ALBERT

Description: Resistance to androgen deprivation therapy, or castration-resistant prostate cancer (CRPC), is often accompanied by metastasis and is currently the ultimate cause of prostate cancer-associated deaths in men. Recently, secondary hormonal therapies have led to an increase of neuroendocrine prostate cancer (NEPC), a highly aggressive variant of CRPC. Here, we identify that high levels of cell surface receptor Trop2 are predictive of recurrence of localized prostate cancer. Moreover, Trop2 is significantly elevated in CRPC and NEPC, drives prostate cancer growth, and induces neuroendocrine phenotype. Overexpression of Trop2 induces tumor growth and metastasis while loss of Trop2 suppresses these abilities in vivo. Trop2-driven NEPC displays a significant up-regulation of PARP1, and PARP inhibitors significantly delay tumor growth and metastatic colonization and reverse neuroendocrine features in Trop2-driven NEPC. Our findings establish Trop2 as a driver and therapeutic target for metastatic prostate cancer with neuroendocrine phenotype and suggest that high Trop2 levels could identify cancers that are sensitive to Trop2-targeting therapies and PARP1 inhibition.

Description: Accurate identification of prostate cancer in frozen sections at the time of surgery can be challenging, limiting the surgeon’s ability to best determine resection margins during prostatectomy. We performed desorption electrospray ionization mass spectrometry imaging (DESI-MSI) on 54 banked human cancerous and normal prostate tissue specimens to investigate the spatial distribution of a wide variety of small metabolites, carbohydrates, and lipids. In contrast to several previous studies, our method included Krebs cycle intermediates (m/z

Description: Background: Pre-leukemic hematopoietic stem cells (HSC) have been implicated in AML (Jan et al STM 2012) and also for several lymphoid leukemias including ALL, HCL, and CLL. Separately, relapse of ALL following CD19 CAR-T cell therapy has been associated with lymphomyeloid lineage switch. Finally, healthy persons with clonally expanded HSCs are at increased risk of hematologic malignancies including lymphomas, and in mouse DLBCL models we previously demonstrated the oncogenic sufficiency of BCL6 overexpression in HSC (Green et al 2014 Nat Comm). Nevertheless, the cellular origin of DLBCL in the majority of patients is not definitively known. We sought to investigate the presence of mutations found in DLBCL within matched HSCs. Methods: We deeply genotyped somatic mutations in diagnostic biopsy tissues of 16 patients with DLBCL using CAPP-Seq to a median sequencing depth of 1100x (Newman et al 2014 Nat Med; Scherer et al 2015 ASH). We then profiled each patient for evidence implicating HSCs using somatic mutation lineage tracing, in either direct or indirect fashion. For direct evaluation, we used highly purified, serially FACS-sorted HSCs from grossly uninvolved bone marrow (BM) (n=5; Fig 1a-b). For indirect assessment, we either profiled serial tumor biopsies (n=13), or interrogated sorted cells from terminally differentiated blood lineages (n=7), including peripheral CD3+ T cells, CD14+ Monocytes, and B cells expressing a light-chain discordant to that of tumor isotype. HSCs and differentiated lineages were then interrogated by direct genotyping, using 3 highly sensitive orthogonal quantitative methods, including Myd88 L265P droplet digital PCR (n=6), BCL6 translocation breakpoint qPCR (n=4), and DLBCL CAPP-Seq profiling of 268 genes (n=5). We used the theoretical limit of detection (LOD) genotyping performance for CAPP-Seq (0.001%, Newman et al 2016 Nat Biotech), and established analytical sensitivity of our custom MYD88 ddPCR via limiting dilution (~1%). These LODs met or exceeded the expected limit of sorting impurity by FACS (~1%). For 6 patients experiencing one or more DLBCL relapse, we deeply profiled 13 serial tumor biopsies by CAPP-Seq, and then assessed overlap in somatic mutations and VDJ sequences in biopsy pairs as additional indirect evidence implicating HSCs. Results: We obtained a median of ~2000 sorted HSCs and ~1700 sorted cells from differentiated lineages, and genotyped each population using one or more of the 3 direct genotyping methods described above. Three patients with sufficient cell numbers were profiled both by CAPP-Seq and either ddPCR (n=2) or qPCR (n=1). Surprisingly, we found no evidence implicating HSCs either directly or indirectly in any of the 16 patients, regardless of the assay employed or the cell types/lineages genotyped (e.g., Fig 1b). In 2 patients with MYD88 L265P mutations, we found evidence for MYD88+ B-cells with discordant light chains by ddPCR (~0.1%) potentially implicating common lymphoid precursors (CLPs), but found no evidence for similar involvement of T-cells or monocytes. In 6 DLBCL patients experiencing relapse, tumor pairs profiled by CAPP-Seq (median depth 957) shared 93% of somatic mutations (75-100%, Fig 1c). Such pairs invariably shared clonal IgH VDJ rearrangements (4/4, 100%), thus implicating a common progenitor arising in later stages of B-cell development, not HSCs. Conclusions: We find no evidence to implicate HSCs in the derivation of DLBCL. While formal demonstration of absence of pre-malignant HSCs in DLBCL would require overcoming practical and technical limitations (including number of available HSCs, sorting purity, and genotyping sensitivity), the pattern of shared somatic alterations at relapse makes this highly unlikely. We speculate that unlike lymphoid leukemias, the cell-of-origin for most DLBCLs reside later in B-lymphopoiesis, beyond CLPs. Figure. (a) HSC sorting from BM by FACS (b) Allele frequencies of mutations found by CAPP-Seq in an examplary DLBCL case (x-axis) compared to the same variants in HSCs (y-axis). (c) Phylogenetic trees of DLBCL patients experiencing relapse (n=6) with tumor pairs sequenced by CAPP-Seq. Shown are the evolutionary distances between (i) germline and common inferrable progenitor (CIP) illustrating the fraction of shared mutations between tumor pairs, and (ii) CIP and both diagnostic (tumor 1) and relapse tumors (tumor 2) indicating unique mutations to each tumor. Figure. (a) HSC sorting from BM by FACS (b) Allele frequencies of mutations found by CAPP-Seq in an examplary DLBCL case (x-axis) compared to the same variants in HSCs (y-axis). (c) Phylogenetic trees of DLBCL patients experiencing relapse (n=6) with tumor pairs sequenced by CAPP-Seq. Shown are the evolutionary distances between (i) germline and common inferrable progenitor (CIP) illustrating the fraction of shared mutations between tumor pairs, and (ii) CIP and both diagnostic (tumor 1) and relapse tumors (tumor 2) indicating unique mutations to each tumor. Disclosures Newman: Roche: Consultancy. Levy:Kite Pharma: Consultancy; Five Prime Therapeutics: Consultancy; Innate Pharma: Consultancy; Beigene: Consultancy; Corvus: Consultancy; Dynavax: Research Funding; Pharmacyclics: Research Funding. Diehn:Novartis: Consultancy; Quanticel Pharmaceuticals: Consultancy; Roche: Consultancy; Varian Medical Systems: Research Funding.

