Clinicopathologic analysis of nodal T-follicular helper cell lymphomas, a multicenter retrospective study from China

Background Nodal T-follicular helper cell lymphomas (nTFHLs) represent a new family of peripheral T-cell lymphomas (PTCLs), and comparative studies of their constituents are rare. Methods This study retrospectively enrolled 10 patients with nTFHL-F and 30 patients with nTFHL-NOS diagnosed between December 2017 and October 2023 at six large comprehensive tertiary hospitals; 188 patients with nTFHL-AI were diagnosed during the same period at the First Affiliated Hospital of Zhengzhou University for comparison. Results Compared with nTFHL-AI, nTFHL-NOS patients exhibited better clinical manifestations, lower TFH expression levels, and a lower Ki-67 index. However, no differences in clinicopathological features were observed between nTFHL-F and nTFHL-AI patients as well as nTFHL-NOS patients. According to the survival analysis, the median OS for patients with nTFHL-NOS, nTFHL-AI, and nTFHL-F were 14.2 months, 10 months, and 5 months, respectively, whereas the median TTP were 14 months, 5 months, and 3 months, respectively. Statistical analysis revealed differences in TTP among the three subtypes(P=0.0173). Among the population of patients receiving CHOP-like induction therapy, there were significant differences in the OS and TTP among the nTFHL-NOS, nTFHL-AI, and nTFHL-F patients (P=0.0134, P=0.0205). Both the GDPT and C-PET regimens significantly improved the ORR, OS, and PFS in nTFHL patients. Conclusion There are significant differences in the clinical manifestations, pathology, and survival outcomes among the three subtypes of nTFHLs. However, further research with a larger sample size, and involving clinical pathology and molecular genetics is needed to determine the distinctive biological characteristics of these tumors.


Introduction
Follicular helper T (TFH) cells are a subset of CD4-positive T cells crucial for germinal center (GC) formation, high-affinity antibody production, and memory B-cell development (1).According to the 2016 revision of the World Health Organization (WHO) classification of lymphoid neoplasms, TFH-originated nodal peripheral T-cell lymphomas (nPTCLs) were classified based on the expression of at least two or three TFH markers, such as CD279/PD1, CD10, BCL-6, CXCL13, and ICOS.These tumors were categorized as angioimmunoblastic T-cell lymphoma (AITL), follicular peripheral T-cell lymphoma (F-PTCL), or nodal peripheral T-cell lymphoma with TFH phenotype (nPTCL-TFH) (2).Studies have shown that AITL, F-PTCL, and nPTCL-TFH express the same TFH-related antigens and exhibit similar molecular abnormalities (3)(4)(5)(6).Furthermore, gene mutations commonly associated with AITL, such as RHOA, TET2, and DNMT3A, have also been identified in patients with F-PTCL and nPTCL-TFH (7)(8)(9).Currently, the classification of this family is based mainly on histopathological features.According to the 5th edition of the WHO Classification of Hematolymphoid Tumors: Lymphoid Neoplasms, nTFHLs were renamed the nTFHL angioimmunoblastic type (nTFHL-AI), nTFHL follicular type (nTFHL-F), and nTFHL not otherwise specified (nTFHL-NOS) (10).However, because of the rarity of nTFHL-F and nTFHL-NOS, research comparing the clinical and pathological features of the three subtypes of nTFHLs is limited.In this study, we analyzed the pathological and clinical features of the three subtypes of nTFHLs.

Study population
The data from patients with nTFHL-F or nTFHL-NOS were obtained from six large comprehensive tertiary hospitals in China spanning from December 2017 to October 2023 (Supplementary Table S1 for participating institutions).The clinical data are summarized in Table 1.Patients with nTFHL-AI diagnosed at the First Affiliated Hospital of Zhengzhou University during the same period were also included for comparison.The follow-up cutoff date was October 30, 2023.The study was conducted in accordance with the Declaration of Helsinki and approved by the ethics review board.

