Introduction

Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma (NHL), accounting for almost 40% of patients. With the introduction of molecular assessment, DLBCL has been recognized as a conceptual diagnosis with various subtypes. While the first-line treatment has not been changed, complete remission with first-line treatment being 60% [1–3] remains as the main concern, and identification of high-risk patients who may poorly response to first-line treatment is now the main goal of care [4, 5].

While the classification of DLBCL is a still evolving concept, CD5 expression in DLBCL has been recognized as a distinct entity with conflicting importance. CD5 positivity has been observed with a range of 5% and 22% in newly diagnosed DLBCL [6, 7]. In DLBCL patients who had CD5 expression, this population, unlike the general population of DLBCL patients, LDH enzyme increase, extra nodal involvement, bone marrow involvement, poor ECOG (Eastern Cooperative Oncology Group) performance score, poor international prognostic score, and female tendency were observed to be more frequent. As expected, worse prognosis was observed in patients with CD5 positivity ( +) DLBCL, as well as a higher relapse rate were observed compared to CD5 negative ( −) DLBCL patients [7, 8].

Since CD5 is a T lymphocyte marker and not essential in the diagnosis of DLBCL, it is often neglected in the diagnostic work up leading to conflicting outcomes in terms of its importance. We aimed to determine the frequency of CD5 expression and its association with disease outcomes in DLBCL.

Methods

The study was conducted by the Lymphoma Scientific Subcommittee of Turkish Society of Hematology, including 16 centers among 9 cities that represent multiple geographic areas of Turkey. Diagnostic pathology reports of DLBCL patients between 2015 and 2022 were evaluated in a retrospective manner.

Primary central nervous system (CNS) lymphoma patients were excluded due to poor prognosis and their divergent first-line treatment. Within 2469 patients, CD5 positivity was observed in 169 patients. Complete data of 131 CD5-positive patients and 129 CD5-negative patients were available. Patients’ age, gender, date of diagnosis, stage at diagnosis (Ann-Arbor), ECOG performance evaluation, LDH levels, R-IPI and NCCN-IPI scores, extra nodal and bone marrow involvement, treatments modalities, response to treatment, and follow-up were recorded from the patient files. Pre-treatment samples of all patients were evaluated.

This study was approved by Trakya University Faculty of Medicine Ethical Committee (TUTF-BAEK2020/439).

Pathological assessment and definitions

As a routine practice, pathological assessment of lymph nodes was performed on formalin-fixed, paraffin-embedded tissue sections. A panel of selected antibodies was used by the pathologists: CD20, CD3, CD5, CD10, BCL-2, BCL-6, MUM1, FOXP-1, Ki-67, and MYC proto-oncogene protein (MYC). A cutoff level for dim to strong expression of CD5 was ≥ 50% of tumor cells for considering as immunopositivity. Patients were grouped as germinal center and non-germinal center DLBC based on the Hans algorithm using CD10, BCL-6, and MUM-1 [9]. The cutoffs for positive MYC or BCL-2 results were ≥ 40% and ≥ 50% of cells (Fig. 1) [10, 11].

Fig. 1
figure 1

Many of the cases showed large pleomorfic cell groups with vacuolar cytoplasm and vesicular nucleus (A). Tumor cells were positive for CD20, MUM1, C-MYC, and CD5. High Kİ67 proliferative level was observed on most of the cases (B)

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Fluorescence in situ hybridization (FISH) was performed on cases using LSI dual-color break-apart probes for MYC and BCL6, and dual-color, dual-fusion probe for BCL2/IGH. Positive cases for rearrangements had positive signal in ≥ 15% of nuclei examined.

Statistics

Normality assumption was assessed using Shapiro–Wilk test. Student’s t test and one-way analysis of variance were used to compare independent groups. Tukey post hoc test was used to perform multiple comparisons. The log-rank test using the Kaplan–Meier method was performed for univariate survival analysis. The Cox proportional hazards model with a forward selection method was used to identify the prognostic significance of risk factors on survival. Descriptive statistics were expressed as mean and standard deviation for numerical variables and frequency and percentage for categorical variables. All statistical analyses were performed using SPSS version 20 (SPSS software, IBM Corporation, Armonk, NY).

