Objective quantification of BCL2 protein by multiplex immunofluorescence in routine biopsy samples of diffuse large B-cell lymphoma demonstrates associations with survival and BCL2 gene alterations

Purpose Diffuse large B-cell lymphoma (DLBCL) is curable in 60% of patients treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). MYC translocations, with or without BCL2 translocations, have been associated with inferior survival in DLBCL. We investigated whether expression of MYC protein, with or without BCL2 protein expression, could risk-stratify patients at diagnosis. Patients and Methods We determined the correlation between presence of MYC and BCL2 proteins by immunohistochemistry (IHC) with survival in two independent cohorts of patients with DLBCL treated with R-CHOP. We further determined if MYC protein expression correlated with high MYC mRNA and/or presence of MYC translocation. Results In the training cohort (n = 167), MYC and BCL2 proteins were detected in 29% and 44% of patients, respectively. Concurrent expression (MYC positive/BCL2 positive) was present in 21% of patients. MYC protein correlated with presence of high MYC mRNA and MYC translocation (both P < .001), but the latter was less frequent (both 11%). MYC protein expression was only associated with inferior overall and progression-free survival when BCL2 protein was coexpressed (P < .001). Importantly, the poor prognostic effect of MYC positive/BCL2 positive was validated in an independent cohort of 140 patients with DLBCL and remained significant (P < .05) after adjusting for presence of high-risk features in a multivariable model that included elevated international prognostic index score, activated B-cell molecular subtype, and presence of concurrent MYC and BCL2 translocations. Conclusion Assessment of MYC and BCL2 expression by IHC represents a robust, rapid, and inexpensive approach to risk-stratify patients with DLBCL at diagnosis.

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BCL2 Overexpression Associated With Chromosomal Amplification in Diffuse Large B-Cell Lymphoma

Blood ◽

10.1182/blood.v90.3.1168 ◽

1997 ◽

Vol 90 (3) ◽

pp. 1168-1174 ◽

Cited By ~ 144

Author(s):

Outi Monni ◽

Heikki Joensuu ◽

Kaarle Franssila ◽

Juha Klefstrom ◽

Kari Alitalo ◽

...

Keyword(s):

B Cell ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Comparative Genomic ◽

Chain Gene ◽

Large B Cell Lymphoma ◽

Bcl2 Protein ◽

High Level ◽

Hodgkin's Lymphomas ◽

Large B Cell

Abstract Gene activation by translocation between an oncogene and an immunoglobulin heavy-chain gene, which leads to increased expression of the oncoprotein, is a well-known mechanism in the genesis of B-cell lymphomas. In contrast, the role of gene amplification in activation of oncogenes in non-Hodgkin's lymphomas is poorly characterized. To study the BCL2 amplification we performed comparative genomic hybridization (CGH), Southern blot hybridization, Western analysis, immunohistochemistry, metaphase fluorescence in situ hybridization, and chromosome analysis on 26 cases of diffuse large B-cell lymphoma (large noncleaved cell lymphoma). The gain or high-level amplification of 18q was found in eight tumors (31%) by CGH, and Southern analysis revealed BCL2 amplification in these cases, but not in the cases with normal chromosome 18 or t(14; 18)(q32; q21). Western immunoblot analysis and immunohistochemistry revealed a high-level expression of BCL2 protein in the cases with BCL2 amplification and t(14; 18)(q32; q21). However, translocation (14; 18)(q32; q21) was not detected in any of the cases with BCL2 amplification. Therefore, our results suggest that amplification of the BCL2 gene is an important mechanism for BCL2 protein overexpression in diffuse large B-cell lymphoma.

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PD-L1 gene alterations identify a subset of diffuse large B-cell lymphoma harboring a T-cell–inflamed phenotype

Blood ◽

10.1182/blood-2018-10-879015 ◽

2019 ◽

Vol 133 (21) ◽

pp. 2279-2290 ◽

Cited By ~ 28

Author(s):

James Godfrey ◽

Sravya Tumuluru ◽

Riyue Bao ◽

Michael Leukam ◽

Girish Venkataraman ◽

...