Histopathology and immunohistochemistry
Expert hematopathologists from the pathology department of each participating center reviewed the pathological specimens, including slides stained with hematoxylin-eosin (H&E), immunohistochemistry, and in situ hybridization for Epstein-Barr virus encoded RNA (EBER-ISH), at the time of enrollment to confirm the diagnosis.The diagnosis of nTFHL-AI was defined as the destruction of lymph nodal structure accompanied by dendritic hyperplasia of high endothelial venules (HEVs), the proliferation and expansion of follicular dendritic cell (FDC) networks, and a polymorphic inflammatory background containing histiocytes, plasma cells, and eosinophils (Figure 1).The diagnosis of nTFHL-F required a follicular growth pattern and FDC meshwork associated with follicles without extrafollicular FDC expansion.The follicles were filled with abnormal T cells expressing TFH markers (Figure 2).The diagnosis of nTFHL-NOS was assigned to CD4+ nPTCL with a TFH phenotype that did not meet the criteria for nTFHL-AI and nTFHL-F.This condition usually refers to the absence of dendritic hyperplasia of HEVs, proliferative expansion of the FDC network, and a complex inflammatory background (Figure 3).Comprehensive diagnostic criteria were applied, incorporating clinical presentation, morphological features, and immunohistochemical findings.Genetic rearrangement testing was performed when diagnostic uncertainty persisted to facilitate a definitive diagnosis (2,(10)(11)(12).
The specimen was fixed in 3.7% neutral formaldehyde, routinely deparaffinized, embedded in paraffin, and cut into 3 mm sections for histological examination using hematoxylin and eosin staining.Immunohistochemical staining was performed using the following antibodies: CD3, CD20, CD5, CD4, CD8, CD10, CD21, BCL-6, CXCL13, PD1, CD30, and Ki-67.The basic information of the antibodies is summarized in the Supplementary Table S2.Positive results were determined as follows: PD-1 exhibited cytoplasmic brown-yellow staining, CD10 showed cytoplasmic/ membranous brown-yellow staining, bcl-6 exhibited nuclear brown-yellow staining, and CXCL-13 exhibited perinuclear punctate brown-yellow granular staining.The positivity for TFH markers was defined as the expression in at least 10% of tumor cells.

EBER detection by in situ hybridization
EBV status was determined by ISH to detect EBV-encoded RNA 1 and 2 (EBER1/2s) using peroxidase-labeled probes ISH-7001UM (Beijing Zhongshan Golden Bridge Biotechnology, Beijing, China).Tissues from an EBV-positive NK/T cell lymphoma  were used as a positive control, and PBS buffer as a negative control in place of primary antibody or hybridization solution.Positive cells (PCs) were defined as the presence of moderate or higher intensity staining (brown/dark brown) in the nucleus.The number of PCs was recorded in high-power fields (HPFs).we defined EBERpositive neoplastic cells per high-power field (HPF) exceeded 50 (> 50/HPF) as EBER(+).Samples with scattered (the number of positive neoplastic cells in the highest density region was less than 50/HPF), inadequate, or equivocal positive neoplastic cells (a cutoff value < 50/HPF) were as EBER (-).

T-cell and B-cell clonality analysis
Polymerase chain reaction (PCR) amplification of T-cell receptor genes and immunoglobulin gene rearrangements were performed for detecting monoclonality when necessary following the BIOMED-2 protocol as described previously (13).DNA was extracted from the QIAamp DNA Mini Kit (QIAGEN, Hamburg, Germany) following the manufacturer's protocol.Commercial BIOMED-2 multiplex PCR kits (Righton Gene, Shanghai, China) were used for PCR amplification of TCR and IG gene rearrangements.After separation by capillary electrophoresis, PCR products were subjected to GeneScan analysis on an ABI 9700Genetic Analyzer (Applied Biosystems, CA, USA) and analyzed using the GeneMapper software (Version 4.0; Applied Biosystems, CA, USA).

