Significance of MYC/BCL2 Double Expression in Diffuse Large B-cell Lymphomas: A Single-center Observational Preliminary Study of 88 Cases

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1 Original Article Significance of MYC/BCL Double Expression in Diffuse Large B-cell Lymphomas: A Single-center Observational Preliminary Study of 88 Cases Chutima Pinnark 1 ; Jerasit Surintrspanont ; Thiamjit Chaichana 1 ; Jutamas Wongphoom ; Yuda Chongpison 3 ; Shanop Shuangshoti 1 ; Thamathorn Assanasen 1 Chutima Pinnark 1 Department of Pathology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand. Department of Pathology, King Chulalongkorn Memorial Hospital, Bangkok, Thailand. 3 Research Affairs, Center for Excellence in Biostatistics, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand. * Address Correspondence to author: Chutima Pinnark Department of Pathology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand. chutima.pinnark@gmail.com Received: December 0, 017 Revision received: December 1, 017 Accepted after revision: February 1, 018 BKK Med J 018;14(1): Abstract OBJECTIVES: To study the prevalence of MYC/BCL double expression in diffuse large B-cell lymphomas (DLBCLs) and its prognostic value. MATERIAL AND METHODS: This is a retrospective observational study, which includes patients diagnosed with DLBCLs at King Chulalongkorn Memorial Hospital from 013 to 014. The slides were reviewed, and MYC and BCL immunostains were scored according to Revised WHO 016 Classification. Clinical data were collected from medical records. Patients were divided into two groups: double expression (DE) and non-double expression (NDE). Survivals and hazard ratios (HR) were calculated. RESULTS: Eighty-eight patients were included in the study and 40 (46%) had double expression. The mean age was 60±16 years old; 40.9% were male and 59.1% were female. Eight patients were excluded from the survival analysis due to incomplete clinical data. Of the remaining 80 patients, there were 34 (4.5%) DE and 46 (57.5%) NDE. Median overall survival time () and median progression-free survival time () tended to be lower in the DE group but was not statistically significant (Log-rank test, p = 0.16). Multivariate analysis identified 4 confounders: sex, cell of origin (COO), risk group and addition of rituximab to the standard treatment. Adjusted HRs of the DE group were 1.1 (95%CI , p = 0.57) for and 1.0 (95%CI , p = 0.58) for. In the germinal center subgroup (GCB) of DLBCL, patients with DE had HRs of 4.33 (95%CI , p =, significance level of p < 0.10) for and 4.61 (95%CI , p = ) for ; but, these were not significant in the non-gcb subgroup. CONCLUSION: MYC/BCL double expression (DE) is significantly associated with poorer prognosis than non-double expression (NDE) among DLBCLs with GCB phenotype. MYC/BCL double expression should be reported in the pathological diagnosis of DLBCLs. Keywords: MYC, BCL, double expression, co-expression, diffuse large B-cell lymphoma, prognosis. Diffuse large B-cell lymphomas (DLBCLs) is the most common subtype of non-hodgkin lymphoma (NHL). 1 In Thailand, DLBCLs accounts for 67% of NHL. The cancer behaves aggressively with heterogeneous outcome. Several prognostic and predictive factors have been used in clinical practice for DLBCLs, including Ann Arbor staging, 3 International Prognostic Index (IPI), 4 age-adjusted IPI, 5 International Extranodal Lymphoma Study Group (IELSG) risk score, 6 and cell of origin (COO), which is either germinal center B-cell (GCB) and non-gcb. 7,8 Studies in the last decade demonstrated that double-hit B-cell lymphomas have poorer prognosis than those without double-hit. 9,10 The Revised WHO Classification of Lymphoma 016 includes high- grade B-cell lymphoma (HGBL), with MYC and BCL and/or BCL6 rearrangement as a new subtype of B-cell lymphoma. 11 Some studies have showed that the detection of protein expression by immunohistochemical study (IHC) is comparable to molecular study of gene rearrangement; however, this method has not yet been accepted to classify this subtype of lymphoma MYC/BCL expression is the most important because it accounts for the majority of lymphoma with double expression and is associated with poorer prognosis in patients with DLBCLs. 13,14 16 The Bangkok Medical Journal Vol. 14, No. 1; February 018 ISSN (online)/ (print)

