前言：特发性炎性肌病(idiopathic inflammatory myopathies, IIMs)是一种病因和发病机制均未十分清楚的系统性的自身免疫病。其临床表现多种多样，异质性强，且常累及多种脏器，容易合并恶性肿瘤。既往文献报道肌炎特异性抗体(myositis specific autoantibodies, MSAs)能将肌炎患者区分为更同质的群体，在判断IIMs的临床类型、疗效反应及预后等方面均具有重要意义。本研究分为三部分，第一部分拟探讨与肌炎特异性抗体{抗核基质蛋白(NXP)-2}抗体关联的临床表型，明确其与疾病活动度的相关性；第二部分旨在系统探讨各MSAs与IIMs合并恶性肿瘤的关联性；第三部分在第二部分研究结果的基础上，探讨TIF1-γ基因的遗传改变和差异表达在肿瘤相关性肌炎(cancer-associated myositis, CAM)发病中的可能机制。

第一部分 抗NXP-2抗体相关性肌病的临床表型研究

目的：本研究旨在探讨特发性炎性肌病的患者血清抗NXP-2抗体水平与肌炎活动度及钙化严重程度之间的相关性。

方法：纳入IIMs患者709例。采用包被有MORC3蛋白的自制酶联免疫吸附试剂盒检测患者单份及前后连续随访获得的多份血清中抗NXP-2抗体水平。将抗NXP-2抗体阳性的肌炎患者分为两组：合并钙化组和未合并钙化组。分析抗NXP-2抗体水平与肌炎各器官特异的活动度评分、血清肌酸激酶水平以及钙化严重程度之间的相关性。采用非参数Mann-Whitney U检验、Fisher确切概率法、Spearman相关系数分析及混合线性回归模型进行统计学分析。

结果：横断面研究中共纳入56例IIMs患者，其中未合并钙化组38例，合并钙化组18例。研究发现，抗NXP-2抗体水平仅在未合并钙化组中与医生总体活动度评分、肌肉病变活动度评分及血清肌酸激酶水平呈正相关，而在合并钙化组患者中未观察到该结果。进一步对上述具有重复测量资料的患者数据进行纵向的相关性分析。发现在未合并钙化组，抗NXP-2抗体水平与上述三者指标的联系较前更加紧密，且还发现抗体水平亦与一般情况活动度评分、皮肤病变活动度评分和胃肠道病变活动度评分呈中等相关；而在合并钙化组，抗NXP-2抗体水平仅与医生总体活动度评分及一般情况活动度评分呈弱相关。有趣的是，在未合并钙化组的患者中观察到血清抗NXP-2抗体水平可随着疾病的缓解滴度转阴，亦可随着疾病的复发而复现。抗NXP-2抗体水平与钙化严重程度不相关。

结论：抗NXP-2抗体水平与疾病活动相关，尤其见于未合并钙化组，可作为IIMs疾病活动的评价指标。抗NXP-2抗体水平与疾病活动度之间的关联性在合并与未合并钙化组中的不同提示两组患者临床表型及可能参与的致病机理不同。

第二部分 肌炎特异性抗体与肌炎合并恶性肿瘤的关联性研究

目的：本研究旨在系统的探讨了IIMs血清不同肌炎特异性抗体与肌炎合并恶性肿瘤的相关性。

方法：采用免疫印迹以及酶联免疫吸附法检测血清抗Mi-2α、抗Mi-2β、抗转录中介因子(TIF1)-γ、抗NXP-2、抗小泛素样修饰活化酶(SAE)-1、抗黑色素分化相关基因(MDA)-5、抗信号识别颗粒(SRP)、抗组氨酰- tRNA 合成酶(Jo-1)、抗苏氨酰- tRNA 合成酶(PL-7)、抗丙氨酰- tRNA 合成酶(PL-12)、抗亮氨酰- tRNA 合成酶(OJ)、抗甘氨酰- tRNA 合成酶(EJ)以及抗3-羟基-3-甲基-辅酶A还原酶蛋白(HMGCR)抗体；通过长期随访记录IIMs患者是否合并恶性肿瘤及其结局。采用标化发生比(SIR)评估不同抗体类型下IIMs患者合并恶性肿瘤的风险；分析不同抗体类型下肌炎起病与肿瘤确诊之间的时间间隔有无差异以及不同抗体类型下罹患肿瘤的预后差异。采用Kruskal-Wallis检验，c2检验及Log-rank检验进行统计学分析。

结果：617例IIMs患者中共有72例患者合并恶性肿瘤，阳性率为11.7%；其中抗TIF1-γ抗体组38例、抗NXP2抗体组3例、抗SAE1抗体组4例、抗合成酶抗体组10例（抗Jo-1抗体5例，抗PL-12抗体3例，抗PL-7抗体1例，抗EJ 抗体1例)、抗MDA5抗体、抗HMGCR抗体、抗SRP抗体各1例以及抗体全阴性组14例。与中国同年龄同性别一般人群进行比较，抗TIF1-γ 抗体 (SIR=17.28, 95% CI: 11.94 ~ 24.14)，抗NXP2 抗体(SIR=8.14, 95% CI: 1.63 ~ 23.86)，抗SAE1抗体(SIR=12.92, 95% CI: 3.23 ~ 32.94)，以及抗体全阴性组 (SIR=3.99, 95% CI: 1.96 ~ 7.14) 发生恶性肿瘤的风险明显升高；不同抗体类型与合并恶性肿瘤类型没有显著相关性(P>0.05)；各组肌炎起病与肿瘤确诊时间间隔分别为+0.19 年（抗TIF1-γ抗体）（加号代表肌炎起病早于恶性肿瘤确诊时间，减号代表肌炎起病晚于恶性肿瘤确诊时间)、+0.50 年（抗NXP2抗体）、+0.46年（抗SAE1抗体）、-3.00 年（抗Jo-1抗体）、+0.25年 （抗PL-12抗体）、+0.67年（抗体全阴性组），差异无统计学意义(P>0.05)；比较不同抗体类型下肌炎合并恶性肿瘤的预后，差异无统计学意义(P>0.05)；可将肌炎合并恶性肿瘤分为为肌炎相关性恶性肿瘤组与恶性肿瘤与肌炎不相关组。相较于恶性肿瘤与肌炎不相关组，肌炎相关性恶性肿瘤组患者预后要明显更差，调整年龄和性别等因素后的全因死亡率风险比是10.8(95%CI: 1.38-84.5,P=0.02)。

结论：除了传统认为的抗TIF1-γ抗体外，抗NXP-2抗体、抗SAE1抗体以及抗体全阴性组合并恶性肿瘤的风险亦明显升高，临床尤应对此类患者注意筛查恶性肿瘤；此外，在部分抗HMGCR抗体、抗Jo-1抗体和抗PL-12抗体阳性的患者中恶性肿瘤的发生亦可能与肌炎有关。发现与肌炎相关性恶性肿瘤密切相关的肌炎特异性抗体为进一步阐明肌炎合并恶性肿瘤的机制提供了线索。

第三部分 肿瘤相关抗原TIF1-γ诱导的交叉免疫应答在肌炎合并恶性肿瘤发生机制中的初步探讨

目的：探索TIF1-γ基因的遗传改变和差异表达在CAM发病中的可能机制。

方法：获取6例抗以及5例阴性CAM患者的肿瘤组织及血液标本，应用目标区域捕获测序技术，比对分析TRIM33基因在肿瘤细胞中的突变形式。采用免疫组化法检测CAM患者的肿瘤组织、肌肉组织以及皮肤组织中TIF1-γ蛋白表达水平。

结果：6例抗TIF1-γ抗体阳性的CAM患者的肿瘤组织中有4例发生了体细胞突变，4例发生了杂合性缺失（4例中包含2例未检测体细胞突变的患者）；而在5例抗TIF1-γ阴性的CAM患者中仅检测到1例发生了体细胞突变，所有患者均未检测到杂合性缺失现象。相较抗TIF1-γ抗体阴性组，抗TIF1-γ抗体阳性组中TRIM33基因变异概率明显增高，差异具有统计学意义（100% vs 20%, P=0.03）。4例抗TIF1-γ阳性的CAM患者具有肌肉标本，在其肌肉组织中均检测到TIF1-γ蛋白表达；3例抗TIF1-γ抗体阴性的CAM患者中有1例患者的肌肉组织中检测到了该蛋白表达；无论抗TIF1-γ抗体阳性亦或是阴性，皮肤组织中均可检测到TIF1-γ蛋白表达。

结论：抗TIF1-γ抗体阳性的CAM患者的肿瘤组织更容易发生TRIM33基因错义变异，从而产生突变的TIF-γ蛋白，可能诱导机体产生抗TIF1-γ蛋白的免疫应答；肌肉及皮肤组织中TIF1-γ蛋白的表达为成为上述免疫应答的靶器官提供了基础。研究结果为肿瘤相关抗原TIF1-γ诱导交叉免疫应答导致肌炎提供了部分证据支持。