Description: Background and Methods DLBCL patients exhibit striking heterogeneity in therapeutic responses, yet current methods to assess residual disease have shown suboptimal performance. Circulating tumor DNA (ctDNA) is a promising noninvasive biomarker for disease burden. While High-Throughput Sequencing of immunoglobulin genes (Ig-HTS) has shown utility for ctDNA detection in DLBCL, only a small number of tumor markers are profiled, limiting its sensitivity and broad applicability (Kurtz DM, Blood 2015 and Roschewski M, Lancet Oncology 2015). Here, we studied DLBCL patients using CAPP-Seq, a recently developed approach for ultrasensitive assessment of ctDNA that profiles multiple genomic regions across hundreds of kilobases in a single HTS assay (Newman AM, Nat. Med. 2014). Results We designed a 250kb DLBCL CAPP-Seq panel targeting 1053 regions from 268 genes, incorporating recurrent single nucleotide variants (SNVs), insertions/deletions (indels) and rearrangements, and evaluated its genotyping performance on diverse tumors from 63 patients (Table 1). We detected somatic mutations in 98% of patients (median: 143 variants/pt), including AID-related events in well-known hotspots. Moreover, we detected 100% of BCL2/BCL6 rearrangements and most MYC translocations previously identified by FISH (n = 30). Biopsy-confirmed tumor mutations were detectable with 99.3% specificity in 97% of pretreatment plasma samples (n = 37) with a range of 0.007% to 32% mean fractional abundance. Median recovery rates for both SNVs and fusions in plasma exceeded 85%, indicating robust tumor genotyping performance. Furthermore, in most patients, paired allelic fractions between tumor biopsies and pretreatment plasma samples were significantly correlated (P 〈 0.05), demonstrating that ctDNA can accurately mirror the mutational profiles found in tumor tissues. We next evaluated the utility of direct biopsy-free genotyping of pretreatment plasma using a novel ctDNA genotyping approach. We noninvasively identified variants in 97% of cases (n=37), and confirmed a median of 95% of SNVs per cfDNA sample in paired tumor biopsies, indicating that plasma DNA is an effective surrogate for direct tumor genotyping. Having established the technical performance in pretreatment cfDNA, we next assessed utility of CAPP-Seq for minimal residual disease (MRD) detection (Figure 1). Plasma samples at time points of complete response (n = 22) were collected from 10 patients, all of whom ultimately relapsed. Each plasma sample was then analyzed for evidence of biopsy-confirmed mutations from pretreated tumors. Strikingly, ctDNA was detected in 9 of 10 patients (90%), including 3 of 3 isolated CNS relapses, with fractional abundances as low as 0.004%. Among patients with CAPP-Seq detectable MRD, the median time between first detection and relapse was 162 days, and all patients whose MRD levels were uniformly below the detection limit had blood collections 〉8 months prior to relapse. Conclusions To summarize, we validated robust technical performance of a novel approach for noninvasive tumor genotyping in DLBCL using pretreatment plasma, and for MRD surveillance. Given the demonstrated advantages of CAPP-Seq in the setting of radiographic remission, we envision its utility for improved relapse prediction. Moreover, we anticipate that this biopsy-free genotyping approach will have applications for monitoring subclonal dynamics and emergent mutations in response to therapy. FS, DMK and AMN contributed equally. Disclosures Newman: Roche: Consultancy. Klass:Roche: Employment. Diehn:Roche: Consultancy. Alizadeh:Genentech: Consultancy; Celgene: Consultancy; Roche: Consultancy.

Description: Key Points DLBCL can be detected in the blood by immunoglobulin high-throughput sequencing (Ig-HTS) with high specificity. Although DLBCL can be detected in leukocytes or plasma by Ig-HTS, plasma has greater sensitivity and more accurately reflects disease.

Description: Mast cells (MCs) promote a wide range of localized and systemic inflammatory responses. Their involvement in immediate as well as chronic inflammatory reactions at both local and distal sites points to an extraordinarily powerful immunoregulatory capacity with spatial and temporal versatility. MCs are preferentially found in close proximity to both vascular and lymphatic vessels. On activation, they undergo a biphasic secretory response involving the rapid release of prestored vasoactive mediators followed by de novo synthesized products. Many actions of MCs are related to their capacity to regulate vascular flow and permeability and to the recruitment of various inflammatory cells from the vasculature into inflammatory sites. These mediators often work in an additive fashion and achieve their inflammatory effects locally by directly acting on the vascular and lymphatic endothelia, but they also can affect distal sites. Along these lines, the lymphatic and endothelial vasculatures of the host act as a conduit for the dissemination of MC signals during inflammation. The central role of the MC-endothelial cell axis to immune homeostasis is emphasized by the fact that some of the most effective current treatments for inflammatory disorders are directed at interfering with this interaction.

