citation Format: Shirish Gadgeel, Chris Karlovich, Vlada Melnikova, Lecia V. Sequist, D. Ross Camidge, Heather Wakelee, Maurice Perol, Geoffrey R. Oxnard, Karena Kosco, Cecile Rose T. Vibat, Elaina Mann, Shannon Matheny, Lindsey Rolfe, Mitch Raponi, Mark G. Erlander, Karen Reckamp. Assessment of EGFR mutations in matched urine, plasma and tumor tissue in NSCLC patients treated with rociletinib (CO-1686). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A31.