“A Retro-Prospective Observational Study to Determine the Proportion of Double Expressors in Diffuse Large B-Cell Lymphomas, Not Otherwise Specified (DLBCL, NOS) in a Tertiary Care Setting.”

Dr. Pallavi Rangan* ¹, Mandolkar M ², Kulkarni P ³

1,2,3. Department of Pathology, Deenanath Mangeshkar Hospital and Research Centre, Pune, Maharashtra, India.

Corresponding Author: Dr. Pallavi Rangan, Department of Pathology, Deenanath Mangeshkar Hospital and Research Centre, Pune, Maharashtra, India.

Copy Right: © 2022 Dr. Pallavi Rangan, This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Received Date: September 24, 2022

Published Date: October 01, 2022

 

Abstract

“A retro-prospective observational study to determine the proportion of double expressors in diffuse large b-cell lymphomas, not otherwise specified (dlbcl, nos) in a tertiary care setting.”

Aim: To study the proportion of cases of Double Expressors (DE) in Diffuse Large B-Cell Lymphomas, not otherwise specified (DLBCL, NOS) in a tertiary care setting.

Objectives

1. To determine the proportion of cases of Double Expressors which show coexpression of BCL-2 and c-myc proteins by Immunohistochemistry in cases of DLBCL, NOS.

2. To characterize the patients according to their age-groups, gender and transformation from a lower grade lymphoma and to report these numbers.

3. To correlate, when possible, with the results of Fluorescent In-Situ Hybridization (FISH) to detect cases that harbor a c-myc translocation also showing a BCL2 translocation therefore belonging in the category of Double- Hit (DH) lymphoma.

4. To associate between the diagnosis of DE status and the clinical outcome of the patient at a minimum follow-up period of 1-year post diagnosis (regression of primary tumor, appearance of new lesions and CNS metastases or death.)

Materials and Methods: Specimens from nodal and extra-nodal sites diagnosed as Diffuse Large B-Cell Lymphoma, not otherwise specified (DLBCL, NOS) preserved as paraffin blocks were subjected to Immunohistochemical (IHC) testing for determination of c-myc and BCL2 protein expression. The data was collected from the Amrita HIS and MRD viewer. In co-ordination with the clinical co-guide, the outcome of the patients at a minimum follow-up of 1-year post diagnosis was documented in terms of clinical status and imaging.

Observations & Results: 85/127 cases were noted to be Double Expressors and represented 66.9% of the total cases and 32/127 were noted to be Triple Expressors represented 25.1% of the total cases of DLBCL, NOS.

Using the Hans algorithm, it was noted that 66 cases were of the GCB phenotype (66%) and 34 cases were of the ABC phenotype (34%.) Double Expressors (78.78%) and Triple Expressors (31.81%) were seen more commonly in the cases with GCB phenotype. 8 cases of transformations from low grade non-Hodgkin’s lymphoma were noted and 4 (50%) were known cases of Follicular lymphoma. 16 cases underwent testing by FISH. 7/16 (43.7%) cases were Double Expressors for BCL2 and c-myc by IHC. 2/7 (28.5%) cases were positive for a genetic aberration of c-myc gene, and were thus considered to be Double Hit Lymphomas. In our study, 19% of the total cases of DLBCL, NOS was the proposed population proportion of Double Hit Lymphomas. The clinical evaluation of patients at 1 year post diagnosis revealed that DLBCL, NOS cases of the GCB phenotype showed a better response to chemotherapy and had a better prognosis, as compared to their ABC phenotype counterparts. Post chemotherapy, the highest rate of relapse of 25% was seen in the group showing strong >70% staining for c- myc.

Conclusion

The results are reliable enough to predict the proportion of Double Expressors and Triple Expressors among total number of DLBCL, NOS cases. These cases should ideally be investigated with the help of FISH or PCR for detection of genetic aberrations. This study also predicts the proportion of Double Hit Lymphomas among Double Expressor Lymphomas.

Cases of DLBCL, NOS should also be screened according to the intensity of c-myc staining by IHC. The number of cases with genetic aberrations may not always meet the international reporting guidelines for screening for protein over-expression (40% nuclear positivity for c-myc.) The guidelines for reporting c-myc positivity by IHC should be reviewed to include a greater range of subjects. Cases showing c-myc staining > 70% positivity should be screened for CNS lesions to rule out involvement or relapse at the time of diagnosis and regular follow-up visits. If and when resources permit, all cases of DLBCL, NOS can be evaluated for genetic aberrations so that the ideal targeted chemotherapy is received by the patient to improve prognosis.

