Christopher B. Moeder

High HSP90 expression is associated with decreased survival in breast cancer.

The heat shock protein HSP90 chaperones proteins implicated in breast cancer progression, including Her2/neu. HSP90-targeting agents are in clinical trials for breast cancer. HSP90 expression is high in breast cancer cell lines, yet no large studies have been conducted on expression in human tumors and the association with clinical/pathologic variables. Tissue microarrays containing 10 cell lines and primary specimens from 655 patients with 10-year follow-up were assessed using our automated quantitative analysis (AQUA) method; we used cytokeratin to define pixels as breast cancer (tumor mask) within the array spot and measured HSP90 expression within the mask using Cy5-conjugated antibodies. We similarly assessed estrogen receptor, progesterone receptor, and Her2/neu expression. HSP90 expression was more variable in human tumors than in cell lines (P < 0.0001). High HSP90 expression was associated with decreased survival (P = 0.0024). On multivariable analysis, high HSP90 expression remained an independent prognostic marker. High HSP90 expression was associated with high Her2/neu and estrogen receptor, large tumors, high nuclear grade, and lymph node involvement. Although HSP90 levels were high in all our cell lines, expression in tumors was more variable. High HSP90 expression in primary breast cancer defines a population of patients with decreased survival. Evaluation of HSP90 expression in early-stage breast cancer may identify a subset of patients requiring more aggressive or pathway-targeted treatment. Prospective studies are needed to confirm the prognostic role of HSP90, as well as the predictive role of HSP90 expression in patients treated with HSP90 inhibitors.

Expression patterns and prognostic value of Bag-1 and Bcl-2 in breast cancer.

IntroductionBcl-2 antanogene-1 (Bag-1) binds the anti-apoptotic mediator Bcl-2, and enhances its activity. Bcl-2 and Bag-1 are associated with chemotherapy resistance in cancer cells. Drugs that target Bcl-2 are currently in clinical development. The purpose of the present study was to examine expression patterns of Bag-1 in a large cohort of breast tumors and to assess the association with Bcl-2, estrogen receptor, progesterone receptor and Her2/neu, and other clinical/pathological variables.MethodsTissue microarrays containing primary specimens from 638 patients with 10-year follow-up were employed, and the expression of Bag-1, Bcl-2, estrogen receptor, progesterone receptor and Her2/neu was assessed using our automated quantitative analysis method. We used cytokeratin to define pixels as breast cancer (tumor mask) within the array spot, and we measured biomarker expression within the mask using Cy5 conjugated antibodies.ResultsHigh Bcl-2 expression was associated with improved survival in the entire cohort and in the node-positive subset (P = 0.008 and P = 0.002, respectively). High Bag-1 expression was associated with improved survival in the node-positive subset (P = 0.006). On multivariable analysis, neither Bcl-2 nor Bag-1 retained their independence as prognostic markers. Strong associations were found between Bag-1, Bcl-2, estrogen receptor and progesterone receptor.ConclusionBag-1 and Bcl-2 expression in breast tumors is associated with improved outcome and steroid receptor positivity. Evaluation of Bcl-2 and Bag-1 expression in breast cancer may identify a subset of patients with a favorable prognosis, who might not benefit from chemotherapy or who might benefit from Bcl-2 targeting agents in addition to antihormonal therapy.

Quantitative, fluorescence-based in-situ assessment of protein expression.

As companion diagnostics grow in prevalence and importance, the need for accurate assessment of in situ protein concentrations has increased. Traditional immunohistochemistry (IHC), while valuable for assessment of context of expression, is less valuable for quantification. The lack of rigorous quantitative potential of traditional IHC led to our development of an immunofluorescence-based method now commercialized as the AQUA technology. Immunostaining of tissue samples, image acquisition, and use of AQUA software allow investigators to quickly, efficiently, and accurately measure levels of expression within user-defined subcellular or architectural compartments. IHC analyzed by AQUA shows high reproducibility and demonstrates protein measurement accuracy similar to ELISA assays. The process is largely automated, eliminating potential error, and the resultant scores are exported on a continuous scale. There are now numerous published examples where observations made with this technology are not seen by traditional methods.

Quantitative multiplexed analysis of ErbB family coexpression for primary breast cancer prognosis in a large retrospective cohort.

