Germline copy number variations associated with breast cancer susceptibility in a Japanese population.

Although copy number variations (CNVs) are expected to affect various diseases, little is known about the association between CNVs and breast cancer susceptibility. Therefore, we investigated this relation. Array comparative genomic hybridization was performed to search for candidate CNVs related to breast cancer susceptibility. Subsequent quantitative real-time polymerase chain reaction was carried out for confirmation. We found seven CNV markers associated with breast cancer risk. The means of the relative copy numbers of patients with a history of breast cancer and women in the control group were 0.8 and 1.8 for Hs06535529_cn on 1p36.12 (P < 0.0001), 2.9 and 2.2 for Hs03103056_cn on 3q26.1 (P < 0.0001), 1.2 and 1.8 for Hs03899300_cn on 15q26.3 (P < 0.0001), 1.0 and 1.5 for Hs03908783_cn on 15q26.3 (P < 0.0001), and 1.1 and 1.7 for Hs03898338_cn on 15q26.3 (P < 0.0001), respectively. Interestingly, nine or more copies of Hs04093415_cn on 22q12.3 were found only in 8/193 (4.1 %) patients with a history of breast cancer and in none of the controls (P = 0.0081). Similarly, 12 or more copies of Hs040908898_cn on 22q12.3 were found only in 7/193 (3.6 %) patients with a history of breast cancer and in none of the controls (P = 0.016). A combination of two CNVs resulted in 80.3 % sensitivity, 80.6 % specificity, 82.4 % positive predictive value, and 78.3 % negative predictive value for the prediction of breast cancer susceptibility. These findings may lead to a new means of risk assessment for breast cancer. Confirmatory studies using independent data sets are needed to support our findings.

Aneuploidy predicts outcome in patients with endometrial carcinoma and is related to lack of CDH13 hypermethylation.

PURPOSE: Many investigators have reported that aneuploidy detected by flow cytometry is a useful prognostic marker in patients with endometrial cancer. Laser scanning cytometry (LSC) is a technology similar to flow cytometry but is more feasible for clinical laboratory use. We evaluated the usefulness of DNA ploidy detected by LSC as a prognostic marker in patients with endometrial cancer and investigated genetic and epigenetic factors related to aneuploidy. EXPERIMENTAL DESIGN: Endometrial cancer specimens from 106 patients were evaluated. The methylation status of CDH13, Rassf1, SFRP1, SFRP2, SFRP4, SFRP5, p16, hMLH1, MGMT, APC, ATM, and WIF1 and mutations in the p53 and CDC4 genes were investigated. LSC was carried out to determine DNA ploidy. Fluorescence in situ hybridization was done with chromosome-specific centromeric probes to assess chromosomal instability. RESULTS: Univariate and multivariate analyses revealed that p53 mutation and lack of CDH13 hypermethylation associated positively with aneuploidy. Univariate analysis showed that aneuploidy, chromosomal instability, and lack of CDH13 hypermethylation as well as surgical stage were significantly predictive of death from endometrial cancer. Furthermore, multivariate analysis revealed that stage in combination with either DNA aneuploidy or lack of CDH13 hypermethylation was an independent prognostic factor. CONCLUSION: These results suggest that analysis of DNA ploidy and methylation status of CDH13 may help predict clinical outcome in patients with endometrial cancer. Prospective randomized trials are needed to confirm the validity of an individualized approach, including determination of tumor ploidy and methylation status of CDH13, to management of endometrial cancer patients.

The development of a mini-array for estimating the disease state of gastric adenocarcinoma by array CGH.

BACKGROUND: The treatment strategy usually depends on the disease state in the individual patient. However, it is difficult to estimate the disease state before treatment in many patients. The purpose of this study was to develop a BAC (bacterial artificial chromosome) mini-array allowing for the estimation of node metastasis, liver metastasis, peritoneal dissemination and the depth of tumor invasion in gastric cancers. METHODS: Initially, the DNA copy number aberrations (DCNAs) were analyzed by array-based comparative genomic hybridization (aCGH) in 83 gastric adenocarcinomas as a training-sample set. Next, two independent analytical methods were applied to the aCGH data to identify the BAC clones with DNA copy number aberrations that were linked with the disease states. One of the methods, a decision-tree model classifier, identified 6, 4, 4, 4, and 7 clones for estimating lymph node metastasis, liver metastasis, peritoneal dissemination, depth of tumor invasion, and histological type, respectively. In the other method, a clone-by-clone comparison of the frequency of the DNA copy number aberrations selected 26 clones to estimate the disease states. RESULTS: By spotting these 50 clones together with 26 frequently or rarely involved clones and 62 reference clones, a mini-array was made to estimate the above parameters, and the diagnostic performance of the mini-array was evaluated for an independent set of 30 gastric cancers (blinded - sample set). In comparison to the clinicopathological features, the overall accuracy was 66.7% for node metastasis, 86.7% for liver metastasis, 86.7% for peritoneal dissemination, and 96.7% for depth of tumor invasion. The intratumoral heterogeneity barely affected the diagnostic performance of the mini-array. CONCLUSION: These results suggest that the mini-array makes it possible to determine an optimal treatment for each of the patients with gastric adenocarcinoma.

