Characteristics of CD5-positive diffuse large B-cell lymphoma among Koreans: High incidence of BCL2 and MYC double-expressors.

Aberrant expression of CD5 has been reported in 5-10% of diffuse large B-cell lymphomas (DLBCLs). CD5+ DLBCL had been recognized as an aggressive immunophenotypic subgroup of DLBCL in the 2008 WHO classification of haematolymphoid neoplasm; however, it was eliminated from the list of subgroups of DLBCLs in the revised 2016 classification. Nevertheless, there is much controversy regarding the clinical significance of CD5 expression, and many researchers still assert that this subgroup exhibits an extremely unfavorable prognosis with frequent treatment failure. We retrospectively investigated 405 DLBCLs recruited from three university hospitals in Korea from 1997 to 2013. The clinical profile, immunophenotype, and chromosomal structural alterations of the BCL2 and MYC genes were compared according to CD5 expression. A total of 29 cases of de novo CD5+ DLBCL were identified out of 405 in our series (7.4%). Clinicopathologic correlation was performed in all 29 CD5+ DLBCLs and 166 CD5- DLBCLs which were eligible for full clinical review and further pathologic examination. Compared with CD5- counterparts, CD5+ DLBCLs showed female preponderance, frequent bone marrow involvement, higher lactate dehydrogenase level, advanced Ann Arbor stages and poorer prognosis (all p<0.05). Pathologically, the expression of CD5 positively correlated with that of BCL2, MYC and Ki-67 (all p<0.05). Coexpression of BCL2 and MYC, which is referred to as a double-expressor, was relatively more common in CD5+ DLBCL, whereas translocation or amplification of these genes was very rare. in conclusion, the expression of CD5 is an independent poor prognostic factor of DLBCLs, and this subgroup displays unique clinicopathologic features. Although the exact mechanism remains uncertain, consistent activation of BCL2 and MYC by alternative pathways other than chromosomal translocation may contribute to the pathogenesis.

Introduction

Pathologic diagnosis of malignant lymphoma is based on the application of immunohistochemistry (IHC) using lineage-specific surface markers such as CD3 or CD20. However, aberrant expression of some T-cell markers, of which the most representative is CD5, has been well documented in a subset of B-cell neoplasms [1-3]. In fact, CD5 is an important diagnostic marker of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and mantle cell lymphoma [4].

The expression of CD5 in diffuse large B-cell lymphoma (DLBCL) can be observed in Richter transformation of CLL but can also be found in de novo DLBCLs. Since it was first recognized in 1995 [2], many de novo CD5+ DLBCL cases have been documented, and the overall incidence comprises 5–10% of all DLBCLs [1, 5, 6]. The CD5+ DLBCL had been introduced as an immunophenotypic subgroup of DLBCL in the 2008 WHO classification of haematolymphoid neoplasms, however, the revised 2016 version has omitted designation of the CD5+ subtype. Nevertheless, accumulating evidences suggest that CD5+ DLBCL is a distinctive subgroup which typically presents aggressive clinical features and adverse outcomes [1, 4, 7–11]. Previous studies have confirmed that the prognosis of CD5+ DLBCL is still poor regardless of Rituximab based chemotherapy [1, 9, 10], and even with the salvage stem cell transplantation [12]. To achieve optimal therapeutic responses, better understanding of pathogenic mechanisms and risk stratification are crucial.

To date, most large-scale studies of de novo CD5+ DLBCL have been performed in Japan, and there are only few reports from other Asian countries or Western areas [1, 8, 9, 12–15]. We performed a retrospective study to review detailed characteristics of CD5+ DLBCL among Korean patients, particularly focusing on the relationship to other constitutional prognostic factors, such as cell of origin by IHC, and BCL2 and MYC status.

Materials and methods

Case selection and analysis of the clinical characteristics

Cases diagnosed as DLBCL, not otherwise specified (NOS), were retrieved from three university hospitals (Seoul National University Hospital, Seoul National University Bundang Hospital and Seoul National University Boramae Hospital) in Korea from January 1996 to January 2016. The diagnosis was confirmed by two experienced hematopathologists (HYN and JEK), based on the 2017 WHO classification of Tumours of Haematopoietic and Lymphoid Tissues [4]. Clinical profiles and follow-up data were obtained from electronic medical records.

