Prognostic Role of Absolute Monocyte Count, CD163⁺ Macrophages, and CD8⁺ Lymphocytes in Diffuse Large B-Cell Lymphoma Treated with R-CHOP

Faisal Syarifuddin; Anna Mira Lubis; Agnes Stephanie Harahap; Hamzah Shatri; Wulyo Rajabto; Imam Subekti; Rudy Hidayat; Sukamto Koesnoe

doi:10.12688/f1000research.168760.2

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Research Article

Revised

Prognostic Role of Absolute Monocyte Count, CD163⁺ Macrophages, and CD8⁺ Lymphocytes in Diffuse Large B-Cell Lymphoma Treated with R-CHOP

[version 2; peer review: 2 approved, 1 not approved]

Previously titled : "Prognostic Role of Monocytes, Macrophages, and Lymphocytes in Diffuse Large B-Cell Lymphoma Patients Receiving R-CHOP: A Two-Year Survival Analysis"

Faisal Syarifuddin ¹, Anna Mira Lubis¹, Agnes Stephanie Harahap², [...] Hamzah Shatri³, Wulyo Rajabto¹, Imam Subekti⁴, Rudy Hidayat⁵, Sukamto Koesnoe⁶

Faisal Syarifuddin ¹, Anna Mira Lubis¹, [...] Agnes Stephanie Harahap², Hamzah Shatri³, Wulyo Rajabto¹, Imam Subekti⁴, Rudy Hidayat⁵, Sukamto Koesnoe⁶

PUBLISHED 12 Mar 2026

Author details Author details

¹ Division of Hematology and Medical Oncology, Department of Internal Medicine, Faculty of Medicine, Universitas Indonesia, Rumah Sakit Dr Cipto Mangunkusumo, Central Jakarta, Jakarta, 10430, Indonesia
² Department of Pathological Anatomy, Faculty of Medicine, Universitas Indonesia, Rumah Sakit Dr Cipto Mangunkusumo, Central Jakarta, Jakarta, 10430, Indonesia
³ Division of Psychosomatic and Palliative Medicine, Department of Internal Medicine, Faculty of Medicine, Universitas Indonesia, Rumah Sakit Dr Cipto Mangunkusumo, Central Jakarta, Jakarta, 10430, Indonesia
⁴ Division of Endocrinology, Metabolism, and Diabetes, Department of Internal Medicine, Faculty of Medicine, Universitas Indonesia, Rumah Sakit Dr Cipto Mangunkusumo, Central Jakarta, Jakarta, 10430, Indonesia
⁵ Division of Rheumatology, Department of Internal Medicine, Faculty of Medicine, Universitas Indonesia, Rumah Sakit Dr Cipto Mangunkusumo, Central Jakarta, Jakarta, 10430, Indonesia
⁶ Division of Allergy Immunology, Department of Internal Medicine, Faculty of Medicine, Universitas Indonesia, Rumah Sakit Dr Cipto Mangunkusumo, Central Jakarta, Jakarta, 10430, Indonesia

Faisal Syarifuddin
Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Resources, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Anna Mira Lubis
Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Project Administration, Resources, Supervision, Validation, Writing – Original Draft Preparation

Agnes Stephanie Harahap
Roles: Conceptualization, Formal Analysis, Investigation, Methodology, Resources, Supervision, Validation, Writing – Original Draft Preparation

Hamzah Shatri
Roles: Conceptualization, Investigation, Methodology, Resources, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing

Wulyo Rajabto
Roles: Investigation, Methodology, Resources, Supervision, Validation, Writing – Review & Editing

Imam Subekti
Roles: Data Curation, Formal Analysis, Investigation, Methodology, Resources, Supervision, Validation, Writing – Review & Editing

Rudy Hidayat
Roles: Data Curation, Formal Analysis, Investigation, Resources, Supervision, Validation, Writing – Review & Editing

Sukamto Koesnoe
Roles: Data Curation, Investigation, Methodology, Resources, Supervision, Validation, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the Oncology gateway.

Abstract

Background

Diffuse large B-cell lymphoma (DLBCL) exhibits heterogeneous clinical outcomes, including variations in event-free survival (EFS). Tumor microenvironment (TME) components, particularly absolute monocyte count (AMC), tumor-associated macrophages (TAMs), and tumor-infiltrating lymphocytes (TILs) have been implicated in prognosis, although findings remain inconsistent. This study evaluates the prognostic value of AMC, TAMs (CD163), and TILs (CD8) on two-year EFS in DLBCL patients treated with R-CHOP.

Methods

A retrospective cohort study of 108 DLBCL patients treated from January 2014 to March 2021 was conducted. AMC was obtained from peripheral blood, while CD163 and CD8 expressions were analyzed via immunohistochemistry. Associations with two-year EFS were assessed using hazard ratios (HR), and correlations between AMC and tissue immune markers were evaluated.

Results

High AMC and CD163 expression were significantly associated with poorer two-year EFS (HR = 9.82 and 8.57; both p < 0.001), whereas elevated CD8 expression predicted better outcomes (HR = 0.13; p < 0.001). AMC positively correlated with CD163 (r = 0.577; p < 0.001) and negatively with CD8 (r = –0.599; p < 0.001).

Conclusion

AMC and CD163 are negative prognostic markers, while CD8 is protective. AMC may reflect the immune profile of the TME and serve as a practical prognostic biomarker in DLBCL.

Keywords

DLBCL, monocyte count, CD163, CD8, tumor microenvironment, prognosis

Corresponding author: Faisal Syarifuddin

Competing interests: No competing interests were disclosed.

Grant information: The author(s) declared that no grants were involved in supporting this work.

Copyright: © 2026 Syarifuddin F et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Syarifuddin F, Lubis AM, Harahap AS et al. Prognostic Role of Absolute Monocyte Count, CD163⁺ Macrophages, and CD8⁺ Lymphocytes in Diffuse Large B-Cell Lymphoma Treated with R-CHOP [version 2; peer review: 2 approved, 1 not approved]. F1000Research 2026, 14:814 (https://doi.org/10.12688/f1000research.168760.2) First published: 22 Aug 2025, 14:814 (https://doi.org/10.12688/f1000research.168760.1) Latest published: 12 Mar 2026, 14:814 (https://doi.org/10.12688/f1000research.168760.2)

Revised Amendments from Version 1

In this revised version, several methodological clarifications and reporting improvements have been implemented in response to peer-review feedback. The operational definition of event-free survival (EFS) has been explicitly specified, and the starting point (time zero) for survival analysis has been clearly defined as the initiation of first-cycle R-CHOP therapy. The median follow-up duration, estimated using the reverse Kaplan–Meier method, is now reported.
A patient flow diagram has been added to improve transparency in cohort selection. Exclusion criteria have been further clarified, particularly regarding transformed DLBCL cases. Additional details on immunohistochemical quantification methods, including the measurement approach for CD8 expression, have been provided. Minor numerical inconsistencies and figure legend errors from the previous version have been corrected.
The limitations section has been expanded to acknowledge the single-center design, the use of ROC-derived cutoff values, and the need for external validation in independent cohorts. These revisions strengthen methodological transparency and improve clarity while maintaining the original findings and conclusions.
This revised version includes the addition of one new figure (Figure 1: patient flow diagram) to improve transparency in cohort selection. Minor corrections were made to figure legends and numerical inconsistencies identified in the previous review round. No changes to author names or affiliations are required.

