Abstract

Background: High-grade non-Hodgkin B-cell lymphoma is an aggressive mature B-cell lymphoma that depicts poor treatment response and worse prognosis. The presence of MYC and B-cell lymphoma 2 (BCL2) and/or B-cell lymphoma 6 (BCL6) rearrangements qualifies for triple-hit and double-hit lymphomas (THL/DHL), respectively. We attempted to explore the incidence, distribution, and clinical characteristics of the primary high-grade B-cell lymphoma of the central nervous system (CNS) in our cohort from North India.

Methods: All the histologically confirmed cases of primary CNS diffuse large B-cell lymphoma (PCNS-DLBCL) over a period of 8 years were included. Cases showing MYC and BCL2 and/or BCL6 expression on immunohistochemistry (IHC) (double- or triple-expressor) were further analyzed by fluorescence in situ hybridization for MYC, BCL2 and /or BCL6 rearrangements. The results were correlated with other clinical and pathological parameters, and outcome.

Results: Of total 117 cases of PCNS-DLBCL, there were seven (5.9%) cases of double/triple-expressor lymphomas (DEL/TEL) (six double- and one triple-expressor) with median age of 51 years (age range: 31–77 years) and slight female predilection. All were located supratentorially and were of non-geminal center B-cell phenotype. Only triple-expressor case (MYC+/BCL2+/BCL6+) demonstrated concurrent rearrangements for MYC and BCL6 genes indicating DHL (n = 1, 0.85%), while none of the double-expressors (n = 6) showed MYC, BCL2, or BCL6 rearrangements. The mean overall survival of the DEL/TEL was 48.2 days.

Conclusion: DEL/TEL and DHL are uncommon in CNS; mostly located supratentorially and are associated with poor outcome. MYC, BCL2, and BCL6 IHC can be used as an effective screening strategy for ruling out double/ triple-expressor PCNS-DLBCLs.

Keywords: Central nervous system, Double-hit lymphoma, Fluorescence in situ hybridization, High-grade B-cell non-Hodgkin lymphoma, Immunohistochemistry

INTRODUCTION

Primary central nervous system (CNS) lymphomas are extranodal, malignant non-Hodgkin lymphomas (NHL) that are principally limited to the brain, leptomeninges, eyes or spinal cord, without systemic involvement.[ 12 ] Nearly all these cases (90–95%) have diffuse large B-cell lymphoma (DLBCL) histology and carry a more dismal prognosis than systemic DLBCL.[ 12 ] Using Hans’ algorithm, nodal DLBCLs are classified into two subgroups named germinal center B-cell (GCB) and non-GCB phenotypes where the former carries better prognosis.[ 4 ] However, this classification does not show any prognostic significance in primary CNS DLBCL.[ 9 , 14 , 15 ] The revised World Health Organization classification of hematolymphoid neoplasms (4^th edition; 2016) has reclassified high grade B-cell lymphomas based on MYC, BCL2, and BCL6 gene rearrangements. A small subset of B-cell NHL resembles DLBCL or Burkitt lymphoma or harbors a blastoid morphology and show MYC and BCL2 and /or BCL6 gene rearrangements. These are now known as “high-grade B-cell, double- or triple-hit lymphoma (DHL/THL)”.[ 12 ] This subset of lymphoma shows aggressive behavior and low response to R-CHOP regime, thus carry poor prognosis.[ 10 ] The frequency and prognosis of DHL/THL in CNS has been investigated only by a handful of authors who have reported a very low incidence of DHL/THL in CNS.[ 2 , 3 , 5 , 7 ] In this study, we explored the incidence, distribution, and clinical characteristics of the primary double/triple-expressor and double/THL in the CNS among our cohort in North India.

MATERIALS AND METHODS

All histologically diagnosed cases of primary CNS (PCNSDLBCL) over a period of 8 years (July 2013–June 2021) in our department were evaluated. Approval from the Institute Ethic Committee was obtained (reference no. NK/8003/ Study/864). Majority of the cases were already worked-up for Han’s algorithm as a part of two earlier published studies.[ 8 , 9 ]

Immunohistochemistry (IHC)

For diagnosis, subtyping and double/triple expressor status, the following antibodies were used: Glial fibrillary acidic protein (clone EP672Y, Cell Marque, dilution 1:100), leukocyte common antigen (clone 2B11 + PD7/26, Dako, dilution 1:100), CD3 (Rabbit polyclonal, Cell Marque, dilution 1:500), CD20 (clone L26, Dako, dilution 1:300), CD10 (clone 56C6, Cell Marque, dilution 1:20), B-cell lymphoma 6 (BCL6; cone G1191E/A8, Cell Marque, dilution 1:300), Multiple Myeloma 1 (clone MRQ-43, Cell Marque, dilution 1:300), B-cell lymphoma 2 (BCL2; clone 124, Dako, dilution 1:50), MYC (clone EP121, Cell Marque, dilution 1:50), and Ki-67 (clone SP6, Cell Marque, dilution 1:300). These were performed on Ventana, Biotek automated system with appropriate positive and negative controls run concurrently. Like the previous study, the standard universal cutoffs for positivity were considered.[ 8 , 9 ]

