Search

DLC-001 Study Design (CONSORT diagram)

Supplemental Figure S1. Kaplan-Meier analysis of the progression free survival (PFS) of patients on each treatment arm stratified by BRCA status showed no significant differences.

Grade 3/4 Treatment-Emergent Adverse Events Reported in {greater than or equal to}5% of Patients (Safety Population)

List of Primer Sequences

Supplementary Expression Data. Individual expression values for all 211 genes interrogated in the tumor tissue of 55 patients.

Purpose: Veliparib, a PARP inhibitor, demonstrated clinical activity in combination with oral cyclophosphamide in patients with BRCA-mutant solid tumors in a phase I trial. To define the relative contribution of PARP inhibition to the observed clinical activity, we conducted a randomized phase II trial to determine the response rate of veliparib in combination with cyclophosphamide compared with cyclophosphamide alone in patients with pretreated BRCA-mutant ovarian cancer or in patients with pretreated primary peritoneal, fallopian tube, or high-grade serous ovarian cancers (HGSOC).Experimental Design: Adult patients were randomized to receive cyclophosphamide alone (50 mg orally once daily) or with veliparib (60 mg orally once daily) in 21-day cycles. Crossover to the combination was allowed at disease progression.Results: Seventy-five patients were enrolled and 72 were evaluable for response; 38 received cyclophosphamide alone and 37 the combination as their initial treatment regimen. Treatment was well tolerated. One complete response was observed in each arm, with three partial responses (PR) in the combination arm and six PRs in the cyclophosphamide alone arm. Genetic sequence and expression analyses were performed for 211 genes involved in DNA repair; none of the detected genetic alterations were significantly associated with treatment benefit.Conclusion: This is the first trial that evaluated single-agent, low-dose cyclophosphamide in HGSOC, peritoneal, fallopian tube, and BRCA-mutant ovarian cancers. It was well tolerated and clinical activity was observed; the addition of veliparib at 60 mg daily did not improve either the response rate or the median progression-free survival. Clin Cancer Res; 21(7); 1574–82. ©2015 AACR.

Supplementary Methods, Tables S1 and S2, Figure Legend

Investigator's Choice (IC) Dosing Regimens

Purpose: Randomized, multicenter, open-label, phase 2/3 trial investigating lenalidomide versus investigator's choice (IC) in relapsed/refractory diffuse large B-cell lymphoma (DLBCL).Experimental Design: Patients with DLBCL who received ≥2 prior therapies were stratified by DLBCL subtype [germinal center B-cell (GCB) vs. non-GCB; determined by immunohistochemistry (IHC)] and then randomized 1:1 to lenalidomide (25 mg/day, 21 days of 28-day cycle) or IC (gemcitabine, rituximab, etoposide, or oxaliplatin). Crossover to lenalidomide was permitted for IC-treated patients with radiologically confirmed progressive disease. The primary endpoint was overall response rate (ORR). Progression-free survival (PFS), overall survival, and subtype analysis [GCB vs. activated B-cell (ABC)] using gene expression profiling (GEP) were exploratory endpoints.Results: Stage 1: 102 DLBCL patients (by IHC: non-GCB, n = 54; GCB, n = 48) received ≥1 dose of lenalidomide or IC. Hematologic treatment-emergent adverse events with lenalidomide versus IC included neutropenia (42.6%; 36.4%), anemia (33.3%; 47.3%), thrombocytopenia (24.1%; 43.6%), and leukopenia (5.6%; 12.7%), respectively. Overall, lenalidomide-treated patients had an ORR of 27.5% versus 11.8% in IC (ORRs were similar regardless of IHC-defined DLBCL subtype). Median PFS was increased in patients receiving lenalidomide (13.6 weeks) versus IC (7.9 weeks; P = 0.041), with greater improvements in non-GCB patients (15.1 vs. 7.1 weeks, respectively; P = 0.021) compared with GCB (10.1 vs. 9.0 weeks, respectively; P = 0.550).Conclusions: The clinical benefit of lenalidomide monotherapy in DLBCL patients was more evident in the non-GCB subtype. Exploratory analyses suggest that this preferential benefit was more pronounced in the GEP-defined ABC population, demonstrating a need for additional studies of lenalidomide in DLBCL using GEP subtyping. Clin Cancer Res; 23(15); 4127–37. ©2017 AACR.

Context.—Despite widespread use of formalin-fixed, paraffin-embedded (FFPE) tissue in clinical and research settings, potential effects of variable tissue processing remain largely unknown.Objective.—To elucidate molecular effects associated with clinically relevant preanalytical variability, the National Cancer Institute initiated the Biospecimen Preanalytical Variables (BPV) program.Design.—The BPV program, a well-controlled series of systematic, blind and randomized studies, investigated whether a delay to fixation (DTF) or time in fixative (TIF) affects the quantity and quality of DNA and RNA isolated from FFPE colon, kidney, and ovarian tumors in comparison to case-matched snap-frozen controls.Results.—DNA and RNA yields were comparable among FFPE biospecimens subjected to different DTF and TIF time points. DNA and RNA quality metrics revealed assay- and time point–specific effects of DTF and TIF. A quantitative reverse transcription–polymerase chain reaction (qRT-PCR) assay was superior when assessing RNA quality, consistently detecting differences between FFPE and snap-frozen biospecimens and among DTF and TIF time points. RNA Integrity Number and DV200 (representing the percentage of RNA fragments longer than 200 nucleotides) displayed more limited sensitivity. Differences in DNA quality (Q-ratio) between FFPE and snap-frozen biospecimens and among DTF and TIF time points were detected with a qPCR-based assay.Conclusions.—DNA and RNA quality may be adversely affected in some tumor types by a 12-hour DTF or a TIF of 72 hours. Results presented here as well as those of additional BPV molecular analyses underway will aid in the identification of acceptable delays and optimal fixation times, and quality assays that are suitable predictors of an FFPE biospecimen's fit-for-purpose.

PURPOSEThis trial assessed the utility of applying tumor DNA sequencing to treatment selection for patients with advanced, refractory cancer and somatic mutations in one of four signaling pathways by comparing the efficacy of four study regimens that were either matched to the patient's aberrant pathway (experimental arm) or not matched to that pathway (control arm).MATERIALS AND METHODSAdult patients with an actionable mutation of interest were randomly assigned 2:1 to receive either (1) a study regimen identified to target the aberrant pathway found in their tumor (veliparib with temozolomide or adavosertib with carboplatin [DNA repair pathway], everolimus [PI3K pathway], or trametinib [RAS/RAF/MEK pathway]), or (2) one of the same four regimens, but chosen from among those not targeting that pathway.RESULTSAmong 49 patients treated in the experimental arm, the objective response rate was 2% (95% CI, 0% to 10.9%). One of 20 patients (5%) in the experimental trametinib cohort had a partial response. There were no responses in the other cohorts. Although patients and physicians were blinded to the sequencing and random assignment results, a higher pretreatment dropout rate was observed in the control arm (22%) compared with the experimental arm (6%; P = .038), suggesting that some patients may have had prior tumor mutation profiling performed that led to a lack of participation in the control arm.CONCLUSIONFurther investigation, better annotation of predictive biomarkers, and the development of more effective agents are necessary to inform treatment decisions in an era of precision cancer medicine. Increasing prevalence of tumor mutation profiling and preference for targeted therapy make it difficult to use a randomized phase II design to evaluate targeted therapy efficacy in an advanced disease setting.