See the authors' detailed response to the review by Rafael Pichardo-Rodriguez
See the authors' detailed response to the review by Jenifer Vaughan

Background

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin’s lymphoma, accounting for approximately 30–40% of adult cases. Although the introduction of rituximab-containing immunochemotherapy (R-CHOP) has improved outcomes, clinical prognosis remains heterogeneous, with reported two-year event-free survival (EFS) rates ranging from 39% to 44%.^1–3

Prognostic models such as the International Prognostic Index (IPI)3 and cell-of-origin (COO) classification⁴ have improved risk stratification but do not fully reflect the biological complexity of DLBCL, particularly the role of host immune responses.^4,5

Increasing evidence highlights the importance of the tumor microenvironment (TME), where immune cells may exert either tumor-promoting or tumor-suppressive effects.^5,6 Among immune components of the TME, tumor-associated macrophages (TAMs), especially CD163-positive macrophages, have been associated with immune evasion and adverse outcomes, whereas CD8-positive tumor-infiltrating lymphocytes (TILs) are generally linked to effective anti-tumor immunity and favorable prognosis.^5,7

However, studies evaluating these markers have reported inconsistent results, likely due to heterogeneity in immunohistochemical methods, cutoff definitions, and survival endpoints.^5–9

Peripheral absolute monocyte count (AMC) has also been proposed as a prognostic biomarker in DLBCL. Elevated AMC may reflect systemic immune dysregulation and contribute to an immunosuppressive tumor microenvironment, although its prognostic value remains debated.^10,11 Integrating peripheral immune parameters with tissue-based immune markers may therefore provide a more comprehensive assessment of host–tumor immune interactions. In addition, event-free survival at 24 months has been proposed as a clinically meaningful surrogate endpoint in DLBCL, as early events strongly correlate with long-term disease-related outcomes.^12–14 Accordingly, this study aimed to evaluate the prognostic impact of absolute monocyte count, CD163-positive tumor-associated macrophages, and CD8-positive tumor-infiltrating lymphocytes on two-year event-free survival in patients with diffuse large B-cell lymphoma treated with R-CHOP, and to explore the relationship between peripheral and tissue-based immune markers.

Methods

Study design and setting

This retrospective cohort study analyzed medical record data from patients diagnosed with diffuse large B-cell lymphoma (DLBCL) at Dr. Cipto Mangunkusumo Hospital (RSCM) and the affiliated anatomical pathology laboratory between January 2014 and March 2021. The starting point (time zero) for survival analysis was defined as the date of initiation of first-cycle R-CHOP chemotherapy.

Ethics statement

This retrospective study was approved by the Health Research Ethics Committee of the Faculty of Medicine, Universitas Indonesia and Cipto Mangunkusumo National Central General Hospital (reference number KET-133/UN2.F1/ETIK/PPM.00.02/2023). Given the retrospective nature of the study, written informed consent was obtained from surviving patients whenever feasible. For patients who had died prior to study initiation, the requirement for informed consent was waived by the ethics committee. Patient confidentiality was strictly maintained, and all data were anonymized prior to analysis.

Patient selection

Inclusion criteria were as follows: (a) age ≥18 years; (b) diagnosis of DLBCL according to the World Health Organization (WHO) classification; (c) receipt of an initial R-CHOP chemotherapy regimen consisting of 6–8 cycles, with or without radiotherapy; and (d) completion of chemotherapy with an average delay of no more than one week per cycle. Exclusion criteria included: (a) a history of indolent lymphoma or evidence of transformed DLBCL (e.g., transformation from follicular lymphoma or chronic lymphocytic leukemia), as these represent biologically distinct entities; (b) discordant or multiple histopathological diagnoses; (c) HIV infection; (d) primary central nervous system (CNS) DLBCL; (e) terminal comorbidities (e.g., end-stage renal failure, decompensated liver cirrhosis, or advanced heart failure); and (f) incomplete clinical or pathological data. Patient status was confirmed via telephone contact using contact information recorded in the medical charts. Baseline clinical characteristics collected included demographic data, number of extranodal sites, Ann Arbor stage, serum lactate dehydrogenase (LDH) levels, International Prognostic Index (IPI) score, and cell-of-origin classification based on the Hans algorithm. A flow diagram illustrating patient identification, exclusion criteria, and final cohort assembly is presented in Figure 1.

Figure 1. Study flow diagram of patient selection and follow-up.

Peripheral blood analysis

Absolute monocyte count (AMC) was obtained from peripheral blood samples analyzed using a Sysmex XN-1000 or XN-3000 hematology analyzer (Sysmex Corporation, Kobe, Japan).

Immunohistochemical staining

Formalin-fixed, paraffin-embedded (FFPE) tissue blocks were sectioned at 3–4 μm and mounted on poly-L-lysine–coated slides. Sections were deparaffinized in xylene and rehydrated through a graded alcohol series. Antigen retrieval was performed using Tris–EDTA buffer (pH 9.0) in a decloaking chamber at 98°C for 10 minutes, followed by cooling and washing with phosphate-buffered saline (PBS). Endogenous peroxidase activity was blocked using SeyTek Super Block. Slides were incubated for 30 minutes with Bond™ ready-to-use primary antibodies against CD163 and CD8 (Biocare Medical, Netherlands). After washing, sections were treated with a Val Universal Linker secondary antibody and visualized using a high-contrast diaminobenzidine (DAB) substrate. Counterstaining was performed with Mayer’s hematoxylin, followed by bluing in lithium carbonate and dehydration through graded alcohol and xylene. Slides were coverslipped using entellan. Negative controls were included in each staining batch, while tonsil tissue served as a positive control. For quantitative analysis, five representative high-power fields (HPFs; 400× magnification, area 0.24 mm²) were photographed using a Leica ICC50HD microscope with a camera attachment. Quantification of CD163-positive macrophages and CD8-positive lymphocytes was performed using ImageJ^® software. CD163 expression was quantified as the number of positively stained macrophages per high-power field (cells/HPF). CD8 expression was quantified as the percentage of CD8-positive lymphocytes among total nucleated cells within each high-power field (%). Staining intensity was not incorporated into the analysis to reduce variability related to tissue fixation and processing. All evaluations were independently performed by two blinded investigators to ensure inter-observer reliability.

Event-free survival (EFS)

Event-free survival (EFS) was defined as the time from initiation of R-CHOP chemotherapy to the first occurrence of disease progression, relapse after initial response, initiation of second-line therapy, or death from any cause. Patients without an event were censored at the date of last follow-up.

Statistical analysis

Optimal cutoff values for AMC, CD163, and CD8 were determined using receiver operating characteristic (ROC) curve analysis based on two-year event-free survival. This approach was selected to provide clinically interpretable thresholds while minimizing overfitting in a retrospective cohort. Correlations between AMC, CD163, and CD8 were assessed using Spearman’s correlation test. The median follow-up duration, estimated using a reverse Kaplan–Meier approach, was 20 months. Event-free survival was analyzed using Kaplan–Meier curves and compared using the log-rank test. Univariate and multivariate Cox proportional hazards regression models were employed to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). Variables with a p-value <0.05 in univariate analysis were included in the multivariate model. A two-sided p-value <0.05 was considered statistically significant. Although maximally selected rank statistics have been proposed for determining optimal cutoffs in time-to-event analyses, ROC-based thresholds were selected in this study to provide clinically interpretable values while minimizing model instability in a retrospective cohort with a limited sample size. Future prospective validation using alternative statistical approaches, including maximally selected rank statistics or time-dependent ROC methods, would be valuable. All statistical analyses were performed using SPSS version 20.0 (IBM Corp., Armonk, NY, USA). Missing data were handled using a complete-case analysis approach; variables with missing values were excluded from analyses requiring those parameters, and no data imputation was performed. All data were independently verified by two reviewers to ensure accuracy and completeness.

Results

Patient characteristics

Between January 2014 and March 2021, a total of 108 patients diagnosed with diffuse large B-cell lymphoma (DLBCL) who met the inclusion and exclusion criteria were included in the study. Serum lactate dehydrogenase (LDH) data were missing in nine patients. Among all subjects, 56 (52%) were male, and 72 (66.7%) were younger than 60 years (median age: 53 years; range: 18–88 years). Early-stage disease (Ann Arbor stage I–II) was observed in 60 patients (55.6%), and 89 patients (82%) had 0–1 extranodal sites. Elevated LDH levels (>1× upper limit of normal) were present in 81 patients (81.8%), and 75 patients (75.8%) had a low-risk International Prognostic Index (IPI) score (0–2). Based on the Hans classification, the germinal center B-cell (GCB) subtype was identified in 33 patients (30.6%). During the two-year follow-up period, 58 patients (53.7%) experienced an event, while 46 patients (42.6%) remained event-free. Follow-up information was unavailable for four patients (3.7%). The median values of absolute monocyte count (AMC), CD163 expression, and CD8 expression were 619.2 cells/μL (interquartile range [IQR]: 455.0–759.45), 21.5 cells/HPF (IQR: 14.25–27.0 cells/HPF), and 23% (IQR: 14–39%), respectively. Detailed demographic and clinical characteristics are summarized in Table 1.

Table 1. Demographic and clinical characteristics of study subjects.

