Patients were enrolled from January 2013 to October 2020 from the Department of Hematology, Huadong Hospital (Shanghai, China). A total of 83 patients were diagnosed with high-risk B cell lymphoma, meeting the 2016 WHO diagnostic guidelines for hematopoietic and lymphoid tissue tumors, and all of them underwent pathological type classification by biopsy of lymph nodes and other extranodal tissues. Patients had no vital organ dysfunctions, and their liver, kidney, and heart functions were normal or nearly normal. All patients were intermediate- or adverse-risk (International Prognostic Index (IPI) ≥ 2). For note, 22 patients with IPI = 2 were recurrent, or had cytogenetic aberrations (double-hit lymphoma (MYC/Bcl-2, MYC/Bcl-6) or triple-hit lymphoma (MYC/Bcl-2/Bcl-6) or double-protein expression (MYC/Bcl-2) or triple-protein expression (MYC/Bcl-2/Bcl-6)) and we included these patients into patients with high-risk B cell lymphoma. There were 51 males (61.4%) and 32 females (38.6%). Their median age was 65.9 ± 11.79 years. This study was approved by the ethical committee of Huadong Hospital (approval number: 2021K186) and all patients signed informed consent forms prior to participating in the study.
Patients were given chemotherapy regimens recommended by the National Comprehensive Cancer Network (NCCN) guidelines based on pathological classification, staging, and general conditions. We adjusted the dose of chemotherapy drugs and/or the dosing cycle according to the side effects of treatment and patient tolerance. The first-line chemotherapy for B cell lymphoma is R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone). The second-line regimen is RICE (rituximab, ifosfamide, cisplatin, etoposide), R-ESHAP (rituximab, etoposide, methylprednisolone, cisplatin, cytarabine). If patients couldn’t achieve CR after 6 cycles of R-CHOP chemotherapy, we would choose second-line therapy such as R-ICE or R-ESHAP with modified dose for another two cycles for the elderly fit while ineligible to autologous transplantation. A total of 83 patients who demonstrated an objective response to induction therapies were finally included. Patients who achieve CR or PR after induction therapy from 2013 to 2017 did not receive any maintenance therapy (in observation group), and those from 2018 to 2021 received maintenance therapy (in R2+GM-CSF group). Patients in the R2+GM-CSF group were mostly elderly patients who were not suitable or refused to receive autologous HSCT. They continued to receive the R2 regimen combined with rhGM-CSF (Lenalidomide, 10 mg/d, orally on days 6–15; rituximab 375 mg/m2, intravenous injection on day 6; rhGM-CSF (Amoytop Biotech co) 150 μg/d, subcutaneously on days 1–8) every three months after reaching remission by induction chemotherapy. The patients in the observation group were mostly elderly patients or those who refused to receive autologous HSCT. Patients in the observation group would no longer receive any drugs for maintenance after reaching remission under induction chemotherapy. The flowchart of a clinical trial protocol is shown in Figure 1. There were 39 cases in the R2+GM-CSF group and 44 cases in the observation group.
PBMC collection and Flow cytometry Analysis
Blood was collected from patients in maintenance therapy group and PBMC and immune cells was analyzed before and after each cycle of the maintenance, then compared the difference of WBC, lymphocytes, monocytes, B cells, T cells and NK cells. Eight different surface markers were used in the flow cytometric studies. List mode reanalysis was performed using gating for lymphocytes, monocytes, and myeloid populations, based on side scatter-CD45 histograms. The monoclonal antibodies include isotype controls, and those recognizing CD45, CD3, CD4, CD8, CD19, CD56/16, CD5, and CD7 (BD Horizonä, BD Biosciences, San Jose, CA, USA). The samples were analyzed performed on a Coulter Flow Cytometer (Beckman Coulter, Indianapolis, IN, USA) equipped with a 488 nm Argon laser. The results were reanalyzed using FlowJoä software (FlowJO LLC, Ashland, OR, USA). Flow cytometric data were compared with patient characteristics, including blood counts. The detailed protocol and materials of the flow were shown in Supplemental materials.
Endpoint and follow-up
According to the 2016 NCCN recommended LUGANO evaluation criteria, the efficacy evaluation is divided into complete remission (CR), partial remission (PR), stable disease (SD), and progressive disease (PD). The objective response rate (ORR) is the proportion of CR and PR. All patients were evaluated for efficacy every three cycles during induction treatment period, mainly through positron emission tomography computed tomography (PET/CT) examination of the whole body, ultrasound evaluation or CT scan. For those who did not reach the response of PR or with stable disease or disease progression, the regimen would be changed to the second line therapy. If patients could achieve CR or PR, they would enroll into this study. Patients from 2013 to 2017 were into the observation group (they just stopped chemo and followed up), patients from 2018 to 2021 were into the maintenance therapy group (they just received new R2 regimen as maintenance therapy). The enrolled patients were followed every three months in the first year, every six months in the second year and once a year in the third year. The patients did PET/CT every half a year (every other cycle of maintenance therapy) in the first and the second year and once a year in the third year. For other time, patients received CT scan and ultrasound to follow up. The duration of response (DOR) was defined as the time from the first evaluation of efficacy as complete remission (CR) or partial remission (PR) to the first evaluation of disease progression (PD) or death from any cause.
The follow-up period of this study ended on November 30, 2021. We recorded the data based on the last hospitalization record, out-patient review, and telephone follow-up. OS was defined as the time from the start of randomization to the date of death of the patient due to any cause or the date of the last follow-up. Progression free survival (PFS) was defined as the time from randomization to the date of disease progression or death or last follow-up of patients. In this study, OS and PFS were calculated on a monthly basis. The 83 patients were followed up for a median time of 21.1 ± 14.81 months.
The patients’ characteristics were summarized using the mean and standard deviation for numerical variables and as frequencies with percentages for categorical variables. Differences in patients’ characteristics among the two groups were assessed using Student’s t test for numerical variables and with Fisher’s exact test for categorical variables, including F-tests and Chi-squared tests. The Kaplan–Meier (KM) method was used to estimate unadjusted probabilities of OS and PFS. Unadjusted OS and PFS between subgroups were compared using a Log-rank test. The joint effects and prognostic factors of patient covariates and treatment arms on OS and PFS were assessed using Cox regression models. SPSS 20.0 software (IBM Corp., Armonk, NY, USA) was used to perform all the statistical analyses. A value of P＜0.05 was considered statistically significant.