Data_Sheet_1_Distinct Immunological Landscapes Characterize Inherited and Sporadic Mismatch Repair Deficient Endometrial Cancer.docx

Around 30% of endometrial cancers (EC) are mismatch repair (MMR) deficient, mostly as a consequence of mutations acquired during tumorigenesis, but a significant minority is caused by Lynch syndrome (LS). This inherited cancer predisposition syndrome primes an anti-cancer immune response, even in healthy carriers. We sought to explore the intra-tumoral immunological differences between genetically confirmed LS-associated MMR-deficient (MMRd), sporadic MMR-deficient, and MMR-proficient (MMRp) EC. Endometrial tumors from women with known LS were identified (n = 25). Comparator tumors were recruited prospectively and underwent microsatellite instability (MSI) testing, immunohistochemistry (IHC) for MMR expression and MLH1 methylation testing. Those found to have MLH1 hypermethylation formed the sporadic MMR-deficient group (n = 33). Those found to be mismatch repair proficient and microsatellite stable formed the MMR-proficient group (n = 35). A fully automated monoplex IHC panel was performed on sequential formalin-fixed paraffin-embedded tumor sections to identify CD3+, CD8+, CD45RO+, FoxP3+, and PD-1+ immune cells, and PD-L1 expression by tumor/immune cells. Two independent observers quantified immune marker expression at the tumor center and invasive margin. Mean and overall compartmental T-cell counts generated standard (binary: Low/High) and higher resolution (quaternary: 0–25, 25–50, 50–75, 75–100%) immune scores, which were used as explanatory features in neural network, support vector machine, and discriminant predictive modeling. Overall T-cell counts were significantly different between the three cohorts: CD3+ (p = <0.0001), CD8+ (p = <0.0001), CD45RO+ (<0.0001), FoxP3+ (p = <0.0001), and PD1+ (p = <0.0001), with LS-associated MMR-deficient tumors having highest infiltrations. There were significant differences in CD8+ (p = 0.02), CD45RO+ (p = 0.007), and PD-1+ (p = 0.005) T-cell counts at the invasive margin between LS-associated and sporadic MMR-deficient tumors, but not between sporadic MMR-deficient and MMR-proficient tumors. Predictive modeling could accurately determine MMR status based on CD8+ T-cell counts within the tumor center alone. This study shows that LS-associated and sporadic MMR-deficient EC are distinct immunological entities, which has important implications for treatment and prognosis.

约30%的子宫内膜癌（endometrial cancer, EC）存在错配修复（mismatch repair, MMR）缺陷，其中多数源于肿瘤发生过程中获得的体细胞突变，但仍有相当比例由林奇综合征（Lynch syndrome, LS）引发。林奇综合征是一种遗传性癌症易感综合征，即便在健康携带者体内也会触发抗肿瘤免疫应答。
本研究旨在探究经基因确认的LS相关MMR缺陷型（MMR-deficient, MMRd）、散发性MMR缺陷型与MMR正常型（MMR-proficient, MMRp）子宫内膜癌的瘤内免疫学差异。
已知LS患者的子宫内膜肿瘤样本共纳入25例（n=25）。前瞻性招募对照肿瘤样本，并对其进行微卫星不稳定性（microsatellite instability, MSI）检测、MMR表达免疫组织化学（immunohistochemistry, IHC）及MLH1甲基化检测。其中存在MLH1高甲基化的样本纳入散发性MMR缺陷组（n=33）；错配修复正常且微卫星稳定的样本纳入MMR正常组（n=35）。
采用全自动单重免疫组化试剂盒对连续的福尔马林固定石蜡包埋肿瘤切片进行检测，以识别CD3+、CD8+、CD45RO+、FoxP3+及PD-1+免疫细胞，并检测肿瘤细胞与免疫细胞的PD-L1表达水平。
由两名独立观察者分别在肿瘤中心及侵袭边缘对免疫标志物的表达进行定量分析。
基于平均及分区T细胞计数，可生成两种免疫评分：标准二分法（低/高）及高分辨率四分法（0~25%、25~50%、50~75%、75~100%），并将其作为解释特征应用于神经网络、支持向量机及判别预测建模。
三个队列的总T细胞计数存在显著统计学差异：CD3+（p<0.0001）、CD8+（p<0.0001）、CD45RO+（p<0.0001）、FoxP3+（p<0.0001）及PD-1+（p<0.0001），其中LS相关MMR缺陷型肿瘤的免疫细胞浸润水平最高。
LS相关与散发性MMR缺陷型肿瘤在侵袭边缘的CD8+（p=0.02）、CD45RO+（p=0.007）及PD-1+（p=0.005）T细胞计数存在显著差异，但散发性MMR缺陷型与MMR正常型肿瘤之间无此类差异。
仅通过肿瘤中心的CD8+ T细胞计数，预测建模即可准确判定MMR状态。
本研究表明，LS相关与散发性MMR缺陷型子宫内膜癌属于不同的免疫学亚型，这一发现对患者的治疗与预后评估具有重要指导意义。