Search

Overall sequencing CONSORT diagram. Flow diagram of study patients and analyzed biospecimens processed through and selected for central sequencing using MSK-IMPACT and Guardant360. cfDNA, cell-free DNA; FFPE, formalin-fixed paraffin-embedded; MSK-IMPACT, Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets; QC, quality control.

HER2 mutations define a subset of metastatic breast cancers with a unique mechanism of oncogenic addiction to HER2 signaling. We explored activity of the irreversible pan-HER kinase inhibitor neratinib, alone or with fulvestrant, in 81 patients with HER2-mutant metastatic breast cancer. Overall response rate was similar with or without estrogen receptor (ER) blockade. By comparison, progression-free survival and duration of response appeared longer in ER+ patients receiving combination therapy, although the study was not designed for direct comparison. Preexistent concurrent activating HER2 or HER3 alterations were associated with poor treatment outcome. Similarly, acquisition of multiple HER2-activating events, as well as gatekeeper alterations, were observed at disease progression in a high proportion of patients deriving clinical benefit from neratinib. Collectively, these data define HER2 mutations as a therapeutic target in breast cancer and suggest that coexistence of additional HER signaling alterations may promote both de novo and acquired resistance to neratinib.Significance:HER2 mutations define a targetable breast cancer subset, although sensitivity to irreversible HER kinase inhibition appears to be modified by the presence of concurrent activating genomic events in the pathway. These findings have implications for potential future combinatorial approaches and broader therapeutic development for this genomically defined subset of breast cancer.This article is highlighted in the In This Issue feature, p. 161

Supplementary data

Background Cell-free DNA (cfDNA) analysis using peripheral blood represents an exciting, minimally invasive technology for cancer diagnosis and monitoring. The reliability of testing is dependent on the accuracy and sensitivity of specific molecular analyses to detect tumor-associated genomic variants and on the quantity and quality of cfDNA available for testing. Specific guidelines for standardization and design of appropriate quality programs focused specifically on cfDNA isolation are lacking, as are standardized quality control reagents. Content This report describes and illustrates quality control and quality assurance processes, supported by generation of in-house quality control material, to ensure the reliability of the preanalytical phase of cfDNA analysis. Summary We have developed a robust quality program to support high-volume automated cfDNA extraction from peripheral blood by implementing processes and procedures designed to monitor the adequacy of specimen collection, specimen stability, efficiency of cfDNA extraction, and cfDNA quality.

HER2 mutations define a subset of metastatic breast cancers with a unique mechanism of oncogenic addiction to HER2 signaling. We explored activity of the irreversible pan-HER kinase inhibitor neratinib, alone or with fulvestrant, in 81 patients with HER2-mutant metastatic breast cancer. Overall response rate was similar with or without estrogen receptor (ER) blockade. By comparison, progression-free survival and duration of response appeared longer in ER+ patients receiving combination therapy, although the study was not designed for direct comparison. Preexistent concurrent activating HER2 or HER3 alterations were associated with poor treatment outcome. Similarly, acquisition of multiple HER2-activating events, as well as gatekeeper alterations, were observed at disease progression in a high proportion of patients deriving clinical benefit from neratinib. Collectively, these data define HER2 mutations as a therapeutic target in breast cancer and suggest that coexistence of additional HER signaling alterations may promote both de novo and acquired resistance to neratinib. Significance: HER2 mutations define a targetable breast cancer subset, although sensitivity to irreversible HER kinase inhibition appears to be modified by the presence of concurrent activating genomic events in the pathway. These findings have implications for potential future combinatorial approaches and broader therapeutic development for this genomically defined subset of breast cancer. This article is highlighted in the In This Issue feature, p. 161