Keyword(s):

T Cell ◽

B Cell ◽

Immune Escape ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Data Set ◽

Large B Cell Lymphoma ◽

Programmed Death ◽

Large B Cell ◽

Gene Alterations

Abstract Programmed death-ligand 1 (PD-L1) expression on malignant cells is a dominant immune escape mechanism across a variety of human cancers. A unique genetic mechanism underlying PD-L1 upregulation has been uncovered in classical Hodgkin lymphoma (cHL), in which copy gains of the chromosomal region (9p24.1) containing the programmed death-1 (PD-1) ligands PD-L1 and PD-L2 are recurrently observed. While chromosome 9p24.1 copy-number alterations are ubiquitous in cHL, they also occur in diffuse large B-cell lymphoma (DLBCL), albeit with a lower incidence. Here, fluorescence in situ hybridization was used to identify DLBCLs harboring PD-L1 gene alterations, thereby enabling a characterization of the immunogenomic landscape of these lymphomas. Among 105 DLBCL cases analyzed, PD-L1 alterations were identified in 27%. PD-L1 alterations were highly enriched among non–germinal center DLBCLs and exhibited robust PD-L1 protein expression. These lymphomas were heavily infiltrated by clonally restricted T cells and frequently downregulated human leukocyte antigen expression. RNA sequencing of PD-L1–altered DLBCLs revealed upregulation of genes involved in negative T-cell regulation and NF-κB pathway activation, while whole-exome sequencing identified frequent mutations in genes involved in antigen presentation and T-cell costimulation. Many of these findings were validated in a large external data set. Interestingly, DLBCL patients with PD-L1 alterations had inferior progression-free survival following front-line chemoimmunotherapy; however, in the relapsed/refractory setting, PD-L1 alterations were associated with response to anti-PD-1 therapy. Collectively, our results indicate that PD-L1 alterations identify a unique biological subset of DLBCL in which an endogenous antilymphoma immune response has been activated, and that is associated with responsiveness to PD-1 blockade therapy.

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Pd-l1 gene alterations to identify a subset of diffuse large B cell lymphoma that harbor a T cell inflamed phenotype.

Journal of Clinical Oncology ◽

10.1200/jco.2018.36.15_suppl.7566 ◽

2018 ◽

Vol 36 (15_suppl) ◽

pp. 7566-7566

Author(s):

James K. Godfrey ◽

Sonali M. Smith ◽

Sravya Tumuluru ◽

Justin Paul Kline ◽

James McElherne ◽

...

Keyword(s):

T Cell ◽

B Cell ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Large B Cell Lymphoma ◽

Large B Cell ◽

Gene Alterations

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Outcomes of diffuse large b-cell lymphoma with MYC and/or BCL2 protein expression treated with intensive chemotherapy.

Journal of Clinical Oncology ◽

10.1200/jco.2016.34.15_suppl.7563 ◽

2016 ◽

Vol 34 (15_suppl) ◽

pp. 7563-7563

Author(s):

Amir Issa ◽

Vishwanath Sathyanarayanan ◽

Michelle A. Fanale ◽

Yasuhiro Oki ◽

Fredrick B. Hagemeister ◽

...

Keyword(s):

Protein Expression ◽

B Cell ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Intensive Chemotherapy ◽

Large B Cell Lymphoma ◽

Bcl2 Protein ◽

Large B Cell

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The t(14;18)(q32;q21) Characterizes a Subset of Patients with Diffuse Large-B Cell Lymphoma of Germinal Center Origin with Poor Outcome: Report From the International DLBCL Rituximab-CHOP Consortium Program Study

Blood ◽

10.1182/blood.v118.21.949.949 ◽

2011 ◽

Vol 118 (21) ◽

pp. 949-949 ◽

Cited By ~ 2

Author(s):

Carlo Visco ◽

Alexander Tzankov ◽

Zijun Y. Xu-Monette ◽

Roberto N. Miranda ◽

Emanuele S. G. d'Amore ◽

...

Keyword(s):