Clinical data collection
Clinical data were collected through the use of case report forms.The clinical data included age, sex, B symptoms, Eastern Cooperative Oncology Group (ECOG) performance status, immune inflammatory-related symptoms, Ann Arbor stage, extranodal involvement sites, the International Prognostic Index (IPI), the prognostic index for peripheral T-cell lymphoma unspecified (PIT), treatment methods, treatment response, survival status, and cause of death.The recorded laboratory data included baseline complete blood cell count, albumin level, globulin level, serum lactate dehydrogenase (LDH) level, C-reactive protein (CRP) level, serum immunoglobulin level, Coombs test, and ANA test.Bone marrow involvement was diagnosed through bone marrow biopsy.The involvement of the spleen and other extranodal sites was determined using diagnostic tools such as CT, enhanced CT, and PET-CT.Treatment response was evaluated according to the 2014 Lugano classification criteria (14).The response was categorized as complete remission (CR), partial remission (PR), stable disease (SD), or progressive disease (PD).The objective response rate (ORR) was defined as the proportion of patients who achieved PR and CR.

Statistical analysis
Quantitative data are expressed as either the mean ± standard deviation (SD) or median with interquartile range (IQR).Intergroup comparisons were conducted using Student's t test, the Mann−Whitney U test, or the Kruskal−Wallis test, as appropriate.Categorical data are reported as percentages or proportions, and comparisons were made using either Pearson's chi-square test or Fisher's exact test.Overall survival (OS) was defined as the time interval from diagnosis to either death from any cause or the last follow-up.The time to disease progression (TTP) was defined as the duration from the initiation of treatment to disease progression.Progression-free survival (PFS) was defined as the period from the commencement of treatment to either disease progression or death from any cause.Survival analysis was conducted utilizing the Kaplan−Meier method, and the log-rank test was used to compare survival rates between the two groups.A P value < 0.05 was considered to indicate statistical significance.The statistical analysis was conducted using SPSS software, specifically version 26.0.

Patient characteristics
Table 2 summarizes the baseline characteristics of the entire study population.The nTFHL-NOS patients were younger at disease onset (62.5 vs. 59.0 years, P=0.134) than the nTFHL-AI patients were, and they presented less frequently with ECOG score > 1, B symptoms, rash, and serosal effusion.Laboratory and imaging tests revealed a decreased incidence of anemia, decreased albumin levels, elevated globulin levels, elevated LDH levels, elevated CRP levels, increased polyclonal serum immunoglobulin levels, and advanced disease stage.Most patients demonstrated low to intermediate risk according to both the IPI and the PIT score, and these differences were statistically significant.However, there were no statistically significant differences in clinical presentation, laboratory or imaging findings, staging, IPI score, or PIT score between the nTFHL-F cohort and the nTFHL-AI cohort as well as the nTFHL-NOS cohort.

Immunohistochemistry and in situ hybridization
Similarly, compared with the nTFHL-AI subgroup, the nTFHL-NOS subgroup displayed relatively lower expression levels of TFH markers, particularly for BCL-6, CD10, and CXCL13.Additionally, the nTFHL-NOS subgroup had a lower median Ki67 index (Table 4).

