Array comparative genomic hybridization analysis of flat epithelial atypia (DIN1a) and lobular intraepithelial neoplasia. Homo sapiens

Low grade flat ductal intraepithelial neoplasia (DIN1a, flat epithelial atypia) is one of the earliest morphologically recognizable neoplastic lesions of the breast. Frequently, it occurs in association with lobular intraepithelial neoplasia (LIN). The aim of this study was to elucidate chromosomal aberrations in these early neoplastic breast lesions using array comparative genomic hybridization (CGH) analysis. Laser capture microdissection of 12 archival formalin-fixed, paraffin-embedded specimens harbouring both foci of DIN1a as well as LIN was performed. All analyzed cases of DIN1a and LIN showed chromosomal gains and losses. The aberration encountered most often was loss on 16q in 7 DIN1a (70%) and 10 LIN (91%) cases. Regarding changes in chromosome 1, four DIN1a (40%) and 7 LIN (64%) cases showed a gain on 1q. The results of our study show concurrent chromosomal aberrations of 1q gains and 16q losses in several cases with coexisting LIN and low grade flat DIN. These aberrations are known to be common in low grade invasive ductal carcinomas as well as more advanced (conventional) types of low grade DIN (low grade ductal carcinoma in-situ). Our results raise the possibility of similar molecular-genetic pathways in most of the cases with coexisting LIN and low grade flat DIN. Overall design: In this study, 11 cases of classic lobular intraepithelial neoplasias and 10 cases with several areas of low grade flat DIN (DIN1a or flat epithelial atypia) were analyzed by array CGH. Sections of formalin-fixed paraffin-embedded (FFPE) specimens cut at 7-8µm were mounted on special foil-coated slides (Molecular Devices, San Diego, USA). The sections were then deparaffinized with Xylene, processed in decreasing concentrations of ethanol and stained with haematoxylin. Lasercapture microdissection for both lesions was performed at multiple sites using Veritas Arcturus. For reference-DNA, female mammary tissue without histomorphological changes obtained from reduction mammoplasty specimens was procecessed and laser-microdissected as explained above. Cells were digested in 10µl TE, pH 9, and 0.5µl proteinase K (20mg/ml) for 48h at 55°C. After inactivation of proteinase K at 99°C for 10min, the digest was stored at -20°C. Without any further purification, the complete digest was used for whole genome amplification by means of the WGA (Whole Genome Amplification) kit from Sigma following the manufacturer’s recommendations. Array CGH was performed as described previously. In brief, two µg of amplified tumor and reference DNA were labelled by random priming (BioPrime® Total Genomic Labeling System, Invitrogen, Carlsbad, CA) with Alexa Fluor® 3 and Alexa Fluor® 5, respectively, and hybridized onto a tiling path BAC array, consisting of the human 32k BAC Re-Array Set (BACPAC Resources Center; http://bacpac.chori.org/pHumanMinSet.htm; DNA kindly provided by Pieter de Jong) and a 1Mb Resolution BAC set (clones kindly provided by Nigel Carter, Wellcome Trust Sanger Centre). All protocols are provided in detail on our website (http://www.molgen.mpg.de/~abt_rop/molecular_cytogenetics/) and more information concerning this platform have been submitted to the Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/; GPL5114). For the analysis and visualization of array CGH data, our software-package CGH-PRO was employed. No background subtraction was applied. Raw data were normalized by “Subgrid LOWESS”. For the assessment of copy number gains and losses, we used circular binary segmentation in combination with log2 ratio thresholds of 0.15 and -0.15, respectively.

低级别平坦型导管上皮内瘤变（low grade flat ductal intraepithelial neoplasia，简称DIN1a），又称平坦上皮异型增生（flat epithelial atypia），是乳腺中最早可通过形态学识别的肿瘤性病变之一。该病变常与小叶上皮内瘤变（lobular intraepithelial neoplasia，简称LIN）合并存在。本研究旨在通过阵列比较基因组杂交（array comparative genomic hybridization，简称CGH）分析，阐明这类早期乳腺肿瘤性病变的染色体畸变情况。研究人员对12例同时存在DIN1a与LIN病灶的存档福尔马林固定石蜡包埋（formalin-fixed, paraffin-embedded，简称FFPE）标本进行了激光捕获显微切割（laser capture microdissection，LCM）。所有纳入分析的DIN1a与LIN病例均存在染色体拷贝数的增益与丢失。最常见的染色体畸变为16号染色体长臂缺失，在7例DIN1a病例（占比70%）与10例LIN病例（占比91%）中均检测到该畸变。针对1号染色体的变异，4例DIN1a病例（占比40%）与7例LIN病例（占比64%）存在1号染色体长臂的拷贝数增益。

本研究结果显示，在多例合并存在LIN与低级别平坦型DIN的病例中，均同时存在1q增益与16q缺失的染色体畸变。已知这类畸变在低级别浸润性导管癌，以及更晚期的（经典型）低级别DIN（即低级别导管原位癌（low grade ductal carcinoma in-situ））中均较为常见。本研究结果提示，在多数合并存在LIN与低级别平坦型DIN的病例中，可能存在相似的分子遗传通路。

整体实验设计：本研究通过阵列CGH分析了11例经典型小叶上皮内瘤变病例，以及10例存在多处低级别平坦型DIN（DIN1a或平坦上皮异型增生）病灶的病例。将厚度为7~8μm的福尔马林固定石蜡包埋（FFPE）标本切片贴附于特制的箔包被玻片（美国加利福尼亚州圣地亚哥，Molecular Devices公司）上。随后将切片经二甲苯脱蜡，依次通过梯度乙醇脱水，并以苏木精进行染色。使用Veritas Arcturus设备对两种病变的多个位点进行激光捕获显微切割。参考DNA则取自因乳房缩小整形术获取的、无组织形态学改变的女性乳腺组织，并按照前述步骤进行处理与激光显微切割。将切割得到的细胞置于10μl pH 9的TE缓冲液与0.5μl浓度为20mg/ml的蛋白酶K中，于55℃消化48小时。随后将蛋白酶K于99℃灭活10分钟，消化产物保存于-20℃冰箱。无需额外纯化，直接将全部消化产物按照Sigma公司全基因组扩增（Whole Genome Amplification，简称WGA）试剂盒的操作说明进行全基因组扩增。

阵列CGH实验按照此前报道的方法进行。简言之，取2μg扩增后的肿瘤DNA与参考DNA，分别采用随机引物标记法（BioPrime® 全基因组标记系统，美国加利福尼亚州卡尔斯巴德，Invitrogen公司）以Alexa Fluor® 3与Alexa Fluor® 5进行荧光标记，随后杂交至铺瓦式路径BAC芯片。该芯片包含人类32k BAC重阵列套装（BACPAC资源中心；http://bacpac.chori.org/pHumanMinSet.htm；DNA由Pieter de Jong惠赠）与1Mb分辨率BAC套装（克隆由Nigel Carter、惠康信托桑格研究所惠赠）。所有实验方案的详细内容均可在本团队网站（http://www.molgen.mpg.de/~abt_rop/molecular_cytogenetics/）查阅，有关该芯片平台的更多信息已提交至基因表达综合数据库（Gene Expression Omnibus，简称GEO；http://www.ncbi.nlm.nih.gov/geo/；编号GPL5114）。针对阵列CGH数据的分析与可视化，本研究采用自主开发的CGH-PRO软件包完成。实验未进行背景扣除操作，原始数据通过“亚网格LOWESS法”进行标准化。针对拷贝数增益与丢失的判定，我们采用环状二元分割算法，并分别设置log2比值阈值0.15与-0.15。