Stephen R. Weinstein

Association of A vitamin D receptor polymorphism with sporadic breast cancer development.

Breast cancer is the leading cause of cancer death among Australian women and its incidence is annually increasing. Genetic factors are involved in the complex etiology of breast cancer. The seco‐steroid hormone, 1,25 dihydroxy vitamin D3 can influence breast cancer cell growth in vitro. A number of studies have reported correlations between vitamin D receptor (VDR) gene polymorphisms and several diseases including prostate cancer and osteoporosis. In breast cancer, low vitamin D levels in serum are correlated with disease progression and bone metastases, a situation also noted in prostate cancer and suggesting the involvement of the VDR. In our study, 2 restriction fragment length polymorphisms (RFLP) in the 3′ region (detected by Apa1 and Taq1) and an initiation codon variant in the 5′ end of the VDR gene (detected by Fok1) were tested for association with breast cancer risk in 135 females with sporadic breast cancer and 110 cancer‐free female controls. Allele frequencies of the 3′ ApaI polymorphism showed a significant association (p = 0.016; OR = 1.56, 95% CI = 1.09–2.24) while the TaqI RFLP showed a similar trend (p = 0.053; OR = 1.45, 95% CI = 1.00–2.00). Allele frequencies of the FokI polymorphism were not significantly different (p = 0.97; OR = 0.99, 95% CI = 0.69–1.43) in the study population. Our results suggest that specific alleles of the VDR gene located near the 3′ region may identify an increased risk for breast cancer and justify further investigation of the role of VDR in breast cancer. Int. J. Cancer 83:723–726, 1999.

Association of estrogen receptor and glucocorticoid receptor gene polymorphisms with sporadic breast cancer.

We have utilized a cross‐sectional association approach to investigate sporadic breast cancer. Polymorphisms in 2 candidate genes, ESRα and GRL, were examined in an unrelated breast cancer–affected and age‐matched control population. Several polymorphic regions within the ESRα gene have been identified, and some alleles of these polymorphisms have been found to occur at increased levels in breast‐cancer patients. Additionally, variations in GRL have the potential to disrupt cell transcription and may be associated with cancer formation. We analyzed 3 polymorphisms, from codons 10 (TCT to TCC), 325 (CCC to CCG) and 594 (ACA to ACG) of ESRα, and a highly polymorphic dinucleotide repeat, D5S207, located within 200 kb of the GRL. When allelc frequencies of the codon 594 (exon 8) ESR polymorphism were compared between affected and unaffected populations, a significant difference was observed (p = 0.005). Results from the D5S207 dinucleotide repeat located near GRL also indicated a significant difference between the tested case and control populations (p = 0.001). Allelic frequencies of the codon 10 and codon 325 ESR polymorphisms were not significantly different between populations (p = 0.152 and 0.181, respectively). Our results indicate that specific alleles of the ESR gene (α subtype) and a marker for the GRL gene locus are associated with sporadic breast‐cancer development in the tested Caucasian population and justify further investigation of the role of these and other nuclear steroid receptors in the etiology of breast cancer.

Correlation between BRAF mutation and the clinicopathological parameters in papillary thyroid carcinoma with particular reference to follicular variant.

Mutation of the BRAF gene is common in thyroid cancer. Follicular variant of papillary thyroid carcinoma is a variant of papillary thyroid carcinoma that has created continuous diagnostic controversies among pathologists. The aims of this study are to (1) investigate whether follicular variant of papillary thyroid carcinoma has a different pattern of BRAF mutation than conventional papillary thyroid carcinoma in a large cohort of patients with typical features of follicular variant of papillary thyroid carcinoma and (2) to study the relationship of clinicopathological features of papillary thyroid carcinomas with BRAF mutation. Tissue blocks from 76 patients with diagnostic features of papillary thyroid carcinomas (40 with conventional type and 36 with follicular variant) were included in the study. From these, DNA was extracted and BRAF V600E mutations were detected by polymerase chain reaction followed by restriction enzyme digestion and sequencing of exon 15. Analysis of the data indicated that BRAF V600E mutation is significantly more common in conventional papillary thyroid carcinoma (58% versus 31%, P = .022). Furthermore, the mutation was often noted in female patients (P = .017), in high-stage cancers (P = .034), and in tumors with mild lymphocytic thyroiditis (P = .006). We concluded that follicular variant of papillary thyroid carcinoma differs from conventional papillary thyroid carcinoma in the rate of BRAF mutation. The results of this study add further information indicating that mutations in BRAF play a role in thyroid cancer development and progression.

Single nucleotide polymorphism in hsa-mir-196a-2 and breast cancer risk: a case control study.

microRNAs are small, non-coding RNAs that influence gene expression on a post-transcriptional level. They participate in diverse biological pathways and may act as either tumor suppressor genes or oncogenes. As they may have an effect on thousands of target mRNAs, single-nucleotide polymorphisms in microRNA genes might have major functional consequences, because the microRNAs properties and/or maturation may change. miR-196a has been reported to be aberrantly expressed in breast cancer tissue. Additionally, the SNP rs11614913 in hsa-mir-196a-2 has been found to be associated with breast cancer risk in some studies although not in others. This study evaluated the association between rs11614913 and breast cancer risk in a Caucasian case-control cohort in Queensland, Australia. Results do not support an association of the tested hsa-mir-196a-2 polymorphism with breast cancer susceptibility in this cohort. As there is a discrepancy between our results and previous findings, it is important to assess the role of rs11614913 in breast cancer by further larger studies investigating different ethnic groups.

