Concordant bone marrow involvement of diffuse large B-cell lymphoma represents a distinct clinical and biological entity in the era of immunotherapy.

In diffuse large B-cell lymphoma (DLBCL), the clinical and biological significance of concordant and discordant bone marrow (BM) involvement have not been well investigated. We evaluated 712 de novo DLBCL patients with front-line rituximab-containing treatment, including 263 patients with positive and 449 with negative BM status. Compared with negative BM disease, concordant BM adversely impacted overall and progression-free survival, independent of the International Prognostic Index (IPI) and cell-of-origin classification. Once BM is concordantly involved, poor prognosis was not associated with the extent of BM involvement. Conversely, patients with discordant BM showed favorable overall survival similar to stage I-II DLBCL. A BM-adjusted IPI, using three parameters: concordant BM involvement, age >60 years, and performance status >1, improves the risk stratification for DLBCL with positive BM. Intensive immunochemotherapy seemingly rendered survival benefit for patients with concordant BM, as did rituximab maintenance for the discordant BM group. Frequently revealing adverse clinical and molecular characteristics, patients with concordant BM demonstrated gene expression signatures relevant to tumor cell proliferation, migration and immune escape. In conclusion, clinical and biological heterogeneity is seen in DLBCL with positive BM but concordant BM involvement represents a distinct subset with unfavorable gene signatures, high-risk clinicopathologic features and poor prognosis.

A phase II trial of lenalidomide plus rituximab in previously untreated follicular non-Hodgkin's lymphoma (NHL): CALGB 50803 (Alliance).

BACKGROUND: This multicenter, phase II trial tested the tolerability and efficacy of lenalidomide plus rituximab in patients with previously untreated follicular lymphoma (FL). PATIENTS AND METHODS: Patients with grade 1-3a FL, stage 3-4 or bulky stage 2, FL international prognostic index (FLIPI) 0-2, and no prior therapy were eligible to receive rituximab 375 mg/m2 weekly during cycle 1 and day 1 of cycles 4, 6, 8, and 10, plus lenalidomide 20-25 mg on days 1-21 for twelve 28-day cycles. The primary objectives were to evaluate response rates [complete (CR) and overall] and time to progression. Secondary objectives included toxicity, response according to polymorphisms in FcgR2A and FcgR3A, and changes in circulating pro-angiogenic cells. RESULTS: From October 2010 to September 2011, 66 patients were enrolled. Median age was 53 years, 34 were female, 15 had bulky disease, 21 were FLIPI 0-1, 43 FLIPI 2, and 2 FLIPI 3. One patient withdrew before receiving treatment. Fifty-one patients completed 12 cycles of lenalidomide. Reasons for discontinuation included withdrawal (n = 6), adverse events (n = 6), progression (n = 2). Grade 3-4 hematologic toxicity included neutropenia (21%), lymphopenia (9%), and thrombocytopenia (2%), infection (11%), and rash (8%). Grade 1-2 toxicity included fatigue (78%), diarrhea (37%), rash (32%), and febrile neutropenia in one patient. The overall response rate was 95%; the CR rate was 72% (95% confidence interval, 60% to 83%). With a median follow-up of 5 years, the 2- and 5-year progression-free survival were 86% and 70%, respectively, and the 5-year overall survival was 100%. There was no association between CR rate or PFS and FLIPI, histological grade, bulky disease, FcgR2A/FcgR3A polymorphism, or change in circulating endothelial cell/hematopoietic progenitor cell. CONCLUSION: Lenalidomide plus rituximab was associated with low rates of grade 3-4 toxicity, yielded a CR rate and PFS similar to chemotherapy-based treatment and may represent a reasonable alternative to immunochemotherapy in previously untreated FL. CLINICALTRIALS.GOV IDENTIFIER: NCT01145495.

Transcription factor SPZ1 promotes TWIST-mediated epithelial-mesenchymal transition and oncogenesis in human liver cancer.

