[show abstract][hide abstract] ABSTRACT: We developed a hyperspectral microscopic imaging (HMI) platform that can precisely identify and quantify 10 molecular markers in individual cancer cells in a single pass. The exploitation of an improved separation of circulating tumor cells and the application of HMI provided an opportunity (1) to identify molecular changes in these cells, (2) to recognize the coexpression of these markers, (3) to pose an important opportunity for noninvasive diagnosis, and (4) to use targeted therapy. We balanced the intensity of 10 fluorochromes bound to 10 different antibodies, each specific to a particular tumor marker, so that the intensity of each fluorochrome can be discerned from overlapping emissions. Using 2 touch preps from each primary breast cancer, the average molecular marker intensities of 25 tumor cells gave a representative molecular signature for the tumor despite some cellular heterogeneity. The intensities determined by the HMI correlate well with the conventional 0-3+ analysis by experts in cellular pathology. Because additional multiplexes can be developed using the same fluorochromes but different antibodies, this analysis allows quantification of many molecular markers on a population of tumor cells. HMI can be automated completely, and eventually, it could allow the standardization of protein biomarkers and improve reproducibility among clinical pathology laboratories.
Translational research : the journal of laboratory and clinical medicine. 05/2012; 159(5):366-75.
[show abstract][hide abstract] ABSTRACT: We tested whether assessing the expression of cell cycle related proteins (p53, pRB, p21 and p27) could predict clinical outcomes after radical cystectomy in patients with organ confined urothelial carcinoma of the bladder.
Our study included a development cohort of 272 patients and an external testing cohort of 52 patients with chemotherapy naïve pT1-2N0M0 urothelial carcinoma of the bladder treated with radical cystectomy. Immunohistochemical staining of p53, p27, p21 and pRB was performed on the development cohort of 272 patients and the external testing cohort of 52 patients.
Overall 260 (80.2%) patients had altered expression of at least 1 molecular marker and 105 (32.4%), 95 (29.3%), 44 (13.6%) and 16 (4.9%) had 1 to 4 altered molecular markers, respectively. Addition of the number of altered molecular markers increased the predictive accuracy of the base model for disease recurrence and cancer specific mortality by 15.6% and 14.8%, respectively (p <0.001). The base model included age, gender, pT1 vs pT2 stage, grade, number of lymph nodes removed, lymphovascular invasion and concomitant carcinoma in situ. The combination of molecular markers yielded a predictive accuracy superior to that of any single molecular marker. We developed nomograms for the prediction of recurrence-free and cancer specific survival.
Assessment of the number of altered cell cycle regulatory proteins in the cystectomy specimen improves the prediction of urothelial carcinoma of the bladder recurrence and survival in patients with organ confined disease. A combination of multiple markers is needed to capture the complex biological behavior of urothelial carcinoma of the bladder.
The Journal of urology 12/2011; 187(2):457-62. · 4.02 Impact Factor
[show abstract][hide abstract] ABSTRACT: Study Type - Prognosis (case series) Level of Evidence 4 What's known on the subject? and What does the study add? Insulin-like growth factor II mRNA binding protein 3 (IMP3) is associated with poor outcomes in a variety of malignancies. The role of IMP3 in protate cancer remains poorly understood. IMP3 expression was associated with features of aggressive biology and aggressive prostate cancer recurrence after surgery. Although IMP3 is differentially expressed in patients with features of biologically aggressive prostate cancer, it does not have independent prognostic value in patients treated with RP.
To evaluate the association of insulin-like growth factor II mRNA binding protein 3 (IMP3) with pathological features and outcomes in patients treated with radical prostatectomy (RP).
Immunohistochemical staining for IMP3 was performed on archival tissue microarray specimens from 232 consecutive patients treated with RP for clinically localized disease. None of the patients received neoadjuvant or adjuvant radiation or hormone therapy. IMP3 expression was histologically categorized as normal or abnormal. Disease recurrence was classified as aggressive if metastases were present, post-recurrence prostate-specific antigen (PSA) doubling time was less than 10 months, or if the patients failed to respond to salvage local radiation therapy.
