|Year : 2016 | Volume
| Issue : 4 | Page : 961-970
Prognostic impact of immunohistochemical stratification of diffuse large B-cell lymphoma into germinal and nongerminal subtypes
Nancy Y Asaad, Moshira M Abdel Wahed, Asmaa G Abdou, Marwa M Serag El-Dien, Mona A. H. Mohammed Kora MBBCH
Department of Pathology, Faculty of Medicine, Menoufia University, Shebin El Kom, Egypt
|Date of Submission||04-Apr-2015|
|Date of Acceptance||16-Jun-2015|
|Date of Web Publication||21-Mar-2017|
Mona A. H. Mohammed Kora
Department of Pathology, Menoufia University, Shebin El Kom, 32511
Source of Support: None, Conflict of Interest: None
The aim of this study was to investigate the difference between germinal and nongerminal diffuse large B-cell lymphoma (DLBCL) cases regarding their clinicopathological features, including survival and response to therapy.
DLBCL is the most common subtype of non-Hodgkin's lymphoma in Egypt. It represents about 49% of non-Hodgkin's lymphoma cases according to the latest registry of National Cancer Institute, Cairo University. DLBCL is a heterogeneous group, especially as regards biological behavior, which includes response to chemotherapy and ultimate outcome. Previous studies suggested that classification of DLBCL into germinal and nongerminal subtypes had prognostic importance.
Patients and methods
This retrospective study included 61 de-novo DLBCL cases collected from the archival material of Pathology Department, Faculty of Medicine, Menoufia University, and treated at Menoufia Cancer Institute.
Using Hans' algorithm, 31 (50.8%) cases were of germinal center B-cell (GCB) and 30 (49.8%) cases were of non-GCB subtype. Non-GCB cases showed significant association with splenic involvement (P = 0.01) and larger tumor size (P = 0.03). Overall, 90% of DLBCL cases with poor performance status were of GCB subtype. No significant difference regarding overall survival was noticed between GCB and non-GCB subtypes.
DLBCL should be classified into GCB and non-GCB subtypes because they differ prognostically. Hans' algorthim is a simple and reliable method for the classification of DLBCL, but it should be applied on a large number of cases to be used as a routine method for DLBCL evaluation.
Keywords: BCL6, CD10, diffuse large B-cell lymphoma, Hans' algorthim, MUM-1
|How to cite this article:|
Asaad NY, Abdel Wahed MM, Abdou AG, Serag El-Dien MM, Mohammed Kora MA. Prognostic impact of immunohistochemical stratification of diffuse large B-cell lymphoma into germinal and nongerminal subtypes. Menoufia Med J 2016;29:961-70
|How to cite this URL:|
Asaad NY, Abdel Wahed MM, Abdou AG, Serag El-Dien MM, Mohammed Kora MA. Prognostic impact of immunohistochemical stratification of diffuse large B-cell lymphoma into germinal and nongerminal subtypes. Menoufia Med J [serial online] 2016 [cited 2020 Apr 3];29:961-70. Available from: http://www.mmj.eg.net/text.asp?2016/29/4/961/202495
| Introduction|| |
Malignant lymphoma represents a major health problem throughout the world. It is already the fourth common malignancy in Egypt out of all malignances and is continuing to increase rapidly. In Egypt, it constitutes 11.71% of total malignancies .
The Middle East Cancer Consortium showed that Egyptians and Israeli Jews had the highest rates of lymphoma and non-Hodgkin's lymphoma (NHL) among other Middle East Cancer Consortium populations. Egyptians had higher rates for NHL in age groups 0–14 years with a male-to-female ratio of 1.6: 1 . Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of NHL in Egypt. It represents about 49% of NHL cases according to the latest registry of National Cancer Institute, Cairo University .
NHLs are the 12th most prevalent cancer in Europe. The most frequent lymphoma is DLBCL, with a male-to-female ratio of 1.2: 1, and is more frequent in 61–80-year-old patients .