Background It is possible that the relative lack of progress in treatment outcome among the adolescent and young adult (AYA) group of cancer patients is due to a difference in disease biology compared to the corresponding diseases in younger and older individuals. There is evidence that colon cancer is more aggressive, and has a poorer prognosis in AYA patients than that observed in older adult patients.

Next generation sequencing (NGS) technologies have been widely adapted in clinical oncology by utilizing the profiled genetic mutation information to select patients and to guide the choice of target therapy. To fulfill the regulatory compliance, development of an NGS assay that will be used in clinical trials requires an analytical validation to meet its intend clinical use. NCI-MATCH trial is the largest precision oncology basket trial which uses a single NGS assay (NCI-MATHC NGS assay) to screen the actionable mutations in 6000 patients, who have relapsed/refractory solid tumors and lymphomas after standard systemic treatment, and assigns matched treatment. This article reviews on the critical considerations during development and validation of NGS assays as an investigational device for genomic based clinical trials and provides the experiences from the development of NCI-MATCH NGS assay.

The National Cancer Institute–Molecular Analysis for Therapy Choice (NCI-MATCH) trial is a national signal-finding precision medicine study that relies on genomic assays to screen and enroll patients with relapsed or refractory cancer after standard treatments. We report the analytical validation processes for the next-generation sequencing (NGS) assay that was tailored for regulatory compliant use in the trial. The Oncomine Cancer Panel assay and the Personal Genome Machine were used in four networked laboratories accredited for the Clinical Laboratory Improvement Amendments. Using formalin-fixed paraffin-embedded clinical specimens and cell lines, we found that the assay achieved overall sensitivity of 96.98% for 265 known mutations and 99.99% specificity. High reproducibility in detecting all reportable variants was observed, with a 99.99% mean interoperator pairwise concordance across the four laboratories. The limit of detection for each variant type was 2.8% for single-nucleotide variants, 10.5% for insertion/deletions, 6.8% for large insertion/deletions (gap ≥4 bp), and four copies for gene amplification. The assay system from biopsy collection through reporting was tested and found to be fully fit for purpose. Our results indicate that the NCI-MATCH NGS assay met the criteria for the intended clinical use and that high reproducibility of a complex NGS assay is achievable across multiple clinical laboratories. Our validation approaches can serve as a template for development and validation of other NGS assays for precision medicine.

Background Both single-agent ibrutinib and chlorambucil plus obinutuzumab have shown superior efficacy to chlorambucil monotherapy and are standard first-line treatments in chronic lymphocytic leukaemia. We compared the efficacy of the combination of ibrutinib plus obinutuzumab with chlorambucil plus obinutuzumab in first-line chronic lymphocytic leukaemia or small lymphocytic lymphoma. Methods iLLUMINATE is a multicentre, randomised, open-label, phase 3 trial done at 74 academic and community hospitals in Australia, Canada, Israel, New Zealand, Russia, Turkey, the EU, and the USA in patients with previously untreated chronic lymphocytic leukaemia or small lymphocytic lymphoma, either aged 65 years or older or younger than 65 years with coexisting conditions. Patients were randomly assigned (1:1) using a blocked randomisation schedule, stratified by Eastern Cooperative Oncology Group performance status and cytogenetics, to receive ibrutinib plus obinutuzumab (oral ibrutinib [420 mg once daily continuously] combined with intravenous obinutuzumab [100 mg on day 1,900 mg on day 2,1000 mg on day 8, and 1000 mg on day 15 of cycle 1 and on day 1 of subsequent 28-day cycles, for a total of six cycles]) or chlorambucil plus obinutuzumab (oral chlorambucil [0.5 mg/kg bodyweight on days 1 and 15 of each 28-day cycle for six cycles] combined with the same obinutuzumab regimen). Allocation concealment was achieved using an interactive web response system. Patients and investigators were not masked to treatment assignment. The primary endpoint was progression-free survival assessed by a masked independent review committee in the intention-to-treat population. Safety was assessed in all patients who received at least one dose of study treatment. This study is registered with ClinicalTrials.gov (NCT02264574), and patient enrolment is complete. Findings Between Oct 6, 2014, and Oct 12, 2015, 229 patients were enrolled and randomly assigned to receive ibrutinib plus obinutuzumab (n=113) or chlorambucil plus obinutuzumab (n=116). After a median follow-up of 31.3 months (IQR 29.4-33.2), median progression-free survival was significantly longer in the ibrutinib plus obinutuzumab group (median not reached [95% CI 33.6-non-estimable]) than in the chlorambucil plus obinutuzumab group (19.0 months [15.1-22.1]; hazard ratio 0.23; 95% CI 0.15-0.37; p

Robust and analytically validated assays are essential for clinical studies. We outline an analytical validation study of a targeted next-generation sequencing mutation-detection assay used for patient selection in the National Cancer Institute Molecular Profiling–Based Assignment of Cancer Therapy (NCI-MPACT) trial (NCT01827384). Using DNA samples from normal or tumor cell lines and xenografts with known variants, we assessed the sensitivity, specificity, and reproducibility of the NCI-MPACT assay in five variant types: single-nucleotide variants (SNVs), SNVs at homopolymeric (HP) regions (≥3 identical bases), small insertions/deletions (indels), large indels (gap ≥4 bp), and indels at HP regions. The assay achieved sensitivities of 100% for 64 SNVs, nine SNVs at HP regions, and 11 large indels, 83.33% for six indels, and 93.33% for 15 indels at HP regions. Zero false positives (100% specificity) were found in 380 actionable mutation loci in 96 runs of haplotype map cells. Reproducibility analysis showed 96.3% to 100% intraoperator and 98.1% to 100% interoperator mean concordance in detected variants and 100% reproducibility in treatment selection. To date, 38 tumors have been screened, 34 passed preanalytical quality control, and 18 had actionable mutations for treatment assignment. The NCI-MPACT assay is well suited for its intended investigational use and can serve as a template for developing next-generation sequencing assays for other cancer clinical trial applications.

