Hypermutator-type colorectal carcinomas are microsatellite-stable and have point mutations of the exonuclease domain of the DNA polymerase ε or δ genes (POLE and POLD1, respectively), and an ultrahigh tumor mutational burden (TMB). These tumors may be associated with enhanced antitumor immunity and preferentially afflict younger patients, but this notion awaits validation by accrual of further cases for detailed correlative phenotypic and molecular study. We performed POLE and POLD1 exonuclease domain Sanger sequencing of 271 unselected colorectal carcinomas. We identified two microsatellite-stable tumors with somatic POLE p.P286R variants, both with ultrahigh TMBs as demonstrated by whole exome sequencing. A POLE p.V411L was found in another two microsatellite-stable tumors with ultrahigh TMBs. Two of these four tumors were from young patients (<50 years old, nonsyndromic), and there was seen a prominent T-cell infiltration in three of them. Furthermore, a somatic POLE p.A465T was found in a Lynch-associated tumor, which, hypothetically, might have enhanced TMB (which was the highest of all). In two tumors, a somatic POLE p.V411L and a POLD1 p.E279K, respectively, were found only focally, and TMBs were low. It is commonly assumed that compromise of one allele is sufficient, but this has not been specifically addressed. Therefore, resequencing of the POLE or POLD1 mutations was done with DNA from tumor cells isolated by laser-capture microdissection. This demonstrated that the mutations were monoallelic, and there was no evidence of a "second hit", neither by allelic loss (allelotyping with polymorphic microsatellite markers), nor by promoter methylation (Pyromark CpG assays). Taken together, including at least the more common DNA polymerase mutations in NGS panels allows for straightforward identification of hypermutator-type colorectal carcinomas which often may be "immunoreactive". This is important at least in young patients or when a metastasizing stage of disease has been reached and immune-checkpoint therapy enters deliberation.

中文翻译：

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具有单等位基因 POLE 突变的结直肠癌的高突变负荷：不存在等位基因丢失和基因启动子甲基化。

超突变型结直肠癌是微卫星稳定的，具有 DNA 聚合酶 ε 或 δ 基因（分别为 POLE 和 POLD1）的核酸外切酶结构域的点突变，以及超高肿瘤突变负荷 (TMB)。这些肿瘤可能与增强的抗肿瘤免疫力有关，并优先折磨年轻患者，但这一观点有待通过更多病例的累积进行详细的相关表型和分子研究来验证。我们对 271 个未选择的结直肠癌进行了 POLE 和 POLD1 核酸外切酶域 Sanger 测序。我们鉴定了两个具有体细胞 POLE p.P286R 变体的微卫星稳定肿瘤，两者都具有超高 TMB，如全外显子组测序所证明的。在另外两个具有超高 TMB 的微卫星稳定肿瘤中发现了一个 POLE p.V411L。这四个肿瘤中有两个来自年轻患者（<50 岁，非综合征），其中三个有明显的 T 细胞浸润。此外，在 Lynch 相关肿瘤中发现了体细胞 POLE p.A465T，据推测，该肿瘤可能增强了 TMB（这是所有肿瘤中最高的）。在两个肿瘤中，仅局部发现了体细胞 POLE p.V411L 和 POLD1 p.E279K，并且 TMB 很低。通常假设一个等位基因的妥协就足够了，但这还没有具体解决。因此，对 POLE 或 POLD1 突变的重测序是用激光捕获显微切割法分离的肿瘤细胞的 DNA 完成的。这表明突变是单等位基因的，并且没有“二次打击”的证据，既不是通过等位基因丢失（使用多态性微卫星标记进行等位基因分型），也不是通过启动子甲基化（Pyromark CpG 测定）。总之，至少包括 NGS panel 中更常见的 DNA 聚合酶突变，可以直接识别通常可能具有“免疫反应性”的超突变型结直肠癌。这至少对年轻患者或当疾病已达到转移阶段并且免疫检查点疗法进入审议阶段时很重要。