DLBCL spans a broad age range and is heterogeneous in morphologic appearance, immunophenotype, and biologic behavior, including response to chemotherapy and ultimate outcome .
Management of DLBCL patients was guided by age-adjusted international prognostic index (AAIPI) score, which was considered the best available clinical tool for risk stratification. However, within each subgroup, marked heterogeneity was noticed regarding clinical outcomes. This may be due to biological parameters that are probably not taken into account by this index. Therefore, in the past years, many efforts have been made to find biological prognostic markers able to stratify DLBCL cases into prognostically different groups .
Gene expression profiling (GEP) studies have been performed to divide DLBCL into two major molecular subtypes: germinal center B-cell (GCB) like and non-GCB like. The latter was associated with inferior overall survival. However, to date, GEP has not been routinely feasible. Therefore, an attempt was made to correlate each of these two subtypes with immunohistochemical markers, which are CD10 and BCL6 for GCB type and MUM-1 for non-GCB .
Immunohistochemical expression of CD10, BCL6, and MUM-1 was used to divide DLBCL into GCB and non-GCB subtypes .
| Aim of the Work|| |
The present study aimed to investigate the differences between germinal and nongerminal DLBCL cases with respect to their clinicopathological features, including survival and response to therapy.
| Patients and Methods|| |
Sixty-one de-novo DLBCL cases were included in this selective retrospective study. The cases were retrieved from the archival material of Pathology Department, Faculty of Medicine, Menoufia University. They received treatment in Menoufia Cancer Institute between January 2009 and December 2014.
The clinical data were collected from patients' medical records and included age, sex, primary site of involvement (nodal or extranodal) , extent of nodal involvement (generalized or localized) , Ann-Arbor staging , splenic involvement, bone marrow involvement, lactate dehydrogenase (LDH) values , performance status (PS) , international prognostic index (IPI) score , revised IPI score , AAIPI , and recurrence .
All patients received six cycles of chemotherapy, CHOP-R (cyclophosphamide, hydroxydaunorubicin, oncovin, and prednisolone – rituximab). After the completion of treatment, immediate clinical response was assessed on the basis of the criteria revised by the International Working Formulation Group .
Histopathological examination of hematoxylin and eosin-stained sections was performed, to confirm the diagnosis and evaluate the following:
- Lymph node architecture: This was either partially or totally effaced 
- Capsular invasion and perinodal fat involvement: This was evaluated for evidence of neoplastic invasion 
- Extent of necrosis: This was expressed in percentage
- Histopathological variants of DLBCL: These were centroblastic , immunoblastic , T-cell/histiocyte-rich DLBCL , and anaplastic large cell type 
- Mitotic count .
The streptavidin–biotin amplified system was used for immunostaining. The slides were submitted to subsequent steps of deparaffinization and rehydration. Antigen retrieval was performed by boiling in citrate buffer saline (pH 6) for CD10 and MUM-1 and in EDTA buffer (pH 8) for BCL6, followed by cooling at room temperature. CD10 (Cat. # 081287), BCL6 (Cat. # MS-1114-R7), and MUM-1 (Cat. # MA1–25525) (mouse monoclonal antibodies; LabVision/Neomarkers, Fremont, California, USA) were used. CD10 and BCL6 were received as 7.0 ml ready-to-use form and MUM-1 was received as a 1.0 ml concentrate (diluted by using PBS at a dilution 1: 100). Incubation with primary antibodies was performed overnight at room temperature. Thereafter, the secondary antibody was applied with DAB as a chromogen substrate and Mayer's hematoxylin as a counterstain. Positive control (tonsil) for CD10, BCL6, and MUM-1 and negative control (omitting of primary antibody step) were included in each run of staining.
For all antibodies, cases were considered positive if more than 30% of tumor cells showed true expression (membranous expression for CD10 and nuclear expression for BCL6 and MUM-1) .
