Marianne Tijssen

Human and mouse oligonucleotide-based array CGH

Array-based comparative genomic hybridization is a high resolution method for measuring chromosomal copy number changes. Here we present a validated protocol using in-house spotted oligonucleotide libraries for array comparative genomic hybridization (CGH). This oligo array CGH platform yields reproducible results and is capable of detecting single copy gains, multi-copy amplifications as well as homozygous and heterozygous deletions as small as 100 kb with high resolution. A human oligonucleotide library was printed on amine binding slides. Arrays were hybridized using a hybstation and analysed using BlueFuse feature extraction software, with >95% of spots passing quality control. The protocol allows as little as 300 ng of input DNA and a 90% reduction of Cot-1 DNA without compromising quality. High quality results have also been obtained with DNA from archival tissue. Finally, in addition to human oligo arrays, we have applied the protocol successfully to mouse oligo arrays. We believe that this oligo-based platform using ‘off-the-shelf’ oligo libraries provides an easy accessible alternative to BAC arrays for CGH, which is cost-effective, available at high resolution and easily implemented for any sequenced organism without compromising the quality of the results.

Loss of lamin A/C expression in stage II and III colon cancer is associated with disease recurrence

AIM OF THE STUDY Loss of the nuclear lamina protein lamin A/C (LMNA) has been observed in several human malignancies. The present study aimed to investigate associations between LMNA expression and clinical outcome in colon cancer patients. PATIENTS AND METHODS Clinicopathological data and formalin-fixed paraffin embedded tissues were collected from 370 stage II and III colon cancer patients. Tissue microarrays were constructed, stained for lamin A/C and evaluated microscopically. Microsatellite instability status was determined for 318 tumours. RESULTS Low levels of LMNA expression were observed in 17.8% of colon tumours, with disease recurrence occurring in 45.5% of stage II and III colon cancer patients with LMNA-low expressing tumours compared to 29.6% of patients with LMNA-high expressing tumours (p=0.01). For stage II patients, disease recurrence was observed for 35.7% of LMNA-low compared to 20.3% of LMNA-high expressing tumours (p=0.03). Microsatellite stable (MSS) tumours exhibited more frequently low LMNA expression than microsatellite instable (MSI) tumours (21% versus 9.8%; p=0.05). Interestingly, disease recurrence among LMNA-low and LMNA-high expressing MSS tumours varied significantly for stage III patients who had not received adjuvant chemotherapy (100% versus 37.8%; p<0.01) while no such difference was observed for patients who received adjuvant chemotherapy (46.7% versus 46.0%; p=0.96). CONCLUSION These data indicate that low expression of LMNA is associated with an increased disease recurrence in stage II and III colon cancer patients, and suggest that these patients in particular may benefit from adjuvant chemotherapy.

TPX2 and AURKA promote 20q amplicon-driven colorectal adenoma to carcinoma progression

Background and objective Progression of a colorectal adenoma to invasive cancer occurs in a minority of adenomas and is the most crucial step in colorectal cancer pathogenesis. In the majority of cases, this is associated with gain of a substantial part of chromosome 20q, indicating that multiple genes on the 20q amplicon may drive carcinogenesis. The aim of this study was to identify genes located on the 20q amplicon that promote progression of colorectal adenoma to carcinoma. Design Functional assays were performed for 32 candidate driver genes for which a positive correlation between 20q DNA copy number and mRNA expression had been demonstrated. Effects of gene knockdown on cell viability, anchorage-independent growth, and invasion were analysed in colorectal cancer cell lines with 20q gain. Colorectal tumour protein expression was examined by immunohistochemical staining of tissue microarrays. Results TPX2, AURKA, CSE1L, DIDO1, HM13, TCFL5, SLC17A9, RBM39 and PRPF6 affected cell viability and/or anchorage-independent growth. Chromosome 20q DNA copy number status correlated significantly with TPX2 and AURKA protein levels in a series of colorectal adenomas and carcinomas. Moreover, downmodulation of TPX2 and AURKA was shown to inhibit invasion. Conclusion These data identify TPX2 (20q11) and AURKA (20q13.2) as two genes located on distinct regions of chromosome 20q that promote 20q amplicon-driven progression of colorectal adenoma to carcinoma. Therefore the selection advantage imposed by 20q gain in tumour progression is achieved by gain-of-function of multiple cancer-related genes—knowledge that can be translated into novel tests for early diagnosis of progressive adenomas.