Variable	Total (%)
Gender (n = 108)
Male	56 (52)
Female	52 (48)
Age (Median 53 years, range 18-88 years)
≤60 years	72 (66,7)
>60 years	36 (33,3)
Ann Arbor Staging (n = 108)
I or II	60 (55,6)
III or IV	48 (44,4)
Extranodal lesion (n = 108)
0-1	89 (82)
>1	19 (18)
Serum Lactate Dehydrogenase (LDH) (n = 99)
≤1 x normal	18 (18,2)
>1 x normal	81 (81,8)
IPI Score (n = 99)
Low risk (0-2)	75 (75,8)
High risk (3-5)	24 (24,2)
Protein expression (n = 108)
CD8, Median (IQR)	0,23 (0,14-0,39)
CD163, Median (IQR)	21,5 (14,25-27,0)
AMC (n = 108), Median (IQR)	619,2 (455,0-759,45)
Subtypes based on Hans algorithm (n = 108)
GCB	33 (30.6)
Non-GCB	75 (69,4)
Events in 2 years (n = 108)
Yes	58 (53,7)
No	46 (42,6)
No Information	4 (3,7)

Immunohistological characteristics and optimal cutoff values for AMC, CD163, and CD8

Receiver operating characteristic (ROC) curve analysis was performed to determine optimal cutoff values for AMC, CD163, and CD8 in predicting two-year event-free survival (EFS). The optimal cutoff for AMC was 631 cells/μL, yielding an area under the curve (AUC) of 0.923 (95% CI: 0.866–0.981), with a sensitivity of 82.8% and specificity of 90.0%. For CD163 expression, the optimal cutoff was 23 cells/HPF, with an AUC of 0.931 (95% CI: 0.880–0.983), sensitivity of 75.9%, and specificity of 96.0%. CD8 expression demonstrated an optimal cutoff value of 27.5%, with an AUC of 0.852 (95% CI: 0.773–0.930), sensitivity of 84.5%, and specificity of 84.0%.

ROC curves are presented in Figure 2. Patients were subsequently categorized into high- and low-expression groups according to these cutoff values (Figure 3).

Figure 2. Quantitative ROC curves for 2-year Event-Free Survival: (a) AMC, (b) TAM CD163, and (c) TIL CD8.

Figure 3. CD163 and CD8 expression in DLBCL tissues (100×): (a) Low CD163 expression, (b) High CD163 expression, (c) High CD8 expression, (d) Low CD8 expression.

Correlation between AMC and CD163 or CD8 expression

Spearman’s correlation analysis demonstrated a significant positive correlation between AMC and CD163 expression (r = 0.577, p < 0.001) and a significant negative correlation between AMC and CD8 expression (r = −0.599, p < 0.001). These findings suggest a potential association between peripheral monocyte levels and the immune composition of the tumor microenvironment. Correlation analyses are illustrated in Figure 4.

Figure 4. Correlation between (a) AMC and TAM CD163, and (b) AMC and TIL CD8.

Impact of AMC, CD163, and CD8 on 2-Year EFS

Patients with high AMC levels (≥631 cells/μL) had significantly poorer two-year EFS compared with those with lower AMC levels (HR = 9.82; 95% CI: 4.89–19.70; p < 0.001). Similarly, elevated CD163 expression (≥23 cells/HPF) was associated with inferior EFS outcomes (HR = 8.57; 95% CI: 4.58–16.05; p < 0.001). In contrast, patients with higher CD8 expression (≥27.5%) demonstrated significantly improved EFS (HR = 0.13; 95% CI: 0.06–0.26; p < 0.001). Subgroup analysis based on the Hans cell-of-origin classification showed no significant difference in EFS between the GCB and non-GCB subtypes (HR = 0.75; 95% CI: 0.42–1.35; p = 0.336). In contrast, a high IPI score was significantly associated with reduced EFS (HR = 3.06; 95% CI: 1.77–5.35; p < 0.001). Detailed survival analyses are presented in Table 2 and Figure 5. The median event-free survival time for the overall cohort was 13 months.

Table 2. The relationship of different variables on patient survival.

Variable	Event		HR (CI 95%)	p
Variable	Yes (%)	No (%)	HR (CI 95%)	p
AMC
≥631	48 (90,6)	5 (9,4)	9,817 (4,891-19,703)	<0,001
<631	10 (18,2)	45 (81,8)
CD163
>23	44 (95,7)	2 (4,3)	8,571 (4,576-16,053)	<0,0001
<23	14 (22,6)	48 (77,4)
CD8
≥0,275	9 (19,2)	48 (80,8)	0,128 (0.064-0.256)	<0,001
<0,275	48 (85,7)	8 (14,3)
GCB/nonGCB
GCB	15 (45,5)	18 (54,5)	0,749 (0,416-1,349)	0,336
nonGCB	43 (57,3)	32 (42,7)
IPI
High	22 (91,7)	2 (8,3)	3,075 (1,768-5,349)	<0,001
Low	32 (42,7)	43 (57,3)

Figure 5. Kaplan-Meier survival curves for DLBCL patients based on (a) AMC, (b) TAM CD163, (c) TIL CD8, (d) COO, and (e) IPI score.

Discussion

This study evaluated the prognostic significance of peripheral absolute monocyte count (AMC), CD163-positive tumor-associated macrophages, and CD8-positive tumor-infiltrating lymphocytes in patients with diffuse large B-cell lymphoma treated with R-CHOP. We demonstrated that elevated AMC and increased CD163 expression were significantly associated with inferior two-year event-free survival, whereas higher CD8 infiltration predicted improved outcomes. These findings support the concept that both systemic immune parameters and local tumor microenvironment composition contribute to disease progression in DLBCL. The median age of 53 years in our cohort is consistent with prior Indonesian studies but lower than that reported in Western and East Asian populations, where median ages typically range from the early to mid-60s.^2,9,14 This difference may reflect variations in genetic background, environmental factors, and infectious cofactors such as Epstein–Barr virus. Additionally, the predominance of early-stage disease in our cohort may partly relate to staging practices and limited access to positron emission tomography in resource-constrained settings.^15,16

More than half of the patients experienced an event within two years of treatment initiation, consistent with previous reports indicating that approximately 40–50% of patients with DLBCL experience relapse or treatment failure despite standard immunochemotherapy.^17,18

In this context, early event-free survival has been recognized as a clinically meaningful surrogate endpoint, as events occurring within 24 months strongly correlate with long-term disease-related mortality.¹²^–¹⁴ Because event-free survival in this study incorporated death from any cause as an event, a competing-risk framework was not applied. However, future analyses differentiating disease-specific progression from non–disease-related mortality using competing-risk methods may provide additional granularity in prognostic modeling.

Therefore, identifying accessible biomarkers that predict early events remains highly relevant in clinical practice. Elevated peripheral monocyte count has previously been associated with inferior survival outcomes in DLBCL, potentially reflecting systemic inflammation and monocyte recruitment to the tumor microenvironment.^11,19 Our findings are consistent with this hypothesis, as AMC demonstrated both a strong adverse prognostic impact and a moderate positive correlation with CD163 expression. CD163-positive macrophages represent an immunosuppressive M2-polarized phenotype, which has been implicated in tumor immune evasion, angiogenesis, and resistance to therapy.^19–21

Although cutoff values for CD163 vary across studies, methodological differences in staining techniques, quantification methods, and tissue sampling likely account for this variability.

Conversely, higher CD8-positive T-cell infiltration was associated with improved event-free survival, reinforcing the protective role of cytotoxic lymphocytes in mediating anti-tumor immunity.⁷ The higher CD8 threshold observed in our study compared with some prior reports may reflect the use of whole-section analysis rather than tissue microarrays, which can influence quantification of tumor-infiltrating lymphocytes.^22–24

Notably, the inverse correlation between AMC and CD8 expression suggests a potential imbalance between immunosuppressive macrophage activity and cytotoxic immune responses, underscoring the dynamic and context-dependent interplay within the DLBCL tumor microenvironment.^25,26 The cell-of-origin classification based on the Hans algorithm did not significantly influence two-year event-free survival in our cohort, consistent with several studies questioning its prognostic reproducibility when assessed by immunohistochemistry alone.^20,27,28

In contrast, the International Prognostic Index remained a robust predictor of outcome, reaffirming its clinical utility in risk stratification. These findings suggest that immune-based biomarkers may provide complementary biological insight beyond conventional clinical scoring systems.