Depending on Hans' algorithm : the case was considered GCB type when CD10 was positive or CD10 was negative, BCL6 was positive, and MUM-1 was negative.
The case was considered non-GCB type when CD10 was negative and BCL6 was positive or CD10 was negative, BCL6 was positive, and MUM-1 was positive.
Survival time was calculated in months from the date of diagnosis and ended with the death of the patient or the last follow-up visit. Overall survival time was available for 44/61 (72.13%) DLBCL cases (25 GCB and 19 non-GCB). The range of survival time was 1–105 months with a mean ± SD of 16.25 ± 17.87 months and a median of 9.5 months.
Data were collected, tabulated, and statistically analyzed using a personal computer with statistical package for the social sciences (SPSS Inc, Chicago, Illinois, USA), version 20. c 2 and Fisher's exact tests were used to compare qualitative variables. The Mann–Whitney (U) and Kruskal–Wallis (K) tests were used to compare quantitative variables. Log-rank and Cox regression analysis were used for life-table assessment. P values less than or equal to 0.05 were considered significant .
| Results|| |
The clinicopathological data of the studied DLBCL cases are presented in [Table 1] and [Figure 1].
|Figure 1: Histopathological variants of diffuse large cell B-cell lymphoma. (hematoxylin and eosin, ×400). (a) Centroblastic variant. (b) Immunoblastic variant. (c) Anaplastic variant. (d) T-cell histiocyte-rich cell variant.|
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Immunohistochemical results of CD10, BCL6, and MUM-1 expression
Twenty-eight (28/61, 45.9%) cases were positive for CD10 [Figure 2]. Ten out of 33 (negative for CD10) cases were positive for BCL6 and 23 cases were negative [Figure 3]. Seven out of the 10 cases positive for BCL6 were positive for MUM-1 and three were negative [Figure 4]. Thus, the GCB group included 31/61 (50.8%) cases (28 cases CD10-positve and 31 cases CD10-negative, BCL6-positive, and MUM-1-negative) and the non-GCB group included 30/61 (49.2%) cases (23 cases CD10-negative and BCL6-negative and seven cases CD10-negative, BCL6-positive, and MUM-1-positive) [Figure 5].
|Figure 2: (a) Case of DLBCL showing membranous expression for CD10 in more than 30% of malignant cells. (b) Another case negative for CD10 (immunohistochemistry, ×400). DLBCL, diffuse large B-cell lymphoma.|
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|Figure 3: (a) Case of DLBCL showing nuclear expression for BCL6 in more than 30% of malignant cells. (b) Another case negative for BCL6 (immunohistochemistry, ×400). DLBCL, diffuse large B-cell lymphoma.|
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|Figure 4: (a) Case of DLBCL showing nuclear expression for MUM-1 in more than 30% of malignant cells. (b) Another cases negative for MUM-1 (immunohistochemistry, ×200). DLBCL, diffuse large B-cell lymphoma.|
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|Figure 5: Classification of DLBCL into GCB and non.GCB using Hansf algorthim. DLBCL, diffuse large B.cell lymphoma; GCB, germinal center B.cell.|
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Comparison between GCB and non-GCB groups regarding the studied clinical and pathological parameters
There were significant differences between GCB and non-GCB cases regarding tumor size, splenic involvement, and PS. The non-GCB group had significantly larger tumor sizes compared with the GCB group (P = 0.03). Fourteen out of 19 (14/19, 73.7%) DLBCL cases with splenic involvement were of non-GCB type (P = 0.01). Overall, 90% of cases with poor PS were of the GCB type (P = 0.01) ([Table 2]).
|Table 2 Differences between GCB and non-GCB DLBCL cases regarding the clinicopathological data|
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Comparison between CD10-negative and CD10-positive cases regarding the studied clinical and pathological parameters
CD10-positive cases were significantly associated with small tumor size (P = 0.03), absence of splenic involvement (P = 0.01), and poor PS (P = 0.03) in comparison with negative cases ([Table 3]).
|Table 3 Differences between CD10 negative and positive cases regarding the clinicopathological data|
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Response to therapy
Data on response to therapy were available for 49/61 (80.31%) DLBCL patients (25 GCB and 24 non-GCB). Thirty-six cases out of 49 (73.5%) showed complete response, whereas 13/49 (26.5%) showed partial response and stable disease. There was no significant difference between GCB and non-GCB subtypes regarding response to therapy (P = 0.21).