Backgroud: Idiopathic inflammatory myopathies (IIM) are a systemic autoimmune disease with the etiology and pathogenesis are still not well understood. Their clinical manifestations are diverse and heterogeneous and they often involve a variety of organs and are associated with an increased risk of cancer. The previous studies reported that myositis-specific antibodies (MSAs) could distinguish myositis patients into more homogeneous groups and were of great significance in judging the clinical phenotype, efficacy response and prognosis of IIMs. This study was divided into three parts. The first part was to explore the clinical phenotype related to MSAs (anti-NXP-2 antibodies), and to clarify their relevance to the disease activity index.. The second part was to systematically study the association of MSAs with cancer-associated myositis. The third part was to analyze the influence of genetic alterations and differential expression of transcription intermediary factor 1(TIF1)-γ gene in the pathophysiology of cancer-associated myositis (CAM).

Part I. Differential clinical associations of anti-nuclear matrix protein-2 autoantibodies in patients with idiopathic inflammatory myopathies

Objective: To investigate the associations between anti-NXP-2 autoantibody levels and disease activity as well as calcinosis severity.

Methods: The serum levels of anti-NXP-2 autoantibodies were determined in 709 idiopathic inflammatory myopathies (IIMs) and also serially measured by an in-house enzyme-linked immunosorbent assay using recombinant MORC3. Patients with anti-NXP-2 autoantibodies were divided into two subgroups: with or without calcinosis. The associations of anti-NXP-2 autoantibody levels with organ-specific disease activity, serum creatine kinase (CK) levels, and calcinosis severity were investigated in cross-sectional and longitudinal analyses. Non-parametric Mann-Whitney U test, Fisher’s exact test, Spearman correlation coefficient analysis and mixed linear regression model were used for statistical analysis.

Results: A cross-sectional analysis of 56 patients (38 without calcinosis and 18 with calcinosis) with anti-NXP-2 autoantibodies showed that the levels of anti-NXP-2 autoantibody were positively correlated with the physician global assessment visual analog scale (PGA VAS), muscle VAS, and CK levels in patients without calcinosis, while no such association was found in patients with calcinosis. The longitudinal study revealed strong correlations between the anti-NXP-2 antibody levels and PGA, constitutional, cutaneous, gastrointestinal, muscle VAS, and serum CK levels in patients without calcinosis, but only modest correlation with PGA and constitutional VAS in patients with calcinosis. Of note, in patients without calcinosis, the anti-NXP-2 autoantibodies could turn negative in clinical remission; while reappeared with disease relapse. No association was observed between anti-NXP-2 levels and calcinosis severity.

Conclusion: This study indicated that anti-NXP-2 autoantibodies served as a useful marker for disease activity, especially in patients without calcinosis. The differential associations between anti-NXP-2 autoantibody levels and disease activity suggested a phenotypic difference between patients with and without calcinosis.

Part II. Study on the relationship between myositis-specific autoantibodies profiles and cancer-associated myositis

Objective: Cancer is a significant complication contributing to increased mortality of idiopathic inflammatory myopathies (IIMs), and the association between IIMs and cancer has been extensively reported. Myositis specific autoantibodies (MSAs) can help to stratify patients into more homogeneous groups and may be used as a biomarker for cancer-associated myositis. In this study, we aimed to systematically define the cancer associated MSAs in IIMs.

Methods: Serum anti-Mi-2α, anti-Mi-2β, anti-TIF1-γ, anti-NXP2, anti-SAE1, anti-MDA5, anti-SRP, anti-Jo-1, anti-PL-7, anti-PL-12, anti-OJ, anti-EJ and anti-HMGCR were detected by commercial line dot assays and enzyme-linked immunosorbent assay (ELISA). Through long-term follow-up to understand whether IIMs patients will develop cancer and their outcomes. The cancer risk with different MSAs was estimated by standardized incidence ratio (SIR). Paraneoplastic manifestation, such as the close temporal relationship between myositis onset and cancer diagnoses in patients with different MSAs were also evaluated. Meanwhile, we investigated the prognoses of patients with cancer-associated myositis (CAM) with different MSAs. Kruskal-Wallis test, Chi-square test and Log-rank test were used for statistical analysis.

