Double hit and double expressor B cell lymphomas: Current treatment strategies and impact of hematopoietic cell transplantation

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1 Received: 10 April 2018 Revised: 6 July 2018 Accepted: 16 July 2018 DOI: /acg2.13 INVITED REVIEW Double hit and double expressor B cell lymphomas: Current treatment strategies and impact of hematopoietic cell transplantation Akihiro Ohmoto 1 Shigeo Fuji 2 1 Laboratory of Clinical Genomics, National Cancer Center Research Institute, Tokyo, Japan 2 Department of Hematology, Osaka International Cancer Institute, Osaka, Japan Correspondence: Shigeo Fuji, Department of Hematology, Osaka International Cancer Institute, , Otemae, Chuo-ku, Osakashi, Osaka , Japan (fujishige1231@gmail.com). Abstract High grade B cell lymphoma with rearrangement of MYC and BCL2 and/or BCL6 (double hit lymphoma: DHL) was newly categorized as a subtype in the 2016 revision of the WHO classification of lymphoid neoplasms. DHL is a rare entity accounting for <10% of DLBCL and clinical data of DHL cases are still limited. Standard rituximab incorporated chemotherapy was reported to be underpowered for this intractable disease, and some promising results with intensified regimen including dose adjusted EPOCH R (rituximab, etoposide, vincristine, adriamycin, cyclophosphamide, and prednisone) have been emerging. The benefit of intensified regimen for DHL patients should be determined in randomized trials. The role of consolidative autologous (auto) hematopoietic cell transplantation (HCT) for newly diagnosed cases has been also undetermined. In regards to salvage chemotherapy followed by auto HCT for chemotherapy sensitive relapsed cases, the prognosis seems to be unsatisfactory in patients with DHL, and novel treatment strategies to incorporate effective salvage, auto HCT and maintenance treatment after auto HCT are warranted. Clinical application of allogeneic (allo) HCT has not been established in newly diagnosed and refractory/relapsed (ref/rel) cases. Recently, favorable survival data of allo HCT for ref/rel DHL was reported. To clarify the indication of various treatment strategies, larger scaled studies or new prognostic models for DHL are required. As another topic, clinical investigation of several novel agents such as BCL2 inhibitor is conducted along with DLBCL. Here, we summarize the data relating to DHL focusing on the application of HCT, and also discuss about the combination therapy using novel agents in the setting of HCT. KEYWORDS DA-EPOCH-R regimen, double-expressor lymphoma, double-hit lymphoma, hematopoietic cell transplantation, novel agents 1 INTRODUCTION Akihiro Ohmoto and Shigeo Fuji are contributed equally to this work. The WHO classification of lymphoid neoplasms was revised in 2016, and a new subtype high grade B cell lymphoma with rearrangement of MYC and BCL2 and/or BCL6 was established. 1 This subtype was John Wiley & Sons Ltd Adv Cell Gene Ther. 2018;1:e13. wileyonlinelibrary.com/journal/acg2 1of10

2 2of10 OHMOTO AND FUJI termed as double hit lymphoma (DHL) and was often handled similarly to diffuse large B cell lymphoma (DLBCL). Due to a rarity of DHL, clinical data of DHL cases are limited. 2 In addition, clinical efficacy of standard R CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) regimen is unsatisfactory in patients with DHL. Thus, the development of new treatment strategies including intensified protocol is crucial to overcome the poor prognostic impact of DHL. 3-6 Hematopoietic cell transplantation (HCT) has been chiefly applied in the setting of consolidation after salvage chemotherapy for relapsed DLBCL cases However, clinical efficacy of HCT for DHL has not been fully determined. Thanks to recent advances in basic research, several molecular targeted agents have been introduced into clinical practice for aggressive lymphoma. 11 As the next step, developing combination therapy of HCT and novel agents is attractive to improve the cure rate of DHL. In this article, we summarize the latest evidence about treatment of DHL with a focus on HCT and discuss prospects of HCT in the era of novel agents. 