Intensive immunochemotherapy improves prognosis of young patients with double-expressor lymphoma comparison to non-double-expressor lymphoma: a single-center retrospective cohort study

Background Double-expressor lymphoma (DEL), defined as cases with concurrent MYC and BCL2 proteins overexpression, is associated with poor prognosis when treated with R-CHOP alone. Prognostic data on DEL treated with intensive treatment are limited and controversial. Methods We retrospectively report our experiences about 221 consecutive de-novo diffuse large B cell lymphoma patients and analyze the role of intensive therapies for 65 patients with DEL. Results No significant difference in clinical characteristics and survival outcomes between DEL and non-DEL was observed. Overall, 32 (76.2%) and 20 (87.0%) patients with DEL received R-CHOP and intensive treatment achieved partial response or complete response, respectively. Intensive therapies may improve the prognosis over R-CHOP in patients with DEL (P = 0.029 for PFS, P = 0.026 for OS). Subgroup analysis according to clinical characteristics showed intensive therapies resulted in better prognosis than R-CHOP regimen in DEL patients aged under 60 years (P = 0.004 for PFS; P =0.090 for OS), whereas no significant superiority was found in DEL patients aged over 60 years (P = 0.646 for PFS; P = 0.361 for OS). Conclusions This second Chinese cohort study suggests that intensive immunochemotherapy may have the ability to improve the prognosis of young patients with DEL. based dehydrogenase (LDH), serum β2-microglobulin (β2-MG), initial of and (CT) (PET) the or exact PFS the Kaplan-Meier and log-rank test in subgroups cyclophosphamide, doxorubicin, vincristine, prednisone; PFS: progression-free survival; OS: overall survival; DHL: double-hit lymphoma; CNS: Central nervous system ; IPI: International Prognostic Index; LDH: lactate dehydrogenase; β2-MG: β2-microglobulin; GCB: germinal center B-cell-like; PET: 18 F-fluorodexyglucose positron emission tomography; CT: computed tomography; DA-EPOCH-R: rituximab, etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin; R-HyperC-VAD/MA: rituximab, hyperfractionated cyclophosphamide, vincristine, doxorubicin, dexamethasone, alternating with cytarabine plus methotrexate, R-DHAP rituximab, dexamethasone, cytarabine, and cisplatin; SCT stem cell transplantation; G-CSF: granulocyte colony-stimulating factor; CR: complete response; PR: partial response; PD: progressive disease; SD: stable disease; HR: hazard ratio; CI: confidence interval

subtypes, there has been increasingly more attention in several subgroups of B-cell lymphoma with poor outcomes after RCHOP treatment, especially Double-hit lymphoma (DHL) and Double expressor lymphoma (DEL). DHL is defined as cases with genetic rearrangement for MYC along with BCL2 or BCL6 or both genes, which newly categorized into an individual subtype named "High-grade B-cell lymphoma" with a median OS is only about 13-16.8 months for patients treated with RCHOP (5)(6)(7)(8). DEL is defined as cases with concurrent MYC and BCL2 proteins overexpression, representing 18-34% of patients with DLBCL (9,10). Several research studies have demonstrated an abysmal prognosis for patients with DEL treated with R-CHOP alone, with 5-year PFS and OS rates less than 40% (8)(9)(10). There is an urgent need to dissect clinical characteristics and determine more beneficial therapeutic strategies than RCHOP for patients with DHL and DEL.
Though intensive induction treatment proves significant prognostic improvement for patients with DHL in prior retrospective studies (11)(12)(13), prognostic data on patients with DEL treated with intensive treatment strategies are limited and controversial.
With large unmet medical needs, we conducted this retrospective cohort study, aiming to analyze clinical characteristics and survival outcomes of Chinese patients with DEL comparison to non-DEL and further determine the safety and feasibility of the first-line intensive induction regimens compared to R-CHOP regimen in patients with DEL in subset analyses, which will provide more clinical evidence for better risk-stratified treatment decisions.

Patient selection
This is a retrospective cohort study, including 221 consecutive patients who were diagnosed with de-novo DLBCL and had available pathological specimens at Peking Union Medical College Hospital between January 2015 and December 2018. All cases were diagnosed with clinical symptoms and results of biopsy according to the World Health Organization (WHO) classification criteria. Data on laboratory clinical characteristics were retrieved from medical records, including gender, age, stage based on Ann Arbor staging system (14), IPI score (15), serum lactate dehydrogenase (LDH), serum β2-microglobulin (β2-MG), initial induction regimen, number of extranodal disease sites and CNS involvement evaluated with enhancement computed tomography (CT) scan or 18  Academy of Medical Sciences has approved this retrospective study, in which informed consent was waived, but patient confidentiality was protected.