Protein Expression ◽

B Cell ◽

Germinal Center ◽

Cell Lymphoma ◽

International Prognostic Index ◽

B Cell Lymphoma ◽

Individual Risk ◽

Large B Cell Lymphoma ◽

Bcl2 Protein ◽

Large B Cell

Abstract Abstract 949 Introduction: Diffuse large B cell lymphoma (DLBCL) has a highly variable outcome, and individual risk assessment is largely based on clinical features. Gene expression profiling (GEP) stratifies patients into those with germinal center B-cell (GCB) and activated B-cell subtype (ABC) subtype with different prognoses. These groups have been shown to predict prognosis in patients treated with CHOP or R-CHOP. Conversely, the role of other recognized prognostic markers, such as BCL2 gene abnormalities or Bcl2 expression has been questioned in the new therapeutic era. Materials and Methods: In 438 patients treated with R-CHOP for de novo DLBCL, we analyzed the tumors by immunohistochemistry for Bcl2 protein expression and by interphase fluorescence in situ hybridization (FISH) for BCL2 translocation and other abnormalities. All cases were successfully studied by GEP. The cutoff for Bcl2 protein expression, 60%, used as prognostic factor was determined using receiver operating characteristic curves. Progression-free survival (PFS) and overall survival (OS) were assessed. Results: The t(14;18)(q32;q21) was detected in 82 cases (18.7%) and BCL2 gains occurred in 63 cases (14.3%). Both t(14;18) and BCL2 gains strongly correlated with higher levels of Bcl2 protein expression (p<0.0001 for both). Presence of t(14;18) was associated with the GCB subtype (p<0.0001), whereas BCL2 gains were associated with the ABC subtype (p=0.004). BCL2 gains were not predictive of PFS in any patients' subgroups. Conversely, within the GCB subtype, patients with the t(14;18) displayed a significantly worse outcome compared to GCB patients without t(14;18) with a 5-year PFS of 45% vs 68%, respectively (p<0.0001). Outcome of patients with DLBCL associated with t(14;18) was similar to patients with the ABC subtype (45% vs 48%, p=0.30, Figure 1). No impact of the t(14;18) and BCL2 gains was observed on patients with ABC-DLBCL. Using immunohistochemistry, patients with Bcl2 positive (>60%) tumors had significantly inferior PFS in the GCB subgroup (p=0.03), but not in the ABC subgroup (p=0.54). Multivariate analysis revealed that the presence of the t(14;18), but not Bcl2 protein expression, was independent of the International Prognostic Index in predicting outcome of our patients. Conclusions: Patients with the GCB subtype and t(14;18) exhibit a significantly worse prognosis than patients without t(14;18) when treated with R-CHOP. The assessment of t(14;18) by FISH approach not only functions as a valuable prognosticator for individual risk estimation in GCB-DLBCL patients in addition to the established parameters, but also provides valuable result for therapeutic intervention. Disclosures: No relevant conflicts of interest to declare.

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MYC Gene Single Hit Diffuse Large B Cell Lymphoma Behaves Similarly To MYC/BCL2 Double Hit Lymphoma and Needs Similar Aggressive Treatment

Blood ◽

10.1182/blood.v122.21.4313.4313 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4313-4313 ◽

Cited By ~ 1

Author(s):

Xuan J. Wang ◽

Nishitha M. Reddy ◽

Shaoying Li

Keyword(s):

B Cell ◽

Gene Rearrangement ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Response To Treatment ◽

Clinicopathologic Features ◽

Large B Cell Lymphoma ◽

Bcl2 Protein ◽

Double Hit ◽

Large B Cell

Abstract Introduction Double hit diffuse large B cell lymphoma (DHL) is a heterogeneous group of high grade B-cell lymphoma with concurrent MYC and BCL2 (or other) gene rearrangements. It is well known that DHL has an aggressive clinical course, is resistant to standard R-CHOP chemotherapy, and confers a poor prognosis. Little is known about MYC single hit diffuse large B cell lymphoma (SHL) and how they behave and respond to therapy compared to MYC/BCL2 DHL. The aim of the study was to delineate the characteristics of MYC SHL and compare it to MYC/BCL2 DHL with regards to its clinicopathologic features, especially response to treatment and prognosis. Methods One hundred and fifteen patients diagnosed with large B cell lymphoma between September 2009 and June 2013 at our institution were included in this study. MYC and BCL2 gene rearrangement were confirmed by fluorescence in situ hybridization (FISH) using MYC breakapart probe and BCL2 and IgH dual color dual fusion probes, respectively. BCL6 gene rearrangement was revealed in a subset of cases either by FISH using breakapart probe or karyotype. MYC/BCL2 DHL cases were identified if they had rearrangements of MYC and BCL2. SHL cases were identified if they only had MYC rearrangement. Tumor cells were defined positive for MYC and BCL2 protein expression by immunostain if >40% and 50% of cells showed positive expression, respectively. Patient survival was analyzed using the Kaplan-Meier method and compared using the log-rank test. Fisher's exact test was used to compare the two groups. Results Of the 115 patients, 15 were MYC SHL and 24 were MYC/BCL2 DHL. Four DHL also had BCL6 rearrangement. All cases with karyotype available showed complex karyotype, both in SHL and DHL (Table 1). Of the 15 SHL patients, 9 were male and 6 were female with a median age of 63 years (range 36-78) at diagnosis. Eleven of 14 patients had elevated serum LDH. 10 patients (67%) had bone marrow involvement and 12 (80%) had more than one extranodal sites involved. Thirteen patients (87%) had advanced stage disease (stages III and IV). Most patients (93%) had high intermediate or high risk based on the International Prognostic Index (IPI). By immunostain, MYC was expressed in 12/13 (92%) cases, BCL2 in 9/15 (60%) cases, and MYC and BCL2 were coexpressed in 7/13 (54%) cases. All the above clinicopathologic features were similar between SHL and DHL patients (Table 1, p> 0.05), with the only exception of more prevalent BCL2 expression in DHL patients (p=0.01). All 15 SHL patients received chemotherapy: 2 (13%) received R-CHOP, 2 (13%) received R-Hyper CVAD/Ara-C/MTX, 7 (47%) received R-EPOCH and 4 (27%) received other aggressive regimens. One patient also received stem cell transplant (SCT). Twenty one of the 24 DHL patients had treatment information available and there was no statistically significant difference between the two groups (p=0.25). At a median follow up of 16 months, the median overall survival was 10.4 months for SHL, which was not significantly different from that of DHL (19.9 months; p=0.10; Figure 1). Conclusion Our data suggests that clinicopathologic features of MYC SHL is similar to DHL, including the MYC/BCL2 protein coexpression by immunohistochemistry. Despite similar treatment approach in both groups, the response to treatment and prognosis of patients with MYC SHL is similar to those with DHL. Therefore, both MYC SHL and MYC/BCL2 DHL need to be approached similarly while implementing therapeutic decisions. Novel inhibitors in combination with multi-agent chemotherapy to improve prognosis are urgently needed in this subgroup of lymphoma. Disclosures: No relevant conflicts of interest to declare.

