An imbalance in progenitor cell populations reflects tumour progression in breast cancer primary culture models.

BACKGROUND: Many factors influence breast cancer progression, including the ability of progenitor cells to sustain or increase net tumour cell numbers. Our aim was to define whether alterations in putative progenitor populations could predict clinicopathological factors of prognostic importance for cancer progression. METHODS: Primary cultures were established from human breast tumour and adjacent non-tumour tissue. Putative progenitor cell populations were isolated based on co-expression or concomitant absence of the epithelial and myoepithelial markers EPCAM and CALLA respectively. RESULTS: Significant reductions in cellular senescence were observed in tumour versus non-tumour cultures, accompanied by a stepwise increase in proliferation:senescence ratios. A novel correlation between tumour aggressiveness and an imbalance of putative progenitor subpopulations was also observed. Specifically, an increased double-negative (DN) to double-positive (DP) ratio distinguished aggressive tumours of high grade, estrogen receptor-negativity or HER2-positivity. The DN:DP ratio was also higher in malignant MDA-MB-231 cells relative to non-tumorigenic MCF-10A cells. Ultrastructural analysis of the DN subpopulation in an invasive tumour culture revealed enrichment in lipofuscin bodies, markers of ageing or senescent cells. CONCLUSIONS: Our results suggest that an imbalance in tumour progenitor subpopulations imbalances the functional relationship between proliferation and senescence, creating a microenvironment favouring tumour progression.

Metallothionein 1E is methylated in malignant melanoma and increases sensitivity to cisplatin-induced apoptosis.

DNA methylation plays a major role in cancer by silencing tumour suppressor genes. In melanoma, only a discrete number of methylated genes have been identified so far. After the treatment of melanoma cells with a DNA methyltransferase inhibitor and subsequent transcriptomic profiling, we had identified earlier a cohort of melanoma progression-associated genes regulated by methylation. Here, we identified which of these genes are directly methylated in melanoma cell lines and tissues. First, we examined 16 genes by bisulphite sequencing in the WM793 isogenic cell line model series. Five of these genes (CYBA, FABP5, MT1E, TSPY1 and TAC1) displayed increased methylation in several invasive cell lines compared with the parental WM793 cells, indicating their involvement in progression. Next, we analyzed several matched primary/metastatic tumours using methylation-specific PCR, which revealed that MT1E (one of the five genes assessed) was methylated in the largest proportion of tumours. Examination of a larger cohort of samples showed that 1 of 17 (6%) of the benign naevi, 16 of 43 (37%) primary tumours and 6 of 13 (46%) of the metastases displayed MT1E methylation. In addition, ectopic over-expression of MT1E mediated sensitization to cisplatin-induced apoptosis. Overall, these studies suggest that MT1E is a potential tumour suppressor gene, whose loss may promote resistance to apoptosis-inducing therapies.

Topoisomerase I amplification in melanoma is associated with more advanced tumours and poor prognosis.

In this study, we used array-comparative genomic hybridization (aCGH) and fluorescent in situ hybridization (FISH) to examine genetic aberrations in melanoma cell lines and tissues. Array-comparative genomic hybridization revealed that the most frequent genetic changes found in melanoma cell lines were amplifications on chromosomes 7p and 20q, along with disruptions on Chr 9, 10, 11, 12, 22 and Y. Validation of the results using FISH on tissue microarrays (TMAs) identified TOP1 as being amplified in melanoma tissues. TOP1 amplification was detected in a high percentage (33%) of tumours and was associated with thicker, aggressive tumours. These results show that TOP1 amplification is associated with advanced tumours and poor prognosis in melanoma. These observations open the possibility that TOP1-targeted therapeutics may be of benefit in a particular subgroup of advanced stage melanoma patients.

JAM-A expression positively correlates with poor prognosis in breast cancer patients.