2 DHL AS A NEW SUBTYPE IN REVISED 2016 WHO CLASSIFICATION As for the definite diagnosis of DHL, detecting genetic rearrangement of MYC and BCL2 and/or BCL6 by fluorescent in situ hybridization (FISH) is indispensable. 1 While DHL with MYC and BCL2 rearrangement (MYC/BCL2 DHL) is a major type, DHL with MYC and BCL6 rearrangement (MYC/BCL6 DHL) or THL with these three rearrangements are less common. According to the Mitelman database for 326 patients with DHL/THL, 62% of the cases belonged to MYC/ BCL2 DHL, 8% MYC/BCL6 DHL, and 16% THL, respectively. 12 Pathomorphological feature in each DHL case is various. B cell lymphoma unclassifiable with features intermediate between DLBCL and Burkitt lymphoma (BCL U) or DLBCL are common types, whereas some cases have unique images such as Burkitt lymphoma, follicular lymphoma, or lymphoblastic lymphoma. 5,13-17 Recently revealed mutational profiling separated morphological, immunophenotypic and cytogenetic 108 cases of Burkitt lymphoma, DLBCL or BCL U into three groups: the first group harboring mutations in Burkitt lymphoma associated genes (ID3, TCF3, CCND3, and CMYC); the second group harboring mutations in DLBCL associated genes (BCL2, EZH2, CREBBP1, EP300, SGK1, and MEF2B); the third group sharing these two mutation patterns. 18 Double expressor lymphoma (DEL) is not presented as an independent subtype in the 2016 WHO classification, and described as DLBCL with concurrent MYC and BCL2 expression by immunohistochemistry. 1 According to the previous studies, 21 44% of DLBCL cases belonged to DEL, and this percentage was much higher compared with 2% 11% in DHL. 13,19-25 Here, it should be noted that cutoff values of positivity varied in the range 40% 50% for MYC and 30% 70% for BCL2 in these studies. The 2016 WHO classification recommends cutoff values of 40% for MYC and over 50% for BCL2. 1 The discrepancy of incidence between DHL and DEL indicates that not all DLBCL with concurrent MYC and BCL2 expression has MYC and BCL2 genetic rearrangement. MYC overexpression is explained by not only MYC rearrangement but also other mechanisms such as copy number variation or transcriptional upregulation. 26 While BCL2 overexpression is associated with BCL-2 rearrangement in germinal center B cell (GCB) type DLBCL, the overexpression is caused by NF κb activation or BCL2 gene locus (18q21) amplification in activated B cell (ABC) type DLBCL. 27 Immunohistochemical and cytogenetic analysis for 125 DLBCL series showed that 14% of cases had discordant results between MYC expression and MYC rearrangement. MYC expression had a sensitivity of 53% and a specificity of 96%, and the positive and negative predictive value for MYC rearrangement was 80% and 87%, respectively. 28 In terms of cell of origin classification, it is known that the prevalence is different between DHL and DEL. In the pathological analysis by Green et al., the percentage of GCB type was higher in DHL (91% in DHL vs 27% in DEL), and non GCB type was prevalent in DEL (9% in DHL vs 73% in DEL). 19 Collectively, these results suggest that DHL and DEL are entities with different biological features. Although exploring genetic rearrangement is a gold standard for the diagnosis of DHL, it is disadvantageous in terms of cost, labor, and the need for fresh frozen tissue compared with immunohistochemistry. 29 On the other hand, immunohistochemical and cytogenetic analysis for 117 DLBCL series showed that 6% of the cases were DHL without MYC/BCL 2 co expression, which suggests immunohistochemical screening might overlook a part of DHL. 30 Therefore, some researchers insist that genetic rearrangement of MYC, BCL2, and BCL6, and protein expression of MYC and BCL2 should be assessed for all cases. 31 National Comprehensive Cancer Network Guidelines for DLBCL recommend cost effective FISH analysis of MYC, BCL2, and BCL6 for only GCB DLBCL. 32 Sesques et al. also describes further selective FISH for only GCB DLBCL exhibiting concurrent MYC/BCL 2, reducing the number of subjects by more than 90%. 33 While FISH testing for all newly diagnosed DHL seems practically difficult, the analysis for relapsed DLBCL cases might give clinicians some helpful information to choose the type of HCT: auto HCT or allo HCT. This topic is still under debate and future standardization of diagnostic procedure is needed. 