Genome-wide association studies (GWAS) have proven highly successful in identifying single nucleo... more Genome-wide association studies (GWAS) have proven highly successful in identifying single nucleotide polymorphisms (SNPs) associated with breast cancer (BC) risk. The majority of these studies are on European populations, with limited SNP association data in other populations. We genotyped 51 GWAS-identified SNPs in two independent cohorts of Singaporean Chinese. Cohort 1 comprised 1294 BC cases and 885 controls and was used to determine odds ratios (ORs); Cohort 2 had 301 BC cases and 243 controls for deriving polygenic risk scores (PRS). After age-adjustment, 11 SNPs were found to be significantly associated with BC risk. Five SNPs were present in <1% of Cohort 1 and were excluded from further PRS analysis. To assess the cumulative effect of the remaining 46 SNPs on BC risk, we generated three PRS models: Model-1 included 46 SNPs; Model-2 included 11 statistically significant SNPs; and Model-3 included the SNPs in Model-2 but excluded SNPs that were in strong linkage disequili...
Clear cell sarcoma of kidney (CCSK) is a rare paediatric renal malignant tumour. The majority of ... more Clear cell sarcoma of kidney (CCSK) is a rare paediatric renal malignant tumour. The majority of CCSKs have internal tandem duplications (ITD) of the BCOR gene while a minority has the YWHAE-NUTM2 gene fusion. A third 'double-negative' (DN) category comprises CCSKs with neither BCOR ITD nor YWHAE-NUTM2 fusion. The aim of this study was to characterize 11 histologically diagnosed CCSKs immunohistochemically (with CCND1, BCOR and CCNB3 stains), and genetically. By next-generation sequencing, 10 cases (90.9%) had BCOR exon 15 ITD, with positive BCOR immunoreactivity in 4 (36%) or 8 (72%) cases depending on antibody clone. By RT-PCR, none had the YWHAE-NUTM2 fusion. The DN case had a BCOR-CCNB3 fusion and strong nuclear CCNB3 and BCOR immunoreactivity. Quantitative PCR showed markedly elevated BCOR gene expression in this case whereas BCOR ITD cases had lower elevations. The majority of the CCSKs in our cohort have BCOR ITD, and none have the YWHAE-NUTM2 gene fusion. We verify t...
In 1985 we reported that 11% of a cohort of 151 patients with IgA nephritis (IgAN) had developed ... more In 1985 we reported that 11% of a cohort of 151 patients with IgA nephritis (IgAN) had developed end-stage renal disease (ESRD) after a follow-up period of 5 years. 15 years later, 35% had developed ESRD. We retrieved 125 stored renal biopsy paraffin blocks of the original cohort. From these, 102 patients were included in the present study and scored according to the Oxford classification as 21 specimens with less than 8 glomeruli were excluded and in 2 others, tissue samples were too tiny for a re-block. ESRD was ascertained by linking the study cohort to the Singapore Renal Registry at the National Registry of Diseases Office. Renal survival curves for each of the Oxford MEST lesions: endocapillary proliferation (E) (p < 0.04), segmental glomerulosclerosis (S) (p < 0.05), tubular atrophy/interstitial fibrosis (p < 0.0001) were significantly associated with ESRD. Mesangial hypercellularity, less commonly associated with progressive chronic kidney disease (CKD) in the study...
In this study we have evaluated the use of hypericin ex vivo urine fluorescence cytology as a non... more In this study we have evaluated the use of hypericin ex vivo urine fluorescence cytology as a non-invasive method for detecting early bladder cancers. To date this is the first study reported using this technique with hypericin. Urine samples from patients with early bladder cancers were processed for fluorescence cytology by incubation with hypericin, a novel photosensitizer. Normal urine samples incubated with hypericin served as normal controls. Laser confocal microscopy and spectroscopy was used to detect the fluorescence in the exfoliated low-grade urothelial tumor cells. Fluorescence cytology was considered positive if hypericin fluorescence of the low-grade urothelial tumor cells was detected to be stronger (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;8.5 times) compared to the baseline fluorescence established for normal urine samples. Automated analysis for an objective reproducible outcome appears possible. The possibility of detection of malignant urothelial cells in early cancer makes ex vivo fluorescence cytology promising for routine diagnostic screening.
