Search

Purpura fulminans (PF) is associated with several infections and most commonly with meningococcemia. However, there are only a few reports of this entity in association with toxic shock syndrome toxin-1-producing Staphylococcus aureus. We report a 53-year-old man who presented with fever, progressive hemodynamic instability, multiorgan failure, and thrombocytopenia following lobectomy for a solitary lung metastasis from rectal adenocarcinoma. He developed progressive generalized eruption of nonblanching red, purple, and black macules, papules, and plaques on the trunk and extremities consistent with PF. He died on postadmission day 3. Autopsy examination revealed purulent pleural exudate, which grew toxic shock syndrome toxin-1-producing S. aureus. Premortem and autopsy skin biopsies demonstrated epidermal necrosis, subepidermal bullae, and fibrin thrombi within small cutaneous vessels with minimal perivascular lymphocytic inflammation and without accompanying vasculitis. With this case report, we would like to draw attention to the fact that staphylococcal toxic shock syndrome-associated PF may be highly underrecognized and much more common than reflected in the literature.

Aims Immunohistochemical stains have greatly improved the diagnostic accuracy of renal cell carcinoma (RCC) for primary and distant tumours. We evaluate a marker that has recently been incorporated in clinical practice, PAX-8, in primary and metastatic RCCs. Methods Two distinct tissue microarrays were used, one consisting of over 334 renal tumours, 294 with adjacent normal kidney and the other with 40 matched nephrectomy and metastatic sites of RCC. PAX-8 expression was assessed by a method of quantitative immunofluorescence. Results PAX-8 was positive in 96% (146/152) of normal renal tissue and 83% (227/272) of renal tumours. PAX-8 staining was positive in clear cell, papillary and chromophobe tumours in 80% (165/207), 95% (39/41) and 100% (6/6) of samples, respectively. Overall, intensity of PAX-8 expression was significantly higher in RCC metastatic sites than in the primary site (p=0.0047), however, in matched sites there was no statistically significant difference in the proportion of positive versus negative specimens (p=0.274). Conclusions As the role of molecular markers expands in the diagnostic algorithm, this study confirms that PAX-8 expression is a useful diagnostic marker for RCC. PAX-8 expression was found in the primary tumour and distant sites. Compared with normal tissue and other histological types, clear cell RCC has lower PAX-8 expression and is less frequently positive, therefore, the lack of expression does not exclude a tumour of renal origin.

Activation of the c-Met pathway occurs in a range of malignancies, including papillary renal cell carcinoma (RCC). Its activity in clear cell RCC is less clear. We investigated c-Met expression and inhibition in a large cohort of RCC tumors and cell lines.c-Met protein expression was determined by automated quantitative analysis (AQUA) on a tissue microarray (TMA) constructed from 330 RCC tumors paired with adjacent normal renal tissue. c-Met expression and selective inhibition with SU11274 and ARQ 197 were studied in clear cell RCC cell lines.Higher c-Met expression was detected in all RCC subtypes than in the adjacent normal renal tissue (P0.0001). Expression was highest in papillary and sarcomatoid subtypes, and high-grade and stage tumors. Higher c-Met expression correlated with worse disease-specific survival [risk ratio = 1.36; 95% confidence interval (CI) 1.08-1.74; P = 0.0091] and was an independent predictor of survival, maintained in clear cell subset analyses. c-Met protein was activated in all cell lines, and proliferation (and colony formation) was blocked by SU11274 and ARQ 197.c-Met is associated with poor pathologic features and prognosis in RCC. c-Met inhibition demonstrates in vitro activity against clear cell RCC. Further study of ARQ 197 with appropriate biomarker studies in RCC is warranted.

Purpose: Measurement of autophagy in cancer and correlation with histopathologic grading or clinical outcomes has been limited. Accordingly, we investigated LC3B as an autophagosome marker by analyzing nearly 1,400 tumors from 20 types of cancer, focusing on correlations with clinical outcomes in melanoma and breast cancer. Experimental Design: Staining protocols were developed for automated quantitative analysis (AQUA) using antibodies versus LC3 isoform B (LC3B) and Ki-67. Clinically annotated breast and melanoma tissue microarrays (TMA) and a multitumor array were used. An AQUA program was developed to quantitate LC3B distribution in punctate and diffuse compartments of the cell. Results: LC3B staining was moderate to high in the large majority of tumors. The percentage of area occupied by punctate LC3B was elevated by 3- to 5-fold at high LC3B intensities. In breast cancer and melanoma TMAs, LC3B and Ki-67 showed strong correlations (P < 0.0001), and in multitumor TMAs, mitotic figures were most often seen in tumors with the highest LC3B expression (P < 0.002). In breast cancer, LC3B expression was elevated in node-positive versus node-negative primaries and associated with increased nuclear grade and shortened survival. In a melanoma TMA with no survival data, LC3B levels were highest in nodal, visceral, and cutaneous metastases. Conclusions: The results reveal a common expression of LC3B in malignancy and support emerging evidence that autophagy plays a significant role in cancer progression. High LC3B was associated proliferation, invasion and metastasis, high nuclear grade, and worse outcome. Thus, autophagy presents a key target of therapeutic vulnerability in solid tumors. Clin Cancer Res; 18(2); 370–9. ©2011 AACR.

Purpose As a result of the questionable risk-to-benefit ratio of adjuvant therapies, stage II melanoma is currently managed by observation because available clinicopathologic parameters cannot identify the 20% to 60% of such patients likely to develop metastatic disease. Here, we propose a multimarker molecular prognostic assay that can help triage patients at increased risk of recurrence. Methods Protein expression for 38 candidates relevant to melanoma oncogenesis was evaluated using the automated quantitative analysis (AQUA) method for immunofluorescence-based immunohistochemistry in formalin-fixed, paraffin-embedded specimens from a cohort of 192 primary melanomas collected during 1959 to 1994. The prognostic assay was built using a genetic algorithm and validated on an independent cohort of 246 serial primary melanomas collected from 1997 to 2004. Results Multiple iterations of the genetic algorithm yielded a consistent five-marker solution. A favorable prognosis was predicted by ATF2 ln(non-nuclear/nuclear AQUA score ratio) of more than –0.052, p21WAF1 nuclear compartment AQUA score of more than 12.98, p16INK4A ln(non-nuclear/nuclear AQUA score ratio) of ≤ −0.083, β-catenin total AQUA score of more than 38.68, and fibronectin total AQUA score of ≤ 57.93. Primary tumors that met at least four of these five conditions were considered a low-risk group, and those that met three or fewer conditions formed a high-risk group (log-rank P < .0001). Multivariable proportional hazards analysis adjusting for clinicopathologic parameters shows that the high-risk group has significantly reduced survival on both the discovery (hazard ratio = 2.84; 95% CI, 1.46 to 5.49; P = .002) and validation (hazard ratio = 2.72; 95% CI, 1.12 to 6.58; P = .027) cohorts. Conclusion This multimarker prognostic assay, an independent determinant of melanoma survival, might be beneficial in improving the selection of stage II patients for adjuvant therapy.