3 CLINICAL IMPACTS OF DHL AND DEL As shown in Table 1, retrospective studies demonstrated poor prognosis of DHL patients under various initial regimens. Johnson et al. reviewed 54 patients with MYC/BCL2 DHL and reported that 59% of them died within 6 months after the diagnosis and only 11% survived in remission at a median follow up of 5.3 years MYC/ BCL2 DHL series by Li et al. showed the median overall survival (OS) of 18.6 months. 34 According to the analysis of 49 patients with MYC rearranged aggressive B cell non Hodgkin lymphoma (NHL) by Cohen et al., 59% of the cases had concurrent BCL2 rearrangement,

3 OHMOTO AND FUJI 3of10 TABLE 1 Clinical effects of initial regimen for DHL Author Study design Number of patients Treatments CR rate PFS OS Petrich et al. 3 Retrospective 311 R CHOP (N = 100), R HyperCVAD (N = 65), DA EPOCH R (N = 64), R CODOX M/IVAC (N = 42), Others (N = 40) Oki et al. 4 Retrospective 129 R CHOP (N = 57), R HyperCVAD/MA (N = 34), DA EPOCH R (N = 28), Others (N = 10) Landsburg et al. 5 Retrospective 159 R CHOP (N = 35), R HyperCVAD (N = 32), DA EPOCH R (N = 81), R CODOX M/IVAC (N = 11) Howlett et al. 6 Meta analysis 394 R CHOP (N = 180), DA EPOCH R (N = 91), Others (N = 123) Johnson et al. 13 Retrospective 54 CHOP (N = 23), R CHOP (N = 11), High dose chemotherapy with/ without HCT (N = 6), Palliation (N = 14) Gandhi et al. 16 Retrospective 106 R CHOP (N = 36), DA EPOCH R (N = 33), R HyperCVAD or R CODOX M/IVAC (N = 28) NA 55% (R CHOP 40%, R HyperCVAD/MA 68%, DA EPOCH R: 68%) NA NA Median 10.9 months (R CHOP 7.8 months, Intensive regimen 21.6 months), 40% at 2 years 33% at 2 years (R CHOP 25%, DA EPOCH R 67%, R HyperCVAD 32%) 80% at 3 years (R CHOP 56%, Intensive regimen 88%) Median R CHOP 12.1 months, DA EPOCH R 22.2 months, Others 18.9 months Median 21.9 months, 49% at 2 years 44% at 2 years (R CHOP 41%, DA EPOCH R 76%, R HyperCVAD 44%) 87% at 3 years Median R CHOP 21.4 months, DA EPOCH R 31.4 months, Others 25.2 months NA NA Median CHOP 4.8 months, R CHOP 16.8 months 54% (R CHOP 49%, DA EPOCH R 68%, R HyperCVAD 33%, R CODOX M/IVAC 33%) Median 8.8 months (R CHOP 7.7 months, DA EPOCH R 9.2 months, R HyperCVAD 8.0 months, R CODOX M/IVAC 4.0 months) Median 12.0 months (R CHOP 12.0 months, DA EPOCH R 11.6 months, R HyperCVAD 11.0 months, R CODOX M/ IVAC 6.4 months) Green et al. 19 Retrospective 11 R CHOP (N = 11) 64% 46% at 3 years 46% at 3 years Johnson et al. 20 Retrospective 14 R CHOP (N = 14) NA 18% at 5 years 27% at 5 years Li et al. 34 Retrospective 52 R CHOP (N = 19), R Hyper CVAD (N = 28) Cohen et al. 35 Retrospective 49 R CHOP (N = 17), DA EPOCH R (N = 17), Others (N = 15) Abramson et al. 36 Retrospective 34 R CHOP (N = 15), DA EPOCH R (N = 12), R CODOX M/IVAC (N = 6) Dunleavy et al. 40 Prospective phase II 52 DA EPOCH R (N = 52) NA NA Median 18.6 months, 58% at 1 year 59% Median 16.6 months Median 37.7 months 64% Median 8 months (R CHOP 6 months, DA EPOCH R 21 months, R CODOX M/IVAC 6 months) Median 11.0 months (R CHOP 8 months, DA EPOCH R 34 months, R CODOX M/IVAC 7 months) NA 79% at 14 months 77% at 14 months CR, complete response; DHL, double hit lymphoma; HCT, hematopoietic cell transplantation; NA, not available; OS, overall survival; PFS, progressionfree survival. and the median progression free survival (PFS) and OS in patients with MYC/BCL2 DHL was 8.0 months and 12.5 months, compared with 16.6 months and 37.7 months in all MYC rearranged cases. 35 Green et al. also conducted immunohistochemical and cytogenetic analysis for 193 DLBCL samples and demonstrated that MYC/BCL2 rearrangement detected in 11 cases was a significantly poor

