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ORIGINAL ARTICLE Table of Contents  
Ahead of print publication
Cell of origin classification of diffuse large B-Cell lymphoma


 Department of Pathology, Maulana Azad Medical College, New Delhi, India

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Date of Submission24-Jul-2022
Date of Decision12-Nov-2022
Date of Acceptance17-Nov-2022
Date of Web Publication19-Jan-2023
 

  Abstract 


Context: Diffuse large B-cell lymphoma (DLBCL) is a neoplasm of medium-to-large B lymphoid cells with diffuse growth patterns. Although it is a potentially curable disease, around 40% of the cases are either refractory to primary treatment or relapse. Based on gene expression profiling (GEP), DLBCL can be classified as germinal center B-cell subtype (GCB) and activated B-cell subtype (ABC). About 10%–15% of cases do not convincingly fall into either of the two subtypes and hence remain unclassified. Most widely used and suggested by WHO is Hans algorithm comprising immunohistochemical markers CD10, B-cell lymphoma6 (BCL6), and IRF4/MUM1, which classifies CD10+ and CD10-/BCL6+/MUM1-DLBCL as GCB, while CD10-/BCL6+/MUM1 + and BCL6-DLBCL as non-GCB. Aims: The aim of this study was to classify DLBCL into GCB and non-GCB subtypes using Hans Algorithm. Settings and Design: This was a retrospective study. Materials and Methods: Twenty-eight histologically diagnosed cases of nodal (71.4%), as well as extranodal (28.6%) DLBCL, were taken over the period of 2 years with age ranging between 10 and 65 years with 19 males and 9 females. M: F = 2.1:1. Depending upon the site involved, a primary panel of immunohistochemistry (IHC) markers, namely CD20, CD3, LCA, EMA, and CK, followed by a secondary panel comprising CD10, CD19, CD30, LMP1, BCL2, BCL6, MUM1, MYC, and FOXP1 was used. Results: In this study, it was found that the non-GCB subtype was more common than the GCB subtype in Indian population. Conclusions: Although the gold standard of GEP to assign cells of origin is using RNA microarray analysis, however, due to resource constraints and other limitations such as long turnaround times, IHC is the next acceptable alternative.

Keywords: Cell of origin classification, diffuse large B-cell lymphoma, Hans algorithm


How to cite this URL:
Dhar L, Singh S, Jain SL, Vindal A, Sinha P, Gautam R. Cell of origin classification of diffuse large B-Cell lymphoma. J Microsc Ultrastruct [Epub ahead of print] [cited 2023 Feb 8]. Available from: https://www.jmau.org/preprintarticle.asp?id=368036





  Introduction Top


Diffuse large B-cell lymphoma (DLBCL) is a neoplasm of medium-to-large B lymphoid cells with diffuse growth pattern constituting 25%–35% of non-Hodgkin lymphomas in West[1] and 60%–70% of all BCL in Asia.[2] The incidence is around 62% in India.[3] Around 40% of the cases are either refractory to primary treatment or relapse.[4]

DLBCL can be either nodal or extranodal with approximately 40% being extranodal among which the gastrointestinal tract is most commonly involved.[1] Other sites of involvement are bone, liver, kidneys, adrenal glands, spleen, testes, and thyroid.

Clinically, DLBCL patients are classified into low-risk, intermediate-risk, and high-risk groups based on five parameters such as age, lactate dehydrogenase (LDH) levels, Eastern Cooperative Oncology Group (ECOG) performance, clinical stage, and the presence of any extranodal disease. Gene expression profiling (GEP) classifies DLBCL into two subtypes: (1) germinal center B-cell subtype (GCB) and (2) non-GCB subtype or activated B-cell subtype (ABC) which are independent of the International Prognostic Index (IPI) score. Although RNA microarray analysis is the gold standard for GEP, immunohistochemical algorithms are more economical and easier to perform in resource constraint facilities. The most widely used immunohistochemical algorithm suggested by WHO[1] is Hans algorithm comprising CD10, BCL6, andIRF4/MUM1. CD10 is a marker for germinal center proliferation while BCL6 and MUM1 occur more commonly in the ABC.

Various studies have shown that patients treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) only appear to have poorer progression-free survival (PFS) in ABC subtype DLBCL compared to GCB subtype.[5],[6]

In this study, we classify DLBCL into two subtypes based on Hans Algorithm and compare various studies to see the importance of this classification on the prognosis of patients.

Aims

This study aimed to subclassify DLBCL into its subtypes: (1) GCB type and (2) non-GCB type using immunohistochemistry (IHC) and Hans algorithm.

Settings and design

This was a retrospective study.


