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Supplemental Figure 1: BRAFV600E mutant allele burden in cell-free DNA (cfDNA) of urine and plasma from treatment naïve patients based on BRAFV600E tissue genotype result.

Supplemental Figure 2: Radiographic, histologic, and molecular evaluation of KRASG12S mutant patient with Erdheim-Chester Disease (ECD).

Supplemental Table 3: Plasma and urine cfDNA mutation detection zscores for KRAS (G12A, G12C, G12D, G12R, G12S, G12V, G13D) with tissue biopsy KRASG12 genotyping from patient with Erdheim-Chester Disease (ECD).

Supplemental Table 1: Concordance of initial urinary cell-free DNA (cfDNA) assessment of BRAFV600E mutation with tissue biopsy BRAFV600E result.

Supplemental Table 2: Concordance of initial plasma cell-free DNA (cfDNA) assessment of BRAFV600E mutations with tissue biopsy BRAFV600E result.

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.

Supplementary methods. Supplementary Table S1. Verification of the analytical sensitivity (lower limit of detection) of the KRASG12/G13 mutation-enrichment NGS assay. Supplementary Table S2. KRASG12/G13 mutations in archival tumor tissue, urine cell-free DNA (cfDNA) and plasma cfDNA. Supplementary Table S3. Systemic therapies in patients with serial urine and/or plasma cell-free DNA (cfDNA) collection. Supplementary Figure S1. Workflow and characteristics of the platform used to analyze cell-free DNA in urine and plasma. Supplementary Figure S2. Schematic of the mutation-enrichment next-generatin sequencing (NGS) assay for the detection of KRASG12/13 mutations in cell-free DNA (cfDNA). Supplementary Figure S2. Schematic of the mutation-enrichment next-generatin sequencing (NGS) assay for the detection of KRASG12/13 mutations in cell-free DNA (cfDNA). Supplementary Figure S3. Kaplan-Meier curves of overall survival (OS) based on the number of KRASG12/13 copies in cell-free DNA (cfDNA). Supplementary Figure S4. Kaplan-Meier curves of overall survival (OS) based on the concentration of cell-free DNA (cfDNA).

Background Poly (ADP-ribose) polymerase inhibitors (PARPi) have demonstrated efficacy in treating solid tumors with Homologous Recombination Deficiency (HRD), the inability to repair DNA double-stranded breaks through the Homologous Recombination Repair (HRR) pathway. Specific genetic and epigenetic alterations result in defective HRR function. Bi-allelic loss of BRCA1 or BRCA2 principally drives HRR deficiency. While BRCA1/2 are instrumental to HRR, multiple genes, including PALB2, impact the HRR pathway. PALB2 mutations occur in an estimated 0.97% to 3.66% of solid tumors [AACR GENIE PALB2] and are associated with susceptibility to various cancers. No clinically approved therapies specifically targeting PALB2 currently exist. Emerging evidence suggests that patients with germline or somatic PALB2 mutations may benefit from PARPi treatment, which has potential to be a new tumor agnostic therapy option across a wide range of solid tumors. Methods PAVO is a pan-tumor, single-arm, multicenter Phase-II study assessing the safety and efficacy of niraparib (a PARPi) in patients who harbor a confirmed PALB2 mutation. The study plans to enroll up to 110 adult subjects. Eligible participants must have: locally advanced or metastatic solid tumor(s); confirmed pathogenic or likely pathogenic somatic or germline PALB2 mutation; received all standard of care (SOC) therapy for their tumor type, or are unlikely to derive benefit from SOC therapy in the opinion of the treating physician; ECOG performance status of 0 or 1; life expectancy of ≥ 12 weeks with adequate organ/bone marrow function. Exclusion criteria include a confirmed somatic or germline BRCA1/2 mutation, prior treatment with any PARPi, ovarian or prostate cancer, or rapid progression while on platinum-based therapy in the metastatic setting. Niraparib will be administered in 28 day cycles with daily dosing, as outlined in the protocol. Participants will continue study treatment until documented radiographic progression, unacceptable toxicity, death, or consent withdrawal. The primary study endpoint is objective response rate (ORR), defined as the proportion of participants who have partial or complete response to therapy as assessed by Independent Central Review. Secondary endpoints include DOR, PFS, and CBR. PAVO is sponsored by Tempus with support from GSK (GlaxoSmithKline). The trial opened in March 2022. Tempus molecular data tracking (integrated NGS and EMR data) and the TIME Trial program (rapid match of patients to Just in TIME sites for clinical trials), enable patient identification and prescreening. Enrollment occurs through a combination of TIME and prospective clinical sites, where individualized prescreening models are in development. New site identification and referral of molecularly eligible patients to enrolling centers are ongoing. Clinical Trial Registry: NCT05169437 Citation Format: Tian Zhang, Thomas Weart, Matthias Weiss, Drew Murray, Minaxi Jhawer, Edward Huynh, Shumei Kato, Amy Cummings, Lydia Usha, Arvinder Bhinder, Rajiv Desai, Brad Johnson, Anjali Avadhani, Cecile Rose T. Vibat, Lauren Lopez, Brynna Driscoll, Annajane Ward, Christie K. Rice, Blathnaid Donovan, Scott Sherrin, Mykel Robble, Stephanie O'Leary, Kimberly Blackwell, Amine Aziez, Stephanie Petrone, Kathleen Harnden, Kimberly Strickland, Sonya Reid, Mark Robson, Andrew S. Paulson, Afshin Dowlati. PAVO: A phase-II, open label, single arm study of niraparib in patients with locally advanced/metastatic PALB2 mutated tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT055.