4 4of10 OHMOTO AND FUJI prognostic factor for PFS and OS (3 year PFS: 46% vs 65%, P = 0.01; 3 year OS: 46% vs 75%, P = 0.002). 19 Immunohistochemical studies for cases receiving R CHOP revealed that DEL exhibited poor prognosis. Johnson et al. pathologically reviewed 167 cases of DLBCL and showed that MYC/BCL2 coexpression was associated with poor PFS and OS (P < 0.001), although MYC single expression without co expressed BCL2 was not associated with a poor prognosis. 20 In above mentioned study by Green et al., co expression of MYC/BCL2 as well as MYC/BCL2 rearrangement were poor prognostic factors for PFS and OS (3 year PFS: 39% vs 75%, P < 0.001; 3 year OS: 43% vs 86%, P < 0.001). 19 Hu et al. also analyzed 157 DEL and 309 non DEL cases and demonstrated shorter PFS and OS in patients with MYC/BCL2 DEL than non DEL cases (5 year PFS: 27% vs 73%, P < ; 5 year OS: 30% vs 75%, P < ). Here, adverse impact of co expression of MYC/BCL2 remained in both 241 GCB DLBCL and 225 ABC DLBCL cohort. 21 According to 106 DLBCL series by Perry et al., MYC/BCL2 DEL cases had worse event free survival (EFS) and OS in the entire cohort and in GCB DLBCL cohort (P < 0.001, P < for the entire cohort; P = 0.002, P < for GCB cohort), although prognosis in non GCB cohort was similar between DEL and non DEL. 22 These data consistently highlight the necessity to develop novel treatment strategies for DHL and DEL. 4 INTENSIFIED INDUCTION CHEMOTHERAPY FOR DHL As discussed above, previous studies showed insufficient efficacy of R CHOP regimen for DHL (Table 1). To overcome the poor prognostic impact of DHL, intensified induction chemotherapy is increasingly used in DHL patients. Petrich et al. reviewed 171 DHL patients receiving intensified regimens and 100 patients R CHOP. 3 They reported that complete response (CR) rate was higher with doseadjusted (DA) EPOCH R (rituximab, etoposide, vincristine, doxorubicin, and prednisone) compared with R CHOP or R CODOX M/IVAC (rituximab, cyclophosphamide, vincristine, doxorubicin, and high dose methotrexate, alternating with ifosfamide, etoposide, and cytarabine (AraC)), and PFS was longer with intensive regimen than R CHOP (median PFS: 21.6 months vs 7.8 months, P = 0.001). According to 129 cases of DHL cohort study in MD Anderson Cancer Center, CR rate was better with DA EPOCH R orr Hyper CVAD/MA (rituximab plus hyperfractionated cyclophosphamide, doxorubicin, vincristine, and dexamethasone alternating with high dose methotrexate (MTX), and cytarabine) than R CHOP (68% vs 40%, P = 0.02; 68% vs 40%, P = 0.01), respectively. 4 While EFS and OS were similar between R Hyper CVAD/MA and R CHOP, DA EPOCH R significantly improved EFS compared with R CHOP (hazard ratio (HR): 0.37, P = 0.008). Landsburg et al. analyzed 159 cases with DHL achieving CR and reported that 3 year relapse free survival (RFS) in intensive regimen group (R Hyper CVAD, DA EPOCH R and R CODOX M/IVAC) and R CHOP group was 88% and 56%, respectively (P = 0.002). 5 In 106 DHL case series by Gandhi et al., CR rate with DA EPOCH R was higher than that with R CHOP (68% vs 49%, P = 0.01), and the percentage of primary refractory cases was lower in DA EPOCH R compared with R CHOP (18% vs 46%, P = 0.005) or other intensive regimens (P = 0.03). 16 Another report of 34 cases of DHL by Abramson et al. also found DA EPOCH R regimen as a favorable factor for OS (HR: 0.3), although it was not a significant prognostic factor for PFS. 36 According to the meta analysis for total 394 patients with DHL in 11 studies, median PFS in DA EPOCH R and R CHOP group was 22.2 and 12.1 months, and DA EPOCH R significantly reduced the risk of progression (HR: 0.66, P = 0.03). 6 In terms of prospective data, Cancer and Leukemia Group B (CALGB) or the Spanish PETHEMA Group conducted phase II study of DA EPOCH R in untreated DLBCL patients, reporting CR rate of 84% (CALGB) and 80% (PETHEMA), respectively. 37,38 CALGB successively designed phase III randomized trial of R CHOP vs DA EPOCH R in 524 patients with newly diagnosed DLBCL (CALGB/Alliance 50303). 39 Preliminary analysis of EFS and OS in the entire cohort demonstrated no significant difference between R CHOP and DA EPOCH R group (HR: 1.02, P = 0.89 for EFS; HR: 1.19, P = 0.40 for OS). As for MYCrearranged DLBCL, Dunleavy et al. performed phase II trial of DA EPOCH R in 52 patient cohort including 14 with MYC/BCL2 DHL and reported that PFS at a median follow up of 14 months was 79% in the entire cohort and 87% in DHL. 40 Collectively, it is reasonable to consider DA EPOCH R as the most promising regimen in current clinical practice. The results of prospective studies in DHL patients would determine the efficacy of DA EPOCH R. Central nervous system (CNS) involvement is frequently observed in DHL patients, and even DA EPOCH R regimen is ineffective in not penetrating the CNS fully. 