TRK fusions are found in a variety of cancer types, lead to oncogenic addiction, and strongly predict tumor-agnostic efficacy of TRK inhibition1–8. With the recent approval of the first selective TRK inhibitor, larotrectinib, for patients with any TRK-fusion-positive adult or pediatric solid tumor, to identify mechanisms of treatment failure after initial response has become of immediate therapeutic relevance. So far, the only known resistance mechanism is the acquisition of on-target TRK kinase domain mutations, which interfere with drug binding and can potentially be addressable through second-generation TRK inhibitors9–11. Here, we report off-target resistance in patients treated with TRK inhibitors and in patient-derived models, mediated by genomic alterations that converge to activate the mitogen-activated protein kinase (MAPK) pathway. MAPK pathway-directed targeted therapy, administered alone or in combination with TRK inhibition, re-established disease control. Experimental modeling further suggests that upfront dual inhibition of TRK and MEK may delay time to progression in cancer types prone to the genomic acquisition of MAPK pathway-activating alterations. Collectively, these data suggest that a subset of patients will develop off-target mechanisms of resistance to TRK inhibition with potential implications for clinical management and future clinical trial design. A subset of patients treated with selective TRK inhibitors (including the newly approved larotrectinib) develop off-target resistance mediated by genomic acquisition of MAPK pathway-activating alterations, and may benefit from combined targeted therapy.

Circulating cell-free DNA (cfDNA) from blood plasma of cancer patients can be used to interrogate somatic tumor alterations non-invasively or when adequate tissue is unavailable. We have developed and clinically implemented MSK-ACCESS (Analysis of Circulating cfDNA to Evaluate Somatic Status), an NGS assay for detection of very low frequency somatic alterations in select exons and introns of 129 genes. Analytical validation demonstrated 92% sensitivity in de-novo mutation calling down to 0.5% allele frequency and 99% for a priori mutation profiling. To evaluate the performance and utility of MSK-ACCESS, we report results from the first 681 prospective blood samples (617 patients) that underwent clinical analysis to guide patient management. Somatic mutations, copy number, and/or structural variants were detected in 73% of the samples, and 56% of these circulating-tumor DNA (ctDNA) positive samples had clinically actionable alterations. The utilization of matched white blood cell sequencing allowed retention of somatic alterations while filtering out over 10,000 germline and clonal hematopoiesis variants, thereby greatly enhancing the specificity of the assay. Taken together, our experience illustrates the importance of analyzing a matched normal sample when interpreting cfDNA results and highlights the potential of cfDNA profiling to guide treatment selection, monitor treatment response, and identify mechanisms of treatment resistance.

PURPOSECell-free DNA (cfDNA) analysis offers a noninvasive means to access the tumor genome. Despite limited sensitivity of broad-panel sequencing for detecting low-frequency mutations in cfDNA, it may enable more comprehensive genomic characterization in patients with sufficiently high disease burden. We investigated the utility of large-panel cfDNA sequencing in patients enrolled to a Phase I AKT1-mutant solid tumor basket study.METHODSPatients had AKT1 E17K-mutant solid tumors and were treated on the multicenter basket study (ClinicalTrials.gov identifier: NCT01226316 ) of capivasertib, an AKT inhibitor. Serial plasma samples were prospectively collected and sequenced using exon-capture next-generation sequencing (NGS) analysis of 410 genes (Memorial Sloan Kettering [MSK]-Integrated Molecular Profiling of Actionable Cancer Target [IMPACT]) and allele-specific droplet digital polymerase chain reaction (ddPCR) for AKT1 E17K. Tumor DNA (tDNA) NGS (MSK-IMPACT) was also performed on available pretreatment tissue biopsy specimens.RESULTSAmong 25 patients, pretreatment plasma samples were sequenced to an average coverage of 504×. Somatic mutations were called in 20/25 (80%), with mutant allele fractions highly concordant with ddPCR of AKT1 E17K ( r2= 0.976). Among 17 of 20 cfDNA-positive patients with available tDNA for comparison, mutational concordance was acceptable, with 82% of recurrent mutations shared between tissue and plasma. cfDNA NGS captured additional tumor heterogeneity, identifying mutations not observed in tDNA in 38% of patients, and revealed oncogenic mutations in patients without available baseline tDNA. Longitudinal cfDNA NGS (n = 98 samples) revealed distinct patterns of clonal dynamics in response to therapy.CONCLUSIONLarge gene panel cfDNA NGS is feasible for patients with high disease burden and is concordant with single-analyte approaches, providing a robust alternative to ddPCR with greater breadth. cfDNA NGS can identify heterogeneity and potentially biologically informative and clinically relevant alterations.

