再生不良性貧血中的克隆造血和免疫細胞組成分析與其臨床意義

Abstract

介紹 後天再生不良性貧血 (aplastic anemia, AA) 包含造血幹細胞被破壞及克隆造血 (clonal hematopoiesis, CH)。CH 可能是幹細胞逃避免疫破壞的手段，但有可能促成疾病進展為血液惡性腫瘤，如化生不良症候群 (myelodysplastic syndrome, MDS) 或急性骨髓性白血病 (acute myeloid leukemia, AML)。在免疫反應中，人類白血球抗原 (human leukocyte antigen, HLA) 作為敵我辨識、抗原呈現的重要角色，也許會影響 AA 的發病機制與預後。本研究旨在深入分析重度再生不良性貧血 (severe AA, SAA) 患者的CH，包括PIGA、骨髓血癌相關基因 (myeloid-malignancy-related gene, MR) 體突變、和HLA 基因突變與HLA雜合性缺失 (Loss of Heterozygosity, LOH)，以及免疫細胞組成比例對於 SAA 患者的臨床意義。 方法 本研究蒐集了 129 名經國立臺灣大學醫學院附設醫院診斷的 SAA 患者，蒐集當初診斷時的骨髓染色體分析結果，並取其診斷時冷凍的骨髓樣本，執行次世代定序偵測 MR 基因突變、HLA 基因突變以及LOH，以及用 RNA 定序資料分析骨髓中免疫細胞組成比例，並和病患的預後比對。

結果 我一共蒐集了 129 名 SAA 患者，追蹤時間中位數為 6.6 年。其中 6 名患者 (4.7%) 進展為MDS 或 AML。 52.7% 的患者存在至少一種形式的 CH。其中 13 名 (10%) 診斷時有異常染色體，以第八號染色體三倍體 (Trisomy 8) 最常見 (2.6%)。染色體異常與進展為 MDS 或 AML、治療反應或整體生存期無顯著相關。 26.4% 的患者檢測到 MR 基因體突變。DNMT3A 和 ASXL1 是最常見的突變 (各 4.6%)。PIGA/BCOR/BCORL1 以外的 MR 基因突變與較差存活率相關，然而並未發現 MR 突變狀態與治療反應或 AML/MDS 惡化有顯著相關。在 58 名診斷時有以流式細胞儀檢測有無陣發性血紅素尿症 (paroxysmal nocturnal hemoglobinuria, PNH) 流式細胞儀的患者中，10 名 (17%) 有 PNH 克隆造血 (定義為顆粒球細胞 PNH 比例 > 1%)。PNH 克隆造血陽性組對 ATG 治療表現出稍好的反應趨勢和稍好的存活率傾向。19% 的患者存在 HLA LOH，其中 HLA-A 最常發生 LOH，佔所有 LOH 事件的 44%。本研究觀察到具有兩個或以上 HLA 基因座 LOH 的患者有較快進展為 AML/MDS 和較差存活率的趨勢，但未達統計顯著。而 7.8% 的患者檢測到 HLA 功能喪失性突變，最常發生於 HLA-DRB1 基因座。這些突變與治療反應或存活率無顯著相關性。運用 CIBERSORTx 分析 RNA-seq 結果後發現，與非嚴重型 AA 患者相比，SAA 病人骨髓中 CD8陽性 T 細胞和 自然殺手細胞(NK)的比例較高，而髓系細胞 (myeloid cells, 如顆粒球、單核球) 比例較低。診斷時骨髓中若 CD8陽性 T 細胞和 自然殺手細胞比例較高，其存活率可能較差 (p = 0.26)，惟樣本過少未達統計顯著。有 HLA LOH 的患者骨髓中此二種細胞比例較少，可能暗示 HLA LOH 的確與免疫逃逸現象相關。 結論 再生不良性貧血的病生理其實是非常多樣的，不同類型的克隆造血對其預後的影響仍待釐清。本研究同時評估了 SAA 中多種 CH 機制，並初步揭示了 MR 突變模式、HLA LOH 數量以及骨髓免疫細胞組成比例等的潛在預後影響。未來的研究，若能結合患者的診斷時資料和治療後血液和骨髓樣本的持續追蹤分析，將有助於進一步揭示再生不良性貧血的奧秘，並可能為其複雜的病理機制提供更深入的理解。