15,34 Therefore, as Friedberg recommends, intrathecal therapy with MTX or high dose MTX/AraC combination therapy should be additionally conducted for lowering the risk of CNS relapse THE ROLE OF CONSOLIDATIVE HCT AFTER INDUCTION CHEMOTHERAPY FOR NEWLY DIAGNOSED DHL Another topic for newly diagnosed DHL is the application of consolidative HCT. Previous data about consolidative HCT are summarized in Table 2. Sun et al. reviewed 32 MYC/BCL2 DHL cases including 19 receiving R CODOX M/IVAC followed by HCT and reported that 2 year PFS and OS was 41% and 53% in the entire cohort, and 60% and 82% in HCT cohort. 42 According to another study for 16 patients with MYC/BCL2 DHL receiving DA EPOCH R followed by auto HCT, 2 year PFS, and OS was both 91%. 43 While these data indicate potentiality of consolidative HCT, this approach has failed to show survival benefit in the retrospective comparisons with non HCT cohort. Landsburg et al. compared outcomes between 62 cases receiving auto HCT and 97 without HCT and showed that relapse free survival (RFS) and OS were similar between the 2 groups (3 year RFS: 89% in the HCT group vs 75% in the non HCT group, P = 0.12; 3 year OS: 91% in the HCT group vs 85% in the non HCT group, P = 0.74). 5 Under different induction

5 OHMOTO AND FUJI 5of10 regimen and transplantation type (auto HCT or allo HCT), the studies conducted by Petrich et al., Oki et al. or Gandhi et al. also could not demonstrate survival benefit by consolidative HCT after achieving CR. 3,4,16 As for DEL, subset analysis of randomized phase III SWOG S9704 trial showed patients receiving consolidative auto HCT had better outcomes, although the difference was not significant (2 year OS: 60% in the HCT group vs 18% in the non HCT group, P = 0.25). 44 There is still controversy about the indication of consolidative auto HCT in patients with DHL after intensified induction chemotherapy. As Staton et al. reviews, careful observation without consolidative HCT would be a practical option for cases maintaining CR, and application for selective patients with high risk disease should be individually judged CLINICAL APPLICATION OF HCT FOR REFRACTORY/RELAPSED DHL 6.1 Auto HCT Disease status at auto HCT is one of the most important factors affecting prognosis after HCT. 7,8 The intergroup Collaborative Trial in Relapsed Aggressive Lymphoma (CORAL) study for patients with DLBCL in first relapse or refractory disease identified refractory disease/early relapse from initial therapy as one of poor prognostic factors. 9 Clinical outcomes of auto HCT for refractory/relapsed (ref/rel) cases are listed in Table 3. In the subset analysis of 161 ref/rel DLBCL patients enrolled in the CORAL study, 12 MYC rearranged patients were treated by intensive regimen followed by auto HCT, and the 4 year PFS and OS in these cases was only 14% and 23%, respectively. 46 Herrera et al. also reviewed 117 chemosensitive DLBCL patients receiving auto HCT including 12 DHL and 47 DEL patients. 30 DHL cases had shorter PFS and OS compared with non DHL cases (4 year PFS: 28% vs 57%, P = 0.01; 4 year OS: 25% vs 61%, P = 0.002), and DEL cases also had poorer PFS than no DEL cases (4 year PFS rate: 48% vs 59%, P = 0.05), although the difference was not significant for OS. The prognosis of five cases fulfilling the criteria of both DHL and DEL was dismal (4 year PFS rate: 0%). Standard auto HCT is not able to overcome intractable DHL/DEL, and new approaches incorporating efficient salvage and conditioning regimen, or maintenance treatment after auto HCT should be investigated Allo HCT The role of allo HCT has not been fully established for DHL. Clinical data of allo HCT for ref/rel cases are also listed in Table 3. Recently, Herrera et al. published an interesting data of allo HCT for patients with ref/rel DHL/DEL. 48 Of 78 patients with aggressive B cell NHL, 58% of the cases received previous auto HCT and 49% were refractory to initial regimen. There were no significant differences in PFS and OS between 10 DHL and 68 non DHL cohort (4 year PFS: 40% vs 34%, P = 0.62; 4 year OS: 50% vs 38%, P = 0.46), or between 37 DEL and 41 non DEL cohort (4 year PFS: 30% vs 39%, P = 0.24; 4 year OS: 31% vs 49%, P = 0.17), respectively. In contrast, Kawashima et al. analyzed 37 DEL and 23 non DEL patients undergoing allo HCT and showed that the 2 year PFS and OS were significantly worse in DEL cohort compared with non DEL cohort (2 year PFS: 20% vs 78%, P < 0.001; 2 year OS: 46% vs 77%, P = 0.02). 