BACKGROUND: Assessment of outcome using a single prognostic or predictive marker is the current basis of targeted therapy, but is inherently limited by its simplicity. Multiplexing has provided better classification, but has only been done quantitatively using RNA or DNA. Automated quantitative analysis is a new technology that allows quantitative in situ assessment of protein expression. The authors hypothesized that multiplexed quantitative measurement of ErbB receptor family proteins may allow better prediction of outcome. METHODS: The authors quantitatively assessed the expression of 6 proteins in 4 subcellular compartments in 676 patients using breast carcinoma tissue microarrays. Then using Cox proportional hazards modeling and unsupervised hierarchical clustering, they assessed the prognostic value of the expression singly and multiplexed. RESULTS: Epidermal growth factor receptor (EGFR), HER-2, and HER-3 expression were associated with decreased survival. Multivariate analysis showed high HER-2 and HER-3 expression maintained independence as prognostic markers. Hierarchical clustering of expression data defined a small class enriched for HER-2 expression with 40% 10-year survival, compared with 55% using conventional methods. Clustering also revealed a similarly poor-prognostic subgroup coexpressing EGFR and HER-3 (but low for estrogen receptor, progesterone receptor, and HER-2) with a 42% 10-year survival. CONCLUSIONS: This work shows that the combined analysis of protein expression improved prognostic classification, and that multiplexed models were superior to any single-marker–based method for prediction of 10-year survival. These methods illustrate a protein-based, multiplexed approach that could more accurately identify patients for targeted therapies. Cancer 2009. © 2009 American Cancer Society.

This year, 2008, marks the 10-year anniversary of the development of the modern tissue microarray (TMA). During the last decade, the use of TMAs has grown steadily and accounts for a small but increasing percentage of all cancer biomarker studies performed. The growing popularity of TMA-based studies attests to their benefits in the discovery and validation of new biomarkers. This review will focus on these benefits, but also on the faults of TMAs and the challenges of TMA studies that have been overcome in the last decade. We will also discuss the role of TMAs in the latest revolution in cancer treatment, the use of targeted drug therapy.

Kallikrein-related peptidases, a subgroup of the serine protease enzyme family, are considered important prognostic biomarkers in cancer. In the present study, we sought to determine the prognostic value of kallikrein-related peptidase 6 (KLK6) in ovarian cancer using a novel method of compartmentalized in situ protein analysis. A tissue array composed of 150 advanced stage ovarian cancers, uniformly treated with surgical debulking followed by platinumpaclitaxel combination chemotherapy, was constructed. For evaluation of KLK6 protein expression, we used an immunofluorescence-based method of automated in situ quantitative measurement of protein analysis (AQUA). Mean follow-up time of the cohort was 34.35 months. One hundred and thirty-five of 150 cases had sufficient tissue for AQUA analysis. In univariate survival analysis, low tumor KLK6 expression was associated with better outcome for overall survival over 3 years (P = 0.019). There was no association between tumor KLK6 expression and progression-free survival (P = 0.128). In multivariate survival analysis, adjusting for well-characterized prognostic variables, low tumor KLK6 expression level was one of the most significant predictor variable for overall survival (95% confidence interval, 1.19–3.50; P = 0.009). High tumor KLK6 protein expression is associated with inferior patient outcome in ovarian cancer. KLK6 may represent a promising disease biomarker and therapeutic target in ovarian cancer. (Cancer Sci 2008; 99: 2224–2229)

Recent development of antiangiogenic therapy for renal cell carcinoma (RCC) has significantly improved the treatment of these often refractory tumors. However, not all patients respond to therapy and assays for predicting outcome are needed. As a first step, we analyzed a retrospective cohort of tumors and assessed the ability of VEGF and VEGF receptors (VEGF-R1, -R2 and -R3) to classify tumors. We analyzed tissue microarrays containing 330 RCCs using a novel method of automated quantitative analysis of VEGF and VEGF-R expression by fluorescent immunohistochemistry. Expression of markers was separately quantified within three tissue components: tumor cells, endothelial cells and adjacent normal epithelium. VEGF and VEGF receptors were tightly coexpressed both within tumors and within adjacent normal cells (all P-values

Purpose The variability in scoring of immunohistochemistry, whether a result of true heterogeneity or artifacts in preparation, has led to decreased reliability in companion diagnostics and the recommendation for new standards (eg, the American Society of Clinical Oncology/College of American Pathologists [ASCO-CAP] guidelines). The basis of this problem is the amount of tissue required to be representative of an entire tumor. Because protein expression on tissue microarrays (TMAs) can be rigorously measured and one 0.6-mm spot is equivalent to two to three high-power fields, we used TMAs to assess levels of heterogeneity and to determine optimal representation as a function of outcome. Patients and Methods We analyzed estrogen receptor (ER), progesterone receptor, and human epidermal growth factor receptor 2 (HER-2) expression in two cohorts (n = 676 and n = 152) on a series of four to five separate TMA cores and assessed heterogeneity by linear regression analysis. Minimum, average, and maximum scores were generated for each set, which were then assessed for prognostic and predictive value. Results Each marker shows some heterogeneity, but average r values between 0.7 and 0.8 are seen between TMA spots. Analysis for prognostic value shows that the highest maximum score (of five spots) is the most prognostic for ER, whereas a high HER-2 minimum score is most prognostic for poor outcome and most predictive of response to trastuzumab. Conclusion These results suggest that the representivity required for each biomarker may be a function of its role in tumorigenesis. Furthermore, these results provide scientific basis for the ASCO-CAP guidelines for assessment of HER-2 expression but perhaps suggest that the 30% figure is still too conservative.

Expression of Met, the Hepatocyte Growth Factor receptor, has been shown to have prognostic value in numerous types of cancer including breast, gastric, cervical and head and neck carcinomas. However, traditional analyses of expression have shown variable results and a lack of reproducibility. The AQUA® system is a method of quantitative in situ analysis of protein expression that allows the assessment of reproducibility of both antibodies and assay conditions. Here, we illustrate the necessity for antibody validation when assaying the prognostic value of a potential biomarker. Using five antibodies to the intracellular domain of the Met receptor and 10 cell line controls, we quantitatively assess reproducibility of protein expression. We show that many antibodies are not reproducible at a quantitative level from lot to lot or assay to assay, suggesting new criteria for antibody validation. We also build upon past literature addressing the prognostic value of Met in a cohort of 640 cases of invasive breast cancer on a tissue microarray. We show that high levels of expression of nuclear Met, as determined by antibodies to the intracellular domain and defined as nuclear by subcellular compartmental analysis, is associated with shorter disease-specific survival in breast cancer.