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BCL2 gene aberration as an IPI-independent marker for poor outcome in non-germinal-centre diffuse large B cell lymphoma

Journal of Clinical Pathology ◽

10.1136/jcp.2009.066597 ◽

2009 ◽

Vol 62 (10) ◽

pp. 903-907 ◽

Cited By ~ 16

Author(s):

E C Obermann ◽

M Csato ◽

S Dirnhofer ◽

A Tzankov

Keyword(s):

Protein Expression ◽

B Cell ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Germinal Centre ◽

Individual Risk ◽

Large B Cell Lymphoma ◽

Bcl2 Protein ◽

Worse Prognosis ◽

Large B Cell

Aim:Diffuse large B cell lymphoma (DLBCL) is the most common lymphoid malignancy in the western hemisphere, and is characterised by a highly variable outcome that impedes individual risk assessment. Lacking reliable biomarkers, the international prognostic index (IPI) has been the most reliable factor to predict survival and stratify patients for therapy. The aim of this study was to investigate the frequency and potential prognostic role of BCL2 aberrations on the chromosomal level and the protein level in a large DLBCL collective.Methods:Fluorescence in situ hybridisation (FISH) with commercially available dual-colour break-apart probes and immunohistochemistry were used to assess BCL2 gene abnormalities and bcl2 protein expression on validated tissue microarrays containing 224 well-characterised cases of primary DLBCL.Results:FISH analysis of BCL2 revealed a break in 40/215 cases (19%) and a gain in 66/171 (39%) cases. Only BCL2 gains correlated with bcl2 protein expression (p = 0.001). Presence of any BCL2 gene abnormality, particularly gains, correlated independently of the IPI with a significantly worse prognosis in DLBCL of non-germinal centre (non-GC) phenotype as opposed to DLBCL of non-GC type without this genetic alteration (p = 0.003). DLBCL of germinal centre phenotype did not show this association.Conclusions:Cases of DLBCL of the non-GC type with BCL2 gene aberration are accompanied by a significantly worse prognosis as opposed to cases without such gene abnormalities. It may be helpful to asses BCL2 gene abnormalities by FISH in addition to assessing established parameters for individual risk estimation in DLBCL.

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Clinical Significance of Co-Expression of MYC and BCL2 Protein in Advanced Diffuse Large B-Cell Lymphoma Treated with a Dose-Intensified Immunochemotherapy

Blood ◽

10.1182/blood.v126.23.3940.3940 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3940-3940

Author(s):

Hiromichi Takahashi ◽

Sumiko Kobayashi ◽

Katsuhiro Miura ◽

Daisuke Kurita ◽

Yoshihiro Hatta ◽

...