Across array comparative genomic hybridization: a strategy to reduce reference channel hybridizations.

Array comparative genomic hybridization (array CGH) is widely used for studying chromosomal copy number aberrations (CNAs) on a genome‐wide and high‐resolution scale in heritable disorders and cancers. The aim of this study was to test if the separate channels of dual channel arrays can be interchanged (across array) to either make array CGH more sensitive and cost effective and/or to generate profiles of CNAs and copy number variations (CNVs). Therefore the BT474 breast cancer cell line was compared with a mix of normal reference DNAs hybridized on different arrays and days and DNA copy number profiles were evaluated. Quality was assessed, using regular dual channel array CGH as a standard, using four quality measures, i.e., the median absolute deviation value of chromosome 2, the amplitude of the ERBB2 gene amplification, a deletion on chromosome 9, and the deflection on chromosome 8. The quality of the across array CGH profiles matched or even surpassed the quality of regular dual channel array CGH. In addition, this across array approach was tested for genomic DNA derived from formalin‐fixed paraffin‐embedded tumors tissue samples, resulting in high‐quality copy number profiles, comparable to regular dual channel arrays. Finally, we demonstrated this approach to obtain both CNA and CNV profiles. In summary, across array CGH avoids redundant hybridizations of the same reference material in every experiment either allowing hybridization of two test samples on one array or producing both CNA and CNV profiles simultaneously.

Chromosomal Signatures of a Subset of High-Grade Premalignant Cervical Lesions Closely Resemble Invasive Carcinomas

Cervical cancer develops from precancerous high-grade cervical intraepithelial neoplasia (CIN) harboring a transforming infection with high-risk human papillomavirus, which is characterized by p16(INK4a) overexpression. Once such a lesion has developed, progression toward an invasive squamous cell carcinoma (SCC) may take one or more decades, underlining the heterogeneity of these lesions in terms of duration of existence and progression risk. We performed array-based comparative genomic hybridization (array CGH) on 46 p16(INK4a) immunopositive CIN2/3 lesions to determine whether this heterogeneity is reflected in their chromosomal profiles. Chromosomal profiles of CIN2/3 lesions were related to those of invasive cervical SCC and promoter methylation of CADM1, a tumor suppressor gene known to be functionally involved in the tumorigenic phenotype of cervical cancer cells. Frequent alterations found in CIN2/3 lesions included gains located at chromosome 1, 3, 7, and 20 and losses located at 4, 11, 16, 17, and 19. Unsupervised hierarchical clustering identified two subsets of CIN2/3 lesions, chromosomal profiles of one of which closely resembled invasive SCCs. Gains of 1, 3q, and 20 were characteristic for CIN2/3 lesions with chromosomal signatures resembling carcinomas. In addition, dense promoter methylation of the CADM1 gene was significantly more frequent in these CIN2/3 lesions (P = 0.004). No chromosomal alterations were detected in six CIN1 lesions, five of which were completely p16(INK4a) immunonegative. These findings suggest that biomarkers associated with gains at chromosomes 1, 3q, and 20 are potential hallmarks of advanced p16(INK4a)-positive CIN2/3 lesions with a high short-term risk of progression.