The early detection of carcinoma is very essential for the diagnosis and prognosis of a bladder c... more The early detection of carcinoma is very essential for the diagnosis and prognosis of a bladder cancer patient. In this study we have investigated the use of hypericin as a fluorescent tumour marker and laser confocal microscopy as a diagnostic tool to aid the diagnosis of such cancers. Both cellular and clinical studies have been conducted. In the cellular studies, we have compared two bladder cell lines for the uptake and sub-cellular localization of hypericin. It was found that there was a rapid uptake and clearance of hypericin and significant localization in mitochondria and lysosomes. The study also revealed that there was a time-dependent increase in fluorescence intensity in bladder cells. The optimum localization was found to be 2-4 h post drug incubation. In the clinical study, consisting of 30 patients, both white light and fluorescence cystoscopy were performed after hypericin instillation. Biopsies taken from fluorescing regions were imaged using the confocal microscope. The order of fluorescence was detected to be as follows: normal &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; inflammation &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; grade 1 &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; grade 2 &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; CIS &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; grade 3. It was also found that the fluorescence intensity increased with the stage of the disease thereby enabling the determination of the degree of invasiveness of cancer. This enables the use of hypericin as a prognostic marker and laser confocal microscopy as a tool to aid in diagnosis of bladder cancer.
Bladder cancer is the fourth most common malignant disease worldwide, accounting for 4% of all ca... more Bladder cancer is the fourth most common malignant disease worldwide, accounting for 4% of all cancer cases. In Singapore, it is the ninth most common form of cancer. The high mortality rate in bladder cancer can be reduced by early treatment following pre-cancerous screening. Currently, the gold standard for screening bladder tumors is histological examination of biopsy specimens, which is both invasive and time-consuming. In this study, ex vivo urine fluorescence cytology was investigated to offer an alternative timely and biopsy-free means for detecting bladder cancers. Sediments in patient urine samples were extracted and incubated with a novel photosensitizer, hypericin. Laser confocal microscopy was used to capture the fluorescence images at an excitation wavelength of 488 nm. Images were subsequently processed to single out the exfoliated bladder cancer cells from the other cells based on the cellular size. Intensity histograms of each targeted cell and feature vectors, derived from the histogram moments, were used to represent each sample. A difference in the distribution of the feature vectors of normal and low-grade cancerous bladder cancer cells were observed. A diagnostic algorithm for discriminating between normal and low-grade cancerous cells is elucidated in this report. This study suggests that the fluorescence intensity profiles of hypericin in bladder cells can potentially provide an automated quantitative means of early bladder cancer diagnosis.
Treatment options for primary and metastatic renal cancer are increasing. Accurate data from the ... more Treatment options for primary and metastatic renal cancer are increasing. Accurate data from the pathological examination of renal cancer specimens aid clinicians in stratifying patients for surveillance and adjuvant therapies. This review focuses on biomarkers in diagnosis, prognosis and prediction of the biologic behavior of renal tumors which should be recorded in pathology reports and which are under investigation. Special emphasis is given to the use of immunohistochemical markers in differential diagnosis of various renal tumor subtypes. The relevance of cytogenetic and molecular findings is also discussed. The review includes the 2012 International Society for Urological Pathology Consensus conference recommendations.