Immunohistochemistry (IHC) and Epstein-Barr virus (EBV) detection

To determine the CD5+ subgroup, all DLBCL cases were reexamined and assessed by IHC. IHC was performed using 4 μm sections of paraffin-embedded tissue blocks using the following antibodies BCL2 (M0887, mouse, monoclonal, 1:100; Dako, Carpinteria, CA, USA), BCL6 (LN22, mouse, monoclonal, 1:100; Novocastra, Newcastle, UK), CD3 (M7254, mouse, monoclonal, 1:100; Dako), CD5 (M3641, mouse, monoclonal, 1:100; Dako), CD10 (PA0270, mouse, monoclonal, 1:100; Novocastra), CD20 (M0755, mouse, monoclonal, 1:400; Dako), IRF4/MUM1 (M7259, mouse, monoclonal, 1:100; Dako), Ki-67 (M7240, mouse, monoclonal, 1:100; Dako) and MYC (Y69, rabbit, monoclonal, 1:100; Epitomics, Burlingame, CA, USA). The IHC for antibodies were performed. First, sections were treated with Target Retrieval Solution (Dako, Glostrup, Denmark) at 115˚C for 15 min after inhibiting endogenous peroxidase activity for 30 min with 3% hydrogen peroxidase in methanol for antigen retrieval.Then, immune complexes were detected with the Envision Detection System (Dako) after overnight incubation. Finally, hematoxylin counterstaining was done.

For CD5 IHC, we used the cutoff of more than 50% tumor cells showing immunoreactivity for CD5 of any intensity as positive. Tumor cells with more than 30% staining were considered positive for BCL6, CD10 and IRF4/MUM1 [16]. For BCL2 and MYC, staining of more than 50%, and 40% was used as cutoff, according to the criteria suggested by Johnson et al [17]. EBV in situ hybridization (ISH) was performed using an EBV-encoded RNA (EBER) probe (INFORM EBER Probe; Ventana Medical Systems, Tucson, AZ, USA). The evaluation of IHC and EBV ISH were perfomed by the same two experienced hematopathologists (HYN and JEK), and discordant cases were discussed for consensus.

Detection of IgH/MYC translocation, MYC amplification and IgH/BCL2 translocation

Fluorescence in situ hybridization (FISH) was performed on paraffin embedded tissue block sections according to the manufacturer’s protocol. A Vysis LSI MYC dual-color, break-apart rearrangement probe (Abbott Molecular, Abbott Park, IL, USA) was used to detect MYC translocation, and a Vysis IgH/MYC/CEP 8 Tri-color DF probe (Abbott) was used for amplification. At least 100 cells from each case were assessed for split signals to identify MYC translocation and gene copy number alteration. FISH using a Vysis LSI BCL2 dual-color break-apart rearrangement probe (Abbott Molecular, Abbott Park, IL) was performed to identify BCL2 translocation. The result was considered positive for rearrangement and amplification when >20% of nuclei showed a break-apart signal or extra copies [18].

Statistical analysis

For the association analysis, the Mann–Whitney U-test, Fisher's exact test or Pearson’s chi-square test was performed. Spearman’s ρ was used to assess correlations between variables. Overall survival (OS) was measured from the date of diagnosis to the date of death. Progression-free survival (PFS) was estimated from the date of diagnosis to the date of disease progression, including relapse and death. Univariable Kaplan–Meier survival analysis with log-rank tests was conducted to compare the outcome based on the status of various parameters. Multivariable survival analysis using the Cox proportional hazards model was performed to identify independent prognostic markers. A two-tailed P-value of ≤0.05 was considered statistically significant. All data were analyzed with SPSS software, version 22.0 (SPSS Inc., IBM, Armonk, NY, USA).

Ethics

Ethical approval was obtained by the regional ethics committee in Seoul National University Boramae Hospital, Korea according to the declaration of Helsinki (2014/020, 2014/020/1, and 2014/233) (IRB No. 10-2018-19). Informed consent from participants was not required according to the ethics committee. All data were fully anonymized before accessed.

Results

A total of 405 cases of DLBCL were investigated for CD5 expression on tumor cells, and 30 cases of CD5+ DLBCLs, consisting of one case transformed from CLL and 29 de novo cases (29 of 405, 7.2%), were identified. To compare the clinicopathologic findings between the CD5+ and CD5- groups, a total of 166 CD5-DLBCL, which had no evidence of transformation from low grade lymphoma, were selected, of which detailed clinical data and sufficient tissue for further pathologic studies were available. Clinicopathologic profiles of de novo CD5+ DLBCL (N = 29) and CD5-DLBCL patients are summarized in Table 1. Most CD5- DLBCL patients (21/29, 72.4%) were treated with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone)-based chemotherapy with or without rituximab. Of these, 3 received etoposide in addition to the above combination (R-EPOCH). The remaining 4 patients were managed by supportive care only. Similarly, 129 (77.7%) CD5-DLBCL patients were treated with a CHOP-based regimen. The median follow-up time of de novo CD5+ DLBCL and CD5- DLBCL patients was 13 months (ranging from 1 to 163) and 43 months (ranging from 0 to 261), respectively.

Table 1

Comparison of clinicopathologic features of CD5+ and CD5- DLBCL patients.