 

List of Abbreviations

ABC    : Activated B-Cell

ASCT  : Autologous Stem Cell Transplant

BCL2  : B-Cell lymphoma 2 protein

BCL6  : B-Cell lymphoma 6 protein

CAR-T cell     : Chimeric Antigen Receptor T-Cell

CD       : Cluster of Differentiation

CHOP  : Cyclophosphamide, Hydroxyduanorubicin, Vincristine Sulphate, Prednisone

c-myc  : Cellular myelocytomatosis oncogene

COO    : Cell of Origin

CORAL: Collaborative Trial in Relapsed Aggressive Lymphoma

CNS    : Central Nervous System

CSF     : Cerebrospinal fluid

CNS-IPI: Central Nervous System International Prognostic Index

CT       : Computed Tomography

DAB    : Diaminobenzidine tetrahydrochloride

DE       : Double Expressor

DEL    : Double Expressor Lymphoma

DH      : Double Hit

DHL    : Double Hit Lymphoma

DLBCL: Diffuse Large B-Cell Lymphoma

EBV    : Epstein-Barr Virus

EPOCH: Etoposide, Prednisone, Oncovin, Cyclophosphamide, Hydroxyduanorubicin

FDG-PET: Fluorodeoxyglucose Positron Emission Tomography

FNAB  : Fine Needle Aspiration Biopsy

FISH    : Fluorescent in-Situ Hybridization

GCB    : Germinal Centre B-Cell

GEP     : Gene Expression Profiling

H&E    : Hematoxyline & Eosin

IHC     : Immunohistochemistry

IRF4    : Interferon regulatory factor 4

IPI       : International Prognostic Index

LDH    : Lactate dehydrogenase

MUM1 : Multiple Myeloma Oncogene 1

MRD   : Medical records department

c-myc  : c-myc oncogene

NCCN : National Comprehensive Cancer Network

NHL    : non-Hodgkin’s Lymphoma

NOS    : Not otherwise specified

OS       : Overall survival

PAX    : Paired box family of transcription factors

PCR     : Polymerase chain reaction

PET     : Positron Emission Tomography

PFS      : Progression-free survival

RT-PCR : Real Time Polymerase chain reaction

R-hyper CVAD: Hyperfractionated Cyclophosphamide, Vincristine, Doxorubicin, & Dexamethasone alternating with Methotrexate and Cytarabine

R-CODOX-M/IVAC: Cyclophosphamide, Vincristine, Doxorubicin, and high-dose Methotrexate alternating with Ifosfamide, Etoposide, and Cytarabine

R-C HOP         : Rituximab, Cyclophosphamide, Hydroxyduanorubicin, Oncovin, Prednisone

R-D HAP        : Rituximab, Dexamethasone, High-Dose Cytarabine, and Cisplatin

R-E POCH      : Rituximab, Etoposide, Prednisone, Oncovin, Cyclophosphamide Hydroxyduanorubicin

R-ICE  : Rituximab, Ifosfamide, Carboplatin, and Etoposide

SD       : Standard deviation

SPSS   : Software Package for the Social Sciences

TE       : Triple Expressor

TEL     : Triple Expressor Lymphoma

WHO   : World Health Organization

Introduction

The Rappaport Classification system for lymphomas was introduced in 1958. At that time, it was thought that large cell tumors were not of lymphoid origin. The system highlighted the origin of certain large cell tumors. Treatment was mainly palliative and consisted of nitrogen mustard and anti-metabolites combined with radiation therapy. In the 1980s there was an introduction of a new classification for lymphomas based on clinical course of the disease along with the use of complex combination therapies. The CHOP regimen changed the treatment landscape for Non-Hodgkin’s Lymphomas. Nowadays the WHO classification with over 40 subtypes of Lymphomas exists and with it came a more focused and dedicated approach to clinical research and better drugs. The monoclonal antibody Rituximab showed efficacy in the treatment of NHL in combination with other modalities of chemotherapy, but today, 15 years later this efficacy has been rivalled and exceeded.

Diffuse Large B-Cell Lymphoma (DLBCL) is a clinically and biologically heterogenous disease. DLBCL, NOS constitutes 25-35% of adult Non-Hodgkin Lymphomas in developed countries and higher, in developing countries.[40] DLBCL is a neoplasm of medium or large B lymphoid cells whose nuclei are the same size as, or larger than, those of normal macrophages, or more than twice the size of those of normal lymphocytes, with a diffuse growth pattern. Morphological, biological, and clinical studies divide DLBCLs into morphological variants, molecular sub-types, and distinct disease entities. However, there remain many cases that may be biologically heterogeneous which are classified as

Table No. 1: Classification of Large B-cell lymphomas WHO Classification of Tumors of Haematopoietic and Lymphoid Tissues, 2017 Revised

