Magdalena Benetkiewicz

Frequent genetic differences between matched primary and metastatic breast cancer provide an approach to identification of biomarkers for disease progression.

Breast cancer is a major cause of morbidity and mortality in women and its metastatic spread is the principal reason behind the fatal outcome. Metastasis-related research of breast cancer is however underdeveloped when compared with the abundant literature on primary tumors. We applied an unexplored approach comparing at high resolution the genomic profiles of primary tumors and synchronous axillary lymph node metastases from 13 patients with breast cancer. Overall, primary tumors displayed 20% higher number of aberrations than metastases. In all but two patients, we detected in total 157 statistically significant differences between primary lesions and matched metastases. We further observed differences that can be linked to metastatic disease and there was also an overlapping pattern of changes between different patients. Many of the differences described here have been previously linked to poor patient survival, suggesting that this is a viable approach toward finding biomarkers for disease progression and definition of new targets useful for development of anticancer drugs. Frequent genetic differences between primary tumors and metastases in breast cancer also question, at least to some extent, the role of primary tumors as a surrogate subject of study for the systemic disease.

Microarray‐based survey of CpG islands identifies concurrent hyper‐ and hypomethylation patterns in tissues derived from patients with breast cancer

Maintenance of CpG island methylation in the genome is crucial for cellular homeostasis and this balance is disrupted in cancer. Our rationale was to compare the methylation of CpG islands in tissues (tumor, healthy breast and blood) from patients with breast cancer. We studied 72 genes in 103 samples using microarray hybridization and bisulfite sequencing. We observed tumor specific hyper‐ or hypomethylation of five genes; COL9A1, MT1A, MT1J, HOXA5 and FLJ45983. A general drop of methylation in COL9A1 was apparent in tumors, when compared with blood and healthy breast tissue. Furthermore, one tumor displayed a complete loss of methylation of all five genes, suggesting overall impairment of methylation. The downstream, evolutionary conserved island of HOXA5 showed hypomethylation in 18 tumors and complete methylation in others. This CpG island also displayed a semimethylated state in the majority of normal breast samples, when compared to complete methylation in blood. Distinct methylation patterns were further seen in MT1J and MT1A, belonging to the metallothionein gene family. The CpG islands of these genes are spaced by 2 kb, which shows selective methylation of two structurally and functionally related genes. The promoters of FLJ45983 and MT1A were methylated above 25% in 18 primary and metastatic tumors. Concurrently, there was also >10% methylation of healthy breast tissue in 11 and 5 samples, respectively. This suggests that the methylation process for the latter two genes takes place already in normal breast cells. Our results also point to a considerable heterogeneity of epigenetic disturbance in breast cancer. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045‐2257/suppmat.

Detailed assessment of chromosome 22 aberrations in sporadic pheochromocytoma using array-CGH

Pheochromocytoma is a predominantly sporadic neuroendocrine tumor derived from the adrenal medulla. Previous low resolution LOH and metaphase‐CGH studies reported the loss of chromosomes 1p, 3q, 17p and 22q at various frequencies. However, the molecular mechanism(s) behind development of sporadic pheochromocytoma remains largely unknown. We have applied high‐resolution tiling‐path microarray‐CGH with the primary aim to characterize copy number imbalances affecting chromosome 22 in 66 sporadic pheochromocytomas. We detected copy number alterations on 22q at a frequency of 44%. The predominant finding was monosomy 22 (30%), followed by terminal deletions in 8 samples (12%) and a single interstitial deletion. We further applied a chromosome 1 tiling‐path array in 7 tumors with terminal deletions of 22q and found deletions of 1p in all cases. Our overall results suggest that at least 2 distinct regions on both 22q and 1p are important in the tumorigenesis of sporadic pheochromocytoma. A large proportion of pheochromocytomas also displayed indications of cellular heterogeneity. Our study is to our knowledge the first array‐CGH study of sporadic pheochromocytoma. Future analysis of this tumor type should preferably be performed in the context of the entire human genome using genome‐wide array‐CGH, which is a superior methodological approach. Supplemental material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020‐7136/suppmat/index.html.

High-resolution gene copy number and expression profiling of human chromosome 22 in ovarian carcinomas.