Description: Background: Diffuse large B cell lymphoma (DLBCL) is a clinically heterogeneous disease, where almost half of patients fail to fully respond to therapy and have poor outcomes. Prognostic molecular markers, including translocations in BCL2, BCL6, and MYC, can help identify patients at high risk for treatment failure. However, these markers rely on diagnostic tumor samples which must be obtained invasively and are not universally available. Circulating tumor-specific DNA (ctDNA) could provide a source of material for detection of these translocations directly from the blood. We sought to examine the performance for detection of these translocations from ctDNA, as well as their association with eventual clinical outcomes. Methods: We profiled tumor and plasma samples from patients with DLBCL receiving combination immunochemotherapy at Stanford University and MD Anderson Cancer Center. As the clinical gold standard, tumor samples were assessed with break-apart fluorescence in situ hybridization (FISH) to detect breaks in BCL2, BCL6, and MYC. DNA from tumor and plasma samples was then sequenced using CAPP-Seq, a targeted next-generation sequencing based method for detection of ctDNA (Newman AM et al, Nature Medicine 2014). Translocations were identified from paired-end reads using FACTERA (Newman AM et al, Bioinformatics 2014). We compared the technical performance of our sequencing-based approach to FISH from tumor samples. Finally, we assessed the prognostic value of our method in relation to clinical outcomes. Results: We prospectively enrolled 86 patients with DLBCL as two cohorts, either at Stanford University (n=40) or at MD Anderson Cancer Center (n=46). We first profiled tumor samples from 40 patients (Stanford cohort) for translocations using FISH as part of the clinical standard of care, identifying 24 translocations in total (12 in BCL2, 7 in BCL6, and 5 in MYC). All patients had tumor and plasma available for sequencing. By analyzing DNA from tumor samples with CAPP-Seq, we identified 92% of all translocations previously identified by FISH (11/12 BCL2, 7/7 BCL6, 4/5 MYC). Furthermore, without prior knowledge of tumor genotypes, we were able to identify 79% (19/24) translocations directly from the plasma (10/12 BCL2, 4/7 BCL6, 5/5 MYC). The performance of genotyping translocations from ctDNA improved with increasing tumor burden, such that 95% (19/20) of translocations were detected in samples with 〉16pg/mL (e.g. 5 genome equivalents/mL) of ctDNA. We next validated the performance of noninvasive genotyping for translocations in an independent cohort of 46 pretreatment plasma samples (MD Anderson Cancer Center cohort). In patients with detectable ctDNA, we successfully identified 78% (18/23) of translocations previously seen by FISH. Furthermore, in two patients, we detected translocations previously missed by FISH from FFPE tissue specimens (BCL6-IGH and MYC-IGH). In addition to known translocation partners, we observed several novel partner genes for MYC (e.g. PTMA and 7p15.2) and BCL6 (e.g. IRF1). Importantly, across both cohorts, we identified 82% (9/11) of patients with both MYC and BCL2/BCL6 translocations (double-hit lymphoma, DHL) directly from the plasma without knowledge of the tumor. Moreover, DHL detected noninvasively significantly predicted both progression-free and overall survival (p

