Characterization and gene expression profiling in glioma cell lines with deletion of chromosome 19 before and after microcell-mediated restoration of normal human chromosome 19.

Nearly 10% of human gliomas are oligodendrogliomas. Deletion of chromosome arm 19q, often in conjunction with deletion of 1p, has been observed in 65-80% of these tumors. This has suggested the presence of a tumor suppressor gene located on the 19q arm. Chromosome 19 deletion is also of interest due to the better prognosis of patients with deletion, including longer survival and better response to chemotherapy, compared with patients without deletion. Two glioma cell lines with deletion of 19q were used for chromosome 19 microcell-mediated transfer, to assess the effect of replacing the deleted segment. Complementation with chromosome 19 significantly reduced the growth rate of the hybrid cells compared with the parental cell lines. Affymetrix U133 Plus 2.0 Gene Chip analysis was performed to measure and compare the expression of the chromosome 19 genes in the chromosome 19 hybrid cell lines to the parental cell line. Probes were considered significantly different when a P value <0.01 was seen in all of the cell line comparisons. Of 345 probes within the commonly deleted 19q region, seven genes (APOE, RCN3, FLJ10781, SAE1, STRN4, CCDC8, and BCL2L12) were identified as potential candidate genes. RT-PCR analysis of primary tumor specimens showed that several genes had significant differences when stratified by tumor morphology or deletion status. This suggests that one or more of these candidates may play a role in glioma formation or progression.

TBX2 is preferentially amplified in BRCA1- and BRCA2-related breast tumors.

The chromosome 17q23 region is frequently amplified in breast tumors. Gain of the region is present in 50% of BRCA1-associated breast tumors and 87% of BRCA2-associated breast tumors. The amplification frequency of the RPS6KB1 and TBX2 oncogenes from this amplicon was compared in 27 BRCA1 and BRCA2 mutant breast tumors, 15 breast tumors from high-risk patients with no BRCA1 or BRCA2 mutations, and 62 matched sporadic breast tumor controls. TBX2 was determined to be preferentially amplified and overexpressed in BRCA1 and BRCA2 mutant tumors, whereas RPS6KB1 was not, suggesting a role for TBX2 amplification in the development of BRCA1- and BRCA2-associated breast tumors.

Microsatellite instability in hereditary and sporadic breast cancers.

Sporadic cancers and familial breast cancers are characterized by an increase in genetic instability. Little is known about whether mismatch repair defects accompany this genetic instability. We investigated invasive and/or in situ breast cancers from 30 women with deleterious BRCA1/2 mutations and unclassified variant BRCA1/2 alterations. Forty cases of sporadic breast cancers were also investigated, including 7 medullary carcinomas. Malignant and benign lesions were examined from all cases to better understand tumor progression. Automated immunohistochemistry, with antibodies directed against hMLH1 and hMSH2, was used to screen cases for possible mismatch repair defects. When loss of expression was noted, DNA ploidy was performed by cytomorphometry. DNA, after laser microdissection, was extracted from a majority of familial cases and their corresponding controls, and microsatellite instability analysis was performed. None of the familial or sporadic cases had loss of hMSH2 expression. All but one lesion, a DCIS arising in a deleterious BRCA2 mutation carrier, had loss of hMLH1 expression and a tetraploid profile by image cytomorphometry. There was no MSI in any explored lesions (n = 34), as determined by molecular analysis, including the DCIS with loss of hMLH1 expression. We conclude that DNA mismatch repair defects involving hMLH1 and hMSH2 underexpression are extremely rare events in sporadic and familial breast cancer. Mismatch repair gene mutations may be secondary random events in breast cancer progression.

ERBB2, TBX2, RPS6KB1, and MYC alterations in breast tissues of BRCA1 and BRCA2 mutation carriers.