Differential gene expression in breast cancer cell lines and stroma–tumor differences in microdissected breast cancer biopsies revealed by display array analysis

To examine gene‐expression patterning in late‐stage breast cancer biopsies, we used a microdissection technique to separate tumor from the surrounding breast tissue or stroma. A DD‐PCR protocol was then used to amplify expressed products, which were resolved using PAGE and used as probe to hybridize with representative human arrays and cDNA libraries. The probe derived from the tumor–stroma comparison was hybridized with a gene array and an arrayed cDNA library derived from a GCT of bone; 21 known genes or expressed sequence tags were detected, of which 17 showed differential expression. These included factors associated with epithelial to mesenchymal transition (vimentin), the cargo selection protein (TIP47) and the signal transducer and activator of transcription (STAT3). Northern blot analysis was used to confirm those genes also expressed by representative breast cancer cell lines. Notably, 6 genes of unknown function were restricted to tumor while the majority of stroma‐associated genes were known. When applied to transformed breast cancer cell lines (MDA‐MB‐435 and T47D) that are known to have different metastatic potential, DD array analysis revealed a further 20 genes; 17 of these genes showed differential expression. Use of microdissection and the DD‐PCR array protocol allowed us to identify factors whose localized expression within the breast may play a role in abnormal breast development or breast carcinogenesis.

The clinicopathological roles of alpha-B-crystallin and p53 expression in patients with head and neck squamous cell carcinoma

Aims: The aim of the study was to investigate the expression of alpha‐B‐crystallin and p53 in head and neck squamous cell carcinoma (HNSCC). Methods: Alpha‐B‐crystallin and p53 expressions from 118 HNSCC were studied by immunohistochemistry and correlated with clinicopathological parameters. Results: Alpha‐B‐crystallin expression was seen in 28% (n = 33) of HNSCC. All except one poorly differentiated HNSCC were negative for alpha‐B‐crystallin. p53 expression was seen in 63% (n = 73) of HNSCC and was more common in moderately/poorly differentiated HNSCC (p = 0.034). The proportion of cases with positive staining for either alpha‐B‐crystallin or p53 was different in different anatomical locations in the head and neck. Patients with HNSCC having a high portion of tumour cells expressing p53 had a shorter survival than the other groups (p = 0.032). Conclusion: The expression of p53 and alpha‐B‐crystallin were related to the differentiation and site of the HNSCC. Alpha‐B‐crystallin was not a prognostic marker for HNSCC.

GAEC1 and colorectal cancer : a study of the relationships between a novel oncogene and clinicopathologic features

GAEC1 is a novel gene located at 7q22.1 that was detected in our previous work in esophageal cancer. The aims of the present study are to identify the copy number of GAEC1 in different colorectal tissues including carcinomas, adenomas, and nonneoplastic tissues and characterize any links to pathologic factors. The copy number of GAEC1 was studied by evaluating the quantitative amplification of GAEC1 DNA in 259 colorectal tissues (144 adenocarcinomas, 31 adenomas, and 84 nonneoplastic tissues) using real-time polymerase chain reaction. Copy number of GAEC1 DNA in colorectal adenocarcinomas was higher in comparison with nonneoplastic colorectum. Seventy-nine percent of the colorectal adenocarcinomas showed amplification and 15% showed deletion of GAEC1 (P < .0001). Of the adenomas, 90% showed deletion of GAEC1, with the remaining 10% showing normal copy number. The differences in GAEC1 copy number between colorectal adenocarcinoma, colorectal adenoma, and nonneoplastic colorectal tissue are significant (P < .0001). GAEC1 copy number was significantly higher in adenocarcinomas located in distal colorectum compared with proximal colon (P = .03). In conclusion, GAEC1 copy number was significantly different between colorectal adenocarcinomas, adenomas, and nonneoplastic colorectal tissues. The copy number was also related to the site of the cancer. These findings along with previous work in esophageal cancer imply that GAEC1 is commonly involved in the pathogenesis of colorectal adenocarcinoma.

Semaphorin-plexin signalling genes associated with human breast tumourigenesis.