Immunohistochemistry and FISH
Two experienced morphological and pathological specialists re-stained all formalin-fixed, paraffin-embedded samples and independently reanalyzed the immunohistochemical results, employing the most recent biopsy specimens and a panel of antibodies including MYC (clone Y69; Abcam), BCL2 (clone 124; Dako), BCL6(clone LN22, Dako), CD10 (clone 56C6, Dako) and MUM1(clone MUM1p, Dako). Any discrepancy was resolved by discussion. As previously described (6,16), the positive cut-off of MYC, BCL2, BCL6 protein is 40%, 50%, and 30% expressing cells, respectively. Cases with concurrent positive MYC and BCL2 protein were considered as DEL. Additionally, the GCB and non-GCB subtype of all cases were determined with cell-of-origin (COO) classification according to Hans algorithm based on the histologic results of CD10, BCL6, and MUM1 proteins (17). Furthermore, DHL refers to B-cell lymphoma with genetic rearrangement for MYC and BCL2 and/or BCL6 genes, which newly categorized into a separate category named "High-grade B-cell lymphoma". FISH was conducted to detect the genetic rearrangement in the majority of our samples, applying two different probes to evaluate MYC on 8q24 or BCL6 on 3q27.

Supportive treatment
During treatment, long-lasting granulocyte colony-stimulating factor (G-CSF) and prophylactic antibiotics were applied regularly when peripheral blood leukocyte counts <2.0×10 9 /L. Concentrated red blood cell infusion was conducted when hemoglobin <60 g/L or be complicated by severe cardiopulmonary decompensation. Thrombopoietin was employed when platelet<30 × 10 9 /L, and platelets concentrate were infused when platelet <20 × 10 9 /L.

Treatment evaluation
Treatment evaluation and progression of disease were evaluated with a lumbar puncture, 18Ffluorodexyglucose positron emission tomography (PET), or enhancement computed tomography (CT) scan, complied with revised International Workshop Group response criteria for malignant lymphoma (22). Regular imaging evaluations were conducted at the end of regular treatment courses and every six months after diagnosis. The extra evaluation was performed when physicians suspected disease progressed. PFS duration is defined as the duration between diagnostic date and the date of disease progression, recurrence date, or date of death from any cause. OS duration is described as the interval between the diagnostic date and the date of death or the last follow-up.

Statistical analysis
Data on clinical characteristics were analyzed using Statistical Package for the Social Sciences

Patient characteristics
We reviewed a total of 221 patients in our present study, including 77 (34.8%) patients with DEL and 144 (65.2%) patients with non-DEL. The clinical ，immunohistochemical and FISH characteristics were summarized in Table 1. Overall, the characteristics of patients with DEL were very similar to non-DEL. The median age for all cases was 55 years old (range 13-87). 173 (78.3%) cases were observed at advanced stages (III-IV) at diagnose, and 114 (51.6%) case was found with high IPI score (3)(4)(5). Though the condition of serum β2-MG and rearrangement were not determined in 94 and 78 cases respectively, the majority of cases had elevated serum LDH (122, 55.2%) and serum β2-MG (111, 87.4% in 127, 94 was undetected), respectively. 96 (43.4%) of cases had more than two different extranodal disease sites, and only 3 (1.4%) patients were found with and central nervous system involvement (CNS involvement) at diagnosis. Germinal center B-cell-like (GCB) subtype (134, 60.6%) was relatively more common than the non-GCB subtype, and the proportion of non-GCB in the DEL group was higher than the non-DEL group without significant difference (66.2% vs. 57.6%, P=0.213). Only 143 cases had explicit FISH results, and 8 cases (5.6%) were positive for MYC and BCL2 genetic rearrangement, which is mostly consistent with prior literature.
Generally, there was no significant difference in each baseline clinical characteristic between the DEL group and the non-DEL group.  Treatment-related mortality, defined as deaths due to infection or massive hemorrhage rather than disease progression, was observed in 3 (2.3%) patients received R-CHOP and 1 (2.0%) patients received intensive treatment, respectively.

Survival outcomes of PFS and OS
Generally, there was no statistical difference in survival outcomes between the DEL group and the non-DEL group (P = 0.991 for PFS, P = 0.632 for OS) (Fig. 1A &1B). Median PFS and OS for the DEL group or non-DEL group were undefined because of the limited follow-up time and sample size. Considering the consensus that DEL indicated poor prognosis when treated with R-CHOP than non-DEL, we compare the PFS and OS outcomes between subgroups, including DEL with R-CHOP, DEL with intensive therapy, and non-DEL with RCHOP for further exploration ( Fig.2A &2B).      Table 3 and 4. Nevertheless, we excluded IPI and SCT factors due to their non-independence.