Results

Records of 2469 DLBCL patients were evaluated for the study. CD5 staining was done for all samples and CD5 positivity was observed in 169 patients (6.84%). Complete data of 131 CD5-positive patients and 129 CD5-negative patients were available and considered for further comparisons. In CD5 positivity group, the mean age was 59 years (18–88). Fifty-five percent of the patients were 60 years and older. A 55.7% of the patients were male. In CD5-negative group, the mean age was 64.4 years (25–93). Forty-five percent of the patients were 60 years and older. A 54.2% of the patients were male. In CD5 positivity group, overall survival was 29.8 months (0.1–72.3 months), and it was found to be statistically shorter than CD5-negative patients (OS was 25 months (0.1–70 months)) (p = 0.01) (Fig. 2). Progression-free survival (PFS) was 26.08 months. 41 of 131 (31.3%) patients died during follow-up. General characteristics of patients are summarized in Table 1.

Fig. 2
figure 2

OS comparison in CD5 + and—groups

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Table 1 Characteristic findings of the patients
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In CD5 positivity group 76 patients were non-GCB (61.7%) while 43 patients were GCB (34.9%) and 4 patients were unclassified. In the CD5-negative group, only 36.4% of patients were non-GCB (p = 0.00). When the CD5 -positive group is examined, LDH was observed to be elevated in 65.4% of the patients. Eighty-four patients (66.7%) had Ann-Arbor stage 3–4 disease. Seventy percent of the patients had 1 or more extra nodal involvement. R-IPI score was high in 48% of patients, and NCCN-IPI was intermediate-high or high in 44.1% of the patients. However, in CD5-negative group, only 34.8% of the patients had high LDH values (p = 0.412) and 56.5% of the patients had Ann-Arbor stage 3–4 disease. In addition, only 30% of the patients had extranodal involvement, which was found to be much less than the patients in the CD5 -positive group (p = 0.00). Similarly, the rate of patients with high R-IPI (20.5%) was found to be much lower than those of CD5-positive patients (p = 0.00). In CD5 positivity group, the mean Ki67 was 78.15%. ECOG performance score was observed to be ≥ 2 in 31.25% of the patients. 57% patients had B symptoms (Table 1).

Evaluation of prognostic factors in CD5-positive patients

The mean survival was significantly longer in patients under 60 years (63.41 vs. 42.81 months, p < 0.01). There was no difference in survival between genders (p = 0.891). Patients with B symptom at the time of diagnosis (57%) had a shorter survival (p = 0.002) (Fig. 2). The mean survival of patients with bone marrow involvement was statistically significantly lower (32.03–58.27 months, p < 0.01), likewise in patients with extra nodal involvement (46.90 vs. 66.23 months, p = 0.02) (Figs. 3–4).

Fig. 3
figure 3

Evaluation of survival with prognostic factors (ECOG, age, B symptoms) in CD5 + patients

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Fig. 4
figure 4

Evaluation of survival with prognostic factors (LDH, EN, BM) in CD5 + patients

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Increased LDH was observed to be related with survival (p < 0.01). A 27.5% of the patients had a worse performance status than ECOG 1 and performance was significantly related with survival (p < 0.01). Survival was poorer in patients with both high-intermediate and high risk NCCN-IPI and high R-IPI (> 3) scores (p < 0.01) (Figs. 4–5).

Fig. 5
figure 5

Evaluation of survival with prognostic factors (NCCN-IPI, treatment, D/T expr) in CD5 + patients

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Regarding first-line treatment, 11 patients have received more intense treatment including dose-adjusted EPOCH-R, R-CHOEP, or hyper-CVAD and survival of these patients were similar with patients who received R-CHOP (p = 0.997) (Table 2).

