Primary extranodal diffuse large B-cell lymphoma in the rituximab era: a single center, retrospective analysis

ABSTRACT Objectives To analyse the clinical characteristics and therapeutic response of Chinese patients with primary extranodal diffuse large B-cell lymphoma DLBCL (PE-DLBCL). Methods We analysed the clinical features and outcomes of 197 patients who were newly diagnosed with PE-DLBCL between January 2015 and December 2020. Results The gastrointestinal tract showed the highest rate of involvement (34%), followed by the central nervous system (CNS) and intraocular system (31.5%). The 3-year overall survival (OS) rate was 81% for the entire group and 79% for those with CNS and vitreoretinal involvement. Ann Arbour stage, lactate dehydrogenase level, International Prognostic Index > 2, and complete remission (CR) were significantly related to the survival of patients with PE-DLBCL. The lack of CR was the only independent adverse prognostic factor for OS. Conclusion The clinical outcomes of patients with PE-DLBCL at our centre were encouraging, especially for patients with CNS and vitreoretinal involvement.


Introduction
Malignant lymphomas, which represent a diverse group of diseases, may arise in tissues other than lymph nodes. However, the definition of extranodal lymphomas remains controversial, especially when both lymph nodes and extranodal sites are involved. On the basis of previous studies [1][2][3][4], lymphoma is defined as primarily extranodal if (1) the dominant lesions are present at extranodal sites, and (2) lymph node involvement is only minor or absent. The prevalence of primary extranodal non-Hodgkin's lymphoma (PE-NHL) varies across countries and ranges from 8.7% to 48% [5][6][7][8]. Diffuse large B-cell lymphoma (DLBCL) is the most common (71%−81.3%) pathological subtype of PE-NHL [4,9]. Primary extranodal DLBCL (PE-DLBCL) originates from nearly every extranodal site of the body, especially in the gastrointestinal (GI) tract, followed by the nasopharynx and neck, breast, central nervous system (CNS), skin, bone, thyroid, testis, and rarely other sites [4,10,11].
PE-DLBCLs are well-recognised heterogeneous entities. However, relevant research on these entities is extremely limited. Studies by Shi et al. and Shen et al. showed that the prognosis of extranodal DLBCLs is unsatisfactory [12,13]; however, a large proportion of patients in their studies could not afford rituximab. A 13% improvement in 2-year overall survival (OS) has been reported by combining rituximab with the cyclophosphamide, doxorubicin, vincristine, prednisone (CHOP) regimen in elderly patients [14]. In addition, the 5-year OS was 58% in patients who received the rituximab and CHOP (R-CHOP) regimen versus 45% in those who received CHOP alone [15]. We aimed to identify the improvement in the prognosis of patients with PE-DLBCL in an era in which everyone can afford rituximab. Our centre has also made significant efforts to standardize treatment, ensure intensive induction, and strengthen central prevention. Thus, the main objective of this study was to analyse the specific clinical characteristics, treatment response, and survival of patients with PE-DLBCLs in a single centre in China in this new era.

Patients
Patients with incomplete clinical data and those who were diagnosed at our hospital but did not receive treatment were excluded (n = 25). A total of 644 patients who were newly diagnosed with DLBCL between January 2015 and December 2020 at our hospital were included, and 197 patients diagnosed with PE-DLBCL were evaluated. All biopsy specimens were independently classified by three specialized pathologists according to the WHO classification protocol [16].
Baseline data, such as sex, age, Ann Arbour stage, performance status, extranodal sites, complete blood count (CBC) parameters, and biochemical routine indices, including the levels of liver enzymes, creatinine, serum albumin, and lactate dehydrogenase (LDH), were collected. Essential imaging evaluations, such as positron emission tomography/computed tomography (PET/CT) and whole-body computed tomography (CT), were performed before and after treatment. Patients with lymphoma were staged using the Ann Arbour classification scheme [17] and evaluated according to the International Prognostic Index (IPI) [18]. PE-DLBCL with CNS or gastrointestinal involvement was evaluated using the IELSG score [19] and Lugano classification [20], respectively.