Description: Background: Molecular characterization of tumors currently relies in large part on invasive tissue biopsies, which can often be unavailable or limiting. For example, while biological heterogeneity between diffuse large B-cell lymphoma (DLBCL) patients can be stratified using classification of cell-of-origin (COO), current methods relying on tumor gene expression profiles (GEP) or multi-parametric immunohistochemistry (IHC) require adequate tissue, and can be hampered by modest accuracy and reproducibility. Circulating tumor DNA (ctDNA) is an emerging noninvasive biomarker for tumor detection with potential for DLBCL disease monitoring. Here, we evaluated the utility ofctDNA for COO classification of patients with DLBCL. Methods: We previously built and trained a novel DLBCL COO Naive Bayesian classifier, using somatic alterations in 32 genes and associated with COO subtypes that were previously classified by Affymetrix mRNA GEP of frozen tumors (n=142 Training; Scherer et al., ASH Annual Meeting 2015 / ASCO Annual Meeting 2016). Here, we applied CAPP-Seq, a capture-based targeted high-throughput sequencing (HTS) method (Newman et al., Nat Med 2014), to genetically profile and classify 146 DLBCL patients in independent cohorts. We employed a test/validation framework (n=76 Test, n=70 Validation) to classify patients diagnosed and treated at Stanford University, MD Anderson Cancer Center, or at the University of Eastern Piedmont, Italy. We compared COO classification from pre-treatment plasma samples (n=119), tumor biopsy tissues (n=82, of which 71% were from FFPE), or both (n=41) against the current clinical standard immunohistochemistry method (Hans algorithm, n=117). Kaplan Meier analyses and Cox proportional-hazard models were used to assess clinical utility of biopsy-free and tumor genotyping for prediction of clinical outcomes from time of DLBCL diagnosis. Results: Genotyping of both test and validation cohorts using either tumor or plasma samples allowed COO classification in 100% of patients (GCB=77 / non-GCB=69) with 77% overall concordance to blinded and centralized Hans IHC classification. As expected, histologically transformed (tFL/DLBCL) and double-hit DLBCLs (MYC and BCL2/BCL6 translocated) were strongly biased toward GCB DNA subtype (26/29, 90%), while primary central nervous system lymphomas were almost invariably classified as ABC/non-GCB by ctDNA (6/7, 87%). The concordance between DNA-based COO classification from tumor vs. plasma was 88% (36/41). In the test cohort, COO classification by genotyping from either tissue or plasma was significantly associated with PFS (P=0.003 and 0.02, respectively, Cox proportional-hazard), while classification by Hans criteria failed to show a significant survival difference (P=0.25). The superior outcome of patients with GCB DNA COO subtype was validated when extended to the full cohort (P=0.02, Cox proportional-hazard; P=0.03, log-rank (Figure 1)). We observed a similar, albeit not significant, trend for overall survival. Conclusions: DLBCL COO subtypes can be accurately classified using somatic alterations detectable as circulating tumor DNA. We envision that this approach might help overcome some of the limitations of mRNA GEP and protein/IHC-based methods, including the requirement for invasive biopsies, tissue availability, and suboptimal assay performance. Furthermore, this strategy could support future clinical trials in helping to identify poor-risk groups at diagnosis, to guide future therapy selection, and to improve treatment decisions for patients with DLBCL. Figure 1. Figure 1. Disclosures Newman: Roche: Consultancy. Lovejoy:Roche: Employment. Levy:Kite Pharma: Consultancy; Five Prime Therapeutics: Consultancy; Innate Pharma: Consultancy; Beigene: Consultancy; Corvus: Consultancy; Dynavax: Research Funding; Pharmacyclics: Research Funding. Gaidano:Janssen: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Speakers Bureau; Morphosys: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Speakers Bureau. Diehn:Roche: Consultancy; Novartis: Consultancy; Quanticel Pharmaceuticals: Consultancy; Varian Medical Systems: Research Funding. Alizadeh:Gilead: Consultancy; Celgene: Consultancy; Genentech: Consultancy; Roche: Consultancy.

