Clinicopathological features of adult T‐cell leukemia/lymphoma with T‐follicular helper phenotype

Abstract Aims T‐follicular helper (TFH) cells are effector T‐cells that are crucial for B‐cell selection and differentiation. T‐cell lymphomas derived from TFH cells have distinct characteristics. Additionally, in the World Health Organization (WHO) classification 5th edition, three lymphomas were introduced as independent disease entities with TFH cell origin. We aimed to investigate the clinicopathological features of adult T‐cell leukemia/lymphoma (ATLL) with a TFH phenotype (TFHP). Methods and Results We performed TFH immunohistochemistry analysis of five biomarkers for the biopsy specimen, with TFHP being indicated by a positive result for more than two markers. Among 75 cases of ATLL, 37.3% of them showed TFHP. Compared with cases of ATLL without TFHP, cases of ATLL with TFHP showed higher C‐reactive protein levels (p = 0.0219) and increased high endothelial venule proliferation (p = 0.024). However, there were no significant between‐group differences in overall survival as well as other clinical and morphological findings. Furthermore, there was no significant between‐group difference in TFH markers and FOXP3 expression. Conclusion Some patients with ATLL may present a TFHP, which should not preclude the diagnosis of ATLL. Although presenting a TFHP does not affect prognosis, it is important to identify cases of ATLL with a TFHP since it may inform future treatment strategies.

The former two variants have a poor prognosis, 2,3 with 4-year survival rates of 16.8% and 19.6%, respectively. 2lthough the latter two variants have a relatively good prognosis, approximately 25% of the chronic and smoldering variants transform into leukemia after a long duration. 2,3 follicular helper (TFH) cells are a subset of effector T-cells that are crucially involved in B-cell selection and differentiation to plasma cells and memory B-cells.Furthermore, these cells express anti-programmed cell death protein 1 (PD-1), inducible T-cell co-stimulator (ICOS), CD10, B-cell lymphoma (BCL) 6, chemokine (C-X-C motif) ligand 13 (CXCL13), and CD4. 4 The 2017 WHO classification introduced a new independent subtype of PTCL termed nodal PTCL with TFH phenotype (nPTCL with TFHP), and this category is essentially followed by the WHO 5th edition: nodal TFH cell lymphoma, not otherwise specified (nTFHL-NOS).1 This subtype is characterized by lymphoma cells with a TFHP; however, it is difficult to morphologically distinguish it from PTCL and NOS.Genetic studies have reported mutations in nTFHL-NOS, including in Ten-Eleven Translocation 2, Ras homolog family member A (RHOA), and DNAmethyltransferase 3 alpha.1 Although the role of RHOA remains unknown, its expression in CD4 + T cells induces TFH cell specification, including increased proliferation related to ICOS upregulation.5 RHOA mutations have also been found in 15% of ATLL cases.6 The histological characteristics of ATLL range from small atypical lymphocytes to large ones; accordingly, there remain no established findings for ATLL.7 Therefore, measurement of serum HTLV-1 antibody levels is important in the diagnosis of ATLL.In clinical practice, serum HTLV-1 antibodies are often not examined during histological examination.Therefore, pathologists may encounter cases that were initially diagnosed as nTFHL-NOS but were later shown to be ATLL.However, the characteristics of ATLL with TFHP remain unclear.
We aimed to review the clinicopathological characteristics of ATLL with TFHP.

| Tissue samples
We studied 75 patients diagnosed with ATLL between 2006 and 2012 at the Department of Pathology, Kurume University.][10] We excluded cases of recurrence.This study was approved by the Research Ethics Committee of Kurume University and conducted following the Declaration of Helsinki.

| Morphology and immunohistochemistry
We morphologically examined the paraffin sections of each sample.Lymphoma cells were identified based on their morphological characteristics, including size and variation of the atypical lymphocyte, nucleus, and nucleoli, nuclear shape irregularity, and hyperchromatism with a coarse and irregular distribution.Additionally, morphological variants, including small-, medium-, large-, and anaplastic-cell variants; pleomorphic variants; resembling angioimmunoblastic T-cell lymphoma (AITL: In the WHO 5th edition, the name was changed to nTFHLangioimmunoblastic type (AI), but the definition is different from AITL, so the conventional name AITL is used in the part that introduces previous research.)variants; and Hodgkin-like variants, were classified based on previously reported histological features. 8,11,12For each specimen, the number of eosinophils and high endothelial venules (HEVs) was evaluated at high magnification in three fields at hot spots.

| Statistical analysis
Statistical analyses were conducted using JMP Pro 16 software.Statistical significance was set at p values < 0.05.Overall survival (OS) was defined as the duration from the date of diagnosis to the date of death or last followup.The Kaplan-Meier method was used to estimate the distribution of OS, which was analyzed using the log-rank test.Between-group comparisons of clinicopathological characteristics were performed using the Chi-square test, Fisher's two-sided exact test, or the Mann-Whitney U test.

| OS in ATLL with TFHP
There was no significant difference in OS between cases of ATLL with TFHP (median survival time: 249 days, mean survival days: 582.8 days) and without TFHP (median survival time: 399.5 days, mean survival days: 703.2 days; p = 0.6574; Figure 2).