The epithelial-mesenchymal transition (EMT) is an important process in the progression of cancer. However, its occurrence and mechanism of regulation are not fully understood. We propose a regulatory pathway in which spermatogenic leucine zipper 1 (SPZ1) promotes EMT through its transactivating ability in increasing TWIST1 expression. We compared the expression of SPZ1 and TWIST1 in specimens of hepatocarcinoma cells (HCCs) and non-HCCs. Expression of SPZ1 exhibited a tumor-specific expression pattern and a high correlation with patients' survival time, tumor size, tumor number and progression stage. Moreover, forced expression and knockdown of SPZ1 in hepatoma cells showed that SPZ1 was able to regulate the cellular proliferation, invasion, and tumorigenic activity in a TWIST1-dependent manner in vitro and in vivo. These data demonstrate that SPZ1, a newly dscribed molecule, transactivates TWIST1 promoters, and that this SPZ1-TWIST axis mediates EMT signaling and exerts significant regulatory effects on tumor oncogenesis.

2016 WHO Classification update-What's new in lymphoid neoplasms.

In the 8 years since the publication of the 4th edition of the WHO Classification of Hematopoietic and Lymphoid Tumours in 2008, there has been rapid accumulation of knowledge in the molecular genetics, biology, clinical behavior of many hematologic malignancies. Concepts and models have advanced to the point at which updates were deemed necessary prior to the formal WHO process of publication of a 5th edition as part of the WHO "blue book" series. This overview will focus on lymphoid tumors and highlight important changes and updates to these neoplasms.

Clinicopathologic and molecular characterization of myeloid neoplasms with isolated t(6;9)(p23;q34).

INTRODUCTION: The t(6;9)(p23;q34);DEK-NUP214 [t(6;9)] abnormality is found in 0.7-1.8% of patients with acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS). FLT3-ITD mutations are detected in t(6;9) patients. The t(6;9) abnormality is associated with poor outcomes. We studied the clinicopathologic and molecular profiles of patients with AML/MDS carrying t(6;9). METHODS: We collected clinical data of nine patients with AML/MDS with isolated t(6;9) (median age = 41 years; male/female = 4/5) and genotyped DNAs using whole exome, Sanger, and targeted sequencing. RESULTS: Our cohort was characterized by frequent multilineage dysplasia (56%), absence of phospho-STAT3/STAT5 expression, presence of myeloid markers (CD13, CD33, CD34, CD117, HLA-DR) with an aberrant expression of CD7, and poor outcome (median survival of 20 months). Although basophilia has been described in association with t(6;9), we observed lack of marrow basophilia in our cohort. Molecularly, 83% (5/6) of patients with AML/MDS with t(6;9) were characterized by at least one somatic mutation. Among them, four patients showed multiple mutations. FLT3-ITD mutations were detected in 33% of patients (2/6); 80% (4/5) of mutant patients died even after hematopoietic stem cell transplantation. CONCLUSION: Our data demonstrated that AML/MDS patients with t(6;9) have diverse molecular mutations regardless of the presence of FLT3 mutations, which may contribute to their poor survival outcomes.

Loss of PRDM1/BLIMP-1 function contributes to poor prognosis of activated B-cell-like diffuse large B-cell lymphoma.

PRDM1/BLIMP-1, a master regulator of plasma-cell differentiation, is frequently inactivated in activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL) patients. Little is known about its genetic aberrations and relevant clinical implications. A large series of patients with de novo DLBCL was effectively evaluated for PRDM1/BLIMP-1 deletion, mutation, and protein expression. BLIMP-1 expression was frequently associated with the ABC phenotype and plasmablastic morphologic subtype of DLBCL, yet 63% of the ABC-DLBCL patients were negative for BLIMP-1 protein expression. In these patients, loss of BLIMP-1 was associated with Myc overexpression and decreased expression of p53 pathway molecules. In addition, homozygous PRDM1 deletions and PRDM1 mutations within exons 1 and 2, which encode for domains crucial for transcriptional repression, were found to show a poor prognostic impact in patients with ABC-DLBCL but not in those with germinal center B-cell-like DLBCL (GCB-DLBCL). Gene expression profiling revealed that loss of PRDM1/BLIMP-1 expression correlated with a decreased plasma-cell differentiation signature and upregulation of genes involved in B-cell receptor signaling and tumor-cell proliferation. In conclusion, these results provide novel clinical and biological insight into the tumor-suppressive role of PRDM1/BLIMP-1 in ABC-DLBCL patients and suggest that loss of PRDM1/BLIMP-1 function contributes to the overall poor prognosis of ABC-DLBCL patients.

