Marjan M. Weiss

Comparative genomic hybridisation.

Comparative genomic hybridisation (CGH) is a technique that permits the detection of chromosomal copy number changes without the need for cell culturing. It provides a global overview of chromosomal gains and losses throughout the whole genome of a tumour. Tumour DNA is labelled with a green fluorochrome, which is subsequently mixed (1:1) with red labelled normal DNA and hybridised to normal human metaphase preparations. The green and red labelled DNA fragments compete for hybridisation to their locus of origin on the chromosomes. The green to red fluorescence ratio measured along the chromosomal axis represents loss or gain of genetic material in the tumour at that specific locus. In addition to a fluorescence microscope, the technique requires a computer with dedicated image analysis software to perform the analysis. This review aims to provide a detailed discussion of the CGH technique, and to provide a protocol with an emphasis on crucial steps.

Genomic Alterations in Primary Gastric Adenocarcinomas Correlate with Clinicopathological Characteristics and Survival

Background & aims: Pathogenesis of gastric cancer is driven by an accumulation of genetic changes that to a large extent occur at the chromosomal level. In order to investigate the patterns of chromosomal aberrations in gastric carcinomas, we performed genome‐wide microarray based comparative genomic hybridisation (microarray CGH). With this recently developed technique chromosomal aberrations can be studied with high resolution and sensitivity. Methods: Array CGH was applied to a series of 35 gastric adenocarcinomas using a genome‐wide scanning array with 2275 BAC and P1 clones spotted in triplicate. Each clone contains at least one STS for linkage to the sequence of the human genome. These arrays provide an average resolution of 1.4 Mb across the genome. DNA copy number changes were correlated with clinicopathological tumour characteristics as well as survival. Results: All thirty‐five cancers showed chromosomal aberrations and 16 of the 35 tumours showed one or more amplifications. The most frequent aberrations are gains of 8q24.2, 8q24.1, 20q13.12, 20q13.2, 7p11.2, 1q32.3, 8p23.1–p23.3, losses of 5q14.1, 18q22.1, 19p13.12–p13.3, 9p21.3–p24.3, 17p13.1–p13.3, 13q31.1, 16q22.1, 21q21.3, and amplifications of 7q21–q22, and 12q14.1–q21.1. These aberrations were correlated to clinicopathological characteristics and survival. Gain of 1q32.3 was significantly correlated with lymph node status (p=0.007). Tumours with loss of 18q22.1, as well as tumours with amplifications were associated with poor survival (p=0.02, both). Conclusions: Microarray CGH has revealed several chromosomal regions that have not been described before in gastric cancer at this frequency and resolution, such as amplification of at 7q21–q22 and 12q14.1–q21.1, as well gains at 1q32.3, 7p11.2, and losses at 13q13.1. Interestingly, gain of 1q32.3 and loss of 18q22.1 are associated with a bad prognosis indicating that these regions could harbour gene(s) that may determine aggressive tumour behaviour and poor clinical outcome.

Genomic profiling of gastric cancer predicts lymph node status and survival

Gastric carcinogenesis is driven by an accumulation of genetic changes that to a large extent occur at the chromosomal level. We analysed the patterns of chromosomal instability in 35 gastric carcinomas and their clinical correlations. With microarray competitive genomic hybridization, genomewide chromosomal copy number changes can be studied with high resolution and sensitivity. A genomewide scanning array with 2275 BAC and P1 clones spotted in triplicate was used. This array provided an average resolution of 1.4u2009Mb across the genome. Patterns of chromosomal aberrations were analysed by hierarchical cluster analysis of the normalized log2 tumour to normal fluorescence ratios of all clones, and cluster membership was correlated to clinicopathological data including survival. Hierarchical cluster analysis revealed three groups with different genomic profiles that correlated significantly with lymph node status (P=0.02). Moreover, gastric cancer cases from cluster 3 showed a significantly better prognosis than those from clusters 1 and 2 (P=0.02). Genomic profiling of gastric adenocarcinomas based on microarray analysis of chromosomal copy number changes predicted lymph node status and survival. The possibility to discriminate between patients with a high risk of lymph node metastasis could clinically be helpful for selecting patients for extended lymph node resection.

Determination of amplicon boundaries at 20q13.2 in tissue samples of human gastric adenocarcinomas by high-resolution microarray comparative genomic hybridization.

