NUT midline carcinoma (NMC) is a rare, aggressive poorly differentiated carcinoma genetically defined by NUTM1 gene rearrangement. The purpose of this study was to determine the tumor mutational burden (TMB) and the expression of immunohistochemical (IHC) markers in NMCs that are generally used to identify patients that might benefit from checkpoint immunotherapy. Three cases in a 39-year-old male (case 1) and two 13-year-old females (cases 2, 3) were identified from departmental files, with confirmation by NUT IHC and 15q14 rearrangement by fluorescent in situ hybridization. Normal-tumor paired whole exome sequencing (WES) was applied to determine TMB. IHC for DNA mismatch repair proteins, Programmed cell death ligand 1, programmed cell death 1 (PD1), and CD8 was also performed. WES yielded a TMB of 7.61 and 1.52 per Mbp in the primary and pulmonary metastasis in case 1, respectively, and a TMB of 1.04 per Mbp in the primary tumor of case 2. Programmed cell death ligand 1 tumor proportion score was 20%, 1%, and 0% and combined positive score was 25, 5, and 0 in cases 1, 2, and 3, respectively; PD1 stain counts were 25, 52, and 35 per high-power field and the PD1/CD8 ratio was 95%, 95%, and 99% in cases 1, 2, and 3, respectively. The CD8 count per high-power field was 15, 33, and 30 per high-power field in cases 1, 2, and 3, respectively. Mismatch repair IHCs showed retained staining. Although the number of cases is limited, this study is the first to investigate checkpoint immunotherapy markers in NMCs and the results demonstrate no clear biomarker association. However, the results suggest that, if checkpoint therapy is under consideration, a comprehensive workup utilizing WES and IHC is warranted.

中文翻译：

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NUT 中线癌的肿瘤突变负担和检查点免疫治疗标志物

NUT 中线癌 (NMC) 是一种罕见的侵袭性低分化癌，其基因定义为 NUTM1 基因重排。本研究的目的是确定 NMC 中的肿瘤突变负荷 (TMB) 和免疫组织化学 (IHC) 标志物的表达，这些标志物通常用于识别可能受益于检查点免疫疗法的患者。从部门档案中确定了一名 39 岁男性（病例 1）和两名 13 岁女性（病例 2、3）的三例病例，经 NUT IHC 确认和荧光原位杂交确认 15q14 重排。应用正常肿瘤配对全外显子组测序 (WES) 来确定 TMB。还对 DNA 错配修复蛋白、程序性细胞死亡配体 1、程序性细胞死亡 1 (PD1) 和 CD8 进行了 IHC。WES 的 TMB 为 7.61 和 1。病例 1 的原发灶和肺转移瘤分别为 52/Mbp，病例 2 的原发肿瘤 TMB 为 1.04/Mbp。 程序性细胞死亡配体 1 肿瘤比例评分为 20%、1% 和 0%，合并病例 1、2 和 3 的阳性分数分别为 25、5 和 0；每个高倍视野的 PD1 染色计数为 25、52 和 35，并且病例 1、2 和 3 的 PD1/CD8 比率分别为 95%、95% 和 99%。在病例 1、2 和 3 中，每个高倍视野的 CD8 计数分别为 15、33 和 30。错配修复 IHC 显示保留染色。尽管病例数有限，但这项研究是第一个研究 NMC 中检查点免疫治疗标志物的研究，结果表明没有明确的生物标志物关联。然而，结果表明，如果正在考虑检查点治疗，