Robert Redd

PD-L1 and PD-L2 Genetic Alterations Define Classical Hodgkin Lymphoma and Predict Outcome

PURPOSE Classical Hodgkin lymphomas (cHLs) include small numbers of malignant Reed-Sternberg cells within an extensive but ineffective inflammatory/immune cell infiltrate. In cHL, chromosome 9p24.1/PD-L1/PD-L2 alterations increase the abundance of the PD-1 ligands, PD-L1 and PD-L2, and their further induction through Janus kinase 2-signal transducers and activators of transcription signaling. The unique composition of cHL limits its analysis with high-throughput genomic assays. Therefore, the precise incidence, nature, and prognostic significance of PD-L1/PD-L2 alterations in cHL remain undefined. METHODS We used a fluorescent in situ hybridization assay to evaluate CD274/PD-L1 and PDCD1LG2/PD-L2 alterations in 108 biopsy specimens from patients with newly diagnosed cHL who were treated with the Stanford V regimen and had long-term follow-up. In each case, the frequency and magnitude of 9p24.1 alterations-polysomy, copy gain, and amplification-were determined, and the expression of PD-L1 and PD-L2 was evaluated by immunohistochemistry. We also assessed the association of 9p24.1 alterations with clinical parameters, which included stage (early stage I/II favorable risk, early stage unfavorable risk, advanced stage [AS] III/IV) and progression-free survival (PFS). RESULTS Ninety-seven percent of all evaluated cHLs had concordant alterations of the PD-L1 and PD-L2 loci (polysomy, 5% [five of 108]; copy gain, 56% [61 of 108]; amplification, 36% [39 of 108]). There was an association between PD-L1 protein expression and relative genetic alterations in this series. PFS was significantly shorter for patients with 9p24.1 amplification, and the incidence of 9p24.1 amplification was increased in patients with AS cHL. CONCLUSION PD-L1/PD-L2 alterations are a defining feature of cHL. Amplification of 9p24.1 is more common in patients with AS disease and associated with shorter PFS in this series. Further analyses of 9p24.1 alterations in patients treated with standard cHL induction regimens or checkpoint blockade are warranted.

Prognostic Mutations in Myelodysplastic Syndrome after Stem-Cell Transplantation

BACKGROUND Genetic mutations drive the pathogenesis of the myelodysplastic syndrome (MDS) and are closely associated with clinical phenotype. Therefore, genetic mutations may predict clinical outcomes after allogeneic hematopoietic stem‐cell transplantation. METHODS We performed targeted mutational analysis on samples obtained before transplantation from 1514 patients with MDS who were enrolled in the Center for International Blood and Marrow Transplant Research Repository between 2005 and 2014. We evaluated the association of mutations with transplantation outcomes, including overall survival, relapse, and death without relapse. RESULTS TP53 mutations were present in 19% of the patients and were associated with shorter survival and a shorter time to relapse than was the absence of TP53 mutations, after adjustment for significant clinical variables (P<0.001 for both comparisons). Among patients 40 years of age or older who did not have TP53 mutations, the presence of RAS pathway mutations was associated with shorter survival than was the absence of RAS pathway mutations (P=0.004), owing to a high risk of relapse, and the presence of JAK2 mutations was associated with shorter survival than was the absence of JAK2 mutations (P=0.001), owing to a high risk of death without relapse. The adverse prognostic effect of TP53 mutations was similar in patients who received reduced‐intensity conditioning regimens and those who received myeloablative conditioning regimens. By contrast, the adverse effect of RAS pathway mutations on the risk of relapse, as compared with the absence of RAS pathway mutations, was evident only with reduced‐intensity conditioning (P<0.001). In young adults, 4% of the patients had compound heterozygous mutations in the Shwachman–Diamond syndrome–associated SBDS gene with concurrent TP53 mutations and a poor prognosis. Mutations in the p53 regulator PPM1D were more common among patients with therapy‐related MDS than those with primary MDS (15% vs. 3%, P<0.001). CONCLUSIONS Genetic profiling revealed that molecular subgroups of patients undergoing allogeneic hematopoietic stem‐cell transplantation for MDS may inform prognostic stratification and the selection of conditioning regimen. (Funded by the Edward P. Evans Foundation and others.)