2031 Background: High grade gliomas (HGG) including glioblastoma (GBM) are among the most aggressive brain cancers, with patients exhibiting highly variable treatment responses in both newly diagnosed (ND) and recurrent disease. Temozolomide (TMZ) + radiation therapy is the guideline directed standard of care (SOC) in the ND setting; it has remained relatively unchanged for > 15 years, despite variable patient responses. Current available biomarkers do not inform personalized therapy. Functional drug response testing using patient specific tumor cells may have the potential to inform effective therapy selection, thus advancing functional precision oncology. Progression free survival (PFS) is a meaningful surrogate to overall survival (OS) in GBM and therefore, represents a measure of clinical benefit. Methods: The 3D-PREDICT clinical study (NCT03561207) allows enrollment of HGG patients with ECOG ≤ 3, perhaps representing a more accurate real-world population compared to most clinical studies in the same patient cohort. Tumor tissue was prospectively collected during SOC biopsy/resection and analyzed in an ex vivo cell culture test using a panel of agents prescribed as HGG therapeutics. Results: Data pertain to 56 3D-PREDICT patients who had > 6 months of follow up as of December 31, 2021 or experienced progression/death < 6 months post tissue collection. There were 42 3D-PREDICT patients who had IDH wild type ND GBM and received SOC. PFS analysis of these patients showed 3D ex vivo testing was able to prospectively predict TMZ clinical responders vs. non-responders (Kaplan-Meier, p = 0.039; HR 0.516, 95% CI 0.234,1.137). Test predicted TMZ responders had a relative PFS advantage of 3.7 months. Of the 42 patients, 38 had known MGMT methylation status with 23 patients (60%) being unmethylated. Test predicted responders (9) included unmethylated patients; test predicted non-responders (33) included methylated patients. The data suggest ex vivo testing of patient specific tumor tissue may identify ND HGG patients a priori who respond to TMZ, irrespective of MGMT methylation status. Beyond TMZ response prediction, the test assesses tissue response to 11 additional known HGG therapies and therefore may provide a tool to inform potential alternative treatments to TMZ for ND patients. Also evaluated were 14 3D-PREDICT recurrent HGG patients who received test directed salvage therapy; mean PFS after tissue collection from re-resection was 9.0 months (range 2.3 – 24.7 mo) for 9 patients at first recurrence, and 5.7 months (range 1.8 – 11.4 mo) for 5 patients with 2 to 6 recurrences. These examples demonstrate initial test utilization informing salvage therapy selection, correlated with PFS improvements in recurrent HGG. Conclusions: This functional 3D ex vivo cell culture platform provided survival benefit in analyzed ND and recurrent cohorts. Clinical trial information: NCT03561207.

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., 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% (κ = 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., (undefined), info:eu-repo/semantics/publishedVersion

Li Fraumeni syndrome (LFS) is a hereditary cancer predisposition syndrome caused by germline mutations in TP53. TP53 is the most common mutated gene in human cancer, occurring in 30–50% of glioblastomas (GBM). Here, we highlight a precision medicine platform to identify potential targets for a GBM patient with LFS. We used a comparative transcriptomics approach to identify genes that are uniquely overexpressed in the LFS GBM patient relative to a cancer compendium of 12,747 tumor RNA sequencing data sets, including 200 GBMs. STAT1 and STAT2 were identified as being significantly overexpressed in the LFS patient, indicating ruxolitinib, a Janus kinase 1 and 2 inhibitors, as a potential therapy. The LFS patient had the highest level of STAT1 and STAT2 expression in an institutional high-grade glioma cohort of 45 patients, further supporting the cancer compendium results. To empirically validate the comparative transcriptomics pipeline, we used a combination of adherent and organoid cell culture techniques, including ex vivo patient-derived organoids (PDOs) from four patient-derived cell lines, including the LFS patient. STAT1 and STAT2 expression levels in the four patient-derived cells correlated with levels identified in the respective parent tumors. In both adherent and organoid cultures, cells from the LFS patient were among the most sensitive to ruxolitinib compared to patient-derived cells with lower STAT1 and STAT2 expression levels. A spheroid-based drug screening assay (3D-PREDICT) was performed and used to identify further therapeutic targets. Two targeted therapies were selected for the patient of interest and resulted in radiographic disease stability. This manuscript supports the use of comparative transcriptomics to identify personalized therapeutic targets in a functional precision medicine platform for malignant brain tumors.

