Citations

Abstract BACKGROUND Monocytes and macrophages (mo/mΦ) are part of composition of tumor microenvironment in B-cell non-Hodgkin lymphoma (B-NHL). CD14+HLA-DRlow monocytes are immunosuppressive and this phenotype is promoted by increased expression of IL-10 in lymphoma. Despite their association with poor outcome, monocytes retain phagocytic function particularly in the presence of monoclonal antibodies and can be associated with an improved outcome in patients treated with rituximab. The aim of this study was to determine the prevalence of CD14+HLA-DRlow mo/mΦ in B-NHL tissue compared to normal tissue, and to determine whether their phenotype suggests that they are capable of phagocytic function. METHODS Tissue mo/mΦ were isolated by negative selection from 13 diagnostic B-NHL biopsy specimens and 6 normal tissues using a monocyte enrichment kit. Morphological and immunophenotypic characteristics of isolated mo/mΦ were determined by flow cytometry, immunocytochemistry and cytometry by time-of-flight (CyTOF). The purity of isolated tissue mo/mΦ was more than 95%, being confirmed by Giemsa stain and flow cytometry. RESULTS Increased numbers of CD68+ cells in initial B-NHL biopsies when compared to normal tissue was confirmed by immunohistochemistry and flow cytometry. However, CD14 expression was substantially lower than CD68 expression in the tissues suggesting that many CD68+ mo/mΦ are CD14 negative. Using a monocyte enrichment kit to isolate all mo/mΦ, we found that CD14 negative mo/mΦ constituted half the tissue mo/mΦ. Furthermore, we found by flow cytometry, CyTOF and Giemsa stain that CD14- mo/mΦ constituted 2 populations: a more frequent population of larger cells and a less common population of smaller cells. While all cells expressed CD68, the larger cells had increased expression of CD32, CD64 and HLA-DR. The CD14+ cells typically also expressed CD163 and CD33 and were a subset of the larger monocyte population, while the population of small cells was positive only for CD68 and CD45. Using CD14 and SIRP-α, we could identify 3 populations of mo/mΦ: CD14+SIRP-αhigh, CD14-SIRP-αlow and CD14-SIRP-α- cells. CD14+SIRP-αhigh cells and CD14-SIRP-αlow cells typically constitute the population of larger cells, while CD14-SIRP-α- cells constituted the population of smaller cells. Interestingly, while the CD14-SIRP-α- cells lack the typical phenotypic markers of mo/mΦ, they morphologically have the appearance of monocytic cells and this population appears expanded in lymphoma tissue. CONCLUSIONS We have identified a unique population of small mo/mΦ that have an immature phenotype and lack expression of CD14, SIRP-α, CD163, and other FcγR markers. This subset of mo/mΦ is prevalent in lymphoma and may have limited phagocytic function. This CD68+CD14-SIRP-α- mo/mΦ subpopulation may account for prognostic differences in the outcome of lymphoma patients treated with or without monoclonal antibodies. Citation Format: Ya-Ping Chen, Zhi-Zhang Yang, Jose C. Villasboas, Tammy Price-Troska, Anne J. Novak, Stephen M. Ansell. Expression of CD14 and SIRP-α defines distinct populations of intratumoral monocytes/macrophages in B-cell non-Hodgkin lymphomas. [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 3287.

AbstractSingle nucleotide polymorphisms (SNPs) in FCγ‐receptor genes FCGR3A (rs396991) and FCGR2A (rs1801274) influence the affinity of the Fc portion of anti‐CD20 immunoglobulin G1 monoclonal antibody. Their roles in diffuse large B‐cell lymphoma (DLBCL) treated with rituximab in combination with anthracycline‐based chemotherapy remain controversial. To address this question, we genotyped FCGR2A and FCGR3A SNPs in two prospective DLBCL cohorts from Lymphoma Study Association trials (N = 554) and Iowa/Mayo Specialized Program Of Research Excellence (N = 580). Correlations with treatment response and hematological toxicity were assessed in Lymphoma Study Association. Correlation with event‐free survival (EFS) and overall survival (OS) was performed in both cohorts, followed by a meta‐analysis to increase power. Our study shows the absence of correlation between these SNPs and treatment response. Grades 3 and 4 febrile neutropenia during treatment was more frequently observed in FCGR3A VV (39%) than VF (29%) and FF (32%) carriers (p = 0.04). Our analysis for EFS and OS shows that FCGR3A was not associated with outcome. In a meta‐analysis using an ordinal model, FCGR2A (per R allele) was associated with a better EFS (hazard ratio = 0.87; 95%CI, 0.76–0.99; p = 0.04) and OS (hazard ratio = 0.86; 95%CI, 0.73–1.00; p = 0.05) which was not altered after adjustment for the International Prognostic Index. Overall, our data demonstrate that patients with DLBCL with the low‐affinity FCγRIIA RR had an unexpectedly better outcome than FCγRIIA H carriers. Whether rituximab efficacy is improved in FCγRIIA RR patients due a clearance reduction or other functions of FCγRIIA in DLBCL should be investigated (clinicaltrials.gov identifiers: NCT00135499, NTC00135499 NCT00140595, NCT00144807, NCT00144755, NCT01087424, and NCT00301821). Copyright © 2016 John Wiley & Sons, Ltd.