Classical Hodgkin Lymphoma with Reduced β2M/MHC Class I Expression Is Associated with Inferior Outcome Independent of 9p24.1 Status.

Analysis of Hodgkin lymphomas revealed frequent reduction/loss of antigen-presentation proteins and 9p24.1/PD-L1/PD-L2 alterations. These immune evasion mechanisms had independent prognostic value after frontline therapy and prompt speculation regarding alternative mechanisms of action of PD-1 blockade. In classical Hodgkin lymphoma (cHL), malignant Hodgkin Reed–Sternberg (HRS) cells evade antitumor immunity by multiple mechanisms, including perturbed antigen presentation and enhanced PD-1 signaling. HRS cell expression of the PD-1 ligands is attributable, in part, to copy number alterations of 9p24.1/CD274(PD-L1)/PDCD1LG2(PD-L2). Amplification of PD-L1/PD-L2 is associated with advanced clinical stage and inferior progression-free survival (PFS) following first-line (induction) therapy. The relationships between altered expression of β2-microglobulin (β2M), MHC class I, and MHC class II by HRS cells, PD-L1/PD-L2 amplification, and clinical outcome in cHL are poorly defined. We assessed these variables in diagnostic biopsy specimens from 108 patients with cHL who received uniform treatment and had long-term follow-up and found decreased/absent expression of β2M/MHC class I in 79% (85/108) and decreased/absent expression of MHC class II in 67% (72/108) of cases. Patients with decreased/absent β2M/MHC class I had shorter PFS, independent of PD-L1/PD-L2 amplification and advanced stage. Decreased or absent MHC class II was unrelated to outcome. These results suggest that MHC class I–mediated antigen presentation by HRS cells is an important component of the biological response to standard chemo/radiotherapy. The paucity of β2M/MHC class I expression on HRS cells also prompts speculation regarding alternative mechanisms of action of PD-1 blockade in cHL. Cancer Immunol Res; 4(11); 910–6. ©2016 AACR.

Topological analysis reveals a PD-L1 associated microenvironmental niche for Reed-Sternberg cells in Hodgkin lymphoma

Signaling between programmed cell death protein 1 (PD-1) and the PD-1 ligands (PD-L1, PD-L2) is essential for malignant Hodgkin Reed-Sternberg (HRS) cells to evade antitumor immunity in classical Hodgkin lymphoma (cHL). Copy number alterations of 9p24.1/CD274(PD-L1)/PDCD1LG2(PD-L2) contribute to robust PD-L1 and PD-L2 expression by HRS cells. PD-L1 is also expressed by nonmalignant tumor-associated macrophages (TAMs), but the relationships among PD-L1+ HRS cells, PD-L1+ TAMs, and PD-1+ T cells remain undefined. We used multiplex immunofluorescence and digital image analysis to examine the topography of PD-L1+ and PD-1+ cells in the tumor microenvironment (TME) of cHL. We find that the majority of PD-L1 in the TME is expressed by the abundant PD-L1+ TAMs, which physically colocalize with PD-L1+ HRS cells in a microenvironmental niche. PD-L1+ TAMs are enriched for contacts with T cells, and PD-L1+ HRS cells are enriched for contacts with CD4+ T cells, a subset of which are PD-1+ Our data define a unique topology of cHL in which PD-L1+ TAMs surround HRS cells and implicate CD4+ T cells as a target of PD-1 blockade.

A phase 2 study of Rituximab-Bendamustine and Rituximab-Cytarabine for transplant-eligible patients with mantle cell lymphoma.

