Robert G. Bociek

Expression of PKC-beta or cyclin D2 predicts for inferior survival in diffuse large B-cell lymphoma

We sought to determine whether identification of poor-risk subgroups of diffuse large B-cell lymphoma (DLBCL) using immunohistochemical stains would have practical utility with regard to prognosis and therapeutic decisions. Tissue microarray blocks were created using replicate samples of formalin-fixed, paraffin-embedded tissue from 200 cases of de novo DLBCL. The sections were stained with antibodies to proteins that are expressed by activated or proliferating B cells including MUM1, FOXP1, bcl-2, survivin, protein kinase C-beta (PKC-β), cyclin D2, cyclin D3, and Ki-67. In univariate analysis, tumor expression of cyclin D2 (P=0.025) or PKC-β (P=0.015) was associated with a worse overall survival, whereas none of the other markers was predictive of overall survival. Patients with DLBCL that expressed either cyclin D2 or PKC-β had a 5-year overall survival of only 30% as compared to 52% for those who were negative for both markers (P=0.0019). In multivariate analysis, the expression of cyclin D2 or PKC-β was an independent predictor of poor overall survival (P=0.035). Cyclin D2 and PKC-β expression will be useful in designing a ‘biological prognostic index’ for patients with DLBCL.

MYC and BCL2 protein expression predicts survival in patients with diffuse large B-cell lymphoma treated with rituximab

Diffuse large B‐cell lymphoma (DLBCL) is a heterogeneous disease and “double‐hit” DLBCL, with both MYC and BCL2 translocations has a poor prognosis. In this study, we investigated whether MYC and BCL2 protein expression in tissue would predict survival in DLBCL. The study included 106 cases of de novo DLBCL treated with rituximab and cyclophosphamide, doxorubicin, vincristine and prednisone (R‐CHOP) or CHOP‐like regimens. The results were validated on an independent cohort of 205 DLBCL patients. Patients with low expression of BCL2 (≤30%) and MYC (≤50%) had the best prognosis, whereas those with high BCL2 (>30%) and MYC (>50%) had the worst outcome. In multivariate analysis, the combination of the BCL2 and MYC was an independent predictor of overall survival (OS) and event‐free survival (EFS) (P = 0·015 and P = 0·005, respectively). The risk of death was nine times greater for patients with high BCL2 and MYC compared to those with low expression. High BCL2 and MYC was a strong predictor of poor OS (P < 0·001) and EFS (P = 0·0017) in patients with the germinal centre B‐cell (GCB) type, but not in the non‐GCB type. In DLBCL, high co‐expression of MYC and BCL2 was an independent predictor of poor survival, and could be used to stratify patients for risk‐adapted therapies.

Hedgehog-induced survival of B-cell chronic lymphocytic leukemia cells in a stromal cell microenvironment: a potential new therapeutic target.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by an accumulation of neoplastic B cells due to their resistance to apoptosis and increased survival. Among various factors, the tumor microenvironment is known to play a role in the regulation of cell proliferation and survival of many cancers. However, it remains unclear how the tumor microenvironment contributes to the increased survival of B-CLL cells. Therefore, we studied the influence of bone marrow stromal cell–induced hedgehog (Hh) signaling on the survival of B-CLL cells. Our results show that a Hh signaling inhibitor, cyclopamine, inhibits bone marrow stromal cell–induced survival of B-CLL cells, suggesting a role for Hh signaling in the survival of B-CLL cells. Furthermore, gene expression profiling of primary B-CLL cells (n = 48) indicates that the expression of Hh signaling molecules, such as GLI1, GLI2, SUFU, and BCL2, is significantly increased and correlates with disease progression of B-CLL patients with clinical outcome. In addition, SUFU and GLI1 transcripts, as determined by real-time PCR, are significantly overexpressed and correlate with adverse indicators of clinical outcome in B-CLL patients, such as cytogenetics or CD38 expression. Furthermore, selective down-regulation of GLI1 by antisense oligodeoxynucleotides (GLI1-ASO) results in decreased BCL2 expression and cell survival, suggesting that GLI1 may regulate BCL2 and, thereby, modulate cell survival in B-CLL. In addition, there was significantly increased apoptosis of B-CLL cells when cultured in the presence of GLI1-ASO and fludarabine. Together, these results reveal that Hh signaling is important in the pathogenesis of B-CLL and, hence, may be a potential therapeutic target. (Mol Cancer Res 2008;6(12):1928–36)

B‐cell lymphoma, unclassifiable, with features intermediate between diffuse large B‐cell lymphoma and burkitt lymphoma: study of 39 cases

