Susanne Seitz

Pathology of Ovarian Cancers in BRCA1 and BRCA2 Carriers

Purpose: Germline mutations in the BRCA1 and BRCA2 genes confer increased susceptibility to ovarian cancer. There is evidence that tumors in carriers may exhibit a distinct distribution of pathological features, but previous studies on the pathology of such tumors have been small. Our aim was to evaluate the morphologies and immunophenotypes in a large cohort of patients with familial ovarian cancer. Experimental Design: We performed a systematic review of ovarian tumors from 178 BRCA1 mutation carriers, 29 BRCA2 mutation carriers, and 235 controls with a similar age distribution. Tumors were evaluated by four pathologists blinded to mutation status. Both morphological features and immunochemical staining for p53 and HER2 were evaluated. Results: Tumors in BRCA1 mutation carriers were more likely than tumors in age-matched controls to be invasive serous adenocarcinomas (odds ratio, 1.84; 95% confidence interval, 1.21–2.79) and unlikely to be borderline or mucinous tumors. Tumors in BRCA1 carriers were of higher grade (P < 0.0001), had a higher percentage solid component (P = 0.001), and were more likely to stain strongly for p53 (P = 0.018). The distribution of pathological features in BRCA2 carriers was similar to that in BRCA1 carriers. Conclusions: Use of pathological features can substantially improve the targeting of predictive genetic testing. Results also suggest that BRCA1 and BRCA2 tumors are relatively aggressive and may be expected to have poor prognosis, although this may be treatment dependent.

Strong indication for a breast cancer susceptibility gene on chromosome 8p12-p22: linkage analysis in German breast cancer families

Chromosomal losses involving the short arm of chromosome 8 are frequent in a variety of tumor types, including breast cancer, suggesting the presence of one or more tumor suppressor genes in this region. Previous linkage analysis and studies of loss of heterozygosity (LOH) have suggested the presence of a putative third breast cancer susceptibility gene around D8S505 at 8p12-p22. We have performed linkage analysis in two German breast cancer families, showing negative lod scores with 17q and 13q markers, using seven adjacent microsatellite markers from 8p12-p22. Incorporating LOH data from tumors of the affected family members a maximum cumulative three-point lod score of 3.30 at [circle with M in middle;[equals;0.00 was obtained with D8S137 and D8S131. Our findings considerably strengthen the evidence for a third breast cancer susceptibility locus (BRCA3) mapping to the short arm of human chromosome 8.

Identification of brain- and bone-specific breast cancer metastasis genes

In breast cancer, metastases are relatively widely distributed, with the most common sites being bone, regional lymph nodes, lung, liver, and brain. The detailed mechanism of organ-specific metastasis is poorly understood. In this study, we initiated a search for genes that are implicated in brain or bone metastasis of primary human breast cancer. We generated gene expression profiles of 18 brain and eight bone metastases derived from primary breast tumors. We identified 73 genes differentially expressed between brain and bone metastases. Visualization of the differential gene expression profiles by correspondence and cluster analyses shows that the metastases clearly separate into two distinct groups as an exact reflection of their site of metastasis. Moreover, the analysis of this gene set in primary breast tumors relapsing to either bone or brain allowed accurate categorization of the tumors according to their metastatic site. The identified genes may prove to be excellent markers to predict the site of metastasis in breast cancer patients and could lead to tailor-made therapy to an individual patient.

SASH1: a candidate tumor suppressor gene on chromosome 6q24.3 is downregulated in breast cancer