This article was published in an Elsevier journal. The attached copy is furnished to the author f... more This article was published in an Elsevier journal. The attached copy is furnished to the author for non-commercial research and education use, including for instruction at the author's institution, sharing with colleagues and providing to institution administration. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier's archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright
Aims: We aimed to identify common reasons for second opinion breast pathology referrals at the Pa... more Aims: We aimed to identify common reasons for second opinion breast pathology referrals at the Pathology Department, Singapore General Hospital, focusing on queries and diagnostic issues raised by referring clinicians and pathologists. Methods: Request forms for breast pathology consultations were retrieved from a specialist's correspondence files consisting of pathologists' referrals, and from centralised laboratory records, comprising clinician-initiated referrals. Clinical and histomorphological queries raised by the referrals were collated. Results: Of 299 cases evaluated, clinician-initiated referrals (n ¼ 137, 46%) included requests for review of overall histopathology to confirm carcinoma subtype (n ¼ 47), grade (n ¼ 2), size (n ¼ 4), lymphovascular invasion (n ¼ 1) and confirm hormonal receptor and c-erbB-2 assays (n ¼ 33). Also required were: comparison of recurrent with previous lesions (n ¼ 8); settling discrepant diagnoses between two or more prior pathology reports (n ¼ 4); verification of microinvasion (n ¼ 6), in situ carcinomas (n ¼ 6) or atypical ductal hyperplasias (n ¼ 4); delineation of benign (n ¼ 8) and spindle cell lesions (n ¼ 3); to establish a breast origin of metastatic lesions (n ¼ 5); and distinction of carcinoma from lymphoma (n ¼ 2). Pathologist-initiated referrals (n ¼ 162, 54%) sought arbitration between borderline proliferative lesions (n ¼ 46) and papillary lesions (n ¼ 34); verification of microinvasion (n ¼ 23), stromal lesions (n ¼ 16), and carcinoma subtype (n ¼ 13), especially if the patient was young (n ¼ 5); clarification of metaplastic changes (n ¼ 4) and lobular neoplasia (n ¼ 8); and comparison of fibroepithelial lesions (n ¼ 11). Conclusions: Clinicians sought a second opinion mainly to verify histological diagnoses and report important pathological details for staging and confirmation of hormonal receptor and c-erbB-2 status prior to therapy. Borderline breast lesions are worrisome for both clinicians and pathologists in view of implications for management.
Glutathione S-transferase (GST) is known to play a key role in the detoxification and reduction o... more Glutathione S-transferase (GST) is known to play a key role in the detoxification and reduction of reactive oxygen species (ROS). Thus, we assessed GST activity and GST-pi expression in relation to oxidative stress and apoptosis in breast cancer. Tumor tissues from 32 breast cancer patients were evaluated for GST activity and thiobarbituric acid reactive substances (TBARS) that are by-products of oxidative stress. Four-micron sections of formalin-fixed, paraffin embedded tumors were stained immunohistochemically with anti-GST-pi. Apoptotic cells were detected by in situ end labeling of DNA fragments using a commercial kit. TBARS levels were significantly higher in breast cancers of older patients. GST-pi expression was up-regulated in breast cancers that exhibited higher oxidative stress and associated with higher GST activity. Apoptosis in GST-pi negative tumors was not correlated with GST activity, but GST-pi positive tumors within the same range of oxidative stress showed a reduction in apoptosis as well as an increased GST activity. This correlation was absent in GST-pi positive tumors experiencing higher oxidative stress. GST-pi expression may influence the level of GST activity and delay apoptosis in breast cancer. However, GST-pi expression in tumors with higher levels of oxidative stress may not be sufficient to abrogate the deleterious effects of ROS.
Immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH) are the only tests curre... more Immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH) are the only tests currently approved by the US Food and Drug Administration for classifying which patients will benefit from trastuzumab therapy. The accuracy of these two testing methods can be adversely affected by a variety of pre-analytical, analytical and post-analytical factors. According to the latest published recommendations of the panel of the Joint Committee of the American Society of Clinical Oncology and College of American Pathologists for HER2/neu testing, laboratories performing IHC and FISH for HER2/neu status in breast cancer are now required to have a high concordance of at least 95% between IHC and FISH, significantly higher than that in the published literature. To perform a retrospective analysis of the concordance of IHC and FISH analysis for HER2/neu at Singapore General Hospital and review potential causes of disparity between these two methods. A retrospective review of a total of 106 invasive ductal carcinomas evaluated for HER2/neu at the Department of Pathology, Singapore General Hospital between 2007 and 2008 were included in the study. The initial HER2/neu immunostained slides were reviewed independently without knowledge of FISH results, and concordance between IHC and FISH was determined. Concordance between IHC and FISH assay was excellent and within the published range (104/106=98.1%). The discordant cases represent a well-recognised subset of genetic heterogeneity in HER2/neu, which is known to contribute to positive IHC and negative FISH tests.