The median follow-up was 69.8 months (interquartile range [IQR]: 40.1-99.5). IMP3 expression was abnormal in 42 (18.1%) of 232 patients. IMP3 expression was associated with extracapsular extension (P= 0.020), seminal vesicle invasion (P= 0.024), lymphovascular invasion (P= 0.036) and a high pathological Gleason score (P= 0.009). The 5-year PSA recurrence-free survival for IMP3-negative patients was 83% (standard error [SE]= 3) vs 67% (SE = 8) in IMP3-positive patients (log-rank test, P= 0.015). In a multivariable analysis that adjusted for the effects of surgical margins, extracapsular extension and seminal vesicle invasion, PSA (hazard ratio [HR]: 1.04, P= 0.013), lymph node metastasis (HR: 16.7, P < 0.001) and a high pathological Gleason score (HR 4.3, P= 0.008) were significantly associated with PSA recurrence-free survival, whereas IMP3 expression was not (P= 0.11). Similarly, IMP3 expression was only associated with aggressive recurrence (HR 3.2, P= 0.006).
IMP3 expression is abnormal in approximately one-fifth of prostate cancers. Although IMP3 is differentially expressed in patients with features of biologically aggressive prostate cancer, it does not have an independent prognostic value in patients treated with RP.
BJU International 11/2011; 110(1):63-8. · 3.05 Impact Factor
[show abstract][hide abstract] ABSTRACT: Five years of tamoxifen reduces breast cancer risk by nearly 50% but is associated with significant side effects and toxicities. A better understanding of the direct and indirect effects of tamoxifen in benign breast tissue could elucidate new mechanisms of breast carcinogenesis, suggest novel chemoprevention targets, and provide relevant early response biomarkers for phase II prevention trials. Seventy-three women at increased risk for breast cancer were randomized to tamoxifen (20 mg daily) or placebo for 3 months. Blood and breast tissue samples were collected at baseline and posttreatment. Sixty-nine women completed all study activities (37 tamoxifen and 32 placebo). The selected biomarkers focused on estradiol and IGFs in the blood; DNA methylation and cytology in random periareolar fine-needle aspirates; and tissue morphometry, proliferation, apoptosis, and gene expression (microarray and reverse transcriptase PCR) in the tissue core samples. Tamoxifen downregulated Ets oncogene transcription factor family members ETV4 and ETV5 and reduced breast epithelial cell proliferation independent of CYP2D6 genotypes or effects on estradiol, ESR1, or IGFs. Reduction in proliferation was correlated with downregulation of ETV4 and DNAJC12. Tamoxifen reduced the expression of ETV4- and ETV5-regulated genes implicated in epithelial-stromal interaction and tissue remodeling. Three months of tamoxifen did not affect breast tissue composition, cytologic atypia, preneoplasia, or apoptosis. A plausible mechanism for the chemopreventive effects of tamoxifen is restriction of lobular expansion into stroma through downregulation of ETV4 and ETV5. The human equivalent of murine multipotential progenitor cap cells of terminal end buds may be the primary target.