2 Significance of MYC/BCL Double Expression in Diffuse Large B-cell Lymphomas: A Single-center Observational Preliminary Study of 88 Cases The prevalence and the prognostic value of MYC/BCL double expression has not been studied in Thailand. Therefore, this study was conducted to report the prevalence of DLBCLs with MYC/BCL double expression and to determine whether it has effect upon survival. Abbreviation aaipi Age-adjusted International Prognostic Index CI Confidence Interval C Central nervous system COO Cell of Origin DE Double Expression DH Double hit DH-HGBL High- grade B-cell lymphoma, with MYC and BCL and/or BCL6 rearrangement DLBCL Diffuse large B-cell lymphoma FISH In Situ Hybridization GCB G erminal center B-cell HGBL High-grade B-cell lymphoma HR Hazard ratio HSCT Hematopoietic stem cell transplantation IELSG International Extranodal Lymphoma Study Group IHC Immunohistochemical study IPI International Prognostic Index NDE N ot significant Overall survival Progression-free survival SD Standard deviation WBRT Whole brain radiation Materials and Methods Patients In this retrospective observational study, 88 patients diagnosed with DLBCLs according to the WHO Classification 008 criteria1 at King Chulalongkorn Memorial Hospital (KCMH) from 013 to 014 were retrieved. Eight patients with incomplete clinical data were excluded from the survival analysis. Methods Histologic slides of all cases were reviewed. MYC and BCL immunostains were performed on formalin-fixed paraffin-embedded sections, using anti-c-myc (Y69) rabbit monoclonal primary antibody (Ventana ), and monoclonal mouse anti-human BCL oncoprotein clone 14 ready-to-use (Dako ), respectively. The percentage of MYC and BCL was separately assessed by two pathologists, CP and TA. The positive cutoff values of MYC and BCL according to the WHO Classification of lymphoid neoplasm 016 were 40% and 50%, respectively (Figure 1). Cases with discordance of positive/negative interpretation were reviewed together to reach a consensus. For the remaining cases with concordant interpretation, the mean percentage of positive cells was used for further analysis. The clinical data of all patients were collected from the medical records. The follow-up period was years. The overall survival () and progression-free survival () were recorded. This study was approved by the Institutional Review Board, Faculty of Medicine, Chulalongkorn University, and number 613/59. Figure 1: Diffuse large B-cell lymphoma (DLBCL). Panel A: DLBCL with double expression (DE), MYC+/ BCL+. Panels B-D: DLBCL without double expression (nondouble expression, NDE) (B: MYC+/BCL-, C: MYC-/ BCL+, D: MYC-/BCL-) [Cut-point of positivity; MYC 40%, BCL 50%]. The Bangkok Medical Journal Vol. 14, No.1; February 018 ISSN (online)/ (print) 17

3 Pinnark C, et al. Statistical Analysis Qualitative data were recorded as frequency and percentage. Differences in categorical data were analyzed by Chi-squares test or Fisher s Exact test. Continuous data were recorded as mean and standard deviation, and were compared by t-test. Kaplan-Meier survival curve was used to demonstrate the median survival time. The difference of median survival time was analyzed using Log-rank test. Multivariate analysis was also implemented to detect possible confounders. Then, Cox proportional hazard model was used to calculate the hazard ratio and the adjusted hazard ratio. The effect modification was analyzed, using multivariate analysis and log-likelihood ratio test with p < 0.10 as the cutoff point for statistical significance. This part used Stata/IC 1.1. Other results were displayed as number with 95% confidence interval (CI) and were considered statistically significant if p < Results The prevalence of MYC/BCL Double Expression (DE) There were 88 patients diagnosed with DLBCL from 1 January 013 to 31 December 014; all of which were included in the prevalence study. Fifty-two (59%) cases were biopsy specimens and 36 (41%) cases were excisional specimens. Extranodal non-c sites, lymph node, and C accounted for 46 (5%), 5 (9%) and 17 (19%) cases, respectively. The most common site of extranodal involvement was the gastrointestinal tract, 13 cases (15%) (Figure ). There were 36 (40.9%) male and 5 (59.1%) female patients. The mean age was 60±16 years old (range, years). Five (6.5%) of patients had HIV infection. Forty (46.6%) cases were found to have DE of MYC/BCL. Base of tongue Liver Testis Bone Kidney Breast Bone marrow Media stinum Neck areaa Soft tissue Sininasal Gl tract DE NDE Figure : Sites of extranodal involvement, excluding C. Clinical characteristics of the patients included in the survival analysis Eight patients were excluded from the analysis of survival due to incomplete clinical data; 6 from the double expression (DE) group, and from the non-double expression (NDE) group. The remaining 80 patients were divided into two groups, DE and NDE. The patients characteristics are listed in Table 1. There was no significant difference between the two groups except for sex, i.e., there were significantly more male patients in the DE group than in the NDE group. The detail of the treatments is shown in Table. Eleven patients did not receive chemotherapy because of death before 1 month of follow-up time in 6 cases, end of life care in cases, loss of follow-up in cases, and referral to another hospital in 1 case. Table 1: Characteristics of Diffuse large B-cell Lymphoma patients in the study group, classified by MYC/BCL double expression Category Sex Male Female Age (year); mean (SD) 60 > 60 Organ involvement Nodal Extranodal C Cell of origin* Germinal Non-germinal HIV-related Yes No Stage I II III IV Risk group (IPI/aaIPI/ELSG) Low Intermediate High Treatment Rituximab-based regimen Non Rituximab-based regimen No CMT received Others (HSCT, WBRT) Double expression (n = 34) 19 (55.9) 15 (44.1) 60.5 (15.3) 16 (47.1) 18 (5.9) 13 (38.) 14 (41.) 7 (0.6) 10 (9.4) 4 (70.6) (5.9) 3 (94.1) 1 (.9) 8 (3.6) 3 (8.8) (64.7) 4 (11.8) 14 (41.) 16 (47.0) 1 (35.3) 14 (41.) 6 (17.5) (6.0) n (%) Non-double expression (n = 46) 14 (30.4) 3 (69.6) 59.1 (17.6) 4 (5.) (47.8) 11 (3.9) 5 (54.3) 10 (1.8) 17 (37.0) 9 (63.0) 3 (7) 43 (93) 3 (6.5) 16 (34.8) 3 (6.5) 4 (5.) 10 (1.7) 1 (45.7) 15 (3.6) 18 (39.1) 19 (41.3) 5 (10.9) 4 (8.7) *By Hans algorithm **p for Fisher s Exact test IPI = International Prognostic Index aaipi = Age-adjusted International Prognostic Index IELSG = International Extranodal Lymphoma Study Group HSCT = Hematopoietic stem cell transplantation WBRT= Whole brain radiation p ** 0.57** 0.3** 0.84** 18 The Bangkok Medical Journal Vol. 14, No. 1; February 018 ISSN (online)/ (print)

4 Significance of MYC/BCL Double Expression in Diffuse Large B-cell Lymphomas: A Single-center Observational Preliminary Study of 88 Cases Table: Treatment modalities for the Diffuse large B-cell lymphoma patients. Treatment* Rituximab-based regimen R-CHOP R-DA-EPOCH R-ESHAP R-CVP R-MTX-ifos Non rituximab-based regimen CHOP CVP EPOCH or DA-EPOCH MTX-ifos Others** HSCT WBRT Total *See the details of chemotherapy regimen in the appendix ; **Other regimens = ESHAP and DHAP HSCT = Hematopoietic stem cell transplantation; WBRT = Whole brain radiation n Table 4: Effect modification analysis. Survival Risk group High vs Low & Moderate Treatment Rituximab vs Non-rituximab Cell of origin Germinal vs Non-germinal Risk group High vs Low & Moderate Treatment Rituximab vs Non-rituximab Cell of origin Germinal vs Non-germinal *Significant if p < 0.10;, non-significant DE versus NDE p* Table 3: Hazard ratios of versus Non-double expression in overall survival and progression-free survival. Table 5: Interaction of cell of origin to the effect of double expression on survival of DLBCL comparing to non-double expression. Crude Adjusted Crude Adjusted HR HR % CI % CI p p Survival Germinal origin Non-germinal origin Germinal origin Non-germinal origin DE versus NDE HR 95% Cl p* *Significant if p < 0.10 The Overall Survival (), Progression-Free Survival () and Hazard Ratio (HR) of DE group compared to the NDE group At the end of follow-up ( years), 40/80 patients had died. Twenty patients who passed away were in the DE group (58%) and the remaining 0 were in the NDE group (43%). Both and are shown in Figure 3. The median time was shorter in the DE group (14 months vs. 4 months, Log-rank test, p = 0.16). Multivariate analysis found that sex, COO, risk group and treatment were the confounders. The adjusted