Purpose Ewing sarcoma (ES) and desmoplastic small round cell tumors (DSRCTs) are aggressive sarcomas molecularly characterized by EWSR1 gene fusions. As pathognomonic genomic events in these respective tumor types, EWSR1 fusions represent robust potential biomarkers for disease monitoring. Methods To investigate the feasibility of identifying EWSR1 fusions in plasma-derived cell-free DNA (cfDNA) from patients with ES and DSRCT, we evaluated two complementary approaches in samples from 17 patients with radiographic evidence of disease. The first approach involved identification of patient-specific genomic EWSR1 fusion breakpoints in formalin-fixed, paraffin-embedded tumor DNA using a broad, hybridization capture-based next-generation sequencing (NGS) panel, followed by design of patient-specific droplet digital polymerase chain reaction (ddPCR) assays for plasma cfDNA interrogation. The second approach used a disease-tailored targeted hybridization capture-based NGS panel applied directly to cfDNA, which included EWSR1 as well as several other genes with potential prognostic use. Results EWSR1 fusions were identified in 11 of 11 (100%) ES and five of six (83%) DSRCT cfDNA samples by ddPCR, whereas 10 of 11 (91%) and four of six (67%) were identified by NGS. The ddPCR approach had higher sensitivity, ranging between 0.009% and 0.018%. However, the hybrid capture–based NGS assay identified the precise fusion breakpoints in the majority of cfDNA samples, as well as mutations in TP53 and STAG2, two other recurrent, clinically significant alterations in ES, all without prior knowledge of the tumor genotype. Conclusion These results provide a compelling rationale for an integrated approach using both NGS and ddPCR for plasma cfDNA-based biomarker evaluations in prospective cooperative group studies.

HER2 mutations define a subset of metastatic breast cancers with a unique mechanism of oncogenic addiction to HER2 signaling. We explored activity of the irreversible pan-HER kinase inhibitor neratinib, alone or with fulvestrant, in 81 patients with HER2-mutant metastatic breast cancer. Overall response rate was similar with or without estrogen receptor (ER) blockade. By comparison, progression-free survival and duration of response appeared longer in ER+ patients receiving combination therapy, although the study was not designed for direct comparison. Pre-existent concurrent activating HER2 or HER3 alterations were associated with poor treatment outcome. Similarly, acquisition of multiple HER2-activating events, as well as gatekeeper alterations, were observed at disease progression in a high proportion of patients deriving clinical benefit from neratinib. Collectively these data define HER2 mutations as a therapeutic target in breast cancer and suggest that co-existence of additional HER signaling alterations may promote both de novo and acquired resistance to neratinib.

Correlation between PIK3CA mutations in cell-free DNA and everolimus efficacy in HR + , HER2 − advanced breast cancer: results from BOLERO-2

Circulating tumor cell (CTC) number measured with the CellSearch assay is prognostic for survival in metastatic castration-resistant prostate cancer before and after therapy. Using a standard operating protocol for sample collection, processing, and analysis, we compared detection rates of CellSearch performed using US Food and Drug Administration-cleared methodology with a second positive selection assay, AdnaTest, and a nonselection polymerase chain reaction (PCR)-based (direct detection PCR [DDPCR]) assay in 55 blood samples from 47 men with progressive metastatic castration-resistant prostate cancer. AdnaTest requires processing within 4 hours of the draw and detects KLK3, PSMA, and EGFR transcripts in cells captured on magnetic beads. The DDPCR assay can be processed up to 7 days after a draw and detects KLK2, KLK3, HOXB13, GRHL2, and FOXA1 genes. AdnaTest and DDPCR were considered positive if at least 1 transcript was detected. AdnaTest detected CTCs in 34 samples (62%; 95% confidence interval [CI], 48%-75%), of which 23 (68%) had unfavorable CTC counts by CellSearch. A positive DDPCR result was seen in 38 cases (69%; 95% CI, 55%-81%), including 24 (63%) with unfavorable CellSearch CTC counts. CellSearch found unfavorable CTC counts in 25 samples (45%; 95% CI, 33%-58%). Sensitivities were similar between the AdnaTest and DDPCR assays, and both were more sensitive than CellSearch. Concordance probability estimates (possible values, 0.5-1.0) associating the biomarker result with survival were similar: 0.77 (SE, 0.07) for AdnaTest, 0.72 (SE, 0.08) for DDPCR, and 0.76 (SE, 0.06) for CellSearch. Overall detection rates between the AdnaTest and DDPCR assays were similar, and both were superior to CellSearch. The DDPCR assay required the lowest blood volume, least on-site processing, and longest stability for batch processing.