Loss of 6q or 8p23 is associated with the total number of DNA copy number aberrations in adenoid cystic carcinoma.

We analyzed 10 adenoid cystic carcinomas (ACCs) of the salivary glands by array-based comparative genomic hybridization (a-CGH) using DNA chips spotted with 4,030 bacterial artificial chromosome clones. After the data smoothing procedure was applied, a total of 88 DNA copy number aberrations (DCNAs) were detected. The frequent (≥30%) DCNAs were loss of 6q23-27 and 8p23, and gains of 6p, 6q23, 8p23 and 22q13. High-level gains were detected on 12q15, including MDM2 in two cases. These two cases showed an immunohistochemically high-level (>50%) expression of MDM2 and a low-level expression of p53 (<20%). Furthermore, the total number of DCNAs was significantly greater in ACCs with loss of 6q compared to other ACCs, and in ACCs without the loss of 8p23 compared to other ACCs, respectively. Although limitations exist, a-CGH detected several candidate chromosomal imbalances associated with accumulation of DCNAs in ACCs.

Genomic instability and DNA ploidy are linked to DNA copy number aberrations of 8p23 and 22q11.23 in gastric cancers.

The close relationship between chromosomal instability (CIN) and aneuploidy has been reported. The purpose of this study was to identify genomic aberrations present with CIN and aneuploidy in gastric cancers. FISH and image cytometry were applied to 27 sporadic gastric adenocarcinomas to identify CIN-positive tumors and to determine DNA ploidy, respectively. In addition, array-based comparative genomic hybridization was used to identify bacterial artificial chromosome clones that displayed differences in the frequency of copy number aberrations between CIN-positive and CIN-negative tumors, and between aneuploid and diploid tumors. There were many chromosomal regions with DNA copy number aberrations, some of which were nonrandom aberrations linked to the CIN phenotype and aneuploidy. A copy number loss of 22q11.23 was more frequent in CIN-positive cancers than in others (7/12 vs. 2/15, p<0.01) and in aneuploid cancers than in diploid cancers (8/16 vs. 1/11, p<0.05). The frequency of 22q11.23 loss differed significantly between CIN-positive and aneuploid tumors and between CIN-negative and diploid cancers (7/10 vs. 1/9, p<0.01). In contrast, a DNA copy number gain of 8p23.2 was detected in 6 out of 9 CIN-negative/diploid cancers, but was not detected in CIN-positive/aneuploid cancers (p<0.01). There were no cancers carrying both aberrations (22q11.23 loss and 8p23.2 gain). The present study indicates that a 22q11.23 loss and a 8p23.2 gain are markers for both CIN and aneuploidy. This is the first report describing an inverse relationship between the 22q11.23 loss and 8p23.2 gain in terms of genomic instability and DNA ploidy in gastric cancers.

Identification of DNA copy number aberrations associated with metastases of colorectal cancer using array CGH profiles.

It is important to estimate the biological characteristics of tumors, including the nodal status at the time of diagnosis for optimal treatment of individual cancer patients. Array-based comparative genomic hybridization (aCGH) was performed on 77 sporadic colorectal adenocarcinomas using a chip spotted with 4030 BAC clones. The nodal status was compared with an aCGH profiles depicted using a combination of decision-tree classifier and a Self-Organizing Map (SOM) analysis. Node metastasis was not detected in any of the 6 poorly differentiated adenocarcinomas with a 3q loss. A SOM analysis following the decision-tree classification of the aCGH data allowed for the differentiation in chromosomal regions between high- and low-level decreases in the DNA copy number. Node metastasis was detected in all 5 tumors with the high-level decrease in DNA copy number at Xp, irrespective of the histological type. Node metastasis was also found exclusively in 6 tumors with increase in DNA copy number at the chromosomal region between 11q13.3 and 11q22.3. Copy number aberrations linked to nodal metastasis were identified more collectively by the combination of the decision-tree classifier and a SOM analysis than by the conventional analysis method in aCGH analysis.