Böni R, Matt D, Voetmeyer A, Burg G, Zhuang Z
Department of Dermatology, University Hospital, Zürich, Switzerland.
J Invest Dermatol. 1998 Mar;110(3):215-7. doi: 10.1046/j.1523-1747.1998.00109.x.
Loss of heterozygosity at specific loci in neoplastic cells suggests the presence of a tumor suppressor gene within the deleted region. Using the microdissection technique, loss of heterozygosity has been identified in paraffin-embedded primary melanomas on chromosomes 1p and 9q. Our purpose was to determine loss of heterozygosity in primary cutaneous melanomas and to relate chromosomal alterations with cell morphology and proliferation of the tumor. Two to seven morphologic different areas in 12 primary cutaneous high risk melanomas (Breslow > 1.5 mm), as well as adjacent normal tissue, were microdissected and subjected to single step DNA extraction. Extracted genomic DNA was amplified by polymerase chain reaction using two polymorphic markers on chromosomes 1p (D1S450, between D1S548 and D1S228) and 9q (D9S12). Proliferation was evaluated by MIB-1 (Ki67) immunoreactivity and cell morphology, pigmentation and inflammation surrounding microdissected areas were investigated by microscopic inspection of hematoxylin and eosin stained sections. Twelve of 34 different areas (35%) showed loss of heterozygosity with at least one marker. Two of 32 areas showed loss of heterozygosity with D1S450 (two areas noninformative), seven of 30 with D9S12 (four areas noninformative), and three areas showed loss of heterozygosity at both loci. In three of these cases, analysis of different tumor foci revealed areas with/without loss of heterozygosity. In these cases, the percentage of MIB-I-positive cells was at least four times higher in areas with loss of heterozygosity compared with areas without loss of heterozygosity. Most areas with loss of heterozygosity consisted of small cuboidal to epitheloid cells. Spindle shaped and large anaplastic cells showed loss of heterozygosity less frequently. Neither melanization of tumor cells nor the presence of inflammation had an influence on the frequency of loss of heterozygosity. Primary cutaneous melanomas show intratumoral morphologic and chromosomal heterogeneity. Loss of heterozygosity on chromosomes 1p and 9q correlated with cell proliferation, suggesting that selected cell clones are responsible for tumor progression.
肿瘤细胞中特定位点杂合性的缺失表明在缺失区域存在一个肿瘤抑制基因。运用显微切割技术，已在石蜡包埋的原发性黑色素瘤的1号染色体短臂和9号染色体长臂上检测到杂合性缺失。我们的目的是确定原发性皮肤黑色素瘤中的杂合性缺失情况，并将染色体改变与肿瘤的细胞形态和增殖联系起来。对12例原发性皮肤高危黑色素瘤（ Breslow厚度>1.5 mm）中两到七个形态学不同的区域以及相邻的正常组织进行显微切割，并进行单步DNA提取。提取的基因组DNA通过聚合酶链反应，使用位于1号染色体短臂（D1S450，在D1S548和D1S228之间）和9号染色体长臂（D9S12）上的两个多态性标记进行扩增。通过MIB-1（Ki67）免疫反应性评估增殖情况，并通过苏木精和伊红染色切片的显微镜检查来研究显微切割区域周围的细胞形态、色素沉着和炎症情况。34个不同区域中的12个（35%）显示至少一个标记存在杂合性缺失。32个区域中有2个在D1S450处显示杂合性缺失（2个区域无信息），30个区域中有7个在D9S12处显示杂合性缺失（4个区域无信息），3个区域在两个位点均显示杂合性缺失。在其中3例中，对不同肿瘤灶的分析显示存在/不存在杂合性缺失的区域。在这些病例中，杂合性缺失区域中MIB-1阳性细胞的百分比比无杂合性缺失区域至少高四倍。大多数杂合性缺失区域由小立方形至上皮样细胞组成。梭形和大的间变细胞显示杂合性缺失的频率较低。肿瘤细胞的黑色素化和炎症的存在均未对杂合性缺失的频率产生影响。原发性皮肤黑色素瘤显示肿瘤内的形态学和染色体异质性。1号染色体短臂和9号染色体长臂上的杂合性缺失与细胞增殖相关，提示特定的细胞克隆负责肿瘤进展。