Breast cancer is a significant cause of morbidity and mortality in women with a high incidence of... more Breast cancer is a significant cause of morbidity and mortality in women with a high incidence of recurrence or treatment failure. Growing evidence suggests that cancer stem cells (CSCs) most likely contribute to tumour progression, spread and therapy failure. However, despite extensive research and the tremendous clinical potential of such cells in possible therapeutic management, the real nature of CSCs remains an enigma. In this review, we discuss the fundamental properties and molecular target of CSCs and focus on recent advances regarding the identification of CSC markers with emphasis on breast cancer and the underlying molecular mechanism of CSC phenotypes. We also discuss experimental evidence of targeting molecular pathways in order to modulate breast CSC behaviour in tumourigenesis and the controversies associated with it that potentially weaken the CSC model in breast cancer and other cancers as well.
Background: Chromophobe renal cell carcinoma (chRCC) and renal oncocytoma are two distinct but cl... more Background: Chromophobe renal cell carcinoma (chRCC) and renal oncocytoma are two distinct but closely related entities with strong morphologic and genetic similarities. While chRCC is a malignant tumor, oncocytoma is usually regarded as a benign entity. The overlapping characteristics are best explained by a common cellular origin, and the biologic differences between chRCC and oncocytoma are therefore of considerable interest in terms of carcinogenesis, diagnosis and clinical management. Previous studies have been relatively limited in terms of examining the differences between oncocytoma and chromophobe RCC. Methods: Gene expression profiling using the Affymetrix HGU133Plus2 platform was applied on chRCC (n = 15) and oncocytoma specimens (n = 15). Supervised analysis was applied to identify a discriminatory gene signature, as well as differentially expressed genes. High throughput single-nucleotide polymorphism (SNP) genotyping was performed on independent samples (n = 14) using Affymetrix GeneChip Mapping 100 K arrays to assess correlation between expression and gene copy number. Immunohistochemical validation was performed in an independent set of tumors. Results: A novel 14 probe-set signature was developed to classify the tumors internally with 93% accuracy, and this was successfully validated on an external data-set with 94% accuracy. Pathway analysis highlighted clinically relevant dysregulated pathways of c-erbB2 and mammalian target of rapamycin (mTOR) signaling in chRCC, but no significant differences in p-AKT or extracellular HER2 expression was identified on immunohistochemistry. Loss of chromosome 1p, reflected in both cytogenetic and expression analysis, is common to both entities, implying this may be an early event in histogenesis. Multiple regional areas of cytogenetic alterations and corresponding expression biases differentiating the two entities were identified. Parafibromin, aquaporin 6, and synaptogyrin 3 were novel immunohistochemical markers effectively discriminating the two pathologic entities.
This study aimed to determine the usefulness of a combination of 3 immunohistochemical markers, 3... more This study aimed to determine the usefulness of a combination of 3 immunohistochemical markers, 34β E12, p63 and α-methylacyl coenzyme A racemase (AMACR), for the diagnosis of prostate cancer using tissue microarrays (TMAs) constructed from 91 archival radical prostatectomy specimens derived from the Pathology Department files of Singapore General Hospital, Singapore. Triple immunostaining using a cocktail of these 3 antibodies was performed on TMA sections using the streptavidin-biotin method. When compared with immunohistochemical staining using the individual antibodies, we found that the triple cocktail allowed improved evaluation of basal cells in benign glands, and AMACR allowed simultaneous corroboration of malignant prostatic glands in the same section. We achieved a specificity of 100% with the triple cocktail, and sensitivity was acceptable, at 93.8%. In comparison, specificity and sensitivity of the individual antibodies were 95.5% and 97.3%, 93.3% and 93.8%, 97.0% and 95.6% for p63, 34βE12, and AMACR, respectively. The triple cocktail offers a costeffective way of evaluating abnormal prostatic glandular foci, in addition to maximizing the use of small tissue samples from prostatic needle biopsies.