Univariate survival analysis
There was no significant difference between GCB and non-GCB subtypes regarding overall survival (P = 0.13, log-rank test = 2.25); however, absence of CD10 expression showed better overall survival (P = 0.04, log-rank test = 4.06) compared with positive cases [Figure 6].
|Figure 6: Kaplan-Meier of overall survival for CD10-negative and CD10-positive groups (P = 0.04, log-rank test = 4.06).|
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| Discussion|| |
In this study, we classified DLBCL into GCB and non-GCB subtypes using three primary antibodies – CD10, BCL6, and MUM-1 – according to Hans' algorithm . Many previous studies used Hans' algorithm to classify DLBCL ,,,,,.
Other studies used Choi algorithm, which depends on CD10, BCL6, MUM-1, GCET1, and FOXP1 primary antibodies . Although many algorithms were applied, such as modified Hans' (CD10 and MUM-1) , modified Choi (CD10, MUM-1, GCET1, and FOXP1) , Muris (CD10, MUM-1, and BCL2) , Nyman (MUM-1 and FOXP1) , Tally (CD10, MUM-1, GCET1, and FOXP1) , and Natkunam (LMO2) , Hans' algorithm is still the most valid and commonly used one ,.
Half of our cases were of GCB subtype (50.8%). In previous studies, the percentage of GCB cases ranged from 45.2 to 65% ,,,,,. There is no specific reason known for this different distribution of prognostic subgroups. It may be due to different genetic makeup and specific genetic aberrations pertaining to different populations .
In the present study, we noticed that nongerminal cases carried bad prognostic impact. This was manifested by the association of non-GCB group with large tumor size (P = 0.03). This agreed with the results of Zizhen et al. , who found that the non-GCB primary gastric DLBCL was significantly associated with larger tumor size. However, van Imhoff et al.  stated that most GCB cases were more bulky.
Most nongerminal cases (73.7%) showed splenic involvement (P = 0.01), agreeing with Yeh et al. , who stated that DLBCL presenting initially in bone marrow, liver, or spleen was commonly of a nongerminal immunophenotype. Tanaka et al.  studied three cases of primary splenic lymphoma; two of them (66.6%) were of non-GCB subtype.
PS is a parameter included in the calculation of IPI and AAIPI . Despite the absence of a significant relationship between the GCB and non-GCB subtypes regarding both IPI and AAIPI, 90% of cases with poor PS (2, 3, and 4) were of GCB group (P = 0.01). However, other studies did not show any significant differences between the two groups regarding PS ,,.
Alizadeh and colleagues used GEP to classify DLBCL cases into GCB and non-GCB type. Although they used hundreds of genes to stratify the cases, they concluded that there was residual clinical heterogeneity that cannot be explained by their classification. They could not identify which of the genes that distinguish GCB from non-GCB cases were the most important determinants of response to therapy. In their study, although GCB cases had an overall favorable prognosis, five patients died within the first 2 years of diagnosis. On the other hand, three patients in the non-GCB subgroup were alive 5 years after treatment (despite the poor outcome of most patients in this subgroup) .
DLBCL with translocation of both MYC and BCL2, termed double-hit lymphoma, is characterized by an aggressive clinical course . Moreover, the germinal center subtype patients with isolated BCL2 translocations had significantly worse outcome than the patients without BCL2 rearrangements (P = 0.0002), and their outcome was similar to that of patients with the nongerminal subtype .