Ewing Sarcoma (ES) and Desmoplastic Small Round Cell Tumors (DSRCT) are aggressive sarcomas molecularly characterized byTo investigate the feasibility of identifyingThese results provide a compelling rationale for an integrated approach utilizing both NGS and ddPCR for plasma cfDNA-based biomarker evaluations in prospective cooperative group studies.

Background: The Y537S and D538G mutations in ESR1 are recurrent alterations in metastatic breast cancer (MBC) that promote ligand-independent receptor activation and resistance to estrogen deprivation therapy in laboratory models. The clinical prevalence of these mutations is not well established, but has been reported to be ∼10% by tumor sequencing. Cell free DNA (cfDNA) analysis typically reflects tumor-derived genetic alterations and can be used to characterize a population of patients at a common time point. We hypothesized that cfDNA could be used to detect ESR1 mutation at entry and its clinical impact in a large subset from BOLERO-2 that randomized patients with MBC to Exemestane (EXE) or EXE plus Everolimus (EVE). Methods: Patients with ER+/HER2– MBC with prior exposure to at least 1 non-steroidal aromatase inhibitor (NSAI) were enrolled in BOLERO-2. cfDNA was extracted from 560 baseline plasma samples using QIAamp Circulating DNA kit or QIAsymphony DSP Virus/Pathogen kit. Samples were analyzed by droplet digital PCR for Y537S and D538G. Cox-proportional hazards model was used to assess progression free survival (PFS) in patient subgroups defined by each ESR1 mutation, and the prognostic effect of each ESR1 mutation on overall survival (OS). Results: Of 541 evaluable patients (74.7% of study population), 156 (28.8%) had mutation in ESR1 in D538G (21.1%) and/or Y537S (13.3%) with 30 samples having both mutations. Sequencing of 302 archival tumor specimens (244 primary and 57 metastases) from this study only yielded 4 instances of D538G (1.3%) and 1 Y537S (0.3%). In the overall population, both mutations were poor prognostic factors associated with shorter OS (Table 1). Table 1AlterationNEventsMedian OS (95%CI)HR (95%CI)WT38521732.1 (28.1-36.4) D538G835726.0 (19.2-32.4)1.25 (1.02-1.54)Y537S423020.0 (13.0-29.3)2.31 (1.34-3.97)Double mt302415.2 (10.9-27.4)1.77 (1.31-2.39) PFS results were different for the two mutations. D538G but not Y537S mutation was associated with a shorter PFS with EXE compared to wild type (WT), (hazard ratios, D538G: 1.44 [95%CIs, 1.04-1.99] and Y537S: 0.92 [95%CIs, 0.44-1.93]). The D538G mutant group derived a similar benefit as WT from the addition of EVE to EXE, whereas the Y537S group did not (Table 2). Table 2AlterationGroupNEventsMedian PFS (95%CI)HR (95%CI)WTEXE1281163.9 (2.8-4.2)0.4 (0.31-0.51) EXE/EVE2571728.5 (6.9-9.9) D538GEXE24222.7 (1.4-2.8)0.34 (0.2-0.57) EXE/EVE59455.8 (4.2-8.4) Y537SEXE21164.1 (1.4-6.7)0.98 (0.49-1.94) EXE/EVE21194.2 (1.4-5.4) Conclusions: cfDNA analysis identifies a high rate of the Y537S and D538G ESR1 mutations in ER+ NSAI-treated MBC. As the two mutations may only represent 50-60% of all activating ESR1 mutations, the clinical prevalence of ESR1 mutations in ER+ MBC may be much higher than previously reported. Both mutations appear to be associated with a more aggressive disease biology. Interestingly, these two activating mutations appear to have differential effects on EXE and EVE sensitivity, highlighting new areas for research in ER biology. Citation Format: Chandarlapaty S, Sung P, Chen D, He W, Samoila A, You D, Bhatt T, Patel P, Voi M, Gnant M, Hortobagyi G, Baselga J, Moynahan ME. cfDNA analysis from BOLERO-2 plasma samples identifies a high rate of ESR1 mutations: Exploratory analysis for prognostic and predictive correlation of mutations reveals different efficacy outcomes of endocrine therapy–based regimens. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr S2-07.