49 In their study, 32% of the cases had a history of auto HCT and 35% were chemoresistant at allo HCT. Considering retrospective nature and relatively small sample size, the discordance in two studies might be explained by the difference of patient backgrounds. Favorable TABLE 2 Conslidative HCT for initial DHL and DEL Author Number of patients undergoing consolidative HCT Type of HCT Induction regimen Petrich et al Auto (N = 39), Allo (N = 14) Oki et al Auto (N = 24), Allo (N = 2) Conditioning regimen PFS/EFS of HCT cohort OS of HCT cohort NA NA NA Median not reached R CHOP (N = 2), DA EPOCH R (N = 14), R HyperCVAD/MA (N = 8), Others (N = 2) Landsburg et al Auto (N = 62) R CHOP (N = 8), Intensive regimen (N = 54) Gandhi et al Auto (N = 13). Allo (N = 1) Sun et al Auto (N = 11), Allo (N = 8) NA 68% (2 years) 70% (2 years) NA 89% (3 years) 91% (3 years) Intensive regimen (N = 14) NA NA NA R CODOX M/IVAC (N = 19) Auto: BEAM, ETP, CY, TBI, Allo: CY/TBI 60% (2 years) 82% (2 years) Chen et al Auto (N = 16) DA EPOCH R (N = 14) BEAM (N = 16) 91% (2 years) 91% (2 years) Puvvada et al Auto (N = 5) R CHOP (N = 5) NA 60% (2 years) 60% (2 years) allo, allogeneic; auto, autologous; BEAM, bleomycin, etoposide, cytarabine, and melphalan; CY, cyclophosphamide; DEL, double expressor lymphoma; DHL, double hit lymphoma; EFS, event free survival; ETP, etoposide; HCT, hematopoietic cell transplantation; NA, not available; OS, overall survival; PFS, progression free survival; TBI, total body irradiation.

6 6of10 OHMOTO AND FUJI TABLE 3 Outcomes of auto HCT and allo HCT for refractory/relapsed DHL/DEL Author Cuccuini et al. 46 Number of patients undergoing HCT 12 (MYC rearranged DLBCL) Type of HCT Herrera et al (DHL), 47 (DEL) Auto (N = 59) DHL: CBV (N = 9), R/Z BEAM (N = 3), DEL: CBV (N = 37), R/Z BEAM (N = 10) Herrera et al (DHL), 31 (DEL) Allo (N = 41) DHL: MAC (N = 2), RIC (N = 8), DEL: MAC (N = 7), RIC (N = 24) Kawashima 37 (DEL) Allo (N = 37) MAC (N = 2), et al. 49 RIC (N = 35) Conditioning regimen Donor Source PFS of HCT cohort OS of HCT cohort Auto (N = 12) NA Auto 14% (4 years) 23% (4 years) Auto DHL: PBSC (N = 8), UCB (N = 2), DEL: PBSC (N = 26), BM (N = 3), UCB (N = 2) HLA matched related (N = 12), HLA matched unrelated (N = 6), HLA mismatched related and unrelated (N = 7), Haploidentical (N = 2), UCB (N = 10) DHL 28% (4 years), DEL 48% (4 years) DHL 40% (4 years), DEL 30% (4 years) DHL 25% (4 years), DEL 56% (4 years) DHL 50% (4 years), DEL 31% (4 years) 20% (2 years) 46% (2 years) allo, allogeneic; auto, autologous; BEAM, carmustine. etoposide, cytarabine, melphalan; BM, bone marrow; CBV, carmustine, cyclophosphamide, and etoposide; DEL, double expressor lymphoma; DHL, double hit lymphoma; DLBCL, diffuse large B cell lymphoma; HCT, hematopoietic cell transplantation; MAC, myeloablative conditioning; OS, overall survival; PBSC, peripheral blood stem cell; PFS, progression free survival; R/Z, rituximab or ibritumomab tiuxetan; RIC, reduced intensity conditioning; UCB, umbilical cord blood. survival data by Herrera et al. might indicate the possibility that allo HCT could eliminate adverse impact by DHL/DEL. Considering the dismal outcome of auto HCT in the setting of salvage therapy, it would be reasonable to choose allo HCT in this setting. Larger scaled studies are warranted to verify their results. When clinical application of HCT is discussed, inherent biases in the selection of patients undergoing HCT are unavoidable. 50 In other words, only young and fit cases usually proceed to HCT, which makes the direct comparison between patients who received HCT and those who received another treatment more difficult. HCT specific comorbidity index or pretransplant assessment of mortality score are widely used for evaluating comorbidities of allo HCT in advance. 