Several limitations should be acknowledged. First, disease staging was based on computed tomography rather than positron emission tomography, which may underestimate disease burden and influence risk stratification. Second, serum lactate dehydrogenase data were unavailable in a subset of patients, potentially affecting IPI calculation. Third, additional immune markers such as CD68-positive macrophages or CD4-positive lymphocytes were not assessed due to resource constraints. Finally, the retrospective design introduces the possibility of selection bias and residual confounding. Nevertheless, the consistency of our findings with prior literature supports the relevance of integrated peripheral and tissue-based immune profiling in DLBCL. In addition, the optimal cutoff values were derived from a single-center cohort using ROC-based thresholds, which may be susceptible to optimism bias. Therefore, external validation in independent cohorts is necessary to confirm the robustness and generalizability of these prognostic thresholds.

Conclusions

Higher expression of tumor-associated macrophages (CD163) and elevated absolute monocyte count (AMC) were significantly associated with poorer two-year event-free survival (EFS) in patients with diffuse large B-cell lymphoma (DLBCL) undergoing R-CHOP therapy. In contrast, increased infiltration of CD8+ tumor-infiltrating lymphocytes (TILs) predicted improved survival outcomes. The optimal prognostic cutoff values identified were 631/mm³ for AMC, 23/HPF for CD163, and 27.5%/HPF for CD8. Moderate correlations were observed between AMC and both tissue-based immune markers, with a positive association with CD163 and a negative association with CD8 expression. These findings support the integration of peripheral and tissue-based immune parameters as complementary tools for risk stratification in DLBCL.

Authors’ declaration

- We hereby confirm that all the Figures and Tables in the manuscript are ours. Furthermore, any Figures and images, that are not ours, have been included with the necessary permission for re-publication, which is attached to the manuscript.
- Ethical Clearance: The project was approved by the local ethical committee at University of Indonesia.

Data availabilty

Figshare: Prognostic Role of Monocytes, Macrophages, and Lymphocytes in Diffuse Large B-Cell Lymphoma Patients Receiving R-CHOP: A Two-Year Survival Analysis. figshare. Dataset. https://doi.org/10.6084/m9.figshare.29818244.v1²⁹

The project contains the following underlying data:

• Data.xlsx. (Anonymised data).

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

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16. Batty N, Ghonimi E, Feng L, et al.: The absolute monocyte and lymphocyte prognostic index for patients with diffuse large B-cell lymphoma who receive R-CHOP. Clin. Lymphoma Myeloma Leuk. 2013; 13(1): 15–18. PubMed Abstract | Publisher Full Text | Free Full Text
17. Li YL, Kang-Sheng G, Yue-Yin P, et al.: The lower peripheral blood lymphocyte/monocyte ratio assessed during routine follow-up after standard first-line chemotherapy is a risk factor for predicting relapse in patients with diffuse large B-cell lymphoma. Leuk. Res. 2014; 38(3): 323–328. PubMed Abstract | Publisher Full Text
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19. Rajnai H, Heyning FH, Koens L, et al.: The density of CD8+ T-cell infiltration and expression of BCL2 predicts outcome of primary diffuse large B-cell lymphoma of bone. Virchows Arch. 2014; 464(2): 229–239. PubMed Abstract | Publisher Full Text
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23. Yamauchi T, Tasaki T, Tai K, et al.: Prognostic effect of peripheral blood cell counts in advanced diffuse large B-cell lymphoma treated with R-CHOP-like chemotherapy: a single institution analysis. Oncol. Lett. 2015; 9(2): 851–856. PubMed Abstract | Publisher Full Text | Free Full Text
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Version 2

VERSION 2 PUBLISHED 22 Aug 2025

Author details Author details

Agnes Stephanie Harahap
Roles: Conceptualization, Formal Analysis, Investigation, Methodology, Resources, Supervision, Validation, Writing – Original Draft Preparation

Hamzah Shatri
Roles: Conceptualization, Investigation, Methodology, Resources, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing

Wulyo Rajabto
Roles: Investigation, Methodology, Resources, Supervision, Validation, Writing – Review & Editing

Imam Subekti
Roles: Data Curation, Formal Analysis, Investigation, Methodology, Resources, Supervision, Validation, Writing – Review & Editing

Rudy Hidayat
Roles: Data Curation, Formal Analysis, Investigation, Resources, Supervision, Validation, Writing – Review & Editing

Sukamto Koesnoe
Roles: Data Curation, Investigation, Methodology, Resources, Supervision, Validation, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

The author(s) declared that no grants were involved in supporting this work.

Article Versions (2)

version 2

Revised

Published: 12 Mar 2026, 14:814

https://doi.org/10.12688/f1000research.168760.2

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Published: 22 Aug 2025, 14:814

https://doi.org/10.12688/f1000research.168760.1

© 2026 Syarifuddin F et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Syarifuddin F, Lubis AM, Harahap AS et al. Prognostic Role of Absolute Monocyte Count, CD163⁺ Macrophages, and CD8⁺ Lymphocytes in Diffuse Large B-Cell Lymphoma Treated with R-CHOP [version 2; peer review: 2 approved, 1 not approved]. F1000Research 2026, 14:814 (https://doi.org/10.12688/f1000research.168760.2)

NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article.

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ApprovedThe paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approvedFundamental flaws in the paper seriously undermine the findings and conclusions

Version 2

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PUBLISHED 12 Mar 2026

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Reviewer Report 06 Apr 2026

Teng Fei, Memorial Sloan Kettering Cancer Center, New York, USA

Not Approved

https://doi.org/10.5256/f1000research.196782.r467762

The authors applied statistical methods to assess the prognostic values of AMC, CD163 and CD8 markers for the event-free survival of DLBCL patients treated with R-CHOP. As a statistical reviewer, I would like to point out the following comments and suggestions. To summarize, flaws are observed in the statistical analysis involved in this manuscript, which should be fully addressed to be considered for indexing.

Please justify why it was 2-year EFS that was used to determine the thresholds for the markers of interest.
Please use the wording of “univariable” and “multivariable” regression models, rather than “univariate” and “multivariate”. This would comply with the reporting standard.
Some approaches such as maxstat and time-dependent ROC were mentioned in the method section but not applied for the analyses. Please move the corresponding description to the discussion section as future directions.
Table 1: the title for the second column (Total (%)) is not applicable for the continuous variables.
Figure 4: please make the two panels the same size with the same scale for the x-axis.
Table 2 is confusing. First, it is unclear whether it reports univariable models or multivariable models. Second, it is untypical to report contingency tables for survival models. After checking the sum of numbers from cells of contingency tables, I suppose the results are univariable (108 patients with AMC, but only 99 with ICI). It is then questionable why univariable results, rather than the multivariable ones, are reported to support the main conclusions of this manuscript.
It is stated in the method section that the median follow-up was 20 months. However, no censored observations were reflected in Kaplan-Meier curves from Figure 5, which is contradictory. In addition, there appears to be a strong immortal bias in the population, where all patients survived beyond 6 months, which might be the case but is generally a red flag in statistical review.
Event times are undefined for censored patients in the shared data. I assume the authors used administrative censoring at the end of 2 years, but that would contradict the median follow-up of 20 months.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

No
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

No
Are all the source data underlying the results available to ensure full reproducibility?

No
Are the conclusions drawn adequately supported by the results?