Dybkaer and colleagues proposed a refined classification system based on subset-specific B-cell-associated gene signatures in the normal B-cell hierarchy and found that the centrocyte subtype had a superior prognosis compared with the centroblast subtype of GCB-like DLBCL. The centroblast subtype had a complex genotype, whereas the centrocyte subtype had high TP53 mutation and insertion/deletion frequencies and expressed LMO2, CD58, and stromal-1-signature and major histocompatibility complex class II-signature genes, which are known to have a positive impact on prognosis .
In this study, comparison between GCB and non-GCB cases as regards revised IPI, AAIPI (≤60 and > 60), tumor stage, and LDH values showed no significant differences (P = 0.36, 0.55, 0.33, 0.94, and 0.35, respectively), agreeing with Hans et al. , Berglund et al.  and van Imhoff et al. , in whose studies the two subtypes of DLBCL did not differ with respect to these clinical features.
The differences between GCB and non-GCB groups regarding clinicopathological features may be explained by the fact that DLBCL subgroups had distinct patterns of genomic alterations and particular chromosomal alterations that affect discrete biologic features of the tumors as defined by gene expression signatures. GCB DLBCL appeared to be derived from GCBs, and non-GCB DLBCL may be derived from a post-GCB undergoing plasmacytic differentiation .
Also, advances in molecular genetics have vastly improved our understanding of the biological diversity of DLBCL and have led to the discovery of key oncogenic pathways. In addition to the major molecular designations of GCB and activated B-cell subtypes, next-generation sequencing technologies have unveiled the remarkable complexity of DLBCL .
Regarding response to therapy, this study revealed no significant difference between the two groups (P = 0.21), agreeing with Borovecki et al. , Veelken et al. , and Fu et al. . In contrast, Hassan et al.  stated that GCB gave a better immediate clinical response to chemotherapy compared with non-GCB.
In the present study, there was no significant difference regarding overall survival between germinal and nongerminal DLBCL (P = 0.13). Previous studies agreed with the results of the present study as regards the lack of significance of overall survival ,. However, some studies supported an excellent concordance between subgrouping of DLBCL into germinal and nongerminal subtypes and overall survival .
Several factors may account for this discrepancy, including the studied population, the methodology used, and interobserver discrepancies in the interpretations of the immunostaining slides . Also, the differences may partly be explained by the differences in the cutoff value (10–30%) for a positive staining. In all, 10% is most commonly applied, but many studies found that this level might be too low to subdivide DLBCL into manageable subgroups with similar numbers of patients ,.
CD10 is a membrane-associated, neutral endopeptidase that is expressed in a variety of human tissues but has a restricted expression in the germinal center cells of reactive lymphoid tissues . The current study showed that CD10-negative cases carried poor prognostic impact. This was manifested by the association of CD10-negative cases with increased tumor size (P = 0.03) and involvement of the spleen (P = 0.01). However, CD10-negative cases were associated with good PS (P = 0.03) and longer overall survival on univariate analysis (P = 0.04). However, multivariate analysis revealed that CD10 positivity was not an independent prognostic factor (P = 0.07).
Harada et al.  and Veelken et al.  found that CD10 expression was associated with inferior overall survival. Previous studies using flow cytometry also suggested that CD10 expression in DLBCL may predict inferior survival ,. Other studies noticed no differences in the outcome for patients with DLBCL that express CD10 ,.
Our results contradicted other studies, which found that CD10 expression was associated with improved overall survival ,. However, given the variability of outcomes in these retrospective studies, it is doubtful that CD10 alone can be used to predict survival in DLBCL.
| Conclusion|| |
DLBCL should be classified into GCB and non-GCB subtypes because they prognostically differ. Hans' algorthim is a simple and reliable method for the classification of DLBCL, but it should be applied on a large number of cases to be used as a routine method for DLBCL evaluation. The similarity in the results of dividing DLBCL cases according to Hans' algorithm and by CD10 expression necessitates larger studies and longer follow-ups to verify whether CD10 alone can replace Hans' algorithm.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3]