DLBCL, NOS, which encompasses all cases that do not belong to a specific diagnostic category listed. DLBCL, NOS can be subdivided into germinal centre B-cell (GCB) subtype and activated B-cell (ABC) subtype. c-myc protein is expressed in 30-50% of diffuse large B cell lymphoma (DLBCL) and is associated with concomitant expression of BCL2 in 20% to 35% of cases. DLBCLs with co-expression of c-myc and BCL2 are called double-expressor lymphomas (DELs). There is an overexpression of the c- myc oncogene and BCL2 (≥40% and >50% positive staining by IHC, respectively); whereas double-hit lymphomas (DHLs) have c-myc and BCL2 or BCL6 rearrangement as detected by fluorescence in situ hybridization (FISH) or standard cytogenetics. c-myc/BCL2 double expression is an independent risk factor of DLBCL relapse or progression.

c-myc rearrangement (c-myc+) and DH/THL remains significantly prognostic at the time of relapse. DELs account for approximately one-third of de novo disease and up to 50% of relapsed/refractory (RR) DLBCL. In several studies, DELs were shown to have worse outcomes than other DLBCLs. Poor response to standard chemotherapy CHOP or R-CHOP is seen with DHLs and DELs with a median overall survival of <12 months. To corroborate this, previous studies have shown that when comparing cases treatment with R-CHOP and with R-EPOCH, patients treated with R-EPOCH had a better clinical outcome in terms of median overall survival (OS).[38]

 
Etiology

These tumours usually arise de novo(primary) but can also represent transformation of a less aggressive lymphoma (secondary), such as chronic lymphocytic leukaemia/ small lymphocytic lymphoma, follicular lymphoma, marginal zone lymphoma, or nodular lymphocyte predominant Hodgkin lymphoma. DLBCLs, NOS, occurring in the setting of immunodeficiency are more often EBV-positive than sporadic cases.

Tumor localization

Patients present with nodal or extranodal disease. The most commonly involved extranodal site is the gastrointestinal tract (stomach and ileocaecal region; Other common sites include bone, testes, spleen, Waldeyer ring, salivary glands, thyroid, liver, kidneys, and adrenal glands. Bone marrow involvement in DLBCL can be discordant (low-grade B-cell lymphoma in the marrow, seen in 10- 25% of cases) or concordant (large cell lymphoma in the marrow.) Studies have suggested that FDG- PET is a sensitive technique for detecting concordant bone marrow involvement but is not reliable for discordant disease. The most recent consensus criteria for lymphoma staging indicate that a routine staging bone marrow biopsy is no longer required if FDG-PET is negative.

Morphologic involvement of the peripheral blood by DLBCL is rare. Staging investigations include CT scan with or without PET/CT scan, bone marrow aspirate, and biopsy. Limited stage is defined as stage 1A/2A with non-bulky (<10 cm) disease. CNS-directed investigations, Clinical features There is usually a rapidly enlarging tumour mass at single or multiple nodal or extranodal sites at presentation. Almost half of the patients have stage I or II disease, but the inclusion of PET/CT in the initial staging of DLBCL has resulted in stage migration.

 

Microscopy

Three common and several rare morphological variants have been recognized: Centroblastic, Immunoblastic and Anaplastic.

Immunophenotype

The neoplastic cells typically express pan-B-cell markers such as CD19, CD20, CD22, CD79a, and PAX5. The expression of c-myc and BCL2 varies considerably; In most studies, BCL2 is considered positive if 50% of the tumor cells are positive, and c-myc is considered positive if 40% of the tumor cell nuclei are positive. Co- expression of these two proteins (so-called double-expressors) is more frequent in the ABC subtype.

The Hans algorithm uses three markers to distinguish the GCB from the non-GCB subtype: CD10, BCL6, and IRF4/MUM1 and are each considered positive if 30% of the tumor cells stain positively. CD10 is positive in 30-50% of cases, BCL6 in 60- 90%, and IRF4/MUM1 in 35-65% cases. In normal GCBs expression of IRF4/ MUM1 and BCL6 is mutually exclusive, whereas coexpression of these markers is found in 50% of DLBCLs. BCL2 expression is closely linked to the presence of t (14;18) (q32; q21.3) and is more common in the ABC subtype, but is the result of copy number gains and transcriptional upregulation.

Cell of origin/ postulated normal counterpart

The postulated normal counterparts are peripheral mature B cells of either germinal centre origin (GCB subtype) or germinal centre exit I early plasmablastic or post- germinal centre origin (ABC sub-type), which are associated with survival differences in patients treated with the CHOP chemotherapy regimen plus rituximab (R-CHOP).

Eligibility in newer clinical trials requires the determination of cell of origin status because preliminary data from phase I/II trials suggest that the benefit from the addition of bortezomib, lenalidomide, and ibrutinib to R-CHOP is preferentially seen in the ABC subtype. Therefore, the distinction between the GCB subtype and

the ABC subtype should be made for all cases of DLBCL, NOS, at diagnosis. If gene expression technologies are not available, immunohistochemistry technologies are considered an acceptable alternative. The algorithm used should be specified.