Genome-wide association studies (GWAS) have proven highly successful in identifying single nucleo... more Genome-wide association studies (GWAS) have proven highly successful in identifying single nucleotide polymorphisms (SNPs) associated with breast cancer (BC) risk. The majority of these studies are on European populations, with limited SNP association data in other populations. We genotyped 51 GWAS-identified SNPs in two independent cohorts of Singaporean Chinese. Cohort 1 comprised 1294 BC cases and 885 controls and was used to determine odds ratios (ORs); Cohort 2 had 301 BC cases and 243 controls for deriving polygenic risk scores (PRS). After age-adjustment, 11 SNPs were found to be significantly associated with BC risk. Five SNPs were present in <1% of Cohort 1 and were excluded from further PRS analysis. To assess the cumulative effect of the remaining 46 SNPs on BC risk, we generated three PRS models: Model-1 included 46 SNPs; Model-2 included 11 statistically significant SNPs; and Model-3 included the SNPs in Model-2 but excluded SNPs that were in strong linkage disequili...
Clear cell sarcoma of kidney (CCSK) is a rare paediatric renal malignant tumour. The majority of ... more Clear cell sarcoma of kidney (CCSK) is a rare paediatric renal malignant tumour. The majority of CCSKs have internal tandem duplications (ITD) of the BCOR gene while a minority has the YWHAE-NUTM2 gene fusion. A third 'double-negative' (DN) category comprises CCSKs with neither BCOR ITD nor YWHAE-NUTM2 fusion. The aim of this study was to characterize 11 histologically diagnosed CCSKs immunohistochemically (with CCND1, BCOR and CCNB3 stains), and genetically. By next-generation sequencing, 10 cases (90.9%) had BCOR exon 15 ITD, with positive BCOR immunoreactivity in 4 (36%) or 8 (72%) cases depending on antibody clone. By RT-PCR, none had the YWHAE-NUTM2 fusion. The DN case had a BCOR-CCNB3 fusion and strong nuclear CCNB3 and BCOR immunoreactivity. Quantitative PCR showed markedly elevated BCOR gene expression in this case whereas BCOR ITD cases had lower elevations. The majority of the CCSKs in our cohort have BCOR ITD, and none have the YWHAE-NUTM2 gene fusion. We verify t...
In 1985 we reported that 11% of a cohort of 151 patients with IgA nephritis (IgAN) had developed ... more In 1985 we reported that 11% of a cohort of 151 patients with IgA nephritis (IgAN) had developed end-stage renal disease (ESRD) after a follow-up period of 5 years. 15 years later, 35% had developed ESRD. We retrieved 125 stored renal biopsy paraffin blocks of the original cohort. From these, 102 patients were included in the present study and scored according to the Oxford classification as 21 specimens with less than 8 glomeruli were excluded and in 2 others, tissue samples were too tiny for a re-block. ESRD was ascertained by linking the study cohort to the Singapore Renal Registry at the National Registry of Diseases Office. Renal survival curves for each of the Oxford MEST lesions: endocapillary proliferation (E) (p < 0.04), segmental glomerulosclerosis (S) (p < 0.05), tubular atrophy/interstitial fibrosis (p < 0.0001) were significantly associated with ESRD. Mesangial hypercellularity, less commonly associated with progressive chronic kidney disease (CKD) in the study...
In this study we have evaluated the use of hypericin ex vivo urine fluorescence cytology as a non... more In this study we have evaluated the use of hypericin ex vivo urine fluorescence cytology as a non-invasive method for detecting early bladder cancers. To date this is the first study reported using this technique with hypericin. Urine samples from patients with early bladder cancers were processed for fluorescence cytology by incubation with hypericin, a novel photosensitizer. Normal urine samples incubated with hypericin served as normal controls. Laser confocal microscopy and spectroscopy was used to detect the fluorescence in the exfoliated low-grade urothelial tumor cells. Fluorescence cytology was considered positive if hypericin fluorescence of the low-grade urothelial tumor cells was detected to be stronger (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;8.5 times) compared to the baseline fluorescence established for normal urine samples. Automated analysis for an objective reproducible outcome appears possible. The possibility of detection of malignant urothelial cells in early cancer makes ex vivo fluorescence cytology promising for routine diagnostic screening.