Chemoimmunotherapy followed by autologous stem cell transplantation (ASCT) is a standard therapy for transplant‐eligible patients with newly diagnosed mantle cell lymphoma (MCL). The achievement of complete remission (CR) and minimal residual disease (MRD) negativity are associated with better outcomes. We tested an induction regimen of rituximab/bendamustine followed by rituximab/high‐dose cytarabine (RB/RC). This phase 2 study (NCT01661881) enrolled 23 transplant‐eligible patients aged 42–69, of whom 70% were MCL international prognostic index low‐risk. Patients received three cycles of RB followed by three cycles of RC. The primary endpoint of the trial was the rate of CR after six cycles of therapy, with a rate of 75% considered promising. 96% of patients achieved a CR/unconfirmed CR after treatment, meeting the primary objective. One patient progressed on study, one declined ASCT in CR, and the remaining 21 underwent successful stem cell collection and ASCT. After a median follow‐up of 13 months, the progression‐free survival rate was 96%. Among 15 MRD‐evaluable patients who completed treatment, 93% achieved MRD negativity after RB/RC. In conclusion, RB/RC achieves very high CR and MRD negativity rates in transplant‐eligible patients, with a favourable safety profile. RB/RC warrants further comparative studies, and may become a useful alternative to RCHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone)‐based induction regimens in this patient population.

Phase I/II trial of everolimus in combination with bortezomib and rituximab (RVR) in relapsed/refractory Waldenstrom macroglobulinemia

We examined the combination of the mammalian target of rapamycin inhibitor everolimus with bortezomib and rituximab in patients with relapsed/refractory Waldenstrom macroglobulinemia (WM) in a phase I/II study. All patients received six cycles of the combination of everolimus/rituximab or everolimus/bortezomib/rituximab followed by maintenance with everolimus until progression. Forty-six patients were treated; 98% received prior rituximab and 57% received prior bortezomib. No dose-limiting toxicities were observed in the phase I. The most common treatment-related toxicities of all grades were fatigue (63%), anemia (54%), leucopenia (52%), neutropenia (48%) and diarrhea (43%). Thirty-six (78%) of the 46 patients received full dose therapy (FDT) of the three drugs. Of these 36, 2 (6%) had complete response (90% confidence interval (CI): 1–16). In all, 32/36 (89%) of patients experienced at least a minimal response (90% CI: 76–96%). The observed partial response or better response rate was 19/36 (53, 90 CI: 38–67%). For the 36 FDT patients, the median progression-free survival was 21 months (95% CI: 12–not estimable). In summary, this study demonstrates that the combination of everolimus, bortezomib and rituximab is well tolerated and achieved 89% response rate even in patients previously treated, making it a possible model of non-chemotherapeutic-based combination therapy in WM.

Major Histocompatibility Complex Class II and Programmed Death Ligand 1 Expression Predict Outcome After Programmed Death 1 Blockade in Classic Hodgkin Lymphoma.

Purpose Hodgkin Reed-Sternberg (HRS) cells evade antitumor immunity by multiple means, including gains of 9p24.1/ CD274(PD-L1)/ PDCD1LG2(PD-L2) and perturbed antigen presentation. Programmed death 1 (PD-1) receptor blockade is active in classic Hodgkin lymphoma (cHL) despite reported deficiencies of major histocompatibility complex (MHC) class I expression on HRS cells. Herein, we assess bases of sensitivity to PD-1 blockade in patients with relapsed/refractory cHL who were treated with nivolumab (anti-PD-1) in the CheckMate 205 trial. Methods HRS cells from archival tumor biopsies were evaluated for 9p24.1 alterations by fluorescence in situ hybridization and for expression of PD ligand 1 (PD-L1) and the antigen presentation pathway components-β2-microglobulin, MHC class I, and MHC class II-by immunohistochemistry. These parameters were correlated with clinical responses and progression-free survival (PFS) after PD-1 blockade. Results Patients with higher-level 9p24.1 copy gain and increased PD-L1 expression on HRS cells had superior PFS. HRS cell expression of β2-microglobulin/MHC class I was not predictive for complete remission or PFS after nivolumab therapy. In contrast, HRS cell expression of MHC class II was predictive for complete remission. In patients with a > 12-month interval between myeloablative autologous stem-cell transplantation and nivolumab therapy, HRS cell expression of MHC class II was associated with prolonged PFS. Conclusion Genetically driven PD-L1 expression and MHC class II positivity on HRS cells are potential predictors of favorable outcome after PD-1 blockade. In cHL, clinical responses to nivolumab were not dependent on HRS cell expression of MHC class I.