Clinicopathologic Feature	CD5+ DLBCL(n = 29)	CD5- DLBCL(n = 166)	P-value
Age over 65 yr	15 (51.7%)	59 (35.5%)	0.098
Sex (Male: female)	9:20	103:63	0.002*
Initial diagnosis in lymph node	16 (55.2%)	60 (36.1%)	0.039*
BM involvement	11 (37.9%)	20 (12.1%)	0.010*
Ann Arbor Stage (I-II:III-IV)	10:19	97:69	0.030*
ECOG PS (0–1:2–5)	18:11	132:34	0.060
Elevated LDH	20 (69.0%)	78 (47.0%)	0.028*
>1 Extranodal sites	13 (44.8%)	30 (18.1%)	0.002*
B symptoms	8 (27.6%)	41 (24.7%)	0.689
Bulky tumor	4 (13.8%)	23 (13.9%)	0.980
IPI (0–2:3–5)	11:18	113:53	0.004*
Disease progression	18 (62.1%)	83 (50%)	0.230
CNS relapse	1 (3.7%)	8 (4.8%)	0.745
Treatment			0.065
R-CHOP	18 (62.1%)	78 (47.0%)
CHOP	3 (10.3%)	51 (30.7%)
Other chemotherapy	3 (10.3%)	18 (10.8%)
Other treatment (e.g. Op, RT etc)	0 (0%)	9 (5.4%)
No treatment	4 (13.8%)	2 (1.2%)
PFS, median (range)	10 (0–163)	40 (0–192)	0.003*
OS, median (range)	13 (1–163)	44 (0–261)	0.008*
Hans system (GCB:Non-GCB)	8:19	56:110	0.570
CD10	5 (17.2%)	26 (15.7%)	0.830
BCL6	14 (48.3%)	76 (45.8%)	0.804
MUM1	16 (55.2%)	58 (34.9%)	0.038*
BCL2	17 (58.6%)	50 (30.1%)	0.003*
MYC	9 (33.3%)	7 (4.2%)	<0.001*
MYC/BCL2 double-expressor	8 (27.6%)	5 (3.0%)	<0.001*
Ki-67, median (range) (%)	60 (20–100)	50 (6.6–88.3)	0.007*
MYC rearrangement	2 (6.9%)	6 (3.6%)	0.614

DLBCL, diffuse large B-cell lymphoma; BM, bone marrow; IPI, ECOG, Eastern cooperative group; LDH, lactate dehydrogenase; IPI, international prognostic index; CNS, central nervous system; Op, operation; RT, radiation therapy; PFS, progression-free survival; OS, overall survival; Germinal center B-cell like subgroup.

* P ≤0.05

Compared with CD5- DLBCL, the CD5+ group revealed female preponderance (p = 0.002), more than one extranodal site involvement (p = 0.002), and frequent bone marrow involvement (p = 0.01). In addition, elevated lactate dehydrogenase (LDH, p = 0.028) and a higher international prognostic index (p = 0.004) were more common in CD5+ DLBCL.

Both CD5+ and CD5- DLBCL showed non-germinal center B-cell type predominance according to the Hans algorithm [16]. Compared with CD5- cases, CD5+ DLBCL cases showed more frequent IRF4/MUM1 (p = 0.038), BCL2 (p = 0.003), and MYC (p<0.001) expression and a higher Ki-67 proliferation index (p = 0.007). Concurrent expression of BCL2 and MYC, which is known as double-expressor (DE), was significantly more common in CD5+ cases (8/29 vs. 5/166, p<0.001). Of note, only two (2 of 29 CD5+ DLBCLs, and 2 of 9 MYC overexpressed CD5+ DLBCLs) revealed MYC rearrangement, and no amplification was identified by FISH. BCL2 rearrangement or amplification was not found. (Figs 1 and 2).

Fig 1

Pathologic characteristics of CD5+ DLBCL.

Pleomorphic large cells with many apoptotic features were shown in many cases (A). Tumor cells were positive for CD5 (B), BCL2 (C), IRF4/MUM1 (D). Note the diffuse strong pattern in CD5 IHC. Tumor cells also showed high Ki-67 proliferation index (E).

Fig 2

Representative case showing MYC overexpression and rearrangement.

High expression of MYC protein (A) and presence of MYC gene rearrangement examined by FISH for MYC, break apart probe (B, split signals indicated by arrows).

In the univariable survival analysis, patients with CD5+ DLBCL showed significantly shorter OS (p = 0.025) and PFS (p = 0.033) than those with CD5- tumors (Fig 3), whereas DE lymphomas revealed inferior PFS (p = 0.036) but not OS (p = 0.234) (Fig 4). Additionally, older age (>65 yrs), higher performance status (PS) by the Eastern Cooperative Oncology Group, elevated serum LDH, presence of B symptoms, more than one extranodal involvement and high Ann Arbor stages were all associated with inferior OS and PFS (all p<0.001) (Tables 2 and 3). In the multivariable Cox regression analysis, older age, higher PS, elevated serum LDH and the presence of B symptoms remained independent prognostic factors for OS and PFS, whereas CD5 positivity and DE did not (Tables 2 and 3).