Table 2 Comparison of prognostic markers in CD5 + patients group
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Multivariate analysis was performed by excluding R-IPI and NCCN-IPI, which are among the other data, being ≥ 60 years of age, ECOG ≥ 2 and ≥ 1 extra nodal involvement were related with overall survival as well progressive free survival (Figs. 6, 7, and 8).

Fig. 6
figure 6

Evaluation of progression-free survival (ECOG, Age, EN) with prognostic factors in CD5 + patients

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Fig. 7
figure 7

Multivariate analysis and overall survival (Y-axis is given in logarithmic scale)

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Fig. 8
figure 8

Multivariate analysis and progression free survival (Y-axis is given in logarithmic scale)

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Discussion

As a pan T-cell marker, CD5 is a monomeric type 1 transmembrane glycoprotein. It has both an intracellular and extracellular domain. Although it is frequently expressed from T cells, it is also expressed at a low level by a small subset of naive B lymphocytes [12]. CD5 inhibits signaling downstream of the B-cell receptor and modifies intracellular calcium mobilization. It also suppresses the release of IL-2, resulting in increased production of IL-10, an anti-inflammatory marker and a survival factor for B cells. Additionally, activation of the ERK1/2, PI3K, STAT3, and NFAT2 signal pathways were observed in CD5 positive B cells [7, 12]. CD5-positive B cells may also produce autoantibodies, which may be related with the development of autoimmune events in chronic lymphocytic leukemia [13]. Factors with prognostic relevance including high-LDH levels, bone marrow involvement, presence of extra nodal involvement, high-IPI scores, poor ECOG performance, and CNS recurrence have been more frequently observed in patients with CD5 + DLBCL [8].

The frequency of CD5 positivity is variable in de novo DLBCL. In a retrospective study about the characteristics of CD5 + DLBCL patients in Korea, CD5 positivity was found to be 7.4% [14]. In a multicenter study by Yamaguchi et al. in Japan, this frequency was reported as 10% [15]. However, in large-scale studies in Japan, prevalence rates of CD5 positivity may vary widely (5–22%) [7, 15]. Besides these, in a multicenter large-scale study with data from Western countries, CD5 positivity ratio in DLBCL is (5.5%) [16]. We observed the ratio as 6.84% which is similar in Western countries but dissimilar to East Asian countries. Immunohistochemical CD5 staining not being a routinely performed in all samples may be the reason for the conflicting frequencies across countries and centers. Although, there is a predominance of female patient distribution in CD5 + DLBCL patients in studies from East Asian countries, we have observed a male gender tendency (55.7%) similar with Western countries. Based on the latest classification proposed by World Health Organization and International Consensus Classification Advisory Committee, CD5 positivity was not regarded as a prognostic marker in patients with DLBCL. This may be due to the small percentage of CD5 positivity in this group of patients and may be altered in future classifications [17, 18].

In DLBCL patients, remission rate with current first-line treatment is 50–60%. Based on prognostic scores, the 5-year survival in patients with low-IPI score is 90%, while survival is observed to decrease to 60% in patients with high-IPI scores. Based on the cell of origin, 5-year overall survival (OS) was 48% to 56% in non-GCB subtype, while 73% to 78% in GCB subtype. The 5-year OS in the DHT/THT subtype appears to be very low and nearly 18% [1, 19]. The 5-year OS is 35.5% in CD5 + DLBCL patients, and it has been found to be shorter than general DLBCL patient population [12]. In our study, we observed that the mean survival was 29.8 months in CD5 + DLBCL patients, similar with the literature.

In addition to the shorter OS of CD5 + DLBCL patients, these patients have higher LDH, IPI elevation, advanced Ann-Arbor frequency, poor performance score, and bone marrow involvement at the time of diagnosis compared to CD5-DLBCL patients. CD5 + DLBCL is more often in the non-GCM/ABC phenotype. Also, the presence of double/triple expressor or double/triple hits are more common in these patients at the time of diagnosis [12, 14–16, 20]. Non-GCM/ABC phenotype as well as other factors related with poor prognosis and worse outcomes being more frequent in CD5 positive groups, we may assume that this very group of disease may be regarded as relatively high risk.