Previous low‐resolution studies of chromosome 22 in ovarian carcinoma have suggested its involvement in the development of the disease. We report a high‐resolution analysis of DNA copy number and gene expression of 22q in 18 ovarian carcinomas using a 22q‐specific genomic microarray. We identified aberrations in 67% of the studied tumors, which displayed 3 distinct gene copy number profiles. The majority of the cases (11 of 18) demonstrated heterozygous terminal deletions of various sizes, the smallest of which was 3.5 Mb. The second profile, detected in 3 tumors, revealed the coexistence of heterozygous deletions and different patterns of low‐copy‐number gain that involved the proximal half of 22q. The latter finding has not been reported previously in ovarian carcinoma. One case displayed a continuous deletion encompassing the entire 22q, consistent with monosomy 22. Furthermore, we compared the results with the available data on these tumors by using cDNA microarrays to define the degree of correlation between abnormalities at the DNA level and variation in mRNA expression. By a comparison with the expression data, we were able to identify 21 deleted genes showing low mRNA levels and 12 amplified genes displaying elevated gene expression, several of which play roles in cell cycle control and the induction of apoptosis. Our results indicated significant correlation between DNA copy number aberrations and variation in mRNA expression. We also identified several regions and candidate genes on 22q that should be studied further to determine their role in the development of ovarian cancer.

Chromosome 22 tiling-path array-CGH analysis identifies germ-line- and tumor-specific aberrations in patients with glioblastoma multiforme

Gliomas are common and frequently malignant tumors of the central nervous system. Recurrent allelic losses of chromosome 22 have been reported in gliomas, indicating tumor‐suppressor genes at this location. However, the target genes are still unknown. We applied a high resolution tiling‐path chromosome 22 array to a series of 50 glioblastoma samples, with the aim of investigating the underlying abnormalities in both constitutional and tumor‐derived DNA. We detected hemizygous deletions in 28% of the tumors (14 of 50), with monosomy 22 (10 of 50) being the predominant pattern. The distribution of overlapping hemizygous deletions delineated two putative tumor‐suppressor loci (11.1 and 3.08 Mb in size) across 22q. Most strikingly, we identified two distinct loci affected by regional gains. Both alterations were of germ‐line origin and were unique to samples from patients affected with tumors. Analysis of these two amplified regions revealed the presence of two interesting candidate genes: TOP3B and TAFA5. The TOP3B gene encodes a protein that seems to function in the unlinking of parental strands at the final stage of DNA replication and/or in the dissociation of structures in mitotic cells that could lead to recombination. The TAFA5 gene belongs to a novel family of proteins with similarity to chemokines and brain‐specific expression. The role of the identified candidate loci should be studied further. Our results demonstrated the power of array‐CGH to determine DNA copy number alterations in the context of germ‐line‐ and tumor‐specific aberrations.

High-resolution array-CGH profiling of germline and tumor- specific copy number alterations on chromosome 22 in patients affected with schwannomas

Schwannomas may develop sporadically or in association with NF2 and schwannomatosis. The fundamental aberration in schwannomas is the bi-allelic inactivation of the NF2 gene. However, clinical and molecular data suggest that these tumors share a common pathogenetic mechanism related to as yet undefined 22q-loci. Linkage studies in schwannomatosis, a condition related to NF2, have defined a candidate 22q-locus and excluded the NF2 gene as the causative germline mutation. Thus, analysis of aberrations in schwannomas may lead to the identification of putative gene(s) involved in the development of schwannoma/schwannomatosis. We profiled a series of 88 schwannomas and constitutional DNA using a tiling path chromosome 22 array. Array-CGH is a suitable method for high-resolution discrimination between germline and tumor-specific aberrations. Previously reported frequencies of 22q-associated deletions in schwannomas display large discrepancies, ranging from 30% to 80%. We detected heterozygous deletions in 53% of schwannomas and the predominant pattern was monosomy 22. In addition, three tumors displayed terminal deletions and four harbored overlapping interstitial deletions of various sizes encompassing the NF2 gene. When profiling constitutional DNA, we identified eight loci that were affected by copy number variation (CNV). Some of the identified CNVs may not be phenotypically neutral and the possible role of these CNVs in the pathogenesis of schwannomas should be studied further. We observed a correlation between the breakpoint position, present in tumor and/or constitutional DNA and the location of segmental duplications. This association implicates these unstable regions in rearrangements occurring both in meiosis and mitosis.