Molecular subtypes of diffuse large B cell lymphoma are associated with distinct pathogenic mechanisms and outcomes

Diffuse large B cell lymphoma (DLBCL), the most common lymphoid malignancy in adults, is a clinically and genetically heterogeneous disease that is further classified into transcriptionally defined activated B cell (ABC) and germinal center B cell (GCB) subtypes. We carried out a comprehensive genetic analysis of 304 primary DLBCLs and identified low-frequency alterations, captured recurrent mutations, somatic copy number alterations, and structural variants, and defined coordinate signatures in patients with available outcome data. We integrated these genetic drivers using consensus clustering and identified five robust DLBCL subsets, including a previously unrecognized group of low-risk ABC-DLBCLs of extrafollicular/marginal zone origin; two distinct subsets of GCB-DLBCLs with different outcomes and targetable alterations; and an ABC/GCB-independent group with biallelic inactivation of TP53, CDKN2A loss, and associated genomic instability. The genetic features of the newly characterized subsets, their mutational signatures, and the temporal ordering of identified alterations provide new insights into DLBCL pathogenesis. The coordinate genetic signatures also predict outcome independent of the clinical International Prognostic Index and suggest new combination treatment strategies. More broadly, our results provide a roadmap for an actionable DLBCL classification.Comprehensive integration of mutational and structural alterations in clinically-annotated DLBCL patient samples provides a novel molecular classification of the disease.

Extramedullary Waldenström macroglobulinemia.

Disease assessment in Waldenstrom Macroglobulinemia (WM) is dependent on the percent involvement of B‐cell neoplasm in the bone marrow and IgM paraprotein in the serum. A subset of patients also demonstrates extramedullary involvement, which is infrequently examined. The role of extramedullary involvement in the diagnosis and prognosis of WM is poorly understood. The purpose of this study is to report the characteristics of WM patients with extramedullary disease (EMD). Nine hundred and eight‐five patients with WM were evaluated at one academic center and the presence of EMD was assessed in these patients. Forty‐three (4.4%) patients were identified to have EMD. Nine (21%) patients presented with involvement at WM diagnosis, while 34 (79%) developed EMD post‐therapy for WM. Most frequent EMD sites involved were pulmonary (30%), soft tissue (21%), cerebrospinal fluid (23%), renal (8%), and bone (9%). The median overall survival at 10 years was 79% (95% CI: 57–90%). This is the first study to describe the clinical characteristics, response and overall survival in patients with extramedullary WM. Further studies to define the molecular characteristics of this entity and mechanisms of its development are warranted. Am. J. Hematol. 90:100–104, 2015.

The immunophenotypic spectrum of primary mediastinal large B-cell lymphoma reveals prognostic biomarkers associated with outcome

Primary mediastinal large B‐cell lymphoma (PMBL) is a distinct subtype of diffuse large B‐cell lymphoma (DLBCL) that shows overlap with classical Hodgkin lymphoma (CHL) and a favorable prognosis compared to mediastinal gray‐zone lymphoma (MGZL). We performed immunohistochemistry on initial diagnostic specimens of 49 cases of uniformly treated PMBL to determine the frequency and clinical significance of expression of antigens commonly seen in CHL and MGZL, along with markers previously shown to be prognostic in DLBCL, not otherwise specified. The median age was 37 years with a female:male ratio of 2.3. After a median follow‐up of 78 months, 24% of patients had relapsed or refractory disease and 22% had died; the 5‐year PFS was 70%. Variable CD15 expression was seen in 31% of cases, but was not associated with adverse outcome. Hans cell‐of‐origin, proliferation index, and MYC/BCL2 coexpression were not associated with outcome, while low PDL1 (P = 0.011) and high MUM1 (P = 0.065) staining were each associated with shorter PFS. A biologic risk score (one point each for low PDL1 and high MUM1) stratified patients into three prognostic risk groups for PFS (P = 0.001) and OS (P = 0.032). On separate multivariate models, low PDL1 was independent of R‐IPI risk group for PFS (HR 6.0, P = 0.023), as was a biologic risk score of 2 (HR 5.6, P = 0.011). Incorporation of the biologic risk score sub‐stratified patients within R‐IPI groups for both PFS (P < 0.001) and OS (P < 0.001). In summary, we characterize the immunophenotypic spectrum of PMBL and identify PDL1 and MUM1 as prognostic biomarkers for high‐risk disease. Am. J. Hematol. 91:E436–E441, 2016.