HR was 1.1 (95%CI , p = 0.57). The median time of was also shorter in the DE group (13.5 months vs. to 4 months, Log-rank test, p = 0.16). The adjusted HR was 1.0, (95%CI , p = 0.58). All of them were not statistically significant (Table 3). Effect modification and its interaction with hazard ratio The effect modification of COO, risk group and treatment was done. COO had statistically significant interaction with the HR of both and (Table 4). In GCB phenotype, the DE group had a HR of 4.33 (95%CI , p =, significance level of p < 0.10) for, and 4.61 (95%CI , p =, significance level of p < 0.10) for. In the non-gcb phenotype, the HR was not statistically significant (Table 5, Figure 4 and Figure 5). The Bangkok Medical Journal Vol. 14, No.1; February 018 ISSN (online)/ (print) 19

5 Pinnark C, et al. Kaplan-Meier survival estimates Kaplan-Meier survival estimates Figure 3: Overall survival (left) and progression-free survival (right) of patients with compared to Nondouble expression. Overall Survival (%) Progression-Free Survival (%) Figure 4: Overall survival () and progression-free survival () among patients having DLBCL with germinal phenotype and double expression compared to non-double expression. Overall Survival (%) Progression-Free Survival (%) analysis time Figure 5: Overall survival () and progression-free survival () among patients having DLBCL with non-germinal phenotype and double expression compared to non-double expression. 0 The Bangkok Medical Journal Vol. 14, No. 1; February 018 ISSN (online)/ (print)

6 Significance of MYC/BCL Double Expression in Diffuse Large B-cell Lymphomas: A Single-center Observational Preliminary Study of 88 Cases Discussion To the best of our knowledge, double expression of MYC/ BCL has not been reported in DLBCLs in Thailand. In our cohort of Thai patients, the prevalence of DLBCL with double expression is 46.6%. This figure is higher than those observed in the United States of America (1%) 13 and Denmark (19%) 14 but it is within the range of the Chinese population (30-60%). 15,16 Primary C lymphomas tend to be non-gcb phenotype, which is more common to have double expression. 17 Therefore, one reason that could be attributed to the difference between our prevalence and the Western prevalence is the inclusion of primary C DLBCLs to our study (7 DE and 10 NDE; 7 GCB and 10 non-gcb phenotypes). Ethnicity is also another factor that could play role in the differences. In our study, there were significantly more male patients in the DE group, compared to the NDE group. This might represent selection bias due to small population or an association between DE and sex, which has not been described. Besides gender, other patient characteristics are not significantly different. Some of the previous reports excluded HIV patients from their studies 13,14,18 while some did not mention the HIV status. 15,16 5 cases (6.5%) of our patients were HIV-infected, and such a small proportion had no statistical significance, both as a confounding variable and an effect modifier. Although all of the studies so far have found no effect of DE on the prognosis of patients with DLBCLs, the significance of DE among HIV patients has not yet been explored in a large cohort. The DE group tended to have worse and, as well as adjusted HR for both risk of dying and risk of having progression, but all of these were not statistically significant. Our findings are contradictory to the results of previous studies. 13,14 This could be due to the smaller population and shorter follow-up duration of our study; the previous cohorts that found DE to be significantly associated with worse prognosis enrolled at least 100 patients with a period longer than 3 years of follow-up. A future study with a larger number of patients, longer duration of follow-up and a multicenter study might provide more accurate information. In addition, the prevalence of high grade B-cell lymphoma with double hit of MYC and BCL rearrangement in Thai patients and whether or not double expression can predict the presence of double hit remains to be investigated. From the effect modification model, there is an interesting result with regards to the cell of origin in our cohort. Among those with GCB phenotype, patients with DE were 4.3 times more likely to die and 4.3 times more likely to have progression than those with NDE. Among patients with non-gcb phenotype, the risks of dying and the risk of having progression were similar in both groups. This result is different from the finding of The International DLBCL Rituximab-CHOP Consortium Program in which DE had poorer prognosis in the non-gcb subgroup. 