Laco Jan, Chmelařová Marcela, Vošmiková Hana, Sieglová Kateřina, Bubancová Ivana, Dundr Pavel, Němejcová Kristýna, Michálek Jaroslav, Čelakovský Petr, Mottl Radovan, Sirák Igor, Vošmik Milan, Ryška Aleš
The Fingerland Department of Pathology, Charles University, Faculty of Medicine and University Hospital in Hradec Kralove, Czechia.
Institute for Clinical Biochemistry and Diagnostics, Charles University, Faculty of Medicine and University Hospital in Hradec Kralove, Czechia.
Pathol Res Pract. 2017 Feb;213(2):133-142. doi: 10.1016/j.prp.2016.10.012. Epub 2016 Oct 25.
The aim of the study was detailed clinicopathological investigation of SMARCB1/INI1-deficient sinonasal carcinomas, including molecular genetic analysis of mutational status and DNA methylation of selected protooncogenes and tumor suppressor genes by means of next generation sequencing (NGS) and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). A total of 4/56 (7%) cases of SMARCB1/INI1-deficient carcinomas were detected among 56 sinonasal carcinomas diagnosed over a 19year period using immunohistochemical screening. The series comprised 3 males and 1 female, aged 27-76 years (median 64 years). All tumors arose in the nasal cavity. Three neoplasms were diagnosed in advanced stage pT4. During the follow-up period (range 14-111 months (median 72 months)), three tumors recurred locally, but none of the patients developed regional or distant metastases. Ultimately, two patients died due to the tumor. Microscopically, all tumors consisted of infiltrating nests of polygonal basaloid cells with a variable component of rhabdoid cells with eosinophilic cytoplasm. Immunohistochemically, there was almost diffuse expression of cytokeratins (CK), p16, p40 and p63 in all cases, while expression of CK5/6, CK7 and vimentin was only focal or absent. The detection of NUT gave negative results. In three cases, the absence of SMARCB1/INI1 expression was due to deletion of SMARCB1/INI1 gene. Methylation of SMARCB1/INI1 gene was not found. One tumor harbored HPV18 E6/E7 mRNA. All 12 genes (BRAF, BRCA1, BRCA2, KIT, EGFR, KRAS, NRAS, PDGFRA, PIK3CA, PTEN, RET, and ROS1) tested for mutations using NGS were wild-type. Regarding DNA methylation, all four SMARCB1/INI1-deficient tumors showed methylation of RASSF1 gene by means of MS-MLPA. There was a statistically significant difference in RASSF1 gene methylation between SMARCB1/INI1-deficient and SMARCB1/INI1-positive tumors (p=0.0095). All other examined genes (ATM, BRCA1, BRCA2, CADM1, CASP8, CD44, CDKN1B, CDKN2A, CDKN2B, CHFR, DAPK1, ESR1, FHIT, GSTP1, HIC1, KLLN, MLH1a, MLH1b, RARB, and VLH) were unmethylated. In summary, we described four cases of SMARCB1/INI1-deficient sinonasal carcinoma with detailed clinicopathological data indicating that these tumors can be regarded as a distinct entity with aggressive behaviour. For the first time, we performed analysis of DNA methylation in SMARCB1/INI1-deficient sinonasal carcinomas, reporting on significantly higher methylation of RASSF1 gene in this neoplasm.
本研究的目的是对SMARCB1/INI1缺陷型鼻窦癌进行详细的临床病理研究，包括通过下一代测序（NGS）和甲基化特异性多重连接依赖探针扩增（MS-MLPA）对选定原癌基因和抑癌基因的突变状态和DNA甲基化进行分子遗传学分析。在19年期间诊断的56例鼻窦癌中，通过免疫组化筛查共检测到4/56（7%）例SMARCB1/INI1缺陷型癌。该系列包括3名男性和1名女性，年龄27 - 76岁（中位年龄64岁）。所有肿瘤均发生于鼻腔。3例肿瘤诊断为晚期pT4。在随访期（范围14 - 111个月（中位72个月）），3例肿瘤局部复发，但无一例患者发生区域或远处转移。最终，2例患者死于肿瘤。显微镜下，所有肿瘤均由多边形基底样细胞浸润巢组成，伴有不同比例的具有嗜酸性细胞质的横纹肌样细胞。免疫组化方面，所有病例中细胞角蛋白（CK）、p16、p40和p63几乎呈弥漫性表达，而CK5/6、CK7和波形蛋白的表达仅为局灶性或缺失。NUT检测结果为阴性。3例中，SMARCB1/INI1表达缺失是由于SMARCB1/INI1基因缺失。未发现SMARCB1/INI1基因甲基化。1例肿瘤含有HPV18 E6/E7 mRNA。使用NGS检测的所有12个基因（BRAF、BRCA1、BRCA2、KIT、EGFR、KRAS、NRAS、PDGFRA、PIK3CA、PTEN、RET和ROS1）均为野生型。关于DNA甲基化，所有4例SMARCB1/INI1缺陷型肿瘤通过MS-MLPA显示RASSF1基因甲基化。SMARCB1/INI1缺陷型和SMARCB1/INI1阳性肿瘤之间RASSF1基因甲基化存在统计学显著差异（p = 0.0095）。所有其他检测基因（ATM、BRCA1、BRCA2、CADM1、CASP8、CD44、CDKN1B、CDKN2A、CDKN2B、CHFR、DAPK1、ESR1、FHIT、GSTP1、HIC1、KLLN、MLH1a、MLH1b、RARB和VLH）均未甲基化。总之，我们描述了4例SMARCB1/INI1缺陷型鼻窦癌，并提供了详细的临床病理数据，表明这些肿瘤可被视为具有侵袭性的独特实体。我们首次对SMARCB1/INI1缺陷型鼻窦癌进行了DNA甲基化分析，报告了该肿瘤中RASSF1基因甲基化显著升高。