Fig 3

Kaplan-Meier survival curves of CD5+ and CD5-DLBCL patients.

CD5 positivity was associated with significantly shorter OS (A) and PFS (B).

Fig 4

Kaplan-Meier survival curves of double-expressors compared with others.

DE was associated with inferior PFS (B) but not OS (A).

Table 2

Univariable and multivariable analysis for OS in DLBCL.

Variables	Univariate analysis (OS)		Multivariate analysis (OS)
	OR (95% CI)	P-value	OR (95% CI)	P-value
All DLBCL
Age > 65yr	2.459 (1.622–3.728)	<0.001*	2.111 (1.245–3.579)	0.006*
Performance status	3.473 (2.234–5.401)	<0.001*	2.111 (1.280–3.481)	0.003*
Elevated LDH	2.362 (1.470–3.797)	<0.001*	1.915 (1.080–3.395)	0.026*
B symptom	2.824 (1.828–4.363)	<0.001*	2.206 (1.327–3.664)	0.002*
> 1 Extranodal sites	1.796 (1.113–2.900)	0.017*	1.037 (0.554–1.941)	0.909
Stage	2.260 (1.475–3.464)	<0.001*	1.308 (0.729–2.346)	0.369
Double-expressor	1.465 (0.704–3.049)	0.234	0.583 (0.222–1.531)	0.273
CD5 positivity	1.825 (1.071–3.109)	0.027*	1.374 (0.723–2.612)	0.331
Non-GCB DLBCL
Age > 65yr	2.742 (1.642–4.579)	<0.001*	2.429 (1.260–4.681)	0.008*
Performance status	3.559 (2.062–6.142)	<0.001*	1.369 (0.704–2.664)	0.355
Elevated LDH	2.687 (1.504–4.801)	0.001	1.784 (0.866 = 3.676)	0.116
B symptom	3.026 (1.791–5.115)	<0.001*	2.456 (1.323–4.558)	0.004*
> 1 Extranodal sites	1.952 (1.108–3.438)	0.021*	0.867 (0.375–2.004)	0.739
Stage	2.858 (1.675–4.876)	<0.001*	2.831 (1.486–5.395)	0.002*
Double-expressor	2.189 (0.929–5.154)	0.073	1.181 (0.384–3.635)	0.772
CD5 positivity	2.446 (1.307–4.577)	0.005*	1.766 (0.821–3.798)	0.145

DLBCL, diffuse large B-cell lymphoma; LDH, lactate dehydrogenase; OS, overall survival; OR, odds ratio; CI, confidence interval.

* P ≤0.05

Table 3

Univariable and multivariable ananlysis for PFS in DLBCL.

Variables	Univariate analysis (PFS)		Multivariate analysis (PFS)
	OR (95% CI)	P-value	OR (95% CI)	P-value
All DLBCL
Age > 65yr	25884 (1.738–3.854)	<0.001*	1.918 (1.158–3.176)	0.011*
Performance status	3.288 (2.155–5.018)	<0.001*	2.266 (1.383–3.713)	0.001*
Elevated LDH	2.363 (1.497–3.729)	<0.001*	1.917 (1.100–3.341)	0.022*
B symptom	2.686 (1.766–4.085)	<0.001*	2.191 (1.346–3.569)	0.002*
> 1 Extranodal sites	1.702 (1.079–2.685)	0.022*	0.534 (0.508–1.771)	0.972
Stage	2.134 (1.424–3.199)	<0.001*	1.192 (0.684–2.079)	0.535
Double-expressor	1.722 (0.892–3.325)	0.036*	0.676 (0.264–1.732)	0.414
CD5 positivity	1.748 (1.304–2.954)	0.037*	1.681 (0.873–3.239)	0.121
Non-GCB DLBCL
Age > 65yr	3.186 (1.934–5.250)	<0.001*	2.525 (1.402–4.548)	0.002*
Performance status	3.388 (2.003–5.731)	<0.001*	1.570 (0.822–2.997)	0.171
Elevated LDH	2.861 (1.613–5.075)	<0.001*	1.752 (0.847–3.623)	0.131
B symptom	2.665 (1.604–4.428)	<0.001*	2.612 (1.443–4.728)	0.002*
> 1 Extranodal sites	1.949 (1.131–3.359)	0.023*	0.851 (0.365–1.983)	0.708
Stage	2.598 (1.566–4.311)	<0.001*	2.353 (1.264–4.380)	0.007*
Double-expressor	3.189 (1.500–6.782)	0.003*	1.297 (0.436–3.854)	0.640
CD5 positivity	2.488 (1.347–4.598)	0.004*	2.840 (1.340–6.015)	0.006*

DLBCL, diffuse large B-cell lymphoma; LDH, lactate dehydrogenase; PFS, progression-free survival; OR, odds ratio; CI, confidence interval.

