Clinicopathological findings, prognosis, and Epstein–Barr virus infection in rheumatoid arthritis patients with other iatrogenic immunodeficiency-associated T- and NK-cell lymphoproliferative disorders

Background Other iatrogenic immunodeficiency-associated (OIIA) T- and natural killer (NK)-cell lymphoproliferative disorders (TNK-LPDs) are rare in patients with rheumatoid arthritis (RA). Methods We investigated the clinicopathological characteristics, Epstein–Barr virus (EBV) infection, genetic findings, therapeutic response, and prognostic factors in 21 RA patients with OIIA TNK-LPDs and compared these with those of 39 with OIIA B-cell LPDs (B-LPDs) and 22 with non-OIIA B-LPDs. Results Immunohistologically, 11 patients (52%) showed CD4+ T-LPDs, and 7 had a T follicular helper (TFH) phenotype. The other nine patients (43%) showed CD8+ T-LPDs, and the remaining one (5%) had features of CD3+ CD4− CD8− nasal type TNK-cell lymphoma. CD30+, p53+, and CMYC+ atypical lymphocytes were identified in seven (33%), eight (38%), and five (24%) patients, respectively. In situ hybridisation detected EBV-encoded RNA (EBER) + large atypical lymphocytes in five patients (24%). Nine of 17 patients (53%) showed clonal peaks of TCRγ by polymerase chain reaction. Withdrawal of MTX and biologic drugs was effective in 12 patients (57%), and 8 (38%) received chemotherapies. Two patients with TFH+ or EBV+ CD4+ CD30+ large cell peripheral T-cell lymphoma, one with CD8+ systemic anaplastic large cell lymphoma, and two with systemic EBV+ CD8+ T-cell lymphoma of childhood showed a lethal progressive clinical course within 13 months. Moreover, > 500 U/L LDH, large atypical lymphocytes, expression of CD30, p53, and CMYC, and EBER+ atypical lymphocytes were significantly poor prognostic factors for overall survival (p < 0.05). Median interval from RA onset to OIIA TNK-LPDs was 72 months, which was shorter than 166 months in OIIA B-LPDs (p = 0.003). EBV+ atypical and reactive lymphocytes were frequently found in 15 patients with OIIA TNK-LPDs (71%), in 27 with OIIA B-LPDs (69%), and only in 3 with non-OIIA B-LPDs (14%). Conclusions OIIA TNK-LPDs occurred in early phase of RA, compared with OIIA B-LPDs, and occasionally showed a lethal progressive clinical course. Detection of OIIA TNK-LPD patients with poor prognostic factors is necessary. EBV infection in immunosuppressed patients due to persistent RA, MTX, and biologic drugs may play a role in forming the tumour microenvironment and lymphomagenesis of TNK-LPDs. Supplementary Information The online version contains supplementary material available at 10.1186/s12885-022-10358-0.

In the current study, we subclassified 21 RA patients with OIIA TNK-LPDs into CD4+, CD8+, and CD4− CD8− phenotypes, and identified characteristic histological and immunohistological findings to EBV infection of atypical lymphocytes and scattered lymphocytes in the background, genetic findings, and prognostic factors, which were compared with those of OIIA and non-OIIA B-LPDs. We advocated detection of TNK-LPDs with poor prognostic factors including EBER+ atypical lymphocytes, and examined the etiological role of EBV infection in RA patients with OIIA TNK-LPDs.

Patient selection and clinical findings
Twelve thousand three hundred registered patients with LPDs or lymphomas were reviewed and retrieved retrospectively over the period of 1990 to 2020 at the Department of Pathology, Fukuoka University. Histological classification was performed according to the World Health Organization classification (2017) [1]. We retrospectively reviewed the medical records of the patients who met all the inclusion criteria. The inclusion criteria included patients who met the American College of Rheumatology classification criteria for RA; we selected RA patients who developed LPDs. OIIA LPDs were defined as LPDs occurring in RA patients treated with low-dose MTX, TNF inhibitors, or other biologic drugs [1,9]. Twenty-one RA patients with OIIA TNK-LPDs, 39 RA patients with OIIA B-LPDs, and 22 RA with non-OIIA B-LPDs were analysed. Human immunodeficiency virus (HIV)-positive patients and human T-lymphotropic virus (HTLV)-1-positive carriers were not included in the current study. Corresponding medical records were reviewed to obtain clinical information, including Ann Arbor stage, treatment, and survival. The criteria of LPD regression were if LPDs in RA patients had shrunk or improved following 4-week withdrawal of MTX, biologic drugs, and immunosuppressants, according to the systemic findings including laboratory data and radiological images, and did not require further treatment with chemotherapy [14]. Diagnosis of hemophagocytic lymphohistiocytosis (HLH) was made using the revised HLH-2004 criteria [15].