Comparative genomic hybridization (CGH) of gastric adenocarcinomas frequently shows gains and amplifications of chromosome 20. However, the underlying genetic lesion is unknown and conventional CGH results do not allow specification of the target region. In order to investigate this chromosomal aberration with a higher resolution and sensitivity, microarray‐based CGH was performed with both scanning and high‐resolution arrays of chromosome 20 in a series of 27 gastric adenocarcinomas. Locus‐specific fragments of genomic DNA from bacterial artificial chromosome (BAC) clones were spotted as microarrays. A scanning array contained a set of 27 BAC clones covering chromosome 20q. A high‐resolution array contained 27 overlapping BAC clones at 20q13.2. This high‐resolution array was used to narrow down the amplicon at 20q13.2 in tumours showing amplification of this chromosomal region with the scanning array. Positive copy number changes on chromosome 20q were detected in 12 of 27 cases (44%). These changes included gain of the whole arm of chromosome 20q in 8 of 27 (30%) cases, amplification restricted to 20q12.1 in one case, and amplifications restricted to 20q13 in three cases (11%). The three tumours showing amplification restricted to 20q13 were analysed further using the high‐resolution array. In one tumour, the whole contig was amplified at a constant level. One of the other two tumours had a clear proximal breakpoint, while the other tumour had a clear distal breakpoint within the 20q13.2 region. The proximal and the distal breakpoint were approximately 800 kb apart. In the present study, an amplicon at 20q13.2 has been narrowed down to 800 kb which is likely to harbour one or more putative oncogenes relevant to gastric carcinogenesis, for which ZNF217 and CYP24 are good candidates. Copyright

Best Practice Guidelines for the Use of Next-Generation Sequencing Applications in Genome Diagnostics: A National Collaborative Study of Dutch Genome Diagnostic Laboratories

Next‐generation sequencing (NGS) methods are being adopted by genome diagnostics laboratories worldwide. However, implementing NGS‐based tests according to diagnostic standards is a challenge for individual laboratories. To facilitate the implementation of NGS in Dutch laboratories, the Dutch Society for Clinical Genetic Laboratory Diagnostics (VKGL) set up a working group in 2012. The results of their discussions are presented here. We provide best practice guidelines and criteria for implementing and validating NGS applications in a clinical setting. We introduce the concept of “diagnostic yield” as the main performance characteristic for evaluating diagnostic tests. We recommend that the laboratory procedures, including the tested genes, should be recorded in a publicly available document describing the complete “diagnostic routing.” We also propose that laboratories should use a list of “core disease genes” for specific genetic diseases. This core list contains the essential genes for each disease, and they should all be included in a diagnostic test to establish a reliable and accurate molecular diagnosis. The guidelines will ensure a clear and standardized quality of care provided by genetic diagnostic laboratories. The best practice guidelines and criteria that are presented here were adopted by the VKGL in January 2013.

Barrett's adenocarcinomas resemble adenocarcinomas of the gastric cardia in terms of chromosomal copy number changes, but relate to squamous cell carcinomas of the distal oesophagus with respect to the presence of high-level amplifications

Three different cancers predominantly occur at the gastro‐oesophageal junction: squamous cell carcinomas of the distal oesophagus, adenocarcinomas of the distal oesophagus (Barretts carcinomas), and adenocarcinomas of the gastric cardia. The aim of the present study was to investigate how, and to what extent, Barretts carcinoma differs from adenocarcinoma of the gastric cardia on the one hand and squamous cell carcinoma of the distal oesophagus on the other, with respect to chromosomal aberrations and related gene expression. The present study analysed 14 squamous cell carcinomas, 24 Barretts carcinomas, and 16 carcinomas of the gastric cardia. Comparative genomic hybridization revealed chromosomal abnormalities in all cases. Typical chromosomal aberrations for the squamous cell carcinoma type were gains at 3q and 11q13, and losses at 3p, 4q, 9p, 11q, and 13q. In contrast, typical copy number changes for both cardiac and Barretts adenocarcinomas were gains at 2q, 7p, and 13q, and losses at 17p. High‐level amplification occurred in all three groups, but its frequency in the cardiac carcinomas was lower than in the other two groups. In conclusion, squamous cell carcinomas are characterized by chromosomal aberrations which are distinct from those seen in carcinomas of the gastric cardia and in Barretts adenocarcinomas. With respect to Barretts cancer, the chromosomal aberrations more closely reflect the adenocarcinoma phenotype than the squamous origin of the epithelium. Copyright

The phenotypic spectrum of Schaaf-Yang syndrome: 18 new affected individuals from 14 families.

Purpose:Truncating mutations in the maternally imprinted, paternally expressed gene MAGEL2, which is located in the Prader-Willi critical region 15q11–13, have recently been reported to cause Schaaf-Yang syndrome, a Prader-Willi-like disease that manifests as developmental delay/intellectual disability, hypotonia, feeding difficulties, and autism spectrum disorder. The causality of the reported variants in the context of the patients’ phenotypes was questioned, as MAGEL2 whole-gene deletions seem to cause little or no clinical phenotype.Methods:Here we report a total of 18 newly identified individuals with Schaaf-Yang syndrome from 14 families, including 1 family with 3 individuals found to be affected with a truncating variant of MAGEL2, 11 individuals who are clinically affected but were not tested molecularly, and a presymptomatic fetal sibling carrying the pathogenic MAGEL2 variant.Results:All cases harbor truncating mutations of MAGEL2, and nucleotides c.1990–1996 arise as a mutational hotspot, with 10 individuals and 1 fetus harboring a c.1996dupC (p.Q666fs) mutation and 2 fetuses harboring a c.1996delC (p.Q666fs) mutation. The phenotypic spectrum of Schaaf-Yang syndrome ranges from fetal akinesia to neurobehavioral disease and contractures of the small finger joints.Conclusion:This study provides strong evidence for the pathogenicity of truncating mutations of the paternal allele of MAGEL2, refines the associated clinical phenotypes, and highlights implications for genetic counseling for affected families.Genet Med 19 1, 45–52.