// Parisa Momtaz 1 , James J. Harding 1, 2 , Charlotte Ariyan 1, 2 , Daniel G. Coit 1, 2 , Taha Merghoub 1 , Billel Gasmi 1 , Daoqi You 1 , Agnes Viale 1 , Katherine S. Panageas 1 , Aliaksandra Samoila 1 , Michael A. Postow 1, 2 , Jedd D. Wolchok 1, 2 and Paul B. Chapman 1, 2 1 Memorial Sloan Kettering Cancer Center, New York, New York, USA 2 Weill Cornell Medical College, New York, New York, USA Correspondence to: Paul B. Chapman, email: chapmanp@mskcc.org Keywords: cfDNA, digital PCR, drug cost, relapse-free survival Received: June 07, 2017 Accepted: July 25, 2017 Published: September 16, 2017 ABSTRACT Background: We tested the hypothesis that a 4-month course of adjuvant dabrafenib in stage IIIC BRAF-mutated melanoma would improve 2 year RFS from 24% to 51%, and that tumor-derived cell free DNA (cfDNA) in plasma would correlate with and predict recurrence. Methods: Patients with stage IIIC BRAF V600E/K mutated melanoma who were free of disease after surgical resection received 4 months of adjuvant dabrafenib. Patients were evaluated with imaging at baseline, at the end of cycles 2, 4, 6, then every 3 months until disease relapse or 2 years, whichever came first. Serial blood samples were collected for evaluation of cfDNA at the same time. Results: 21/23 patients enrolled were evaluable; 2 patients withdrew consent during the first week of treatment. The 2 year RFS was 28.6% (95% CI 12-48%). The estimated overall survival at 2 years was 78% (95% CI 51-91%). cfDNA detection had a 53% sensitivity in relapsing patients but cfDNA detection did not provide lead-time advantage over CT scanning. Conclusion: A 4-month course of adjuvant dabrafenib did not result in a detectable improvement in 2-year RFS. cfDNA was less sensitive than standard CT imaging and did not provide a lead-time advantage in detecting relapse.

Background: TRK inhibition is now standard of care for advanced pediatric and adult patients (pts) with TRK fusion solid tumors, regardless of origin. To date, TRK kinase domain mutations are the only known resistance mechanism, and next-generation TRK inhibitors active against these mutations such as LOXO-195 are being developed. We reasoned some pts will develop TRK-independent resistance and hypothesized that these pts will require unique therapeutic approaches. Methods: Paired tumor biopsies and serial cell-free DNA (cfDNA) prospectively collected from pts with TRK fusion-positive cancers treated with first- and next-generation TRK inhibitors before treatment and at progression were sequenced. In parallel, pt-derived and engineered models were analyzed. Results: Alterations involving upstream non-TRK receptor kinases and downstream MAPK pathway members were initially identified in tumors from 3 TRK fusion-positive gastrointestinal (GI) cancer pts who developed resistance to TRK inhibitors. Pt 1 with CTRC-NTRK1 pancreatic cancer developed temporally distinct emergent BRAF V600E and KRAS G12D mutations. Pt 2 with LMNA-NTRK1 colorectal cancer developed temporally distinct KRAS G12A and G12D mutations. Pt 3 with PLEKHA6-NTRK1 cholangiocarcinoma developed focal MET amplification. Phenocopying these clinical observations, pt-derived xenografts and primary cell lines developed BRAF and KRAS mutations following chronic TRK inhibition. Consistently, ectopic expression of these alterations conferred resistance to TRK inhibitors. Given that all 3 index pts had GI cancers, we expanded serial cfDNA sequencing to 5 additional TRK fusion-positive GI disease, identifying 3 with emergent MAPK alterations at progression, bringing the overall frequency of acquired MAPK alterations in GI cancers analyzed to 75% (6/8). To further evaluate whether these emergent alterations induced functional dependence on ERK signaling, pts 1-3 were treated with agents targeting these emergent alterations (dabrafenib + trametinib, LOXO-195 + trametinib, and LOXO-195 + crizotinib, respectively). Pt 1 achieved transient tumor regression, followed by outgrowth of KRAS-mutant disease. Pt 3 achieved a 4.5 months tumor regression. Sequencing at progression in pt 3 identified multiple acquired MET point mutations known to interfere with crizotinib binding. Conclusions: These data suggest that a subset of TRK fusion-positive cancers will develop off-target mechanisms of resistance to TRK inhibition. Relative to other TRK fusion-positive tumors, GI cancers may have a higher propensity for developing these bypass alterations that demonstrate remarkable convergence on ERK signaling. A portion of these mechanisms may be managed with simultaneous targeting of the TRK and MAPK pathways, although additional modeling is required to determine if upfront treatment would confer more durable responses. Citation Format: Emiliano Cocco, Amanda Kulick, Sandra Misale, Rona Yaeger, Pedram Razavi, Helen H. Won, Ryan Ptashkin, Jaclyn F. Hechtman, Eneda Toska, James Cownie, Romel Somwar, Sophie Shifman, Marissa Mattar, S Duygu Selçuklu, Aliaksandra Samoila, Sean Guzman, Brian B. Tuch, Kevin Ebata, Elisa de Stanchina, Rebecca J. Nagy, Richard B. Lanman, Michael F. Berger, Marc Ladanyi, David M. Hyman, Alexander Drilon, Maurizio Scaltriti, Alison M. Schram. Resistance to TRK inhibition mediated by convergent MAP kinase pathway activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-118.