Histology, immunohistology, and detection of EBV
Excised tissue specimens were fixed in 10% formalin to generate formalin-fixed paraffin-embedded (FFPE) samples that were stained with haematoxylin and eosin. For immunohistochemistry, antibodies were applied to the tumour samples using a Leica Bond III-automated stainer (Leica Biosystems, Buffalo Grove, IL, USA), and peroxidase reactions were developed using diaminobenzidine. These antibodies are listed in Table S1. Criteria of small, medium, and large atypical cell sizes were compliant with those of mantle cells, centrocytes, and centroblasts, respectively, in lymphoid follicles. Largecell TNK-LPDs were characterised by ≥50% large lymphoid cells with distinct nucleoli. Small-cell TNK-LPDs consisted of predominantly small-or medium-sized atypical lymphocytes. For all immunostaining, reactions were considered positive when ≥30% of atypical lymphocytes were positively stained. The TFH phenotype was defined by the expression of at least two of the following five antibodies: PD-1, inducible T-cell co-stimulator (ICOS), BCL6, C-X-C motif chemokine ligand (CXCL)13, and CD10 [16]. Assessments of CMYC+, p53+, or programmed cell death-ligand (PD-L) 1+ atypical lymphocytes and the number of PD-L1+ non-neoplastic cells were performed according to the methods reported in our previous study [17]. The presence of EBV infection was determined by in situ hybridisation of EBER+ nuclear signals in ≥50% of atypical lymphoid cells. For EBER staining, tissue sections were hybridised in a solution of 50% formamide containing fluorescein isothiocyanate-labelled EBER oligonucleotides (Leica). Double-staining of lymphocyte markers and EBER was performed to confirm the presence of EBV+ T or B cells.

Detection of RHOA G17V mutation by Sanger sequencing
PCR was performed with AmpliTaq gold (Thermo Fisher Scientific, Waltham, MA, USA) using 40 ng genomic DNA, 0.3 μM primers, and 2 μL AmpliTaq gold master mix. A PCR-amplified product of 244 bp, including the codon for the 17th amino acid, was obtained in 12 TLPD patients, and direct sequencing of these products was performed. The coding DNA position 50G > T mutation of the RHOA gene predicted the change of the wild-type G (Gly) to the mutant type V (Val) [19].

Statistical analysis
The clinicopathological features of patients with OIIA TNK-LPDs, OIIA B-LPDs, and non-OIIA B-LPDs were compared with Fisher's exact test or χ 2 test. Medians were compared with Wilcoxon rank sum test. For the RA patients with LPDs, progression-free survival (PFS) was calculated from the initial diagnosis date to the first date of disease progression or relapse, and overall survival (OS) was calculated from the initial time of diagnosis to the date of last follow-up or death. PFS and OS curves were generated using the Kaplan-Meier method and analysed by the proportional hazards model. A p-value of < 0.05 was considered statistically significant. Statistical analyses were performed using JMP 10 software (SAS Institute, Cary, NC, USA).