Treatment, response, and follow-up
The first-line treatment for patients with PE-DLBCL, except for PCNS-DLBCLs, was the R-CHOP regimen [14] or the rituximab, etoposide, prednisone, vincristine, cyclophosphamide (R-EPOCH) regimen [21] for patients with double-protein expression. The regimen was administered every three weeks over 6-8 cycles. For some patients, methotrexate (MTX; 1 g/m 2 , four times) was added for CNS prophylaxis in induction therapy [22]. Local and systemic therapies were applied in patients with primary vitreoretinal DLBCL (PVR-DLBCL): intravitreal methotrexate injections for one year (400 µg, total 16 injections) and the R2 regimen, which included six cycles of lenalidomide (25 mg on day 1-21) and rituximab (375 mg/m 2 on day 1) [23]. Other cases of PCNS-DLBCL were treated with high-dose methotrexate (3.5 g/m 2 )-based combined chemotherapies. The regimen was administered every three weeks over 6-8 cycles. In some patients, surgery was performed to confirm the diagnosis and reduce the tumour burden. Autologous haematopoietic stem cell transplantation is recommended for relapsed/refractory or high-risk patients.
The response to treatment was assessed according to the International Working Group criteria [24]. Eighteen patients were lost to follow-up by the end of the follow-up period (December 31, 2021).

Ethics approval and consent to participate
All procedures were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and the Helsinki Declaration of 1975. The study was approved by the institutional review board of Peking Union Medical College Hospital. Informed consent was obtained from all patients for inclusion in the study.

Statistical analysis
Outcomes were described as progression-free survival (PFS) and overall survival (OS). PFS was defined as the time from diagnosis to the first disease progression or death for any reason. The OS was calculated from the date of diagnosis to the date of the last follow-up or death for any reason. GraphPad Prism (version 9.0) was used to perform statistical analysis. Univariate analysis and generation of survival curves were performed using Kaplan-Meier and log-rank tests. Variables with a P value < 0.1 were selected for multivariate Cox regression analysis. Differences were evaluated using a two-tailed test; P values < 0.05 were considered statistically significant.

Prognostic factors
The univariate and multivariate analysis results for patients with PE-DLBCL are presented in Table 2. On the basis of the univariate results, Ann Arbour stage, LDH level, IPI > 2, not receiving CR, and double hit were significantly associated with survival. However, in the multivariate analysis, failure to achieve CR after first-line therapy was an independent predictor of OS and PFS in patients with PE-DLBCL. The univariate and multivariate analyses results for PGI-DLBCL are presented in Table 3. According to the univariate results, B symptoms, LDH, IPI > 2, Lugano stage, failure to achieve CR, and double hits were significantly related to the survival of patients with PGI-DLBCL. The independent risk factors for PFS included not receiving CR and Lugano stage IV disease in the multivariate analysis. The risk factors for survival in patients with PCNS-DLBCL and PVR-DLBCL are shown in Table 4.