51,52 In selecting DLBCL patients who are feasible for allo HCT after auto HCT failure, the Center for International Blood and Marrow Transplant Research working group proposed a prognostic model composed of four factors (Karnofsky performance status < 80, chemoresistance, interval from auto HCT to allo HCT shorter than 1 year and myeloablative conditioning). 53 From the point of limited number of DHL patients proceeding to allo HCT, constructing prognostic models for DHL might be difficult. Klyuchnikov et al. proposed algorithm for application of HCT in DLBCL based on chemosensitivity, risk for failure of auto HCT and risk for non relapse mortality (NRM) with allo HCT. 47 It should be validated whether the same approach is also applicable to DHL. As another point, suitable conditioning regimen or donor source for DHL is unknown, and clinical practice is generally founded on approaches for DLBCL. Because of increased NRM, the superiority of myeloablative conditioning over reduced intensity conditioning was not shown in DLBCL patients. 54,55 On the other hand, the association between graft vshost disease and decreased relapse rate was not evident in DLBCL, which suggests anti lymphoma effect might not be robust for DLBCL Further studies of DHL in the setting of allo HCT are warranted. 7 DEVELOPMENTS OF NOVEL AGENTS FOR REFRACTORY/RELAPSED CASES Considering controversial role of HCT for DHL and severe toxicities brought by HCT, patients with ref/rel DHL should be actively enrolled into clinical trials using novel agents. Owing to the nature of rarity, clinical developments for DHL are underway along with DLBCL as a whole. The onset of DHL is biologically explained by BCL2 rearrangement followed by MYC rearrangement, where MYC increases cell proliferation and BCL 2 inhibits apoptosis. 2,14 The approach targeting MYC and BCL 2 as driver genes is attractive, and clinical investigations for BCL 2 inhibitors or agents suppressing MYC are in progress. 14 Preclinical models showed that MYC activated aurora kinases in B cell lymphoma and these kinases were necessary for the maintenance of MYC positive DLBCL, which theoretically explains the usefulness of aurora A kinase inhibitor. 59 In the phase I studies of the first generation BCL 2 inhibitor navitoclax or

7 OHMOTO AND FUJI 7of10 At diagnosis Prognostic scoring system to choose the optimal treatment strategy Efficient screening method to identify DHL cases combining FISH with immunohistochemistry Treatment (Newly diagnosed cases) Prospective trials to optimize the intensive induction regimen including DA-EPOCH-R Prognostic scoring system to identify the high-risk patient who requires consolidative HCT in first remission (Relapsed/refractory cases) Prognostic models to choose allo-hct or auto-hct in chemo-sensitive disease Large-scaled retrospective studies for DHL patients undergoing allo-hct Novel treatment strategies combining HCT with novel agents FIGURE 1 Current discussion topics for DHL. Several unsolved problems about diagnosis and treatment are summarized in this figure. DHL, double hit lymphoma; FISH, fluorescent in situ hybridization; DA EPOCH R, dose adjusted rituximab, etoposide, vincristine, doxorubicin and prednisone; allo HCT, allogeneic hematopoietic cell transplantation; auto HCT, autologous hematopoietic cell transplantation highly selective BCL 2 inhibitor venetoclax as a single agent, overall response rate (ORR) for ref/rel DLBCL patients was 0% and 18%, respectively. 60,61 According to the phase II trial of aurora A kinase inhibitor alisertib by Friedberg et al., ORR was 14% in DLBCL cohort. 62 Efficacy of BCL 2 inhibitors and alisertib as a single agent (ORR less than 20%) is limited in aggressive DLBCL and combination therapies with cytotoxic agents should be tested to achieve sufficient disease control. Lenalidomide and ibrutinib are promising especially for patients with non GCB type DLBCL. Phase I trial of lenalidomide plus DA EPOCH R regimen for 15 DHL/DEL patients in the University of Chicago showed ORR of 75%, and phase II study for the same population is in progress (NCT ). 63 As for MYC positive lymphomas, trials of oral proteasome inhibitor ixazomib plus DA EPOCH R (NCT ) or dual PI3 kinase/histone deacetylase inhibitor CUDC 907 alone (NCT ) are underway. Sauter et al. investigated ibrutinib plus R ICE (rituximab, ifosfamide, carboplatin, and etoposide) regimen for transplant eligible 21 ref/rel DLBCL patients. 