Ansell, Stephen M.

SummaryIbrutinib‐related data in Waldenström macroglobulinaemia (WM) remain sparse, particularly outside of trials. We report on 80 patients [previously treated, n = 67 (84%), treatment‐naïve, n = 13 (16%)] with WM, evaluated consecutively at Mayo Clinic, who received ibrutinib off‐study after its approval in 2015 for WM. Overall response rate (ORR) was 91%; major‐response rate (MRR) was 78%. The median time to first response and best response was 2·9 [95% confidence interval (CI): 2–4] and 5·7 (95% CI: 4–12) months, respectively. The median follow‐up was 19 (95% CI: 14–21) months; 18‐month progression‐free survival (PFS) was 82%. The median time on therapy was 12·5 (95% CI: 9·3–16·7) months, and the median duration‐of‐response was 32 (range: 23–32) months. Twenty‐five patients (31%) had discontinued therapy at last follow‐up (68% due to treatment‐related toxicities) and 18% of patients required dose reduction. Fatigue (12%) and atrial‐fibrillation (11%) were common non‐haematological toxicities. IgM rebound occurred in 36% of patients who abruptly discontinued ibrutinib. Following ibrutinib discontinuation, 84% of patients received subsequent treatment, achieving an ORR of 57% and MRR of 50%. The median PFS from commencement of subsequent salvage therapy was 18 months. Ibrutinib therapy, outside of clinical trials, is effective in WM, but is associated with toxicities and challenges, including IgM rebound and a high drug discontinuation rate for reasons other than disease progression.

AbstractEpidemiologic studies show an increased risk of non‐Hodgkin lymphoma (NHL) in patients with autoimmune disease (AD), due to a combination of shared environmental factors and/or genetic factors, or a causative cascade: chronic inflammation/antigen‐stimulation in one disease leads to another. Here we assess shared genetic risk in genome‐wide‐association‐studies (GWAS).Secondary analysis of GWAS of NHL subtypes (chronic lymphocytic leukemia, diffuse large B‐cell lymphoma, follicular lymphoma, and marginal zone lymphoma) and ADs (rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis). Shared genetic risk was assessed by (a) description of regional genetic of overlap, (b) polygenic risk score (PRS), (c)"diseasome", (d)meta‐analysis.Descriptive analysis revealed few shared genetic factors between each AD and each NHL subtype. The PRS of ADs were not increased in NHL patients (nor vice versa). In the diseasome, NHLs shared more genetic etiology with ADs than solid cancers (p = .0041). A meta‐analysis (combing AD with NHL) implicated genes of apoptosis and telomere length.This GWAS‐based analysis four NHL subtypes and three ADs revealed few weakly‐associated shared loci, explaining little total risk. This suggests common genetic variation, as assessed by GWAS in these sample sizes, may not be the primary explanation for the link between these ADs and NHLs.

Abstract Clinical successes with immune check-point blockers have demonstrated the potency of the immune system in controlling cancers, most strikingly in Hodgkin lymphoma (HL), where overall response rates to PD1/L1 inhibitors approach 90%. Complete or durable responses, however, are uncommon, therefore targeting the PD1/L1 axis alone is not sufficient. Recent work analyzing the spatial arrangement of PD1 and PDL1 expressing cells has given us new insight into the mechanism of action of PD1/L1 inhibitors, however this work limited itself to studying a single check point marker on a subset of cells. We hypothesize that comprehensive profiling of the frequency and spatial arrangement of immune cells in the Hodgkin lymphoma tumor immune microenvironment (TME) will provide new insights into the mechanism of checkpoint blockers and identify novel targets for immune therapy. Until now, multiparameter spatial analysis of the immune microenvironment was limited by technical challenges. Flow and mass cytometry are able to identify immune subsets of interest but spatial information is lost. Multiplex tissue imaging methods are limited to 6-8 simultaneous markers and cannot capture the full complexity of the immune phenotypes. The Fluidigm Hyperion imaging mass cytometry (IMC) system combines a CyTOF mass cytometer with a laser ablation system allowing for 40+ parameter simultaneous immunophenotyping on a single slide of FFPE tissue, with sub-cellular resolution. We have developed a panel of 34 antibodies that allow for comprehensive characterization of CD4, CD8 and myeloid cells components in the TME of Hodgkin lymphoma using IMC. Here we report on our spatial analysis of TIM3 and LAG3 expressing CD4+ lymphocytes. Our data suggests LAG3+CD4+ and TIM3+CD4+ lymphocytes had shorter mean nearest distance to PDL1+Hodgkin Reed-Sternberg (HRS) cells upon comparison to PDL1- HRS cells (t-test, p=1.703e-08,p=1.126e-14). Future studies should explore multiple exhausted marker models that seeks to further understand the presence of simultaneous exhaustion signals in the niche environment. These data suggest that therapies that target TIM3 and/or LAG3 should be tested in Hodgkin Lymphoma and that spatial analysis of immune subsets by IMC should be explored as selective and pharmacodynamic biomarkers. Citation Format: Anthony R. Colombo, Monirath Hav, Erik Gerdtsson, Jose Bisnesto-Villasboas, Stephen Ansell, James Hicks, Peter Kuhn, Akil Merchant. Revisiting immune exhaustion in Hodgkin’s lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1189.