Genome wide array comparative genomic hybridisation analysis of premalignant lesions of the stomach

Background: Gastric cancer is one of the most frequent malignancies in the world, ranking fifth in the Netherlands as a cause of cancer death. Surgery is the only curative treatment for advanced cases, but results of gastrectomy largely depend on the stage of the disease. A better understanding of the mechanisms of progression from a preneoplastic condition through intraepithelial neoplasia to invasive cancer may provide information relevant to designing focused prevention strategies. Methods: Because the pattern of chromosomal aberrations in precursors of gastric cancer is unclear, 11 gastric polyps with intraepithelial neoplasia (three hyperplastic polyps and eight adenomas) were analysed by microarray comparative genomic hybridisation to study chromosomal instability in precursors of gastric cancer. Results: Chromosomal aberrations were detected in all specimens. Adenomas showed no more chromosomal aberrations than did the hyperplastic polyps. The most frequent aberrations were gain of 7q36 and 20q12, and loss of 5q14–q21 in the adenomas, and loss of 15q11–14, 1p21–31, and 21q11–21.2 in the hyperplastic polyps. The most frequent chromosomal aberration in common to both types was loss of 9p21.3. Conclusion: Hyperplastic polyps showed many chromosomal aberrations, confirming that neoplastic transformation can occur in these lesions. These observations are consistent with the existence of two morphologically and genetically distinct pathways to gastric cancer—the hyperplastic polyp pathway and the (intestinal type) adenoma pathway. The relative contribution of each to gastric carcinogenesis in general, and how they compare to patterns of chromosomal aberrations in the more prevalent flat foci of intraepithelial neoplasia remain to be determined.

Rapid quantitative assessment of gastric corpus atrophy in tissue sections.

Background/Aims—Grading of Helicobacter pylori induced atrophic gastritis using the updated Sydney system is severely limited by high interobserver variability. The aim of this study was to set up a quantitative test of gastric corpus mucosal atrophy in tissue sections and test its reproducibility and correlation with the Sydney scores of atrophy. Method—Mucosal atrophy was assessed in 124 haematoxylin and eosin stained corpus biopsy specimens by two experienced gastrointestinal pathologists (EB, JL) according to the updated Sydney system as none (n = 33), mild (n = 33), moderate (n = 33), or pronounced (n = 25). In each specimen, the proportions of glands, stroma, infiltrate, and intestinal metaplasia in the glandular zone were measured as volume percentages using a point counting method. The optimal point sample size, intra-observer and interobserver reproducibility, discriminative power for degrees of atrophy, and correlations with H pylori status were evaluated. Results—Counting 400 points in 200 fields of vision provided the smallest sample size that still had excellent intra-observer and interobserver reproducibility (r ≥ 0.96). Overall, the volume percentage of glands (VPGL), infiltrate (VPI), and stroma (VPS) correlated well with the Sydney scores for atrophy (p ≤ 0.003). However, no differences were found between non-atrophic mucosa and mild atrophy. No correlation was found between age and either the Sydney grade of atrophy or the VPGL or VPS. In non-atrophic mucosa and mild atrophy, H pylori positive cases showed a significantly higher VPI than did H pylori negative cases. A lower VPGL was seen in H pylori positive cases than in H pylori negative cases in the mild atrophy group. VPS did not correlate with H pylori status within each grade of atrophy. Conclusion—Point counting is a powerful and reproducible tool for the quantitative analysis of mucosal corpus atrophy in tissue sections. These data favour the combination of “none” and “mild” atrophy into one category, resulting in a three class grading system for corpus atrophy, when using the updated Sydney system.

Comparative genomic hybridisation as a supportive tool in diagnostic pathology

Aims: Patients with multiple tumour localisations pose a particular problem to the pathologist when the traditional combination of clinical data, morphology, and immunohistochemistry does not provide conclusive evidence to differentiate between metastasis or second primary, or does not identify the primary location in cases of metastases and two primary tumours. Because this is crucial to decide on further treatment, molecular techniques are increasingly being used as ancillary tools. Methods: The value of comparative genomic hybridisation (CGH) to differentiate between metastasis and second primary, or to identify the primary location in cases of metastases and two primary tumours was studied in seven patients. CGH is a cytogenetic technique that allows the analysis of genome wide amplifications, gains, and losses (deletions) in a tumour within a single experiment. The patterns of these chromosomal aberrations at the different tumour localisations were compared. Results: In all seven cases, CGH patterns of gains and losses supported the differentiation between metastasis and second primary, or the identification of the primary location in cases of metastases and two primary tumours. Conclusion: The results illustrate the diagnostic value of CGH in patients with multiple tumours.