Cancer Prevention Research 07/2011; 4(11):1852-62. · 4.89 Impact Factor
[show abstract][hide abstract] ABSTRACT: PURPOSES: To describe the differential tissue expression of tumor-associated trypsin inhibitor (TATI) in normal bladder urothelium, primary urothelial carcinoma of the bladder (UCB) and metastatic UCB and to assess the association of TATI expression with molecular markers commonly altered in UCB and clinical outcomes after radical cystectomy. METHODS: Slides from eight cystectomy patients without cancer, 191 radical cystectomy patients, 20 lymph nodes without metastasis and 40 lymph nodes with UCB were stained. Tissue expression of TATI, cyclin E1, cyclin D1, p53, p21, p27, pRB, Ki-67, Bcl-2, Caspase-3, Survivin and Cyclooxigenase-2 was measured in a tissue microarray. Cancer-specific and recurrence-free survival after radical cystectomy was recorded. RESULTS: TATI was expressed in 100% of patients without cancer, while 71% of radical cystectomy specimens and 90% of lymph node metastases exhibited decreased or no TATI expression. In radical cystectomy specimens, TATI expression decreased with advancing pathologic stage (P < 0.001) and lymphovascular invasion (P = 0.055). In univariate analyses, but not in multivariable Cox proportional hazard regression analyses, decreased TATI expression was associated with increased probability of tumor recurrence and cancer-specific mortality. Decreased TATI expression was correlated with altered expression of Cyclooxigenase-2 (P = 0.005), p21 (P = 0.035) and Ki-67 (P = 0.004). CONCLUSIONS: We found that normal urothelium expresses TATI and that TATI expression decreases with advancing tumor stage. While there was no prognostic benefit to TATI when adjusted for standard clinicopathologic features, it seems to play an important biologic role in UCB pathogenesis and invasion. Its association with markers involved in the cell cycle, proliferation and inflammation serves as hypothesis for molecular interactions.
World Journal of Urology 07/2011; · 2.89 Impact Factor
[show abstract][hide abstract] ABSTRACT: Although the presence of microsatellite instability (MSI) in patients with colorectal cancer (CRC) may have implications for prognosis, therapy, and family counseling, to the authors' knowledge, the prevalence of MSI has not been well described among individuals of Hispanic origin with CRC residing in the United States.
A retrospective cohort study using a hospital-based tumor registry to identify individuals of Hispanic origin who were diagnosed with CRC was conducted. Clinical data and tumor samples were retrieved. Molecular analyses included testing for MSI using a panel of 5 mononucleotide markers (BAT25, BAT26, NR21, NR24, and NR27) in a pentaplex polymerase chain reaction assay, as well as immunohistochemistry for the mismatch repair (MMR) proteins mutL homolog (MLH) 1, mutS homolog (MSH) 2, MSH6, and postmeiotic segregation increased 2 (PMS2) 2 on representative tissue.
A total of 111 individuals of Hispanic origin with CRC were identified. Approximately 41.4% were women, and the median age was 57 years (interquartile range [IQR], 47.1-63.5 years). Eleven patients (9.9%; 95% confidence interval [95% CI], 4.2%-15.6%) had MSI CRC, whereas 14 patients (12.6%; 95% CI, 7.3%-21.8%) had CRC with ≥1 MMR protein abnormality. Ten of 11 individuals with MSI had clinical or molecular characteristics suspicious for Lynch syndrome such as abnormal expression of MSH2 and/or MSH6 (n=7) or age<50 years at the time of diagnosis (n=7).
The prevalence of MSI CRC among Hispanic individuals may be similar to that of other races and ethnicities, but clinicopathological characteristics, including age at diagnosis and pattern of abnormal MMR protein expression, suggest that sporadic MSI CRC may be less common in individuals of Hispanic origin, and that much of the MSI observed in this situation may be attributable to Lynch syndrome. Further exploration of the causes of disparate presentations of CRC by ethnicity and race is warranted.
Cancer 11/2010; 116(21):4965-72. · 5.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: • To investigate androgen receptor (AR) expression in a large series of patients with bladder cancer (BC) because data on a limited number of patients showed that loss of AR expression was associated with invasive BC.
• A total of 472 patients with urothelial bladder carcinoma (UBC) from two institutional centres (Toronto and Dallas) were analysed. Tissue microarrays comprising both non-muscle-invasive UBC (n= 167) and muscle-invasive UBC (n= 305) were accrued and immunohistochemical staining for AR was performed. • We used bright-field microscopy imaging coupled with advanced colour detection software to detect, classify and count stained cellular objects and manual scoring. • Results obtained in Dallas were blindly reviewed and validated in Toronto and samples randomly chosen were further analysed in Rochester, NY, USA.