Abstract Background: Rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) therapy is standard of care for patients with newly diagnosed DLBCL. A majority of patients respond to R-CHOP, however ∼9% will have primary refractory disease and outcomes in these patients are extremely poor. While there is some knowledge of the pharmacogenomic (PGx) determinants of individual agents within R-CHOP, little is known about the PGx determinants of combination therapy. Our study aims to elucidate germline PGx determinants of drug bioavailability and undesirable side chain reactions in DLBCL patients treated with R-CHOP having potential to influence refractory disease. Methods: Newly diagnosed, DLBCL patients treated with R-CHOP between 2002 and 2012 with germline DNA and genotyping data were selected from the Molecular Epidemiology Resource within the Iowa/Mayo Lymphoma SPORE. Refractory disease was defined as either i) a best response of stable or progressive disease or ii) progressive disease by the end of the planned number of cycles of R-CHOP. PGx candidates within known pharmacokinetics and pharmacodynamics (PD) pathways for 11 genes (1,040 single nucleotide polymorphisms (SNPs)) were selected for study (CPT 2012;92(4):414-7). Odds ratios (ORs) and 95% confidence intervals (CI) were used to estimate the association of SNPs with refractory disease, controlling for age, gender, and age-adjusted International Prognostic Index. Results: The median age at diagnosis of 424 of DLBCL patients treated with R-CHOP was 62 years (range 18-92); 45.7% were female; and 58.4% had Ann Arbor Stage III-IV. By the end of initial therapy 40 patients had primary refractory disease. Of the 1,040 SNPs evaluated, 120 (12%) were significantly associated with refractory disease at the 5% significance threshold suggesting novel associations with drug metabolism. The top SNP from each PGx candidate gene are shown in Table, with ALDH5A1 and CYP2B6 showing the most significant associations. Conclusions: While needing replication, we identified associations within two PGx candidate genes that are potentially functionally related to cyclophosphamide PD: alleles within CYP2B6 may have relevance in R-CHOP outcomes due to potential competition with other drug metabolizing enzymes shared with prednisone and vincristine biotransformation in the liver, and alleles withinALDH5A1 may impact a deactivating side chain reaction in B lymphocytes. Table: R-CHOP PGx candidates with known side chain reactionsPGx candidaten SNPsTop SNPChrOR* (95% CI)P-valueMAFABCB1365rs3480093571.86 (1.14 – 3.04)1.32E-020.29ADH1A32rs122996841.66 (0.60 - 4.53)3.27E-010.038ALDH3A116rs2228100171.26 (0.71 - 2.22)4.28E-010.24ALDH5A1192rs946103466.30 (2.43 - 16.4)1.55E-040.030CBR110rs2835266212.10 (0.90 - 4.89)8.76E-020.052CBR356rs45527331212.12 (0.91 - 4.98)8.29E-020.050CYP2B6107rs1038376192.38 (1.44 - 3.94)7.74E-040.27CYP3A421rs2898858372.32 (0.80 - 6.78)1.23E-010.028CYP3A531rs778032871.69 (0.68 - 4.16)2.57E-010.054SLC22A16193rs11217403561.80 (1.06 - 3.03)2.78E-020.21SOD116rs1041740211.35 (0.83 - 2.19)2.26E-010.32Chr=chromosome, MAF=minor allele frequency, *Adjusted for age, sex, and age-adjusted International Prognostic Index Citation Format: Matthew K. Breitenstein, Susan L. Slager, Anne J. Novak, Matthew J. Maurer, Carrie A. Thompson, Umar Farooq, Brian K. Link, Thomas M. Habermann, Curtis L. Olswold, Andrew L. Feldman, Grzegorz S. Nowakowski, Stephen M. Ansell, Thomas E. Witzig, Richard M. Weinshilboum, Liewei Wang, James R. Cerhan. Pharmacogenomic of refractory response to R-CHOP therapy in diffuse large B-cell lymphoma (DLBCL) patients. [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 B143.