  Materials and Methods Top


The study comprised 28 histologically diagnosed cases of nodal (71.4%) as well as extranodal (28.6%) DLBCL that was diagnosed over the period of 2 years, and the retrospective analysis of data was done, and further IHC with the detailed panel using Hans algorithm was performed.

Ethical approval was taken from Maulana Azad medical college Institutional ethical committee with reference number F.no. 18/ IEC/ MAMC/ 2018/ Path dated 29/8/2018.

The biopsy was conducted in the department of surgery and processed in the department of pathology. Paraffin-embedded blocks were made, and H- and E-stained sections were examined for diagnosis. IHC was done on sections on poly-L-lysine-coated slides depending on the site of involvement. A primary panel of IHC markers, namely CD20, CD3, LCA, EMA, and CK, followed by a secondary panel comprising CD10, CD19, CD30, LMP1, BCL2, BCL6, MUM1, MYC, and FOXP1 was used [Table 1].
Table 1: Depicts the antibodies used with their clone and supplier

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Interpretation of immunohistochemistry

Subtyping was done using Hans algorithm

CD10 positive or CD10 negative, BCL6 positive, MUM1 negative as GCB and CD10 negative or BCL6 negative or BCL6 positive, MUM1 positive as Non-GCB [Table 2].
Table 2: Depicts the cutoff and staining character of each antibody

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  Results Top


The patients ranged between 10 and 65 years in age, with a median age of 34 years with 19 males and 9 females; male: female ratio being 2.1:1.

Out of 28 patients, 20 (71.4%) presented with nodal involvement, either single or multiple. The most commonly involved nodes were the cervical and inguinal lymph nodes.

Eight out of 28 patients (28.6%) presented with extranodal involvement, out of which 3/8 (37.5%) patients presented with abdominal pain and distension. On radiological examination, these patients had mesenteric masses. Four out of 8 (50%) of patients came with nonhealing ulcers in the upper aerodigestive tract.

One out of 8 (12.5%) patients had gradually progressive midline neck swelling and had symptoms of dysphagia and change in voice due to involvement.

Three out of 28 (10.7%) patients had a previous history of immunosuppression; 2/3 (66.7%) had old-treated Koch's while 1/3 (33.3%) was HIV positive.

One out of 28 (0.04%) patients also had a previous history of Hodgkin's lymphoma.

The various sites involved are shown in [Figure 1].
Figure 1: Sites involved (n = 28)

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The microphotograph of H- and E-stained sections and positive IHC sections are shown in [Figure 2], [Figure 3], [Figure 4]. IHC interpretation as shown in [Table 3].
Figure 2: (a) H and E stained microphotograph of atypical cells of DLBCL (×40), (b) H and E stained microphotograph of atypical cells of DLBCL (×400), (c) DAB stained Microphotograph showing CD20 positivity in Atypical cells, DLBCL (×40), (d) DAB stained Microphotograph showing CD10 positivity in Atypical cells, DLBCL (×40), (e) DAB stained Microphotograph showing BCL6 positivity in Atypical cells, DLBCL (×40) (f) DAB stained Microphotograph showing MUM1 positivity in Atypical cells, DLBCL (×40) DLBCL: Diffuse large B-cell lymphoma, DAB: Diaminobenzidine, BCL: B-cell lymphoma

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Figure 3: (a) DAB stained Microphotograph showing CD19 positivity in Atypical cells, DLBCL (×40), (b) DAB stained Microphotograph showing MYC positivity in Atypical cells, DLBCL (×40), (c) DAB stained Microphotograph showing BCL2 positivity in Atypical cells, DLBCL (×200), DLBCL: Diffuse Large B-Cell Lymphoma, DAB: Diaminobenzidine, BCL: B-Cell Lymphoma

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Figure 4: (a) DAB stained Microphotograph showing LMP1 positivity in Atypical cells, DLBCL (×40). (b) DAB stained Microphotograph showing CD30 positivity in Atypical cells, DLBCL (×400), (c) DAB stained Microphotograph showing FOXP1 positivity in Atypical cells, DLBCL (×40). DLBCL: Diffuse Large B-Cell Lymphoma, DAB: Diaminobenzidine

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Table 3: Immunohistochemical interpretation and cell of origin classification of cases

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Two cases were positive for BCL6 (12.5%). Out of these two cases, one was CD10-/BCL6+/MUM1-and one was CD10-/BCL6+/MUM1+.

Two cases were positive for both CD10 and MUM1 and three cases were negative for CD10 and MUM1.

Co-expression of BCL6 and MUM1 was seen in one case (3.5%).

Using the Hans algorithm, 13/28 cases (46.4%) were GCB and 15/28 (53.6%) were non-GCB.

8/28 (28.5%) cases were dual expressors for MYC and BCL2. 1/28 (0.04%) case was dual expressor for MYC and BCL6.