Background: Several lines of laboratory evidence support a role of persistent activation of Akt pathway in oropharyngeal squamous cell carcinoma (OSCC) progression. Loss of phosphatase PTEN is one of the proposed mechanisms of Akt activation. We sought to determine the prognostic significance of Akt activation in a cohort of patients with OSCC as well as the association between phosphorylated (activated) Akt and PTEN levels. Methods: Using a novel system of in situ quantitative protein expression analysis (AQUA), we studied the protein expression levels of phosphorylated Akt (p-Akt) and PTEN on a tissue microarray. The array included 79 OSCCs with a mean follow-up of 36 months. Results: Patients with tumors expressing low tumor p-Akt levels had lower 5-year local recurrence rates (5% versus 38%). Additionally, these patients had improved 5-year overall survival rates (45% versus 27%). This survival effect was likely due to disease recurrence, as there was no difference in death without recurrence between low- and high-expressing groups. In adjusted analysis, tumor p-Akt expression was a strong predictor of local recurrence. A significant inverse relationship was found between nuclear p-Akt and nuclear PTEN: Tumors with high nuclear p-Akt had low nuclear PTEN and vice versa. Conclusions: Akt activation in OSCC is associated with adverse patient outcome, indicating that Akt is a promising molecular target in OSCC. PTEN loss may be one of the mechanisms of Akt activation in OSCC. (Cancer Epidemiol Biomarkers Prev 2007;16(3):553–8)

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. (Cancer Epidemiol Biomarkers Prev 2006;15(6):1179-83)

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. Cancer 2006. © 2006 American Cancer Society.

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.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.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/IVI/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).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.

Background: Several lines of evidence support the epidermal growth factor receptor (EGFR) as a molecular target for therapy in head and neck squamous cell carcinomas (HNSCC). Determination of tumor EGFR levels by conventional immunohistochemistry has not always predicted antitumor efficacy. Quantitative assays may provide more accurate assessment of the level of EGFR receptor in the tumor, which may thus provide more reliable prognostic and predictive information. We studied the prognostic value of quantitative assessment of EGFR in oropharyngeal squamous cell cancers treated with radiotherapy.Experimental Design: We studied EGFR protein expression on a tissue microarray composed of 95 oropharyngeal cancer cases using an in situ molecular-based method of quantitative assessment of protein expression (AQUA) and correlated those with clinical and pathologic data. Automated, quantitative analysis uses cytokeratin to define pixels as cancer (tumor mask) within the array spot and measures intensity of EGFR expression using a Cy5-conjugated antibody within the mask. A continuous index score is generated, which is directly proportional to the number of molecules per unit area, and cases were defined as high expressing if they were above the median expression level.Results: The mean follow-up time for survivors was 44.9 months, and for the entire cohort was 34.8 months. Patients with high tumor EGFR expression levels had a local recurrence rate of 58% compared with 17% for patients with low EGFR tumor expression (P < 0.01). Similarly, patients with high nuclear EGFR expression had a local recurrence rate of 54% compared with 21% for patients with low EGFR nuclear expression (P < 0.05). Additionally, patients with high tumor and nuclear EGFR levels had inferior disease-free survival compared with low expressors (19% versus 43% and 19% versus 45%, respectively. P < 0.05 for each). In multivariate analysis adjusting for well-characterized prognostic variables, high tumor and nuclear EGFR expression levels retained their prognostic significance.Conclusion: The AQUA system provides a continuous measurement of EGFR on paraffin-embedded tissue and was able to reveal the association between EGFR expression and outcome expected from the biological role of EGFR. In the future, EGFR AQUA score may be useful in predicting response to EGFR-targeted therapies.

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 ≥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.

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.

Background: β-catenin, depending on subcellular localization, plays a dual role in carcinogenesis: as a signaling factor (in the nucleus) and as an adhesion molecule (in cell membrane). In this study, we sought to determine the role of β-catenin in head and neck carcinogenesis.Methods: First, we studied the incidence of mutations of β-catenin in a cohort of 60 head and neck squamous cell cancers (HNSCC). We subsequently evaluated the protein expression levels of β-catenin in a cohort of oropharyngeal squamous cell cancer tissue microarray using a novel in situ method of quantitative protein analysis and correlated those with cyclin D1 levels and clinical and pathologic data.Results: The mean follow-up time for survivors was 45 months and for all patients was 35 months. We found no mutations in the cohort of 60 HNSCC. β-catenin displayed primarily membranous expression pattern. Patients with high tumor-node-metastasis stage were more likely to have high expression of β-catenin (P = 0.040). Patients with low β-catenin expression had a local recurrence rate of 79% compared with 29% for patients with high β-catenin tumors (P = 0.0021). Univariate Cox regression revealed a hazard ratio for low β-catenin tumors of 3.6 (P = 0.004). Kaplan-Meier analysis showed that patients with low β-catenin expressing tumors trended toward worse 5-year disease-free survival (P = 0.06). In multivariate analysis, only β-catenin expression status was an independent prognostic factor (P = 0.044) for local recurrence. Tumors with high β-catenin had low cyclin D1 and vice versa (P = 0.007).Conclusions: The absence of activating β-catenin mutations combined with the inverse correlation between β-catenin levels with cyclin D1 levels and outcome suggest that β-catenin mainly functions as an adhesion and not signaling molecule in HNSCC.

Despite years of discovery and attempts at validation, few molecular biomarkers achieve acceptance in the clinical setting. Tissue-based markers evaluated by immunohistochemistry suffer from a high degree of inter- and intraobserver variability. One recent advance in this field that promises to automate this process is the development of AQUA, a molecular-based method of quantitative assessment of protein expression. This system integrates a set of algorithms that allows for the rapid, automated, continuous, and quantitative analysis of tissue samples, including the separation of tumor from stromal elements and the subcellular localization of signals. This study uses the AQUA system to assess a recently described prostate cancer biomarker, α-methylacyl-CoA-racemase (AMACR), and to determine the effectiveness of the quantitative measurement of this marker as a means for making the diagnosis of prostate cancer. Using a prostate cancer progression tissue microarray containing a wide range of prostate tissues, AQUA was directly compared to standard immunohistochemical evaluation for AMACR protein expression using the p504s monoclonal antibody. Both methods produced similar results showing AMACR protein expression to be strongest in the clinically localized prostate cancer, followed by the metastatic tumor samples. Benign prostate tissue was categorized as negative for most tissue samples by immunohistochemistry. However, AMACR was detectable using the AQUA system at low levels using the standard 1:25 dilution but also at 1:250 dilution, which is not detectable by light microscopy. The AQUA system was also able to discriminate foamy gland prostate cancers, which are known to have a lower AMACR expression than typical acinar prostate cancers, from benign prostate tissue samples. Finally, a receiver-operating-characteristic curve was plotted to determine the specificity of the AMACR AQUA Z-score (normalized AQUA score) to predict that a given tissue microarray sample contains cancer. The area under the curve was calculated at 0.90 (P < 0.00001; 95% CI, 0.84 to 0.95). At an AMACR AQUA Z-score score of −0.3, 91% of the 70 samples classified as prostate cancer were correctly categorized without the intervention of a pathologist reviewing the tissue microarray slide. In conclusion, the AQUA system provides a continuous measurement of AMACR on a wide range of prostate tissue samples. In the future, the AMACR AQUA Z-score may be useful in the automated screening and evaluation of prostate tissue biomarkers.

