Genomic Analysis of Early Hereditary Diffuse Gastric Cancer

Abstract

Diffuse gastric cancer (DGC) is one of the two main types of stomach cancer. Histologically, DGC can be subdivided into poorly differentiated carcinoma and signet ring cell carcinoma (SRCC). Carriers of germline mutations in the gene encoding E-cadherin (CDH1) are predisposed to DGC (hereditary DGC, HDGC). Essentially all asymptomatic mutation carriers present with occult, multifocal SRCC confined to the gastric mucosa before they develop advanced disease. Initial characterisation of these early HDGC foci (referred to as eHDGC) suggests an indolent nature of these lesions, contrary to their current pathological definition as malignant disease. The main aim of this study was to determine if genomic instability is an early event in HDGC and how it might lead to disease progression. We assessed chromosomal aberrations in the early intramucosal eHDGCs by using array comparative genomic hybridisation (CGH). Taking advantage of possibly the largest eHDGC collection worldwide, we analysed tissue from twelve members of one HDGC family, all carrying the same CDH1 mutation. All specimens were formalin-fixed paraffin-embedded (FFPE), as eHDGC is generally identified in resected stomachs by microscopy due to both its intramucosal location and the small size of the foci. As a result, the first part of this study focussed on the optimisation and establishment of techniques for the genomic analysis of degraded DNA from minimal amounts of laser-capture microdissected (LCM) neoplastic cells. In the first step, we assessed several histological stains for their suitability in identifying neoplastic cells found in eHDGCs, followed by an optimisation of the LCM procedure to obtain consistent dissection of individual cells. Next, we evaluated different DNA extraction parameters to obtain sufficient amounts of DNA for genomic profiling and established a multiplex PCR reaction as a quality control for the prediction of successful profiling. Since no agreement exists on the most suitable platform for the profiling of FFPE-DNA, two different array CGH systems were compared. DNA from FFPE and matched fresh-frozen (FF) tissue was separately analysed on both a SNP-based array (Affymetrix) and on an oligonucleotide-based array (Agilent), and resulting profiles were evaluated for the level of agreement between matched pairs of FFPE and FF samples. On the SNP-array, a substantial increase in apparent copy number alterations was observed in all FFPE tissues relative to their matched FF counterparts. In contrast, FFPE and matched FF genomic profiles obtained via the oligonucleotide array were highly concordant, indicating the reliability of this platform for the analysis of degraded DNA. We then proceeded with the genomic analysis of the early HDGC foci. For all twelve HDGC patients, 30,000 cells from each of three tissue types, signet ring cells (SRC), poorly differentiated cells (PDCs) and non-malignant epithelial cells were dissected and analysed using the Agilent platform. Chromosomal aberrations were found in SRCs and PDCs, and confirmed by multiplex ligation dependent probe amplification (MLPA). No changes were detected that were common to all patients. Accordingly, no common genes, pathways, or miRNAs were identified to be altered in eHDGC, suggesting the aberrations had not yet undergone clonal selection. Importantly, no aneuploidy or other large-scale chromosomal rearrangements were detected. Instead, all aberrations affected small regions (< 4.8 Mb) and were predominantly deletions. Analysis of DNA sequence patterns revealed that essentially all aberrations possess the characteristics of common fragile sites. The presence of genomic instability at the fragile sites in the absence of widespread genomic instability was highly consistent with eHDGC being relatively indolent. We therefore examined by immunohistochemistry the activity of key DNA repair proteins in eHDGC. ATM-H2AX-CHK2 were consistently expressed and activated in all examined eHDGC foci, indicating an intact and activated DNA repair system accompanying genomic instability at fragile sites. Altogether, this study has established the methodology for the successful CGH profiling of FFPE tissue and has provided the first genetic characterisation of the earliest stage of DGC development. This analysis and subsequent immunohistochemical examination has shown that early stage intramucosal DGCs are (unlike advanced DGC) typified by low levels of genomic instability at fragile sites. Moreover, they express an active DNA damage response (DDR), providing a molecular basis for the observed indolence of eHDGC