Primary testicular diffuse large B-cell lymphoma displays distinct clinical and biological features for treatment failure in rituximab era: a report from the International PTL Consortium.

Primary testicular diffuse large B-cell lymphoma (PT-DLBCL) is a unique subtype of DLBCL. The impact of rituximab on survival and patterns of treatment failure in PT-DLBCL patient remain controversial. We analyzed the clinical and biological feature of 280 PT-DLBCL cases, 64% of which were treated with rituximab-containing regimens. Although most (95%) patients achieved complete remission, a continuous risk of relapse was observed. Rituximab significantly reduced the cumulative risk of relapse (P=0.022) and improved both progression-free survival (PFS, P=0.012) and overall survival (OS, P=0.027) of PT-DLBCL patients (5-year PFS, 56% vs 36%; 5-year OS, 68% vs 48%). Central nervous system and contralateral testis were the most common sites of relapse, but other extranodal and nodal sites of relapse were also observed. Most cases of PT-DLBCL had a non-germinal center B-cell like (84%) immunophenotype and an activated B-cell like (86%) gene expression profile (GEP) subtype. The distinctive GEP signature of primary testicular lymphoma was relevant to tumor cell proliferation, dysregulated expression of adhesion molecules and immune response, likely accounting for the poor outcome. Accordingly, forkhead box P1 transcription factor (FOXP1) and T-cell leukemia/lymphoma 1 (TCL1) oncogenic activation were confirmed and predicted a significant trend of poor survival. This study provides valuable observations for better understanding of both clinical and biological features in PT-DLBCL patients.

Identification of Ezrin-Radixin-Moesin proteins as novel regulators of pathogenic B-cell receptor signaling and tumor growth in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is a hematological cancer associated with an aggressive clinical course. The predominant subtypes of DLBCL display features of chronic or tonic B-cell antigen receptor (BCR) signaling. However, it is not known whether the spatial organization of the BCR contributes to the regulation of pro-survival signaling pathways and cell growth. Here, we show that primary DLBCL tumors and patient-derived DLBCL cell lines contain high levels of phosphorylated Ezrin-Radixin-Moesin (ERM) proteins. The surface BCRs in both activated B cell and germinal B cell subtype DLBCL cells co-segregate with phosphoERM suggesting that the cytoskeletal network may support localized BCR signaling and contribute to pathogenesis. Indeed, ablation of membrane-cytoskeletal linkages by dominant-negative mutants, pharmacological inhibition and knockdown of ERM proteins disrupted cell surface BCR organization, inhibited proximal and distal BCR signaling, and reduced the growth of DLBCL cell lines. In vivo administration of the ezrin inhibitor retarded the growth of DLBCL tumor xenografts, concomitant with reduction in intratumor phosphoERM levels, dampened pro-survival signaling and induction of apoptosis. Our results reveal a novel ERM-based spatial mechanism that is coopted by DLBCL cells to sustain tumor cell growth and survival.

MCL-1 and BCL-xL-dependent resistance to the BCL-2 inhibitor ABT-199 can be overcome by preventing PI3K/AKT/mTOR activation in lymphoid malignancies.