Discussion
To our knowledge, this is a cohort of extranodal lymphomas showing fairly good clinical outcomes in the last five years. Thus, the prognosis of PE-DLBCL has improved in the era of rituximab therapy.  Different extranodal involvement sites are associated with distinct clinical characteristics. Our centre treated slightly more female patients than male patients with PE-DLBCL [25][26][27]. Among the patients with PE-DLBCL, 38.1% were in the advanced stage when diagnosed, whereas primary nodal DLBCL was more frequently classified as stage III-IV [25,28,29]. Some patients may show earlier local symptoms due to a tumour mass. In addition, different definitions for primary extranodal lymphoma can also lead to these differences [3,30].
AlShemmari and Lu et al. reported that the gastrointestinal tract is the most common site of extranodal involvement, while involvement of other sites is rare [4,31]. However, patients with CNS and intraocular involvement also accounted for a high proportion of the cases in our study, partly because of the conduct of clinical trials, which will lead to considerable differences in subsequent analyses.
For patients with PCNS-DLBCL, in comparison with the findings of the largest descriptive study in China, the cases treated at our centre showed a better outcome. Surgical excision for intracranial PCNS-DLBCL has been shown to not improve survival [32,33], which is consistent with our results. Systemic chemotherapy combined with intravitreal chemotherapeutic drug injection for patients with primary vitreoretinal DLBCL (PVR-DLBCL) as the initial treatment may achieve a relatively better outcome [23]. Hernandez et al. reported that lenalidomide [34] had a prominent effect on DLBCL, particularly on non-GCB subtypes. Hernandez et al. confirmed that a combination of rituximab and lenalidomide (R2) can lead to a good synergistic effect [35]. The R2 regimen was recently reported to show significant activity in patients with relapsed/refractory PVRL and PCNSL [36]. Our centre began to use the R2-MTX regimen as the first choice for standard induction chemotherapy for newly diagnosed PCNS-DLBCL, and reported an ORR of 100% (NCT 04120350). However, despite advances in initial treatment, more than 1/3 of the patients show disease relapse [37], and there are no uniform treatment guidelines for relapsed and refractory PCNSL (R/R PCNSL). In this study, no statistically significant difference was observed in survival between patients with PVR-DLBCL and PCNS-DLBCL; this may be attributed to the higher relapse rate of PVR-DLBCL, which was mostly associated with central relapse. Bruton tyrosine kinase (BTK) inhibitor [38] and PD-1 blockade [39] therapies show good antitumour activity in R/R PCNSL. Six patients with R/R PCNSL treated with orelabrutinib plus sintilimab achieved PR/CR in this study and are still under follow-up. Thus, with the ongoing development of newer treatment modalities and monitoring tools, these patients have a more promising future.
In the rituximab era, fewer scholars, such as Wang et al., believe that surgical treatment can achieve a better prognosis than conservative treatment [40]. Most studies have shown that surgical treatment plays an increasingly inconspicuous role in the treatment of PGI-DLBCL and is mostly used for definitive diagnosis or clinical emergencies [41,42]. The data from our study also showed no significant difference in the outcomes of patients receiving chemotherapy alone and those treated with surgery combined with chemotherapy. The value of probable prognostic factors such as sex, LDH levels, B symptoms, and the IPI in patients with PGI-DLBCL remains controversial [43][44][45]. In this regard, we agree with previously reported studies that the Lugano stage can effectively predict the prognosis of PGI-DLBCL [27,46].
Although univariate analysis showed differences in IPI, LDH, DH, Ann Arbour stage, and achievement of CR following first-line treatment, achievement of CR was identified as a factor leading to better survival in our study, which was different from the finding in our previous retrospective study [13] and research data from other centers [26]. This can be explained by the selection bias for hospitalized patients and the fact that the increasing availability of alternative immuno-targeted therapies has reduced the differences in outcomes associated with different baseline characteristics. The short follow-up period in our study is another factor that cannot be ignored. To achieve CR in first-line treatment, we administered intensive induction therapy to some patients with double-protein expression, which also proved to yield better efficacy. Simultaneously, the findings also indicate the need for a better prognostic integral model for PE-DLBCL in the future [47][48][49]. Different sites of extranodal involvement are associated with widely varying clinical outcomes [31,50]. Comparative analyses in the largest comprehensive descriptive study in China [12] and the SEER database [51], which includes data for clinical outcomes in relation to the anatomical distribution of DLBCL, showed that the PE-DCLBL patients in our cohort had a better prognosis. However, longer follow-up periods are required to confirm this finding. In our study, primary bone DLBCL and primary thyroid gland DLBCL(PT-DLBCL) were associated with a better prognosis, consistent with previous reports [52][53][54]. Two-thirds of patients with PT-DLBCL underwent total or subtotal thyroidectomy, which has been proven to be associated with significantly improved survival [55]. In other studies, primary testis DLBCL was related to a higher CNS relapse rate and unfavourable outcomes [56,57]. Of the 4/7 patients in our study who received MTX for CNS prophylaxis, which is still controversial [58,59], only one showed central recurrence, and all had favourable clinical outcomes.
In summary, our study demonstrated that different sites of extranodal involvement in patients were associated with different clinical features and outcomes. The clinical outcome of PE-DLBCL at our centre has been greatly improved through standardized treatment, the addition of immune-targeted drugs such as lenalidomide, the use of local plus systemic treatment for PVR-DLBCL in cooperation with the ophthalmology department, and the application of intravenous MTX prophylaxis for central high-risk groups.

Author contributions
JZ analyzed and interpreted the patient data and was a major contributor in writing the manuscript. DBZ and ZW conceived and designed the work that led to the submission, acquired data and had an important role in interpreting the results. WZ gave final approval of the version to be published. All authors read and approved the final manuscript.