64 Of evaluable 20 patients, 18 (90%) achieved response and 16 (80%) proceeded to HCT. Remarkably, all of eight cases with non GCB type achieved CR after completing the first cycle. Clinical trial of ibrutinib targeting DHL cases seemed to be finished in M.D. Anderson Cancer Center (NCT ). This trial assessed clinical effect of ibrutinib maintenance after auto HCT, and the design is novel in incorporating a novel agent into auto HCT and trying to intensify disease control after HCT. Besides moleculartargeted agents, antiprogrammed death 1 monoclonal antibody nivolumab or infusion of anti CD19 chimeric antigen receptor T cells exhibited ORR of 36% and 67% for ref/rel DLBCL, and additional investigation for DHL is expected in the future. 65,66 8 CONCLUSION AND PROSPECTS FOR THE FUTURE The revised WHO 2016 classification of lymphoid neoplasms highlights DHL as a rare and intractable subtype. As a brief summary, current discussion topics of DHL are described in Figure 1. In the first place, clinical data of DHL cases alone are limited. In addition, antitumor effect of standard R CHOP regimen is insufficient for DHL, and promising intensive regimen including DA EPOCH R are Newly diagnosed DHL Relapsed/refractory DHL DA-EPOCH-R Novel agents Salvage regimen Disease status at HCT PS Comorbidity Age Consolidative auto/allo-hct Observation Auto-HCT Allo-HCT Novel agents Novel agents FIGURE 2 Our proposed treatment algorithm for DHL with a focus on HCT. As for newly diagnosed cases, DA EPOCH R regimen without consolidative HCT is the commonest approach. Patients with high risk disease are candidates for consolidative auto/allo HCT. As for ref/rel cases, salvage chemotherapy followed by auto HCT or allo HCT is considered according to the several factors such as disease status at HCT or comorbidity. In clinical trial settings, novel agents are expected to combine with salvage chemotherapy or as maintenance treatment after auto HCT or allo HCT to improve the cure rate of DHL. DHL, double hit lymphoma; ref/rel, refractory/relapsed; DA EPOCH R, dose adjustedrituximab, etoposide, vincristine, doxorubicin and prednisone; auto HCT, autologous hematopoietic cell transplantation; allo HCT, allogeneichematopoietic cell transplantation; PS, performance status

8 8of10 OHMOTO AND FUJI currently examined as initial treatment. Standard auto HCT for relapsed cases seems to be inefficient, requiring new strategies to overcome intractable DHL. Although it is still controversial, favorable survival data of allo HCT have just been reported for ref/rel DHL cases, and larger scaled studies are expected to clarify the position of allo HCT. In the era of novel agents, it is focused how to combine HCT with these agents aiming at better prognosis. Clinical trials of ibrutinib plus R ICE salvage regimen or ibrutinib maintenance after auto HCT are examples of this challenge, and further trials are strongly warranted. Our proposed treatment algorithm based on available data are shown in Figure 2. In the future, the clinical decision to choose auto HCT or allo HCT for DHL or feasibility of HCT for elderly patients should be discussed based on clinical trials. CLINICAL IMPLICATIONS While there have been no established treatment strategies for newly diagnosed DHL/DEL, initial intensive chemotherapy such as DA EPOCH R regimen without consolidative HCT is commonly used. In relapsed/refractory cases, salvage chemotherapy followed by auto HCT or allo HCT are practically applied. CONFLICTS OF INTERESTS The authors declare no competing financial interests. ETHICS STATEMENT The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to. No ethical approval was required as this is a review article. ORCID Akihiro Ohmoto REFERENCES 1. Swerdlow SH, Campo E, Pileri SA, et al. The 2016 revision of the World Health Organization classification of lymphoid neoplasms. Blood. 2016;127: Rosenthal A, Younes A. High grade B cell lymphoma with rearrangements of MYC and BCL2 and/or BCL6: double hit and triple hit lymphomas and double expressing lymphoma. 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