Background Emerging clinical data show prediction of response to therapies targeting specific genetic aberrations have unexpectedly variable outcomes. This multicenter, double-blind, randomized trial opened in 2013 to compare response rates (RR) between 2 groups of patients (pts) identified to have an actionable mutation of interest (aMOI) in one of 3 genetic pathways (DNA repair, PI3K, or RAS/RAF/MEK): group A) Pts treated with agent(s) targeting one selected pathway (experimental arm-A) and B) Pts treated with agent(s) not targeting that pathway (control arm-B). Based on the data available at the time, the aMOI selection criteria encompassed alterations throughout the entire selected pathway instead of specific genetic changes. Methods Primary objective is to compare the RR (CR and PR) and 4 months PFS between treatments arms A and B. A CLIA-certified genetic analysis of a fresh tumor biopsy was performed at entry. Pts with an aMOI were randomized 2:1 to arm A vs. B. Study drugs were: 1) DNA repair-a) veliparib & temozolomide (VT); b) AZD1775 & carboplatin (AC); pts with p53 mutations were preferentially selected for AC; 2) PI3K- everolimus (E); 3) RAS- trametinib(T). At disease progression, Arm B pts could cross over to their target arm (A). Results 193 pts underwent biopsies; >90% of samples completed DNA sequencing. 96 pts (50%) had an aMOI and were randomized to treatment. Cohort VT had insufficient accrual on the experimental arm to be evaluable. AC, E and T cohorts were closed due to futility. Enrollment rate after treatment assignment was 77% for Arm A and 53%, for arm B. Attrition analysis between arms A and B are ongoing. Conclusions The increasing availability of genetic sequencing and bias toward expected benefit of highly specific treatment agents may account for the large number pt withdrawal from Arm A. This imbalance made comparison of arms A and B uninterpretable. The trial has been amended to employ a non-randomized design to complete the assessment of VT’s activity. Analysis of the aMOIs are ongoing to develop a more stringent selection criteria for future precision medicine trials. Clinical trial identification NCT01827384. Editorial acknowledgement Christina Rosenberger, PhD. Legal entity responsible for the study NCI. Funding NCI. Disclosure S. Kummar: Advisory / Consultancy: Corvus Pharmaceuticals; Advisory / Consultancy: MedTree; Advisory / Consultancy: Nodus Therapeutics; Advisory / Consultancy: Genentech; Advisory / Consultancy: ShangPharma Innovation; Advisory / Consultancy: Seattle Genetics; Travel / Accommodation / Expenses: Bayer; Shareholder / Stockholder / Stock options: Dhrishti Inc.; Research grant / Funding (institution): Bristol-Myers Squibb; Research grant / Funding (institution): Dynavax; Research grant / Funding (institution): Pfizer; Research grant / Funding (institution): Loxo; Research grant / Funding (institution): Corvus Pharmaceuticals; Research grant / Funding (institution): Plexxikon; Research grant / Funding (institution): Jounce Therapeutics; Research grant / Funding (institution): ADC Therapeutics; Research grant / Funding (institution): Advenchen Laboratories; Research grant / Funding (institution): Incyte; Research grant / Funding (institution): Taiho Pharmaceutical. N. Moore: Licensing / Royalties: Nestle Nutrition. P. Williams: Licensing / Royalties, I was a co-inventor of the DLBCL cell of origin patent recently filed by the NIH: NIH; Research grant / Funding (institution): Illumina. K.P.S. Raghav: Advisory / Consultancy: Bayer; Travel / Accommodation / Expenses: TRACON Pharma; Honoraria (self): Bayer; Honoraria (self): Eisai. F. Meric-Bernstam: Advisory / Consultancy: Genentech; Advisory / Consultancy: Inflection Biosciences; Advisory / Consultancy: Pieris Pharmaceuticals; Advisory / Consultancy: Clearlight Diagnostics; Advisory / Consultancy: Darwin Health; Advisory / Consultancy: Samsung Bioepis; Advisory / Consultancy: Spectrum Pharmaceuticals; Advisory / Consultancy: Aduro Biotech; Advisory / Consultancy: Origimed; Advisory / Consultancy: Xencor; Advisory / Consultancy: Debiopharm Group; Advisory / Consultancy: Mersana; Honoraria (self): Sumitomo Group; Honoraria (self): Dialectica; Research grant / Funding (self): Novartis; Research grant / Funding (self): AstraZeneca; Research grant / Funding (self): Taiho Pharmaceutical; Research grant / Funding (self): Genentech; Research grant / Funding (self): Calithera Biosciences; Research grant / Funding (self): Debiopharm Group. S. Leong: Shareholder / Stockholder / Stock options: Antares Pharmaceuticals; Shareholder / Stockholder / Stock options: Spectrum Pharmaceuticals; Honoraria (self): Lilly; Research grant / Funding (institution): Deciphera; Research grant / Funding (institution): Karyopharm Therapeutics; Research grant / Funding (institution): Bristol-Myers Squibb; Research grant / Funding (institution): Lilly. S. Waqar: Research grant / Funding (institution): Spectrum Pharmaceuticals; Research grant / Funding (institution): Lilly; Research grant / Funding (institution): Pfizer; Research grant / Funding (institution): Genentech/Roche; Research grant / Funding (institution): Daiichi Sankyo; Research grant / Funding (institution): Newlink Genetics; Research grant / Funding (institution): EMD Serono; Research grant / Funding (institution): Puma Biotechnology; Research grant / Funding (institution): Novartis; Research grant / Funding (institution): Xcovery; Research grant / Funding (institution): Synermore biologics; Research grant / Funding (institution): Celgene; Research grant / Funding (institution): Vertex; Research grant / Funding (institution): Bristol-Myers Squibb; Research grant / Funding (institution): Stem CentRx; Research grant / Funding (institution): Hengrui Therapeutics; Research grant / Funding (institution): Checkpoint Therapeutics; Research grant / Funding (institution): Ignyta; Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): ARIAD. All other authors have declared no conflicts of interest.

Signaling by human hepatocyte growth factor (hHGF) via its cell surface receptor (MET) drives mitogenesis, motogenesis and morphogenesis in a wide spectrum of target cell types and embryologic, developmental and homeostatic contexts. Oncogenic pathway activation also contributes to tumorigenesis and cancer progression, including tumor angiogenesis and metastasis, in several prevalent malignancies. The HGF gene encodes full-length hHGF and two truncated isoforms known as NK1 and NK2. NK1 induces all three HGF activities at modestly reduced potency, whereas NK2 stimulates only motogenesis and enhances HGF-driven tumor metastasis in transgenic mice. Prior studies have shown that mouse HGF (mHGF) also binds with high affinity to human MET. Here we show that, like NK2, mHGF stimulates cell motility, invasion and spontaneous metastasis of PC3M human prostate adenocarcinoma cells in mice through human MET. To identify target genes and signaling pathways associated with motogenic and metastatic HGF signaling, i.e., the HGF invasive program, gene expression profiling was performed using PC3M cells treated with hHGF, NK2 or mHGF. Results obtained using Ingenuity Pathway Analysis software showed significant overlap with networks and pathways involved in cell movement and metastasis. Interrogating The Cancer Genome Atlas project also identified a subset of 23 gene expression changes in PC3M with a strong tendency for co-occurrence in prostate cancer patients that were associated with significantly decreased disease-free survival.

The two major subtypes of diffuse large B cell lymphoma (DLBCL)—activated B cell–like (ABC) and germinal center B cell–like (GCB)—arise by distinct mechanisms, with ABC selectively acquiring mutations that target the B cell receptor (BCR), fostering chronic active BCR signaling(1). The ABC subtype has a ∼40% cure rate with currently available therapies, which is worse than the rate for GCB DLBCL, and highlights the need for ABC subtype-specific treatment strategies(2). We hypothesized that ABC, but not GCB, DLBCL tumors would respond to ibrutinib, an inhibitor of BCR signaling. In a phase 1/2 clinical trial that involved 80 subjects with relapsed or refractory DLBCL, ibrutinib produced complete or partial responses in 37% (14/38) of those with ABC DLBCL, but in only 5% (1/20) of subjects with GCB DLBCL (P = 0.0106). ABC tumors with BCR mutations responded to ibrutinib frequently (5/9; 55.5%), especially those with concomitant myeloid differentiation primary response 88 (MYD88) mutations (4/5; 80%), a result that is consistent with in vitro cooperation between the BCR and MYD88 pathways. However, the highest number of responses occurred in ABC tumors that lacked BCR mutations (9/29; 31%), suggesting that oncogenic BCR signaling in ABC does not require BCR mutations and might be initiated by non-genetic mechanisms. These results support the selective development of ibrutinib for the treatment of ABC DLBCL.