Breast cancer risk is greatly increased in women who carry mutations in the BRCA1 or BRCA2 genes. Because breast cancer initiation is different between BRCA1/2 mutation carriers and women who do not carry mutations, it is possible that the mechanism of breast cancer progression is also different. Histopathologic and genetic studies have supported this hypothesis. To test this hypothesis further, we utilized a large cohort of women who underwent therapeutic mastectomy (TM) and contralateral prophylactic mastectomy (PM). From this cohort, we developed case groups of women with a family history of breast cancer with BRCA1/2 deleterious mutations, with unclassified variant alterations, and with no detected mutation and matched these cases with sporadic controls from the same TM and PM cohort. Fluorescence in situ hybridization was performed on paraffin sections by use of dual-color probes for ERBB2/CEP17, MYC/CEP8, TBX2/CEP17, and RPS6KB1/CEP17. All malignant and benign lesions, including putative precursor lesions, were studied. The invasive cancers from deleterious mutation carriers had a higher prevalence of duplication of MYC (P = 0.006) and TBX2 (P = 0.0008) compared to controls and a lower prevalence of ERBB2 amplification (P = 0.011). Coduplication of MYC and TBX2 was common in the in situ and invasive lesions from the deleterious mutation carriers. The odds ratio of having a BRCA1/2 mutation is 31.4 (95% CI = 1.7-569) when MYC and TBX2 are coduplicated but ERBB2 is normal. Unclassified variant carriers/no mutation detected and sporadic controls had a similar prevalence of alterations, suggesting that hereditary patients with no deleterious mutations follow a progression pathway similar to that of sporadic cases. With the exception of one atypical ductal hyperplasia lesion, no putative precursor lesion showed any detectable alteration of the probes tested. There was no significant intratumoral heterogeneity of genetic alterations. Our data confirm that a specific pattern of genomic instability characterizes BRCA1/2-related cancers and that this pattern has implications for the biology of these cancers. Moreover, our current and previous results emphasize the interaction between phenotype and genotype in BRCA1/2-related breast cancers and that a combination of morphologic features and alterations of ERBB2, MYC, and TBX2 may better define mechanisms of tumor progression, as well as determine which patients are more likely to carry BRCA1/2 mutations.

Pathologic characteristics of breast parenchyma in patients with hereditary breast carcinoma, including BRCA1 and BRCA2 mutation carriers.

BACKGROUND: BRCA1 and BRCA2 alterations are associated with an increased risk of developing breast carcinoma. The authors hypothesized that the progression of breast neoplasia may differ between patients with hereditary disease and patients with nonhereditary disease and that this difference in progression may be visualized by studying the prevalence of precursor lesions and neoplastic lesions. METHODS: The authors developed two case cohorts of high-risk patients with a strong family history of breast carcinoma who underwent prophylactic mastectomy. The first cohort was comprised of women who underwent therapeutic mastectomy and contralateral prophylactic mastectomy, and the second cohort was comprised of women who underwent bilateral prophylactic mastectomy. Patients without a family history of breast carcinoma who underwent unilateral or bilateral prophylactic mastectomy were selected as a control group. DNA from peripheral blood leukocytes was screened for BRCA1 and BRCA2 mutations. The available pathologic materials were reviewed independently by two pathologists, and all neoplastic and precursor lesions were identified and classified. Proliferation activity was assessed using MIB-1 immunohistochemistry on all available lesions from the unilateral mastectomy cohort. RESULTS: The 28 women from the unilateral cohort with deleterious BRCA1/2 mutations had a lower prevalence of proliferative fibrocystic changes (PFC) (7%) compared with their matched control group (25%) (P = 0.075) and with patients who had a family history but no BRCA1/2 mutation (22-33%). None of the 11 deleterious mutation carriers from the bilateral cohort (0%) had PFC compared with 36% of women in the matched control group (P = 0.03). There was no major difference in the prevalence of other precursor lesions (including in situ carcinoma) in either cohort. Invasive carcinomas from the deleterious mutation carriers in the unilateral cohort were of higher grade compared with the control group (P = 0.003) and patients without a mutation (P < 0.0001) but were of similar grade compared with carriers of unclassified variant BRCA1/2 alterations (P = 0.20). Neoplastic lesions from the deleterious mutation carriers in the unilateral cohort had higher MIB-1 proliferation indices compared with other patients with and without a family history of breast carcinoma. CONCLUSIONS: The current data suggest that the progression rate of breast neoplasia is accelerated in women who carry BRCA1/2 deleterious mutations compared with other patients who have breast carcinoma with or without a family history. This increased progression rate should be taken into account when considering the surveillance of asymptomatic women.