Overexpression of anti-apoptotic BCL-2 family members is a hallmark of many lymphoid malignancies, including chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL) that can be targeted with small molecule inhibitors. ABT-199 is a rationally designed BCL-2 homology (BH)-3 mimetic that specifically binds to BCL-2, but not to MCL-1 and BCL-xL. Although the thrombocytopenia that occurs with navitoclax treatment has not been a problem with ABT-199, clinical trials in CLL could benefit by lowering the ABT-199 concentration through targeting other survival pathways. In this study, we investigated the mechanisms of resistance that develops to ABT-199 therapy by generating ABT-199-resistant (ABT199-R) cell lines via chronic exposure of NHL cell lines to ABT-199. Acquired resistance resulted in substantial AKT activation and upregulation of MCL-1 and BCL-xL levels that sequestered BIM. ABT199-R cells exhibited increased MCL-1 stability and failed to activate BAX in response to ABT-199. The ABT-199 acquired and inherent resistant cells were sensitized to treatment with ABT-199 by inhibitors of the PI3K, AKT, and mTOR pathways, NVP-BEZ235 and GS-1101. NVP-BEZ235, a dual inhibitor of p-AKT and mTOR, reduced MCL-1 levels causing BIM release from MCL-1 and BCL-xL, thus leading to cell death by BAX activation. The PI3Kδ inhibitor GS-1101 (idelalisib) downregulated MCL-1 and sensitized ABT199-R cells through AKT-mediated BAX activation. A genetic approach, through siRNA-mediated down-regulation of AKT, MCL-1, and BCL-xL, significantly decreased cell survival, demonstrating the importance of these cell survival factors for ABT-199 resistance. Our findings suggest a novel mechanism that modulates the expression and activity of pro-survival proteins to confer treatment resistance that could be exploited by a rational combination therapeutic regimen that could be effective for treating lymphoid malignancies.

PRPF8 defects cause missplicing in myeloid malignancies.

Mutations of spliceosome components are common in myeloid neoplasms. One of the affected genes, PRPF8, encodes the most evolutionarily conserved spliceosomal protein. We identified either recurrent somatic PRPF8 mutations or hemizygous deletions in 15/447 and 24/450 cases, respectively. Fifty percent of PRPF8 mutant and del(17p) cases were found in AML and conveyed poor prognosis. PRPF8 defects correlated with increased myeloblasts and ring sideroblasts in cases without SF3B1 mutations. Knockdown of PRPF8 in K562 and CD34+ primary bone marrow cells increased proliferative capacity. Whole-RNA deep sequencing of primary cells from patients with PRPF8 abnormalities demonstrated consistent missplicing defects. In yeast models, homologous mutations introduced into Prp8 abrogated a block experimentally produced in the second step of the RNA splicing process, suggesting that the mutants have defects in proof-reading functions. In sum, the exploration of clinical and functional consequences suggests that PRPF8 is a novel leukemogenic gene in myeloid neoplasms with a distinct phenotype likely manifested through aberrant splicing.

Addition of rituximab to chemotherapy overcomes the negative prognostic impact of cyclin E expression in diffuse large B-cell lymphoma.

BACKGROUND: High levels of cyclin E (CCNE) are accompanied by shorter survival in cyclophosphamide, hydroxydaunorubicin, oncovin and prednisone (CHOP)-treated diffuse large B-cell lymphomas (DLBCL), independent of the international prognostic index (IPI). Data on the prognostic role of CCNE in the 'rituximab (R)-era' are lacking. METHODS: To test reproducibility and applicability of observations from the 'pre-R era' to the 'R era', we examined the prognostic role of CCNE expression by immunohistochemistry in 1579 DLBCL on tissue microarrays (TMA); 339 patients were treated by CHOP and 635 by R-CHOP. RESULTS: 1209 samples (77%) were evaluable; failures were due to missing TMA punches and fixation artefacts. Mean expression of CCNE was 13% (0-85%); applying a cut-off of >16%, 382 DLBCL (31%) were positive. CCNE did not correlate with any of the known variables (IPI, primary site, cell of origin, proliferation, and BCL2- or C-MYC rearrangements). We were able to reproduce data suggesting an IPI- and response to therapy independent, negative prognostic impact of CCNE in CHOP-treated DLBCL patients: CCNE-positive cases had a median survival of 16 months compared with 57 months in negative ones (p=0.012). In R-CHOP-treated patients the prognostic impact of CCNE was abrogated and only IPI, cell of origin and response to therapy had a prognostic significance. CONCLUSIONS: Addition of R to CHOP overcomes the negative prognostic impact of CCNE in DLBCL. Thus, R not only prolongs survival in DLBCL but also serves a cautionary note that prognostic factors should not be transferred into the 'R era' without proper scientific studies.

