Diffuse large B-cell lymphoma (DLBCL) is the most common high-grade non-Hodgkin lymphoma accounting for 30–40% of B-cell non-Hodgkin lymphomas (1). DLBCL has considerable biologic, molecular, and clinical heterogeneity resulting in different responses to therapy (2). DLBCL is a potentially curable disease with an overall 60–70% chance of achieving durable complete remission (CR) with the currently used standard first-line immunochemotherapy. However, 30–40% of patients are either refractory to first-line treatment or experience relapse and eventually will die of disease progression (3). Patients achieving remission lasting for 24 months or more from the end of first-line treatment have favorable prognosis at long term (4–6). The diagnosis and subtyping of DLBCL have come far to date, from morphological assessment of tissue slide to now, where numerous ancillary tests are a prerequisite, including immunophenotyping performed by immunohistochemistry (IHC), cytogenetics, and detailed molecular testing to classify the disease based on cell of origin (COO). With the advent of novel therapeutic options, it is expected that molecular subtyping of DLBCL at diagnosis will allow prognostic stratification of patients into distinct subgroups which could provide preclinical rationale for therapeutic targeting the involved pathways and paving the application of personalized treatment. The original DLBCL molecular classification using DNA microarray-based technology was initially described by Alizadeh et al. (7). This technology allowed the analysis of transcriptional gene expression pattern, and distinguished two major COO subtypes of DLBCL: germinal center B-cell like (GCB) and activated B-cell like (ABC). This resulted in the molecular subtyping of 80–85% of all DLBCL cases. Treated with standard immunochemotherapy, the ABC subgroup had poorer 5-year survival compared to the GCB group (16% vs. 76%, respectively).

Hans (8), Choi (9), and Tally (10) are the most established algorithms that are based on IHC to assess COO with the Hans algorithm being the most commonly used in clinical practice. However, the clinical significance of COO subtyping using IHC remains controversial, and the assessment of COO by gene expression profiling rather than IHC is better associated with the prognosis of DLBCL (11).

Translocations, gene rearrangements, and protein expression patterns including but not limited to BCL6, BCL2, and MYC have been shown to have prognostic implications.

The human proto-oncogene BCL6 was identified from chromosomal breaks at 3q27, is expressed predominantly in germinal center B-cells (12, 13), and functions as a sequence-specific transcriptional repressor (14, 15). Mutation or translocation of BCL6 gene has been implicated in the pathogenesis of certain subtypes of DLBCL (12). BCL6 is frequently affected by chromosomal translocations, occurring more frequently in ABC DLBCL. There is no consensus on the effect of BCL6 translocation on prognosis of DLBCL, especially in the rituximab era, with studies showing either favorable (16, 17), neutral (18, 19), or unfavorable outcomes (20).

The BCL2 protein is a central regulator of the mitochondrial apoptotic pathway and its gene is located on the chromosome arm 18q21 (21). The primary function of BCL2 is to promote cell survival by inhibiting apoptosis (22–24). The BCL2 gene is overexpressed in many cancers including DLBCL and is usually associated with drug resistance (25). In DLBCL, the ratio of BCL2-positive cases is highly variable, ranging from 20% to 80% across studies that used IHC (26, 27). Some studies showed BCL2 protein expression as a marker of poor prognosis in GCB subtype of DLBCL (26, 28), while others reported it to be a marker of poor prognosis in ABC-DLBCL, only (29).

Chromosomal translocations dysregulating MYC (8q24) were reported in 5–15% of GCB DLBCL cases (17), while MYC protein detection in 5–40% of DLBCL cases (30–32). MYC gene translocation was associated with a very poor outcome in DLBCL (33–36). MYC translocation in DLBCL is usually associated with other gene abnormalities, e.g., BCL2 or BCL6 rearrangements (37, 38). Some studies show that about 30% of DLBCL co-express high levels of MYC and BCL2 proteins, which cases are called ‘double expressor’ DLBCL (DEL) (39).

Although substantial knowledge has accumulated about prognostication in DLBCL, a lot of questions have remained unanswered. The main objective of this study was to describe the distribution of and to test the prognostic ability of IHC- and interphase fluorescence in situ hybridization (FISH)-based molecular markers in a Hungarian cohort of newly diagnosed DLBCL patients from the rituximab era.

This multicenter, retrospective study was approved by the Committee of Science and Research Ethics (ETT-TUKEB) under reference number 50268-8/2017.