The age and gender distribution at the time of diagnosis is variable. In studies from East Asian countries, the frequency of female patients is significantly higher [12, 14, 15]. However, there is no difference in gender distribution in Western countries [16]. In our data, it was observed that male patients were in the majority (55.7%). In the study conducted on CD5 + DLBCL patient data from western countries, it is observed that 76.7% of CD5 + patients are over 60 years old [16]. But, in a single-center study in China, 53.3% of CD5 + DLBCL patients were below the age of 60 [12]. In our data, the rate of patients, 60 years and older, are higher (55%) (Table 3).and – patients group

Table 3 The Comparison with literature data of presence of prognostic markers in CD5 + and – patients group
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Central nervous system relapse is reported to be more frequent in CD5 + DLBCL patients. In the study by Zhang et al., it was observed that CNS involvement in relapsed DLBCL patients was 35% in CD5 + patients, (19% in CD5 −) [20]. Similarly, in the study by Thakral B. et al., CNS relapsed was 33.3% in CD5 + and 15.6% in CD5 − patients [21]. We observed that 19 patients have relapsed and only 4 patients had CNS relapsed (3% of all patients, 21% of all relapses). CNS prophylaxis was not a routine component of the main treatment in all centers and CNS relapse was not related with the use of prophylaxis.

In our data, 64.6% of the patients had high LDH levels (13.4% \(\ge\) threefold). Twenty-three percent of the patients had bone marrow involvement, 70% had extra nodal involvement, and 57% of the patients had B symptoms. Advanced Ann-Arbor stage disease was observed in 66.6% of patients, in addition to the higher frequency of all these factors that have impact on prognosis for CD5 + DLBCL patients. In a study with a small number of patients from Taiwan, the presence of CD5 + was observed as the only adverse prognostic factor determined in multivariate analysis [22].

Increased OS with the introduction of rituximab seems to be valid also for CD 5 + patients. A study by Hyo R et al. showed that addition of rituximab to chemotherapy similarly increased OS and progression-free survival (PFS) [23]. A study by Miyazaki K. et al. revealed that addition of rituximab to chemotherapy in CD 5 + DLBCL patients significantly increases OS [24]. However, even if rituximab is included in the treatment, a prospective study by Tzankov A et al. reported a negative effect of the presence of CD5 in multiparameter analyzes in patients receiving R-CHOP [25]. In a retrospective study, in which DLBCL patients treated with R-EPOCH chemotherapy, with a 28.5-month median follow-up, 37.5% of CD5 + patients had died (9.6% in CD5 −) [21]. In the study conducted by Zhang F. et al. to evaluate the effect of intensive treatment on survival, the effectiveness of da-EPOCH-R and R-CHOP treatments were compared. It was observed that PFS and OS were better in both CD5 + and CD5 − groups in patients who received da-EPOCH-R in short-term follow-up. In the long-term follow-up, this was not observed for OS in CD5 + patients. Also, in another study with a small group of patients with short-term follow-up, we can say that the da-EPOCH-R combined with high-dose methotrexate regimen had a better survival in the CD5 + patient group [26]. We also observed that the first-line treatment of 11 patients was a more intensive treatment (da-EPOCH-R, R-CHOEP, hyper-CVAD). The mean survival of these patients compared to patients who received R-CHOP as first-line therapy was similar. The low number of patients receiving intensive treatment seems to be an important limiting factor in this comparison.

Limitations

The study had two important limiting factors. First, we were able to access to a limited extent to survival statistics of CD5 − control group. The second important limitation was the study being retrospective.

Study highlight

– Due to its prognostic effect, CD5 must be applied immunohistochemically for all DLBCL patients.

– CD5 status and CNS relapse needs to be evaluated in further studies.

– Considering the data in the literature and the data in our study, the different evaluation of CD5 + patients seems to be an indisputable fact.

– With the survival and literature information, we think there is an unmet need for this subgroup of patients with low survival expectancy. Thus, we need new studies with novel treatment modalities to be combined or to replace the standard of care therapies.