Background Next-generation sequencing (NGS) assays are highly complex tests that can vary substantially in both their design and intended application. Despite their innumerous advantages, NGS assays present some unique challenges associated with the preanalytical process, library preparation, data analysis, and reporting. According to a number of professional laboratory organization, control materials should be included both during the analytical validation phase and in routine clinical use to guarantee highly accurate results. The SeraseqTM Solid Tumor Mutation Mix AF10 and AF20 control materials consist of 26 biosynthetic DNA constructs in a genomic DNA background, each containing a specific variant or mutation of interest and an internal quality marker at 2 distinct allelic frequencies of 10% and 20%, respectively. The goal of this interlaboratory study was to evaluate the Seraseq AF10 and AF20 control materials by verifying their performance as control materials and by evaluating their ability to measure quality metrics essential to a clinical test. Methods Performance characteristics were assessed within and between 6 CLIA-accredited laboratories and 1 research laboratory. Results Most laboratories detected all 26 mutations of interest; however, some discrepancies involving the internal quality markers were observed. Conclusion This interlaboratory study showed that the Seraseq AF10 and AF20 control materials have high quality, stability, and genomic complexity in variant types that are well suited for assisting in NGS assay analytical validation and monitoring routine clinical applications.

Purpose: Randomized, multicenter, open-label, phase 2/3 trial investigating lenalidomide versus investigator's choice (IC) in relapsed/refractory diffuse large B-cell lymphoma (DLBCL). Experimental Design: Patients with DLBCL who received ≥2 prior therapies were stratified by DLBCL subtype [germinal center B-cell (GCB) vs. non-GCB; determined by immunohistochemistry (IHC)] and then randomized 1:1 to lenalidomide (25 mg/day, 21 days of 28-day cycle) or IC (gemcitabine, rituximab, etoposide, or oxaliplatin). Crossover to lenalidomide was permitted for IC-treated patients with radiologically confirmed progressive disease. The primary endpoint was overall response rate (ORR). Progression-free survival (PFS), overall survival, and subtype analysis [GCB vs. activated B-cell (ABC)] using gene expression profiling (GEP) were exploratory endpoints. Results: Stage 1: 102 DLBCL patients (by IHC: non-GCB, n = 54; GCB, n = 48) received ≥1 dose of lenalidomide or IC. Hematologic treatment-emergent adverse events with lenalidomide versus IC included neutropenia (42.6%; 36.4%), anemia (33.3%; 47.3%), thrombocytopenia (24.1%; 43.6%), and leukopenia (5.6%; 12.7%), respectively. Overall, lenalidomide-treated patients had an ORR of 27.5% versus 11.8% in IC (ORRs were similar regardless of IHC-defined DLBCL subtype). Median PFS was increased in patients receiving lenalidomide (13.6 weeks) versus IC (7.9 weeks; P = 0.041), with greater improvements in non-GCB patients (15.1 vs. 7.1 weeks, respectively; P = 0.021) compared with GCB (10.1 vs. 9.0 weeks, respectively; P = 0.550). Conclusions: The clinical benefit of lenalidomide monotherapy in DLBCL patients was more evident in the non-GCB subtype. Exploratory analyses suggest that this preferential benefit was more pronounced in the GEP-defined ABC population, demonstrating a need for additional studies of lenalidomide in DLBCL using GEP subtyping. Clin Cancer Res; 23(15); 4127–37. ©2017 AACR.

The assignment of diffuse large B-cell lymphoma into cell-of-origin (COO) groups is becoming increasingly important with the emergence of novel therapies that have selective biological activity in germinal center B cell–like or activated B cell–like groups. The Lymphoma/Leukemia Molecular Profiling Project's Lymph2Cx assay is a parsimonious digital gene expression (NanoString)–based test for COO assignment in formalin-fixed paraffin-embedded tissue (FFPET). The 20-gene assay was trained using 51 FFPET biopsies; the locked assay was then validated using an independent cohort of 68 FFPET biopsies. Comparisons were made with COO assignment using the original COO model on matched frozen tissue. In the validation cohort, the assay was accurate, with only 1 case with definitive COO being incorrectly assigned, and robust, with >95% concordance of COO assignment between 2 independent laboratories. These qualities, along with the rapid turnaround time, make Lymph2Cx attractive for implementation in clinical trials and, ultimately, patient management.

The National Cancer Institute-Molecular Analysis for Therapy Choice (NCI-MATCH) trial is a national signal-finding precision medicine study that relies on genomic assays to screen and enroll patients with relapsed or refractory cancer after standard treatments. We report the analytical validation processes for the next-generation sequencing (NGS) assay that was tailored for regulatory compliant use in the trial. The Oncomine Cancer Panel assay and the Personal Genome Machine were used in four networked laboratories accredited for the Clinical Laboratory Improvement Amendments. Using formalin-fixed paraffin-embedded clinical specimens and cell lines, we found that the assay achieved overall sensitivity of 96.98% for 265 known mutations and 99.99% specificity. High reproducibility in detecting all reportable variants was observed, with a 99.99% mean interoperator pairwise concordance across the four laboratories. The limit of detection for each variant type was 2.8% for single-nucleotide variants, 10.5% for insertion/deletions, 6.8% for large insertion/deletions (gap ≥4 bp), and four copies for gene amplification. The assay system from biopsy collection through reporting was tested and found to be fully fit for purpose. Our results indicate that the NCI-MATCH NGS assay met the criteria for the intended clinical use and that high reproducibility of a complex NGS assay is achievable across multiple clinical laboratories. Our validation approaches can serve as a template for development and validation of other NGS assays for precision medicine.

Although next-generation sequencing technologies have been widely adapted for clinical diagnostic applications, an urgent need exists for multianalyte calibrator materials and controls to evaluate the performance of these assays. Control materials will also play a major role in the assessment, development, and selection of appropriate alignment and variant calling pipelines. We report an approach to provide effective multianalyte controls for next-generation sequencing assays, referred to as the control plasmid spiked-in genome (CPSG). Control plasmids that contain approximately 1000 bases of human genomic sequence with a specific mutation of interest positioned near the middle of the insert and a nearby 6-bp molecular barcode were synthesized, linearized, quantitated, and spiked into genomic DNA derived from formalin-fixed, paraffin-embedded–prepared hapmap cell lines at defined copy number ratios. Serial titration experiments demonstrated the CPSGs performed with similar efficiency of variant detection as formalin-fixed, paraffin-embedded cell line genomic DNA. Repetitive analyses of one lot of CPSGs 90 times during 18 months revealed that the reagents were stable with consistent detection of each of the plasmids at similar variant allele frequencies. CPSGs are designed to work across most next-generation sequencing methods, platforms, and data analysis pipelines. CPSGs are robust controls and can be used to evaluate the performance of different next-generation sequencing diagnostic assays, assess data analysis pipelines, and ensure robust assay performance metrics.