The IHC slides were independently evaluated by two pathologists, who were blinded to the clinical data. The cutoff for MYC, BCL6, and BCL2 positivity were taken as 40%, 50%, and 50%, respectively. Cases showing MYC and BCL2 and/or BCL6 expression (double- or triple-expressor lymphoma, DEL/TEL) were further analyzed by fluorescence in situ hybridization (FISH) for MYC, BCL2, and BCL6 rearrangements.

FISH

FISH was performed on formalin-fixed, paraffin-embedded tissue for MYC, BCL2, and BCL6 rearrangements using Dual Color Break Apart Probe (Abbott/Vysis). The signals were analyzed on 3–4 μm sections of all respective blocks by fluorescent microscope (Olympus BX53) using appropriate filters and images were assessed by Genesis software (version 8.1.0.47741). Overlapped nuclei, ill-defined borders, or degenerated nuclei were excluded from evaluation. Minimum 100 interphase nuclei were scored. A positivity for rearrangement was defined as >15% of tumor cells with a split or single red signal.

RESULTS

A total of 117 histopathologically confirmed PCNS-DLBCL cases were included in this study. The median age of presentation was 55 years (age range: 19–85 years) with male-to-female ratio of 1.65:1. Ninety-five cases were located in the supratentorial compartment (81.19%). Using Hans’ algorithm, the cases were divided into non-GCB (n = 96; 82.05%), GCB (n = 14; 11.96%), and unclassified (n = 7; 5.98%) categories.

MYC, BCL2, and BCL6 expression by IHC

There were six double-expressor and one TEL. Among the double-expressor cases (n = 6), the overexpression for both MYC and BCL2 protein (MYC+/BCL2+) was seen in five cases [ Figures 1a - c ], whereas single case exhibited MYC and BCL6 overexpression (MYC+/BCL6+). None of these double-expressors showed any MYC, BCL2, or BCL6 rearrangements on FISH analysis [ Figure 1d ]. Only one case demonstrated co-expression of all these three markers (MYC+/BCL2+/BCL6+) and thus qualified for TEL [ Figures 2a - f ]. The median age of presentation of DEL/TEL was 51 years (age range: 31– 77 years), and there were four female and three male patients [ Table 1 ]. All the seven patients were HIV negative and immunocompetent. All were supratentorial in location and belonged to non-GCB phenotype (based on Hans’ algorithm).

Figure 1:

Primary double-expressor central nervous system lymphoma (Case 1). (a) Strong nuclear positivity for MYC (>40%; peroxidase; ×200). (b) B-cell lymphoma 2 (BCL2) showing diffuse cytoplasmic expression (>50%; peroxidase; ×200). (c) B-cell lymphoma 6 (BCL6) showing no positivity (peroxidase; ×200). (d) No MYC rearrangement on fluorescence in situ hybridization. Furthermore, no rearrangements for BCL2 and BCL6 genes (images not provided).

Figure 2:

Primary double-hit central nervous system lymphoma (Case 4). (a) High-grade B-cell lymphoma with starry-sky growth pattern and centroblastic morphology (hematoxyline and eosin; ×200). (b) CD20 showing diffuse membranous positivity (peroxidase; ×200). (c) Diffuse nuclear positivity for multiple myeloma 1 (>30%; peroxidase; ×200). (d) Diffuse nuclear positivity for MYC (>40%; peroxidase; ×200). (e) Variable nuclear positivity for B-cell lymphoma 6 (BCL6) (>50%; peroxidase; ×200). (f) Diffuse cytoplasmic positivity for B-cell lymphoma 2 (BCL2) (>50%; peroxidase; ×200). (g-i) Fluorescence in situ hybridization using dual color break apart probe shows concurrent rearrangements for MYC (g) and BCL6 (h) genes but no BCL2 gene rearrangement (i).

MYC, BCL2, and BCL6 rearrangements by FISH

FISH for rearrangement was performed on these seven cases of DEL/TEL. The triple-expressor case (MYC+/BCL2+/BCL6+) demonstrated concurrent rearrangement for MYC and BCL6 indicating DHL [ Figures 2g - h ]; however, there was no BCL2 rearrangement [ Figure 2i ]. Briefly, this was a young female, who presented with left frontal mass. The biopsy showed features of DLBCL with centroblastic morphology and was of non-GCB phenotype. She received chemotherapy regime (high-dose methotrexate) but succumbed to her illness after 90 days.