• The AR were positively expressed in 61/472 (12.9%) bladder tumours. No statistically significant difference in AR expression between men and women was observed. • Only 9.0% of non-muscle-invasive BC expressed the AR compared with 15.1% of muscle-invasive tumours (P= 0.059). The highest percentage of AR positivity (28.9% of cases) was found in T2 tumours. • There was no statistically significant difference in death from BC, time to death, or time to recurrence between AR-positive and AR-negative cases.
• In contrast to previous reports, based on our large BC series, we did not observe a decrease in AR protein expression in bladder tumours with increased pathological stage. Our data do not suggest that loss of AR expression is gender-related nor is it associated with invasive BC.
BJU International 11/2010; 108(1):24-30. · 3.05 Impact Factor
[show abstract][hide abstract] ABSTRACT: to test whether the expression of human epidermal growth factor receptor 2 (HER-2) is of prognostic value in a contemporary cohort of patients with urothelial carcinoma of the urinary bladder (UCB).
tissue microarrays of 198 patients were constructed and immunohistochemical stainings were performed on the primary tumours and on lymphatic nodal metastases. All patients were treated with radical cystectomy (RC) and regional lymphadenectomy for UCB. HER-2 expression was assessed using continuous HER-2 expression scores (ranging from 0.1 to 3.9) generated using an automated cellular imaging system. Scores of ≥ 1.0 in at least 10% of tumour cells were regarded as HER-2 positive. We correlated HER-2 scores with pathological and clinical variables, including disease recurrence and cancer-specific mortality.
of 198 patients undergoing RC with lymphadenectomy, there was HER-2 positivity in 55 primary tumours (27.8%) compared with 44.2% of the evaluable positive lymph nodes (P < 0.001). HER-2 positivity was significantly associated with the presence of lymphovascular invasion (LVI; P= 0.026). With a median (range) follow-up of 35.4 (1.3-176.1) months, 101 patients (51.0%) had UCB recurrence and 82 patients (41.4%) died from the disease. In multivariable analyses that adjusted for the effects of pathological tumour stage, grade, LVI, lymph node metastasis and adjuvant chemotherapy, HER-2 positive patients were at increased risk for both UCB recurrence (hazard ratio [HR] 1.955, P= 0.003) and UCB-specific mortality (HR 2.066, P= 0.004) compared with patients with negative HER-2 expression.
a positive HER-2 status is associated with aggressive UCB and provides independent prognostic information for UCB recurrence and mortality. Assessment of HER-2 status can be used to identify patients at high risk of disease progression who may benefit from adjuvant HER-2-targeted mono- or combined therapy after RC.
BJU International 10/2010; 106(8):1216-22. · 3.05 Impact Factor
[show abstract][hide abstract] ABSTRACT: The clinical utility of urine markers in urothelial cancer (UC) surveillance is not established. We previously evaluated the use of fluorescence in situ hybridization (FISH) in managing patients with atypical cytology at risk for UC. This study evaluates its role in patients with negative cytology with a history of UC.
Between June 2007 and January 2009, every patient with a history of UC who underwent cystoscopy and cytology with UroVysion test were identified. A comprehensive chart review was performed on each patient with negative cytology.
The population comprised 142 patients undergoing cancer surveillance; 111 patients with negative cystoscopy, 19 with equivocal cystoscopy, and 12 with positive cystoscopy. In patients with negative cystoscopy, there was cancer in only 1 of 111 patients. UroVysion could detect the only patient with UC with sensitivity of 100% and had a negative predictive value (NPV) of 100%. In patients with equivocal cystoscopy, it detected 2 tumors that would be missed by cytology. There were 4 false negative results (sensitivity 33.3% and NPV 66.7%). In patients with obvious lesion on cystoscopy, there were 9 false negative results (sensitivity 10% and NPV 18.2%).
Few patients with negative cystoscopy and negative cytology have cancer. Patients with equivocal and positive cystoscopy and negative cytology frequently have cancer and the UroVysion FISH assay was not helpful in these cases. The cost-effectiveness of the FISH assay needs to be assessed prior to widespread use in patients with negative cytology.
[show abstract][hide abstract] ABSTRACT: We tested whether the altered immunohistochemical expression of angiogenesis related markers is associated with outcomes of patients with urothelial carcinoma of the bladder, and assessed the correlation of angiogenesis related markers with molecular markers commonly altered in urothelial bladder carcinoma.
Vascular endothelial growth factor, basic fibroblast growth factor and thrombospondin 1 expression data were collected, as were microvessel density data. Immunohistochemical staining was performed on specimens from 204 patients treated with radical cystectomy for urothelial carcinoma of the bladder. We also stained serial sections of the specimens for cyclin E1, cyclin D1, p53, p21, p27, pRB, Ki-67, Bcl-2, caspase-3, survivin and cyclooxygenase-2. We measured time to disease recurrence and cancer specific mortality, as well as the association with clinical and pathological features and other molecular markers.
The altered expression of vascular endothelial growth factor (over expression), basic fibroblast growth factor (over expression) and thrombospondin 1 (decreased expression) was 86%, 79% and 63%, respectively. Median microvessel density was 20. All 4 markers were associated with established clinicopathological features of aggressive urothelial carcinoma of the bladder (such as stage, lymphovascular invasion and lymph node metastasis) and other molecular markers. On multivariable analyses that adjusted for standard pathological features basic fibroblast growth factor and thrombospondin 1 were independent predictors of disease recurrence (HR 3.6, p = 0.002 and HR 2.2, p = 0.001, respectively) and cancer specific mortality (HR 2.8, p = 0.02 and HR 2.3, p = 0.003, respectively). When all 4 markers were included in 1 model basic fibroblast growth factor and thrombospondin 1 retained their independent association with disease recurrence (HR 2.9, p = 0.014 and HR 1.8, p = 0.022, respectively) and only thrombospondin 1 was independently associated with cancer specific mortality (HR 1.9, p = 0.031).
Angiogenesis related molecular markers are commonly altered in urothelial carcinoma of the bladder, making them a target for therapy. Down-regulation of thrombospondin 1 and up-regulation of basic fibroblast growth factor are independent predictors of clinical outcomes of patients with urothelial carcinoma of the bladder.
The Journal of urology 03/2010; 183(5):1744-50. · 4.02 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cervical dysplasia and tumorigenesis have been linked with numerous chromosomal aberrations. The goal of this study was to evaluate 35 genomic regions associated with cervical disease and to select those which were found to have the highest frequency of aberration for use as probes in fluorescent in-situ hybridization.
The frequency of gains and losses using fluorescence in-situ hybridization were assessed in these 35 regions on 30 paraffin-embedded cervical biopsy specimens. Based on this assessment, 6 candidate fluorescently labeled probes (8q24, Xp22, 20q13, 3p14, 3q26, CEP15) were selected for additional testing on a set of 106 cervical biopsy specimens diagnosed as Normal, CIN1, CIN2, CIN3, and SCC. The data were analyzed on the basis of signal mean, % change of signal mean between histological categories, and % positivity.
The study revealed that the chromosomal regions with the highest frequency of copy number gains and highest combined sensitivity and specificity in high-grade cervical disease were 8q24 and 3q26. The cytological application of these two probes was then evaluated on 118 ThinPrep samples diagnosed as Normal, ASCUS, LSIL, HSIL and Cancer to determine utility as a tool for less invasive screening. Using gains of either 8q24 or 3q26 as a positivity criterion yielded specificity (Normal +LSIL+ASCUS) of 81.0% and sensitivity (HSIL+Cancer) of 92.3% based on a threshold of 4 positive cells.
The application of a FISH assay comprised of chromosomal probes 8q24 and 3q26 to cervical cytology specimens confirms the positive correlation between increasing dysplasia and copy gains and shows promise as a marker in cervical disease progression.