Introduction Acquired aplastic anemia (AA) involves the destruction of hematopoietic stem cells (HSCs) and clonal hematopoiesis (CH). CH may serve as a mechanism for HSCs to evade immune attack and can potentially promote disease progression to hematologic malignancies, such as myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). The human leukocyte antigen (HLA) system plays a crucial role in immune pathway and thus may influence the pathogenesis and prognosis of AA. This study aims to conduct an in-depth analysis of CH, including somatic mutations in PIGA, myeloid-malignancy-related genes (MR), and HLA and loss of heterozygosity (LOH), and their clinical significance. Comparison in the composition of BM immune cells between the patients with severe aplastic anemia (SAA) and non-severe AA (NSAA) was also performed by RNA-seq data. Materials and Methods This study recruited 129 patients diagnosed with SAA at the National Taiwan University Hospital. In addition to chromosome analysis, cryopreserved bone marrow samples obtained at diagnosis were subjected to next-generation sequencing to detect PIGA, MR and HLA gene mutations, and LOH of HLA. RNA-seq data was also performed to analyze bone marrow immune cell composition. These findings were then correlated with the patients' prognoses. Results A total of 129 SAA patients were included in this study, with a median follow-up time of 6.6 years. Among them, 6 patients (4.7%) progressed to MDS or AML. 52.7% of patients presented with at least one form of CH. Among these, 13 patients (10%) had chromosomal abnormalities at diagnosis, with trisomy 8 being the most common (2.6%). The presence or absence of chromosomal abnormalities did not correlate with treatment response, progression to MDS or AML, or overall survival. Somatic mutations in PIGA and MR genes were detected in 26.4% of patients. DNMT3A and ASXL1 were the most frequently mutated genes (4.6% each). Excluding PIGA/BCOR/BCORL1, MR gene mutations were associated with poorer survival rates; however, there was no significant association between MR mutation status and treatment response or progression to AML/MDS. Among the 58 patients who underwent flow cytometry analysis to detect paroxysmal nocturnal hemoglobinuria (PNH) clone at diagnosis, 10 (17%) had a PNH clone (PNH granulocyte proportion > 1%). The PNH-positive group showed a trend towards a slightly better response to antithymocyte globulin (ATG) treatment and a trend for better survival. HLA LOH was present in 19% of patients, with HLA-A being the most frequent locus for LOH, accounting for 44% of all LOH events. This study observed that patients with LOH at two or more HLA loci had a trend towards faster progression to AML/MDS and poorer survival, though this did not reach statistical significance. Furthermore, 7.8% of patients had loss-of-function mutations in HLA genes, most commonly in the HLA-DRB1 locus. These mutations showed no significant correlation with treatment response or survival. Using CIBERSORTx to analyze RNA-seq results, relative to patients with NSAA, the bone marrow of the SAA cohort was enriched in CD8-positive T cells and NK cells but depleted of myeloid cells, and this was potentially associated with poorer survival (p = 0.26), although was not statistically significant because of the small sample size. Patients with HLA LOH had lower proportions of T cells and NK cells in their bone marrow, possibly suggesting that HLA LOH is indeed related to immune evasion phenomena. Conclusion Aplastic anemia is a highly heterogeneous disease, and the prognostic impact of different types of clonal hematopoiesis remains to be clarified. This study concurrently evaluated multiple mechanisms of CH in SAA and provided preliminary insights into the potential prognostic implications of MR mutation patterns, the number of HLA LOH loci, and the composition of bone marrow immune cells. Future research, by integrating patients' diagnostic data with continuous follow-up analysis of blood and bone marrow samples post-treatment, will help to further unravel the mysteries of aplastic anemia and may provide a deeper understanding of its complex pathological mechanisms.