Double-Expressor Phenotype (BCL-2/c-MYC Co-expression) of Diffuse Large B-Cell Lymphoma and Its Clinicopathological Correlation

Introduction Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease, the spectrum of which is increasing with time. The 2016 World Health Organization (WHO) update on hematopoietic tumors recognized a prognostic subgroup of DLBCL called double-expressor DLBCL. Double-expressor DLBCL is defined by the co-expression of c-MYC and BCL-2 by using immunohistochemical (IHC) studies. To our knowledge, very few studies have looked into the pathological features of this newly defined prognostic category of DLBCL; therefore, in this study we evaluated the frequency of the double-expressor phenotype of DLBCL and its association with other clinicopathological parameters. Methods We conducted a retrospective observational study in the Department of Histopathology, Liaquat National Hospital and Medical College, from November 2017 till December 2020. Pathological and clinical records were retrieved from departmental archives. All cases diagnosed as DLBCL were included in the study. More than 40% c-MYC expression in the presence of more than 50% BCL-2 expression was defined as double-expressor DLBCL. Results The mean age of the patients was 52.1±16.9 years. The mean Ki67 index was 73.0±17.0%. A total of 48.6% cases were of germinal center B-cell-like (GCB) subtype, and 59.6% cases were nodal. Double-expressor phenotype was noted in 35.8% of DLBCL cases. A significant association of double-expressor phenotype was noted with age, gender, Ki67 index and subtype of DLBCL. Double-expressor DLBCL had a higher mean age than non-double-expressor DLBCL. Similarly, double-expressor DLBCL had a higher Ki67 index. Moreover, double-expressor phenotype was associated with non-GCB subtype DLBCL. Conclusion We found a high proportion of double-expressor phenotype DLBCL in our population. Moreover, double-expressor phenotype DLBCL was associated with female gender, higher age, higher Ki67 and non-GCB subtype. The association of double-expressor DLBCL with a high Ki67 index and non-GCB subtype confers a poor prognostic significance of this variant of DLBCL, requiring more aggressive therapy.


Introduction
Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease, the spectrum of which is increasing with time [1]. Gene expression profiling studies have distinguished two subtypes of DLBCL, namely, germinal center B-cell-like (GCB) and activated B-cell-like (ABC) subtypes. While molecular studies are considered the gold standard for this subtyping, immunohistochemical (IHC) studies are considered surrogate for the subtyping of DLBCL into GCB and non-GCB types. Studies have shown the prognostic significance of this subtyping, with the GCB subtype being prognostically better than the non-GCB subtype [2]. The 2016 World Health Organization (WHO) update on hematopoietic tumors recognized another prognostic subgroup of DLBCL called double-expressor DLBCL. Double-expressor DLBCL is defined by the co-expression of c-MYC and BCL-2 by using IHC studies. Double-expressor DLBCL is prognostically poorer than the non-double expressor phenotype DLBCL; however, it is better than double-hit and triple-hit B-cell lymphomas, prognostically. Double-hit and triple-hit B-cell lymphomas are high-grade B-cell lymphomas defined by the genetic rearrangements of c-MYC and BCL-2 and/or BCL-6, accordingly, by targeted fluorescence in situ hybridization (FISH) studies. Double-hit and triple-hit lymphomas are currently not considered subtypes of DLBCL as they prognostically behave differently than DLBCL. DLBCL is the most common non-Hodgkin's lymphoma in Pakistan [3]. To our knowledge, very few studies have looked into the pathological features of this newly defined prognostic category of DLBCL; therefore, in this study we evaluated the frequency of the double-expressor phenotype of DLBCL and its association with other clinicopathological parameters.

Materials And Methods
We conducted a retrospective observational study in the Department of Histopathology, Liaquat National Hospital and Medical College, from November 2017 till December 2020. Pathological and clinical records were retrieved from departmental archives. All cases diagnosed as DLBCL were included in the study. A panel of IHC stains was applied to diagnose DLBCL, including CD20, PAX5, CD3, CD5, SOX11, cyclin D1, CD10, and CD23. If differential diagnosis included carcinoma or melanoma, then S100 and pan-cytokeratin IHC stains were included in the initial IHC panel. Further IHC studies were performed to subcategorize DLBCL. The Hans algorithm was used to subcategorize DLBCL into GCB and non-GCB subtypes. GCB subtype was defined as more than 30% expression of CD10, or more than 30% expression of BCL-6 in the absence of MUM1 and CD10 expression. All other immunophenotypic expressions (CD10-/MUM1+/BCL-6+, CD10-/MUM1+/BCL-6-, CD10-/MUM1-/BCL-6-) were labeled as non-GCB DLBCL. More than 40% c-MYC expression in the presence of more than 50% BCL-2 expression was defined as double-expressor DLBCL ( Figure 1). Data analysis was performed using the Statistical Package for the Social Sciences, version 26.0 (IBM Corp., Armonk, NY). Chi-square test, independent t-test, and Fisher's exact test were used to check the association. p-values < 0.05 were considered as significant.