Description: Abstract 584 The Human Germinal center Associated Lymphoma (HGAL) gene is exclusively expressed in germinal center (GC) B-lymphocytes and GC-derived lymphomas. In patients with diffuse large B-cell lymphomas (DLBCL), HGAL expression identifies a subgroup of patients with biologically distinct tumors associated with improved survival. Our previous in vitro studies demonstrated that HGAL decreases spontaneous and chemoattractant-induced cell motility by activating the RhoA signaling pathway and by directly interacting and augmenting F-actin and myosin II binding. However, the major function of HGAL in GC lymphocytes remains largely unknown. Based on our previous observation of tyrosine phosphorylation of a modified ITAM motif in the HGAL by Lyn, we hypothesized that HGAL may be involved in B-cell receptor (BCR) signaling. Indeed, following BCR stimulation of two GCB-like lymphoma cell lines (Raji and VAL), we observed marked reduction of Syk, Btk and PLCγ phosphorylation upon knockdown of endogenous HGAL by specific but not control siRNAs. Concordantly, HGAL knockdown in BCR-stimulated Raji cells reduced Ca2+ mobilization and decreased NFAT transcriptional activity as analyzed by a luciferase reporter assay. HGAL expression in the BCR-stimulated HBL1 lymphoma cell line (lacking endogenous HGAL protein) resulted in increased Syk, Btk and PLCγ phosphorylation. Syk plays a major role in coupling BCR activation to downstream effectors. Endogenous HGAL was detected in immunoprecipitates of endogenous Syk and vice versa. Nanoscope microscopy studies confirmed co-localization of HGAL and Syk proteins in cell membranes, which was enhanced following BCR stimulation. In BCR-stimulated cells, Syk kinase activity was markedly increased following addition of HGAL protein as measured by an in vitro Syk kinase activity assay. To comprehensively examine HGAL effects on immune system and BCR signaling, we generated a transgenic mouse model in which HGAL is expressed under the control of the mouse Ly-6E.1 promoter in Sca1+ hematopoietic stem cells and progenitors of C57BL/6 × CBA mice. The Sca1-HGAL transgenic mice showed normal embryonic and post natal development, and at 8 weeks of age demonstrated normal lymphoid development without any significant changes in the major hematopoietic compartments (bone marrow (BM), spleen, thymus and peripheral lymph nodes) and in peripheral blood. They also exhibited normal GC development in response to a T-cell dependent antigen immunization. In contrast, at 12 months of age the Sca1-HGAL mice developed a decrease in BM immature B-cells at the expense of recirculating B-cells (B220+IgDhi) compared to the age-matched normal littermates, suggesting a defect in B-cell lymphopoiesis. All the Sca1-HGAL transgenic mice became ill from approximately 12 months of age and all died between 12 to 22 months of age with statistically shorter survival as compared to the wild type controls. Analysis of these animals showed massive splenomegaly with marked white pulp hyperplasia and presence of multiple, frequently contiguous nodules predominantly composed of polyclonal follicular (B220+CD21intCD23hi) B lymphocytes. Extra-lymphatic infiltration by similar B lymphocytes was observed in the liver, lungs and kidneys of Sca1-HGAL mice with advanced disease. IgG isotype titers in these animals tended to be higher than in the wild-type controls, reaching a statistically significant difference for the IgG1 isotype. Follicular hyperplasia in the Sca1-HGAL transgenic mice is likely attributable to increased RhoA activation and enhanced BCR signalling manifested by increased Syk phosphorylation, Ca2+ mobilization and in vitro B cell proliferation following BCR stimulation, in agreement with similar data observed in human DLBCL cell lines expressing HGAL. Gene expression profiling of lymphoid tissues confirmed significantly enhanced BCR signalling and RhoA pathway activation in Sca1-HGAL transgenic mice, corresponding to similar pathway activation in human lymphoma cell lines over-expressing HGAL. Overall, our findings demonstrate that HGAL, specifically expressed in GC B cells, enhances responsiveness to antigens by stimulating Syk kinase activity that without appropriate regulation may lead to lymphoproliferation. Further studies are needed to examine the role of HGAL in the pathogenesis of GC-derived lymphomas. Disclosures: No relevant conflicts of interest to declare.