Citations

Abstract Background: Copanlisib is a novel pan-Class I phosphatidylinositol-3-kinase (PI3K) inhibitor with potent preclinical inhibitory activity against both PI3K-δ and PI3K-α isoforms. A phase II study of copanlisib in patients with relapsed/refractory indolent or aggressive lymphoma reported promising activity in the indolent NHL group (Dreyling et al., ENA 2014). The objective of this study is to evaluate the efficacy and safety of copanlisib in patients with indolent B-cell NHL relapsed after or refractory to standard therapy. Methods: Patients meeting the following criteria are eligible for enrollment: histologically confirmed diagnosis of indolent B-cell NHL, with follicular lymphoma (FL) grade 1-2-3a, marginal zone lymphoma (MZL; splenic, nodal, or extra-nodal), small lymphocytic lymphoma (SLL) with absolute lymphocyte count < 5 × 109/L at the time of diagnosis and at study entry, or lymphoplasmacytoid lymphoma/Waldenström macroglobulinemia (LPL/WM), and who have previously received ? 2 prior lines of therapy with rituximab and an alkylating agent and must be refractory to the last treatment with rituximab (refractory defined as progressing or not responding within 6 months of the last course of treatment). Patients will be randomized in 2:1 manner to receive 60 mg of copanlisib administered intravenously on days 1, 8 and 15 of a 28-day cycle or placebo administered on the same schedule until disease progression or intolerable toxicity. Patients in the placebo arm will be allowed to cross-over to receive follow-up treatment with copanlisib, after confirmed disease progression. Dose reductions due to toxicities will be allowed. Radiologic tumor assessment will be performed every 12, 16 or 24 weeks for years 1, 2, and 3, respectively. The primary endpoint will be progression-free survival (PFS). Secondary objectives include overall objective response rate (ORR), duration of response (DOR), time to progression (TTP), complete response rate (CRR), overall survival (OS), time to deterioration as well as time to improvement in disease-related symptoms. Exploratory objectives will include secondary PFS after first progression in placebo-treated patients who cross over to receive copanlisib and time to improvement and the time to deterioration in disease-related symptoms - physical (DRS-P) of at least 3 points as measured by the FLymSI-18 questionnaire. Approximately 189 patients who meet the eligibility criteria will be randomized 2:1, with approximately 126 patients in the copanlisib monotherapy arm and approximately 63 patients in placebo arm. The study is planned to detect a 132% increase in median PFS in copanlisib versus placebo (i.e. to detect a hazard ratio of 0.43), using a stratified log-rank test. This study is currently enrolling patients (NCT02369016). Citation Format: Grzegorz S. Nowakowski, Igor Gorbatchevsky, Florian Hiemeyer, Lisa Cupit, Barrett H. Childs. A randomized, double-blind phase III study of phosphatidylinositol-3 kinase alpha/delta inhibitor copanlisib versus placebo in patients with rituximab-refractory indolent non-Hodgkin's lymphoma (iNHL): CHRONOS-2. [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 CT084.