Comprehensive gene expression profiling and immunohistochemical studies support application of immunophenotypic algorithm for molecular subtype classification in diffuse large B-cell lymphoma: a report from the International DLBCL Rituximab-CHOP Consortium Program Study.

Gene expression profiling (GEP) has stratified diffuse large B-cell lymphoma (DLBCL) into molecular subgroups that correspond to different stages of lymphocyte development-namely germinal center B-cell like and activated B-cell like. This classification has prognostic significance, but GEP is expensive and not readily applicable into daily practice, which has lead to immunohistochemical algorithms proposed as a surrogate for GEP analysis. We assembled tissue microarrays from 475 de novo DLBCL patients who were treated with rituximab-CHOP chemotherapy. All cases were successfully profiled by GEP on formalin-fixed, paraffin-embedded tissue samples. Sections were stained with antibodies reactive with CD10, GCET1, FOXP1, MUM1 and BCL6 and cases were classified following a rationale of sequential steps of differentiation of B cells. Cutoffs for each marker were obtained using receiver-operating characteristic curves, obviating the need for any arbitrary method. An algorithm based on the expression of CD10, FOXP1 and BCL6 was developed that had a simpler structure than other recently proposed algorithms and 92.6% concordance with GEP. In multivariate analysis, both the International Prognostic Index and our proposed algorithm were significant independent predictors of progression-free and overall survival. In conclusion, this algorithm effectively predicts prognosis of DLBCL patients matching GEP subgroups in the era of rituximab therapy.

Phase II trial of galiximab (anti-CD80 monoclonal antibody) plus rituximab (CALGB 50402): Follicular Lymphoma International Prognostic Index (FLIPI) score is predictive of upfront immunotherapy responsiveness.

BACKGROUND: This phase II CALGB trial evaluated the activity and safety of an extended induction schedule of galiximab (G) plus rituximab (R) in untreated follicular lymphoma (FL). PATIENTS AND METHODS: Patients with previously untreated FL (grades 1, 2, 3a) received 4 weekly infusions of G + R, followed by an additional dose every 2 months four times. International Workshop Response Criteria were used to evaluate response. RESULTS: Sixty-one patients were treated and antibody infusions were well tolerated. The overall response rate (ORR) is 72.1% (95% confidence interval 59.2% to 82.9%): 47.6% complete response (CR)/unconfirmed complete response (CRu) and 24.6% partial response. At a median follow-up time of 4.3 years (range, 0.3-5.3 years) median progression-free survival (PFS) is 2.9 years. Notably, Follicular Lymphoma International Prognostic Index (FLIPI) correlated with ORR, CR rate, and PFS, and the low-risk FLIPI group (n = 12) achieved a 92% ORR, 75% CR/CRu rate, and 75% 3-year PFS. CONCLUSIONS: An extended induction schedule of G + R in previously untreated FL is well tolerated and appears particularly efficacious in those patients with low-risk FLIPI scores. In addition, this trial served as the initial platform for additional CALGB 'doublet' combination regimes of rituximab plus other novel targeted agents.

CpG methylation patterns and decitabine treatment response in acute myeloid leukemia cells and normal hematopoietic precursors.

The DNA hypomethylating drug decitabine maintains normal hematopoietic stem cell (HSC) self-renewal but induces terminal differentiation in acute myeloid leukemia (AML) cells. The basis for these contrasting cell fates, and for selective CpG hypomethylation by decitabine, is poorly understood. Promoter CpGs, with methylation measured by microarray, were classified by the direction of methylation change with normal myeloid maturation. In AML cells, the methylation pattern at maturation-responsive CpGs suggested at least partial maturation. Consistent with partial maturation, in gene expression analyses, AML cells expressed high levels of the key lineage-specifying factor CEBPA, but relatively low levels of the key late-differentiation driver CEBPE. In methylation analysis by mass spectrometry, CEBPE promoter CpGs that are usually hypomethylated during granulocyte maturation were significantly hypermethylated in AML cells. Decitabine-induced hypomethylation was greatest at these and other promoter CpGs that are usually hypomethylated with myeloid maturation, accompanied by cellular differentiation of AML cells. In contrast, decitabine-treated normal HSCs retained immature morphology, and methylation significantly decreased at CpGs that are less methylated in immature cells. High expression of lineage-specifying factor and aberrant epigenetic repression of some key late-differentiation driver genes distinguishes AML cells from normal HSCs, and could explain the contrasting differentiation and methylation responses to decitabine.

