早中期肢端型皮肤恶性黑素瘤肿瘤内部异质性与基因拷贝数变异及单核苷酸变异相关性研究

Malignant melanoma (MM) has significant intratumoral heterogeneity. A comparative analysis of clinical phenotypes (based on color, degree of proliferation), different pathological states and genetic characteristics within the primary tumor will help us understand the patterns and mechanisms through which MM develops, as well as promote diagnostics and molecularly targeted therapies. However, due to technical limitimations, few correlative studies have been performed to investigate pathological and genetic heterogeneities underlying MM. As MM tumor cells are scattered, studies of the heterogeneity within the tumor at Phase III or earlier stages are rare. .In this study, the advanced single-cell sequencing technolgoy will be used in cooperation to study the most common MM in China, the acral type MM at or earlier than phase III. Specific tumor cell subpopulations from phenotyically different areas within the same lesion will be identifiyed by immunohistochemistry and isolated by laser capture microdissection. These cell subpopulations will then be analyzed on a single-cell sequencing platform for gene copy number variations (CNV) and single nucleotide variation (SNV) to characterize each tumor case's genetic heterogeneity. Furthermore, comparative analyses of characteristics in pathology, CNV and SNV in phenotypically different areas of the same tumor will be performed to uncover their correlations and establish tumor phylogenetic trees, thereby providing new insights into the development of early diagnosis and targeted therapies.

恶性黑素瘤（malignant melanoma，MM）具有显著的肿瘤内部异质性，比较研究MM原发灶内不同表型（依据色泽、增生程度）、不同病理表现的肿瘤细胞的遗传学特征，通过分析其中的共性和异质性，有助于了解MM发生发展的规律和机制，对于诊断和分子靶向治疗也有指导意义。然而限于技术，既往少有MM病灶内部组织学和遗传学异质性的比较研究，由于肿瘤细胞散在分布，更少见针对III期以内MM的肿瘤内部异质性研究。.本研究利用成熟合作的单细胞测序新技术，拟针对III期以内肢端型皮肤恶性黑素瘤（中国常见肢端型），选取原发灶内不同表型区域组织，通过激光捕获显微切割分别获取微量肿瘤细胞，从基因拷贝数变异（CNV）与单核苷酸变异（SNV）入手，进行恶性黑素瘤肿瘤内部异质性比较研究，总结不同表型区域里肿瘤细胞的病理、CNV、SNV特征表现，归纳相关性，建立肿瘤进化树，为MM早期诊断和靶向治疗提供借鉴。

肢端恶性黑素瘤是中国人好发的黑素瘤亚型，从临床表现、分子特征到治疗方法选择都与高加索人群高发的浅表扩散型黑素瘤有显著差异。比较研究肢端黑素瘤原发灶内不同表型（依据色泽、增生程度）区域及不同病理表现的肿瘤细胞遗传学特征，分析异质性，有助于了解这种中国人高发肿瘤发生发展的规律和机制，对于诊断和分子靶向治疗也有指导意义。限于技术和病例分布人群特点，既往少有针对早中期肢端黑素瘤病灶内部组织学和遗传学异质性的比较研究。.针对早中期肢端黑素瘤细胞巢小且分散，本项目组解决了既往获取纯肿瘤细胞及微量DNA进行全外显子测序的瓶颈问题。具体方法是利用激光捕获显微切割技术，从组织切片中获取微小肿瘤细胞巢（含20余个细胞），然后应用Malbac技术（一种为单细胞基因测序开发的高效全基因组扩增方法）扩增每组细胞，从而准确描绘出该肿瘤巢中细胞的基因拷贝数变异（CNV）和单核苷酸变异（SNV）情况。.研究在4个层面比较了基于CNV与SNV的异质性：同患者黑素瘤和色素痣之间，不同患者黑素瘤之间，同一黑素瘤病灶不同表型之间，以及相同临床表型且在病理下相邻的肿瘤细胞巢之间。研究发现上述4个层面上都存在CNV与SNV显著差异，异质性明显。基于异质性与临床和病理表型的相关性，项目组还成功构建了肢端恶性黑色素瘤细胞的进化树，发现其在肿瘤早期呈现为分支进化模式。研究结果提示了肢端型恶性黑素瘤肿瘤细胞的主干变异特征及不同临床表型的分支变异特征，初步描绘了肿瘤细胞的进化路径。.研究结果有助于寻找不同进展阶段肢端恶性黑素瘤细胞的遗传学变异特征，进而作为分子诊断标记指导临床精确分期和靶向治疗。肿瘤内部多层次的显著异质性也进一步提示未来治疗应该是多维度和多靶向。