Furthermore, 4/28 (14.2%) cases were found to be CD30 positive, out of which 3/28 (10.7%) were LMP1 positive, hence were classified under Epstein–Barr virus (EBV)-positive DLBCL, not otherwise specified.


  Discussion Top


DLBCL is a neoplasm of medium-to-large B lymphoid cells. The nuclear size if the tumor cells are more than twice the size of a normal lymphocyte. Moreover, a diffuse pattern of growth is seen.[1]

DLBCL can either be primary that arises de novo or they can be secondary. Secondary DLBCL may occur due to the transformation of other less aggressive tumors such as chronic lymphocytic leukemia (2%–8%), follicular lymphoma (25%–35%), nodal marginal zone lymphoma (3.8%–13%),[7] and nodular lymphocyte predominant Hodgkin's lymphoma (3%–5%).[1] In this study, only one patient (1/28 cases; 3.57%) had a previous history of Hodgkin's lymphoma, making it the single case of secondary DLBCL.

Immunodeficiency is one of the risk factors of DLBCL, in which EBV positivity can be seen in 3%–10% of cases.[1] 3/28 (10.7%) cases in this study had a previous history of immunosuppression (TB and HIV). 3/28 (10.7%) were positive for LMP1 making them EBV-positive DLBCL.[1]

DLBCL can be either nodal or extranodal with approximately 40% being extranodal among which the gastrointestinal tract is most commonly involved.[1] Other sites of involvement are bone, liver, kidneys, adrenal glands, spleen, testes, and thyroid. In this study, 71.5% of cases were nodal while 28.5% of cases were extranodal DLBCL.

The IPI classification of DLBCL is based on five criteria which include age, LDH levels, ECOG performance, clinical stage, and the presence of extranodal disease. They are divided as low-, intermediate-, and high-risk groups. However, this fails to predict the PFS based on tumor biology, leading to the need for the cell of origin classification. The most economic and uncomplicated approach for cell of origin (COO) subclassification of DLBCL is IHC and various algorithms based on IHC. Hans algorithm, suggested by WHO has a sensitivity of 85%–90% and specificity of 52%–82%.[8]

The Hans algorithm subclassifies DLBCL into GCB and non-GCB. The non-GCB DLBCL can either be ABC or unclassified.

CD10 is expressed in 30%–50% DLBCL cases and is a marker of germinal center differentiation and hence seen in the GCB subtype. The most common translocation occurring in DLBCL is the rearrangement of 3q27 involving BCL6 and it occurs more commonly in the ABC subtype. It is expressed in 60%–90% of cases. MUM1 (also called IRF4) expression is necessary for the proliferation of B lymphocytes that occurs in response to signals from antigen receptors.[9] It is seen in 35%–65% of ABC DLBCL cases which is responsible for the proliferation of the tumor cells. Co-expression of BCL6 and MUM1 can be seen in up to 50% of cases of DLBCL. FOXP1 is expressed in 20% of DLBCL (non-GCB subtype), while BCL2 gene translocation is observed in 20%–30% of DLBCL, more commonly the GCB subtype. MYC rearrangement is seen in 8%–14% of cases and can be seen both in the GCB and ABC subtypes.

Cases which have both MYC and BCL2/BCL6 expressions are called double expressors. However, cases with both MYC and BCL2/BCL6 translocation are called double-hit lymphomas. Double-hit Lymphomas belong to a separate category of high-grade BCLs and not DLBCL. In this study, 8/28 (28.5%) were double expressor for MYC and BCL2 and 1/28 (0.04%) cases were double expressor for MYC and BCL6; which makes a total of 9/28 (32.1%) cases as double expressors. Other important markers to distinguish between GCB and non-GCB are GCET1 expression which is seen in 40%–50% of cases and is highly correlated with the GCB subtype and LMO2 expression which is seen in 45% of cases, also highly correlating with GCB subtype.

Subclassification of DLBCL aids in deciding the chemotherapy with the most favorable outcome and hence the prognosis. Various studies have suggested that the NF-κB pathway remains active and there is increased expression of NF-κB gene signaling in the ABC subtype and hence is one of the important factors in its pathogenesis.[5],[6] The prognosis of ABC is worse than that of GCB. A study conducted by Lenz et al. in 2008 showed that the 5-year PFS rate with R-CHOP therapy was 74% in GCB DLBCL and 40% in ABC DLBCL.[10] Other studies showed that therapy with dose-adjusted (DA)-EPOCH-R had better overall survival (OS) in ABC DLBCL ranging between 56% and 67%, the OS being 100% in GCB DLBCL.[11]

Another study conducted in 2009 by Dunleavy et al. showed that the addition of bortezomib enhances the chemotherapy effectiveness in ABC DLBCL but has no role in GCB DLBCL. Treatment with bortezomib-DA-EPOCH in case of relapsed or refractory ABC DLBCL had a median OS of 10.8 months, an overall response rate and complete response rate of 83% and 42%, respectively.[12] Hence, drugs such as etoposide, bortezomib, lenalidomide, ibrutinib, vincristine, doxorubicin, cyclophosphamide, and prednisone along with R-CHOP have improved the clinical outcome in ABC-DLBCL.