18 Sesques and Johnson 19 recently reviewed DLBCLs and high-grade B-cell lymphomas (HGBL) and their correlation with DE, double hit (DH) and mechanism of gene rearrangement of MYC and BCL. They demonstrated that most DLBCLs with DE and GCB phenotype have DH caused by gene translocation, which is the same mechanism of DH in HGBL. Therefore, some DLBCLs with GCB phenotype and DE are in fact HGBL. This could explain why DE has poorer prognosis among patients with GCB phenotype. Furthermore, FISH for MYC and BCL translocation should be performed in DLBCLs with GCB phenotype and DE because it is the most cost-effective method to identify patients with DH-HGBL. This suggests that, in addition to the cell of origin, DE should be considered as one of the important biological risk factors that should be included in the pathological report. Conclusion The prevalence of MYC/BCL double expression DLBCLs seem to be higher in Thai patients compared to American and Danish, but comparable to Chinese. alone was not statistically associated with prognosis in our cohort. However, for the GCB phenotype subgroup, those with DE carried poorer outcome. References 1. Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of tumours of haematopoietic and lymphoid tissues. Lyon, France: IARC Press; Bunworasate U, Siritanaratanakul N, Khuhapinant A, et al. A nationwide prospective multicenter study of clinical features and outcomes of non-hodgkin lymphoma in Thailand: an analysis of 939 Cases. Blood 011;118: Cheson BD, Fisher RI, Barrington SF, et al. 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7 Pinnark C, et al. 9. Aukema SM, Siebert R, Schuuring E. et al. Double-hit B-cell lymphomas. Blood 011;117(8): Snuderl M, Kolman OK, Chen YB, et al. B-cell lymphomas with concurrent IGH-BCL and MYC rearrangements are aggressive neoplasms with clinical and pathologic features distinct from Burkitt lymphoma and diffuse large B-cell lymphoma. Am J Surg Pathol 010;34(3): Swerdlow SH, Campo E, Pileri SA, et al. The 016 revision of the World Health Organization classification of lymphoid neoplasms. Blood 016;17(0): Green TM, Nielsen O, de Stricker K, et al. High levels of nuclear MYC protein predict the presence of MYC rearrangement in diffuse large B-cell lymphoma. Am J Surg Pathol 01;36(4): Johnson NA, Slack GW, Savage KJ, et al. Concurrent Expression of MYC and BCL in Diffuse Large B-Cell Lymphoma Treated With Rituximab Plus Cyclophosphamide, Doxorubicin, Vincristine, and Prednisone. J Clin Oncol 01;30(8): Green TM, Young KH, Visco C, et al. Immunohistochemical double-hit score is a strong predictor of outcome in patients with diffuse large B-cell lymphoma treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone. J Clin Oncol 01;30(8): Jing Wang, Min Zhou, Jing-Yan Xu, et al. Combination of BCL- and MYC protein expression improves high-risk stratification in diffuse large B-cell lymphoma. Onco Targets Ther 015;8: Xia B, Zhang L, Guo SQ, et al. Coexpression of MYC and BCL- predicts prognosis in primary gastrointestinal diffuse large B-cell lymphoma. World J Gastroenterol 015;1(8): Hattab EM, Martin SE, Al-Khatib SM, et al. Most primary central nervous system diffuse large B-cell lymphomas occurring in immunocompetent individuals belong to the nongerminal center subtype: a retrospective analysis of 31 cases. Mod Pathol 010;3: Hu S, Xu-Monette ZY, Tzankov A, et al. MYC/BCL protein coexpression contributes to the inferior survival of activated B-cell subtype of diffuse large B-cell lymphoma and demonstrates high-risk gene expression signatures: a report from The International DLBCL Rituximab-CHOP Consortium Program. Blood 013;11(0): Sesques P, Johnson NA. Approach to the diagnosis and treatment of high-grade B-cell lymphomas with MYC and BCL and/or BCL6 rearrangements. Blood 017;19(3):80-8. The Bangkok Medical Journal Vol. 14, No. 1; February 018 ISSN (online)/ (print)