Treatment and follow-up

One case of DEL was lost to follow-up. Remaining six patients received standard chemotherapy (high-dose methotrexate [poly-/monochemotherapy] + rituximab). Five patients (including the DHL) died, and one was alive at last follow-up [ Table 1 ]. The mean overall survival of DEL/TEL was 48.2 days.

Table 1:

The clinicopathological features and outcomes of double- and/or triple-expresser primary central nervous system lymphoma of this study.

DISCUSSION

DHL is a subtype of high-grade B-cell NHL that shows MYC rearrangement and additional rearrangement of known oncogenes. BCL2 acts as the most often co-rearranged gene (75%), followed by CCND1 (13%), BCL6 (10%), and BCL3 (2%).[ 1 ] The incidence of DHL/THL increases with the patient age. Most of the nodal DHL patients are in the advanced stage (III/IV) and have median overall survival of 7–19 months.[ 6 ] Thirunavukkarasu et al.[ 13 ] from our institute studied a large series of 109 systemic high-grade B-cell NHL and observed an incidence rate of 11.9% DHL among the North Indian population where all these cases had MYC and BCL2 rearrangements. However, we registered a single (0.85%) case of primary CNS DHL in the same population with almost similar number of cases. DEL, although not a distinct subset of DLBCL, accounts for 20–30% of DLBCL cases, and carries poor prognosis.[ 11 ] In comparison to the systemic DLBCL, the prognostic significance of DEL/TEL and DHL/THL in the CNS is inconclusive, as the number of these cases are very sparse.

In this study, we came across a single case of DHL (MYC/ BCL6 concurrent rearrangement) in a young female, who presented with left frontal mass. The biopsy showed features of DLBCL with centroblastic morphology which was of non-GCB phenotype. She received chemotherapy regime (high dose methotrexate) but succumbed to her illness after 90 days of follow-up. Only five cases of DHL have been documented in the CNS till date, while there is no report of THL [ Table 2 ].[ 2 , 3 , 5 , 7 ] In 2008, Cady et al.[ 3 ] documented the first case of DHL in a case series of 75 PCNS-DLBCL that showed translocations for MYC and BCL6 genes. Five years later, Brunn et al.[ 2 ] examined 50 cases of PCNS-DLBCL and found single case showing concurrent rearrangements for MYC and BCL6. In addition to this, Nosrati et al.[ 5 ] screened 78 cases of PCNS DLBCL and found another case of DHL with MYC and BCL2 rearrangements, that belonged to GCB phenotype. Recently, two more cases of DHL were reported, where both showed concurrent translocations of MYC and BCL6 genes.[ 7 ] Here, the authors, like our study, screened only the double or TEL (n = 12) by FISH and detected 2 DHL cases. Likewise, we also discovered single case of DHL with MYC/BCL6 concurrent rearrangement out of seven double/triple-expressor cases. IHC for MYC, BCL2, and BCL6 is a useful screening method to recognize the DHL/THL.[ 13 ] Thus, compared to nodal DHLs, where MYC and BCL2 rearrangement is the commonest combination, MYC and BCL6 rearrangement is more common in PCNS DHLs. In nodal DLBCL, almost all cases of MYC/ BCL2 rearranged DHL are of GCB phenotype, whereas MYC/BCL6 rearranged DHL mostly belong to non-GCB phenotype. In the CNS also, MYC/BCL6 rearranged DHL mostly belong to the non-GCB type.[ 5 , 7 ] Similar to DHL, the incidence of DEL is also not well described in the CNS. We found 5.9% cases were DEL, which is much less compared to their frequency in nodal DLBCL, which varies from 20% to 30%.[ 11 ] The majority of the systemic DEL cases show MYC and BCL2 expression, and we also observed a similar trend in the CNS. The DEL cases in this study had a poor outcome with mean overall survival of 48.2 days, compared to median survival of 675 days in non-GCB PCNS-DLBCL as documented in our previous study.[ 9 ] The prognosis and treatment protocol of primary CNS DEL/DHL is difficult to determine due to extremely low number of documented cases.

Table 2:

Clinicopathological features of double hit primary central nervous system high grade B cell lymphoma reported in the literature.

CONCLUSION

Primary DEL and DHL are extremely rare in the CNS. Compared to the systemic DHL where MYC/BCL2 rearrangement is seen in the majority, CNS DHL cases mostly show MYC/BCL6 rearrangement. In this study, DEL and DHL in CNS uniformly showed poor outcome. IHC for MYC, BCL2, and BCL6 is an effective screening strategy to exclude the DEL and DHL cases in PCNS-DLBCL, since such cases may require more aggressive therapy. Thus, we recommend performing this panel of IHC in all cases of PCNS-DLBCL, even if FISH is not available.

Declaration of patient consent

The Institutional Review Board (IRB) permission obtained for the study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

Disclaimer

The views and opinions expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Journal or its management. The information contained in this article should not be considered to be medical advice; patients should consult their own physicians for advice as to their specific medical needs.

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