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 Mary's 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 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 Conclusions: Trovagene HPV test's 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 population's 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.

Recurrent high-grade glioma is a challenging disease process, without consensus on effective second-line therapy options. Individualized, patient-specific, biologically-based data is desirable in driving therapeutic decision-making. Patients with recurrent high-grade glioma and planned surgical re-resection at our institution were prospectively enrolled into the 3D-PREDICT study. Tissue was collected at the time of surgery for ex vivo 3D cell culture assays comprising a panel of agents commonly used for high-grade glioma, including chemotherapies and targeted therapies used in other solid cancers. In all cases, therapeutic agent selection was guided by the neuro-oncologist’s clinical judgement, factoring the patient’s age, performance status, comorbidities, toxicities/side effect profile of potential agents, and drug accessibility, plus ex-vivo drug response RESULTS: We present 3 cases in which the selection of agents was influenced by the tissue-derived 3D cell culture results; treatment led to clinical response observed in terms of progression free survival, quality of life, and pharmacologic tolerability. In Case 1, a patient with recurrent anaplastic astrocytoma was treated with a BRAF inhibitor for 12 months with excellent tolerability and no radiographic progression. Case 2 demonstrates the use of combination bevacizumab and irinotecan after disease progression subsequent to standard treatment. This patient had local radiographic control for 7 months, tolerating the regimen well. In Case 3, an individual with recurrent glioblastoma was treated with combination carboplatin and etoposide based on assay response prediction to both agents; treatment has been tolerated well with radiographic stability at 6 months while maintaining good performance status. This case series represents our institutional experience of utilizing patient-specific, ex-vivo tissue-derived cell drug response profiling to guide choice of therapy for recurrent high-grade glioma patients. Using individualized, tumor-specific drug sensitivity data to guide these decisions is representative of the ongoing paradigm shift into the realm of individualized medicine to improve outcomes in cancer patients.

BackgroundPersonalized medicine requires accurate molecular profiling for targeted therapy decisions. Insufficient tissue yield or tumor heterogeneity frequently limits the correct tissue biomarker determination. As a noninvasive complement to traditional tissue biopsies, liquid biopsies detect and track cancer driver mutations from biofluids (e.g., blood, urine). Here we present the analytical validation of Target Selector™ ctDNA assays capable of single mutant DNA copy detection.MethodsThe Target Selector ctDNA assay applies a patented Switch-Blocker technology to suppress amplification of background (wild-type) WT alleles, while allowing specific amplification of very low frequency mutant alleles. In contrast to allele specific enrichment technologies like ddPCR, one Switch-Blocker inhibits amplification of a DNA target up to 15 bp in length (e.g., one Switch-Blocker covers all KRAS exon 2, codon 12 and 13 variants). Target enrichment is achieved through a quantitative PCR reaction; subsequent DNA sequencing confirms mutation identity. Analytical validation with cancer cell line DNA was conducted by three independent operators using five instruments across five days.ResultsA total of 3086 samples were tested on EGFR, BRAF and KRAS Target Selector ctDNA assays, with EGFR WT as a reference. All assays showed >99% analytical sensitivity and specificity. Single mutant copy detection is confirmed by experimental data and theoretical estimates. In the presence of 14000 WT DNA copies, limits of detection were: EGFR Del19, 0.01%; EGFR L858R, 0.02%; EGFR T790M, 0.01%; BRAF V600E, 0.01%; KRAS G12C, 0.02%. Inter- and intra-assay analyses showed r2>0.94, suggesting consistent performance among operational variables. Healthy donor samples (100 tests) showed clinical specificity at >99%. Finally, Target Selector clinical experience data of >2200 patient samples is consistent with published tissue mutation prevalence.ConclusionsHighly sensitive Target Selector ctDNA assays with single mutant copy detection and limit of detection at 0.02% or better enable accurate molecular profiling vital for disease management.

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.

Background: Molecular profiling of tumors provides information key for devising personalized therapeutic strategies for managing disease in cancer patients. A liquid biopsy is emerging as a sensitive means to evaluate biomarker status without the complications and costs associated with surgical biopsies, particularly for patients unable or unwilling to undergo invasive procedures. Materials and Methods: Patient blood specimens were collected in Biocept’s proprietary Blood Collection Tubes for liquid biopsy testing in Biocept’s CLIA-certified, CAP-accredited laboratory. Dual Circulating Tumor Cell (CTC) and circulating tumor DNA (ctDNA) platforms were utilized to detect ALK or ROS1 gene rearrangements by FISH, or EGFR mutations, respectively. Results: Described are three metastatic NSCLC patients for which liquid biopsy guided the selection of a targeted therapy when a standard tissue biopsy was inadequate to assess biomarker status. All three patients were prescribed EGFR Tyrosine Kinase Inhibitor (TKI) treatment after an activating EGFR mutation was detected via liquid biopsy. One patient exhibited a complete response for approximately two years. Two patients received osimertinib following emergence of the EGFR T790M resistance mutation, which was also detected via a liquid biopsy. Conclusion: Liquid biopsy analyses of ctDNA and CTCs can complement tumor testing, identifying potential drivers of a patient’s cancer. Clinical utility of liquid biopsy is demonstrated where the first line and subsequent targeted treatments were prescribed based on the identification of genomic alterations in blood. Each patient received a therapeutic benefit that significantly extended survival and enhanced their quality of life.