Abstract BACKGROUND Genomic intra- and intertumor heterogeneity is one main reason for relapse and resistance to therapy. There is a shortage of studies characterizing the level of genomic intertumoral heterogeneity of known cancer genes in multiple longitudinal biopsies of individual patients. Additionally, a workflow to deconvulate the intermixture of tumor and stromal components is missing. To overcome these limitations and to decipher the genomic heterogeneity and clonal evolution, we developed a method and applied it on matched (primary-recurrence/metastasis) non-squamous, non-small cell lung cancers (NSCLC). METHODS Multiparameter Ploidy Profiling (MPP) comprises the isolation of nuclei from frozen or formalin and paraffin embedded (FFPE) tissues, followed by multiparamter flow sorting. Sorted populations were subjected to genomic profiling by high resolution array comparative genomic hybridization (aCGH) and massively parallel sequencing (MPS). DAPI allowed to seperate populations by ploidy and anti-TTF1 antibodies was used to control for tumor nuclei. Array-CGH, combined with ploidy, was used to retrieve genome wide copy numbers. The Comprehensive Cancer Panel that covers all-exons of 409 cancer genes was applied on all sorted tumor and stromal populations to detect somatic mutations and their variant allelic frequency (VAF). RESULTS MPP was successfully applied to 44 frozen or FFPE tissue specimens from 19 patients. Array-CGH and MPS of TTF1-negative, normal cells were concordant to germline controls. Sequencing revealed that 50% of mutations are shared between primary tumors and metastases. Except for one patient, mutations with VAF>0.3 are shared between primary and metastasis. The variant allelic frequency was significantly higher in shared mutations than mutations that were private to one tumor. Besides common activating mutations in EGFR and KRAS we found biallelic inactivation in tumor suppressor genes like KEAP1, NF1, STK11 and TP53. Two clonal evolutionary patterns were found: 1) early and 2) late divergence. Matched tumors without any shared mutations were classified as unrelated primary tumors. CONCLUSION The power of MPP is to increase the precision of downstream analysis, due to the sorting of pure populations of tumor cells. It permitted to infer the clonal evolution of tumor populations with unprecedented confidence. The low level of genomic heterogeneity of mutations with VAF>0.3 in this study is in line with recent data from Zhang et al., who showed that the poor precision of low depth sequencing (<277x) contributes to an overestimation of genomic heterogeneity (Zhang et al., Science 2014). Integrational analysis of ploidy, chromosomal aberrations and mutations in 409 cancer genes allowed to draw the evolutionary picture of each patient's disease. MPP appears as a promising tool for identification of genomic vulnerabilities that could be exploited for tailored treatment. Citation Format: Thomas Lorber, Tanja Dietsche, Valeria Perrina, Darius Juskevicius, Arthur Krause, David Mueller, Michael Barrett, Christian Ruiz, Lukas Bubendorf. Clonal evolution and genomic tumor heterogeneity in non-small cell lung cancer deciphered by multiparameter ploidy profiling. [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 158.

Abstract Background: Genomic tumor heterogeneity is one reason for recurrence and resistance to cancer therapy. It is still not known what leads to the relapse or the emergence of metastasis in individual patients. To contribute to answer this question, we investigated the evolutionary patterns in squamous cell carcinoma of the lung (SCC) in 10 patients with multiple matched tumors (primary vs recurrence/metastasis). Methods: To identify the genomic profile, frozen tissues from SCC patients were subjected to multiparameter ploidy profiling (MPP). Tumor populations were sorted based on ploidy (DNA-content using DAPI) and p40 was used to control for diploid tumor populations. Subsequent genomic profiling by array comparative genomic hybridization (aCGH) and targeted massive parallel sequencing (Ion Torrent Comprehensive Cancer Panel with an all-exon coverage of 409 cancer genes) was applied. Results: The usage of MPP enabled the determination of the clonal composition of the SCC and define genomic characteristics. Due to flow sorting the tumor populations were effectively isolated and existence of distinct clonal populations was indicated. Aneuploid and diploid tumors were successfully revealed using the additional marker p40. Therefore, the absence of non-malignant cells increased the precision in the genomic analyses Conclusion: The study uncovered the composition of human SCC in the sense of different clonal populations, which harbor private genomic aberrations and mutations confirmed by aCGH and NGS. Even if our data is still preliminary we provide evidence that applying MPP increases the precision of aCGH and sequencing analysis. Genomic analyses of sorted pure tumor population allows to reconstruct the clonal evolution and facilitates the understanding of tumor heterogeneity and their evolutionary pattern, which could have potentially an impact on therapeutic response or metastasis development. Citation Format: Arthur Krause, Thomas Lorber, Valeria Perrina, Tanja Dietsche, Michael Barrett, Christian Ruiz, Lukas Bubendorf. The analysis of the genomic evolution of squamous cell carcinoma of the lung. [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 155.

Abstract Diagnostic testing is an integral component of patient evaluation in the emergency department ( ED ). Emergency clinicians frequently use diagnostic testing to more confidently exclude “worst‐case” diagnoses rather than to determine the most likely etiology for a presenting complaint. Increased utilization of diagnostic testing has not been associated with reductions in disease‐related mortality but has led to increased overall healthcare costs and other unintended consequences (e.g., incidental findings requiring further workup, unnecessary exposure to ionizing radiation or potentially nephrotoxic contrast). Shared decision making ( SDM ) presents an opportunity for clinicians to discuss the benefits and harms associated with diagnostic testing with patients to more closely tailor testing to patient risk. This article introduces the challenges and opportunities associated with incorporating SDM into emergency care by summarizing the conclusions of the diagnostic testing group at the 2016 Academic Emergency Medicine Consensus Conference on SDM . Three primary domains emerged: 1) characteristics of a condition or test appropriate for SDM , 2) critical elements of and potential barriers to SDM discussions on diagnostic testing, and 3) financial aspects of SDM applied to diagnostic testing. The most critical research questions to improve engagement of patients in their acute care diagnostic decisions were determined by consensus.