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.

Nuclear to non-nuclear Pmel17/gp100 expression (HMB45 staining) as a discriminator between benign and malignant melanocytic lesions

HMB45 is a mouse monoclonal antibody raised against Pmel17/gp100, a melanoma-specific marker, which is routinely used in the diagnosis of primary cutaneous malignant melanoma. The standard expression pattern for a positive HMB45 staining result on immunohistochemistry is based upon the results of chromogenic-based methods. We re-evaluated the patterns of HMB45 staining across the 480-core ‘SPORE melanoma progression array’ containing lesions representing the spectrum of melanocytic lesions ranging from thin nevus to visceral metastasis using the fluorescence-based staining technique and automated quantitative analysis (AQUA) of the obtained digital images. The methods validated the expected cytoplasmic HMB45 staining pattern in 70/108 malignant lesions and in the epithelial components of nevus specimens. However, the fluorescence-based approach revealed a nuclear gp100 localization present in the dermal component of all nevi that was not seen before. This nuclear localization could not be observed on routine chromogenic stains, because the standard hematoxylin nuclear counterstain overwhelms the weak nuclear HMB45 stain. The thin (0.450±0.253) and thick (0.513±0.227) nevi had strongly positive mean ln(nuclear/non-nuclear AQUA score ratios), which are significantly higher than those from the group of malignant lesions (P<0.0001). This finding was reproduced on a smaller but independent progression array composed of nevi and melanomas from the Yale Pathology archives (P<0.01). The odds ratio associated with a sample being a nevus was 2.24 (95% CI: 1.87–2.69, P<0.0001) for each 0.1 unit increase of the ln(nuclear/non-nuclear AQUA score ratio) to yield an ROC curve with 0.93 units of area and a simultaneously maximized sensitivity of 0.92 and specificity of 0.80 for distinguishing benign nevi from malignant melanomas. On the basis of this preliminary study, we propose that the ratio of nuclear to non-nuclear HMB45 staining may be useful for diagnostic challenges in melanocytic lesions.

Association between the nuclear to cytoplasmic ratio of p27 and the efficacy of adjuvant polychemotherapy in early breast cancer

BACKGROUND The purpose of this study was to evaluate the prognostic and predictive value of p27 expression in patients with early breast cancer. PATIENTS AND METHODS Quantitative immunofluorescence assays for p27 were done on a tissue microarray that included 823 samples from patients randomized between anthracycline-based chemotherapy and no chemotherapy. Quantification of p27 was done using the AQUA® system (HistoRx, Inc., Branford, CT). Both p27 nuclear expression and the nuclear to cytoplasmic ratio were assessed. RESULTS Nuclear p27 expression was not predictive for the efficacy of anthracycline-based chemotherapy [adjusted P=0.18 for disease-free survival (DFS)] nor prognostic [95% confidence interval (CI) 0.99-1.01, P=0.49]. However, p27 nuclear/cytoplasmic ratio was predictive for the efficacy of adjuvant chemotherapy (adjusted P=0.016 DFS). The adjusted hazard ratio (HR) for relapse associated with adjuvant chemotherapy was 0.56 (95% CI 0.37-0.84, P=0.005) and 1.06 (95% CI 0.76-1.47, P=0.74) for patients with high and low nuclear/cytoplasmic ratio, respectively. p27 N/C ratio was prognostic in patients treated with chemotherapy (HR for relapse or death for a 1 unit increase in p27 N/C ratio was 0.30, 95% CI 0.12-0.77) but not in the untreated arm (HR for relapse or death was 1.27, 95% CI 0.58-2.8). CONCLUSIONS This study did not confirm the role of p27 nuclear expression as a prognostic parameter. However, the p27 nuclear/cytoplasmic ratio was predictive in patients treated with anthracycline-based chemotherapy.

A Quantitative Immunofluorescence Assay (AQUA) Suggests Significant Misclassification (15%) of Estrogen Receptor Status in Breast Cancer.

Introduction: Estrogen Receptor (ER) is arguably the most powerful predictive marker in breast cancer. However, a recent incident in Canada revealed a strikingly high false-negative rate and raised awareness of the current limitations in our measurement of ER. The current clinical standard is both subjective and qualitative. Even though guidelines are about to be issued to standardize this assay, there is very little data on the misclassification rate in current practice in the US.Hypothesis: Our hypothesis is that the use of a quantitative assay for ER on a series of retrospective collections may reveal both the level and significance of the misclassification rate.Method: Cell lines with a range of ER expression levels were analyzed by quantitative western blotting in parallel with IF/AQUA analysis (r2 = 0.865), in order to create standard curves for assessment of absolute ER protein concentration in tissue. The optimized assay was then used to quantify ER protein expression in a large cohort of archival breast cancer samples from Yale (1962-1982, n =617).Results: Using a set of standard curves with recombinant ER and cell line controls, we developed a standardized method for quantifying ER as an absolute concentration (pg ER per μg total protein) in formalin-fixed tissue on TMAs. This ER AQUA assay has a range in sensitivity from 50 pg/μg to 1500pg/μg total protein. Quantification of ER protein expression on the Yale archival cohort revealed a unimodal distribution with 49.8% of cases above the 50pg/ug threshold and thus defined as positive. When compared to pathologist performed conventional ER classification, we found a false negative rate of 6.65% and a false positive rate of 10.2%, for a total misclassification rate of 16.6%. Although no response data is available on that cohort, data is under analysis on 4 other cohorts with endocrine therapy treatment information, including an independent Yale cohort, SWOG 9313, NSABP B14, and the TEAM trial.Conclusion: We have developed a quantitative method to measure absolute levels of ER in breast tissue. Use of this assay on a series of cohorts suggests a misclassification rate in the 15% range. The significance of this level of misclassification with respect to response to endocrine therapy is currently under study. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4068.

Predictive value of p27 for the benefit of adjuvant anthracycline-based chemotherapy in early breast cancer.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #6063 Background: p27 is a cyclin-dependent kinase (cdk) inhibitor that plays a role in cell cycle regulation. The expression of this protein, assessed by immunohistochemistry (IHC), has prognostic value for overall survival in breast cancer patients (Porter P.L., J Natl Cancer Inst., 2007). Based on this, we hypothesized that p27 could be a predictive factor for the efficacy of adjuvant anthracycline (A)-based chemotherapy in early breast cancer. Methods: Tumor samples from 823 patients, included at Institut Gustave Roussy (IGR) in two randomized trials comparing an A-based chemotherapy with no adjuvant treatment, were used to construct a tissue microarray. We previously reported predictive values of ER, Her2 expression and molecular subclassification using the same tissue array (Conforti R., Ann Oncol., 2007). Nuclear and cytoplasmic expression of p27 was assessed by the AQUA system. A p27 ratio of nuclear and cytoplasmic expression (p27 N/C ratio) was calculated for each tumor sample. The prognostic and predictive value of the continuous value of p27 N/C ratio was assessed by a Cox regression model adjusted for treatment, age, tumor grade, stage and ER expression (IHC) and stratified by trial. Results: p27 was assessable in 715 primary tumors. The p27 N/C ratio was significantly different according to the molecular subclasses (p=0.001) and was higher in Her2- versus Her2+ and in EGFR+ versus EGFR- tumors (p=0.0075 and p<0.0001 respectively). There was an almost significant interaction between p27 N/C ratio and disease-free survival in the adjusted Cox model (p=0.052). The adjusted hazard ratios (HR) of relapse or death of a unit increase in p27 N/C ratio was HR = 1.27 (95% CI = [0.58-2.81], p = 0.55) in the control arm and HR = 0.30 (95% CI = [0.12-0.77], p= 0.01) in the A-arm. For overall survival, the adjusted interaction p-value was equal to 0.08. The adjusted HR of death of a unit increase in p27 N/C ratio was HR=1.67 (95% CI = [0.61-1.73], p=0.32) in the control arm and HR=0.32 (95% CI = [0.10-0.99], p=0.05) in the A-arm. Conclusion: p27 N/C ratio, determined by the AQUA system, was borderline predictive for the efficacy of adjuvant A-based chemotherapy. However, this study is hampered by a relatively low number of events, a low dose of anthracycline, and multi-hypotheses testing (overall 20 biomarkers tested in different studies). Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6063.