The cell-cell adhesion protein junctional adhesion molecule-A (JAM-A) influences epithelial cell morphology and migration. As migration is required for tumor cell invasion and metastasis, we sought to elucidate the role of JAM-A in invasive breast cancer. A breast cancer tissue microarray was analyzed for JAM-A protein expression, in parallel with analysis of JAM-A gene expression data from a breast cancer clinical dataset. Our data demonstrate a novel association between JAM-A gene and protein upregulation and poor prognosis in breast cancer. To mechanistically dissect this process, we used lentiviral technology to stably knock down JAM-A gene expression by shRNA in MCF7 breast cancer cells, which express high-endogenous levels of JAM-A. We also antagonized JAM-A function in wild-type MCF7 cells using an inhibitory antibody that blocks JAM-A dimerization. Knockdown or functional antagonism of JAM-A decreased breast cancer cell migration in scratch-wound assays. Reductions in beta1-integrin protein levels were observed after JAM-A-knockdown in MCF7 cells, suggesting a mechanism for reduced motility after loss of JAM-A. Consistent with this hypothesis, tissue microarray analysis of beta1-integrin protein expression in invasive breast cancer tissues revealed a trend toward high beta1-integrin protein levels being indicative of poor prognosis. Twenty-two percent of patients were observed to coexpress high levels of JAM-A and beta1-integrin protein, and MDA-MB-231 breast cells stably overexpressing JAM-A showed an increase in beta1-integrin protein expression. Our results are consistent with a previously unreported role for JAM-A overexpression as a possible mechanism contributing to progression in primary breast cancer; and a potential therapeutic target.

Urinary insulin-like growth factor 2 identifies the presence of urothelial carcinoma of the bladder.

OBJECTIVE: To examine urinary insulin-like growth factor 2 (IGF-2) levels in patient urine samples and determine the potential of IGF-2 as a marker for the presence of urothelial carcinoma of the bladder (UCB). PATIENTS AND METHODS: The current gold standard for diagnosis of UCB is cystoscopy and cytological analysis. The identification of an accurate urine marker for UCB with the potential to replace unnecessary cystoscopy would benefit patients with UCB and others investigated after detecting haematuria. In the present study, we analysed 65 urine samples, and optimized an enzyme-linked immunosorbent assay-based approach to measure urinary levels of IGF-2. RESULTS: Based on a threshold of 5.4 ng/mL, patients with UCB have significantly elevated levels of urinary IGF-2 (P = 0.009) and this difference remained significant after adjustment for age and sex (P = 0.04). Sensitivity and specificity values of 80% and 52%, respectively, were determined for urinary IGF-2 alone and when combined with nuclear matrix protein 22 (NMP22; an approved biomarker for detection of UCB). There was a positive correlation between urinary IGF-2 levels and NMP22 levels in patient urine samples and the combined assay improved the detection of UCB (sensitivity 85% and specificity 52%). CONCLUSION: Substantiated evidence has identified IGF-2 as a valuable marker for UCB. In addition, the novel observations of the present study have shown that aberrant levels of IGF-2 occurring in the presence of UCB, can now be determined through a simple and inexpensive urine assay.

Recurrence of urothelial carcinoma of the bladder: a role for insulin-like growth factor-II loss of imprinting and cytoplasmic E-cadherin immunolocalization.

PURPOSE: This study documents the frequency of insulin-like growth factor-II (IGF-II) loss of imprinting (LOI) in a series of 87 bladder tissues. E-cadherin (CDH1) immunolocalization was also investigated due to the known redistribution of this adherence protein to the cytoplasm following exogenous exposure to IGF-II. EXPERIMENTAL DESIGN: Informative IGF-II cases were identified following DNA-PCR amplification and subsequent sequencing of the transcribable ApaI RFLP in exon 9 of IGF-II. Similar approaches using primer-specific cDNA templates identified the imprinting status of IGF-II in these informative cases. CDH1 cellular localization was assessed on a tissue microarray platform of 114 urothelial carcinoma of the bladder (UCB) cases (70 pT(a) noninvasive and 44 pT(1) lamina propria invasive) using the commercially available Novocastra antibody. RESULTS: IGF-II LOI was evident in 7 of 17 (41%) UCB tumors and 4 of 11 (36%) tumor-associated normal urothelial samples. Two of four pT(1) grade 3 tumors, the subject of much debate concerning their suitability for radical cystectomy, showed LOI at the IGF-II locus. In those tumors showing IGF-II LOI, 4 of 7 (57%) displayed concomitant CDH1 cytoplasmic staining. In contrast, only 3 of 10 (30%) IGF-II maintenance of imprinting tumors had concomitant CDH1 cytoplasmic localization. UCB cell lines displaying cytoplasmic CDH1 immunolocalization expressed significantly higher levels of IGF-II (CAL29, HT1376, and RT112) compared with RT4, a cell line displaying crisp membranous CDH1 staining. Finally, cytoplasmic CDH1 staining was an independent predictor of a shorter time to recurrence independent of tumor grade and stage. CONCLUSIONS: We suggest that CDH1 cytoplasmic immunolocalization as a result of increased IGF-II levels identifies those nonmuscle invasive presentations most likely to recur and therefore might benefit from more radical nonconserving bladder surgery.