BACKGROUND Aberrant activation of the β-catenin signaling pathway has been implicated in several malignancies, including breast carcinoma. Recently, it was shown that p53 down-regulated β-catenin in a complex fashion. The authors examined the expression of β-catenin, key members of its signaling pathway, and p53 in a large cohort of breast tumors. METHODS The authors conducted an immunohistochemical analysis of the expression of β-catenin, upstream modulators (HER-2/neu, Met, and epidermal growth factor receptor [EGFR]), downstream target genes (cyclin D1 and matrix metalloproteinase-7 [MMP7]), and p53 on a tissue microarray of 346 lymph node-negative breast carcinomas. The results were correlated with one another and with standard clinicopathologic parameters. RESULTS β-Catenin expression was observed in the membrane and/or cytoplasm without any significant nuclear expression. HER-2/neu and EGFR were observed on the membrane in 21% and 6% of tumors, respectively, and Met stained in a membrane/cytoplasm distribution in 28% of cases. Cyclin D1 was expressed in the nucleus and MMP7 was expressed in the cytoplasm in 26% and 75% of tumors, respectively. Nuclear expression of p53 was noted in 31% of tumors. When each marker was analyzed separately, only p53 and Met demonstrated a significant correlation with survival. However, patients who had tumors that coexpressed high levels of β-catenin and p53 had markedly worse overall survival (P = 0.0026). In multivariate analysis, only tumor size, Met, and the coexpression of β-catenin and p53 retained statistical significance. CONCLUSIONS The current findings support a potential synergistic effect of abnormal β-catenin regulation and p53 status in the pathogenesis and natural history of lymph node-negative breast carcinoma. Furthermore, the results show that a combined analysis of multiple markers, notably β-catenin and p53, may enhance the prognostic capabilities compared with individual markers. Cancer 2004. © 2004 American Cancer Society.

Purpose: Macrophage colony-stimulating factor-1 receptor (CSF-1R) is a transmembrane tyrosine kinase receptor, which is abnormally expressed in invasive breast cancer. Small cohort studies have demonstrated that increased expression of CSF-1R is associated with ipsilateral breast cancer recurrence. Correlation with survival has not been reported. Our aim was to further evaluate the role of CSF-1R in breast cancer, by studying the expression of CSF-1R in a large cohort of clinical specimens. Experimental Design: Tissue microarrays containing 301 node-negative and 280 node-positive cases were used. Immunohistochemical staining was performed and correlated with overall survival, nodal status, and other clinicopathological data. Results: CSF-1R expression was strongly associated with nodal status. Of the node-negative cases, 114 (38.9%) stained positive for CSF-1R, whereas 189 (67.5%) of the node-positive cases expressed CSF-1R (P < 0.0001). CSF-1R expression is also associated with larger tumor size (P = 0.02). Positive staining was strongly associated with decreased survival (P = 0.0003). Among node-negative patients, CSF-1R expression was associated with decreased overall survival (P = 0.045), whereas among node-positive patients, it was not (P = 0.47). In multivariate analysis, CSF-1R was not independent of nodal status as a predictor of survival. Conclusions: CSF-1R expression is a strong predictor of poor outcome in nonmetastatic breast cancer. It is significantly more frequently expressed in patients with nodal involvement. Among the node-negative patients, it has a stronger association with survival than among the node-positive patients. Our findings support other preclinical findings that CSF-1R may be involved in local invasion and metastasis. Thus, this receptor may be an effective target for therapeutic agents.

The recent development of tissue microarrays-composed of hundreds of tissue sections from different tumors arrayed on a single glass slide-facilitates rapid evaluation of large-scale outcome studies. Realization of this potential depends on the ability to rapidly and precisely quantify the protein expression within each tissue spot. We have developed a set of algorithms that allow the rapid, automated, continuous and quantitative analysis of tissue microarrays, including the separation of tumor from stromal elements and the sub-cellular localization of signals. Validation studies using estrogen receptor in breast carcinoma show that automated analysis matches or exceeds the results of conventional pathologist-based scoring. Automated analysis and sub-cellular localization of beta-catenin in colon cancer identifies two novel, prognostically significant tumor subsets, not detected by traditional pathologist-based scoring. Development of automated analysis technology empowers tissue microarrays for use in discovery-type experiments (more typical of cDNA microarrays), with the added advantage of inclusion of long-term demographic and patient outcome information.

Tissue microarrays are a method of relocating tissue from conventional histologic paraffin blocks in a manner that tissue from multiple patients or blocks can be seen on the same slide. This is done by using a needle to biopsy a standard histologic section and placing the core into an array on a recipient paraffin block. This technique allows maximization of tissue resources by analysis of small core biopsies of blocks, rather than complete sections. Using this technology, a carefully planned array can be constructed using cases from pathology tissue block archives, and a 20-year survival analysis can be done on a cohort of 600 or more patients using only a few microliters of antibody in a single experiment. Furthermore, this cohort can be analyzed thousands of times with different reagents as a result of judicious sectioning of the array block. This review describes this process and discusses the issues of representative sampling in heterogeneous lesions, the issue of antigen preservation, and some technical strategies and methods of array construction. In summary, this technique can provide a highly efficient, high-throughput mechanism for evaluation of protein expression in large cohorts. It has the potential for allowing validation of new genes at a speed comparable to the rapid rate of gene discovery afforded by DNA microarrays.

Background Sorafenib was the first Food and Drug Administration approved anti-angiogenic therapy for renal cell carcinoma (RCC). Currently, there are no validated predictive biomarkers for sorafenib. Our purpose was to determine if sorafenib target expression is predictive of sorafenib sensitivity. Methods We used an automated, quantitative immunofluorescence-based method to determine expression levels of sorafenib targets VEGF, VEGF-R1, VEGF-R2, VEGF-R3, c-RAF, B-RAF, c-Kit, and PDGFR-β in a cohort of 96 patients treated with sorafenib. To measure vasculature in the tumor samples, we measured microvessel area (MVA) by CD-34 staining. Results Of the markers studied, only high MVA was predictive of response (p = 0.005). High MVA was associated with smaller primary tumors (p = 0.005). None of the biomarkers studied was predictive of overall or progression-free survival. Using the Bonferroni adjustment correcting for 9 variables with an alpha of 0.05, MVA remained significantly associated with sorafenib response. Conclusions Our results suggest that high MVA in tumor specimens might be associated with a greater likelihood of response to therapy. Further studies are needed to confirm these results in additional patients and in patients receiving other VEGF-R2 inhibitors, as MVA might be useful to improve patient selection for VEGF-R2 inhibitors.