Background: The current analysis was performed to evaluate the impact of PIK3CA hotspot mutations on everolimus efficacy in BOLERO-2 participants, using cell-free DNA (cfDNA) from plasma samples collected at the time of patient randomisation. Methods: PIK3CA H1047R, E545K, and E542K mutations in plasma-derived cfDNA were analysed by droplet digital PCR (ddPCR). Median PFS was estimated for patient subgroups defined by PIK3CA mutations in each treatment arm. Results: Among 550 patients included in cfDNA analysis, median PFS in everolimus vs placebo arms was similar in patients with tumours that had wild-type or mutant PIK3CA (hazard ratio (HR), 0.43 and 0.37, respectively). Everolimus also prolonged median PFS in patients with PIK3CA H1047R (HR, 0.37) and E545K/E542K mutations (HR=0.30) with a similar magnitude. Conclusions: Mutation analysis of plasma-derived cfDNA by ddPCR suggests that PFS benefit of everolimus was maintained irrespective of PIK3CA genotypes, consistent with the previous analysis of archival tumour DNA by next-generation sequencing.

The accessibility of tumor-derived cell-free DNA (cfDNA) in blood plasma provides a means to non-invasively profile somatic mutations in solid tumor patients. Clinical applications include longitudinal monitoring of disease burden and acquired drug resistance, identification of clinically relevant alterations and mutation signatures, and detection of minimal residual disease. However, the low fraction of tumor-derived cfDNA in plasma in many patients requires assays and bioinformatics methods that are much more sensitive than have been used for traditional tissue-based analysis. The design of our cfDNA NGS panel is based on prospectively-collected clinical sequencing data obtained from more than 20,000 patients at Memorial Sloan Kettering Cancer Center using MSK-IMPACT, a custom 468-gene sequencing test authorized by the FDA for somatic mutation profiling. Exons harboring hotspot mutations, clinically actionable mutations, and elevated somatic mutation rates were selected for inclusion in the cfDNA panel. Additional non-coding content was included to enable optimal detection of selected copy number alterations, regions of loss of heterozygosity, rearrangement breakpoints, and microsatellite instability. Altogether the panel contains 208 kilobases of sequence from 129 cancer genes. Ultra-deep sequencing and unique molecular indexing enable PCR-generated replicate sequences to be collapsed into error-free consensus sequences, thereby facilitating the high-confidence detection of mutations present at low allele fractions. We developed an open source bioinformatics tool, Marianas, for collapsing PCR replicates into consensus sequences and computing associated quality and performance metrics. Marianas incorporates many empirically derived features that lead to significant noise reduction. It efficiently processes a bam file with 20,000X coverage in 20 minutes on a single processor. We benchmarked the performance of Marianas against other available tools for collapsing and consensus base calling. The relative contributions of sources of error such as barcode contamination and sample cross-talk during PCR and sequencing were also quantified. We found that using unique dual sample indexes in multiplexed sequencing runs was essential to suppress these sources of noise. Applying these aggregated methods to analyze plasma cfDNA samples obtained from patients across a range of solid tumor types and disease stages, we were able to reliably detect clinically relevant mutations with variant allele fractions below 0.003, including subclonal mutations associated with acquired drug resistance. This approach, when applied prospectively on clinical specimens, has the potential to facilitate diagnosis, prognosis, and treatment selection in an era of precision oncology. Citation Format: Juber Patel, Maysun Hasan, Fanli Meng, Xiaohong Jing, Dilmi Perera, Jonathan Reichel, Erika Gedvilaite, Julie Yang, Maha Shady, Sandeep Raj, Preethi Srinivasan, Ian Johnson, Jiashi Wang, Mirna Jarosz, Aliaksandra Samoila, Agnes Viale, Bob Li, Pedram Razavi, Dana Tsui, Michael Berger. Development and optimization of a comprehensive high-sensitivity NGS cancer assay and bioinformatics pipeline for plasma cfDNA profiling [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 5598.