* P ≤0.05

Among non-GCB type DLBCL, CD5 expression was associated with inferior OS (p = 0.005) and PFS (p = 0.004), while DE was associated only with shorter PFS (p = 0.003) in univariable analysis. In multivariable analysis, CD5 positivity was an independent prognostic factor for poor PFS (p = 0.006), but not OS (p = 0.145) (Tables 2 and 3).

Discussion

In this study, we demonstrated that CD5 expression was associated with aggressive clinical features and poor survival in DLBCL. Pathologically, the expression of CD5 correlated with BCL2 and MYC positivity, and concurrent expression of these two proteins (DE) was more frequently found in CD5+ DLBCL, whereas chromosomal translocation or amplification was rarely noted. We suggest that CD5+ DLBCL is a distinct immunophenotypic subtype, and its pathogenesis and biologic nature are possibly related to alternative activation of MYC or BCL2 proteins other than gene rearrangement.

The overall incidence of CD5+ DLBCL in our Korean cases accounted for 7.2%, which is similar to previous series from Japan and other Western countries [1, 8, 9, 13–15]. Most of our series were de novo cases, because the occurrence of CLL/SLL and mantle cell lymphoma is extremely low in the Korean population, occupying only 2.1% and 1.3% of malignant lymphoma, respectively [19]. Many previous studies identified CD5 expression as an independent prognostic factor regardless of rituximab use [1, 7, 9, 10, 13, 20, 21]. Others also reported that more intensive chemotherapy other than R-CHOP or stem cell transplantation did not overcome its dismal outcome [12, 15, 22]. In the present study, CD5+ DLBCL showed female predominance, higher IPI and Ann Arbor stages, and other aggressive clinical features, which are in line with previous reports [1, 5, 7–9, 13, 14, 20, 21, 23]. These clinical characteristics are also commonly noted in double hit lymphoma (DHL) or DE cases [17, 24, 25]; both have been known as high-risk lymphomas although the clinical behaviors of DE are not as much aggressive as DHLs [26]. In particular, there is considerable pathologic overlap between DE lesions and CD5+ DLBCL regarding cell of origin and complicated mutational events [1, 9, 27–29]. Therefore, we paid special attention to the relationship of CD5 with BCL2 or MYC expression due to the aforementioned resemblance between CD5+ DLBCL and DE lymphomas. We found a strong association with CD5 positivity and BCL2 expression, which was reported in previous series [9, 13, 15, 23], and a correlation of CD5 with MYC, which has not been widely investigated before. However, only 2 (6.9%) cases of MYC rearrangement were detected and no MYC amplification or BCL2 rearrangement was identified in contrast to high levels of protein expression. Although there have been few reports, most revealed scarce mutations in the MYC gene in CD5+ DLBCL [9, 12]. Alteration of STAT3 and nuclear factor-kappaB (NF-kB) pathway was suggested for a possible explanation for overexpression of BCL2 in CD5+ DLBCL [9]. Similar genetic alterations have also been demonstrated in recent studies suggesting that the pathology of DE lymphomas reflect cumulative mutations involving B-cell receptor signaling and NF-kB [28, 30, 31]. In DE tumors, alternative transcriptional mechanisms, posttranscriptional, or posttranslational pathways involving miRNA, histone binding proteins, and ubiquitination were proposed as the basis of MYC activation [32-35]. Although direct evidence of CD5 expression influencing BCL2 or MYC activation cannot be presented in the present study, the aggressive biologic behavior of CD5+ DLBCL might be related to the higher frequency of DE in this group.

CD5 is a glycoprotein mainly expressed on the membrane of mature T cells and is only dimly expressed on a subset of late stage hematogones/normal B-lineage precursors [36-38]. Most CD5+ DLBCLs may originate from early B-cells prior to the germinal center stage with some exceptions [39]. Recently published paper by a Japanese group revealed a significantly lower prevalence of MYD88 and CD79B gene mutations in CD5+ DLBCL in contrast to other extranodal DLBCLs [40]. These findings support the idea that the cellular origin of CD5+ DLBCL might be different from that of ordinary DLBCL. Although the cellular origin is unclear, expression of CD5 is known to induce interleukin-10 production and has anti-apoptotic function maintaining B-cell survival [41], which is attributed to the aggressive biology of CD5+ DLBCL. Currently, there is no specific guideline on management of CD5+ DLBCL, although many cases are controlled under high risk stratification. Based on recent studies suggesting that MYC upregulates immune checkpoint pathways such as programmed death-ligand 1 (PD-L1) [42], immunotherapy can be added to CD5+ DLBCL patients overexpressing MYC.