In this study, it was found that the non-GCB subtype was more common than the GCB subtype (53.6% vs. 46.4%) in Indian population. This was in concordance with a retrospective study conducted by Vaid et al. in Indian population using molecular subtyping in 198 cases of DLBCL from 2009 to 2017 where it was found that 45.5% of cases were GCB and 54.5% were ABC DLBCL. This further necessitates the subtyping of DLBCL so as to ameliorate the prognosis and OS of the patients.[13]

GEP helps identifying the various subtypes of DLBCL. Although the gold standard of GEP to assign COO is using RNA microarray analysis, however, due to resource constraints and other limitations such as long turnaround times, IHC is the next acceptable alternative.


  Conclusion Top


In a resource constraint institute and place, immunohistochemical panels and various algorithms such as WHO suggested Hans algorithm can help subclassifying DLBCL and help improve treatment and prognosis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Swerdlow S, Campo E, Harris N, Jaffe E, Pileri S, Steun H, et al. WHO Classificatiom of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed. Lyon: IARC; 2017.  Back to cited text no. 1
    
2.
Flowers CR, Fedewa SA, Chen AY, Nastoupil LJ, Lipscomb J, Brawley OW, et al. Disparities in the early adoption of chemoimmunotherapy for diffuse large B-cell lymphoma in the United States. Cancer Epidemiol Biomarkers Prev 2012;21:1520-30.  Back to cited text no. 2
    
3.
Chen Y, Han T, Iqbal J, Irons R, Chan WC, Zhu X, et al. Diffuse large B-cell lymphoma in Chinese patients: Immunophenotypic and cytogenetic analyses of 124 cases. Am J Clin Pathol 2010;133:305-13.  Back to cited text no. 3
    
4.
Nair R, Arora N, Mallath MK. Epidemiology of non-Hodgkin's lymphoma in India. Oncology 2016;91 Suppl 1:18-25.  Back to cited text no. 4
    
5.
Dunleavy K, Wilson WH. Appropriate management of molecular subtypes of diffuse large B-cell lymphoma. Oncology (Williston Park) 2014;28:326-34.  Back to cited text no. 5
    
6.
Miyazaki K. Treatment of diffuse large B-Cell lymphoma. J Clin Exp Hematop 2016;56:79-88.  Back to cited text no. 6
    
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Alderuccio J, Lossos I. Prognostic factors and risk of transformation in marginal zone lymphoma. Ann Lymphoma 2020;4:6.  Back to cited text no. 7
    
8.
Riedall P, Smith S. Should we use cell of origin and Dual protein expression in treating DLBCL. Chron lymphoma Myeloma Leuk 2017;8:91-7.  Back to cited text no. 8
    
9.
Alizadeh AA, Eisen MB, Davis RE, Ma C, Lossos IS, Rosenwald A, et al. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature 2000;403:503-11.  Back to cited text no. 9
    
10.
Lenz G, Wright G, Dave SS, Xiao W, Powell J, Zhao H, et al. Stromal gene signatures in large-B-cell lymphomas. N Engl J Med 2008;359:2313-23.  Back to cited text no. 10
    
11.
Wilson WH, Jung SH, Porcu P, Hurd D, Johnson J, Martin SE, et al. A Cancer and Leukemia Group B multi-center study of DA-EPOCH-rituximab in untreated diffuse large B-cell lymphoma with analysis of outcome by molecular subtype. Haematologica 2012;97:758-65.  Back to cited text no. 11
    
12.
Dunleavy K, Pittaluga S, Czuczman MS, Dave SS, Wright G, Grant N, et al. Differential efficacy of bortezomib plus chemotherapy within molecular subtypes of diffuse large B-cell lymphoma. Blood 2009;113:6069-76.  Back to cited text no. 12
    
13.
Vaid A, Karanth S, Khurana A, Sood N, Sen A, Dheeraj G, et al. Difference in clinic-pathological features, prognostic features and treatment outcomes between molecular subtypes among Indian cohort with diffuse large B-cell lymphoma- single centre experience. Blood 2019;134:5350-3.  Back to cited text no. 13
    

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Correspondence Address:
Lity Dhar,
Room No. 62, Ground Floor, Pathology Block, Maulana Azad Medical College, Bahadur Shah Zafar Marg, New Delhi - 110 002
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jmau.jmau_66_22



    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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