11500Background: E17K mutation of AKT1 is a recurrent, oncogenic event in solid tumors where treatment (Tx) with AKT inhibition has shown promise. Little is known about the significance of genomic ...

519Background: PIK3CA is the most commonly mutated gene in metastatic HR+ breast cancer but its impact on the clinical efficacy of mTOR inhibitor EVE and endocrine therapies is not fully established. We analyzed cfDNA for PIK3CA hotspot mutations to determine their impact on the clinical benefit of exemestane (EXE) or EXE plus EVE from the BOLERO-2 trial that randomized patients (pts) to these two treatment arms (2:1). Methods: Baseline plasma samples were analyzed by ddPCR for H1047R, E545K, and E542K mutations (~85% of activating PIK3CAmutations). Cox-proportional hazards model was used to assess PFS in pt subgroups defined by all or domain specific mutations separately (E545K/E542K for helical and H1047R for kinase domain). Results: 550 of 724 pts (76% trial population) had evaluable genotype results. 238 (43.3%) mutations were identified with frequencies of 25.3%, 11.3% and 7.5% for H1047R, E545K and E542K, respectively. Among 247 pts with PIK3CA genotype derived from sequencing matched archival tumor...

e22185 Background: Detection of cell-free DNA (cfDNA, 100-300 base pairs) in blood and body fluids is of growing interest for non-invasive diagnosis and monitoring of disease in cancer patients. Th...

5083 Background: Serum cytokines have been proposed as immunologic biomarkers of clinical responses based on their role in tumor biology. Recently, two whole blood mRNA signatures, which included immunomodulatory gene transcripts, were found to be predictive of survival in CRPC. This study explores serum cytokines, measured with analytically valid assays, as prognostic biomarkers in CRCP. Methods: Serum was collected from 75 progressive CRPC patients treated at MSKCC. A panel of 10 cytokines (M-CSF, IFN-γ, TNF-α, IL-1 β, IL-4, IL-5, IL-6, IL-10, IL-12 and IL-13) was measured in a CLIA-certified laboratory by clinically validated ELISAs. To create a risk group classification based on the 10 cytokines, a regression tree methodology was used with the intent to maximize the survival differences between risk groups. In addition to the cytokine risk groups, PSA, LDH, albumin, hemoglobin and CTC enumeration were also independently prognostic. The Cox model was developed to determine the factors that jointly predicted survival. The concordance probability estimate (CPE) was used to determine the discriminatory power of this model. Results: Among the 10 cytokines, M-CSF (stimulates monocytes and macrophages) and IL-10 (suppresses T-cell-mediated anti-tumor responses), were most predictive of overall survival in a 3 risk-group model. The relative risk for log CTC was 1.78 (95% CI 1.43 – 2.21). The combination of the cytokine risk groups and CTC enumeration provided the best discriminatory power for predicting survival, yielding a discrimination index measured by the CPE equal to 0.77 (se = 0.03). Conclusions: A risk group classification, based on two serum cytokines, M-CSF and IL-10, and log CTC measured by analytically valid assays, predicted survival in patients with progressive CRPC. These cytokines may reflect the biology within the tumor microenvironment, and may also serve as biomarker for clinical benefit. Independent validation in a similar cohort of patients is ongoing. [Table: see text]