In summary, CD5+DLBCL is a distinct subgroup of DLBCL, characterized by aggressive clinical courses that can easily be missed in routine clinical practice. To date, this is the largest study of CD5+ DLBDLs in Korea, and we confirmed that CD5 is an easily available, cost-effective prognostic biomarker for DLBCL. Although the exact mechanism of CD5 expression and its connection to BCL2 or MYC and DE requires further investigation at the molecular level, our results will provide a basis for new therapeutic regimens including small molecule inhibitors that block MYC or BCL2 and even immunotherapy.

Clinicopathologic dadta of the CD5+ and CD5- DLBCL patients included in the analysis.

(XLSX)

Click here for additional data file.

18 Sep 2019

PONE-D-19-18460

Characteristics of CD5-positive diffuse large B-cell lymphoma among Koreans: high incidence of BCL2 and MYC double-expressors

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Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: The authors detail the clinicopathologic characteristics of CD5-positive diffuse large B-cell lymphoma (DLBCL) in Korea. The manuscript is well-written and the data are well presented. The manuscript would benefit from some clarifying details, particularly to aspects of the Methods and Results section, as detailed below.

1. Consider adding the following terms to the Keywords if space permits: cell of origin, MYC, BCL2

2. Please clarify the statement in the Abstract and Introduction that “CD5+ DLBCL had been introduced as a unique immunophenotypic subgroup of DLBCL in the 2008 WHO classification of haematolymphoid neoplasms, however, the revised 2016 version has omitted the designation of the CD5+ subtype.” In reviewing the text of the 2008 WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues (4th edition), CD5+ DLBCL is not listed as a separate variant, subgroup, subtype or entity in Table 10.14 or in the overall Table of Contents, similar to the 2016/17 WHO Classification (revised 4th edition).

3. The authors state that the diagnosis of DLBCL was confirmed by two experienced hematopathologists. Was this also true for evaluation of immunohistochemical stains requiring semiquantitative analysis, such as BCL2, BCL6, CD10, MUM1, MYC and Ki67? How were these assessed and, if by more than one evaluator, how were discrepancies resolved, particularly if they fell close to positive/negative cut-offs?

4. A cut-off of 30% was chosen for BCL2 with reference to the Hans et al, Blood 2004 paper (reference 18) detailing the Hans algorithm to determine cell of origin in DLBCL. However, since BCL2 does not constitute part of the Hans algorithm, it would be more appropriate to use a cut-off for BCL2 that is typically used in the assessment of double-expressor phenotype, such as 50%, as supported by Johnson et al, J Clin Oncol 2012, which the authors cite as a basis for the MYC cut-off of 40%.

5. What cut-offs for positivity/negativity were used for scoring FISH in determining rearrangement and amplification? This should be detailed in the Methods section.

6. The authors exclude 1 case of CD5+ DLBCL that was transformed from chronic lymphocytic leukemia (CLL). Were the 166 CD5-negative DLBCL cases also de novo (i.e. were cases transformed from underlying low-grade lymphoma also excluded in the comparison group)? If so, this should be clearly stated. If not, this should be considered since cases of transformed DLBCL may behave differently from de novo cases.

7. Among the clinicopathologic features that differed significantly between CD5+ and CD5-negative cases was bone marrow involvement. Is it possible to review these cases to see whether there were any differences in patterns of marrow involvement between CD5+ and CD5-negative cases? Given the rarity of de novo CD5+ DLBCL, its prognostic significance and the fact that CD5 expression may not be routinely assessed by immunohistochemistry, any information about pattern of marrow involvement that might be a clue to CD5 positivity would be of practical utility to pathologists in helping to make this diagnosis.

8. On page 11, lines 185-186, the data provided for double-expressor phenotype in CD5+ vs. CD5-negative cases (9/29 vs. 7/166) is incorrect; these values are for MYC single expression as shown in Table 1 on page 10. Please correct.

Reviewer #2: The authors described clinicopathologic characteristics of CD5+ DLBCL among Koreans which should be the largest cohort in that area. Although this immunohistochemical subtype has been removed from the newest WHO classification of hematolymphoid neoplasm, it is still worthy of consideration in the clinical practice. It is well designed retrospective study and the manuscript was relatively well written. However, the authors should answer to the following inquires before publication.

Question 1.

What is the authors' suggested reasons that the incidence of CNS relapse in this cohort is far lower in comparison with Japanese's?

Question 2.

Are there any possibilities that CD5+DLBCL in this study might be high grade B cell lymphoma, not otherwise specified (not double hit lymphoma)?

Question 3

How frequent is double expressors (DE) among DLBCL? It is better to compare the incidence of DE and CD5+DLBCL to discuss possible link between them.