The early detection of carcinoma is very essential for the diagnosis and prognosis of a bladder c... more The early detection of carcinoma is very essential for the diagnosis and prognosis of a bladder cancer patient. In this study we have investigated the use of hypericin as a fluorescent tumour marker and laser confocal microscopy as a diagnostic tool to aid the diagnosis of such cancers. Both cellular and clinical studies have been conducted. In the cellular studies, we have compared two bladder cell lines for the uptake and sub-cellular localization of hypericin. It was found that there was a rapid uptake and clearance of hypericin and significant localization in mitochondria and lysosomes. The study also revealed that there was a time-dependent increase in fluorescence intensity in bladder cells. The optimum localization was found to be 2-4 h post drug incubation. In the clinical study, consisting of 30 patients, both white light and fluorescence cystoscopy were performed after hypericin instillation. Biopsies taken from fluorescing regions were imaged using the confocal microscope. The order of fluorescence was detected to be as follows: normal &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; inflammation &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; grade 1 &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; grade 2 &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; CIS &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; grade 3. It was also found that the fluorescence intensity increased with the stage of the disease thereby enabling the determination of the degree of invasiveness of cancer. This enables the use of hypericin as a prognostic marker and laser confocal microscopy as a tool to aid in diagnosis of bladder cancer.
Bladder cancer is the fourth most common malignant disease worldwide, accounting for 4% of all ca... more Bladder cancer is the fourth most common malignant disease worldwide, accounting for 4% of all cancer cases. In Singapore, it is the ninth most common form of cancer. The high mortality rate in bladder cancer can be reduced by early treatment following pre-cancerous screening. Currently, the gold standard for screening bladder tumors is histological examination of biopsy specimens, which is both invasive and time-consuming. In this study, ex vivo urine fluorescence cytology was investigated to offer an alternative timely and biopsy-free means for detecting bladder cancers. Sediments in patient urine samples were extracted and incubated with a novel photosensitizer, hypericin. Laser confocal microscopy was used to capture the fluorescence images at an excitation wavelength of 488 nm. Images were subsequently processed to single out the exfoliated bladder cancer cells from the other cells based on the cellular size. Intensity histograms of each targeted cell and feature vectors, derived from the histogram moments, were used to represent each sample. A difference in the distribution of the feature vectors of normal and low-grade cancerous bladder cancer cells were observed. A diagnostic algorithm for discriminating between normal and low-grade cancerous cells is elucidated in this report. This study suggests that the fluorescence intensity profiles of hypericin in bladder cells can potentially provide an automated quantitative means of early bladder cancer diagnosis.
Treatment options for primary and metastatic renal cancer are increasing. Accurate data from the ... more Treatment options for primary and metastatic renal cancer are increasing. Accurate data from the pathological examination of renal cancer specimens aid clinicians in stratifying patients for surveillance and adjuvant therapies. This review focuses on biomarkers in diagnosis, prognosis and prediction of the biologic behavior of renal tumors which should be recorded in pathology reports and which are under investigation. Special emphasis is given to the use of immunohistochemical markers in differential diagnosis of various renal tumor subtypes. The relevance of cytogenetic and molecular findings is also discussed. The review includes the 2012 International Society for Urological Pathology Consensus conference recommendations.