Background : Rituximab (RTX) is commonly used to treat Waldenström's macroglobulinemia (WM). Treatment options are limited for patients (pts) who fail rituximab therapy. Single-agent ibrutinib (ibr) is highly active in WM and is approved in the United States and Europe for WM. In the phase 3 iNNOVATE study, ibr plus RTX resulted in significantly longer PFS and higher response rates vs RTX alone, both among TN and previously treated pts with WM (Dimopoulos MA, et al. N Engl J Med. 2018). Previous reports from the open-label, single-agent substudy showed sustained responses and a manageable toxicity profile in RTX-refractory WM, with a median follow-up of 18.1 mo (Dimopoulos MA, et al. Lancet Oncol. 2016). Follow-up data from both the randomized portion (median follow-up of 30.4 mo) and substudy (median follow-up of 38.7 mo) of iNNOVATE are presented. Methods : Pts had confirmed, symptomatic WM requiring treatment. Pts in the randomized portion (Arms A and B) with prior RTX therapy required a response (≥MR) to their last RTX-based regimen. Pts were randomized to daily 420 mg ibr (Arm A; IR) or placebo (Arm B; R) plus RTX (375 mg/m2/week IV at weeks 1-4 and 17-20). In Arm C, pts who failed to achieve ≥MR or relapsed Results : A total of 150 pts were randomized to Arms A and B (n=75/arm; Table 1). Median age was 69 y. High IPSSWM was reported in 38% of pts; 45% of pts were treatment naïve. With prolonged follow-up and a median duration of treatment of 29.5 mo with IR and 15.5 mo with R, investigator-assessed major response rates (≥PR) were 77% with IR vs 33% with R (P In Arm C, 31 pts received single-agent ibr (Table 1). Median age was 67 y. Most (71%) pts had ≥3 prior therapies; all patients were RTX-refractory and 90% had prior treatment with cyclophosphamide. High IPSSWM was reported in 42% of pts. With longer follow-up, median investigator-assessed PFS was NR (95% CI: 27.6-NR); estimated 36-mo PFS was 61%. Major response rate by investigator assessment was 77% and ORR was 90%. The estimated 36-mo OS was 84%. Improved Hb level was seen in 71% of pts. Treatment was ongoing in 52% of pts. Median duration of ibr treatment was 37.0 mo, with no major hemorrhagic or atrial fibrillation events reported. Grade ≥3 AEs occurred in 74% of pts and 39% had serious AEs. No fatal AEs were reported. Conclusions : IR showed continued superiority over R, in treatment-naïve and previously treated pts with WM, regardless of genotypic factors. Similarly, in heavily pretreated, RTX-refractory pts with follow-up >3 y, single-agent ibr was highly active and response improved over time. Alone or in combination, ibr had a manageable safety profile and no new safety signals were identified with longer follow-up. Disclosures Buske: Roche: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Bayer: Research Funding. Tedeschi:Gilead: Consultancy; AbbVie: Consultancy; Janssen: Consultancy, Speakers Bureau. Trotman:Beigene: Research Funding; Celgene: Research Funding; Janssen: Research Funding; PCYC: Research Funding; Roche: Research Funding; Jassen: Research Funding. García-Sanz:Spanish Government: Research Funding; Pharmacyclics: Research Funding; BMS: Consultancy, Honoraria; Hospira: Research Funding; Gilead: Research Funding; Amgen Inc.: Research Funding; Incyte: Consultancy; Takeda: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses. MacDonald:Roche: Honoraria; Gilead: Honoraria; Janssen: Honoraria; Lundbeck: Honoraria; Seattle Genetics: Honoraria. Leblond:Abbvie: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Amgen: Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Sandoz: Honoraria; Gilead: Honoraria, Speakers Bureau. Herbaux:Gilead Sciences, Inc.: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Tam:Beigene: Honoraria, Other: Travel funding; Roche: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria, Travel funding; Gilead: Honoraria; AbbVie: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Roche: Honoraria; Beigene: Honoraria, Other: Travel funding; Pharmacyclics: Honoraria; Janssen: Honoraria, Research Funding. Palomba:Seres: Honoraria, Patents & Royalties; Juno: Honoraria, Patents & Royalties; Merck: Consultancy; Pharmacyclics: Consultancy; Therakos: Consultancy. Matous:Celgene: Consultancy, Honoraria, Speakers Bureau. Shustik:Amgen: Consultancy; Takeda: Consultancy; Celgene: Consultancy. Kastritis:Takeda: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Other: Travel, Accommodations, Expenses; Genesis Pharma: Consultancy; Prothena: Consultancy. Treon:Johnson & Johnson: Consultancy; Janssen: Consultancy, Other: Travel, Accommodations, Expenses; BMS: Research Funding; Pharmacyclics: Consultancy, Other: Travel, Accommodations, Expenses, Research Funding. Lih:Abbvie: Employment, Equity Ownership; Abbvie/Pharmacyclics: Other: Travel, Accommodations, Expenses. Li:Pharmacyclics: Employment; Abbvie: Equity Ownership; BMS: Equity Ownership; Pfizer: Equity Ownership; Abbott: Equity Ownership; Amgen: Equity Ownership; Gilead: Equity Ownership; Biogen: Equity Ownership; Celgene: Equity Ownership; Medivation: Equity Ownership; Merck: Equity Ownership; Exelixis: Equity Ownership; Juno: Equity Ownership; Isis: Equity Ownership; Aduro: Equity Ownership; Merrimack: Equity Ownership. Salman:Pharmacyclics LLC: Employment, Equity Ownership; Abbvie: Equity Ownership. Graef:Abbvie: Equity Ownership, Patents & Royalties; Pharmacyclics: Employment, Other: Leadership, Patents & Royalties. Dimopoulos:Celgene: Honoraria; Janssen: Honoraria; Takeda: Honoraria; Amgen: Honoraria; Bristol-Myers Squibb: Honoraria.

Using transcripts initiated at a chromosomally integrated retrovirus-based promoter to perturb gene expression randomly in human prostate cancer cells, we isolated cell clones resistant to taxane lethality and discovered the role of a previously uncharacterized gene,txr1,in this phenotype. We show thattxr1impedes taxane-induced apoptosis in tumor cells by transcriptionally down-regulating the production of thrombospondin-1 (TSP-1)—known earlier for both its anti-angiogenic and proapoptotic actions. Decrease of Txr1 or treatment with TSP-1 or TSP-1 mimetic peptide sensitized cells to taxane cytotoxicity by activating signaling through the CD47 receptor (also known as the integrin-associated protein), whereas interference with CD47 function reduced taxane-induced cell death. Cellular abundance of Txr1 and TSP-1 varied inversely, and alteration of the level of both proteins correlated highly with taxol resistance in 13 of 19 NCI-60 cancer cell lines. Our results reveal a hitherto unsuspected mechanism of taxane resistance, elucidate the role oftxr1in this resistance, and identifytxr1as a regulator of TSP-1 production and an agent for its chemotherapeutic modulation.

A complex programme of regulation governs gene expression during development of the morphologically and biochemically complex eubacterial genus Streptomyces. Earlier work has suggested a model in which 'higher level' pleiotropic regulators activate 'pathway-specific' regulators located within chromosomal gene clusters encoding biosynthesis of individual antibiotics. We used mutational analysis and adventitious overexpression of key Streptomyces coelicolor regulators to investigate functional interactions among them. We report here that cluster-situated regulators (CSRs) thought to be pathway-specific can also control other antibiotic biosynthetic gene clusters, and thus have pleiotropic actions. Surprisingly, we also find that CSRs exhibit growth-phase-dependent control over afsR2/afsS, a 'higher level' pleiotropic regulatory locus not located within any of the chromosomal gene clusters it targets, and further demonstrate that cross-regulation by CSRs is modulated globally and differentially during the S. coelicolor growth cycle by the RNaseIII homologue AbsB. Our results, which reveal a network of functional interactions among regulators that govern production of antibiotics and other secondary metabolites in S. coelicolor, suggest that revision of the currently prevalent view of higher-level versus pathway-specific regulation of secondary metabolism in Streptomyces species is warranted.