Abstract Ligand–receptor complexes play a central role in mediating a range of processes in immunology and cancer biology. The ability to directly quantify the fraction of receptors occupied by a ligand in a given biospecimen, as opposed to assessing the concentration of ligand and receptor separately, could provide an additional and valuable clinical and research tool for assessing whether receptors are occupied by a ligand. To address this need, a biomarker platform was developed to quantify the fraction of receptors occupied by a ligand using pairs of RNA aptamers, where one aptamer binds preferentially to the unoccupied receptor and the other to the ligand–receptor complex. Bound aptamer was quantified using RT-qPCR colorimetric probes specific for each aptamer. The binding ratio of aptamer correlated with the fraction of receptors occupied by a ligand. This assay, termed as LIRECAP (LIgand–REceptor Complex-binding APtamer) assay, was used to determine the fraction of soluble CD25 occupied by IL2 in the serum from subjects with B-cell lymphoma. No correlation was found between the type of lymphoma and total soluble CD25 or IL2 independently. In contrast, the fraction of soluble CD25 occupied by IL2 was significantly higher in follicular lymphoma patient serum compared with diffuse large B-cell lymphoma patient serum. We conclude that this technology has the potential to serve as a high-throughput biomarker platform to quantify the fraction of receptors occupied by a ligand.

Abstract In this month's CME article, an analysis of over 1300 patients with diffuse large B-cell lymphoma (DLBCL), Wang et al demonstrate that the approximately 13% of patients with concurrent indolent lymphoma at diagnosis had the same prognosis as those patients with isolated DLBCL.