Results: A total of 72 patients in 617 IIMs patients had cancer, with a positive rate of 11.7%. Among these 72 individuals, 38 tested positive for anti-TIF1-γ, three for anti-NXP2, four for anti-SAE1, 10 for anti-aminoacyl-tRNA-synthetase (anti-ARS) antibodies (included five anti-Jo-1, three anti-PL-12, one anti-PL-7 and one anti-EJ antibodies); one each for anti-MDA5, anti-HMGCR, and anti-SRP, 14 for MSAs- (patients who carried none of these MSAs, hereinafter referred as the “MSAs-” group). Compared with the general Chinese population, IIMs patients with anti-TIF1-γ antibodies (SIR=17.28, 95% CI: 11.94 to 24.14); anti-NXP2 antibodies (SIR=8.14, 95% CI: 1.63 to 23.86); or anti-SAE1 antibodies (SIR=12.92, 95% CI: 3.23 to 32.94), or who were MSAs-negative (SIR=3.99, 95% CI: 1.96 to 7.14) faced an increased risk for cancer. There was no association between specific MSAs subtypes and certain types of cancer (p>0.05). The median duration of IIMs at cancer-diagnosis did not differ significantly between the groups: +0.19 years in the anti-TIF1-γ group (a plus sign signifies cancer developing after myositis onset; a minus sign signifies cancer developing before myositis); +0.5 years in the anti-NXP2 group; +0.46 years in the anti-SAE1 group; -3.00 years in the anti-Jo-1 group; +0.25 years in the anti-PL-12 group; and +0.67 years in the “MSAs-” group (p>0.05). Myositis complicated with cancer can be classified as CAM and cancer-unrelated to myositis. There were no prognostic differences among the cancer-associated myositis (CAM) patients from different MSAs subgroups. However, in comparison to those with cancer-unrelated to myositis, CAM had a worse prognosis, with an age- and sex-adjusted Cox hazard ratio (HR) of 10.8 [95%CI: 1.38-84.5, P = 0.02] for all-cause mortality.

Conclusion: Our study demonstrates, in what is to our knowledge the largest population examined to date, that anti-NXP2 antibodies, anti-SAE antibodies, MSAs^-, and previously reported anti-TIF1-γ antibodies, are all associated with an increased risk of cancer in IIMs patients. Moreover, our data suggest that in some cases, anti-HMGCR, anti-Jo-1 and anti-PL-12 antibodies production might also be driven by malignancy. This can aid in the etiologic research of paraneoplastic myositis and clinical management.

Part III. A preliminary study on the mechanism of cross immunity induced by tumor-associated antigen TIF1-γ in cancer-associated dermatomyositis.

Objective: To investigate the possible mechanisms of genetic alteration and differential expression of transcription intermediary factor 1(TIF1)-γ in the development of CAM.

Methods: Paired tumor tissues and blood samples from 6 anti-TIF1-γ-positive CAM patients and 5 anti-TIF1-γ-negative CAM were obtained. The target region capture and sequencing technique was used to analyze the mutation type of TRIM33 in cancer cells. The protein level of TIF1-γ in tumor, muscle and skin tissues of CAM patients was detected by immunohistochemistry.

Results: In the tumors of 6 anti-TIF1-γ-positive CAM patients, four cases had somatic mutations and 4 cases exhibited loss of heterozygosity (these four cases included two cases that did not contain a detectable somatic mutation). Only 1 of the 5 anti-TIF1-γ-negative CAM patients had somatic mutations and none of patients exhibited loss of heterozygosity. Compared with the anti-TIF1-γ-negative group, the mutation probability of TRIM33 was significantly higher in the anti-TIF1-γ-positive group, and the difference was statistically significant (100% vs 20%, P=0.03). Four cases of anti-TIF1-γ-positive CAM patients had muscle specimens and TIF-1-γ protein was expressed in their muscle tissues. Meanwhile, the protein was expressed in one of three anti-TIF1-γ-negative CAM patients. Notably, TIF1-γ protein was expressed in the skin tissue regardless of whether the anti-TIF1-γ antibody is positive or negative.

Conclusion: The tumor tissue of anti-TIF1-γ-positive CAM patients is more susceptible to gene mutation of TRIM33, thus inducing the body to develop an immune response against TIF1-γ to produce the mutated TIF-γ protein, and the expression of TIF1-γ protein in muscle and skin tissue may become the target of the above immune response. The results provide some evidence that myositis may be caused by the tumor associated antigen TIF-γ-induced cross-immune responses.