High resolution analysis of DNA copy-number aberrations of chromosomes 8, 13, and 20 in gastric cancers

DNA copy-number gains of chromosomes 8q, 13q, and 20q are frequently observed in gastric cancers. Moreover gain of chromosome 20q has been associated with lymph node metastasis. The aim of this study was to correlate DNA copy-number changes of individual genes on chromosomes 8q, 13q, and 20q in gastric adenocarcinomas to clinicopathological data. DNA isolated from 63 formalin-fixed and paraffin-embedded gastric adenocarcinoma tissue samples was analyzed by whole-genome microarray comparative genomic hybridization and by multiplex ligation-dependent probe amplification (MLPA), targeting 58 individual genes on chromosomes 8, 13, and 20. Using array comparative genomic hybridization, gains on 8q, 13q, and 20q were observed in 49 (77.8%), 25 (39.7%), and 49 (77.8%) gastric adenocarcinomas, respectively. Gain of chromosome 20q was significantly correlated with lymph node metastases (p = 0.05) and histological type (p = 0.02). MLPA revealed several genes to be frequently gained in DNA copy number. The oncogene c-myc on 8q was gained in 73% of the cancers, while FOXO1A and ATP7B on 13q were both gained in 28.6% of the cases. Multiple genes on chromosome 20q showed gains in more than 60% of the cancers. DNA copy-number gains of TNFRSF6B (20q13.3) and ZNF217 (20q13.2) were significantly associated with lymph node metastasis (p = 0.02) and histological type (p = 0.02), respectively. In summary, gains of chromosomes 8q, 13q, and 20q in gastric adenocarcinomas harbor DNA copy-number gains of known and putative oncogenes. ZNF217 and TNFRSF6B are associated with important clinicopathological variables, including lymph node status.

High-Resolution Array Comparative Genomic Hybridization in Sporadic and Celiac Disease–Related Small Bowel Adenocarcinomas

Purpose: The molecular pathogenesis of small intestinal adenocarcinomas is not well understood. Understanding the molecular characteristics of small bowel adenocarcinoma may lead to more effective patient treatment. Experimental Design: Forty-eight small bowel adenocarcinomas (33 non–celiac disease related and 15 celiac disease related) were characterized for chromosomal aberrations by high-resolution array comparative hybridization, microsatellite instability, and APC promoter methylation and mutation status. Findings were compared with clinicopathologic and survival data. Furthermore, molecular alterations were compared between celiac disease–related and non–celiac disease–related small bowel adenocarcinomas. Results: DNA copy number changes were observed in 77% small bowel adenocarcinomas. The most frequent DNA copy number changes found were gains on 5p15.33-5p12, 7p22.3-7q11.21, 7q21.2-7q21.3, 7q22.1-7q34, 7q36.1, 7q36.3, 8q11.21-8q24.3, 9q34.11-9q34.3, 13q11-13q34, 16p13.3, 16p11.2, 19q13.2, and 20p13-20q13.33, and losses on 4p13-4q35.2, 5q15-5q21.1, and 21p11.2-21q22.11. Seven highly amplified regions were identified on 6p21.1, 7q21.1, 8p23.1, 11p13, 16p11.2, 17q12-q21.1, and 19q13.2. Celiac disease–related and non–celiac disease–related small bowel adenocarcinomas displayed similar chromosomal aberrations. Promoter hypermethylation of the APC gene was found in 48% non–celiac disease–related and 73% celiac disease–related small bowel adenocarcinomas. No nonsense mutations were found. Thirty-three percent of non–celiac disease–related small bowel adenocarcinomas showed microsatellite instability, whereas 67% of celiac disease–related small bowel adenocarcinomas were microsatellite unstable. Conclusions: Our study characterized chromosomal aberrations and amplifications involved in small bowel adenocarcinoma. At the chromosomal level, celiac disease–related and non–celiac disease–related small bowel adenocarcinomas did not differ. A defect in the mismatch repair pathways seems to be more common in celiac disease–related than in non–celiac disease–related small bowel adenocarcinomas. In contrast to colon and gastric cancers, no APC nonsense mutations were found in small bowel adenocarcinoma. However, APC promoter methylation seems to be a common event in celiac disease–related small bowel adenocarcinoma. Clin Cancer Res; 16(5); 1391–401