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.

Erdheim-Chester disease (ECD) is a rare histiocytosis with a high prevalence of BRAF V600E mutation (>50% of patients). Patients with BRAF-mutant ECD can respond to BRAF inhibitors. Unfortunately, the lack of adequate archival tissue often precludes BRAF testing. We hypothesized that cell-free DNA (cfDNA) from plasma or urine can offer an alternative source of biologic material for testing. We tested for BRAF V600E mutation in cfDNA from the plasma and urine of 6 ECD patients. In patients with available archival tissue, the result of BRAF mutation analysis was concordant with plasma and urine cfDNA results in all 3 patients (100% agreement, kappa 1.00). In all 6 patients, BRAF mutation analysis of plasma and urine cfDNA was concordant in 5 of 6 patients (83% agreement, kappa 0.67). Testing for BRAF V600E mutation in plasma and urine cfDNA should be further investigated as an alternative to archival tissue mutation analysis.

Background: Liquid biopsy has recently emerged as a minimally invasive and cost effective means to assess cancer biomarkers without the risk of surgical biopsy complications. Using a simple blood draw, circulating tumor cell (CTC) analysis is capable of providing information with implications for diagnostics, prognosis, and treatment decisions of various carcinomas. Serial CTC measurements can aid monitoring disease progression or response to therapy. Biocept's proprietary TargetSelector™ technology captures CTCs and provides assessment of enumeration, protein expression by immunofluorescence, and gene amplifications or translocations via FISH. There is limited data on clinical utility of CTCs in germ cell tumors (GCT). We aim to evaluate the prognostic and predictive significance of CTCs in refractory GCT in an ongoing clinical trial of brentuximab vedontin and bevacizumab in refractory CD 30+ GCT (NCT02988843). CTC enumeration will be performed at baseline, prior to cycle 3 of treatment, and at progression to monitor treatment response in blood from patients with refractory testicular cancer. Methods: Peripheral whole blood samples from refractory testicular cancer patients are collected into Biocept CEE-Sure™ blood collection tubes that are validated to preserve CTCs for up to 96 hours. Biocept's TargetSelector™ platform utilizes a proprietary antibody capture cocktail and microchannel enabling enrichment, enumeration, and CTC analyses which were performed at Biocept's CLIA-certified and CAP accredited laboratory. Results: From a patient with heavily pretreated refractory testicular cancer, serial blood collections were obtained at baseline and prior to cycle 3 (approximately six week interval). Two CTCs were detected at baseline. Prior to cycle 3, there was 1 CTC (corresponding to tumor marker response and CT scan showing stable disease). At progression after cycle 4, the CTC count increased to 4, corresponding with disease progression in tumor markers and CT scan. In this patient, the CTC capture utilizing the TargetSelector™ CTC technology was very predictive of clinical response and progression. Conclusions: Clinical application of Biocept's TargetSelector™ CTC technology enables the sensitive detection of CTCs in testicular cancer. Longitudinal CTC assessment implemented in the clinical setting can be used to assess drug responsiveness and follow disease progression in this rare disease, as well as in more common solid tumor cancers. The TargetSelector™ liquid biopsy platform provides an economical, non-invasive, and reliable means to arm physicians with valuable information for disease management and patient care. Ongoing collection of blood samples and data analysis from patients being enrolled on the study will provide us a better understanding of the potential clinical use of this novel technology in GCT patients. Citation Format: Shilpa Gupta, Benjamin L. Maughan, Cecile Rose T. Vibat, Veena M. Singh. TargetSelector™ CTC technology demonstrates clinical utility in monitoring treatment response in germ cell tumors (testicular cancer) [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 5597.

Initial diagnostic biopsy procedures often yield insufficient tissue for molecular testing, and invasive surgical biopsies can be associated with significant cost as well as risk to the patient. Liquid biopsy offers an alternative and economical means for molecular characterization of tumors via a simple peripheral blood draw. This case report describes the ability of liquid biopsy to detect an ALK translocation where tissue analysis by fluorescence in situ hybridization was negative for the genetic alteration. Identification of an ALK rearrangement in circulating tumor cells from a blood specimen led to sequential targeted therapies that included crizotinib followed by alectinib. The patient demonstrated outstanding clinical response during treatment with each of the prescribed ALK inhibitors. This case demonstrates the clinical utility of Biocept’s liquid biopsy to detect actionable biomarkers by surveying the systemic landscape of a patient’s disease where identification of the same genetic drivers may be missed in analyses of heterogeneous tumor tissue.