Abstract Background: Excess adiposity has been associated with lymphomagenesis, possibly mediated by increased cytokine production causing a chronic inflammatory state. The relationship between obesity, cytokine polymorphisms, and selected mature B-cell neoplasms is reported. Method: Data on 4,979 cases and 4,752 controls from nine American/European studies from the InterLymph consortium (1988–2008) were pooled. For diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), joint associations of body mass index (from self-reported height and weight) and 12 polymorphisms in cytokines IL1A (rs1800587), IL1B (rs16944, rs1143627), IL1RN (rs454078), IL2 (rs2069762), IL6 (rs1800795, rs1800797), IL10 (rs1800890, rs1800896), TNF (rs1800629), LTA (rs909253), and CARD15 (rs2066847) were investigated using unconditional logistic regression. BMI-polymorphism interaction effects were estimated using the relative excess risk due to interaction (RERI). Results: Obesity (BMI ≥ 30 kg/m2) was associated with DLBCL risk [OR = 1.33; 95% confidence interval (CI), 1.02–1.73], as was TNF-308GA+AA (OR = 1.24; 95% CI, 1.07–1.44). Together, being obese and TNF-308GA+AA increased DLBCL risk almost 2-fold relative to those of normal weight and TNF-308GG (OR = 1.93; 95% CI, 1.27–2.94), with a RERI of 0.41 (95% CI, −0.05–0.84; Pinteraction = 0.13). For FL and CLL/SLL, no associations with obesity or TNF-308GA+AA, either singly or jointly, were observed. No evidence of interactions between obesity and the other polymorphisms were detected. Conclusions: Our results suggest that cytokine polymorphisms do not generally interact with BMI to increase lymphoma risk but obesity and TNF-308GA+AA may interact to increase DLBCL risk. Impact: Studies using better measures of adiposity are needed to further investigate the interactions between obesity and TNF-308G>A in the pathogenesis of lymphoma. Cancer Epidemiol Biomarkers Prev; 24(7); 1061–70. ©2015 AACR.

Abstract Objectives and Rationale: Structural variants (SV) including large copy number aberrations (CNV), translocations, inversions, and large insertions and deletions (INDEL) play a critical role in tumorigenesis and progression. In fact, we now know that tumors can be categorized according to the size of mutations harbored. In several cancers, including ovarian and breast cancer, the large structural mutations, rather than single site mutations, play a dominate role in tumor etiology. Therefore, it's critical to implement reliable algorithm for the detection of structural variants in DNA sequencing data. Mate-pair sequencing is a protocol specifically implemented for detection of the whole-genome level structural variants. It requires less sequencing depth therefor is cost effective, and enables the detection of CNVs, translocations, and inversions simultaneously. However, so far there has been no reliable bioinformatics pipeline for the analyses of the mate-pair sequencing data. Methods: Our novel algorithm, the SnowShoes-SV, is an exhaustive search algorithm designed specifically for mate-pair DNA sequencing data analyses. It calls the SVs based on disconcordant read pairs. The false SVs are filtered according to the following criteria: (i) the number of the supporting read pairs; (ii) the lack of reads from control data that implicate SV at the same region; (iii) the mappability and uniqueness of the region based on data from the ENCODE project; (iv) consistencies of the mapping orientations of the supporting read pairs; (v) the similar sizes between sequencing library and the two end read clusters. Results: Using a set of samples previously genotyped by aCGH, the SnowShoes-SV successfully detected all known CNVs and other SVs. It also identified copy number neutral translocations and inversions previously not identified by aCGH. In addition, the algorithm nominated novel SVs which are to be validated by PCR. Conclusions: SnowShoes-SV is a highly sensitive and specific algorithm for SV detection from the mate-pair DNA sequencing data. Citation Format: Yan W Asmann, Chen Wang, Brian M Necela, Xianfeng Chen, Jean-Pierre A Kocher, Matthew J Maurer, Thomas M Habermann, Susan L Slager, Andrew L Feldman, Anne J Novak, James R Cerhan, Edith A Perez, E Aubrey Thompson. An exhaustive algorithm for detecting copy number aberrations and large structural variants in whole-genome mate-pair sequencing data [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-07-03.

SummaryDeubiquitinase enzymes (DUBs) of the proteasomal 19S regulatory particle are emerging as important therapeutic targets in several malignancies. Here we demonstrate that inhibition of two proteasome‐associated DUBs (USP14 and UCHL5) with the small molecule DUB inhibitor b‐AP15, results in apoptosis of human Waldenström macroglobulinaemia (WM) cell lines and primary patient‐derived WM tumour cells. Importantly, b‐AP15 produced proteotoxic stress and apoptosis in WM cells that have acquired resistance to the proteasome inhibitor bortezomib. In silico modelling identified protein residues that were critical for the binding of b‐AP15 with USP14 or UCHL5 and proteasome enzyme activity assays confirmed that b‐AP15 does not affect the proteolytic capabilities of the 20S proteasome β‐subunits. In vitro toxicity from b‐AP15 appeared to result from a build‐up of ubiquitinated proteins and activation of the endoplasmic reticulum stress response in WM cells, an effect that also disrupted the mitochondria. Focused transcriptome profiling of b‐AP15‐treated WM cells revealed modulation of several genes regulating cell stress and NF‐κB signalling, the latter whose protein translocation and downstream target activation was reduced by b‐AP15 in vitro. This is the first report to define the effects and underlying mechanisms associated with inhibition of USP14 and UCHL5 DUB activity in WM tumour cells.

Key Points BCL2 mutations in FL correlate with activation-induced cytidine deaminase expression and frequently alter the amino acid sequence of the protein. Mutations in the BCL2 coding sequence at diagnosis are associated with shortened time to transformation and earlier death due to lymphoma.