B‐cell lymphoma, unclassifiable (B‐UCL), with features intermediate between diffuse large B‐cell lymphoma and Burkitt lymphoma, is a poorly characterized entity. Therefore, we investigated cases of B‐UCL treated by the Nebraska Lymphoma Study Group (NLSG). We searched the NLSG registry for years 1985–2010 for cases of B‐UCL. Immunohistochemical stains and fluorescence in situ hybridization studies for MYC, BCL2 and BCL6 gene rearrangements were performed. Among the 39 cases studied, 54% were male and 46% were female, with a median age of 69 years. The majority of patients presented with advanced‐stage disease (62%) and had high (3–5) International Prognostic Index (IPI) scores (54%). The median overall survival (OS) was only 9 months and the 5‐year OS was 30%. Patients with low IPI scores (0–2) had a better survival than those with high scores (3–5). The cases were genetically heterogeneous and included 11 ‘double‐hit’ lymphomas with rearrangements of both MYC and BCL2 or BCL6. None of the immunohistochemical or genetic features was predictive of survival. This B‐cell lymphoma is a morphologically‐recognizable entity with a spectrum of genetic abnormalities. New and better treatments are needed for this aggressive lymphoma.

Cardiac magnetic resonance imaging for the assessment of the myocardium after doxorubicin-based chemotherapy.

Objectives:Doxorubicin is associated with a cumulative dose–dependent nonischemic cardiomyopathy. Cardiac magnetic resonance imaging (cMRI) is able to examine both structural and functional components of the myocardium. Our aim was to assess the myocardial changes in non-Hodgkin lymphoma patients undergoing doxorubicin-based chemotherapy using cMRI. Materials and Methods:cMRI examination was performed before and 3 months after chemotherapy. Experienced investigators interpreted each cMRI, and were blinded to all data. Left ventricular ejection fractions (LVEF), cardiac deformation, and delayed gadolinium enhancement (GD-DE) were quantified for each cMRI. The change between LVEF, GD-GE, and cardiac deformation parameters were compared between the 2 cMRI studies. A &Dgr; LVEF≥10% was considered clinically relevant. The findings of GD-GE or changes in myocardial strain were analyzed as independent variables. Results:All 10 patients enrolled received a cumulative dose of doxorubicin of 300 mg/m2. A comparison of pretreatment and posttreatment cMRI demonstrated 5 (50%) patients with a ≥10% decrease in LVEF (median, −8.4%; range, 1% to −17%; P=0.004). Three patients had at least 1 new or progressive segment of GD-DE. The global circumferential strain was significantly lower in patients after treatment, as compared with values before treatment (P=0.018) and to normal controls (P=0.046). Patients after treatment also had significantly lower global longitudinal strain than controls (P=0.035), and longitudinal strain values that tended to decrease compared with pretreatment values (P=0.073). Discussion:Our data suggests that cMRI has the ability to assess both early structural and functional myocardial changes in association with doxorubicin-based chemotherapy.

Lymph node‐induced immune tolerance in chronic lymphocytic leukaemia: a role for caveolin‐1

Emerging evidence indicates that the tumour microenvironment (TME) regulates the behaviour of chronic lymphocytic leukaemia (CLL). However, the precise mechanism and molecules involved in this process remain unknown. Gene expression profiles of CLL cells from lymph node (LN), bone marrow (BM) and peripheral blood (PB) indicate overexpression of a tolerogenic signature in CLL cells in lymph nodes (LN‐CLL). Based on their role in B cell biology, the progression of CLL, or immune regulation, a few genes of this 83‐gene signature were selected for further analyses. We observed a significant correlation between the clinical outcomes and the expression of CAV1 (P = 0·041), FGFR1 isoform 8 (P = 0·032), PTPN6 (P = 0·031) and ZWINT (P < 0·001). CAV1, a molecule involved in the regulation of tumour progression in other cancers, was seven‐fold higher in LN‐CLL cells compared to BM‐ and PB‐CLL cells. Knockdown of CAV1 expression in CLL cells resulted in significantly decreased migration (P = 0·016) and proliferation (P = 0·04). When CAV1 was knocked down in B and T cell lines, we observed an inability to form immune synapses. Furthermore, CAV1 knockdown in CLL cells impaired their ability to form immune synapses with autologous T lymphocytes and allogeneic, healthy T cells. Subsequent analyses of microarray data showed differential expression of cytoskeletal genes, specifically those involved in actin polymerization. Therefore, we report a novel role for CAV1 in tumour‐induced immunosuppression during the progression of CLL.