Results
A total of 109 DLBCL cases were included in the study. The mean age of the patients was 52.1±16.9 years. The mean Ki67 index was 73.0±17.0%. A total of 48.6% cases were of GCB subtype, and 59.6% cases were nodal. Double-expressor phenotype was noted in 35.8% of DLBCL cases ( Table 1).

Discussion
In this study, we found that a significant proportion of DLBCL had co-expression of BCL-2 and c-MYC conferring a double-expressor phenotype according to the WHO classification. Moreover, double-expressor phenotype DLBCL was associated with female gender, higher age, higher Ki67 index and non-GCB subtype.
Some studies have proposed that the prognosis of c-MYC/BCL-2 double-expressor phenotype is worse than other subtypes of DLBCL [4,5], but other studies had mixed results [6,7]. Nagib et al. studied doubleexpressor DLBCL and found that it was associated with an overall decreased disease-free survival [8]. Naseem et al. studied the frequency and prognosis of double-expressor DLBCL and found that the frequency of c-MYC/BCL-2 co-expression was 14%, with a median survival of 10 months [9]. Conversely, we found a higher frequency of double-expressor phenotype in our study (35.8%).
It is recommended to differentiate double-expressor DLBCL from other DLBCL subtypes as they have poor clinical behavior and need more aggressive interventions [10,11]. Some recent studies have confirmed that a high Ki67 proliferation index and c-MYC/BCL-2 co-expression in DLBCL were independently associated with poor clinical outcomes [2,12,13]. Factors such as age, sex, and proliferation index and their relationship with DLBCL subtypes should be studied in detail to fully understand the importance of each factor separately, as it might have significant clinical implications.
Owing to the expression of cMYC, the differential diagnosis of double-expressor DLBCL also includes Burkitt's lymphoma (BL). However, BL is characterized by intermediate-sized lymphoid cells with almost 100% Ki67 index, positive expression with CD10 and BCL-6, and lack of expression of BCL-2, although the same immuophenotype can be encountered in DLBCL and differentiation is sometimes difficult to make in the absence of molecular/genetic analysis.
Standard therapy for DLBCL is rituximab, cyclophosphamide, hydroxydaunorubicin (doxorubicin), Oncovin (vincristine), and prednisone (R-CHOP). Owing to the aggressive nature of double-expressor and double-hit lymphoma, more aggressive regimens than R-CHOP like EPOCH (etoposide, vincristine, doxorubicin, with cyclophosphamide and prednisone) were suggested; however, the prognosis remains dismal. Alternatively, novel-targeted agents, directly or indirectly inhibiting BCL-2 and cMYC, are being investigated and results are encouraging [14].
Our study was limited owing to the small sample size and retrospective study design. Moreover, clinical follow-up data were not available to compare survival between double-expressor and non-double expressor phenotypes of DLBCL in our study. In addition, molecular studies were not performed to evaluate genetic rearrangements of c-MYC and BCL-2.

Conclusions
In this study, we evaluated the co-expression of BCL-2 and c-MYC in DLBCL designated as the doubleexpressor phenotype DLBCL. We noted that a significant proportion of DLBCL in our study had doubleexpressor phenotype. We also found that double-expressor DLBCL had a higher Ki67 index than the nondouble-expressor DLBCL. An association of double-expressor phenotype was also noted with non-GCB-type DLBCL. As previous studies have confirmed a poor prognostic significance of a high Ki67 index and non-GCB type in DLBCL, an association of double-expressor DLBCL with non-GCB phenotype and a high Ki67 index portends a poor prognostic significance of double-expressor DLBCL. Future studies are warranted to evaluate the difference in disease-free survival of double-expressor DLBCL and other subtypes of DLBCL.

Additional Information Disclosures
Human subjects: Consent was obtained by all participants in this study. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.