Discussion
Double-expressor lymphoma (DEL) is a relatively common subgroup of DLBCL with poor prognosis compared to non-DEL when treated with R-CHOP regimen. Patients with DEL were more likely to be associated with higher tumor-proliferation property and inferior response rates to R-CHOP therapy (24). Intensive treatment, especially DA-EPOCH-based regimen, demonstrated promising results in DHL, relapsed or refractory DLBCL, and subgroups of DLBCL with high proliferation (25,26). The optimal therapeutic strategies for patients with DEL remain a significant challenge because previous results have shown great controversies on intensive immunochemotherapeutic regimens recently. This study demonstrated that intensive treatment, especially DA-EPOCH-R, was associated with higher response rates and better survival outcomes than the R-CHOP regimen in DEL patients and might have the ability to eliminate the inferior prognosis of DEL over non-DEL to some extent.
To our knowledge, this is the second report analyzing the survival of a consecutive cohort of Chinses patients with double-expressor DLBCL treated with intensive therapies or R-CHOP, but our findings and conclusions differ from the first Chinese cohort study conducted by Xinyu Zhang (27). We found the majority of patients with DLBCL in our study had some characteristics including advanced stage, aged under 60, elevated LDH and β2-MG, extranodal disease sites<2 at diagnosis and there was no significant difference between two groups, which means patients with DEL was not more severe than non-DEL at baseline. In our cohort, 77 (34.8%) and 5.6% DLBCL cases were associated with the DEL subgroup and DHL subgroup, respectively.
66.2% DEL cases had a non-GCB subtype, which is consistent with the past findings (63%) ( (29), the PFS superiority of intensive treatment in our study might analogize long-time OS superiority to some extent.
Interestingly significant prognostic superiority of intensive treatment was not observed in the first Chinese cohort and some other unplanned subset analyses from trials, potentially due to their limited sample sizes and lower proportions of GCB subtype, which proved superior survival than non-GCB for patients with DLBCL (30). Additionally, given that their patients were mainly diagnosed before 2016, more application of advanced supportive treatment in recent years might significantly improve the safety and prognosis of intensive regimens in our study. Other possible reasons for the discrepancy in outcomes include selection bias caused by DHL, potentially different proportions of patients received radiotherapy or SCT. More importantly, different genetic subtype distribution between races, such as MCD, in Hesperian and Chinese (31,32).
As for frontline SCT, it seemingly resulted in less prognostic benefits than intensive therapies for patients with DEL due to no significant comparisons, which may prove the opinion that DLBCL may be a "one-shot" disease. Kawashima (2018) retrospectively demonstrated the inferior prognosis of DEL compared to non-DEL, even after allogeneic hematopoietic cell transplantation. The definite benefits of SCT and intensive therapies for patients with DEL should be determined by larger randomized trials. Furthermore, due to the increased toxicity and treatment-related complications of intensive treatment, overall consideration of age, comorbidities, tolerance of the initial chemotherapy, performance status, and marrow function are necessary to make individualized therapy before conducting SCT or induction therapy. Adequate supportive treatment is a prerequisite for the safety of the intensive treatment. Although hematological toxicities were more common in patients with intensive treatment, it does not necessarily increase the incidence of neutropenic fever and treatment-related mortality, possibly due to more active application of prophylactic antibiotics and long-lasting G-CSF in patients with intensive regimens, which significantly reduced the duration of neutropenia. inhibitor) (36) and several advanced immunotherapies, such as checkpoint inhibitors and cellular immunotherapies (37). The combination of new targeted drugs with R-CHOP regimen or intensive regimens may show survival superiority than conventional intensive treatment in the future and bring more treatment opportunities for patients with DEL. Though this is a promising therapeutic strategy, before there is sufficient medical evidence, we still recommend intensive chemotherapy for patients with DEL, especially for young patients.

Conclusions
In conclusion, clinical characteristics were comparable between DEL and non-del. The results of this study suggest that intensive immunochemotherapy may improve the prognosis of patients with double-expressor lymphoma aged under 60 years, which should be considered for future prospective clinical trials. Academy of Medical Sciences has approved this retrospective study, in which informed consent was waived, but patient confidentiality was protected.

Consent for publication
Consent was obtained from all cases included in the study.