Phase I trial of 90Y-ibritumomab tiuxetan in patients with relapsed B-cell non-Hodgkin's lymphoma following high-dose chemotherapy and autologous stem cell transplantation

Between January 2001 and September 2005, 19 patients with progressive B-cell non-Hodgkins lymphoma were treated with a cohort-specific dose of yttrium-90 ibritumomab tiuxetan (0.10 – 0.20 mCi/kg) to determine appropriate dosing in patients who had previously received high-dose chemotherapy and autologous stem cell transplantation (ASCT). Patients were required to have adequate end organ function and bone marrow status. Patients had been treated with a median of three prior therapies (range, 1 – 9). The median time from ASCT to radioimmunotherapy was 28 months. Hematologic toxicities were dose-limiting and included grade 3 – 4 thrombocytopenia (53%), neutropenia (32%), and anemia (21%). The majority of grade 3 – 4 events occurred at the 0.2 mCi/kg dose level. Nine patients responded (complete response, complete response unconfirmed, or partial response) to the therapy. At a median follow-up of 37 months, the 1-year event-free and overall survival rates were 26% and 57%, respectively. A dose of 0.2 mCi/kg ibritumomab tiuxetan is safe and effective for patients with progressive disease after high-dose chemotherapy and ASCT.

The addition of rituximab reduces the incidence of secondary central nervous system involvement in patients with diffuse large B‐cell lymphoma

This is a retrospective analysis of the frequency of CNS relapses in DLBCL patients treated prior to and during the rituximab era. The addition of rituximab to chemotherapy significantly decreases the frequency of CNS relapses.

Isolated MYC cytogenetic abnormalities in diffuse large B-cell lymphoma do not predict an adverse clinical outcome

In this study, we investigated the significance of MYC, BCL2 and BCL6 gene abnormalities in a cohort of 205 diffuse large B-cell lymphoma (DLBCL) patients studied by conventional and/or fluorescence in situ hybridization cytogenetic analysis. Combining these methods, 172 cases (84%) were classified as MYC−, 17 (8%) were MYC+/BCL2−/BCL6−, and 16 (8%) were double/triple-hit lymphomas (i.e. MYC+/BCL2+, MYC+/BCL6+, or MYC+/BCL2+/BCL6+). We found a significant difference in event-free survival (EFS) among the three groups (p = 0.02), with the double/triple-hit group having the worst EFS. Patients who were MYC+, but BCL2− and BCL6−, had the best EFS. We conclude that patients with MYC+ DLBCL, but without BCL2 or BCL6 abnormalities, do not have a worse outcome when compared to those who are MYC−. However, patients with double/triple-hit DLBCL have a very poor outcome and should be treated with aggressive or novel therapies.

The utility of lactate dehydrogenase in the follow up of patients with diffuse large B-cell lymphoma

Background Serum lactate dehydrogenase is a non-specific marker for lymphoma whose prognostic significance is well established for both indolent and aggressive lymphomas at the time of diagnosis. The performance characteristics of this enzyme in predicting relapse in patients with diffuse large B-cell lymphoma has not been well studied. Methods This study compared serum lactate dehydrogenase levels in 27 patients with diffuse large B-cell lymphoma who relapsed after sustaining a complete response versus 87 patients who did not relapse. For relapsed patients, the serum lactate dehydrogenase level at relapse was compared with the level three months before (considered baseline). For non-relapsed patients, the last two levels during follow-up were compared. For statistical analysis the T-test was used to compare differences in mean values between groups. The sensitivity, specificity, positive and negative predictive values for serum lactate dehydrogenase in detecting relapse compared to confirmatory imaging were calculated. Results At relapse, only 33% patients had increases in serum lactate dehydrogenase above the upper limit of normal. The mean increase was 1.2-fold above the upper limit of normal for relapsed vs. 0.83 for those who did not relapse (p-value = 0.59). The mean increase in serum lactate dehydrogenase, from baseline, was 1.1-fold in non-relapsed vs. 1.3 in relapsed patients (p-value = 0.3). The likelihood ratio of relapse was 4.65 for patients who had 1.5-fold increases in serum lactate dehydrogenase above baseline (p-value = 0.03). The sensitivity, specificity, positive and negative predictive values of 1.5-fold increases for detecting relapse, compared to clinical and imaging findings were 0.18, 0.95, 0.55, and 0.79, respectively. Conclusion A 1.5-fold increase in serum lactate dehydrogenase, over a period of 3 months, is associated with increased likelihood of relapse from diffuse large B-cell lymphoma.