This article was published in an Elsevier journal. The attached copy is furnished to the author f... more This article was published in an Elsevier journal. The attached copy is furnished to the author for non-commercial research and education use, including for instruction at the author's institution, sharing with colleagues and providing to institution administration. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier's archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright
Aims: We aimed to identify common reasons for second opinion breast pathology referrals at the Pa... more Aims: We aimed to identify common reasons for second opinion breast pathology referrals at the Pathology Department, Singapore General Hospital, focusing on queries and diagnostic issues raised by referring clinicians and pathologists. Methods: Request forms for breast pathology consultations were retrieved from a specialist's correspondence files consisting of pathologists' referrals, and from centralised laboratory records, comprising clinician-initiated referrals. Clinical and histomorphological queries raised by the referrals were collated. Results: Of 299 cases evaluated, clinician-initiated referrals (n ¼ 137, 46%) included requests for review of overall histopathology to confirm carcinoma subtype (n ¼ 47), grade (n ¼ 2), size (n ¼ 4), lymphovascular invasion (n ¼ 1) and confirm hormonal receptor and c-erbB-2 assays (n ¼ 33). Also required were: comparison of recurrent with previous lesions (n ¼ 8); settling discrepant diagnoses between two or more prior pathology reports (n ¼ 4); verification of microinvasion (n ¼ 6), in situ carcinomas (n ¼ 6) or atypical ductal hyperplasias (n ¼ 4); delineation of benign (n ¼ 8) and spindle cell lesions (n ¼ 3); to establish a breast origin of metastatic lesions (n ¼ 5); and distinction of carcinoma from lymphoma (n ¼ 2). Pathologist-initiated referrals (n ¼ 162, 54%) sought arbitration between borderline proliferative lesions (n ¼ 46) and papillary lesions (n ¼ 34); verification of microinvasion (n ¼ 23), stromal lesions (n ¼ 16), and carcinoma subtype (n ¼ 13), especially if the patient was young (n ¼ 5); clarification of metaplastic changes (n ¼ 4) and lobular neoplasia (n ¼ 8); and comparison of fibroepithelial lesions (n ¼ 11). Conclusions: Clinicians sought a second opinion mainly to verify histological diagnoses and report important pathological details for staging and confirmation of hormonal receptor and c-erbB-2 status prior to therapy. Borderline breast lesions are worrisome for both clinicians and pathologists in view of implications for management.
Glutathione S-transferase (GST) is known to play a key role in the detoxification and reduction o... more Glutathione S-transferase (GST) is known to play a key role in the detoxification and reduction of reactive oxygen species (ROS). Thus, we assessed GST activity and GST-pi expression in relation to oxidative stress and apoptosis in breast cancer. Tumor tissues from 32 breast cancer patients were evaluated for GST activity and thiobarbituric acid reactive substances (TBARS) that are by-products of oxidative stress. Four-micron sections of formalin-fixed, paraffin embedded tumors were stained immunohistochemically with anti-GST-pi. Apoptotic cells were detected by in situ end labeling of DNA fragments using a commercial kit. TBARS levels were significantly higher in breast cancers of older patients. GST-pi expression was up-regulated in breast cancers that exhibited higher oxidative stress and associated with higher GST activity. Apoptosis in GST-pi negative tumors was not correlated with GST activity, but GST-pi positive tumors within the same range of oxidative stress showed a reduction in apoptosis as well as an increased GST activity. This correlation was absent in GST-pi positive tumors experiencing higher oxidative stress. GST-pi expression may influence the level of GST activity and delay apoptosis in breast cancer. However, GST-pi expression in tumors with higher levels of oxidative stress may not be sufficient to abrogate the deleterious effects of ROS.
Immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH) are the only tests curre... more Immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH) are the only tests currently approved by the US Food and Drug Administration for classifying which patients will benefit from trastuzumab therapy. The accuracy of these two testing methods can be adversely affected by a variety of pre-analytical, analytical and post-analytical factors. According to the latest published recommendations of the panel of the Joint Committee of the American Society of Clinical Oncology and College of American Pathologists for HER2/neu testing, laboratories performing IHC and FISH for HER2/neu status in breast cancer are now required to have a high concordance of at least 95% between IHC and FISH, significantly higher than that in the published literature. To perform a retrospective analysis of the concordance of IHC and FISH analysis for HER2/neu at Singapore General Hospital and review potential causes of disparity between these two methods. A retrospective review of a total of 106 invasive ductal carcinomas evaluated for HER2/neu at the Department of Pathology, Singapore General Hospital between 2007 and 2008 were included in the study. The initial HER2/neu immunostained slides were reviewed independently without knowledge of FISH results, and concordance between IHC and FISH was determined. Concordance between IHC and FISH assay was excellent and within the published range (104/106=98.1%). The discordant cases represent a well-recognised subset of genetic heterogeneity in HER2/neu, which is known to contribute to positive IHC and negative FISH tests.