Phosphoproteins and the dawn of functional phenotyping.

Phosphorylation is one of the most important processes in cell signal transduction. Detection of phosphorylated proteins in cancer tissue is useful for prognosis and diagnosis, and it might be very helpful in monitoring treatment using targeted therapy. For these reasons, the in situ quantitative measurement and subcellular localization of phosphoproteins will likely be important. However, phosphoproteins are extremely labile, a likely explanation for inconsistent or contradictory reports. Thus, the development of new paradigms for tissue handling, immunostaining, and quality control are needed.

Serious pulmonary toxicity in patients with Hodgkin's lymphoma with SGN-30, gemcitabine, vinorelbine, and liposomal doxorubicin is associated with an FcγRIIIa-158 V/F polymorphism.

BACKGROUND: Based on in vitro synergistic cytotoxicity when anti-CD30 antibodies are combined with gemcitabine, the Cancer and Leukemia Group B conducted a double-blind, randomized, phase II trial of SGN-30 with gemcitabine, vinorelbine, and pegylated liposomal doxorubicin (GVD) in patients with relapsed Hodgkin's lymphoma. PATIENTS AND METHODS: In part 1 of the trial, 16 patients received SGN-30 with GVD to assess the safety of the combination. In part 2, patients were randomly allocated to SGN-30 (n = 7) or placebo (n = 7) with GVD to determine overall response rate (ORR). RESULTS: ORR in all 30 patients was 63% (65% with SGN-30 plus GVD, n = 23, and 57% with placebo plus GVD, n = 7). Median event-free survival was 9.0 months, with no difference between the two arms. Grades 3-5 pneumonitis occurred in five patients receiving SGN-30 and GVD, leading to premature closure of the trial. All five patients with pulmonary toxicity had a V/F polymorphism in the FcγRIIIa gene (P = 0.008). CONCLUSIONS: Together with historical data demonstrating a 2% incidence of pulmonary events with GVD, these results indicate that SGN-30 cannot safely be administered concurrently. The risk of pneumonitis with SGN-30 and GVD is greatest in patients with an FcγRIIIa V/F polymorphism.

Prognostic value of regulatory T cells, lymphoma-associated macrophages, and MUM-1 expression in follicular lymphoma treated before and after the introduction of monoclonal antibody therapy: a Southwest Oncology Group Study.

BACKGROUND: The purpose was to examine the prognostic impact of features of tumor cells and immune microenvironment in patients with follicular lymphoma treated with and without anti-CD20 monoclonal antibody therapy. PATIENTS AND METHODS: Tissue microarrays were constructed from archived tissue obtained from patients on three sequential Southwest Oncology Group (SWOG) trials for FL. All three trials included anthracycline-based chemotherapy. Anti-CD20 monoclonal antibodies were included for patients in the latter two trials. Immunohistochemistry was used to study the number and distribution of cells staining for forkhead box protein P3 (FOXP3) and lymphoma-associated macrophages (LAMs) and the number of lymphoma cells staining for myeloma-associated antigen-1 (MUM-1). Cox proportional hazards regression was used to evaluate the association between marker expression and overall survival (OS). RESULTS: The number or pattern of infiltrating FOXP3 cells and LAMs did not correlate with OS in sequential SWOG studies for FL. The presence of MUM-1 correlated with lower OS for patients who received monoclonal antibody but not for those treated with chemotherapy alone. CONCLUSIONS: Immune cell composition of lymph nodes did not correlate with OS in this analysis of trials in FL. The mechanism of the observed correlation between MUM-1 expression and adverse prognosis in patients receiving monoclonal antibody therapy requires confirmation.