SummaryBackgroundMatrix metalloproteinase‐9 is a proteolytic enzyme whose expression is increased in ulcerative colitis.AimTo evaluate the safety and efficacy of GS‐5745, a fully humanised anti‐matrix metalloproteinase‐9 monoclonal antibody, in moderately‐to‐severely active ulcerative colitis.MethodsWe randomised 74 patients with ulcerative colitis to treatment with single or multiple ascending intravenous or subcutaneous doses of GS‐5745 or placebo. Multiple‐dose cohorts received either IV infusions (0.3, 1.0, 2.5 or 5.0 mg/kg GS‐5745 or placebo) every 2 weeks (three total IV infusions) or five weekly SC injections (150 mg GS‐5745 or placebo). The primary outcomes were the safety, tolerability and pharmacokinetics of escalating single and multiple doses of GS‐5745. Exploratory analyses in the multiple‐dose cohorts included clinical response (≥3 points or 30% decrease from baseline in Mayo Clinic score and ≥1 point decrease in the rectal bleeding subscore or a rectal bleeding subscore ≤1) and clinical remission (a complete Mayo Clinic score ≤2 with no subscore >1) at Day 36. Biological effects associated with a clinical response to GS‐5745 were explored using histological and molecular approaches.ResultsTwenty‐three of the 42 patients (55%) receiving multiple doses of GS‐5745 had adverse events, compared with 5/8 patients (63%) receiving placebo. GS‐5745 showed target‐mediated drug disposition, approximately dose‐proportional increases in maximum plasma concentration and more than dose‐proportional increases in the area under the plasma drug concentration‐time curve. Clinical response occurred in 18/42 patients (43%) receiving GS‐5745 compared with 1/8 patients (13%) receiving placebo. Clinical remission occurred in 6/42 patients (14%) receiving GS‐5745 and 0/8 (0%) receiving placebo. Patients with a clinical response to GS‐5745 had reductions in matrix metalloproteinase‐9 tissue levels (mean 48.9% decrease from baseline compared with a mean 18.5% increase in nonresponders, P = 0.008) significant improvements in histopathology scores (confirmed with three separate histological disease activity indices), as well as changes in colonic gene expression that were consistent with reduced inflammation.ConclusionThis phase 1 trial provides preliminary evidence for the safety and therapeutic potential of GS‐5745 in the treatment of ulcerative colitis.