e23036 Background: Liquid biopsy is a minimally invasive and cost effective way to assess cancer biomarkers without the risk of surgical biopsy complications. Circulating tumor cell (CTC) analysis from body fluids can provide critical information towards early detection, prognosis and treatment decisions. Accurate CTC evaluations require optimal cell preservation. Cell lysis, DNA degradation, or membrane alterations compromise CTC analyses and accurate diagnoses. This work compares Biocept’s proprietary CEE-Sure BCT and Saccomanno's Cytology Fixative largely used for sputum collection. Methods: One million BT474 (HER2 amplified) or H3112 (ALK re-arranged) cells were spiked into 500 µl medium; 500 µl of CEE-Sure or Saccomanno fixative was added. Tubes were stored at 4°C for 1 day, 1 week, or 1 month. Cells were centrifuged, resuspended, and counted (Celigo). Around 150 cells in 15 µl of RPMI medium were flowed into Biocept's microfludic system for cell capture; recovery (%) was calculated. Captured cells were subjected to fluorescent in situ hybridization (FISH) analyses for qualitative signal evaluation. Results: As similar results were observed for both cell lines and all time points, combined data will be shown. Median cell recovery after CEE-Sure incubation was 14.1% (range 1.7–44%, n = 12) vs 5.4% (range 0.07–26.9%, n = 12) in Saccomanno's fixative. Median cell capture of ~150 cells fed into Biocept’s microchannel was 96% (range 72-98%) for CEE-Sure vs 82% (range 21-96%) for Saccomanno. Paired t-tests showed significant differences for both recovery and capture. FISH signals from CEE-Sure samples were qualitatively rated Fair to Good, while Saccomanno samples had Poor to Fair, grainy, non-specific signals. Conclusions: This preliminary work shows consistently higher cell recovery, better cell membrane maintenance, and higher quality FISH signals for samples stored in Biocept's CEE-Sure vs Saccomano’s fixative. With liquid biopsy testing gaining rapid traction, maximal cell stability during the transport and storage are crucial. Additional fixative comparison is ongoing in various patient specimen types. These results support expansion of molecular analyses in sputum samples enriched for lung epithelial cells.

e23033 Background: 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. Biocept's 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 received at Biocept from 1Mar 2016 to 4Jan 2017 from late stage NSCLC patients. Methods: Blood was collected in Biocept OncoCEE BCT validated to preserve DNA ≤ 8 days. TargetSelector was used to detect ctDNA L858R, Del19 and T790M.EGFR allele copy numbers for wild type and each mutant were calculated. The prevalence of each mutation was compared to US averages. Results: Del19, L858R, and T790M mutations were detected in 12.9%, 8.5%, and 9.9% of the analyzed blood samples, respectively. This is concordant with US averages, which are 10% for each mutation. Median copy numbers/ml of blood were 30 for Del19 (range: 1 – 91974), 15 for L858R (range: 1 – 91200), and 10 for T790M (range: 1 – 137360). The median wild type EGFR copy number detected/ml blood was 2304 (range: 8 – 2498725). In ~80% of T790M cases, ≥ 1 concomitant activating mutation was detected. Conclusions: Biocept's TargetSelector detects EGFR mutations (Del19, L585R, and T790M) at a very high level of sensitivity down to 1 mutant copy/ml in advanced NSCLC patients at frequencies consistent with cited US rates. Moreover, the underlying activating mutation was detected in ~80% of T790M cases.

e23037 Background: EGFR mutations are the most frequent targetable genomic alterations in non-small cell lung cancer (NSCLC) patients (pts). While tissue biopsy remains the standard for assessing of EGFR mutation status, it is invasive and not always feasible. Liquid biopsy is a minimally invasive alternative. Biocept’s proprietary TargetSelector system evaluates circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) in blood. We aimed to clinically validate the accuracy of EGFR-specific TargetSelector in NSCLC pts. Methods: At three time points (T0: baseline before TKI, T1: during EGFR-TKI therapy, T2: after progression), blood samples were collected in Biocept OncoCEE BCT validated to preserve DNA up to 8 days. These samples were interrogated for three EGFR mutations: exon 19 deletions (Del 19), L858R, and T790M. The objectives are to assess detection sensitivity of liquid biopsy using EGFR mutation status vs the tissue as gold standard and to evaluate whether the detection sensitivity changes with EGFR-TKI therapy. Results: A total of 53 study pts were enrolled (male, 21; female, 32). The mean age was 70.6 (range: 46 – 90). Most pts had stage IV disease (43, 81.1%) and lung adenocarcinoma (48, 90.6%). 26 (49.1%) pts had EGFR mutations in tumor tissue: Del 19, 13; L858R, 8; T790M, 6; other, 8. Detection sensitivity for sensitizing EGFR mutations (Del 19 and L858R) at T0, T1, and T2 was 60.0% (6/10), 33.3% (5/15), and 33.3% (1/3), respectively. There was no statistical difference in CTC counts between activating EGFR mutation-positive and -negative pts (mean CTC count: 10.5 vs 20.1; p = 0.11 by two-sided t-test). Detection sensitivity for T790M was 33.3% (2/6) and 5 of 6 pts were receiving T790M directed therapy (3, rociletinib; 2, osimertinib) at the time of blood draw. Two pts – one patient before initiation of EGFR-TKI and the other during treatment with erlotinib – were found to have T790M mutations only in blood and not in tissue. Conclusions: Activating EGFR mutation detection may decrease during the course of TKI therapy, possibly due to treatment response. Further research with an expanded sample size and serial collections are needed to evaluate this finding, and to investigate possible implications of the presence of T790M only in blood.