Table 1 summarizes the clinical characteristics of the 247 patients included. The median age at the time of diagnosis was 65 years (range: 19–91 years), 65.2% of patients were >60 years, 46.6% were male, 42.9% had an elevated serum lactate dehydrogenase and 74.1% had an advanced (stage III/IV) disease. The majority (94.7%) were treated with R-CHOP or similar regimens, the most common salvage therapies were the R-ICE and R-DHAP protocols.

The clinical and genetic heterogeneity of DLBCL still presents challenges in predicting response to treatment and prognosis. The 2016 revision of the World Health Organization classification of lymphoid neoplasms made it mandatory to classify the molecular subgroups of DLBCL into GCB and ABC subtypes (37) and this remains unchanged in the WHO 5^th edition published in 2023 (38). However, 10–15% of DLBCL cases cannot be categorized into one of the two COO groups and are termed unclassified DLBCL using gene expression studies. In the current study, we used IHC and FISH techniques on samples from DLBCL patients to classify rituximab-treated patients in many ways including by COO. Then, we interpreted survival data in this context.

In our cohort of patients, the COO phenotype failed to predict prognosis, which is surprising knowing that some studies have demonstrated significantly better survival for the GCB group (47, 48). However, the prognostic value of COO remains controversial, and other authors did not report any differences in overall prognosis based on COO, in line with our observations (49–51). Table 6 . summarizes the COO distribution and survival data of DLBCL patients according to the immunophenotyping results reported in our study and other published reports. The controversy of the literature may be explained by many factors including but not restricted to the retrospective nature of the studies with a mixed pool of patients receiving various treatment regimens, in addition to a relatively short follow-up period (52).

Factors that were independently associated with EFS in the multivariate analysis were IPI, high MIB-1 (>90%), and BCL6 expression. According to our results, BCL6 protein overexpression carries a positive prognostic effect on OS and EFS. BCL6 protein expression is considered as a hallmark of GC origin in DLBCL and it is associated with favorable outcome reported by some studies, consistent with our results (8, 19, 53–56). However, our findings demonstrated no significant association between BCL6 protein expression and BCL6 translocation. In our study, the frequency of BCL6 gene rearrangement was 21.4% and most of the cases were stratified into the non-GCB group according to the Hans algorithm. The association between the BCL6 gene alteration and non-GCB phenotype was confirmed by other studies as well (17, 20, 57, 58). In our study, BCL6 rearrangement had no prognostic impact on OS or EFS. Other studies yielded conflicting results showing that BCL6 rearrangement was associated with a worse outcome (20, 59–63), reporting no significant associations at all (64–67), or implying an association with favorable outcomes (68, 69).

BCL2, MUM1, and MYC protein expressions did not emerge as independent prognostic variables in multivariate analysis. Unlike the high proliferation index (>90%) detected by MIB-1 antibody, which proved to be an independent predictor of good prognosis regarding OS and EFS. We did not find any significant difference in OS and EFS of DEL cases compared to the non-DEL cases.

Our finding suggesting no impact of BCL2 expression on the prognosis of patients with DLBCLis in line with the results of some previous publications (8, 70–73). However, other researchers have observed adverse outcomes with BCL2 protein overexpression (17, 26, 27, 74). In addition, in our study, there was no significant association between BCL2 protein expression and BCL2 gene aberrations.

Finally, even in the rituximab era, there are still significant differences in OS and EFS across the IPI groups.

Our study has several strengths and limitations. The major strength of our work includes the size and coverage of the study population (247 DLBCL cases from 7 Hungarian centers), allowing a detailed and representative survival analysis. The major limitation of our work is the study’s retrospective design, which, as reflected by the number of excluded patients, resulted in lack of a full dataset in some analyses.

Successful integration of predictive and prognostic tools in DLBCL trials requires combination of clinical and molecular factors. Our study provides additional support to previously reported publications that DLBCL is characterized by heterogeneous molecular features and clinical outcomes. Based on our findings, only the IPI, BCL6 expression by IHC, and high (>90%) MIB-1 expression and not the other markers analyzed (CD10, BCL2, MUM1, MYC positivity, and gender) are independent predictors of OS and EFS in DLBCL. We did not find any difference in survival by GCB vs. non-GCB subtypes. These findings may improve prognostication in DLBCL and can contribute to designing further research in the area. However, considering the limitations of our study, these findings should be validated in prospective series.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

The studies involving humans were approved by The Committee of Science and Research Ethics (ETT-TUKEB) under reference number 50268-8/2017. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

HA, IV-N, BA, and LP designed the research; NF and ZS analyzed data; HA, BA, and ZS drafted the manuscript; HA, LP, BA, IV-N, ZR, AG, BK, LK and ZS reviewed and approved the final version of the manuscript. All authors contributed to the article and approved the submitted version.