| Pathological characteristics of ATLL with TFHP
We performed between-group comparisons of pathological characteristics (Tables 2 and 3).Among cases of ATLL with TFHP, there were five (

| DISCUSSION
T-follicular helper cells are a subset of T-cells whose function has been examined since the early 2000s.In nonneoplastic lymph nodes, TFH cells are localized in the germinal center of the lymph follicle and are crucially involved in selecting B-cells, inducing antibody production, directing B-cell differentiation, and class switching of Bcells. 4Since the discovery of TFH cells, there have been reported cases of PTCLs with phenotypic features of TFH.nTFHL-AI, nTFHL-follicular type, and nTFHL-NOS have been listed as lymphomas with a TFH cell origin according to the WHO classification of tumors of hematopoietic and lymphoid tissue, 5th edition. 1 Although the precise role of RHOA remains unknown, expression of RHOA in CD4 + T cells induces TFH cell specification including increased proliferation related to ICOS upregulation. 5dult T-cell leukemia/lymphoma is generally considered to have a poor prognosis, and it has widely varying histological characteristics.There are cases that are composed of small-sized atypical lymphocytes to composed of large-sized ones.Additionally, some cases of ATLL are similar to the AITL, anaplastic large cell lymphoma, and Hodgkin lymphoma.The diagnostic clue for ATLL is confirming the positivity of serum HTLV-1 antibody. 1,7,11,12ecently, it has been reported that some ATLL cases are positive for TFH markers, but no study has examined their comprehensive clinical characteristics. 13In this study, in addition to the pathological analysis of ATLL with TFHP, clinical characteristics and prognosis were also examined.
In both groups, the most common morphological variant was the large-cell variant, followed by the pleomorphic and medium-cell variants.These three variants accounted for 96.5% and 91.5% of cases of ATLL with and without TFHP, respectively.Accordingly, we observed no betweengroup difference in the tumor morphology.
Regarding stromal cells in AITL, which is the most studied tumor derived from TFH cells, various cytokines are secreted.Specifically, angiopoietin and vascular endothelial growth factor are secreted to induce proliferation, while interleukin (IL)-5 is secreted to F I G U R E 2 Overall survival (OS) in adult T-cell lymphoma/ leukemia with T-follicular helper phenotype (TFHP).OS was compared between TFHP cases and non-TFHP cases, but no significant difference was observed (p = 0.6574).
promote inflammation, including eosinophil proliferation. 1 Accordingly, we assessed HEV proliferation and the number of eosinophils, which are representative infiltrative inflammatory cells.There was significantly higher HEV proliferation in ATLL with TFHP than in ATLL without TFHP.There was no significant betweengroup difference in the eosinophil counts.Consistent with our findings, a previous study reported no  difference in eosinophil infiltration between AITL and PTCL unspecified (currently known as PTCL, NOS). 14hese histological features were generally the same as those of the previously reported study. 13egarding FOXP3, the normal counterpart cell of ATLL is considered to be CD4 + CD25 + FOXP3 + T regulatory cells (Treg), and these cells are of a different lineage from TFH cells.While FOXP3 is considered a key molecule in Treg cells, 15 previous studies have reported that ATLL do not always express FOXP3. 8Additionally, RHOA mutations, which are important for differentiation into T-follicular helper cells, have been reported in approximately 15% of ATLL cases. 5,6Therefore, it was hypothesized that part of ATLL might be TFH cells.If the expression of TFH markers and the expression of FOXP3 are inversely correlated, it suggests the possibility that a subset of ATLL is of TFH cell origin.However, we did not observe a relationship between the expression of TFH markers and FOXP3.
Regarding clinical presentation, cases of ATLL with TFHP showed increased CRP levels and a tendency to develop hypercalcemia.Regarding prognosis, there was no significant between-group difference in OS.We observed no significant between-group differences in other clinical parameters.Although ATLL with TFHP showed similar immunohistochemical characteristics, including TFH markers, as AITL, we observed no between-group differences in the presence of hepatosplenomegaly, skin rash, and pleural effusion, which are the clinical characteristics of nTFHL-AI.nTFHL-AI involves immune activation and may be related to autoimmune disorders, including the presence of autoantibodies, hemolytic anemia, and hypergammaglobulinemia; however, we observed no significant between-group difference. 16ontrastingly, we observed a significant between-group difference in CRP levels.CRP is an acute-phase protein produced in the liver, with its synthesis being induced by cytokines such as IL1β, IL6, and tumor necrosis factor (TNF).Lymphoma cells in AITL have been shown to produce TNFα and IL6. 14,17Furthermore, lymphoma cells in ATLL with TFHP may produce these inflammatory cytokines; however, this was not investigated in this study since it deviated from the study objective.Although a previous study reported that the prognosis is worse with higher CRP levels, the prognosis of the TFHP group with high CRP levels was not significantly different from that without TFHP in our study. 18This may be due to the number of cases in this study not statistically sufficient.It is hoped that similar cases will be accumulated and re-examined in a larger cohort.
There has been recent progress in the stratification of treatment for PTCL.Identified effective drugs include 5-azacytidine and romidepsin, which have shown a higher overall response rate and CR rate in lymphoma with TFHP than without. 19Therefore, the treatment of ATLL should be stratified according to the presence of the TFHP.

| CONCLUSION
Adult T-cell leukemia/lymphoma may present with TFH markers, which does not preclude the diagnosis of ATLL but does not affect the prognosis.However, it may be important to stratify treatment interventions for ATLL according to the presentation of TFHP.

Figure 3
Figure3shows the number of positive TFH markers and their FOXP3 expression rates in lymphoma cells.The vertical axis shows the percentage of FOXP3-positive cells among the lymphoma cells, while the horizontal axis shows the number of positive TFH markers.There was no relationship between the expression of TFH markers and FOXP3.

T A B L E 2 T A B L E 3 F I G U R E 3
Abbreviations: HEV, high endothelial venule; HPF, high power field.
Clinical characteristics of ATLL patients in the present study.