Discussion
Seven of our examined 21 RA patients (33%) had OIIA CD4+ TFH+ T-LPDs, and 9 (43%) had complicated CD8+ T-LPDs. EBER+ atypical lymphocytes, scattered EBER+ small lymphocytes, and some large B lymphocytes were detected in six of seven patients with CD4+ TFH+ T-LPDs and in five with CD8+ T-LPDs. A previous study demonstrated that 19 of 28 RA patients with OIIA T-LPDs (68%) showed histological features of CD4+ TFH+ AITL with scattered EBER+ lymphocytes in the background [8]. In RA patients, increased TFH cells and decreased regulatory T cells in the peripheral blood were significantly correlated with the disease activity of RA (p < 0.05), and TFH cells were frequently found in the germinal centres of the involved synovial tissue [20]. In RA patients treated with MTX and biologic drugs, EBV-specific effector memory CD8+ T cells were positively correlated with increased EBV viral load in the peripheral blood [21]. It was suggested that abnormal proliferation of CD4+ TFH cells and CD8+ T cells was due to persistent RA activity and EBV infection found in the immunosuppressive states of RA patients treated with MTX and biologic drugs.
In the current study, five of 21 RA patients with OIIA TNK-LPDs (24%) showed a lethal progressive clinical course within 13 months, having features of TFH+ PTCL, EBV+ CD4+ CD30+ large-cell PTCL-NOS, EBV− CD8+ sALCL, and sEBV+ CD8+ CD30+/− TCLs. Four reported RA patients treated with MTX or MTX and Janus kinase inhibitors suffered from sALCL and EBV+ CD30+ large-cell PTCL, and the three ALK− patients died of the disease within months, while the remaining patient with ALK+ sALCL is alive at 24 months [7,11,12,22]. Parakkal et al. [13] reported that 3 RA patients treated with MTX and TNF inhibitors for 1.2 to 5 years as well as 22 patients with IBD receiving TNF inhibitors and azathioprine had hepatosplenic TCL in the FDA database, and 2 RA patients died of the disease. The current and previous studies confirmed that RA patients treated with MTX and TNF inhibitors occasionally had complicated T-LPDs with a lethal progressive clinical course.   Our two examined RA patients (Case Nos 15, 16) treated with MTX-complicated CD8+ T-LGL showed indolent clinicopathological features without chemotherapy [1]. Schwaneck et al. [10] reported that RA patients with mainly CD8+ and occasionally CD4+ T-LGLs mostly showed an indolent clinical course following rituximab (anti-CD20) and anti-IL6 receptor Ab treatment in addition to continuous MTX therapy and/ or TNF inhibitors. They suggested that RA-associated T-LGL occurred independently of MTX therapy for RA, and probably due to TNF inhibitors, because of decreasing circulating T-LGLs after cessation of TNF inhibitors. Andrade et al. [23] reported that one RA patient treated with MTX and TNF inhibitor (etanercept) showed CD4+ CD8+ TCL with HLH and scattered EBV+ cells and received prompt chemotherapy, remaining alive at 24 months. The current study demonstrated that two RA patients (Case Nos. 13,14) treated with MTX-complicated sEBV+ CD8+ TCL died within 4 months, and another patient treated with MTX and TNF inhibitor (infliximab) (Case No. 18) showed CD8+ T-LPD with features of EBV− HLH. Even in post-transplant LPDs, only a few patients showed sEBV+ CD8+ TCL with a lethal progressive clinical course and CD8+ T-LPDs with features of HLH [24][25][26]. These findings demonstrated that T-LGL with an indolent clinical course was occasionally found, and rare types of lethal and curable EBV+/− CD8+ TCL or T-LPDs with HLH were also detected in RA patients treated with MTX and TNF inhibitors.
The current study demonstrated that clonal peaks of TCRγ were detected in 9 of 17 RA patients with OIIA TNK-LPDs (53%). Only one patient among our examined seven with TFH+ TCL and T-LPDs (14%) showed the RHOA p.G17V mutation. Twenty-one of 47 RA patients with OIIA B-LPDs (45%) showed rearrangements of the immunoglobulin heavy chain (IGH) gene in FFPE specimens by PCR [27], and the clonality was associated with poor recurrence-free survival (p = 0.05), but not with OS. It was suggested that frequent spontaneous regression of TNK-LPDs as well as B-LPDs by MTX withdrawal in RA patients occurred due to abnormal but not overt neoplastic proliferation of lymphocytes. However, seven RA patients with MTX-associated TNK-LPDs (33%) died of the disease. Notably, ≥ 500 U/L LDH was a significant poor prognostic factor of PFS and OS in RA patients with OIIA TNK-LPDs (p < 0.05). Careful follow-up of serum LDH as well as sIL2R is necessary to detect the progression of OIIA TNK-LPDs in the early-phase of the disease. Histologically, p53 expression in 57 patients with TNKCL was positively correlated with TP53 mutation variant allele frequency, and p53 was a significant poor prognostic factor (p = 0.009) [28]. CMYC expression in patients with aggressive-type adult T-cell leukaemia/lymphoma (ATLL) was significantly higher than that in patients with smouldering and chronic types (p < 0.01) [29]. Because large-sized, CD30+, p53+, CMYC+, and EBER+ atypical lymphocytes were identified as poor prognostic factors in RA patients with OIIA TNK-LPDs (p < 0.05), timely treatment with chemotherapy and biologic drugs is necessary for TNK-LPD patients with a progressive clinical course having some of the identified prognostic factors.
EBER+ atypical lymphocytes and scattered EBER+ lymphocytes were frequently found in the15 examined patients with OIIA TNK-LPDs (71%), and the findings have been also reported in RA patients with OIIA TNK-LPDs (89%) and B-LPDs (63, 45%), but rarely in RA patients with non-OIIA B-LPDs (8%) [3,4,8]. Ejima-Yamada et al. [30] reported that spontaneous regression of LPDs by withdrawal of MTX and biologic drugs was frequently found in 15 of 21 RA patients with OIIA EBV+ B-LPDs (71%), as opposed to none of 17 OIIA EBV− B-LPDs (0%) (p < 0.01). Another recent study demonstrated that high EBV real-time PCR value, improvement of lymphocyte counts in the peripheral blood, and patients with EBER+ cells in the involved tissues were significantly associated with the spontaneous regression of LPDs by withdrawal of MTX and biologic drugs in 34 RA patients with LPDs (p = 0.003, p = 0.036, and p = 0.032, respectively) [31]. These findings highly suggest that tumour microenvironments and lymphomagenesis caused by EBV in RA patients occurred in the immunosuppressive states caused by MTX, THF inhibitors, and other biologic drugs. Bayda et al. [32] demonstrated that strong expression of EBV-produced Bam-HI A rightward transcripts (BART s) and early lytic gene, BNLF2, were frequently found in 14 AITL patients compared with 21 other types of T-, B-and Hodgkin lymphomas by next-generation sequencing for EBV transcriptomes, and the two genes contributed to immune escape and survival of the infected cells. Furthermore, frequent deletion of BART of EBV in peripheral blood mononuclear cells was detected in 10 of 23 patients with TNKCL (44%) and 10 of 14 EBV+ large B-cell lymphoma (71%), but not in 15 with infectious mononucleosis and 32 with post-transplant LPDs [33]. To determine the detailed influence of EBV infection, it is necessary to examine the high expression or deletion of EBV BART and BNLF2 genes in peripheral blood mononuclear cells and tumour tissues between RA patients with OIIA LPDs having regression and no regression of LPDs by withdrawal of MTX and biologic drugs.
This study has some limitations. It was a retrospective study based on small numbers of rare TNK-LPDs in RA patients treated with different types of therapies for LPDs. Hence, it was difficult to confirm the definitive prognostic factors and etiological roles of EBV in TNK-LPDs of RA patients.