Nanobodies (Nbs) or single-domain antibodies are among the smallest and most stable binder scaffolds known. In vitro display is a powerful antibody discovery technique used worldwide. We describe the first adaptation of in vitro mRNA/cDNA display for the rapid, automatable discovery of Nbs against desired targets, and use it to discover the first ever reported nanobody against the human full-length glucose transporter, GLUT-1. We envision our streamlined method as a bench-top platform technology, in combination with various molecular evolution techniques, for expedited Nb discovery.

Patients with Langerhans cell histiocytosis (LCH) and Erdheim–Chester disease (ECD) have a high frequency of BRAFV600E 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 BRAFV600E 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 KRASG12S-mutant ECD and dynamically tracked disease burden in patients treated with a variety of therapies. These results indicate that cfDNA BRAFV600E 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 BRAFV600E-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 BRAFV600E mutation and monitor response to therapy in these disorders. Cancer Discov; 5(1); 64–71. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 1

A kinetic reverse transcription–polymerase chain reaction (RT-PCR)-based assay is described that can discriminate and quantitate differentially spliced mRNAs. This assay should be generally applicable for high-throughput quantitation of differentially spliced transcripts. The utility of this method was assessed for spliced transcripts encoded by the human Na+-K+-2Cl− cotransporter gene hNKCC1. Evidence is presented that the NKCC1 isoform of the human Na+-K+-2Cl− cotransporter is differentially spliced analogous to that recently described for the mouse Na+-K+-2Cl− cotransporter gene BSC2. The nucleotide sequences of the two human splice variants predict Na+-K+-2Cl− cotransporter proteins differing only in length. Stable transfectants expressing these human splice variants, designated NKCC1a or NKCC1b, were constructed. Both splice variants produce functional Na+-K+-2Cl− cotransporters in vivo. The abundance of NKCC1 mRNA and patterns of differential splicing in 10 different tissue types and three cell lines were quantitated using the kRT-PCR assay. The results showed that the total amount of NKCC1 mRNA varied by more than 30-fold in the human tissues and cell lines examined. The ratio of NKCC1a/NKCC1b varied nearly 70-fold among these same tissues and cell lines suggesting that differential splicing of the NKCC1 transcript may play a regulatory role in human tissues.

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.

e15011Background: Mutant KRAS is a predictive biomarker for mCRC patient treatment selection. Recent technological advances enable quantification of mutational load in circulating tumor (ct) DNA, w...

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.

A number of gene replacements at different loci were constructed using covalently closed circular (ccc) plasmid DNA in the recB21 recC22 sbcB15 sbcC201 mutant of Escherichia coli (JC7623). Selected constructs representing deletions and insertion mutations formed from double-crossover events involving the ccc plasmid molecules and the genome were confirmed by Southern blots, and the frequency of double-crossover events was evaluated. It is reported that such mutants may be constructed without linearizing plasmid DNA, as described previously.