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Reviewer #1: No

Reviewer #2: Yes: Gyeongsin Park

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25 Sep 2019

Dear Dr. Joseph S Pagano:

Academic Editor

PLOS ONE,

Thank you and the reviewers for taking time to check the manuscript and suggest considerate comments. We have made some corrections and clarifications in the manuscript after going over the reviewer’s comments. And we’ve re-attached the manuscript file without blue highlighting along with the “manuscript with track change” file. We hope that the manuscript suffices publication in your journal.

In the following paragraphs, we address specific points in detail:

Reviewer #1:

1. Consider adding the following terms to the Keywords if space permits: cell of origin, MYC, BCL2

Response: We agree with your idea and added the three keywords you suggested above,.

2. Please clarify the statement in the Abstract and Introduction that “CD5+ DLBCL had been introduced as a unique immunophenotypic subgroup of DLBCL in the 2008 WHO classification of haematolymphoid neoplasms, however, the revised 2016 version has omitted the designation of the CD5+ subtype.” In reviewing the text of the 2008 WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues (4th edition), CD5+ DLBCL is not listed as a separate variant, subgroup, subtype or entity in Table 10.14 or in the overall Table of Contents, similar to the 2016/17 WHO Classification (revised 4th edition).

Response: We’ve checked 2008 WHO classification of haematolymphoid neoplasms again, and confirmed that CD5+ DLBCL was listed as a separate immunophenotypic subgroup in Table 10.14. Although it was not designated as a separate entity, CD5+ DLBCL was listed as immunophenotypic variant of DLBCL, NOS, due to the aggressive behavior reported mainly by Japanese pathologists. And accumulating data from other Asian and Western countries also support this. However, we admit that using the word “distinct” in the abstract section and “unique” in the introduction section may cause confusion to the authors, thus we modified the statement. The same table cited in the literature is shown below to reassure this statement.

(Ponz et al. J Hematopathol 2009.2:83-87.)

3. The authors state that the diagnosis of DLBCL was confirmed by two experienced hematopathologists. Was this also true for evaluation of immunohistochemical stains requiring semiquantitative analysis, such as BCL2, BCL6, CD10, MUM1, MYC and Ki67? How were these assessed and, if by more than one evaluator, how were discrepancies resolved, particularly if they fell close to positive/negative cut-offs?

Response: Since this is a retrospective study, IHC slides were reevaluated by the same hematopathologists (HYN and JEK) consecutively, with discussion of discrepancies. All cases reached consensus including those with the value near the cut-offs. We added this detail in the method section.

4. A cut-off of 30% was chosen for BCL2 with reference to the Hans et al, Blood 2004 paper (reference 18) detailing the Hans algorithm to determine cell of origin in DLBCL. However, since BCL2 does not constitute part of the Hans algorithm, it would be more appropriate to use a cut-off for BCL2 that is typically used in the assessment of double-expressor phenotype, such as 50%, as supported by Johnson et al, J Clin Oncol 2012, which the authors cite as a basis for the MYC cut-off of 40%.

Response: We appreciate you for raising this important issue. We’ve re-revaluated according to the criteria suggested by Johnson et al., which used 50% as the cut-off for BCL2. In CD5+ DLBCLs, one case was revised to negative BCL2 expression. In CD5- DLBCLs, we found 3 cases revised to negative expression. Nevertheless, there was no change in the number of double expressors (DE) in both CD5+ and CD5- cases because all DE cases showed diffuse BCL2 expression far exceeding 50%.

5. What cut-offs for positivity/negativity were used for scoring FISH in determining rearrangement and amplification? This should be detailed in the Methods section.

Response: We scored at least 100 cells, and the case was considered positive if 20% or more of the nuclei exhibited rearrangements or extra copies. The similar cut off was applied in other previous studies (Quesada et al. Modern Pathology 2017;30:1688-1697., Huang et al. Diagnostic Pathology 2019;14:81.)

6. The authors exclude 1 case of CD5+ DLBCL that was transformed from chronic lymphocytic leukemia (CLL). Were the 166 CD5-negative DLBCL cases also de novo (i.e. were cases transformed from underlying low-grade lymphoma also excluded in the comparison group)? If so, this should be clearly stated. If not, this should be considered since cases of transformed DLBCL may behave differently from de novo cases.

Response: Yes. All 166 cases of CD5 (-) DLBCL had no histologic and clinical evidence of transformation from precedent low grade lymphoma. We designed this study to reduce the bias as best as we could by including only de novo CD5 (-) cases. The incidence of SLL/CLL or follicular lymphoma is low in Korea, occupying 1.3% and 7.1% of total lymphomas, respectively, according to the most recent data (Jung HR et al, Classification of malignant lymphoma subtypes in Korean patients; a report of 4th nationwide study, Journal of Hematopathology 2019). Actually, transformed DLBCLs are extremely rare in Korean population.