Background:Diffuse Large B Cell Lymphoma (DLBCL) is characterized by both biologic and genetic heterogeneity. While significant therapeutic advances have been made, nearly 30% of cases fail frontline treatment. Recent data suggest that 5–10% of DLBCL can be categorized as double/triple hit lymphoma (DH/TH), defined as having concurrent MYC, BCL2 and/or BCL6 gene rearrangements, identified by fluorescence in‐situ hybridization (FISH) probes. According to the 2017 revision of the World Health Organization classification of lymphoid neoplasms, DH/TH DLBCL is now considered a high grade B‐cell lymphoma (HGBL). While gene rearrangements represent significant genetic events in cancer, copy number alterations (CNAs) also play an important role, often through genomic amplification of oncogenes or loss of tumor suppressors. Despite their similarities, the contributions of CNAs in MYC, BCL2, and BCL6, alone, or in combination with gene rearrangements, are largely unknown in DLBCL and could identify a group of previously uncharacterized patients that may have high expression of MYC, BCL2 and/or BCL6 driven by alternative mechanisms.Aims:The goal of the study is to define the landscape and prognostic significance of gene rearrangements and copy gains in MYC, BCL2, and BCL6 in a cohort of newly diagnosed DLBCL.Methods:A total of 176 cases of DLBCL with both FISH and CNA data were available for the study. FISH was performed on formalin‐fixed paraffin‐embedded sections using break‐apart probes to MYC, BCL2, and BCL6 in the Mayo Clinic Department of Lab Medicine and Pathology. Somatic copy number alterations were identified using whole exome sequencing and the Affymetrix OncoScan® FFPE assay on patient tumor FFPE tissue, and were interrogated using Nexus Copy Number 9.0 software. All patients were treated with immunochemotherapy and prospectively followed for event‐free and overall survival.Results:FISH identified 3 groups of previously described patients, DH/TH or HGBL (n = 11), translocation positive (single MYC, BCL2, BLC6, or double BCL2/BLC6, n = 86), and translocation negative (n = 79). Expanding upon the translocation positive subgroup, copy number analysis identified patients (19 of 86, 22%) with copy number gains in a second or third gene resulting in genomic hits at both MYC and BCL2/6; we classified these as alternative‐double/triple‐hit (A‐DH/TH). An example of this would be a MYC translocation plus a BCL2 and/or BCL6 gain (n = 5), or a BCL2 and/or BCL6 translocation plus a copy gain in MYC (n = 14). Similar interrogation of a publically available dataset (n = 250), revealed similar proportions of these patient groups in an independent cohort of DLBCL, validating our findings. Cell of origin classification of the A‐DH/TH cases revealed that they were 53% GCB. A‐DH/TH patients, in comparison to the translocation‐positive/copy‐gain‐negative cases, were at greater risk of an event (Cox HR = 1.5, 95% CI = 0.71–3.0) and the cases trended towards a worse overall survival (log‐rank P = 0.065; Cox HR = 2.0, 95% CI = 0.94–4.3). Patients with copy‐gains alone had similar outcomes as those with no copy‐gain or translocations, suggesting a central role for at least one translocation event in predicting outcome.Summary/Conclusion:Our data suggest that use of CNA data in combination with traditional FISH can identify a unique subset of DLBCL, A‐DH/TH, that are missed by standard clinical practice. More importantly, we find that these patients do poorly and may benefit from more aggressive treatment, although we do acknowledge that our population size was small and further validation is needed.image

Background:With an expanding array of novel treatment options in follicular lymphoma (FL), we largely lack effective biomarkers to predict response in this molecularly heterogeneous disease. Activating mutations focused on components of the nutrient‐sensing arm of the mTORC1 pathway (RRAGC, ATP6V1B2, ATP6AP1), are particularly enriched in FL, occurring in ∼30% of cases. Meanwhile, clinical mTOR inhibitors (mTORi) have shown some promise, with response rates of 50–60% in relapsed/refractory FL.Aims:We therefore sought to determine whether gene mutation status ‐ particularly in genes regulating the mTOR pathway – may represent biomarkers of mTORi‐response in FL.Methods:We retrospectively analyzed pre‐treatment tumor samples from relapsed/refractory FL patients treated on two phase II clinical trials with single agent mTOR inhibitors, everolimus (NCT00436618) or temsirolimus (NCT00290472), with well characterized clinical outcomes. In total, 21 patients had available biopsies, consisting of 15 everolimus‐ and 6 temsirolimus‐treated cases.Tumor DNA extracted from formalin fixed paraffin embedded (FFPE) tissue was subjected to targeted sequencing using a 22 gene panel comprising genes recurrently mutated in FL, and PI3K/AKT/mTOR pathway genes mutated in germinal center lymphomas. Libraries were prepared using capture‐based target enrichment (Nonacus), and sequenced on the NextSeq550 (Illumina) with a mean coverage depth of x700. A stringent bioinformatic pipeline was adopted with variants called with VarScan2, and a 10% variant allele frequency (VAF) threshold was employed to filter out sequencing artefacts due to the FFPE material. 100% (15/15) of a selection of variants were validated by Sanger sequencing. Staining for pS6 was undertaken on tissue slides to examine downstream mTOR activation by immunohistochemistry.Results:Overall response rate (CR and PR) was 48% (10/21). Mutational analysis showed no significant association between clinical response and mTORC1‐pathway mutations (mut) taken individually or collectively; although, absolute numbers of mutations in these genes were low, including RRAGC (responder: 0/10 mut vs non‐responder: 1/11 mut), ATP6AP1 (responder: 1/10 vs non‐responder: 1/11), and ATP6V1B2 (responder: 1/10 vs non‐responder 3/11). Notably, no difference in pS6 staining was seen between responders and non‐responders.However, we found mutations in the epigenetic regulator CREBBP to be enriched in the responder group (100% (10/10) responders vs 54.5% (6/11) non‐responders, p = 0.04). This effect was more pronounced when considering only mutations within the catalytic histone acetyltransferase (HAT) domain of CREBBP, with HAT mutations seen in 100% (10/10) responders, and 27.3% (3/11) non‐responders (p = 0.001).Summary/Conclusion:Mutations in the mTOR pathway did not predict for response to mTOR inhibition in our study. The enrichment of CREBBP mutations in responders, and particularly those affecting the catalytic HAT domain, points to CREBBP as a potential biomarker of response which warrants validation in trials of drugs modulating the PI3K/AKT/mTOR axis. More broadly our data may lend support to a distinct clinical and biological behavior of CREBBP HAT domain‐mutant disease that also warrants further evaluation. Taken together, our findings highlight the potential opportunities afforded by undertaking correlative studies, and underlines the need to incorporate prospective biomarker discovery into clinical trials in order to help focus future therapeutic strategies more rationally.