No

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Biostatistics

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

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Reviewer Report 30 Mar 2026

Rafael Pichardo-Rodriguez, Instituto de Investigaciones en Ciencias Biomédicas (INICIB), Universidad Ricardo Palma, Lima, Peru

Approved

https://doi.org/10.5256/f1000research.196782.r467144

I have no further comments; I ... Continue reading

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PUBLISHED 22 Aug 2025

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Reviewer Report 20 Nov 2025

Rafael Pichardo-Rodriguez, Instituto de Investigaciones en Ciencias Biomédicas (INICIB), Universidad Ricardo Palma, Lima, Peru

Approved with Reservations

https://doi.org/10.5256/f1000research.185964.r430460

Thank you for the opportunity to review this manuscript, which evaluates prognostic biomarkers in diffuse large B-cell lymphoma. The study addresses a clinically relevant and timely topic; however, there are several methodological and reporting issues that should be addressed before ... Continue reading

Operational definition of EFS
The Methods section does not specify the operational definition of event-free survival (EFS). While EFS is an increasingly used endpoint in lymphoma, its interpretation requires caution since it is a heterogeneous surrogate outcome. I recommend that the authors clearly define EFS and consider, in subsequent analyses, a competing-risk approach to differentiate disease progression/recurrence from non–disease-related mortality.
Estimation and reporting of median follow-up
The manuscript does not describe how the median follow-up time was estimated, nor does it report the actual value. This parameter is essential to determine whether the observation period was long enough to capture meaningful clinical events. The authors should specify the method used (e.g., reverse Kaplan–Meier) and include the corresponding median follow-up.
Flow diagram (CONSORT/REMARK)
A patient flow diagram is not provided. Such a diagram is crucial to visualize the selection process, exclusions, and losses to follow-up. The authors should include a flowchart following the CONSORT or STROBE guidelines as appropriate.
Definition of follow-up period and time zero
The starting point (time zero) for follow-up is not defined. Clarifying whether this corresponds to the date of diagnosis, initiation of treatment, or completion of therapy is critical for reproducibility and for appropriate interpretation of time-to-event analyses.
Handling of missing data
The manuscript does not mention whether missing data were present or how they were handled. The authors should state whether they performed data imputation, complete-case analysis, or any other strategy, as this can influence the validity of their findings.
Exclusion criteria
The exclusion of “indolent lymphomas” is mentioned, yet this group would not typically be included in a cohort restricted to DLBCL. Instead, it would be more relevant to specify whether transformed DLBCL cases (e.g., from CLL or follicular lymphoma) were excluded, since these represent a distinct biological entity.
Determination of cut-off values for continuous biomarkers
There is no standardized methodology for defining cut-off points of continuous biomarkers in DLBCL. For time-dependent outcomes, I recommend using Maximally Selected Rank Statistics with the maxstat package in R to determine optimal thresholds, although this is left to the authors’ discretion.

In summary, this manuscript investigates clinically meaningful and novel biomarkers in Lymphoma. With the suggested methodological clarifications and improvements, the study could be considerably strengthened and would merit reconsideration in a second review round.
Thank you again for the opportunity to evaluate this valuable work, and I commend the authors for their contribution to the field of lymphoma research

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

No
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

No

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Hemato-Oncology, Lymphoma.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Author Response 12 Mar 2026

Faisal Syarifuddin, Division of Hematology and Medical Oncology, Department of Internal Medicine, Faculty of Medicine, Universitas Indonesia, Rumah Sakit Dr Cipto Mangunkusumo, Central Jakarta, 10430, Indonesia

12 Mar 2026

Author Response
We sincerely thank the reviewer for the thorough and insightful methodological evaluation of our manuscript. We greatly appreciate the constructive recommendations, which have substantially improved the clarity, transparency, and rigor ... Continue reading
We sincerely thank the reviewer for the thorough and insightful methodological evaluation of our manuscript. We greatly appreciate the constructive recommendations, which have substantially improved the clarity, transparency, and rigor of our study. The revisions are summarized below:

The operational definition of event-free survival (EFS) has now been explicitly defined in the Methods section as the time from initiation of first-cycle R-CHOP chemotherapy to the first occurrence of disease progression, relapse after response, initiation of second-line therapy, or death from any cause. We also clarified the rationale for selecting two-year EFS as a clinically meaningful endpoint based on established literature. Additionally, we have acknowledged in the Discussion that future studies may benefit from competing-risk modeling to distinguish disease-specific progression from non–disease-related mortality.

The median follow-up duration is now reported (20 months), estimated using the reverse Kaplan–Meier method. The method used for estimation has been explicitly stated in the Statistical Analysis section.

A patient flow diagram has been added (Figure 1) to transparently illustrate patient identification, exclusions, and final cohort assembly, in alignment with STROBE/REMARK recommendations.

The starting point (time zero) for follow-up has been clearly defined as the initiation date of first-cycle R-CHOP chemotherapy to ensure reproducibility and appropriate interpretation of time-to-event analyses.

Handling of missing data has now been explicitly described. A complete-case analysis approach was used, and no data imputation was performed. The extent of missing LDH data is now reported in the Results section.

The exclusion criteria have been clarified to explicitly state that transformed DLBCL cases (e.g., transformation from follicular lymphoma or chronic lymphocytic leukemia) were excluded, as these represent biologically distinct entities.

Regarding the determination of cut-off values for continuous biomarkers, we have clarified that receiver operating characteristic (ROC) analysis based on two-year EFS was selected to provide clinically interpretable thresholds while minimizing model instability in a retrospective cohort of limited sample size. We have acknowledged in the Statistical Analysis and Discussion sections that alternative approaches, such as maximally selected rank statistics, may be considered in future prospective validation studies.

The limitations section has been expanded to acknowledge the single-center design, potential optimism bias associated with ROC-derived thresholds, and the need for external validation in independent cohorts.

We are grateful for the reviewer’s careful assessment and believe that these revisions have substantially strengthened the methodological transparency and overall quality of the manuscript.
We sincerely thank the reviewer for the thorough and insightful methodological evaluation of our manuscript. We greatly appreciate the constructive recommendations, which have substantially improved the clarity, transparency, and rigor of our study. The revisions are summarized below:

The operational definition of event-free survival (EFS) has now been explicitly defined in the Methods section as the time from initiation of first-cycle R-CHOP chemotherapy to the first occurrence of disease progression, relapse after response, initiation of second-line therapy, or death from any cause. We also clarified the rationale for selecting two-year EFS as a clinically meaningful endpoint based on established literature. Additionally, we have acknowledged in the Discussion that future studies may benefit from competing-risk modeling to distinguish disease-specific progression from non–disease-related mortality.

The median follow-up duration is now reported (20 months), estimated using the reverse Kaplan–Meier method. The method used for estimation has been explicitly stated in the Statistical Analysis section.

A patient flow diagram has been added (Figure 1) to transparently illustrate patient identification, exclusions, and final cohort assembly, in alignment with STROBE/REMARK recommendations.

The starting point (time zero) for follow-up has been clearly defined as the initiation date of first-cycle R-CHOP chemotherapy to ensure reproducibility and appropriate interpretation of time-to-event analyses.

Handling of missing data has now been explicitly described. A complete-case analysis approach was used, and no data imputation was performed. The extent of missing LDH data is now reported in the Results section.

The exclusion criteria have been clarified to explicitly state that transformed DLBCL cases (e.g., transformation from follicular lymphoma or chronic lymphocytic leukemia) were excluded, as these represent biologically distinct entities.

Regarding the determination of cut-off values for continuous biomarkers, we have clarified that receiver operating characteristic (ROC) analysis based on two-year EFS was selected to provide clinically interpretable thresholds while minimizing model instability in a retrospective cohort of limited sample size. We have acknowledged in the Statistical Analysis and Discussion sections that alternative approaches, such as maximally selected rank statistics, may be considered in future prospective validation studies.

The limitations section has been expanded to acknowledge the single-center design, potential optimism bias associated with ROC-derived thresholds, and the need for external validation in independent cohorts.

We are grateful for the reviewer’s careful assessment and believe that these revisions have substantially strengthened the methodological transparency and overall quality of the manuscript.
Competing Interests: I have no competing interests Close
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COMMENTS ON THIS REPORT

Author Response 12 Mar 2026

Faisal Syarifuddin, Division of Hematology and Medical Oncology, Department of Internal Medicine, Faculty of Medicine, Universitas Indonesia, Rumah Sakit Dr Cipto Mangunkusumo, Central Jakarta, 10430, Indonesia

12 Mar 2026

Author Response
We sincerely thank the reviewer for the thorough and insightful methodological evaluation of our manuscript. We greatly appreciate the constructive recommendations, which have substantially improved the clarity, transparency, and rigor ... Continue reading
We sincerely thank the reviewer for the thorough and insightful methodological evaluation of our manuscript. We greatly appreciate the constructive recommendations, which have substantially improved the clarity, transparency, and rigor of our study. The revisions are summarized below:

The operational definition of event-free survival (EFS) has now been explicitly defined in the Methods section as the time from initiation of first-cycle R-CHOP chemotherapy to the first occurrence of disease progression, relapse after response, initiation of second-line therapy, or death from any cause. We also clarified the rationale for selecting two-year EFS as a clinically meaningful endpoint based on established literature. Additionally, we have acknowledged in the Discussion that future studies may benefit from competing-risk modeling to distinguish disease-specific progression from non–disease-related mortality.