Global analysis of gene expression by using DNA microarrays is employed increasingly to search for differences in biological properties between normal and diseased tissue. In such studies, expression that deviates from defined thresholds commonly is used for creating genetic signatures that characterize disease vs. normality. Although it is axiomatic that the threshold parameters applied to microarray analysis will alter the contents of such genetic signatures, the extent to which threshold choice can affect the fundamental conclusions made from microarray-based studies has not been elucidated. We used gabriel (Genetic Analysis By Rules Incorporating Expert Logic), a platform of knowledge-based algorithms for the global analysis of gene expression, together with conventional statistical approaches, to examine the sensitivity of conclusions to threshold choice in recently published microarray-based studies. An analysis of the effects of threshold decisions in one of these studies [Ramaswamy, S., Ross, K. N., Lander, E. S. & Golub, T. R. (2003) Nat. Genet. 33, 49–54], which arrived at the important conclusion that the metastatic potential of primary tumors is encoded by the bulk of cells in the tumor, is the focus of this article. We discovered that support for this conclusion highly depends on the threshold used to create gene expression signatures. We also found that threshold choice dramatically affected the gene function categories represented nonrandomly in signatures. Our results suggest that the robustness of biological conclusions made by using microarray analysis should be routinely assessed by examining the validity of the conclusions by using a range of threshold parameters.

The ability to investigate the transcription of thousands of genes concurrently by using DNA microarrays offers both major scientific opportunities and significant analytical challenges. Here we describe gabriel , a rule-based system of computer programs designed to apply domain-specific and procedural knowledge systematically and uniformly for the analysis and interpretation of data from DNA microarrays. gabriel 's problem-solving rules direct stereotypical tasks, whereas domain-specific knowledge pertains to gene functions and relationships or to experimental conditions. Additionally, gabriel can learn novel rules through genetic algorithms, which define patterns that best match the data being analyzed and can identify groupings in gene expression profiles preordered by chromosomal position or by a nonsupervised algorithm such as hierarchical clustering. gabriel subsystems explain the logic that underlies conclusions and provide a graphical interface and interactive platform for the acquisition of new knowledge. The present report compares gabriel 's output with published findings in which expert knowledge has been applied post hoc to microarray groupings generated by hierarchical clustering.

Next Generation Sequencing (NGS) technologies are used to detect somatic mutations in tumors and study germ line variation. Most NGS studies use DNA isolated from whole blood or fresh frozen tissue. However, formalin-fixed paraffin-embedded (FFPE) tissues are one of the most widely available clinical specimens. Their potential utility as a source of DNA for NGS would greatly enhance population-based cancer studies. While preliminary studies suggest FFPE tissue may be used for NGS, the feasibility of using archived FFPE specimens in population based studies and the effect of storage time on these specimens needs to be determined. We conducted a study to determine whether DNA in archived FFPE high-grade ovarian serous adenocarcinomas from Surveillance, Epidemiology and End Results (SEER) registries Residual Tissue Repositories (RTR) was present in sufficient quantity and quality for NGS assays. Fifty-nine FFPE tissues, stored from 3 to 32 years, were obtained from three SEER RTR sites. DNA was extracted, quantified, quality assessed, and subjected to whole exome sequencing (WES). Following DNA extraction, 58 of 59 specimens (98%) yielded DNA and moved on to the library generation step followed by WES. Specimens stored for longer periods of time had significantly lower coverage of the target region (6% lower per 10 years, 95% CI: 3-10%) and lower average read depth (40x lower per 10 years, 95% CI: 18-60), although sufficient quality and quantity of WES data was obtained for data mining. Overall, 90% (53/59) of specimens provided usable NGS data regardless of storage time. This feasibility study demonstrates FFPE specimens acquired from SEER registries after varying lengths of storage time and under varying storage conditions are a promising source of DNA for NGS.

We have developed an informatics system, GeneMed, for the National Cancer Institute (NCI) molecular profiling-based assignment of cancer therapy (MPACT) clinical trial (NCT01827384) being conducted in the National Institutes of Health (NIH) Clinical Center. This trial is one of the first to use a randomized design to examine whether assigning treatment based on genomic tumor screening can improve the rate and duration of response in patients with advanced solid tumors. An analytically validated next-generation sequencing (NGS) assay is applied to DNA from patients’ tumors to identify mutations in a panel of genes that are thought likely to affect the utility of targeted therapies available for use in the clinical trial. The patients are randomized to a treatment selected to target a somatic mutation in the tumor or with a control treatment. The GeneMed system streamlines the workflow of the clinical trial and serves as a communications hub among the sequencing lab, the treatment selection team, and clinical personnel. It automates the annotation of the genomic variants identified by sequencing, predicts the functional impact of mutations, identifies the actionable mutations, and facilitates quality control by the molecular characterization lab in the review of variants. The GeneMed system collects baseline information about the patients from the clinic team to determine eligibility for the panel of drugs available. The system performs randomized treatment assignments under the oversight of a supervising treatment selection team and generates a patient report containing detected genomic alterations. NCI is planning to expand the MPACT trial to multiple cancer centers soon. In summary, the GeneMed system has been proven to be an efficient and successful informatics hub for coordinating the reliable application of NGS to precision medicine studies.

Purpose: Veliparib, a PARP inhibitor, demonstrated clinical activity in combination with oral cyclophosphamide in patients with BRCA-mutant solid tumors in a phase I trial. To define the relative contribution of PARP inhibition to the observed clinical activity, we conducted a randomized phase II trial to determine the response rate of veliparib in combination with cyclophosphamide compared with cyclophosphamide alone in patients with pretreated BRCA-mutant ovarian cancer or in patients with pretreated primary peritoneal, fallopian tube, or high-grade serous ovarian cancers (HGSOC). Experimental Design: Adult patients were randomized to receive cyclophosphamide alone (50 mg orally once daily) or with veliparib (60 mg orally once daily) in 21-day cycles. Crossover to the combination was allowed at disease progression. Results: Seventy-five patients were enrolled and 72 were evaluable for response; 38 received cyclophosphamide alone and 37 the combination as their initial treatment regimen. Treatment was well tolerated. One complete response was observed in each arm, with three partial responses (PR) in the combination arm and six PRs in the cyclophosphamide alone arm. Genetic sequence and expression analyses were performed for 211 genes involved in DNA repair; none of the detected genetic alterations were significantly associated with treatment benefit. Conclusion: This is the first trial that evaluated single-agent, low-dose cyclophosphamide in HGSOC, peritoneal, fallopian tube, and BRCA-mutant ovarian cancers. It was well tolerated and clinical activity was observed; the addition of veliparib at 60 mg daily did not improve either the response rate or the median progression-free survival. Clin Cancer Res; 21(7); 1574–82. ©2015 AACR.