Background: Radiographic imaging in patients with mCRC is the current standard of care for monitoring responses. At least 40% of patients with mCRC have tumor associated KRAS mutations, and these may be detected in tumor DNA in plasma and urine. The aim of this study was to correlate the dynamics of KRAS mutational load in urinary and plasma ctDNA with clinical responses in patients with mCRC. Methods: In a blinded biomarker study of 30 metastatic patients CRC receiving chemotherapy or surgical treatment for resectable liver metastases, interim analysis was conducted in 13 patients (7 harboring KRAS G12/13 mutation in the tumor tissue, 6 wild-type for KRAS by tissue). For KRAS-positive patients, urine and plasma specimens were obtained pre-treatment, 2 weeks after initiation of chemotherapy, and subsequently at monthly intervals. Urine ctDNA was extracted using a method that preferentially isolates short fragmented ctDNA. Quantitative analysis of KRAS G12/13 mutation load in both urine and plasma was performed with the same validated KRAS G12/13 assay, which utilizes a wild-type blocker PCR enrichment followed by a next generation sequencing-based detection (MiSeq) with standardized reporting of mutant copies per 10^5 genome equivalents. Results: In an interim analysis, patients enrolled had pre-treatment urine and plasma specimens. In addition, a subset of patients (6) had longitudinal collections for up to 17 months. Eleven of 13 patients had liver-dominant metastatic disease. All KRAS tissue positive patients were receiving FOLFOX; all KRAS tissue-negative patients were receiving FOLFIRI plus cetuximab. In all KRAS G12/13 tissue positive patients with liver metastatic disease, a urinary ctDNA KRAS mutation concordant with tissue was identified; in 2 patients with non-liver metastatic disease, KRAS mutation was discordant or unidentifiable suggesting different biology in these patients. Plasma ctDNA KRAS mutation concordant with tissue was identified in 6 of 7 patient. Overall, the dynamics of ctDNA KRAS mutation burden in urine showed stronger concordance with clinical course and lack of temporal fluctuations as compared to the dynamics of ctDNA KRAS in plasma. A significant decrease in urinary ctDNA KRAS signal was observed as early as 2 weeks on chemotherapy, and this molecular response correlated with subsequently documented radiographic response. In 1 patient with documented progressive disease, an increase in urinary ctDNA KRAS G12/13 signal was observed 2 months prior to radiographic progression. Finally, 5 of 6 patients who tested negative for KRAS G12/13 mutations by tumor biopsy also tested negative in urine and plasma; 1 additional tissue KRAS wild-type patient tested positive for KRAS G12D mutation by urine and plasma ctDNA. Conclusions: In a proof of concept study, urine ctDNA was shown to carry a concordant KRAS mutation with tissue of mCRC patients. ctDNA analysis identified additional KRAS G12/13 mutation positive patient in whom KRAS mutations were undetected by the tumor test suggesting tumor heterogeneity and/or inadequate biopsy. The dynamics of ctDNA KRAS mutational load in urine correlated with radiographic responses, especially in liver-dominant metastatic patients, suggesting that urinary ctDNA may be a valuable method for monitoring treatment responses in patients with mCRC. Citation Format: Afsaneh Barzi, Vlada Melnikova, Cecile Rose T. Vibat, Saege Hancock, Yan Ning, Dana Agafitei, Mark G. Erlander, Heinz-Josef Lenz. Use of urinary circulating tumor DNA (ctDNA) KRAS for monitoring treatment response in patients with metastatic colorectal cancer (mCRC). [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 B4.

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

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

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

8081 Background: Acquisition of the EGFR T790M resistance mutation is a hallmark of disease progression in patients with metastatic EGFR mutant lung adenocarcinoma treated with anti-EGFR inhibitors...

11048 Background: Tumor heterogeneity and clonal selection contribute to resistance to molecular targeted therapies. Dynamic tracking of urine cfDNA mutations can offer a noninvasive tool for monit...

4022 Background: Median overall survival (OS) time of patients with unresectable PC varies widely. Diagnostic tools are presently lacking to predict outcomes. The majority of pancreatic tumors have...

3594 Background: Over half of patients with CRC will develop liver metastases. Surgical resection greatly improves outcomes in these patients. Non-invasive markers are needed to better monitor trea...

e19092 Background: Radiographic imaging in lung adenocarcinoma patients is not always reliable and a non-tissue based approach for monitoring treatment responses or minimal residual disease is desi...

288 Background: Non-resectable pancreatic cancer patients have a wide range of median time for overall survival (OS). Currently there is a lack of diagnostic tools to predict patient outcome at diagnosis. KRAS mutations are present in the vast majority of pancreatic tumors. The study objective was to determine if quantitative baseline and longitudinal monitoring of KRAS mutations from plasma circulating tumor DNA (ctDNA) could be used to stratify patients for predicting outcome. Methods: Plasma was prospectively collected from the Danish BIOPAC study for non-resectable pancreatic cancer patients undergoing treatment with gemcitabine or FOLFIRINOX. Feasibility of monitoring ctDNA KRAS mutations was assessed in 10 patients with long OS (median 493 days; range 360-1031) and 10 patients with short OS (median 66 days; range 21-136). KRAS G12A/C/D/R/S/V, and G13D mutations were PCR enriched, sequenced by massively parallel deep sequencing, quantitated and standardized by reporting number of copies detected per 105 genome equivalents (GE). Results: In a pilot study of 20 patients, all 18 patients with evaluable DNA had detectable KRAS mutations. Of 18 patients, 12 had baseline plus longitudinal time points (7 short, 5 long OS). Mutant KRAS copies were higher for short OS (median=994; range 0-34305 copies/105 GE) vs. with long OS (median 196; range, 34-278 copies/105 GE). Longitudinally, KRAS mutation levels remained mostly low with long OS (last time point median 204; range 8-873 copies/105 GE) vs. short OS where levels increased or remained high (median 2363; range 71-47919 copies/105 GE). Identical KRAS mutations were consistently detected for a given patient with short OS. However, long OS patients had variable KRAS mutations in longitudinal analysis. Conclusions: High levels of ctDNA KRAS mutations at diagnosis and post-treatment elevation of KRAS mutations were more associated with short OS. Different levels of KRAS mutation at diagnosis may predict patient outcome and could reflect distinct underlying tumor biology. Expansion of this prospective-retrospective biomarker cohort will be reported.