Loss of heterozygosity (LOH) and in silico expression analysis were applied to identify genes significantly downregulated in breast cancer within the genomic interval 6q23–25. Systematic comparison of candidate EST sequences with genomic sequences from this interval revealed the genomic structure of a potential target gene on 6q24.3, which we called SAM and SH3 domain containing 1 (SASH1). Loss of the gene-internal marker D6S311, found in 30% of primary breast cancer, was significantly correlated with poor survival and increase in tumor size. Two SASH1 transcripts of approximately 4.4 and 7.5 kb exist and are predominantly transcribed in the human breast, lung, thyroid, spleen, placenta and thymus. In breast cancer cell lines, SASH1 is only expressed at low levels. SASH1 is downregulated in the majority (74%) of breast tumors in comparison with corresponding normal breast epithelial tissues. In addition, SASH1 is also downregulated in tumors of the lung and thyroid. Analysis of the protein domain structure revealed that SASH1 is a member of a recently described family of SH3/SAM adapter molecules and thus suggests a role in signaling pathways. We assume that SASH1 is a new tumor suppressor gene possibly involved in tumorigenesis of breast and other solid cancers. We were unable to find mutations in the coding region of the gene in primary breast cancers showing LOH within the critical region. We therefore hypothesize that other mechanisms as for instance methylation of the promoter region of SASH1 are responsible for the loss of expression of SASH1 in primary and metastatic breast cancer.

Decreased expression of angiogenesis antagonist EFEMP1 in sporadic breast cancer is caused by aberrant promoter methylation and points to an impact of EFEMP1 as molecular biomarker

EGF‐containing fibulin‐like extracellular matrix protein 1 (EFEMP1) was recently described as an antagonist of angiogenesis. Motivated by a strong dependence of tumor growth and metastasis on angiogenesis, we investigated the role of EFEMP1 in human breast cancer. We applied RNA microarray expression analysis and quantitative real‐time PCR (QRT) in a total of 45 sporadic breast cancer tissues and found EFEMP1 down‐regulation in 59% and 61% of the analyzed tissues, respectively. This down‐regulation was confirmed on protein level. Immunohistochemistry in 211 breast cancer tissues resulted in reduced or even abolished EFEMP1 expression in 57–62.5% of the tumors. Bisulphite genomic sequencing in breast cancer cell lines and primary breast cancer tissues revealed promoter methylation as the major cause of this down‐regulation. Furthermore, analysis of 203 clinically well characterized primary breast cancers displayed a significant correlation of reduced EFEMP1 protein expression with poor disease‐free (p = 0.037) and overall survival (p = 0.032), particularly in those node‐positive patients who received adjuvant anthracycline‐based chemotherapy, but not in those treated by either cyclophosphamide‐methotrexate‐5‐fluorouracil (CMF) or Tamoxifen. In summary, the presented data demonstrate for the first time the reduced EFEMP1 expression on RNA and protein level in a substantial number of sporadic breast carcinomas and its correlation with epigenetic alterations. Furthermore, these data point towards a possible predictive impact of EFEMP1 expression in primary breast cancer.

ST18 is a breast cancer tumor suppressor gene at human chromosome 8q11.2

We have identified a gene, ST18 (suppression of tumorigenicity 18, breast carcinoma, zinc-finger protein), within a frequent imbalanced region of chromosome 8q11 as a breast cancer tumor suppressor gene. The ST18 gene encodes a zinc-finger DNA-binding protein with six fingers of the C2HC type (configuration Cys-X5-Cys-X12-His-X4-Cys) and an SMC domain. ST18 has the potential to act as transcriptional regulator. ST18 is expressed in a number of normal tissues including mammary epithelial cells although the level of expression is quite low. In breast cancer cell lines and the majority of primary breast tumors, ST18 mRNA is significantly downregulated. A 160 bp region within the promoter of the ST18 gene is hypermethylated in about 80% of the breast cancer samples and in the majority of breast cancer cell lines. The strong correlation between ST18 promoter hypermethylation and loss of ST18 expression in tumor cells suggests that this epigenetic mechanism is responsible for tumor-specific downregulation. We further show that ectopic ST18 expression in MCF-7 breast cancer cells strongly inhibits colony formation in soft agar and the formation of tumors in a xenograft mouse model.