The NCI MATCH is an efficacy signal-finding trial to identify molecular alterations that can be matched to targeted treatments in tumor biopsies from patients (pts) with refractory solid tumors or lymphomas.br />METHODS: An Ion Torrent Oncomine panel sequencing assay and a binary PTEN immunohistochemistry assay were employed. NCI-MATCH opened in August 2015. A protocol-directed pause in screening for interim analysis occurred in November 2015 with goals to assess tumor biopsy feasibility and quality, performance of the CLIA-accredited laboratories, any unanticipated concerns with the study, the match rate for the first activated 10 arms and for planned arms, the spectrum of molecular abnormalities identified, histologic tumor types and demographics. RESULTS: Between 8-12-15 and 11-11-15, 795 pts enrolled for screening and 739 biopsies were submitted. Biopsies were submitted from community (2/3) and academic (1/3) sites. Sequencing was completed on 645 of these specimens (87%). Median turn around time was 27 days but increased from 14 days in the first month to 36 days in the last month, correlating with marked increase in weekly accrual. The highest toxicity (Grade 3) possibly related to biopsy was < 1%. The most frequent tumor types sequenced were colorectal (13%), breast (13%), ovarian (11%), non-small cell lung (7.4%), endometrial (7%), pancreatic (5%), head/neck (5%) esophageal/gastric (4%), cholangiocarcinoma and neuroendocrine (3% each), and small cell lung, prostate, bladder, and unknown primary ( CONCLUSIONS: Accrual was brisk and biopsies were feasible and safe with adequate tumor yield and results. The match rate was lower than predicted for the first 10 treatment arms. Accrual of less common tumor histologic types exceeded expectations. Results from the interim analysis will inform the types of treatment and predicted match rate for additional MATCH arms. NCI-MATCH will re-open with increased capacity and additional treatment arms with a match rate goal of at least 20%. Citation Format: Barbara A. Conley, Robert Gray, Alice Chen, Peter O’Dwyer, Carlos Arteaga, Brent Coffey, David Patton, Shuli Li, Lisa M. McShane, Larry Rubinstein, Robert Comis, Jeffrey Abrams, Paul M. Williams, Chih-Jian Lih, Stanley Hamilton, Edith Mitchell, James Zwiebel, Keith Flaherty, NCI MATCH team. NCI-molecular analysis for therapy choice (NCI-MATCH) clinical trial: interim analysis. [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 CT101.

Background Clinicians have long appreciated the distinct phenotype of systemic juvenile idiopathic arthritis (SJIA) compared to polyarticular juvenile idiopathic arthritis (POLY). We hypothesized that gene expression profiles of peripheral blood mononuclear cells (PBMC) from children with each disease would reveal distinct biological pathways when analyzed for significant associations with elevations in two markers of JIA activity, erythrocyte sedimentation rate (ESR) and number of affected joints (joint count, JC). Methods PBMC RNA from SJIA and POLY patients was profiled by kinetic PCR to analyze expression of 181 genes, selected for relevance to immune response pathways. Pearson correlation and Student's t-test analyses were performed to identify transcripts significantly associated with clinical parameters (ESR and JC) in SJIA or POLY samples. These transcripts were used to find related biological pathways. Results Combining Pearson and t-test analyses, we found 91 ESR-related and 92 JC-related genes in SJIA. For POLY, 20 ESR-related and 0 JC-related genes were found. Using Ingenuity Systems Pathways Analysis, we identified SJIA ESR-related and JC-related pathways. The two sets of pathways are strongly correlated. In contrast, there is a weaker correlation between SJIA and POLY ESR-related pathways. Notably, distinct biological processes were found to correlate with JC in samples from the earlier systemic plus arthritic phase (SAF) of SJIA compared to samples from the later arthritis-predominant phase (AF). Within the SJIA SAF group, IL-10 expression was related to JC, whereas lack of IL-4 appeared to characterize the chronic arthritis (AF) subgroup. Conclusions The strong correlation between pathways implicated in elevations of both ESR and JC in SJIA argues that the systemic and arthritic components of the disease are related mechanistically. Inflammatory pathways in SJIA are distinct from those in POLY course JIA, consistent with differences in clinically appreciated target organs. The limited number of ESR-related SJIA genes that also are associated with elevations of ESR in POLY implies that the SJIA associations are specific for SJIA, at least to some degree. The distinct pathways associated with arthritis in early and late SJIA raise the possibility that different immunobiology underlies arthritis over the course of SJIA.

The National Cancer Institute is developing a national repository of patient-derived cancer models (PDMs) comprised of[T] clinically-annotated patient-derived xenografts (PDXs); patient-derived tumor cell cultures (PDCs, including conditionally-reprogrammed tumor cell cultures) prepared from primary and metastatic tumors, circulating tumor cells (CTCs), and/or PDXs; tumor cell lysates, DNA, and RNA; and cancer-associated fibroblast cell lines (CAFs, autologous when possible) to serve as a resource for academic discovery efforts and public-private partnerships for drug discovery. NCI will provide a long-term home for >1000 PDX and PDC models, each produced from tissues and blood supplied by NCI-designated Cancer Centers and NCI-supported clinical trials networks. The effort is targeting the collection of tumors that are less prevalent in current resources, such as: small cell lung cancer, prostate cancer, bladder cancer, pancreatic cancer, head and neck cancers, as well as sarcomas and melanomas. The goals of the project are: (1) to develop a minimum of ∼50 unique patient models (both PDXs and PDCs) per disease such that the size of each molecularly-characterized subgroup is useful for subsequent validation and/or efficacy studies; (2) to perform comprehensive pre-competitive molecular characterization of patient samples and earliest passage PDXs and PDCs that includes the NCI-MPACT mutation panel, WES, RNASeq, copy number determination, histology, growth curves, and pilot proteomic/phospho-proteomic studies; and (3) to make all models and associated pre-clinical and clinical data available through a publicly available website. To date, over 1700 specimens from 1100 patients have been received for the development of PDMs; the overall ‘take' rate for PDXs originating from solid tumors is 70% with >170 assessable models and another 270 early passage tumors currently in evaluation. As expected, based on collection priorities, tumors of genitourinary, digestive, head and neck, musculoskeletal, respiratory, and skin origin are the major histological sites of origin for our PDX models. In addition, over 90 conditionally-reprogrammed cell lines have been expanded from both 18-gauge needle biopsies and surgical resections, and have passed initial quality control procedures; many of these cell cultures have a matched PDX. Over 150 CAF lines have been developed following repeated (>10) purification steps using flow cytometry and are in the process of quality control procedures that demonstrate complete lack of growth in NOD-SCID gamma IL2 receptor null (NSG) mice; of these CAFs, we have developed matched pairs of PDCs and CAFs from the same patient in 16 cases. To evaluate the potential utility of the NCI PDM Repository, we have prospectively ‘entered' 22 models in a pre-clinical trial for which eligibility (based on actionable mutations) and treatment arms are identical to those in the NCI-MPACT study (NCT01827384). Multiple objective responses (significant improvement in overall survival) have been observed in all arms of the study and in a variety of models. WES and RNASeq analysis have proven essential to explain the therapeutic responses that we have observed. We are also evaluating the relationship of in vitro and in vivo activity for the NCI-MPACT drug panel in the models where concurrent PDCs and PDXs have been produced. A web site has been developed that will provide annotated information on the models (such as DNA sequence, gene expression, prior therapy) to investigators to assist in the distribution of the contents of the repository to the research community. We expect to be able to begin distribution in late spring of 2016. Citation Format: James H. Doroshow, Melinda Hollingshead, Yvonne Evrard, P. Mickey Williams, Vivekananda Datta, Biswajit Das, Chih-Jian Lih, Dianne Newton. NCI patient derived models repository. [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 PL08-02.