MAL promoter hypermethylation as a novel prognostic marker in gastric cancer

T-lymphocyte maturation associated protein, MAL, has been described as a tumour-suppressor gene with diagnostic value in colorectal and oesophageal cancers, and can be inactivated by promoter hypermethylation. The aim of this study was to analyse the prevalence of MAL promoter hypermethylation and the association with mRNA expression in gastric cancers and to correlate methylation status to clinicopathological data. Bisulphite-treated DNA isolated from formalin-fixed and paraffin-embedded samples of 202 gastric adenocarcinomas and 22 normal gastric mucosae was subjected to real-time methylation-specific PCR (Q-MSP). Two regions within the MAL promoter (M1 and M2) were analysed. In addition, 17 frozen gastric carcinomas and two gastric cancer cell lines were analysed both by Q-MSP and real-time RT–PCR. Methylation of M1 and M2 occurred in 71 and 80% of the gastric cancers, respectively, but not in normal gastric mucosa tissue. Hypermethylation of M2, but not M1, correlated with significantly better disease-free survival in a univariate (P=0.03) and multivariate analysis (P=0.03) and with downregulation of expression (P=0.01). These results indicate that MAL has a putative tumour-suppressor gene function in gastric cancer, and detection of promoter hypermethylation may be useful as a prognostic marker.

Gastric cancers of Western European and African patients show different patterns of genomic instability

BackgroundInfection with H. pylori is important in the etiology of gastric cancer. Gastric cancer is infrequent in Africa, despite high frequencies of H. pylori infection, referred to as the African enigma. Variation in environmental and host factors influencing gastric cancer risk between different populations have been reported but little is known about the biological differences between gastric cancers from different geographic locations. We aim to study genomic instability patterns of gastric cancers obtained from patients from United Kingdom (UK) and South Africa (SA), in an attempt to support the African enigma hypothesis at the biological level.MethodsDNA was isolated from 67 gastric adenocarcinomas, 33 UK patients, 9 Caucasian SA patients and 25 native SA patients. Microsatellite instability and chromosomal instability were analyzed by PCR and microarray comparative genomic hybridization, respectively. Data was analyzed by supervised univariate and multivariate analyses as well as unsupervised hierarchical cluster analysis.ResultsTumors from Caucasian and native SA patients showed significantly more microsatellite instable tumors (p < 0.05). For the microsatellite stable tumors, geographical origin of the patients correlated with cluster membership, derived from unsupervised hierarchical cluster analysis (p = 0.001). Several chromosomal alterations showed significantly different frequencies in tumors from UK patients and native SA patients, but not between UK and Caucasian SA patients and between native and Caucasian SA patients.ConclusionsGastric cancers from SA and UK patients show differences in genetic instability patterns, indicating possible different biological mechanisms in patients from different geographical origin. This is of future clinical relevance for stratification of gastric cancer therapy.

DNA Quality Assessment for Array CGH by Isothermal Whole Genome Amplification

Background: Array Comparative Genomic Hybridization (array CGH) is increasingly applied on DNA obtained from formalin-fixed paraffin-embedded (FFPE) tissue, but in a proportion of cases this type of DNA is unsuitable. Due to the high experimental costs of array CGH and unreliable methods for DNA quality testing, better prediction methods are needed. The aim of this study was to accurately determine the quality of FFPE DNA input in order to predict quality of array CGH outcome. Material and Methods: DNA quality was assessed by isothermal amplification and compared to array CGH quality on 59 FFPE gastric cancer samples, one FFPE colorectal cancer sample, two FFPE normal uvula samples, one fresh frozen and six FFPE HNSCC samples. Gastric cancer DNA was also quality tested by β-globin PCR. Results: Accurate prediction of DNA quality using the isothermal amplification was observed in the colorectal carcinoma, HNSCC and uvula samples. In gastric cancer samples, the isothermal amplification was a more accurate method for selecting good quality DNA for array CGH compared to using PCR product lengths. The isothermal amplification product was used for array CGH and compared to the results achieved using non-amplified DNA in four of the samples. DNAs before and after amplification yielded the same segmentation patterns of chromosomal copy number changes for both the fresh DNA sample and the FFPE samples. Conclusion: The efficiency of isothermal DNA amplification is a reliable predictor for array CGH quality. The amplification product itself can be used for array CGH, even starting with FFPE derived DNA samples.