INTRODUCTION Gene expression profiling has enabled us to demonstrate the heterogeneity of breast cancers. The potential of a tumour to grow and metastasise is partly dependant on its ability to initiate angiogenesis or growth and remodelling of new blood vessels, usually from a pre-existing vascular network, to ensure delivery of oxygen, nutrients, and growth factors to rapidly dividing transformed cells along with access to the systemic circulation. Cell-cell signalling of semaphorin ligands through interaction with their plexin receptors is important for the homeostasis and morphogenesis of many tissues and has been widely studied for a role in neural connectivity, cancer, cell migration and immune responses. This study investigated the role of four semaphorin/plexin signalling genes in human breast cancers in vivo and in vitro. MATERIALS AND METHODS mRNA was extracted from formalin fixed paraffin embedded archival breast invasive ductal carcinoma tissue samples of progressive grades (grades I-III) and compared to tissue from benign tumours. Gene expression profiles were determined by microarray using the Affymetrix GeneChip® Human Genome U133 Plus 2.0 Arrays and validated by Q-PCR using a Corbett RotorGene 6000. Following validation, the gene expression profile of the identified targets was correlated with those of the human breast cancer cell lines MCF-7 and MDA-MD-231. RESULTS The array data revealed that 888 genes were found to be significantly (p≤0.05) differentially expressed between grades I and II tumours and 563 genes between grade III and benign tumours. From these genes, we identified four genes involved in semaphorin-plexin signalling including SEMA4D which has previously been identified as being involved in increased angiogenesis in breast cancers, and three other genes, SEMA4F, PLXNA2 and PLXNA3, which in the literature were associated with tumourigenesis, but not directly in breast tumourigenesis. The microarray analysis revealed that SEMA4D was significantly (P=0.0347) down-regulated in the grade III tumours compared to benign tumours; SEMA4F, was significantly (P=0.0159) down-regulated between grades I and II tumours; PLXNA2 was significantly (P=0.036) down-regulated between grade III and benign tumours and PLXNA3 significantly (P=0.042) up-regulated between grades I and II tumours. Gene expression of SEMA4D was validated using Q-PCR, demonstrating the same expression profile in both data sets. When the sample set was increased to incorporate more cases, SEMA4D continued to follow the same expression profile, including statistical significance for the differences observed and small standard deviations. In vitro the same pattern was present where expression for SEMA4D was significantly higher in MDA-MB-231 cells when compared to MCF-7 cells. The expression of SEMA4F, PLXNA2 and PLXNA3 could not be validated using Q-PCR, however in vitro analysis of these three genes revealed that both SEMA4F and PLXNA3 followed the microarray trend in expression, although they did not reach significance. In contrast, PLXNA2 demonstrated statistical significance and was in concordance with the literature. DISCUSSION We, and others, have proposed SEMA4D to be a gene with a potentially protective effect in benign tumours that contributes to tumour growth and metastatic suppression. Previous data supports a role for SEMA4F as a tumour suppressor in the peripheral nervous system but our data seems to indicate that the gene is involved in tumour progression in breast cancer. Our in vitro analysis of PLXNA2 revealed that the gene has higher expression in more aggressive breast cancer cell types. Finally, our in vitro analysis on PLXNA3 also suggest that this gene may have some form of growth suppressive role in breast cancer, in addition to a similar role for the gene previously reported in ovarian cancer. From the data obtained in this study, SEMA4D may have a role in more aggressive and potentially metastatic breast tumours. CONCLUSIONS Semaphorins and their receptors, the plexins, have been implicated in numerous aspects of neural development, however their expression in many other epithelial tissues suggests that the semaphorin-plexin signalling system also contributes to blood vessel growth and development. These findings warrant further investigation of the role of semaphorins and plexins and their role in normal and tumour-induced angiogenesis in vivo and in vitro. This may represent a new front of attack in anti-angiogenic therapies of breast and other cancers.

Expression of glucocorticoid and progesterone nuclear receptor genes in archival breast cancer tissue.

BackgroundPrevious studies in our laboratory have shown associations of specific nuclear receptor gene variants with sporadic breast cancer. In order to investigate these findings further, we conducted the present study to determine whether expression levels of the progesterone and glucocorticoid nuclear receptor genes vary in different breast cancer grades.MethodsRNA was extracted from paraffin-embedded archival breast tumour tissue and converted into cDNA. Sample cDNA underwent PCR using labelled primers to enable quantitation of mRNA expression. Expression data were normalized against the 18S ribosomal gene multiplex and analyzed using analysis of variance.ResultsAnalysis of variance indicated a variable level of expression of both genes with regard to breast cancer grade (P = 0.00033 for glucocorticoid receptor and P = 0.023 for progesterone receptor).ConclusionStatistical analysis indicated that expression of the progesterone nuclear receptor is elevated in late grade breast cancer tissue.

Lipomeningioma: case report and review of the literature

We present a case of histologically confirmed lipomeningioma, the first to our knowledge reported in Australia. A 61-year-old man presented with seizures and confusion, and was found to have a non-enhancing left extra axial temporo-parietal lesion on CT and MRI scan. On MRI, the mass lesion showed hyper-intensity on the T1 weighted images, hypo-intensity on fat suppressed T2 weighted images and no enhancement with intravenous gadolinium, indicating a mass consisting predominantly of fatty tissue. A subsequent CT also showed the mass lesion to be hypodense with Hounsfield units indicating fatty tissue. A durally based tumour with high fat content macroscopically was excised at craniotomy under ultrasound guidance. Post-operative recovery was uneventful. Histology demonstrated a meningioma with high lipid content in the form of mature adipocytes and without atypical features. While not exceedingly rare, fewer than 30 cases of lipomeningioma, lipomatous meningioma, or lipidised meningioma have been reported in the world literature.