Progesterone receptor B (PRB) promoter hypermethylation in sporadic breast cancer: progesterone receptor B hypermethylation in breast cancer.

INTRODUCTION: Oestrogen receptor alpha (ER alpha) is traditionally measured on all breast tumour specimens to identify those patients more likely to respond to anti-oestrogens. Progesterone receptor (PR) status has contributed useful information in defining more responsive subgroups. PR negativity may be a marker for increased signalling through growth factor receptor tyrosine kinase pathways. Progesterone acts through two PRs, PRA and PRB. PRB, the functionally active PR, can be silenced by promoter hypermethylation. METHODS: Following DNA and RNA extraction from 94 breast carcinomas, the methylation status of the PRB promoter was assessed by sodium bisulphite modification and methylation sensitive PCR (MSP). A quantitative realtime PCR analysis (QRTPCR) was used to determine the levels of PRB mRNA expression. Protein expression was evaluated immunohistochemically with a commercially available PRB antibody. RESULTS: 76% of the primary breast carcinoma samples demonstrated a methylated band for PRB. PRB methylation significantly compromised total PR immunohistochemistry (IHC) expression (P = 0.03). PRB mRNA correlated positively with total PR IHC (r = 0.58, P = 0.04), ER alpha IHC (P = 0.02), and tumour grade (P = 0.01). PRB protein expression was significantly associated with a number of favourable prognostic variables including smaller (P = 0.004) lower grade (P = 0.007), ER alpha IHC positive tumours (P < 0.001), and tumours with a low Nottingham Prognostic Index (NPI) (P = 0.0008). PRB mRNA levels were significantly associated with better overall survival (P = 0.04) in a univariate analysis. CONCLUSION: The majority of tumours were methylated for PRB. This did not directly compromise PRB expression suggesting that other factors may down regulate the PR gene. When PRB was expressed, it correlated with good prognostic markers and better overall survival.

Molecular basis for estrogen receptor alpha deficiency in BRCA1-linked breast cancer.

BACKGROUND: BRCA1-mutant breast tumors are typically estrogen receptor alpha (ER alpha) negative, whereas most sporadic tumors express wild-type BRCA1 and are ER alpha positive. We examined a possible mechanism for the observed ER alpha-negative phenotype of BRCA1-mutant tumors. METHODS: We used a breast cancer disease-specific microarray to identify transcripts that were differentially expressed between paraffin-embedded samples of 17 BRCA1-mutant and 14 sporadic breast tumors. We measured the mRNA levels of estrogen receptor 1 (ESR1) (the gene encoding ER alpha), which was differentially expressed in the tumor samples, by quantitative polymerase chain reaction. Regulation of ESR1 mRNA and ER alpha protein expression was assessed in human breast cancer HCC1937 cells that were stably reconstituted with wild-type BRCA1 expression construct and in human breast cancer T47D and MCF-7 cells transiently transfected with BRCA1-specific short-interfering RNA (siRNA). Chromatin immunoprecipitation assays were performed to determine if BRCA1 binds the ESR1 promoter and to identify other interacting proteins. Sensitivity to the antiestrogen drug fulvestrant was examined in T47D and MCF-7 cells transfected with BRCA1-specific siRNA. All statistical tests were two-sided. RESULTS: Mean ESR1 gene expression was 5.4-fold lower in BRCA1-mutant tumors than in sporadic tumors (95% confidence interval [CI] = 2.6-fold to 40.1-fold, P = .0019). The transcription factor Oct-1 recruited BRCA1 to the ESR1 promoter, and both BRCA1 and Oct-1 were required for ER alpha expression. BRCA1-depleted breast cancer cells expressing exogenous ER alpha were more sensitive to fulvestrant than BRCA1-depleted cells transfected with empty vector (T47D cells, the mean concentration of fulvestrant that inhibited the growth of 40% of the cells [IC40] for empty vector versus ER alpha: >10(-5) versus 8.0 x 10(-9) M [95% CI = 3.1 x 10(-10) to 3.2 x 10(-6) M]; MCF-7 cells, mean IC40 for empty vector versus ER alpha: >10(-5) versus 4.9 x 10(-8) M [95% CI = 2.0 x 10(-9) to 3.9 x 10(-6) M]). CONCLUSIONS: BRCA1 alters the response of breast cancer cells to antiestrogen therapy by directly modulating ER alpha expression.

Contribution of DNA and tissue microarray technology to the identification and validation of biomarkers and personalised medicine in breast cancer.