Purpose Anti-angiogenic therapies are among the most commonly used drugs in renal cell carcinoma. Tumor vascularity, defined by microvessel area, may be associated with response to these drugs. Clinical studies suggest that metastatic sites are more responsive than primary tumors. Our purpose was to characterize microvessel area (MVA) in matched primary and metastatic samples and in samples of different histologies. Methods We employed a method of automated, quantitative analysis of in situ tumor components to identify the area of CD-34 staining endothelial cells within renal cell carcinoma tumors. MVA was assessed in corresponding primary and metastatic samples from 34 patients, as well as in 334 primary nephrectomy specimens with variable histologies. Results MVA measurements from different parts of the same tumor correlated well (R = 0.75), indicating that MVA was fairly uniform within a tumor. While MVA was slightly higher in primary tumors than corresponding metastatic sites, the difference was not statistically significant (P = 0.1). MVA in paired primary and metastatic samples correlated moderately well (R = 0.36). MVA was higher in clear cell than papillary histology and oncocytomas (P Conclusions Lack of significant differences MVA in matched primary and metastatic samples suggests that both types of tumors should respond to anti-angiogenic drugs. This should be confirmed on additional cohorts. Given the small cohort, future predictive biomarker studies entailing MVA measurements should include specimens from both sites. Clear cell carcinomas are more vascular than other histologic subtypes, which may explain the higher response rates to anti-angiogenic therapies in clear cell tumors.

Pre-analytic variables, specifically cold ischemic time, have been implicated as key variables in the measurement of proteins by immunohistochemistry. To determine the significance and magnitude of antigenic loss due to pre-analytic variables, we have compared protein antigenicity in core needle biopsies, with essentially no cold ischemic time, to that in routinely processed tumor resection specimens. Two cohorts of matched core needle biopsies and tumor resections were collected with 20 matched pairs and 14 matched pairs, respectively. Both series were analyzed by quantitative immunofluorescence using the AQUA® method. Antibodies to phospho-ERK, total ERK, phospho-AKT, total AKT, phospho-S6K1, total S6K1, Estrogen Receptor, Ki67, cytokeratin and GAPDH were measured. Detection levels for all phospho-epitopes were significantly decreased in tumor resections compared to biopsies while no significant change was seen in the corresponding total proteins. Of the other four proteins examined, Estrogen Receptor and cytokeratin showed significant loss of antigenicity. This data suggests that measurement of phospho-protein antigenicity in formalin-fixed tissue by immunological methods is dramatically affected by pre-analytic variables. This study suggests that core needle biopsies are more accurate for assessment of tissue biomarkers.

A subset of cells, tentatively called cancer stem cells (CSCs), in breast cancer have been associated with tumor initiation, drug resistance, and tumor persistence or aggressiveness. They are characterized by CD44 positivity, CD24 negativity, and/or ALDH1 positivity in flow cytometric studies. We hypothesized that the frequency or density of these cells may be associated with more aggressive tumor behavior. We borrowed these multiplexed, flow-based methods to develop an in situ method to define CSCs in formalin-fixed paraffin-embedded breast cancer tissue, with the goal of assessing the prognostic value of the presence of CSCs in breast cancer. Using a retrospective collection of 321 node-negative and 318 node-positive patients with a mean follow-up time of 12.6 years, we assessed TMAs using the AQUA method for quantitative immunofluorescence. Using a multiplexed assay for ALDH1, CD44, and cytokeratin to measure the coexpression of these proteins, putative CSCs appear in variable sized clusters and in 27 cases (of 490), which showed significantly worse outcome (log rank P = 0.0003). Multivariate analysis showed that this marker combination is independent of tumor size, histological grade, nodal status, ER-, PR,- and HER2-status. In this cohort, ALDH1 expression alone does not significantly predict outcome. We conclude that the multiplexed method of in situ identification of putative CSCs identifies high risk patients in breast cancer.

Angiogenesis is required for progression and metastasis of melanoma. Analysis of angiogenic molecules in benign and malignant tissues may allow identification of markers useful for prediction of sensitivity to antiangiogenic agents. We hypothesized that differential expression of vascular endothelial growth factor (VEGF) and its receptors VEGF-R1, VEGF-R2, and VEGF-R3 would be higher in melanomas than nevi and higher in advanced melanoma. Using automated quantitative analysis, we quantified VEGF, -R1, -R2 and -R3 expression in melanoma tissue microarrays composed of 540 nevi and 468 melanoma specimens (198 primaries, 270 metastases). VEGF, VEGF-R1, VEGF-R2, and VEGF-R3 expression was significantly higher in melanomas than nevi by unpaired t tests (P < .0001). VEGF-R2 expression was higher in metastatic specimens (P < .0001), but VEGF-R3 expression was higher in primaries (P < .0001). VEGF was coexpressed with all 3 receptors when assessed by Spearman's rank correlation. VEGF, VEGF-R1, VEGF-R2, and VEGF-R3 expression is higher in melanomas than nevi. Higher expression of VEGF-R2 was found in metastases versus primaries, supporting the idea that selection for an angiogenic phenotype in metastatic melanoma is conferred via up-regulation of VEGF-R2. However, higher expression of VEGF-R3 was seen on primary lesions, potentially implicating this receptor in initiation of lymphatic tumor spread. Clinical trials using antiangiogenic agents in melanoma should include correlative assays of VEGF, VEGF-R1, VEGF-R2, and VEGF-R3 as biomarkers of response to therapy, preferably using quantitative methods such as automated quantitative analysis. Such assessments could assist with evaluation of these molecules as therapeutic targets in melanoma, ultimately facilitating improved selection of patients for treatment.

SummaryKallikrein-related peptidases, a subgroup of the serine protease enzyme family, are considered to be important prognostic biomarkers in cancer. In this study we sought to determine the prognostic value of kallikrein-related peptidase 8 (KLK8,hK8,KLK-8) in ovarian cancer using a novel method of compartmentalised in situ protein analysis. A tissue array composed of 150 advanced stage ovarian cancers, uniformly treated with surgical debulking followed by platinum-paclitaxel combination chemotherapy, was constructed. For the evaluation of kallikrein-related peptidase 8 protein expression, we used an immunofluorescence-based method of automated in situ quantitative protein analysis (AQUA). Mean follow-up time of the cohort was 34.35 months. One hundred twenty-six of 150 cases had sufficient tissue for AQUA analysis. There were significant correlations between tumour mask KLK8 protein expression levels and clinicopathological variables, including grade (p=0.0011), residual disease (p=0.0063) and clinical response to chemotherapy(p=0.0346). In univariate survival analysis there was a significant correlation between KLK8 tumour mask expression and five years progression-free survival, meanwhile it was not associated with five-year overall survival (p =0.0694). Specifically, low KLK8 expression correlated with better outcome (top vs. bottom quartile, p=0.0319). In multivariate survival analysis, adjusting for well-characterised prognostic variables, tumour KLK8 expression level retained its prognostic significance for progression-free survival (95%CI: 0.341–1.027, p=0.045). The possibility that KLK8 may be a suitable candidate as a diagnostic and prognostic marker warrants further investigation.

ProblemWe previously proposed that head and neck squamous cell carcinoma (HNSCC) presents in three histopathologically similar but molecularly distinct types where p16 expression combined with high-risk Human Papillomavirus (HPV) presence defines three distinct subgroups: Class I (HPV-), Class II (HPV16+/p16-), Class III (HPV16+/p16+). In this study we sought to further explore and validate this classification schema.MethodsParaffin-embedded specimens from 77 patients with OSCC classified into the 3-class model based on p16 expression and HPV DNA presence were previously queried for expression of 14 target proteins with known involvement in tumor progression using AQUA(tm). We calculated pairwise associations by Spearman correlation and performed hierarchical clustering analysis to assess global expression patterns of class I, II and III tumors.ResultsHierarchical clustering analysis demonstrated class I and class II tumors displayed similar clustering patterns. Class III tumors (HPV+/p16+) displayed uniq...

Microvessel density may be one measure of tumor associated angiogenesis but is methodologically difficult to standardize and reproduce. We used our automated quantitative image analysis system, AQUA, to more objectively assess microvessel area. Cytokeratin and CD31 were used to create tumor and vessel compartments respectively with AQUA. Microvessel area was defined as CD31 compartment area normalized to the tissue spot area (CD31 area/area of entire tissue spot). Consecutive breast cancer whole sections were stained with CD31 to compare pathologist-based microvessel density with AQUA microvessel area. Microvessel areas of 3-fold redundant tissue microarrays of 652 primary breast cancers were also assessed. CD34 and factor VIII-related antigen were also tested. There was nearly linear correlation between pathologist's microvessel density and AQUA microvessel area with regression coefficient R = 0.846. On the redundant arrays, of the 67% evaluable cases, 52% were microvessel area high and 48% low with good reproducibility of scores (Spearman rho 0.551). AQUA microvessel area was associated with larger tumors, node positivity, and estrogen receptor negativity, with 20 year survival at the univariate and multivariate levels (P < .0001 and P = .0121, respectively). CD34 or factor VIII-related antigen were more heterogenous, had poor association with CD31, and did not correlate with outcome. AQUA-based microvessel area was significantly correlated with both standard breast cancer prognostic parameters as well as with clinical outcome. In the future, it may also allow the use of the AQUA-based algorithms to quantify the expression of angiogenic biomarkers to either tumor or microvessel area-specific compartments.

Background Kallikreins, a subgroup of the serine protease enzyme family, are considered important prognostic biomarkers in cancer. Here, we sought to determine the prognostic value of kallikrein 7 (hk7) in ovarian cancer using a novel method of compartmentalized in situ protein analysis. Patients and methods A tissue array composed of 150 advanced-stage ovarian cancers, uniformly treated with surgical debulking followed by platinum–paclitaxel (Taxol) combination chemotherapy, was constructed. For evaluation of kallikrein 7 protein expression, we used an immunofluorescence-based method of automated in situ quantitative measurement of protein analysis (AQUA). Results Mean follow-up time of the cohort was 34.35 months. One hundred and twenty eight of 150 cases had sufficient tissue for AQUA. In univariate survival analysis, low tumor hk7 expression was associated with better outcome for overall survival (OS) and disease-free survival in 3 years (P values 0.032 and 0.037, respectively). In multivariate survival analysis, adjusting for well-characterized prognostic variables, low tumor hk7 expression level was the most significant predictor variable for OS (95% confidence interval 0.125–0.729, P = 0.007). Conclusions High tumor hk7 protein expression is associated with inferior patient outcome in ovarian cancer. hk7 may represent a promising prognostic factor in ovarian cancer.

Vascular endothelial growth factor has been shown to be up-regulated in breast cancers. Vascular endothelial growth factor receptors, VEGFR-1 and VEGFR-2, are the principal mediators of its effects. Together with VEGFR-1 and VEGFR-2, neuropilin-1 may act as a coreceptor for vascular endothelial growth factor. Although vascular endothelial growth factor exerts important effects on endothelial cells, VEGFRs are likely present on tumor cells as well. We used AQUA to analyze tumor-specific expression of vascular endothelial growth factor, VEGFR-1, VEGFR-2, and neuropilin-1 on a large cohort of breast cancer tissue microarray. Two-fold redundant arrays were constructed from 642 cases of primary breast adenocarcinomas. Automated image analysis with AQUA (Automated Quantitative Analysis) was then performed to determine a quantitative expression score. Scores from redundant arrays were normalized and averaged. Kaplan-Meier survival analysis showed that high levels of vascular endothelial growth factor, VEGFR-1, VEGFR-2, and neuropilin-1 were all significantly associated with survival (Miller Siegmeund corrected P = .0020, .0160, and .0320, respectively). In addition, vascular endothelial growth factor and neuropilin-1 retained a significant association with survival independent of other standard prognostic factors. Vascular endothelial growth factor, VEGFR-1 and -2, and neuropilin-1 are expressed to varying degrees in primary breast cancers and have prognostic significance. Further study of the functional significance of this finding is warranted as well as the prognostic value of these biomarkers in other tumor microenvironment-specific compartments (eg, vessels).

To investigate the role of small intestinal carcinoid tumor-derived fibrotic mediators, TGFbeta1 and CTGF, in the mediation of fibrosis via activation of an "intestinal" stellate cell.GI carcinoid tumors were collected for Q RT-PCR analysis of CTGF and TGFbeta1. Markers of stellate cell desmoplasia were identified in peritoneal fibrosis by immunohistochemistry and stellate cells cultured from fresh resected fibrotic tissue. CTGF and TGFbeta1 were evaluated using quantitative tissue array profiling (AQUA analysis) in a GI carcinoid tissue microarray (TMA) with immunostaining and correlated with clinical and histologically documented fibrosis. Serum CTGF was analyzed using a sandwich ELISA assay.Message levels of both CTGF and TGFbeta1 in SI carcinoid tumors were significantly increased (2-fold, P0.05) versus normal mucosa and gastric (non-fibrotic) carcinoids. Activated stellate cells and markers of stellate cell-mediated fibrosis (vimentin, desmin) were identified in histological fibrosis. An intestinal stellate cell was immunocytochemically and biochemically characterized and its TGFbeta1 (10-7M) initiated CTGF transcription response (3-fold, P0.05) demonstrated. In SI carcinoid tumor patients with documented fibrosis, TMA analysis demonstrated higher CTGF immunostaining (AQUA Score: 92 +/- 8; P0.05), as well as elevated TGFbeta1 (90.6 +/- 4.4, P0.05). Plasma CTGF (normal 12.5 +/- 2.6 ng/mL) was increased in SI carcinoid tumor patients (31 +/- 10 ng/mL, P0.05) compared to non-fibrotic GI carcinoids (15 ng/mL).SI carcinoid tumor fibrosis is a CTGF/TGFbeta1-mediated stellate cell-driven fibrotic response. The delineation of the biology of fibrosis will facilitate diagnosis and enable development of agents to obviate its local and systemic complications.

Epidemiologic and preclinical studies suggest that cyclooxygenase-2 (Cox-2) may promote tumor growth and spread by affecting angiogenesis and apoptosis in breast cancer. Using a tissue microarray (TMA), we analyzed the expression and subcellular localization of Cox-2 by AQUA and X-tile, our algorithms for quantitative analysis of protein expression and determination of optimal cutpoints. Our TMA consisted of 669 Stage I-III primary breast cancers. The total tumor and subcellular expression of Cox-2 were then correlated with clinicopathologic factors and with survival. Cox-2 expression appeared higher in malignant than in benign tissue and was predominantly membrane/cytoplasmic (i.e. non-nuclear). X-tile determines an optimum cutpoint on a training set then uses this cutpoint on a validation set. This cutpoint was 19.3 (top 44 percent defined as positive) with high nonnuclear Cox-2 expressers having significantly worse survival. Cox-2 expression also was inversely associated with estrogen receptor (ER) and progesterone receptor (PR), and directly associated with nuclear grade. Multivariate analysis showed that Cox-2 remained a significant prognostic factor for survival independent of tumor size, nodal status, ER, Her2/neu, and grade. In summary, Cox-2 is overexpressed in breast neoplasms, is associated with other markers of poor prognosis, and is significantly associated with worse survival independent of known prognostic factors. Furthermore, AQUA and X-tile analysis suggest an optimal cutpoint that may be helpful in future investigations of Cox-2 and specifically, in studies looking at its expression as a predictive biomarker in clinical trials of Cox-2 inhibitors in breast cancer.

Immunohistochemical analyses (IHC) of biomarkers are extensively used for tumor characterization and as prognostic and predictive measures. The current standard of single slide analysis assumes that one 5 microM section is representative of the entire tumor. We used our automated image analysis technology (AQUA) using a modified IHC technique with fluorophores to compare estrogen receptor (ER) expression in multiple blocks/slides from cases of primary breast cancer with the objective of quantifying tumor heterogeneity within sections and between blocks. To normalize our ER scores and allow slide-to-slide comparisons, 0.6 microm histospots of representative breast cancer cases with known ER scores were assembled into a 'gold standard array' (GSA) and placed adjacently to each whole section. Overall, there was excellent correlation between AQUA scores and the pathologist's scores and reproducibility of GSA scores (mean linear regression R value 0.8903). Twenty-nine slides from 11 surgical cases were then analyzed totaling over 2000 AQUA images. Using standard binary assignments of AQUA (>10) and pathologist's (>10%) scores as being positive, there was fair concordancy between AQUA and pathologist scores (73%) and between slides from different blocks from the same cases (75%). However using continuous AQUA scores, agreement between AQUA and pathologist was far lower and between slides from different blocks from the same cases only 19%. Within individual slides there was also significant heterogeneity in a scattered pattern, most notably for slides with the highest AQUA scores. In sum, using a quantitative measure of ER expression, significant block-to-block heterogeneity was found in 81% of cases. These results most likely reflect both laboratory-based variability due to lack of standardization of immunohistochemistry and true biological heterogeneity. It is also likely to be dependent on the biomarker analyzed and suggests further studies should be carried out to determine how these findings may affect clinical decision-making processes.

Background: The deleted in colorectal cancer (DCC) protein, the product of DCC tumor suppressor gene, is frequently altered in cancer. Preclinical data demonstrate that DCC regulates b-catenin levels. Here, we sought to determine the association of DCC with b-catenin protein levels, clinicopathological parameters and patient outcome in ovarian cancer using a method of in situ compartmentalized protein analysis. Methods: A tissue array composed of 150 advanced-stage ovarian cancers, treated with surgical debulking and platinum–paclitaxel (Taxol) combination chemotherapy, was constructed. For evaluation of protein expression, we used an immunofluorescence-based method of automated in situ quantitative measurement of protein analysis (AQUA). Results: One hundred and twelve patients (74%) had sufficient tissue for AQUA. The median follow-up time for the entire cohort was 33 months. Patients with low nuclear DCC expression had a 3-year progression-free survival (PFS) rate of 0% compared with 33% of those with high DCC expression (P = 0.0067). In multivariate analysis, low nuclear DCC expression level retained its prognostic significance for PFS. Between DCC and b-catenin, a significant relationship was found, where tumors with low DCC had low b-catenin and vice versa (P = 0.003). Conclusions: Low nuclear DCC levels predict for poor patient outcome in epithelial ovarian cancer. DCC may exert its antitumor function, in part, through regulation of b-catenin levels.

To use differential gene expression of candidate markers to discriminate benign appendiceal carcinoids (APCs) from malignant and mixed cell APCs.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.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.CgA message was elevated (1000-fold, P0.05) in all tumor types. NAP1L1 was elevated (10-fold, P0.03) in both malignant and goblet cell adenocarcinoids compared with normal and incidental lesions (P0.006). MAGE-D2 and MTA1 message were significantly elevated (10-fold, P0.01) 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, P0.05) in the appendicitis-associated and malignant appendiceal carcinoids but was significantly decreased (10-fold, P0.05) 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.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.

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.

The role of β-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 β-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 β-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 β-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 β-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. (Cancer Res 2006; 66(10): 5487-94)

Chromogranin A (CgA), present in secretory dense core granules,1 is widely used as an immunohistochemical (IHC) marker of neuroendocrine tumors (NETs). In addition, since CgA is cosecreted with the amines and peptides that are present in the neurosecretory granules, it is also useful as a serum marker.2 Indeed, since the majority of peptide-producing endocrine neoplasms including gastrointestinal (GI) carcinoids secrete CgA, it is considered a definitive NE tumor marker for diagnosis and therapeutic evaluation.3 Observations that CgA serum or plasma levels reflect tumor load and may be an independent marker of prognosis in patients with midgut carcinoids have led to its widespread clinical utility in NET disease.4 A substantial body of data, however, has accumulated indicating that NE cells are also present in many tumors of nonendocrine origin.5–9 Although the reasons for this are unknown, it has been proposed that this observation may reflect neuronal dedifferentiation of such lesions.10 Although there are limited data available concerning serum levels of CgA in subjects with nonendocrine tumors, elevated levels have been reported in association with prostatic,11,12 non-small cell lung,13 ovarian, pancreatic, and colonic neoplasia.14 The sensitivity of detecting NE cells in non-NE tumors is dependent on the tumor type, the NE marker used, and the detection technique (IHC, serum analysis or PCR of NE markers). At this time, analysis of gene levels by PCR is the most sensitive method for the detection of cell-specific markers.15 Our hypothesis is that since CgA is a marker that specifically identifies gastrointestinal NETs, a more sensitive method for detection of this marker using a PCR approach would: 1) be more effective than conventional immunohistochemistry, 2) alter tumor staging, and 3) differentiate GI NETs from phenotypically indeterminate tumors and GI adenocarcinomas. We used a real-time quantitative reverse transcription-PCR (Q RT-PCR) approach to identify the presence of NET cells (by CgA) in primary tumors and metastases, and confirmed the expression, utility, and specificity of this marker. We then examined protein expression of CgA immunohistochemically to assess the utility of protein assessment and compared this with the real-time PCR technique.

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.

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.

Purpose: This study was designed to provide a comprehensive assessment on the role of β1,6-branched oligosaccharides in the metastasis and outcome of breast carcinoma. Generation of these structures on N-glycans is initiated by β1,6-N-acetylglucosaminyltransferase V and used by both myeloid cells and cancer cells in systemic migration. Experimental Design: Tissue microarrays of >700 tumors (>400 patients; 30-year follow-up data) were stained through lectin histochemistry with leukocytic phytohemagglutinin (LPHA), a selective marker for β1,6-branched oligosaccharides. Node-negative and node-positive primary tumors and patient-matched lymph node metastases were scored by blinded observers. Results: Metastases stained at significantly greater intensities than did the patient-matched primary tumors (P < 0.0001), demonstrating for the first time that the abundance of β1,6-branched oligosaccharides was directly associated with breast carcinoma nodal metastasis. Multivariate analyses revealed that β1,6-branched oligosaccharides in primary tumors were a predictor of poor outcome, most notably in node-negative tumors, where an LPHA staining score of 3+ gave a risk factor of 3.3, independent of tumor size, nuclear grade, or patient age (P = 0.007). Conclusions: The data firmly establish a role for β1,6-N-acetylglucosaminyltransferase V activity and β1,6-branched oligosaccharides in breast carcinoma metastasis, and reemphasize the involvement, although poorly understood, of aberrant glycosylation in tumor progression.

BACKGROUND Microsatellite instability (MSI) with concomitant mutations in the coding region of transforming growth factor-beta type II receptor (TGFβRII) 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 TGFβRII 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 TGFβRII and MSH2. RESULTS Mutational examination of the TFGβRII 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 TGFβRII immunostaining using AQUA demonstrated that TGFβRII 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 TGFβRII, 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 TGFβRII but were associated with the retention of mismatch repair protein function and a microsatellite-stable phenotype. Cancer 2005. © 2004 American Cancer Society.

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. 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. 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). 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.

The addition of B-cell lymphoma 2 (Bcl-2) antisense to dacarbazine in the treatment of metastatic melanoma demonstrates improved response rates and progression-free survival when compared with dacarbazine alone. Studies on small cohorts of melanoma patients have shown variability in Bcl-2 expression (60%–96% positive). We performed quantitative analysis of Bcl-2 expression in a large patient cohort to assess the association with outcome. Tissue microarrays containing intact melanoma specimens representing 402 patients (339 with associated survival data) were analyzed with our AQUA system for automated quantitative analysis. Automated, quantitative analysis uses S100 to define pixels as melanoma (tumor mask) within the array spot and measures intensity of Bcl-2 expression using a Cy5 conjugated antibody within the mask. A continuous index score is generated, which is directly proportional to the number of molecules per unit area. Scores were divided into quartiles and correlated with clinical variables. High Bcl-2 expression was associated with better outcome in the entire cohort and among metastatic specimens only (P = 0.004 and P = 0.015, respectively). Expression was higher in primary than in metastatic specimens (P < 0.0001). There was no association between Bcl-2 expression and Breslow depth or Clark level. The diverse results within the literature may be due to use of small cohorts or variability in staining technique. These results suggest studies are needed to evaluate the association between quantitative assessment of Bcl-2 expression and response to Bcl-2 targeting therapy toward the goal of improved response rates to these drugs.

The incidence of cutaneous malignant melanoma continues to increase every year, and this disease remains the leading cause of skin cancer death in industrialized countries. Despite the aggressive nature of advanced melanoma, there are no standard biological assays in clinical usage that can predict metastasis. This may be due, in part, to the inadequacy of reproducible assessment of protein expression using traditional immunohistochemistry. We have previously described a novel method of quantitative assessment of protein expression (AQUA) with the continuity and accuracy of an ELISA assay but with maintenance of critical spatial information. Here, we modify this technology for the evaluation of protein expression in melanoma. Using a tissue microarray cohort of 405 melanoma lesions and 17 normal skin samples, we analyzed expression of HDM2, the human homologue of murine double minute 2 with automated quantitative analysis. We show that expression levels in the nucleus are significantly higher in primary melanomas than in metastatic lesions. Furthermore, high levels of expression are predictive of better outcome. This study demonstrates that quantitative assessment of protein expression is useful in melanoma to validate potential tissue biomarkers and suggests that human homologue of murine double minute 2 may be a valuable prognostic tool for management of malignant melanoma.

Melanoma is among the most chemotherapy-resistant malignancies. Numerous new agents have been developed that target specific molecules on cancer cells, including the monoclonal antibody trastuzumab, which targets Her2/neu and has been very beneficial in the treatment of breast cancer. There are conflicting reports in the literature about Her2/neu expression in melanoma specimens, but all of the cohorts studied have been small. We therefore examined Her2/neu expression in a very large cohort of melanoma specimens in order to determine the value of exploring trastuzumab therapy for melanoma patients. Immunohistochemical staining was performed on two tissue microarrays, together containing 600 intact specimens. Expression was evaluated semi-quantitatively and correlated with tumour stage and other clinicopathological data. Of the 600 specimens in the cohort, 31 patients (5.2%) had positive Her2/neu expression. Among the primary cutaneous specimens (n=269), 7% had positive Her2/neu staining, while 3.6% of the recurrent or metastatic specimens (n=331) had positive Her2/neu staining (P=0.06). Among the primary lesions there was no significant correlation between Her2/neu expression, Clark level and ulceration; however, Her2/neu expression was associated with lesions with a Breslow depth of < 2 mm (P=0.05). Using this very large cohort of melanoma specimens, we found only a few cases with aberrant Her2/neu expression, many of them being primary cutaneous lesions rather than recurrent or metastatic lesions. Our findings suggest that drugs that specifically target Her2/neu are not likely to be useful for the treatment of metastatic melanoma or as adjuvant therapy for melanoma patients at high risk for recurrence.