The median follow-up duration is now reported (20 months), estimated using the reverse Kaplan–Meier method. The method used for estimation has been explicitly stated in the Statistical Analysis section.

A patient flow diagram has been added (Figure 1) to transparently illustrate patient identification, exclusions, and final cohort assembly, in alignment with STROBE/REMARK recommendations.

The starting point (time zero) for follow-up has been clearly defined as the initiation date of first-cycle R-CHOP chemotherapy to ensure reproducibility and appropriate interpretation of time-to-event analyses.

Handling of missing data has now been explicitly described. A complete-case analysis approach was used, and no data imputation was performed. The extent of missing LDH data is now reported in the Results section.

The exclusion criteria have been clarified to explicitly state that transformed DLBCL cases (e.g., transformation from follicular lymphoma or chronic lymphocytic leukemia) were excluded, as these represent biologically distinct entities.

Regarding the determination of cut-off values for continuous biomarkers, we have clarified that receiver operating characteristic (ROC) analysis based on two-year EFS was selected to provide clinically interpretable thresholds while minimizing model instability in a retrospective cohort of limited sample size. We have acknowledged in the Statistical Analysis and Discussion sections that alternative approaches, such as maximally selected rank statistics, may be considered in future prospective validation studies.

The limitations section has been expanded to acknowledge the single-center design, potential optimism bias associated with ROC-derived thresholds, and the need for external validation in independent cohorts.

We are grateful for the reviewer’s careful assessment and believe that these revisions have substantially strengthened the methodological transparency and overall quality of the manuscript.
We sincerely thank the reviewer for the thorough and insightful methodological evaluation of our manuscript. We greatly appreciate the constructive recommendations, which have substantially improved the clarity, transparency, and rigor of our study. The revisions are summarized below:

The operational definition of event-free survival (EFS) has now been explicitly defined in the Methods section as the time from initiation of first-cycle R-CHOP chemotherapy to the first occurrence of disease progression, relapse after response, initiation of second-line therapy, or death from any cause. We also clarified the rationale for selecting two-year EFS as a clinically meaningful endpoint based on established literature. Additionally, we have acknowledged in the Discussion that future studies may benefit from competing-risk modeling to distinguish disease-specific progression from non–disease-related mortality.

The median follow-up duration is now reported (20 months), estimated using the reverse Kaplan–Meier method. The method used for estimation has been explicitly stated in the Statistical Analysis section.

A patient flow diagram has been added (Figure 1) to transparently illustrate patient identification, exclusions, and final cohort assembly, in alignment with STROBE/REMARK recommendations.

The starting point (time zero) for follow-up has been clearly defined as the initiation date of first-cycle R-CHOP chemotherapy to ensure reproducibility and appropriate interpretation of time-to-event analyses.

Handling of missing data has now been explicitly described. A complete-case analysis approach was used, and no data imputation was performed. The extent of missing LDH data is now reported in the Results section.

The exclusion criteria have been clarified to explicitly state that transformed DLBCL cases (e.g., transformation from follicular lymphoma or chronic lymphocytic leukemia) were excluded, as these represent biologically distinct entities.

Regarding the determination of cut-off values for continuous biomarkers, we have clarified that receiver operating characteristic (ROC) analysis based on two-year EFS was selected to provide clinically interpretable thresholds while minimizing model instability in a retrospective cohort of limited sample size. We have acknowledged in the Statistical Analysis and Discussion sections that alternative approaches, such as maximally selected rank statistics, may be considered in future prospective validation studies.

The limitations section has been expanded to acknowledge the single-center design, potential optimism bias associated with ROC-derived thresholds, and the need for external validation in independent cohorts.

We are grateful for the reviewer’s careful assessment and believe that these revisions have substantially strengthened the methodological transparency and overall quality of the manuscript.
Competing Interests: I have no competing interests Close
Report a concern

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Reviewer Report 12 Nov 2025

Jenifer Vaughan, University of the Witwatersrand J, Johannesburg, Gauteng, South Africa

Approved

https://doi.org/10.5256/f1000research.185964.r427565

This is a well written article which has assessed the prognostic influence of elevated peripheral blood monocyte counts along with CD163 expressing macrophages and CD8+ lymphocytes within the tumour microenvironment of DLBCL. The findings recapitulate those of previous studies, ... Continue reading

The title is somewhat misleading, as the study focused on specific subsets of macrophages and lymphocytes, and not on all macrophages and lymphocytes. It would be better to specify that CD163+ macrophages and CD8+ lymphocytes were evaluated This should be applied in the abstract as well.
It is stated that all of the participants gave written informed consent, but this was a retrospective study, and some of the participants had demised before the study commenced. Please clarify how this was possible.
Under the immunohistochemical staining methods, it is stated that CD8 was reported as a percentage of stained T-cells. How were all the T-cells identified if only a CD8 stain was used?
There is an error in the 1^st paragraph of the Results, where it is stated that four patients remained event free (it should be 46 patients according to Table 1).
In the following line of the same paragraph, there is an error in the number of numbers of CD163 positive cells (reported as a percentage and not cells/HPF).
In Table 1, units should be included for the numbers provided for the AMC, CD8 and CD163.
There is an error in the legend for Figure 2; it is stated that (d) shows low CD8 expression, but the image appears to show high CD8 expression?
Why was 2 year EFS looked at and not the more overall meaningful survival statistic of overall survival?
In the 2^nd paragraph of the Discussion, it is stated that Extranodal involvement was seen in 27% of patients. This information is not included in the Results section?
Similarly, in the 4^th paragraph of the Discussion, it is stated that the median survival time was 13 months, but this too was not included in the Results section. It is not a good practice to introduce new results in the Discussion.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Tumour microenvironement of DLBCL, HIV-associated lymphoma

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

CITE

Report a concern

Author Response 12 Mar 2026

Faisal Syarifuddin, Division of Hematology and Medical Oncology, Department of Internal Medicine, Faculty of Medicine, Universitas Indonesia, Rumah Sakit Dr Cipto Mangunkusumo, Central Jakarta, 10430, Indonesia

12 Mar 2026

Author Response
We sincerely thank the reviewer for the constructive feedback, which has significantly improved the clarity and methodological transparency of our manuscript. The revisions are summarized below:
- The title
... Continue reading
We sincerely thank the reviewer for the constructive feedback, which has significantly improved the clarity and methodological transparency of our manuscript. The revisions are summarized below:

The title has been revised to explicitly specify CD163⁺ macrophages and CD8⁺ lymphocytes to accurately reflect the immune subsets evaluated. Corresponding changes have been made in the Abstract for consistency.

The Ethics section has been clarified to state that written informed consent was obtained from surviving patients whenever feasible, while consent for deceased patients was waived in accordance with ethics committee approval.

The immunohistochemical quantification method has been revised to clarify that CD8 expression was measured as the percentage of CD8-positive lymphocytes among total nucleated cells within each high-power field using ImageJ® software, correcting the previous ambiguity.

The typographical error regarding the number of event-free patients has been corrected (46 patients). The unit for CD163 expression has been standardized as cells/HPF throughout the manuscript.

Units for AMC (cells/µL), CD163 (cells/HPF), and CD8 (%) have been added in Table 1 to improve clarity.

The figure legend has been corrected to accurately reflect CD8 expression levels.

Extranodal involvement and the median event-free survival time (13 months) are now explicitly reported in the Results section to avoid introducing new findings in the Discussion.

The rationale for selecting two-year event-free survival (EFS24) as the primary endpoint has been clarified, emphasizing its established relevance as a clinically meaningful surrogate marker in DLBCL.

We greatly appreciate the reviewer’s thoughtful comments, which have strengthened the overall quality and transparency of the manuscript.
We sincerely thank the reviewer for the constructive feedback, which has significantly improved the clarity and methodological transparency of our manuscript. The revisions are summarized below:

The title has been revised to explicitly specify CD163⁺ macrophages and CD8⁺ lymphocytes to accurately reflect the immune subsets evaluated. Corresponding changes have been made in the Abstract for consistency.

The Ethics section has been clarified to state that written informed consent was obtained from surviving patients whenever feasible, while consent for deceased patients was waived in accordance with ethics committee approval.