Abstract Introduction: Triple negative breast carcinoma (TNBC) is a subtype of breast cancer with a paucity of therapeutic targets and a poor prognostic phenotype. In a retrospective cohort, we sought to determine the prevalence of the amplicon targeting the 9p24.1 locus, resulting in over-expression of PD-L1, PD-L2, and JAK2 (PDJ), and potentially actionable therapeutic targets. We then probed the genomic landscape of these PDJ positive tumors and identified co-occurring copy number aberrations, including focal amplifications and homozygous deletions. The presence of the PDJ amplicon and selected co-occurring aberrations provide a unique description of a clinically relevant subtype of TNBC. Methods: We evaluated fresh frozen and formalin-fixed paraffin embedded tumor samples from 64 patients with triple negative breast cancer whom underwent definitive surgical resection. Clinical annotation was available in 60 of the samples. Tumor populations (diploid, tetraploid, and aneuploidy) were sorted from each biopsy using DNA content flow cytometry. Each sorted sample was interrogated with oligonucleotide array comparative genomic hybridization (aCGH). All microarray slides were scanned using an Agilent 2565C DNA scanner and the images were analyzed with Agilent Feature Extraction version 10.7. The aCGH data was assessed with a series of QC metrics then analyzed using an aberration detection algorithm. Results: We detected a high level (log2ratio greater than or equal to 2) amplicon targeting 9p24.1 in 18 of 64 patients (28%) genomic profiles with triple negative breast carcinoma. In the PDJ positive population, we detected 8 of 18 patients (44%) with co-amplification targeting myc at 8q24, 3 of 18 patients (17%) with co-amplifications targeting EGFR at 7p11, 3 of 18 patients (17%) with co-amplifications targeting PIK3CA. These co-occurring genomic events in PDJ positive tumors may provide clinically actionable targets. Other selected amplifications detected included NOTCH3, KRAS, RUNX1, TUBAL3, FGFR2, AKT1, AKT2, YPEL2, PBXL7, KIT. We detected PTEN homozygous deletion in 2 of 18 patients (11%) PDJ positive tumors. Other homozygous deletions identified in the genomic landscape included FAT1, SOX3, Park2, TNFAIP3, GPC3, RB1, and CREBBP. Conclusions: In our retrospective analysis, the amplification of chromosome 9p24.1 involving PD-L1, PD-L2, and JAK2 is present in approximately 28% of triple negative breast cancer patients. The genomic landscape of these PDJ positive TNBCs include recurring high-level focal amplifications and targeted homozygous deletions of clinically relevant genes. The clinical implications of these data are under current investigation using model systems and are in early phase clinical trials. The efficacy of immune checkpoint inhibitors including nivolumab, pembrolizumab, and JAK2 inhibitors including ruxolitinib, in PDJ positive triple negative breast carcinoma is intriguing and remains to be elucidated. Citation Format: Gawryletz CD, Anderson KS, Cunliffe HE, Northfelt DW, McCullough AE, Lenkiewicz E, Malasi S, Pockaj BA, Barrett MT. The genomic landscape of PD-L1, PD-L2, Jak2 (PDJ) amplified triple negative breast carcinoma. [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 P6-03-13.

Abstract Background A subset of triple negative breast cancers (TNBC) has somatic amplification of chromosome 9p24.1, encoding PD-L1, PD-L2, and JAK2 (the "PDJ" amplicon). The JAK/STAT pathway promotes malignant transformation and proliferation, and the JAK1/JAK2 inhibitor ruxolitinib is being evaluated in early phase clinical trials in breast cancer. This study was designed to measure the frequency of PDJ copy number gain in existing TNBC cell lines, and determine the impact of targeted JAK2 inhibition on cellular proliferation. Methods: Copy number alterations were measured in nine TNBC cell lines (MDA-MB-453, MDA-MB-436, BT549, MDA-MB231, MFM-223, MDA-MB-468, H5578T, MDA-MB-157, HCC1937) by array comparative genomic hybridization (aCGH). JAK2 and pSTAT3 expression was measured by immunoblot in four of the TNBC lines (HCC1937, MDA-MB-468, MDA-MB-231, MDA-MB-436) and compared with MCF10A, SK-BR3, and T47D. To selectively inhibit JAK2, lentiviral vectors encoding five different shRNA targeting JAK2 were generated. Ruxolitinib treatment was performed at 25 uM for 48 hrs and 72 hrs. Cell proliferation was measured by CellTiter-Glo. Results: All TNBC cell lines had an elevated level of baseline protein expression of JAK2 and pSTAT3 compared to SK-BR3, T47D, and MCF10A. Of the nine TNBC cell lines, only MDA-MB-436 and BT549 had copy number gain of 9p24.1 (log2ratio<1). Four independent shRNA efficiently silenced over 90% of JAK2 expression in MDA-MB-231. The inhibition of JAK2 in MDA-MB-231 was associated with a decrease in both JAK2 and pSTAT3 protein expression and reduction in cell proliferation compared to control shRNA after 96 hrs (p<0.0001). Inhibition of proliferation was also observed for both MDA-MB-231 and MDA-MB-436 after ruxolitinib treatment. Conclusion These results demonstrate that existing TNBC cell lines have varying copy number alterations of chromosome 9p24.1 encoding JAK2, and targeted JAK2 inhibition in TNBC inhibits cell proliferation. Citation Format: Chen M, Andreozzi M, Gonzalez-Malerva L, Eaton S, Pockaj B, Barrett MT, Anderson KS. JAK2 copy number and targeted JAK2 inhibition of TNBC cell lines. [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 P5-04-19.