Discussion
PTCLs encompasse a heterogeneous group of tumors.Recently, as TFH cell has gained increased recognition, a subset of lymphomas originating from nodal TFH cells has been identified within PTCLs.In contrast to other T-cell subsets, TFH cells undergo multistep differentiation to acquire a unique phenotype characterized by the expression of PD-1, BCL-6, CXCL13, CD10, and ICOS (12).TFH cells located in the B-cell zone of lymph nodes interact with B cells in the GC, promoting B-cell growth, antibody formation, and immunoglobulin class switching.nTFHLs exhibit unique histopathological features and clinical symptoms associated with immune dysregulation.A prime example is nTFHL-AI, which manifests with systemic symptoms, including generalized lymphadenopathy, hepatosplenomegaly, rash, polyserositis, and elevated serum globulin levels.However, the survival and prognostic characteristics of nTFHLs are better than those of PTCL, NOS (15,16).Since the 4th edition of the WHO Classification of Hematolymphoid Tumors identified three types of nTFHLs, the rarity of the two new subtypes has led to a scarcity of large-scale studies delineating their biological boundaries.The 5th classification continues to designate these types as provisional subtypes, warranting further research.Our study represents the first and largest dataset analysis directly comparing the three subtypes of nTFHLs (10).
In recent years, studies have demonstrated that, compared with nTFHL-AI, nTFHL-NOS is indicative of a relatively indolent subtype (16).Our findings corroborate this statement, as our study revealed that patients with nTFHL-NOS exhibited milder clinical manifestations, fewer laboratory and imaging abnormalities, less advanced-stage disease, and improved treatment response and  survival.Furthermore, several case reports have substantiated these findings (17)(18)(19)(20)(21).Despite the absence of significant differences in clinical features between nTFHL-AI patients and nTFHL-F patients as well as nTFHL-NOS patients, patients with nTFHL-F exhibited worse survival outcomes, possibly indicating a more unfavorable prognostic subtype.The IPI and PIT scores retain their significance in assessing the prognosis of nTFHLs (22).
Treatment for nTFHLs currently relies on the principles of PTCLs treatment, with CHOP-like regimens being commonly used for induction treatment.However, the response rates and duration of response to CHOP-like regimens are poor, particularly in nTFHL-F patients, for whom only a 29% ORR was observed.Therefore, there is a crucial need to explore new regimens suitable for treating nTFHLs.Advances in molecular biology, epigenetics, and the immunological microenvironment in nTFHLs have led to the study of targeted therapy against nTFHLs' antibodies, TCR signaling pathways, as well as epigenetic mutations, and immune therapy aimed at regulating the immunological microenvironment (23,24).Notably, targeted therapy against epigenetic mutations has shown significant benefits, as observed in various clinical studies.Belinostat combined with CHOP or Chidamide combined with CHOP achieved an ORR of 90% in untreated AITL patients.Azacitidine achieved an ORR of 75% and a median PFS of 15 months in R/R AITL patients.In a multicenter phase II study, Azacitidine and Romidepsin achieved an ORR of 80% (12/15) and a CRR of 60% (9/15) in T-FHCL patients.Our center has also conducted multiple clinical trials (25,26) showing promising results with certain regimens, such as "GDPT" (gemcitabine, cisplatin, prednisone, thalidomide) and "C-PET" (chidamide plus prednisone, etoposide, and thalidomide), which significantly improved disease remission rates and prolonged disease remission duration in nTFHL patients.Furthermore, ongoing research on the new regimen " Das + AZA + CEP " (Dasatinib, Azacitidine, Etoposide, Cyclophosphamide, Prednisone) for AITL treatment and the exploration of other new regimens aim to further enhance the therapeutic efficacy of this disease family.
Compared with nTFHL-AI, nTFHL-NOS exhibited lower expression levels of TFH signal, which is consistent with findings from previous studies (15,16,27).Additionally, further observation revealed that nTFHL-NOS patients had a lower median Ki67 index, indicating decreased proliferative activity.Recent studies have revealed that the predominant gene mutations in nTFHL-AI include RHOA, TET2, DNMT3A, and IDH2 R172 mutations, among others, whereas nTFHL-F and nTFHL-NOS exhibit distinct mutation rates.IDH2 R172 , in particular, appears to be exclusive to nTFHL-AI (5,(27)(28)(29).Furthermore, studies have demonstrated a correlation between IDH2 and the tumor microenvironment (TME) in patients with nTFHL-AI (7,30), suggesting a role for epigenetic mutations in the variation of clinicopathological features.Similarly, nTFHL-F is characterized by distinct ITK-SYK fusion genes (31) known to induce malignant PTCL (32).However, due to limitations in the available research data, further investigations of molecular genetics were not conducted.Future research should prioritize the enhancement of molecular and epigenetic investigations of the three subtypes to reveal their underlying pathology and clinical characteristics in greater detail.
Ultimately, notable distinctions exist in terms of clinical manifestations, pathology, and survival outcomes among the three types of nTFHLs.To gain a deeper understanding of the pathological basis and clinical characteristics of the three types of tumors, additional case studies and molecular genetic confirmation are needed.

TABLE 1
Characteristics of nTFHL-F and nTFHL-NOS patients.

TABLE 2
Characteristics of all patients.

TABLE 4
IHC and EBER-ISH characteristics of all patients.

TABLE 3
ORR of all patients' induction therapy.