Background:Recent large next generation sequencing studies have redefined the genomic landscape of diffuse large B‐cell lymphoma (DLBCL). While many of the more common genetic variants, primarily mutations, have been biologically characterized, little is known about newly emerging genetic aberrations. In particular, somatic copy number alterations remain poorly studied due to the large genomic regions they cover and the number of genes involved. This, in combination with lack of remission or early relapse in a portion of DLBCL, opens new opportunities to explore yet unidentified genetic contributions to lymphomagenesis.Aims:The goal of this study was to identify variants associated with failure to achieve event free survival at 24 months (EFS24) and define their biologic significance in DLBCL.Methods:Newly diagnosed DLBCL treated with standard immunochemotherapy were included in this study (n = 246). Copy number data was generated by using the molecular inversion probe OncoScan™ FFPE Assay Kit (Affymetrix, Santa Clara, CA, USA). OncoScan OSCHP files were analyzed using Nexus Copy Number 9.0 software (Biodiscovery, El Segundo, USA). Data interpretation and copy number calling was done using the human reference genome GRCh37/hg19. Files were analyzed using the Nexus FASST2‐Segmentation algorithm, which is based on a Hidden Markov Model approach for calling genetic events. For functional analysis, SOCS6 was overexpressed and studied in GCB‐DLBCL cell lines.Results:Upon review of genomic copy number data from DLBCL cases (n = 246), loss of 18q22.1‐q23 was a significant event in our cohort (p = 0.018), is found in 11% (n = 27) of DLBCL, and is enriched in germinal center DLBCL (GCB‐DLBCL) patients (18/107, 17%, p < 0.05) compared to activated B‐cell (ABC)/non‐GCB DLBCL patients (1/93, 1%). Furthermore, GCB‐DLBCL patients with a loss of 18q22.1‐q23 were significantly more likely to fail EFS24 (50%, p < 0.05) compared to GCB‐DLBCL patients without the loss (29%). Genes located at 18q22.1‐q23 include, TMX3, DOK6 and SOCS6. SOCS family proteins have been shown to play an important role in tumor biology, although little is known about SOCS6. SOCS6 gene expression is downregulated in a variety of solid tumors and is hypermethylated in some hematological malignancies. RNASeq data from DLBCL suggest variable expression across tumors. Using a panel of GCB cell lines we found that SOCS6 expression was low to negative, and publically available cell line data suggests this region is hypermethylated. Expression of SOCS6 was higher in a panel of mantle cell lymphoma and ABC‐DLBCL cell lines and this again correlated with their cell line methylation data, where no hypermethylation was found in those cell lines. Together, this data suggests that downregulation of SOCS6 through genomic loss or hypermethylation may have biologic importance. To explore the role of SOCS6, we restored expression in GCB cell lines and immuno‐fluorescence staining suggest localization to the nucleus and cytoplasm, as previously reported in other cell types. Studies to explore the role of SOCS6 in cytokine signaling and STAT family protein regulation are ongoing.Summary/Conclusion:Using genome copy number data from 246 DLBCL, we have found that loss of 18q22.1‐q23 (p = 0.018) is associated with GCB‐DLBCL and failure to achieve EFS24. Furthermore, we have identified that SOCS6 may have clinical and biologic significance in GCB‐DLBCL.