The immunohistochemical quantification method has been revised to clarify that CD8 expression was measured as the percentage of CD8-positive lymphocytes among total nucleated cells within each high-power field using ImageJ® software, correcting the previous ambiguity.

The typographical error regarding the number of event-free patients has been corrected (46 patients). The unit for CD163 expression has been standardized as cells/HPF throughout the manuscript.

Units for AMC (cells/µL), CD163 (cells/HPF), and CD8 (%) have been added in Table 1 to improve clarity.

The figure legend has been corrected to accurately reflect CD8 expression levels.

Extranodal involvement and the median event-free survival time (13 months) are now explicitly reported in the Results section to avoid introducing new findings in the Discussion.

The rationale for selecting two-year event-free survival (EFS24) as the primary endpoint has been clarified, emphasizing its established relevance as a clinically meaningful surrogate marker in DLBCL.

We greatly appreciate the reviewer’s thoughtful comments, which have strengthened the overall quality and transparency of the manuscript.
Competing Interests: I have no competing interests Close
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COMMENTS ON THIS REPORT

Author Response 12 Mar 2026

Faisal Syarifuddin, Division of Hematology and Medical Oncology, Department of Internal Medicine, Faculty of Medicine, Universitas Indonesia, Rumah Sakit Dr Cipto Mangunkusumo, Central Jakarta, 10430, Indonesia

12 Mar 2026

Author Response
We sincerely thank the reviewer for the constructive feedback, which has significantly improved the clarity and methodological transparency of our manuscript. The revisions are summarized below:
- The title
... Continue reading
We sincerely thank the reviewer for the constructive feedback, which has significantly improved the clarity and methodological transparency of our manuscript. The revisions are summarized below:

The title has been revised to explicitly specify CD163⁺ macrophages and CD8⁺ lymphocytes to accurately reflect the immune subsets evaluated. Corresponding changes have been made in the Abstract for consistency.

The Ethics section has been clarified to state that written informed consent was obtained from surviving patients whenever feasible, while consent for deceased patients was waived in accordance with ethics committee approval.

The immunohistochemical quantification method has been revised to clarify that CD8 expression was measured as the percentage of CD8-positive lymphocytes among total nucleated cells within each high-power field using ImageJ® software, correcting the previous ambiguity.

The typographical error regarding the number of event-free patients has been corrected (46 patients). The unit for CD163 expression has been standardized as cells/HPF throughout the manuscript.

Units for AMC (cells/µL), CD163 (cells/HPF), and CD8 (%) have been added in Table 1 to improve clarity.

The figure legend has been corrected to accurately reflect CD8 expression levels.

Extranodal involvement and the median event-free survival time (13 months) are now explicitly reported in the Results section to avoid introducing new findings in the Discussion.

The rationale for selecting two-year event-free survival (EFS24) as the primary endpoint has been clarified, emphasizing its established relevance as a clinically meaningful surrogate marker in DLBCL.

We greatly appreciate the reviewer’s thoughtful comments, which have strengthened the overall quality and transparency of the manuscript.
We sincerely thank the reviewer for the constructive feedback, which has significantly improved the clarity and methodological transparency of our manuscript. The revisions are summarized below:

The title has been revised to explicitly specify CD163⁺ macrophages and CD8⁺ lymphocytes to accurately reflect the immune subsets evaluated. Corresponding changes have been made in the Abstract for consistency.

The Ethics section has been clarified to state that written informed consent was obtained from surviving patients whenever feasible, while consent for deceased patients was waived in accordance with ethics committee approval.

The immunohistochemical quantification method has been revised to clarify that CD8 expression was measured as the percentage of CD8-positive lymphocytes among total nucleated cells within each high-power field using ImageJ® software, correcting the previous ambiguity.

The typographical error regarding the number of event-free patients has been corrected (46 patients). The unit for CD163 expression has been standardized as cells/HPF throughout the manuscript.

Units for AMC (cells/µL), CD163 (cells/HPF), and CD8 (%) have been added in Table 1 to improve clarity.

The figure legend has been corrected to accurately reflect CD8 expression levels.

Extranodal involvement and the median event-free survival time (13 months) are now explicitly reported in the Results section to avoid introducing new findings in the Discussion.

The rationale for selecting two-year event-free survival (EFS24) as the primary endpoint has been clarified, emphasizing its established relevance as a clinically meaningful surrogate marker in DLBCL.

We greatly appreciate the reviewer’s thoughtful comments, which have strengthened the overall quality and transparency of the manuscript.
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Jenifer Vaughan, University of the Witwatersrand J, Johannesburg, South Africa
Rafael Pichardo-Rodriguez, Instituto de Investigaciones en Ciencias Biomédicas (INICIB), Universidad Ricardo Palma, Lima, Peru
Teng Fei, Memorial Sloan Kettering Cancer Center, New York, USA

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10 Views

06 Apr 2026 | for Version 2

Teng Fei, Memorial Sloan Kettering Cancer Center, New York, USA

10 Views Cite this report Responses(0)

Not Approved

Please justify why it was 2-year EFS that was used to determine the thresholds for the markers of interest.
Please use the wording of “univariable” and “multivariable” regression models, rather than “univariate” and “multivariate”. This would comply with the reporting standard.
Some approaches such as maxstat and time-dependent ROC were mentioned in the method section but not applied for the analyses. Please move the corresponding description to the discussion section as future directions.
Table 1: the title for the second column (Total (%)) is not applicable for the continuous variables.
Figure 4: please make the two panels the same size with the same scale for the x-axis.
Table 2 is confusing. First, it is unclear whether it reports univariable models or multivariable models. Second, it is untypical to report contingency tables for survival models. After checking the sum of numbers from cells of contingency tables, I suppose the results are univariable (108 patients with AMC, but only 99 with ICI). It is then questionable why univariable results, rather than the multivariable ones, are reported to support the main conclusions of this manuscript.
It is stated in the method section that the median follow-up was 20 months. However, no censored observations were reflected in Kaplan-Meier curves from Figure 5, which is contradictory. In addition, there appears to be a strong immortal bias in the population, where all patients survived beyond 6 months, which might be the case but is generally a red flag in statistical review.
Event times are undefined for censored patients in the shared data. I assume the authors used administrative censoring at the end of 2 years, but that would contradict the median follow-up of 20 months.

Is the work clearly and accurately presented and does it cite the current literature?

Partly
Is the study design appropriate and is the work technically sound?

No
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

No
Are all the source data underlying the results available to ensure full reproducibility?

No
Are the conclusions drawn adequately supported by the results?

No

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Biostatistics

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3 Views

30 Mar 2026 | for Version 2

Rafael Pichardo-Rodriguez, Instituto de Investigaciones en Ciencias Biomédicas (INICIB), Universidad Ricardo Palma, Lima, Peru

3 Views Cite this report Responses(0)

Approved

I have no further comments; I congratulate the authors on their effort.

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Hemato-Oncology, Lymphoma.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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17 Views

20 Nov 2025 | for Version 1

Rafael Pichardo-Rodriguez, Instituto de Investigaciones en Ciencias Biomédicas (INICIB), Universidad Ricardo Palma, Lima, Peru

17 Views Cite this report Responses(1)

Approved With Reservations

Operational definition of EFS
The Methods section does not specify the operational definition of event-free survival (EFS). While EFS is an increasingly used endpoint in lymphoma, its interpretation requires caution since it is a heterogeneous surrogate outcome. I recommend that the authors clearly define EFS and consider, in subsequent analyses, a competing-risk approach to differentiate disease progression/recurrence from non–disease-related mortality.
Estimation and reporting of median follow-up
The manuscript does not describe how the median follow-up time was estimated, nor does it report the actual value. This parameter is essential to determine whether the observation period was long enough to capture meaningful clinical events. The authors should specify the method used (e.g., reverse Kaplan–Meier) and include the corresponding median follow-up.
Flow diagram (CONSORT/REMARK)
A patient flow diagram is not provided. Such a diagram is crucial to visualize the selection process, exclusions, and losses to follow-up. The authors should include a flowchart following the CONSORT or STROBE guidelines as appropriate.
Definition of follow-up period and time zero
The starting point (time zero) for follow-up is not defined. Clarifying whether this corresponds to the date of diagnosis, initiation of treatment, or completion of therapy is critical for reproducibility and for appropriate interpretation of time-to-event analyses.
Handling of missing data
The manuscript does not mention whether missing data were present or how they were handled. The authors should state whether they performed data imputation, complete-case analysis, or any other strategy, as this can influence the validity of their findings.
Exclusion criteria
The exclusion of “indolent lymphomas” is mentioned, yet this group would not typically be included in a cohort restricted to DLBCL. Instead, it would be more relevant to specify whether transformed DLBCL cases (e.g., from CLL or follicular lymphoma) were excluded, since these represent a distinct biological entity.
Determination of cut-off values for continuous biomarkers
There is no standardized methodology for defining cut-off points of continuous biomarkers in DLBCL. For time-dependent outcomes, I recommend using Maximally Selected Rank Statistics with the maxstat package in R to determine optimal thresholds, although this is left to the authors’ discretion.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

No
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Partly
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

No

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Hemato-Oncology, Lymphoma.