Completion of the human genome project has revolutionised translational medicine. High-throughput technology now permits investigators to systematically interrogate the genome, transcriptome, proteome and metabolome. It is expected that these advances will eventually be translated into new more sensitive diagnostic tests and less toxic therapeutics. A major shift is expected in clinical oncology over the next few decades as we start to move away from currently practiced, population-based approaches to personalised medicine. In this emerging approach, the molecular and pathophysiological characteristics of an individual patient and tumour will be measured and tailored therapeutic regimens will be administered based on these profiles. One of the key steps in this process will be the identification and validation of biomarkers. Whilst great advances have been made in the discovery of putative biomarkers, disappointingly few have been translated into clinically applicable assays. It is widely believed that this is due to a lack of well-designed, thorough validation studies. Here, we review the role of DNA microarrays and tissue microarrays in the validation of biomarkers in breast cancer, with emphasis on their potential application to determine mode of personalised therapy in the future.

MRNA stability and overexpression of fatty acid synthase in human breast cancer cell lines.

BACKGROUND: Elevation of fatty acid synthase (FAS) in human cancers is often associated with increased tumor aggression. The basic genetic mechanisms leading to increased enzyme content in cancer cells were investigated using cell lines derived from human metastatic breast carcinomas (T47D, Zr75 and SKBr3) and normal human breast epithelium (184A1). MATERIALS AND METHODS: Western analysis, Northern blotting, [2-(14)C]malonyl-CoA incorporation assays, nuclear run-off transcription assays, mRNA decay assays, and poly(A) tail assays were used to measure and compare transcription rates of the FAS gene among the four cell lines. RESULTS: By Western analysis, FAS levels in T47D were 2.6 times lower than ZR75 and SKBr3, but 6.7 times greater than non-neoplastic 184A1 cells. FAS mRNA levels and specific activity correlated with protein content. In contrast, relative rates of FAS gene transcription were significantly higher in non-neoplastic 184A1 cells than T47D, ZR75 and SKBr3. Stability of message was investigated to explain this discrepancy. The half-life of FAS mRNA in 184A1 cells was 5.6 h, or 4-5-fold less than ZR75 and SKBr3. Poly(A) tail assays showed that FAS mRNA species from 184A1 cells tended to be longer than those of breast cancer cell lines (500-1500 nt versus 500-800 nt, respectively). CONCLUSION: Breast cancer cell lines contained significantly more FAS enzyme, message and activity than non-neoplastic 184A1 cells. Yet, 184A1 cells exhibited higher rates

Multiple markers for melanoma progression regulated by DNA methylation: insights from transcriptomic studies.

The incidence of melanoma is increasing rapidly, with advanced lesions generally failing to respond to conventional chemotherapy. Here, we utilized DNA microarray-based gene expression profiling techniques to identify molecular determinants of melanoma progression within a unique panel of isogenic human melanoma cell lines. When a poorly tumorigenic cell line, derived from an early melanoma, was compared with two increasingly aggressive derivative cell lines, the expression of 66 genes was significantly changed. A similar pattern of differential gene expression was found with an independently derived metastatic cell line. We further examined these melanoma progression-associated genes via use of a tailored TaqMan Low Density Array (LDA), representing the majority of genes within our cohort of interest. Considerable concordance was seen between the transcriptomic profiles determined by DNA microarray and TaqMan LDA approaches. A range of novel markers were identified that correlated here with melanoma progression. Most notable was TSPY, a Y chromosome-specific gene that displayed extensive down-regulation in expression between the parental and derivative cell lines. Examination of a putative CpG island within the TSPY gene demonstrated that this region was hypermethylated in the derivative cell lines, as well as metastatic melanomas from male patients. Moreover, treatment of the derivative cell lines with the DNA methyltransferase inhibitor, 2'-deoxy-5-azacytidine (DAC), restored expression of the TSPY gene to levels comparable with that found in the parental cells. Additional DNA microarray studies uncovered a subset of 13 genes from the above-mentioned 66 gene cohort that displayed re-activation of expression following DAC treatment, including TSPY, CYBA and MT2A. DAC suppressed tumor cell growth in vitro. Moreover, systemic treatment of mice with DAC attenuated growth of melanoma xenografts, with consequent re-expression of TSPY mRNA. Overall, our data support the hypothesis that multiple genes are targeted, either directly or indirectly, by DNA hypermethylation during melanoma progression.

Development and preliminary evaluation of the VPS ReplaySuite: a virtual double-headed microscope for pathology.