Keyword(s):

B Cell ◽

Research Funding ◽

De Novo ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Intermediate Risk ◽

Chugai Pharmaceutical ◽

Large B Cell Lymphoma ◽

Bcl2 Protein ◽

Large B Cell

Abstract Background Recent studies have shown that the concurrent expression of MYC and BCL2 protein evaluated by immunohistochemistry (IHC) in patients with de novo diffuse large B-cell lymphoma (DLBCL) is associated with worse survival when treated with standard R-CHOP, but the effect of intensive chemotherapies for such patients is unknown. Thus, we evaluated the impact of the co-expression of MYC and BCL2 protein among patients with advanced DLBCL, who were treated with a dose-intensive immunochemotherapy followed by up-front autologous stem cell transplantation (ASCT). Patients and Methods This is a retrospective analysis of patients with de novo DLBCL, who were categorized into high/high-intermediate risk by the age-adjusted International Prognostic Index (aaIPI). They were consecutively treated with the R-Double-CHOP regimen, consisting of rituximab (375 mg/m2, day -2), cyclophosphamide (750 mg/m2, day 1, 2), doxorubicin (50 mg/m2, day 1, 2), vincristine (1.4 mg/m2 [maximum 2.0 mg/body], day 1) and prednisolone (50 mg/m2, day 1-5) followed by consolidative high-dose chemotherapies at our institution from 2001 to 2013. MYC and BCL2 protein were measured by IHC assay using formalin-fixed paraffin-embedded tissue specimens for all available cases. Cut-off values of positivity for MYC and BCL2 protein were set as 40% and 50% of stained tumor cell, respectively. Lymphomas showing concurrent positivity for MYC and BCL2 protein were defined as "Double expressor lymphoma (DEL)". Results A total of 40 patients with a median 53-years (range 19-68) of age were analyzed. Twenty-one patients were at high risk and the other 19 patients were at high-intermediate risk by aaIPI. Cell of origin (COO) subtypes classified by Hans algorithm consisted of 14 germinal center B-cell (GCB) type and 26 non-GCB type. Totally, 10 (25%) patients were categorized into DEL. The overall response (OR) and the complete response (CR) rates to R-Double-CHOP for all patients were 93% and 83%, respectively. The OR and the CR rates were not significantly different between the DEL group and the non-DEL group (100% vs 90%, and 80% vs 83%, respectively). The proportion of patients proceeding to ASCT was not significantly different among these groups (50% vs 60%). With a median 52 months (range 3-155) of follow-up, the 3-year progression-free survival (PFS) and the overall survival (OS) rates for all patients were 55% and 72%, respectively (Figure a, b). Both the PFS and the OS were significantly worse in the DEL group than in the non-DEL group (Figure c, d). As for aaIPI and COO subtyping, either high/high-intermediate risk or GCB/non-GCB subtype were not significantly associated with the outcome of PFS or OS. Conclusion The concurrent expression of MYC/BCL2 protein in advanced DLBCL was associated with shorter remission duration and worse survival despite similar susceptibility to the treatment when a dose-intensive immunochemotherapy was applied. Our findings suggest that patients with advanced DEL may not benefit from dose-intensified therapies, and therefore need highly discrete strategies. Disclosures Miura: Astellas Pharma Inc.: Honoraria; Celgene K.K.: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; CHUGAI PHARMACEUTICAL CO. LTD: Honoraria; Kyowa Hakko Kirin CO., Ltd, Japan: Honoraria; Meiji Seika Pharma: Honoraria; Janssen Pharmaceutical K.K.: Honoraria. Hatta:Kyowa Hakko Kirin CO., Ltd, Japan: Honoraria; CHUGAI PHARMACEUTICAL CO. LTD: Honoraria; Celgene K.K.: Honoraria. Iriyama:Brystol-Myers K.K.: Honoraria. Takei:Kyowa Hakko Kirin CO., Ltd, Japan: Research Funding; Bristol-Myers K.K.: Research Funding; Nippon Kayaku Co.: Research Funding; Shionogi & Co.: Research Funding; Meiji Seika Pharma: Research Funding; Astellas Pharma Inc.: Research Funding; Janssen Pharmaceutical K.K.: Research Funding; TEIJIN PHARMA LIMITED: Research Funding; CSL Behring K.K: Research Funding; Japan Blood Products Organization: Research Funding; Sumitomo Dainippon Pharma Co.: Research Funding; TORII, PHAMACEUTICAL CO: Research Funding; Alexion Pharmaceuticals: Research Funding; YAKULT HONSHA CO., Ltd.: Research Funding; Taisho Toyama Pharmaceutical Co., Ltd.: Research Funding; TAIHO PHARMACEUTICAL CO., Ltd.: Research Funding; CHUGAI PHARMACEUTICAL CO. LTD: Research Funding.

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