Frequent STAT3 Mutations in CD8+ T Cells from Patients with Pure Red Cell Aplasia
Authors: Kawakami Toru , Sekiguchi Nodoka , Kobayashi Jun , Imi Tatsuya , Matsuda Kazuyuki , Yamane Taku , Nishina Sayaka , Senoo Yasushi , Sakai Hitoshi , Ito Toshiro , Koizumi Tomonobu , Hirokawa Makoto , Nakao Shinji , Nakazawa Hideyuki , Ishida Fumihiro 
Published: October 2019

Background: Dysregulation of T-cell mediated immunity is responsible for acquired pure red cell aplasia (PRCA). Although STAT3 mutations are frequently detected in patients with T-cell large granular lymphocytic leukemia (T-LGLL), which is often complicated by PRCA and is also reported to be associated with acquired aplastic anemia (AA) and myelodysplastic syndrome (MDS), whether STAT3-mutated T cells are involved in the pathophysiology of PRCA and other types of bone marrow failure (BMF) remains unknown.

Methods: Patients with AA, AA-paroxysmal nocturnal hemoglobinuria (AA-PNH) syndrome, MDS or PRCA were enrolled in this study. We performed STAT3 mutation analyses of the peripheral blood mononuclear cells using an allele-specific PCR (AsPCR) to detect STAT3 Y640F or D661Y mutations and amplicon sequencing using primers covering entire coding region of STAT3. CD4+ T cells, CD8+ T cells or granulocytes were sorted, if possible, and also subjected to the analyses. The T-cell receptor (TCR) Vβ repertoire was analyzed using whole blood samples by flow cytometry.

Results: A total of 124 patients including PRCA (n=42), AA (n=54), AA-PNH (n=7) and MDS (n=21) were enrolled. The subtypes of PRCA were as follows: idiopathic, n= 15, T-LGLL-associated, n=13; thymoma or thymic cancer-associated, n=7; autoimmune disease-associated, n=5; adverse drug reactions, n=1; and human parvovirus B19 infection complicated by T-LGLL, n=1. As an initial step in the STAT3 mutational analysis, we screened all patients with an AsPCR. Among the 42 patients with PRCA, 3 (10%) of 29 patients without T-LGLL and 6 (46%) of 13 patients with T-LGLL were positive for mutations. In contrast, none of the patients with AA, MDS or AA-PNH were positive (P value= 0.000031). Then, we examined MNC-derived DNA from 73 patients (PRCA, n=42 and AA, n=31) using amplicon sequencing. In this sequencing analysis, the median depth of coverage was 6,219x (range; 1,065-14,188). No STAT3 mutations were detected in 31 AA patients. In contrast, 15 (36%) PRCA patients possessed STAT3 mutations. The variant allele frequency of STAT3 mutations ranged from 0.0057 to 0.489. In all of the 7 patients studied, the STAT3 mutations were restricted to sorted CD8+ T cells. Three patients were negative for STAT3 mutations in MNCs but found to be positive when sorted CD8+ T cells were analyzed. The prevalence of STAT3 mutation in idiopathic, thymoma-associated, autoimmune disorder-associated and T-LGLL-associated PRCA was 33% (5/15), 29% (2/7), 20% (1/5), and 77% (10/13), respectively. In total, STAT3 mutations were detected in 8 of 29 (28%) PRCA patients without T-LGLL and 10 of 13 (77%) PRCA patients with T-LGLL. When TCRVβ repertoires of CD8+ T cells sorted from 3 STAT3 mutation(+) PRCA patients without T-LGLL were analyzed, skewed TCRVb repertoires were evident in all patients, and STAT3 mutations were detected in skewed TCRVβ fractions from 2 patients whose samples were available for cell sorting. The STAT3-mutation(+) patients were younger (median age of 63 years vs 73 years, P= 0.026) and less responsive to cyclosporine (CsA) (46% [6/13] vs 100% [8/8], P= 0.0092) in comparison to STAT3-mutation(-) patients. Of note, 4 of 8 STAT3 mutation(+) patients who had been refractory to CsA were treated with CY and all of them responded well, whereas none of 8 STAT3 mutation(-) patients required secondary treatment with CY owing to a sustained response to CsA.

Discussion: This study is the first to reveal frequent STAT3 mutations in PRCA patients, even in thouse without T-LGLL, using a large cohort of patients. The presence of STAT3-mutated CD8+ T cells may be unique background of PRCA, irrespective of disease etiology. Poor response to CsA in STAT3 mutation(+) patients suggests that STAT3-mutated CD8+ T cells may be less sensitive to the inhibitory effects of CsA than non-mutated CD8+ T cells. In contrast, our analyses failed to detect STAT3 mutations in any of 52 AA patients, suggesting that STAT3 mutation(+) T cells had little impact on the development of AA in Japanese patients.

Conclusion: STAT3 mutations are frequently detected in the CD8+ T cells of PRCA patients, regardless of the presence of T-LGLL. The identification of STAT3 mutations may be useful for appropriately managing patients with PRCA.

Figure. Figure.

Disclosures

Nakao: Novartis: Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria.