Biomedical subjects

Robert L Camp

Publications and source records attributed to Robert L Camp.

At least 19 recordsLinked to original sources

PubMed2006

Classification of breast cancer using genetic algorithms and tissue microarrays.

PURPOSE: A multitude of breast cancer mRNA profiling studies has stratified breast cancer and defined gene sets that correlate with outcome. However, the number of genes used to predict patient outcome or define tumor subtypes by RNA expression studies is variable, nonoverlapping, and generally requires specialized technologies that are beyond those used in the routine pathology laboratory. It would be ideal if the familiarity and streamlined nature of immunohistochemistry could be combined with the rigorously quantitative and highly specific properties of nucleic acid-based analysis to predict patient outcome. EXPERIMENTAL DESIGN: We have used AQUA-based objective quantitative analysis of tissue microarrays toward the goal of discovery of a minimal number of markers with maximal prognostic or predictive value that can be applied to the conventional formalin-fixed, paraffin-embedded tissue section. RESULTS: The minimal discovered multiplexed set of tissue biomarkers was GATA3, NAT1, and estrogen receptor. Genetic algorithms were then applied after division of our cohort into a training set of 223 breast cancer patients to discover a prospectively applicable solution that can define a subset of patients with 5-year survival of 96%. This algorithm was then validated on an internal validation set (n=223, 5-year survival=95.8%) and further validated on an independent cohort from Sweden, which showed 5-year survival of 92.7% (n=149). CONCLUSIONS: With further validation, this test has both the familiarity and specificity for widespread use in management of breast cancer. More generally, this work illustrates the potential for multiplexed biomarker discovery on the tissue microarray platform.

Algorithms↗

PubMed2006

Expression of tumor necrosis factor--related apoptosis-inducing ligand receptors 1 and 2 in melanoma.

PURPOSE: The proapoptotic receptors tumor necrosis factor--related apoptosis-inducing ligand receptor 1 (TRAIL-R1) and TRAIL-R2 are targets of drugs in clinical development, and receptor expression levels may be important determinants of sensitivity to receptor agonists. We assessed TRAIL-R1 and TRAIL-R2 expression patterns in a large cohort of melanomas and benign nevi. EXPERIMENTAL DESIGN: We analyzed tissue microarrays containing 546 melanomas and 540 nevi using our automated quantitative method to measure protein levels in situ (AQUA). The system uses S100 to define pixels as melanoma (tumor mask) within the array spot and measures intensity of TRAIL-receptor expression using Cy5-conjugated antibodies within the mask. AQUA scores were correlated with clinical and pathologic variables. RESULTS: TRAIL-R1 and TRAIL-R2 expression was higher in melanomas than in nevi (P < 0.0001), and higher in primary than in metastatic specimens (P = 0.0031 and P < 0.0001, respectively). TRAIL-R1 and TRAIL-R2 expression exceeding the 95th percentile for nevi was found in 19% and 74% of melanoma specimens, respectively. Although on univariate analysis, high TRAIL-R2 expression correlated with increased survival (P = 0.0439), it was not associated with survival within the primary or metastatic subcohorts. TRAIL-R1 expression was not associated with survival. CONCLUSIONS: TRAIL-R1 and TRAIL-R2 expression is higher in malignant melanocytes than in their benign counterparts, suggesting that these receptors might be effective therapeutic targets in melanoma. Expression is higher in early-stage disease than in metastatic specimens, and expression exceeding that found in nevi is found in a substantially larger fraction of melanomas for TRAIL-R2 compared with TRAIL-R1. Assessment of baseline tumor TRAIL receptor expression may be important in analysis of clinical trials involving TRAIL receptor agonists.

Gene Expression Regulation↗

PubMed2006

Quantitative in situ analysis of beta-catenin expression in breast cancer shows decreased expression is associated with poor outcome.

The role of beta-catenin in breast cancer and its prognostic value is controversial. The prognostic value had been assessed previously in a series of nonquantitative immunohistochemical studies with conflicting results. In efforts to clarify the relationship between beta-catenin protein expression and breast cancer prognosis, we have assessed a retrospective 600 case cohort of breast cancer tumors from the Yale Pathology archives on tissue microarrays. They were assessed using automated quantitative analysis (AQUA) with a series of array-embedded cell lines for which the beta-catenin concentration was standardized by an ELISA assay. The expression levels of the standard clinical markers HER2, estrogen receptor (ER), progesterone receptor (PR), and Ki-67 were also assessed on the same cohort. X-tile software was used to select optimal protein concentration cutpoints and to evaluate the outcome using a training set and a validation set. We found that low-level expression of membranous beta-catenin is associated with significantly worse outcome (38% versus 76%, 10-year survival, validation set log-rank P = 0.0016). Multivariate analysis of this marker, assessed in a proportional hazards model with tumor size, age, node status, nuclear grade, ER, PR, HER2, and Ki-67, is still highly significant with a hazard ratio of 6.8 (P < 0.0001, 95% confidence interval, 3.1-15.1). These results suggest that loss of beta-catenin expression at the membrane, as assessed by objective quantitative analysis methods, may be useful as a prognostic marker or may be part of a useful algorithm for prognosis in breast cancer.

Algorithms↗

PubMed2006

Vascular endothelial growth factor, FLT-1, and FLK-1 analysis in a pancreatic cancer tissue microarray.

BACKGROUND: Measures of vascular endothelial growth factor (VEGF) expression in pancreatic cancer typically have been qualitative or semiquantitative. The objective of this study was to use a series of algorithms called AQUA that quantitatively assesses protein expression on tissue microarrays (TMAs) to compare in situ expression of VEGF and its primary receptors, VEGF receptor 1 (FLT-1) and VEGF receptor 1 (FLK-1), on a pancreatic cancer TMA. METHODS: TMAs were constructed by arraying 1.5-mm cores from 76 samples of pancreatic adenocarcinoma (1996-2002) that were obtained from the archives of the Yale Department of Pathology. The staining for AQUA was similar to standard immunohistochemistry and involved antigen retrieval and the application of primary antibodies, but with epifluorescence detection. Slides were counterstained with 4',6-diamidino-2-phenylindole for nuclear visualization and cytokeratin for membrane visualization. The primary antibodies used were VEGF, FLT-1, FLK-1, and cytokeratin. RESULTS: Disease stage was highly prognostic for outcome, as expected. Total amounts of VEGF and its receptors were assessed within the tumor mask and were divided into quartiles. Kaplan-Meier survival curves showed that VEGF and FLK-1 were not associated clearly with outcome. However, the expression of FLT-1 was correlated significantly, and the patients who had tumors with the lowest expression FLT-1 levels had the worst survival (P = .0038). In multivariate analysis, FLT-1 expression was an independent prognostic factor for overall survival (P = .0044). CONCLUSIONS: VEGF and its 2 principal receptors were expressed to varying degrees in tumors of the pancreas. A significant association was found between low expression of FLT-1 and both poor prognosis and advanced stage, suggesting that tumor expression of this VEGF receptor is a marker of less aggressive disease.

Adenocarcinoma↗

PubMed2006

Utility of molecular genetic signatures in the delineation of gastric neoplasia.

BACKGROUND: Current techniques to define gastric neoplasia are limited but molecular genetic signatures can categorize tumors and provide biological rationale for predicting clinical behavior. We identified three gene signatures: Chromogranin A (CgA), MAGE-D2 (adhesion), and MTA1 (metastasis) that define gastrointestinal (GI) carcinoids and hypothesize that their expression can delineate gastric neoplasia. This strategy provides a molecular basis to define neuroendocrine gastric carcinoids (GCs), neuronal stromal tumors (GISTs), or epithelial cell (gastric adenocarcinomas [GCAs])-derived tumors. METHODS: Total RNA was isolated from 38 GCs: Type I/II (n = 7), Type III/IV (n = 6), GISTs (n = 12), GCAs (n = 13), and normal mucosa (n = 12). Quantitative reverse transcriptase polymerase chain reaction (Q RT-PCR) gene expression was quantified against glyseraldehyde-3-phosphate dehydrogenase (GAPDH) and CgA and MTA1 protein expression levels were analyzed by immunohistochemical analyses of a gastric neoplasia microarray. RESULTS: CgA was elevated in Type I/II (10-fold; P < .01) and Type III/IV (100-fold, P < .005), decreased in GISTs (100-fold, P < .03), and unchanged in GCAs. MAGE-D2 was 5-10-fold elevated (P < .05) in Type III/IV, GISTs, and GCAs but not in Type I/II tumors. MTA1 (> 5-fold, P < .01) was elevated in GCs (Type III/IV>I/II, P < .05), in GISTs (> 4-fold, P < .05), and GCAs. CgA protein levels were elevated in GCs (P < .005) but not in GISTs and GCAs. MTA1 levels were elevated in all tumors (P < .02) compared with normal, and especially with tumor invasion (P < .05). CONCLUSION: CgA discriminates GCs from other gastric neoplasms; overexpression of MAGE-D2 and MTA1 differentiate Type III/IV from Type I/II GCs. GISTs share similar expression patterns with Type III/IV GCs but have decreased CgA. MTA1 is a marker of tumor invasion.

Adaptor Proteins, Signal Transducing↗

PubMed2006

The role of genetic markers--NAP1L1, MAGE-D2, and MTA1--in defining small-intestinal carcinoid neoplasia.

BACKGROUND: Standard clinical and immunohistochemical methods cannot reliably determine whether a small intestinal carcinoid (SIC) is indolent or aggressive. We hypothesized that carcinoid malignancy could be defined by using quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) and immunohistochemical approaches that evaluate potential marker genes. METHODS: Candidate marker gene expression (nucleosome assembly protein 1-like 1 [NAP1L1], melanoma antigen D2 [MAGE-D2], and metastasis-associated protein 1 [MTA1]) identified by Affymetrix transcriptional profiling was examined by QRT-PCR in SIC, liver, and lymph node (LN) metastases, colorectal carcinomas, and healthy tissues. Immunohistochemical expression levels of MTA1 were analyzed quantitatively by a novel automated quantitative analysis in a tissue microarray of 102 gastrointestinal carcinoids and in a breast/prostate carcinoma array. RESULTS: Affymetrix transcriptional profiling identified three potentially useful malignancy-marker genes (out of 1709 significantly altered genes). By QRT-PCR, NAP1L1 was significantly (P < .03) overexpressed in SIC compared with colorectal carcinomas and healthy tissue. Increased levels (P < .05) were identified in both liver and LN metastases. Levels in colorectal carcinomas were the same as in healthy mucosa. MAGE-D2 and MTA1 were increased (P < .05) in primary tumors and metastases and overexpressed in carcinomas. Automated quantitative analysis demonstrated the highest levels of MTA1 immunostaining in malignant primary SICs and in metastases to the liver and LN. These were significantly increased (P < .02) compared with nonmetastatic primary tumors. MTA1 was overexpressed in breast and prostate carcinomas (P < .05). CONCLUSIONS: SICs overexpress the neoplasia-related genes NAP1L1 (mitotic regulation), MAGE-D2 (adhesion), and MTA1 (estrogen antagonism). The ability to determine the malignant potential of these tumors and their propensity to metastasize provides a biological rationale for the management of carcinoids and may have prognostic utility.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Molecular classification identifies a subset of human papillomavirus--associated oropharyngeal cancers with favorable prognosis.

PURPOSE: We sought to determine the prevalence of biologically relevant human papillomavirus (HPV) in oropharyngeal squamous cell carcinoma (OSCC). Retinoblastoma (Rb) downregulation by HPV E7 results in p16 upregulation. We hypothesized that p16 overexpression in OSCC defines HPV-induced tumors with favorable prognosis. METHODS: Using real-time polymerase chain reaction for HPV16, we determined HPV16 viral load in a cohort of 79 OSCCs annotated with long-term patient follow-up. A tissue microarray including these cases was also analyzed for p53, p16, and Rb utilizing in situ quantitative protein expression analysis. Seventy-seven tumors were classified into a three-class model on the basis of p16 expression and HPV-DNA presence: class I, HPV-, p16 low; class II, HPV+, p16 low; and class III, HPV+, p16 high. RESULTS: Sixty-one percent of OSCCs were HPV16+; HPV status alone was of no prognostic value for local recurrence and was barely significant for survival times. Overall survival was improved in class III (79%) compared with the other two classes (20% and 18%; P = .0095). Disease-free survival for the same class was 75% versus 15% and 13% (P = .0025). The 5-year local recurrence was 14% in class III versus 45% and 74% (P = .03). Only patients in class III had significantly lower p53 and Rb expression (P = .017 and .001, respectively). Multivariable survival analysis confirmed the prognostic value of the three-class model. CONCLUSION: Using this system for classification, we define the molecular profile of HPV+ OSCC with favorable prognosis, namely HPV+/p16 high (class III). This study defines a novel classification scheme that may have value for patient stratification for clinical trials testing HPV-targeted therapies.

Adult↗

PubMed2006

Q RT-PCR detection of chromogranin A: a new standard in the identification of neuroendocrine tumor disease.

OBJECTIVE: Message and protein expression of CgA was examined to evaluate the sensitivity of a PCR-based approach in the detection of covert neuroendocrine (NE) tissue. SUMMARY BACKGROUND DATA: Immunohistochemical (IHC) measurement of chromogranin A (CgA) discriminates gastrointestinal (GI) carcinoids from epithelial tumors. IHC is, however, an insensitive technique to identify micrometastases or delineate subpopulations of NE cells. METHODS: CgA gene expression was examined by Q-RT PCR in GI carcinoids (small intestinal and metastases, n=17, gastric, n=5), appendiceal tumors (n=10), and adenocarcinomas (gastric, n=5, colorectal, n=6). CgA protein expression levels were quantitatively analyzed following IHC by automated quantitative analysis (AQUA) in 2 tissue microarrays (GI carcinoid and GI adenocarcinoma). RESULTS: CgA gene was overexpressed (P<0.001) in GI carcinoids compared with GI adenocarcinomas and normal mucosa. Elevated levels (P<0.00001) were also identified in carcinoid liver and lymph node (LN) metastases. CgA levels were higher (approximately 2-4-fold) in NE appendiceal carcinoids than in adenocarcinoids, but in GI adenocarcinomas were identical to normal mucosa. Histologically normal lymph nodes expressed detectable CgA message in 30% of cases. CgA protein levels were highest in primary GI carcinoids and in liver metastases and significantly elevated (P<0.005) compared with nonmetastatic lesions. Expression in liver and LN metastases was significantly elevated (P<0.000001) compared with normal. Analysis of mRNA by Q-RT PCR was >200-fold more sensitive than by IHC. CONCLUSIONS: Overexpression of CgA mRNA and protein in GI carcinoids can identify metastatic cells; thus, PCR for CgA can be used to identify micrometastases not evident by light microscopy or IHC as well as define tumors of ambivalent morphologic phenotype. The use of this sensitive strategy to assess NETs and apparently normal LNs and liver may be of future utility in defining therapeutic strategy.

Adolescent↗

PubMed2006

Genetic differentiation of appendiceal tumor malignancy: a guide for the perplexed.

OBJECTIVE: To use differential gene expression of candidate markers to discriminate benign appendiceal carcinoids (APCs) from malignant and mixed cell APCs. SUMMARY BACKGROUND DATA: Controversy exists in regard to the appropriate surgical management of APCs since it is sometimes difficult to predict tumor behavior using traditional pathologic criteria. We have identified 5 differentially expressed genes (a mitosis-regulatory gene NAP1L1, an adhesin MAGE-D2, an estrogen-antagonist, the metastasis marker MTA1, the apoptotic marker NALP, and chromogranin A) that define gut neuroendocrine cell behavior. METHODS: Total RNA was isolated using TRIzol reagent from 42 appendiceal samples, including appendiceal carcinoids identified at exploration for appendicitis (no evidence of metastasis; n = 16), appendicitis specimens (n = 11), malignant appendiceal tumors (> 1.5 cm, evidence of metastatic invasion; n = 7), and mixed (goblet) cell appendiceal adenocarcinoids (n = 3), normal appendiceal tissue (n = 5), and 5 colorectal cancers. Gene expression (CgA, NAP1L1, MAGE-D2, MTA1, and NALP1) was examined by Q-RT PCR (Applied Biosystems) and quantified against GAPDH. RESULTS: CgA message was elevated (> 1000-fold, P < 0.05) in all tumor types. NAP1L1 was elevated (> 10-fold, P < 0.03) in both malignant and goblet cell adenocarcinoids compared with normal and incidental lesions (P < 0.006). MAGE-D2 and MTA1 message were significantly elevated (> 10-fold, P < 0.01) in the malignant and goblet cell adenocarcinoid tumors but not in the appendicitis-associated carcinoids or normal mucosa. The apoptotic marker, NALP1, was overexpressed (> 50-fold, P < 0.05) in the appendicitis-associated and malignant appendiceal carcinoids but was significantly decreased (> 10-fold, P < 0.05) in the goblet cell adenocarcinoids. Elevated CgA transcript and protein levels indicative of a carcinoid tumor were identified in one acute appendicitis sample with no histologic evidence of a tumor. CONCLUSIONS: These data demonstrate that malignant APCs and goblet cell adenocarcinoids have elevated expression of NAP1L1, MAGE-D2, and MTA1 compared with appendiceal carcinoids identified at surgery for appendicitis. This and the differences in NALP1 gene expression (decreased in goblet cell adenocarcinoids) provide a series of molecular signatures that differentiate carcinoids of the appendix. CgA identified all appendiceal tumors as well as covert lesions, which may be more prevalent than previously recognized. The molecular delineation of malignant appendiceal tumor potential provides a scientific basis to define the appropriate surgical management as opposed to morphologic assessment alone.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Evaluation of the prognostic value of cellular inhibitor of apoptosis protein in epithelial ovarian cancer using automated quantitative protein analysis.

PURPOSE: The cellular inhibitor of apoptosis protein (cIAP) is regarded as an important prognostic biomarker in cancer. Here, we sought to determine the prognostic value of cIAP protein levels in epithelial ovarian cancer using a novel method of compartmentalized in situ protein analysis. METHODS: A tissue array composed of 150 advanced-stage ovarian cancers, treated with surgical debulking followed by platinum/paclitaxel-based combination chemotherapy, was constructed. For evaluation of protein expression, we used an immunofluorescence-based method of automated in situ quantitative measurement of protein analysis. RESULTS: The mean follow-up time for the entire cohort was 34.4 months. Patients with tumors bearing high cIAP membranous expression had a 3-year survival rate of 31% compared with 73% for patients with low cIAP expressing tumors (P = 0.0020). In multivariable analysis, adjusting for well-characterized prognostic variables, low membranous cIAP expression level was the only significant prognostic factor for overall survival. CONCLUSIONS: Our results indicate that cIAP protein levels have prognostic value in ovarian cancer patients. Modulation of cIAP levels may improve clinical outcome in ovarian cancer.

Automation↗

PubMed2005

Automated quantitative analysis (AQUA) of in situ protein expression, antibody concentration, and prognosis.

BACKGROUND: Disparate results in the immunohistochemistry literature regarding the relationship between biomarker expression and patient outcome decrease the credibility of tissue biomarker studies. We investigated whether some of these disparities result from subjective optimization of antibody concentration. METHODS: We used the automated quantitative analysis (AQUA) system and various concentrations of antibodies against HER2 (1 : 500 to 1 : 8000 dilutions), p53 (1 : 50 to 1 : 800 dilutions), and estrogen receptor (ER; 1 : 100 and 1 : 1000 dilutions) to assess expression of HER2 and p53 in a tissue microarray containing specimens from 250 breast cancer patients with long-term survival data available. HER2 expression in the tissue microarray was also assessed by conventional immunohistochemistry. Relative risk (RR) of disease-specific mortality was assessed for every cutpoint with the X-tile program. Cumulative disease-specific survival was assessed by the Kaplan-Meier method. All statistical tests were two-sided. RESULTS: For HER2 and p53 and an optimal cutpoint, when a high antibody concentration (i.e., 1 : 500 dilution) was used with the AQUA system, low expression was associated with poorer survival than high expression; however, when a low antibody concentration (i.e., 1 : 8000 dilution) was used, high expression was associated with poorer survival. For example, for a 1 : 8000 dilution of HER2 antibody and high expression defined as the top 15% of HER2 expression, high HER2 expression was associated with increased disease-specific mortality (RR = 1.98, 95% confidence interval [CI] = 1.21 to 3.23; P = .007), compared with low expression. However, for a 1 : 500 dilution of HER2 antibody and high expression defined as the top 85% of HER2 expression, high HER2 expression was associated with decreased disease-specific mortality (RR = 0.47, 95% CI = 0.29 to 0.76; P = .002), compared with low HER2 expression. CONCLUSIONS: Biomarker antibody concentration appears to dramatically affect the apparent relationship between biomarker expression and outcome.

Adult↗

PubMed2005

Effect of epidermal growth factor receptor expression level on survival in patients with epithelial ovarian cancer.

BACKGROUND: Several lines of laboratory evidence support the epidermal growth factor receptor (EGFR) as an adverse prognostic indicator in ovarian cancers. However, different methods of immunohistochemical assessment have yielded conflicting results. Here, we sought to determine the prognostic value of EGFR in ovarian cancer using a novel method of compartmentalized in situ protein analysis. METHODS: A tissue array composed of 150 advanced-stage ovarian cancers uniformly treated, with surgical debulking followed by platinum-paclitaxel combination chemotherapy, was constructed. For evaluation of EGFR protein expression, we used an immunofluorescence-based method of automated in situ quantitative measurement of protein analysis (AQUA). RESULTS: Mean follow-up time for the entire cohort was 34.4 months. Eighty-one of 150 cases had sufficient tissue for AQUA analysis. High tumor EGFR expression was associated with poor outcome for overall survival (P=0.0001) and disease-free survival (P=0.0005) at 3 years. In multivariable analysis, adjusting for well-characterized prognostic variables, EGFR expression status was the most significant prognostic factor for disease-free and overall survival. CONCLUSION: The conflicting results in the literature regarding the prognostic value of EGFR may be due to the technical difficulties inherent in assessing EGFR with immunocytochemistry. In the present study, we show that measurement of EGFR protein levels in ovarian cancer using AQUA is feasible and can give important prognostic information.

Carcinoma↗

PubMed2005

Subcellular localization and protein levels of cyclin-dependent kinase inhibitor p27 independently predict for survival in epithelial ovarian cancer.

PURPOSE: p27 protein is regarded as a valuable prognostic biomarker in cancer with a potential use as a molecular target. However, different methods of immunohistochemical assessment have yielded conflicting results. Here, we sought to determine the prognostic value of p27 in ovarian cancer using a novel method of compartmentalized in situ protein analysis. EXPERIMENTAL DESIGN: A tissue array composed of 150 advanced stage ovarian cancers uniformly treated, with surgical debulking followed by platinum-paclitaxel combination chemotherapy, was constructed. For evaluation of p27 protein expression, we used an immunofluorescence-based method of automated in situ quantitative measurement of protein analysis [automated quantitative analysis (AQUA)]. RESULTS: The mean follow-up time of the patients was 34.3 months. Patients with low Fédération Internationale des Gynaecologistes et Obstetristes stage were more likely to have low nuclear p27 expression (P = 0.008). Low nuclear p27 expression was associated with improved 3-year overall survival (66% versus 20%, P = 0.0047) and disease-free survival (27% versus 12%, P = 0.022). In multivariable analysis, adjusting for well-characterized prognostic variables, low nuclear p27 expression level was the most significant prognostic factor for both disease-free and overall survival. CONCLUSIONS: Our results indicate that quantitative assessment of nuclear p27 expression level by automated in situ quantitative analysis is a strong predictor for outcome in ovarian cancer.

Adult↗

PubMed2005

Evaluating the expression and prognostic value of TRAIL-R1 and TRAIL-R2 in breast cancer.

PURPOSE: The cell surface receptors tumor necrosis factor-related apoptosis-inducing ligand receptor 1 (TRAIL-R1) and TRAIL-R2 transmit apoptotic signals, and agents that activate these receptors are in clinical development. We sought to determine the expression and prognostic value of TRAIL-R1 and TRAIL-R2 in early-stage breast cancer. EXPERIMENTAL DESIGN: Tissue microarrays containing specimens from 655 breast cancer patients with 20-year follow-up were employed and evaluated with our automated quantitative analysis (AQUA) system. The system uses cytokeratin to define pixels as breast cancer (tumor mask) within the array spot, and measures intensity of TRAIL receptor expression using Cy5 conjugated antibodies within the mask. AQUA scores were correlated with clinical and pathologic variables. TRAIL-R1 and TRAIL-R2 expression were similarly studied on 95 unmatched normal breast specimens. RESULTS: TRAIL-R1 expression was not associated with survival. High TRAIL-R2 expression strongly correlated with decreased survival (P = 0.0005). On multivariate analysis, high TRAIL-R2 expression remained an independent prognostic marker, as did nodal status and tumor size. High TRAIL-R2 expression correlated strongly with lymph node involvement (P = 0.0003). TRAIL-R2 expression was stronger in malignant specimens than in normal breast epithelium (P < 0.0001). CONCLUSIONS: High TRAIL-R2 expression was independently associated with decreased survival in breast cancer. The biological basis and the sensitivity of high TRAIL-R2 expressing cells to TRAIL agonists and/or chemotherapy are subject to further investigation. Evaluation of TRAIL-R2 expression in early-stage breast cancer may identify a subset of patients requiring more aggressive or pathway-targeted adjuvant treatment. Clinical trials involving TRAIL-R2 agonists should stratify patients based on TRAIL-R2 expression.

Adult↗

PubMed2005

Using a xenograft model of human breast cancer metastasis to find genes associated with clinically aggressive disease.

Metastasis is the primary cause of death from breast cancer. A xenograft model was used to identify genes potentially involved with metastasis, comparing expression in the poorly metastatic GI101A human breast cancer cell line and a highly metastatic variant, GILM2. cDNA microarray analyses of these isogenic variants were done using 16K Operon 70-mer oligonucleotide microarray slides. Differentially expressed genes were identified by ANOVA, and differences of > or =2.5-fold were found for 106 genes. Changes in protein or RNA expression were confirmed for 10 of 12 genes. Three markers, heat shock protein 70 (HSP-70), chemokine (C-X-C motif) ligand 1 (CXCL-1), and secreted leukocyte protease inhibitor (SLPI), were studied further with breast cancer tissue microarrays using a novel method of automated quantitative analysis. This uses cytokeratin to define pixels as breast cancer (tumor mask) within the tissue array spot and then measures intensity of marker expression using a cyanine 5-conjugated antibody within the mask. Scores were correlated with clinicopathologic variables. High HSP-70 expression and high nuclear CXCL-1 expression in primary tumors were both associated with decreased survival (P = 0.05 and 0.027, respectively). Expression of each marker was strongly associated with lymph node involvement (P = 0.0002, 0.008, 0.0012, and 0.012 for HSP-70, nuclear CXCL-1, cytoplasmic CXCL-1, and SLPI, respectively). Identification of genes associated with metastasis in experimental models may have clinical implications for the management of breast cancer, because some of these are associated with lymph node metastasis and survival and might be useful as prognostic markers or molecular targets for novel therapies.

Animals↗

PubMed2005

Automated quantitative analysis of E-cadherin expression in lymph node metastases is predictive of survival in invasive ductal breast cancer.

PURPOSE: The tumor suppressor adhesion molecule E-cadherin is believed to have an anti-invasive role in breast cancer. Lymph node involvement is the best prognostic marker known, yet there is variability in outcome among node-positive patients. We investigated the relationship between E-cadherin expression in primary invasive ductal tumors and corresponding nodal metastases, and determined the prognostic value of E-cadherin expression in node-positive breast cancer. EXPERIMENTAL DESIGN: Membrane E-cadherin expression was studied by immunohistochemical staining of tissue microarrays with fluorescent-labeled antibodies. An objective method of automated quantitative analysis (AQUA) was used. AQUA uses cytokeratin to define pixels as breast cancer (tumor mask) within the array spot, and measures E-cadherin expression using a Cy5-conjugated antibody within the mask. RESULTS: We employed a tissue microarray containing 207 primary and matched nodal metastases suitable for AQUA analysis. There was no significant difference in mean staining intensity between the primary and nodal specimens (P = 0.8). A scattergram was generated which identified a subset of patients (25%) with high E-cadherin expression in nodal metastases, and this top quartile had improved survival (P = 0.028). On univariate analysis, increased E-cadherin expression in nodal metastases was strongly associated with improved survival (P = 0.007), whereas expression in primary tumors was not (P = 0.13). On multivariate analysis, nodal E-cadherin expression retained its independent association with survival, as did tumor size and HER2/neu status. CONCLUSIONS: Strong E-cadherin expression in lymph node metastases was highly predictive of improved survival. This suggests that expression of adhesion molecules at metastatic sites portends less aggressive tumor behavior.

Breast Neoplasms↗

PubMed2005

Cyclin d1 is a valuable prognostic marker in oropharyngeal squamous cell carcinoma.

BACKGROUND: The current tumor-node-metastasis system is inadequate to accurately classify patients in terms of prognosis. Thus, with the availability of recently developed molecular tools, considerable interest lies in discovering prognostic markers in order to guide treatment decisions. In this study, we sought to determine the prognostic significance of the cell cycle regulator cyclin D1 in oropharyngeal squamous cell carcinoma (OSCC). EXPERIMENTAL DESIGN: We studied the protein expression levels of cyclin D1 on a tissue microarray composed of 63 OSCCs with long-term follow-up data available. Protein expression was analyzed with an automated in situ quantitative (AQUA) method which allows preservation of tissue morphology while quantifying protein expression in paraffin-embedded tissue. RESULTS: The mean follow-up time was 35 months. High cyclin D1 nuclear expression was associated with increased 5-year local recurrence rate (48% versus 15%), inferior 5-year disease-free survival (16% versus 58%), and inferior 5-year overall survival (17% versus 53%). In multivariate Cox regression, high nuclear cyclin D1 expression was an independent predictor for local recurrence, disease-free survival, and overall survival at 5 years. CONCLUSIONS: Our results indicate that quantitative assessment of nuclear cyclin D1 expression level by automated in situ quantitative analysis is a strong predictor for outcome in OSCC. Thus, cyclin D1 may be a potential target for molecular intervention in patients with oropharyngeal squamous cell cancer.

Adult↗

PubMed2005

Microsatellite instability and gene mutations in transforming growth factor-beta type II receptor are absent in small bowel carcinoid tumors.

BACKGROUND: Microsatellite instability (MSI) with concomitant mutations in the coding region of transforming growth factor-beta type II receptor (TGFbetaRII) results in an aberrant growth-regulatory phenotype in colorectal carcinomas. The authors postulated that a similar mechanism occurred during the malignant evolution of small bowel carcinoid tumors. METHODS: Mutational analysis of two coding regions in the TGFbetaRII gene associated with MSI and BAT-26 within intron 5 of the mismatch repair gene, hMSH2, was undertaken in small bowel carcinoids (n = 14), lymph node metasasis (n = 1) and liver metastases (n = 5). Quantitative PCR analysis [TAQMAN, Applied Biosystems, Foster City, CA] was then undertaken to examine gene alterations in mismatch repair genes (hMLH1 and hMSH2) in small bowel carcinoids (n = 7) and matched normal mucosa (n = 5). Staining was then analyzed using quantitative tissue array profiling (AQUA analysis) in a small bowel EC carcinoid tissue microarray (n = 55 tumors) with immunostaining against TGFbetaRII and MSH2. RESULTS: Mutational examination of the TFGbetaRII gene and BAT-26 demonstrated that MSI was not present in any carcinoid material. Q RT-PCR analysis demonstrated statistically significant increased message levels of hMSH2 but not hMLH1 in carcinoid tumors. Quantitative analysis of membrane TGFbetaRII immunostaining using AQUA demonstrated that TGFbetaRII expression was down-regulated (P < 0.0002) in thirty-three primary small bowel carcinoids that exhibited lymph node and liver metastases compared to normal mucosa. AQUA analysis of nuclear MSH2 immunostaining demonstrated no differences for MSH2 between normal tissue and carcinoid tumor metastasis. Small bowel carcinoids characterized by variable expression of TGFbetaRII, did not exhibit MSI and had no differences in MSH2 expression. CONCLUSIONS: The molecular events leading to the formation of carcinoid tumors in the small bowel were different from those resulting in epithelial carcinomas. The usually slow-growing and relatively nonaggressive carcinoid tumors had variable expression of TGFbetaRII but were associated with the retention of mismatch repair protein function and a microsatellite-stable phenotype.

Adult↗