654 Background: Acquisition of point mutations in KRAS gene is causally associated with the onset of development of a resistance to anti-EGFR therapy in colorectal cancer. Newly acquired KRAS mutations can be detected in blood plasma months before radiographic detection. The objective of this study was to demonstrate feasibility of an ultrasensitive non-invasive method for detection of KRAS mutations in urine and plasma of patients with advanced colorectal cancer. Methods: Archived, matched urine and plasma samples (stored between 3-5 years prior to ctDNA extraction) from 20 treatment naïve, advanced stage cancer patients with known tumor tissue KRAS mutations determined by an accredited clinical laboratory, were used in a retrospective setting for a blinded concordance study. KRAS status in urine and plasma was compared to that in tumor tissue in order to assess clinical sensitivity of the ctDNA assay. An ultrashort-amplicon (31bp) assay for KRAS mutation enrichment and detection in highly fragmented urinary and plasma ctDNA was developed. The assay detected 1 copy of KRASG12A/C/D/R/S/V or G13D mutant allele in a background of wild-type DNA with a verified analytical sensitivity of 0.007% (7 copies per ~100,000 genome equivalents). Results: In a pilot study of 20 advanced stage colorectal cancer patients, 15 of 16 evaluable archived urine samples (94%) had KRAS mutation that was concordant with tissue biopsy. Of 20 archived plasma samples evaluated, 19 (95%) displayed the KRAS mutation concordant with tumor tissue. Of 16 paired urine and plasma samples, 15 (94%) had concordant KRAS mutation calls. Conclusions: This study demonstrates high clinical sensitivity (≥94%) of concordant KRAS mutation detection between urine, plasma and tissue specimens from advanced colorectal cancer patients. Early detection and monitoring of acquired KRAS mutations in circulating tumor DNA, and in particular urinary ctDNA, opens the possibility of a new paradigm for a truly non-invasive method of individualized care for colorectal cancer patients.

Ataxia telangiectasia (A-T) is an autosomal recessive disease caused by loss of function of the serine/ threonine protein kinase ATM (ataxia telangiectasia mutated). A-T patients have a 250-700-fold increased risk of developing lymphomas and leukemias which are typically highly invasive and proliferative. In addition, a subset of adult acute lymphoblastic leukemias and aggressive B-cell chronic lymphocytic leukemias that occur in the general population show loss of heterozygosity for ATM. To define the specific role of ATM in lymphomagenesis, we studied T-cell lymphomas isolated from mice with mutations in ATM and/or p53 using cytogenetic analysis and mRNA transcriptional profiling. The analyses identified genes misregulated as a consequence of the amplifications, deletions and translocation events arising as a result of ATM loss. A specific recurrent disruption of the granzyme gene family locus was identified resulting In an aberrant granzyme B/C fusion product. The combined application of cytogenetic and gene expression approaches identified specific loci and genes that define the pathway of initiation and progression of lymphoreticular malignancies in the absence of ATM.

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

e22175 Background: Detection and monitoring of oncogenic mutations in urinary cell-free (cf) DNA opens the possibility of a new paradigm for a truly non-invasive method of individualized care for m...

11012 Background: Histiocytosis patients (pts) have a high frequency of BRAFV600E mutations and respond to RAF inhibition but low tumor content and stromal contamination make detection in tissue bi...

The trabecular meshwork (TM) of the eye plays a central role in modulating intraocular pressure by regulating aqueous humor outflow, although the mechanisms are largely unknown. We and others have shown previously that aqueous humor outflow facility is modulated by conditions that alter TM cell volume. We have also shown that the Na-K-Cl cotransport system is a primary regulator of TM cell volume and that its activity appears to be coordinated with net efflux pathways to maintain steady-state volume. However, the cellular mechanisms that regulate cotransport activity and cell volume in TM cells have yet to be elucidated. The present study was conducted to investigate the hypothesis that intracellular Cl concentration ([Cl]i) acts to regulate TM cell Na-K-Cl cotransport activity, as has been shown previously for some other cell types. We demonstrate here that the human TM cell Na-K-Cl cotransporter is highly sensitive to changes in [Cl]i. Our findings reveal a marked stimulation of Na-K-Cl cotransport activity, assessed as ouabain-insensitive, bumetanide-sensitive K influx, in TM cells following preincubation of cells with Cl-free medium as a means of reducing [Cl]i. In contrast, preincubation of cells with media containing elevated K concentrations as a means of increasing [Cl]i results in inhibition of Na-K-Cl cotransport activity. The effects of reducing [Cl]i, as well as elevating [Cl]i, on Na-K-Cl cotransport activity are concentration dependent. Furthermore, the stimulatory effect of reduced [Cl]i is additive with cell-shrinkage-induced stimulation of the cotransporter. Our studies also show that TM cell Na-K-Cl cotransport activity is altered by a variety of Cl channel modulators, presumably through changes in [Cl]i. These findings support the hypothesis that regulation of Na-K-Cl cotransport activity, and thus cell volume, by [Cl]i may participate in modulating outflow facility across the TM.