X染色体灭活方式检测在诊断头颈鳞状细胞癌颈淋巴微转移中的作用

目的探讨X染色体灭活检测肿瘤细胞克隆来源在诊断头颈鳞癌颈淋巴结微转移中的作用。方法对20例临床N0M0头颈部鳞癌包括10例术后病理诊断明确的颈淋巴结微转移癌和10例可疑颈淋巴结微转移癌，通过组织显微切割和蛋白酶K消化技术获得肿瘤组织DNA，在限制性酶切和PCR扩增后观察肿瘤细胞雄激素受体在X染色体上的标志，明确X染色体灭活情况；通过比较原发癌和转移灶或可疑微转移灶细胞的检测结果鉴定转移部位细胞与原发癌肿瘤细胞的克隆同源性，进而对颈淋巴转移情况做出正确诊断。结果10例不同程度表达肿瘤细胞表面标志、病理诊断明确的颈淋巴结转移癌与其对应的原发癌均为单克隆来源，并且具有相同的X染色体灭活方式，提示两者之间具有相同的克隆来源，证明颈淋巴结转移癌来自原发癌；10例原发灶不同程度地表达表皮生长因子受体（epidermal growth factor receptor，EGFR）和角蛋白，但颈淋巴结内可疑微转移灶不表达EGFR和角蛋白的可疑颈淋巴结微转移癌，X染色体灭活分析发现6例可疑转移灶细胞与原发癌细胞具有克隆同源性，证实为颈淋巴结内微转移；其余4例两者间不具有相同的细胞克隆来源，排除淋巴结转移癌。结论采用x染色体灭活法检测肿瘤细胞的克隆来源对诊断头颈肿瘤颈淋巴结微转移具有很好的应用前景和潜在临床实用价值。

Role of clonality analysis by X-chromosome inactivation in the diagnosis of cervical lymph node occult micrometastasis from squamous carcinoma of the head and neck

Objective To investigate the role of clonality analysis by X-chromosome inactivation in the diagnosis of cervical lymph node metastasis from squamous carcinoma of the head and neck. Methods Twenty cases of clinical N0M0 squamous carcinoma of the head and neck with either pathologically confirmed or suspected occult micrometastsis in the cervical lymph node were recruited. Interested DNA samples were procured through tissue microdissection and one-step proteinase K digestion, and the clonality analysis was carried out by means of restriction enzyme digestion and amplification of human androgen receptor markers(HUMURA) to check out the status of X-chromosome inactivation. The clonal origin of the primary tumor cells and the interested cell clones in the cervical lymph node was traced by X-chromosome inactivation,and the diagnosis of cervical lymph node micrometastasis was either confirmed or ruled out. Results Tumor cells from both primary and metastatic lesions were monoclonal and identical in clonal origin in 10 patients with pathologically confirmed cervical lymph node metastasis, whose metastatic tumor cells expressed EGF receptor. For 10 patients with suspected micrometastasis in the neck nodes, whose focused lesions did not expressed any EGF receptor protein by immunohistochemistry, the identical and monoclonal origin between the primary tumor and the suspected metastatic lesion in the neck node was confirmed in 6 patients, and the polyclonal origin of the neck node lesions was revealed in other 4 patients. The diagnosis of micrometastasis in the neck node was thus ascertained in 6 and ruled out in 4 suspected cases. Conclusions Examination of X-chromosome inactivation pattern is a useful method for identification of the neck node occult micrometastasis from squamous carcinoma of the head and neck.