Conclusions
We investigated the clinicopathological characteristics and prognostic factors of 21 RA patients with OIIA TNK-LPDs. Immunohistologically, seven CD4+ TLPD patients (33%) had TFH+ atypical lymphocytes, nine (43%) showed CD8+ T-LPDs, and five (24%) had EBER+ large, atypical lymphocytes. Two patients with TFH+ and EBV+ CD4+ CD30+ PTCL and three with CD8+ ALK− sALCL and sEBV+ CD8+ TCL showed a lethal progressive clinical course within 13 months. Furthermore, ≥ 500 U/L LDH, large, atypical lymphocytes, and CD30, p53, CMYC, and EBER expression in atypical lymphocytes were identified as significant poor prognostic factors for OS (p < 0.05). Detection of OIIA TNK-LPDs with poor prognostic factors is required to improve patient outcome. EBER+ atypical and reactive lymphocytes were found in the 15 patients with TNK-LPDs (71%) as well as in 27 of the examined 39 OIIA B-LPDs (69%) but was rare in 22 non-OIIA B-LPDs (14%). EBV infection in the immunosuppressive state resulting from persistent RA and treatment with MTX, TNF inhibitors, and other biologic drugs may play a role in forming the tumour microenvironment and lymphomagenesis of TNK-LPDs.