BACKGROUND: Advances in computing and telecommunications have resulted in the availability of a range of online tools for use in pathology training and quality assurance. The majority focus on either enabling pathologists to examine and diagnose cases, or providing image archives that serve as reference material. Limited emphasis has been placed on analysing the diagnostic process used by pathologists to reach a diagnosis and using this as a resource for improving diagnostic performance. METHODS: The ReplaySuite is an online pathology software tool that presents archived virtual slide examinations to pathologists in an accessible video-like format, similar to observing examinations with a double-headed microscope. Delivered through a customized web browser, it utilises PHP (Hypertext PreProcessor) to interact with a remote database and retrieve data describing virtual slide examinations, performed using the Virtual Pathology Slide (VPS). To demonstrate the technology and conduct a preliminary evaluation of pathologists opinions on its potential application in pathology training and quality assurance, 70 pathologists were invited to use the application to review their own and other pathologists examinations of 10 needle-core breast biopsies and complete an electronic survey. 9 pathologists participated, and all subsequently completed an exit survey. RESULTS: Of those who replayed an examination by another pathologist, 83.3% (5/6) agreed that replays provided an insight into the examining pathologists diagnosis and 33.3% (2/6) reconsidered their own diagnosis for at least one case. Of those who reconsidered their original diagnosis, all re-classified either concordant with group consensus or original glass slide diagnosis. 77.7% (7/9) of all participants, and all 3 participants who replayed more than 10 examinations stated the ReplaySuite to be of some or great benefit in pathology training and quality assurance. CONCLUSION: Participants conclude the ReplaySuite to be of some or of great potential benefit to pathology training and quality assurance and consider the ReplaySuite to be beneficial in evaluating the diagnostic trace of an examination. The ReplaySuite removes temporal and spatial issues that surround the use of double-headed microscopes by allowing examinations to be reviewed at different times and in different locations to the original examination. While the evaluation set was limited and potentially subject to bias, the response of participants was favourable. Further work is planned to determine whether use of the ReplaySuite can result in improved diagnostic ability.

Sclerosing polycystic sialadenopathy: a rare cause of recurrent tumor of the parotid gland.

We present a case report of a woman with a recurrent parotid mass that was histologically confirmed to be sclerosing polycystic sialadenopathy, a rarely reported cause of salivary gland tumor. This tumor measured 11.8 x 6 cm, the largest such lesion yet reported. Histologic analysis revealed foci of atypical intraductal acinic cell proliferation and necrosis severe enough to amount to intraductal carcinoma in situ. Despite its large size, it was successfully excised, leaving the patient with only moderate facial nerve dysfunction postoperatively.

Development and evaluation of the virtual pathology slide: a new tool in telepathology.

BACKGROUND: The Virtual Pathology Slide is an interactive microscope emulator that presents, via the Internet or CD-ROM, a complete 15.53 mm x 11.61 mm digitalized tissue section. The Virtual Pathology Slide mimics the use of a microscope in both the stepwise increase in magnification (from 16x up to 2000x) and in lateral motion in the X and Y Cartesian directions. This permits a pathologist to navigate to any area on a slide, at any magnification, similar to a conventional microscope. OBJECTIVE: The aim of this study was to assess the diagnostic accuracy and acceptability of the Virtual Pathology Slide. METHODS: Ten breast needle core biopsies were randomly selected and presented to 17 pathologists or trainee pathologists with at least 2 years experience in pathology practice. Participants were required to examine each case online and provide a diagnostic classification using online feedback forms. The recorded data permitted examination of interobserver variability and user satisfaction. RESULTS: Agreement between original glass-slide diagnosis and consensus diagnosis using the Virtual Pathology Slide was reached in 9 out of 10 slides. Percentage concordance for slides lay in the range of 35.3% to 100% with an average percentage concordance between slides of 66.5%. The average Kappa statistics for interobserver agreement was 0.75 while average percentage concordance amongst participants was 66.5%. Participants looked at an average of 22 fields of view while examining each slide. Confidence: 81.25% of the participants indicated confidence using the Virtual Pathology Slide to make a diagnostic decision, with 56.25% describing themselves as "reasonably confident," 18.75% as "confident," and 6.25% as "very confident." Ease of use: 68.75% reported the system as "easy" or "very easy" to use. Satisfaction: 87.5% of participants expressed satisfaction with image quality, with 43.75% describing the image quality as "adequate," 25% describing it as "good," and 18.75% describing the image quality as "excellent." Pathologists with a working bandwidth greater than 20 kilobits per second found the download speed of the Virtual Pathology Slide "adequate" or better. CONCLUSIONS: Results from this study show that the Virtual Pathology Slide can be used to make a correct diagnostic decision, and that the system is a realistic alternative to dynamic telepathology.