Genetic profiling

Chromosomal translocations: 30% of cases show re-arrangement of the 3q27 region involving BCL6, which is the most common translocation in DLBCL and occur commonly in the ABC subtype. Translocation of the BCL2 gene occurs in 20-30% of DLBCL cases commonly in the GCB subtype. It is present in about 40% of cases and is closely associated with BCL2 and CD10 protein expression. c-myc rearrangement is observed in 8-14% of cases evenly distributed between the GCB subtype and the ABC subtype; half of the DLBCL cases that harbour a c-myc translocation also show a BCL2 and/or BCL6 translocation, (Double-Hit Lymphoma.) Cases with typical DLBCL morphology and isolated c-myc translocation belong in the category of DLBCL, NOS. Most DLBCL, NOS cases with a c-myc translocation are also Double Expressers (i.e are positive for both c-myc and BCL2 protein.)

Prognostic and predictive factors

Clinical features: The 5-year progression-free and overall survival rates are about 60% and 65%, respectively. Stage and patient age are factors that affect survival. The International Prognostic Index (IPI) incorporates five clinical variables including age, lactate dehydrogenase (LDH), number of extranodal sites, Ann Arbor stage, and Eastern Cooperative Oncology Group (ECOG) performance status were used to risk stratify and identify 4 discrete risk categories. [39] Other prognostic factors associated with inferior outcome include tumour bulk (~ 10 cm), male sex, vitamin D deficiency, low body mass index, elevated serum free light chains, monoclonal serum lgM proteins, low absolute lymphocyte/monocyte count, and concordant (but not discordant) bone marrow involvement. A study published by Zhou Z, Sehn LH, Rademaker AW, Gordon LI, LaCasce AS, Crosby-Thompson A et. al. in Blood concluded that the National Comprehensive Cancer Network (NCCN) IPI is a robust and useful tool to stratify prognostically relevant subgroups of DLBCL patients in the current era of rituximab-based therapy. Compared with the IPI and other modifications of the IPI, it better incorporates 2 known continuous prognostic variables, age and LDH, in a rational way that is both simple to apply and valid in the rituximab era. With its enhanced capacity to discriminate risk groups, it has value in treatment planning and in discussions of prognosis. Its utility can also be found in stratification of future randomized clinical trials. Because there is continued enthusiasm for defining a high-risk group in the R-CHOP era, the NCCN-IPI will be useful in identifying candidates for novel approaches including in post remission therapies such as intensification with autologous stem cell transplant or consolidation/maintenance with new targeted agents. [37] Immunophenotyping: BCL2 and BCL6 are biomarkers of which the reported prognostic effect was altered by the addition of Rituximab to the CHOP chemotherapy regimen. Predictive markers include those for determination of Cell of Origin (i.e GCB subtype vs ABC subtype) being tested for phase III clinical trials and markers of the presence of relevant oncogene translocations. The double-expression of c-myc and BCL2 proteins is found in approximately 30% of all cases of DLBCL, NOS and is associated with inferior survival according to studies. Double-expression status also predicts increased risk of CNS relapse in DLBCL, NOS, and is independent of the CNS International Prognostic Index (CNS-IPI.)

Genetics: The presence of a BCL2 translocation is associated with inferior outcome in GCB DLBCL in patients treated with R-CHOP. BCL2 copy-number gain predicts inferior survival in the ABC subtype. Translocation of BCL6 is more frequent in ABC subtype DLBCL, and in some studies it has been associated with improved survival. c-myc translocations occur in about 8-14% of DLBCL, NOS cases and are associated with inferior survival. Most studies have confirmed that c-myc and BCL2 Double-hit lymphomas are much more common among GCB subtype and are associated with inferior survival. c-myc and BCL6 translocations are more common in the ABC subtype.

Aims & Objectives

Aims:

1. To study the proportion of cases of Double Expressors (DE) in Diffuse Large B-Cell Lymphomas, not otherwise specified (DLBCL, NOS) in a tertiary care setting.

 

 

Objectives:

1. 2. To determine the proportion of cases of Double Expressors which show co- expression of BCL2 and c-myc proteins in cases of DLBCL, NOS.
2. To characterize the patients according to their age-groups, gender, date of diagnosis and transformation from a lower grade lymphoma and to report these numbers.
3. To correlate, when possible, with the results of Fluorescent In-Situ Hybridization (FISH) to detect cases that harbor a c-myc translocation also showing a BCL2 translocation therefore belonging in the category of Double-Hit (DH) lymphoma.
4. To associate between the diagnosis of DE status and the clinical outcome of the patient at a minimum follow-up period of 1-year post diagnosis (regression) of primary tumor, appearance of new lesions and CNS metastases or death.

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