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Author Response

12 Mar 2026

Faisal Syarifuddin, Division of Hematology and Medical Oncology, Department of Internal Medicine, Faculty of Medicine, Universitas Indonesia, Rumah Sakit Dr Cipto Mangunkusumo, Central Jakarta, 10430, Indonesia

We sincerely thank the reviewer for the thorough and insightful methodological evaluation of our manuscript. We greatly appreciate the constructive recommendations, which have substantially improved the clarity, transparency, and rigor of our study. The revisions are summarized below:

The operational definition of event-free survival (EFS) has now been explicitly defined in the Methods section as the time from initiation of first-cycle R-CHOP chemotherapy to the first occurrence of disease progression, relapse after response, initiation of second-line therapy, or death from any cause. We also clarified the rationale for selecting two-year EFS as a clinically meaningful endpoint based on established literature. Additionally, we have acknowledged in the Discussion that future studies may benefit from competing-risk modeling to distinguish disease-specific progression from non–disease-related mortality.
The median follow-up duration is now reported (20 months), estimated using the reverse Kaplan–Meier method. The method used for estimation has been explicitly stated in the Statistical Analysis section.
A patient flow diagram has been added (Figure 1) to transparently illustrate patient identification, exclusions, and final cohort assembly, in alignment with STROBE/REMARK recommendations.
The starting point (time zero) for follow-up has been clearly defined as the initiation date of first-cycle R-CHOP chemotherapy to ensure reproducibility and appropriate interpretation of time-to-event analyses.
Handling of missing data has now been explicitly described. A complete-case analysis approach was used, and no data imputation was performed. The extent of missing LDH data is now reported in the Results section.
The exclusion criteria have been clarified to explicitly state that transformed DLBCL cases (e.g., transformation from follicular lymphoma or chronic lymphocytic leukemia) were excluded, as these represent biologically distinct entities.
Regarding the determination of cut-off values for continuous biomarkers, we have clarified that receiver operating characteristic (ROC) analysis based on two-year EFS was selected to provide clinically interpretable thresholds while minimizing model instability in a retrospective cohort of limited sample size. We have acknowledged in the Statistical Analysis and Discussion sections that alternative approaches, such as maximally selected rank statistics, may be considered in future prospective validation studies.
The limitations section has been expanded to acknowledge the single-center design, potential optimism bias associated with ROC-derived thresholds, and the need for external validation in independent cohorts.

We are grateful for the reviewer’s careful assessment and believe that these revisions have substantially strengthened the methodological transparency and overall quality of the manuscript.

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9 Views

12 Nov 2025 | for Version 1

Jenifer Vaughan, University of the Witwatersrand J, Johannesburg, Gauteng, South Africa

9 Views Cite this report Responses(1)

Approved

The title is somewhat misleading, as the study focused on specific subsets of macrophages and lymphocytes, and not on all macrophages and lymphocytes. It would be better to specify that CD163+ macrophages and CD8+ lymphocytes were evaluated This should be applied in the abstract as well.
It is stated that all of the participants gave written informed consent, but this was a retrospective study, and some of the participants had demised before the study commenced. Please clarify how this was possible.
Under the immunohistochemical staining methods, it is stated that CD8 was reported as a percentage of stained T-cells. How were all the T-cells identified if only a CD8 stain was used?
There is an error in the 1^st paragraph of the Results, where it is stated that four patients remained event free (it should be 46 patients according to Table 1).
In the following line of the same paragraph, there is an error in the number of numbers of CD163 positive cells (reported as a percentage and not cells/HPF).
In Table 1, units should be included for the numbers provided for the AMC, CD8 and CD163.
There is an error in the legend for Figure 2; it is stated that (d) shows low CD8 expression, but the image appears to show high CD8 expression?
Why was 2 year EFS looked at and not the more overall meaningful survival statistic of overall survival?
In the 2^nd paragraph of the Discussion, it is stated that Extranodal involvement was seen in 27% of patients. This information is not included in the Results section?
Similarly, in the 4^th paragraph of the Discussion, it is stated that the median survival time was 13 months, but this too was not included in the Results section. It is not a good practice to introduce new results in the Discussion.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Tumour microenvironement of DLBCL, HIV-associated lymphoma

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Respond to this report

Responses (1)

Author Response

12 Mar 2026

Faisal Syarifuddin, Division of Hematology and Medical Oncology, Department of Internal Medicine, Faculty of Medicine, Universitas Indonesia, Rumah Sakit Dr Cipto Mangunkusumo, Central Jakarta, 10430, Indonesia

We sincerely thank the reviewer for the constructive feedback, which has significantly improved the clarity and methodological transparency of our manuscript. The revisions are summarized below:

The title has been revised to explicitly specify CD163⁺ macrophages and CD8⁺ lymphocytes to accurately reflect the immune subsets evaluated. Corresponding changes have been made in the Abstract for consistency.
The Ethics section has been clarified to state that written informed consent was obtained from surviving patients whenever feasible, while consent for deceased patients was waived in accordance with ethics committee approval.
The immunohistochemical quantification method has been revised to clarify that CD8 expression was measured as the percentage of CD8-positive lymphocytes among total nucleated cells within each high-power field using ImageJ® software, correcting the previous ambiguity.
The typographical error regarding the number of event-free patients has been corrected (46 patients). The unit for CD163 expression has been standardized as cells/HPF throughout the manuscript.
Units for AMC (cells/µL), CD163 (cells/HPF), and CD8 (%) have been added in Table 1 to improve clarity.
The figure legend has been corrected to accurately reflect CD8 expression levels.
Extranodal involvement and the median event-free survival time (13 months) are now explicitly reported in the Results section to avoid introducing new findings in the Discussion.
The rationale for selecting two-year event-free survival (EFS24) as the primary endpoint has been clarified, emphasizing its established relevance as a clinically meaningful surrogate marker in DLBCL.

We greatly appreciate the reviewer’s thoughtful comments, which have strengthened the overall quality and transparency of the manuscript.

View more View less

Competing Interests

I have no competing interests

Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

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Prognostic Role of Absolute Monocyte Count, CD163⁺ Macrophages, and CD8⁺ Lymphocytes in Diffuse Large B-Cell Lymphoma Treated with R-CHOP

Abstract

Background

Methods

Results

Conclusion

Keywords

Revised Amendments from Version 1

Background

Methods

Study design and setting

Ethics statement

Patient selection

Figure 1. Study flow diagram of patient selection and follow-up.

Peripheral blood analysis

Immunohistochemical staining

Event-free survival (EFS)

Statistical analysis

Results

Patient characteristics

Table 1. Demographic and clinical characteristics of study subjects.

Immunohistological characteristics and optimal cutoff values for AMC, CD163, and CD8

Figure 2. Quantitative ROC curves for 2-year Event-Free Survival: (a) AMC, (b) TAM CD163, and (c) TIL CD8.

Figure 3. CD163 and CD8 expression in DLBCL tissues (100×): (a) Low CD163 expression, (b) High CD163 expression, (c) High CD8 expression, (d) Low CD8 expression.

Correlation between AMC and CD163 or CD8 expression

Figure 4. Correlation between (a) AMC and TAM CD163, and (b) AMC and TIL CD8.

Impact of AMC, CD163, and CD8 on 2-Year EFS

Table 2. The relationship of different variables on patient survival.

Figure 5. Kaplan-Meier survival curves for DLBCL patients based on (a) AMC, (b) TAM CD163, (c) TIL CD8, (d) COO, and (e) IPI score.

Discussion

Conclusions

Authors’ declaration

Data availabilty

References

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