Analysis of DLC-1 expression in human breast cancer

The chromosome region 8p12-p22 shows frequent allelic loss in many neoplasms, including breast cancer (BC). The DLC-1 gene, located on 8p21-p22, might be a candidate tumor suppressor gene in this region. To evaluate the involvement of DLC-1 in breast carcinogenesis we studied DLC-1 mRNA expression in a panel of 14 primary human BC and the corresponding normal breast cells as well as 8 BC cell lines. Low levels or absence of DLC-1 mRNA were observed in 57% of primary BC and 62.5% of BC cell lines, respectively. We could not find any correlation between DLC-1mRNA expression and deletions at the DLC-1 locus. Transfection of the gene into DLC-1 deficient T-47D cells raised the DLC-1 mRNA level and resulted in inhibition of cell growth and reduced colony-forming capacity. Our results indicate a role of DLC-1 in BC carcinogenesis.

Deletion mapping and linkage analysis provide strong indication for the involvement of the human chromosome region 8p12-p22 in breast carcinogenesis.

We have identified a high frequency of loss of heterozygosity (LOH) on the human chromosome region 8p12-p22 in a panel of microdissected familial (86% LOH) and sporadic (74% LOH) breast tumours. The two most frequently deleted regions were defined around marker D8S133 and in a broader centromeric region bounded by markers D8S137 and D8S339. We cannot unequivocally characterize the 8p12-p22 loss as an early or a late event in breast carcinogenesis. In parallel, we have performed linkage analysis in four German breast cancer families. A location score greater than 13.67 corresponding to a LOD score of 2.97 at the marker D8S137 has been obtained. Our results considerably strengthen the evidence for a breast cancer susceptibility gene(s) located on the short arm of the chromosome region at 8p12-p22.

Identification of microsatellite instability and mismatch repair gene mutations in breast cancer cell lines

At present, there is conflicting evidence whether microsatellite instability (MSI) plays a role in the pathogenesis of breast cancer. Here we describe for the first time an MSI+ phenotype in two breast cancer cell lines, CAL51 and MT‐3, resembling that observed in colorectal cancers. These cell lines are characterized by near‐diploid and hyperdiploid karyotypes, respectively. We detected MSI in these cell lines within two non‐coding (BAT‐25 and BAT‐26) and within coding repeat sequences of genes known to be mutated in MSI+ cancer (TGFBR2, IGF2R, BAX). We provide evidence that the inactivation of MMR genes is responsible for MSI in these cell lines.

Mutation analysis and mRNA expression of trail-receptors in human breast cancer

The chromosome region 8p12‐p22 shows frequent allelic loss in a variety of human malignancies, including breast cancer (BC). The tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL)‐receptors TRAIL‐R1, ‐R2, ‐R3 and ‐R4 are located on 8p21‐p22 and might be candidate tumor suppressor genes in this region. To evaluate the involvement of TRAIL receptors in breast carcinogenesis, we have analyzed the entire coding region of TRAIL‐R2 and the death domain (DD) regions of TRAIL‐R1 and ‐R4 for the detection of somatic mutations in a series of breast tumors, lymph node metastases and BC cell lines. Overall, we detected 1, 11 and 3 alterations in the TRAIL‐R1, ‐R2 and ‐R4 genes, respectively. Although functional studies have not yet been performed, we assume that most of these alterations do not alter the function of TRAIL‐receptors. Additionally, we analyzed individuals from BC families for the detection of TRAIL‐R2 germline mutations. One alteration has been found in the Kozak consensus motif at position ‐4 with respect to the translation initiation AUG [1‐4 (C→A)]. We further studied the mRNA expression of TRAIL and the 4 TRAIL receptors. In BC cell lines, a strongly decreased mRNA expression of TRAIL, TRAIL‐R1, ‐R3 and ‐R4 was found, whereas the expression of TRAIL‐R2 was only slightly reduced. In breast tumors, a 1.2–3.6‐fold reduction of mRNA signals of the 5 genes was observed. No correlation was found between the expression level of TRAIL and the receptor mRNAs and clinicopathologic variables and between the expression of TRAIL‐R2 and TP53 mutation status and loss of heterozygosity (LOH) at 8p21‐p22. Taken together, we cannot exclude the involvement of TRAIL‐receptors in BC. Our mutation studies indicate that DD receptor mutations occur at low frequency and are not the primary cause for the altered mRNA expression of TRAIL and TRAIL‐receptors in BC.