Breast cancer is a significant cause of morbidity and mortality in women with a high incidence of... more Breast cancer is a significant cause of morbidity and mortality in women with a high incidence of recurrence or treatment failure. Growing evidence suggests that cancer stem cells (CSCs) most likely contribute to tumour progression, spread and therapy failure. However, despite extensive research and the tremendous clinical potential of such cells in possible therapeutic management, the real nature of CSCs remains an enigma. In this review, we discuss the fundamental properties and molecular target of CSCs and focus on recent advances regarding the identification of CSC markers with emphasis on breast cancer and the underlying molecular mechanism of CSC phenotypes. We also discuss experimental evidence of targeting molecular pathways in order to modulate breast CSC behaviour in tumourigenesis and the controversies associated with it that potentially weaken the CSC model in breast cancer and other cancers as well.
Background: Chromophobe renal cell carcinoma (chRCC) and renal oncocytoma are two distinct but cl... more Background: Chromophobe renal cell carcinoma (chRCC) and renal oncocytoma are two distinct but closely related entities with strong morphologic and genetic similarities. While chRCC is a malignant tumor, oncocytoma is usually regarded as a benign entity. The overlapping characteristics are best explained by a common cellular origin, and the biologic differences between chRCC and oncocytoma are therefore of considerable interest in terms of carcinogenesis, diagnosis and clinical management. Previous studies have been relatively limited in terms of examining the differences between oncocytoma and chromophobe RCC. Methods: Gene expression profiling using the Affymetrix HGU133Plus2 platform was applied on chRCC (n = 15) and oncocytoma specimens (n = 15). Supervised analysis was applied to identify a discriminatory gene signature, as well as differentially expressed genes. High throughput single-nucleotide polymorphism (SNP) genotyping was performed on independent samples (n = 14) using Affymetrix GeneChip Mapping 100 K arrays to assess correlation between expression and gene copy number. Immunohistochemical validation was performed in an independent set of tumors. Results: A novel 14 probe-set signature was developed to classify the tumors internally with 93% accuracy, and this was successfully validated on an external data-set with 94% accuracy. Pathway analysis highlighted clinically relevant dysregulated pathways of c-erbB2 and mammalian target of rapamycin (mTOR) signaling in chRCC, but no significant differences in p-AKT or extracellular HER2 expression was identified on immunohistochemistry. Loss of chromosome 1p, reflected in both cytogenetic and expression analysis, is common to both entities, implying this may be an early event in histogenesis. Multiple regional areas of cytogenetic alterations and corresponding expression biases differentiating the two entities were identified. Parafibromin, aquaporin 6, and synaptogyrin 3 were novel immunohistochemical markers effectively discriminating the two pathologic entities.
This study aimed to determine the usefulness of a combination of 3 immunohistochemical markers, 3... more This study aimed to determine the usefulness of a combination of 3 immunohistochemical markers, 34β E12, p63 and α-methylacyl coenzyme A racemase (AMACR), for the diagnosis of prostate cancer using tissue microarrays (TMAs) constructed from 91 archival radical prostatectomy specimens derived from the Pathology Department files of Singapore General Hospital, Singapore. Triple immunostaining using a cocktail of these 3 antibodies was performed on TMA sections using the streptavidin-biotin method. When compared with immunohistochemical staining using the individual antibodies, we found that the triple cocktail allowed improved evaluation of basal cells in benign glands, and AMACR allowed simultaneous corroboration of malignant prostatic glands in the same section. We achieved a specificity of 100% with the triple cocktail, and sensitivity was acceptable, at 93.8%. In comparison, specificity and sensitivity of the individual antibodies were 95.5% and 97.3%, 93.3% and 93.8%, 97.0% and 95.6% for p63, 34βE12, and AMACR, respectively. The triple cocktail offers a costeffective way of evaluating abnormal prostatic glandular foci, in addition to maximizing the use of small tissue samples from prostatic needle biopsies.
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