7. Among the clinicopathologic features that differed significantly between CD5+ and CD5-negative cases was bone marrow involvement. Is it possible to review these cases to see whether there were any differences in patterns of marrow involvement between CD5+ and CD5-negative cases? Given the rarity of de novo CD5+ DLBCL, its prognostic significance and the fact that CD5 expression may not be routinely assessed by immunohistochemistry, any information about pattern of marrow involvement that might be a clue to CD5 positivity would be of practical utility to pathologists in helping to make this diagnosis.

Response: In Korea, bone marrow specimens are processed in the clinical laboratory medicine clinic which is a different department in every hospital. For reviewing the bone marrow slides, we have to ask help to clinical laboratory medicine doctors in three different hospitals, which would be very hard and time consuming. It would be appreciated if you would understand our situation.

8. On page 11, lines 185-186, the data provided for double-expressor phenotype in CD5+ vs. CD5-negative cases (9/29 vs. 7/166) is incorrect; these values are for MYC single expression as shown in Table 1 on page 10. Please correct.

Response: We corrected the typographical error (9/29 --> 8/29, 7/166 --> 5/166).

Reviewer #2:

1. What is the authors' suggested reasons that the incidence of CNS relapse in this cohort is far lower in comparison with Japanese's?

Response: Japanese studies reported 13 to 14.5% CNS relapse rate (Yamaguchi et al. Haematologica 2008; 93:1195-1202., Miyazaki et al. Annals of Oncology 2011;22: 1601–1607.). Notably, only one case of CD5+ DLBCL showed CNS relapse in our series. Kong et al. (J Korean Med Sci 2004; 19: 815-9) have also described 5 cases of de novo CD5+ DLBCL in Korean patients, all of whom showed bone marrow involvement but the authors did not mention the presence of CNS relapse. CXCR4/CXCL12 axis or downregulated ECM and cell adhesion related genes have been suggested as the potential causes for frequent CNS relapse in CD5+ DLBCL (Xu-Monette et al. Oncotarget 2015;6 (8):5615-5633., Barretina et al. Ann Hematol 2003; 82:500-505.), and further molecular studies may help in unraveling this discrepancy between Japanese and Koeran CD5+ DLBCL.

2. Are there any possibilities that CD5+DLBCL in this study might be high grade B cell lymphoma, not otherwise specified (not double hit lymphoma)?

Response: According to the revised 4th edition of WHO classification, high grade B-cell lymphoma is diagnosed when clinically aggressive mature B-cell lymphoma shows features intermediate between DLBCL and Burkitt lymphoma (BL) or appears blastoid morphology. This category is referred to B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL category in 2008 WHO classification. By definition, MYC, BCL2 and/or BCL6 rearrangements should not be found, and cases which can be morphologically diagnosed as DLBCL are excluded from this category. In our series, the morphology of CD5+ DLBCL cases clearly falls on classic DLBCL category and did not harbor blastoid appearance. Therefore, CD5+ DLBCL in our study should stay in DLBCL category.

3. How frequent is double expressors (DE) among DLBCL? It is better to compare the incidence of DE and CD5+DLBCL to discuss possible link between them.

Response: In the present study, DE was found in 6.7% (13/195) of all DLBCL. In CD5+ DLBCL, 27.6% (8/29) were classified as DE, while 3% (5/166) were categorized as DE in CD5- DLBCL. Although the overall incidence of DE in our cohort is lower than previous studies (Johnson et al. J Clin Oncol 2012, Bogusz et al. 23% PLoS One. 2017; 12(2): e0172364), the association of CD5 expression and DE seems reliable. As we mentioned in our manuscript, altered STAT3 and NF-kB pathway is assumed to be an explanation for overexpression of BCL2 in CD5+ DLBCL. This is so far the first series that shed light of the association of MYC and CD5 expression, and further studies are required to uncover the mechanism of MYC activation in CD5+ DLBCL.

Editor’s comments:

Response: We corrected minor occurrences of overlapping text (paraphrase with citation of references) according to the editor’s advice. We submitted raw data of this study as a supplementary file. And we cited a new reference regarding the incidence of SLL/CLL and mantle cell lymphoma in Korea because it is so far the most recent data.

Submitted filename: Reponse to Reviewers.docx

Click here for additional data file.

9 Oct 2019

Characteristics of CD5-positive diffuse large B-cell lymphoma among Koreans: high incidence of BCL2 and MYC double-expressors

PONE-D-19-18460R1

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Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: Thank you, authors, for carefull responses with adequate correction to review comments.

I remind tnat the authors are supposed to keep ethics on research and publications.

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Reviewer #1: No

Reviewer #2: Yes: Gyeongsin Park

15 Oct 2019

PONE-D-19-18460R1

Characteristics of CD5-positive diffuse large B-cell lymphoma among Koreans: high incidence of BCL2 and MYC double-expressors

Dear Dr. Kim:

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