Systemic juvenile idiopathic arthritis (SJIA) is a chronic autoinflammatory condition. The association with macrophage activation syndrome, and the therapeutic efficacy of inhibiting monocyte-derived cytokines, has implicated these cells in SJIA pathogenesis. To characterize the activation state (classical/M1 vs. alternative/M2) of SJIA monocytes, we immunophenotyped monocytes using several approaches. Monocyte transcripts were analyzed by microarray and quantitative PCR. Surface proteins were measured at the single cell level using flow cytometry. Cytokine production was evaluated by intracellular staining and ELISA. CD14(++)CD16(-) and CD14(+)CD16(+) monocyte subsets are activated in SJIA. A mixed M1/M2 activation phenotype is apparent at the single cell level, especially during flare. Consistent with an M2 phenotype, SJIA monocytes produce IL-1β after LPS exposure, but do not secrete it. Despite the inflammatory nature of active SJIA, circulating monocytes demonstrate significant anti-inflammatory features. The persistence of some of these phenotypes during clinically inactive disease argues that this state reflects compensated inflammation.

We have isolated and sequenced a Drosophila genomic DNA that encodes the entire coding region of the laminin B1 chain. The genomic DNA sequenced spans 11,787 bp, including a 1.1-kb 5'-flanking region, 5 exons, 4 introns, and a 1.4-kb 3'-flanking region. The open reading frame is within the two largest exons, the exons 3 and 4, while the first two and the last exons are much smaller and are untranslated. The structure of the Drosophila laminin B1 gene is similar to the Drosophila laminin B2 gene. Their exon-intron lengths and Eco RI, Pst I restriction maps are quite conserved. Both of their open reading frames are very compact, and their first introns are much larger than all of the rest of the introns. These results are consistent with the suggestion that the B1 and B2 genes could be derived from an ancestral gene. The similarity of the proximal 5'-flanking regions of the Drosophila B1 and B2 genes is 46.6%. Also, similar sequences of transcriptional regulatory elements, even though not site conserved, are found in both proximal 5'-flanking regions of the B1 and B2 genes. When transfected into Drosophila SL-2 cells, pCAT plasmid containing 1,048 bp of 5'-flanking region shows a strong expression of chloramphenicol acetyltransferase (CAT) activity. The deletion clones that contain sequences between nucleotides -462 to +150, and -282 to +150 all show strong CAT activity. These results suggest that this 5'-flanking promoter region may contain DNA sequences that can promote the expression of the laminin B1 gene.

TNF-α plays an important role in immune regulation, inflammation, and autoimmunity. Chronic TNF exposure has been shown to down-modulate T cell responses. In a mouse T cell hybridoma model, TNF attenuated T cell receptor (TCR) signaling. We have confirmed that chronic TNF and anti-TNF exposure suppressed and increased T cell responses, respectively. In adult TCR (BDC2.5) transgenic nonobese diabetic mice, DNA microarray analysis of global gene expression in BDC2.5 CD4 + T cells in response to chronic TNF or anti-TNF exposure showed that genes involved in functional categories including T cell signaling, cell cycle, proliferation, ubiquitination, cytokine synthesis, calcium signaling, and apoptosis were modulated. Genes such as ubiquitin family genes, cytokine inducible Src homology 2-containing genes, cyclin-dependent kinase inhibitors p21, p57, calmodulin family genes (calmodulin-1, -2, and -3) and calcium channel voltage-dependent, N type α1B subunit (CaV2.2) were induced by TNF, whereas Vav2, Rho GTPase-activating protein, calcium channel voltage-dependent, L type α1C subunit (CaV1.2), IL-1 receptor-associated kinase-1 and -2, and IL enhancer binding factor 3 were reduced by TNF. Genes such as CaV1.2 and proliferating cell nuclear antigen, repressed by TNF, were induced by anti-TNF treatment. Further, we showed that chronic TNF exposure impaired NF-κB and adaptor protein 1 transactivation activity, leading to T cell unresponsiveness. Thus, our results present a detailed picture of transcriptional programs affected by chronic TNF exposure and provide candidate target genes that may function to mediate TNF-induced T cell unresponsiveness.

Background Glioblastoma multiforme (GBM) is the most common primary intracranial tumor and despite recent advances in treatment regimens, prognosis for affected patients remains poor. Active cell migration and invasion of GBM cells ultimately lead to ubiquitous tumor recurrence and patient death. To further understand the genetic mechanisms underlying the ability of glioma cells to migrate, we compared the matched transcriptional profiles of migratory and stationary populations of human glioma cells. Using a monolayer radial migration assay, motile and stationary cell populations from seven human long term glioma cell lines and three primary GBM cultures were isolated and prepared for expression analysis. Results Gene expression signatures of stationary and migratory populations across all cell lines were identified using a pattern recognition approach that integrates a priori knowledge with expression data. Principal component analysis (PCA) revealed two discriminating patterns between migrating and stationary glioma cells: i) global down-regulation and ii) global up-regulation profiles that were used in a proband-based rule function implemented in GABRIEL to find subsets of genes having similar expression patterns. Genes with up-regulation pattern in migrating glioma cells were found to be overexpressed in 75% of human GBM biopsy specimens compared to normal brain. A 22 gene signature capable of classifying glioma cultures based on their migration rate was developed. Fidelity of this discovery algorithm was assessed by validation of the invasion candidate gene, connective tissue growth factor (CTGF). siRNA mediated knockdown yielded reduced in vitro migration and ex vivo invasion; immunohistochemistry on glioma invasion tissue microarray confirmed up-regulation of CTGF in invasive glioma cells. Conclusion Gene expression profiling of migratory glioma cells induced to disperse in vitro affords discovery of genomic signatures; selected candidates were validated clinically at the transcriptional and translational levels as well as through functional assays thereby underscoring the fidelity of the discovery algorithm.

There is growing interest in the application of molecular profiling, including sequencing, genotyping, and/or mRNA expression profiling, to the analysis of patient tumors with the objective of applying these data to inform therapeutic choices for patients with advanced cancers. Multiple clinical trials that are attempting to validate this personalized or precision medicine approach are in various stages of development and execution. Although preliminary data from some of these efforts have fueled excitement about the value and utility of these studies, their execution has also provoked many questions about the best way to approach complicating factors such as tumor heterogeneity and the choice of which genetic mutations to target. This commentary highlights some of the challenges confronting the clinical application of molecular tumor profiling and the various trial designs being utilized to address these challenges. Randomized trials that rigorously test patient response to molecularly targeted agents assigned based on the presence of a defined set of mutations in putative cancer-driving pathways are required to address some of the current challenges and to identify patients likely to benefit from this approach.

TPS2642 Background: There is growing interest in matching targeted agents to genetic aberrations found in tumors of patients in early phase trials. MPACT is a randomized trial designed to assess wh...

The eubacterial species Streptomyces coelicolor proceeds through a complex growth cycle in which morphological differentiation/development is associated with a transition from primary to secondary metabolism and the production of antibiotics. We used DNA microarrays and mutational analysis to investigate the expression of individual genes and multigene antibiotic biosynthetic pathways during these events. We identified expression patterns in biosynthetic, regulatory, and ribosomal protein genes that were associated highly specifically with particular stages of development. A knowledge-based algorithm that correlates temporal changes in expression with chromosomal position identified groups of contiguous genes expressed at discrete stages of morphological development, inferred the boundaries of known antibiotic synthesis gene loci, and revealed novel physical clusters of coordinately regulated genes. Microarray analysis of RNA from cells mutated in genes regulating synthesis of the antibiotics actinorhodin (Act) and undecylprodigiosin (Red) identified proximate and distant sites that contain putative ABC transporter and two-component system genes expressed coordinately with genes of specific biosynthetic pathways and indicated the existence of two functionally and physically discrete regulons in the Red pathway.