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DIRAS3 is an imprinted tumor suppressor gene that is downregulated in 60% of human ovarian cancers. Re-expression of DIRAS3 at physiological levels inhibits proliferation, decreases motility, induces autophagy, and regulates tumor dormancy. Functional inhibition of autophagy with choroquine in dormant xenografts that express DIRAS3 significantly delays tumor regrowth after DIRAS3 levels are reduced, suggesting that autophagy sustains dormant ovarian cancer cells. This study documents a newly discovered role for DIRAS3 in forming the autophagosome initiation complex (AIC) that contains BECN1, PIK3C3, PIK3R4, ATG14, and DIRAS3. Participation of BECN1 in the AIC is inhibited by binding of BECN1 homodimers to BCL2. DIRAS3 binds BECN1, disrupting BECN1 homodimers and displacing BCL2. Binding of DIRAS3 to BECN1 increases the association of BECN1 with PIK3C3 and ATG14, facilitating AIC activation. Amino acid starvation of cells induces DIRAS3 expression, reduces BECN1-BCL2 interaction and promotes autophagy, whereas DIRAS3 depletion blocks amino acid starvation-induced autophagy. In primary ovarian cancers, punctate expression of DIRAS3, BECN1, and the autophagic biomarker MAP1LC3 are highly correlated (P < 0.0001), underlining the clinical relevance of these mechanistic studies. Punctate expression of DIRAS3 and MAP1LC3 was detected in only 21–23% of primary ovarian cancers but in 81–84% of tumor nodules found on the peritoneal surface at second-look operations following primary chemotherapy. This reflects a 4-fold increase (P < 0.0001) in autophagy between primary disease and post-treatment recurrence. We suggest that DIRAS3 not only regulates the AIC, but induces autophagy in dormant, nutrient-deprived ovarian cancer cells that remain after conventional chemotherapy, facilitating their survival.

BACKGROUND: Microtubule-associated proteins (MAPs) endogenously regulate microtubule stability. Here, the prognostic value of stathmin, a destabilizing protein, was assessed in combination with MAP-tau, a stabilizing protein, in order to evaluate microtubule stabilization as a potential biomarker. METHODS: Stathmin and MAP-tau expression levels were measured in a breast cancer cohort (n ¼ 651) using the tissue microarray format and quantitative immunofluorescence (AQUA) technology, then correlated with clinical and pathological characteristics and disease-free survival. RESULTS: Univariate Cox proportional hazard models indicated that high stathmin expression predicts worse overall survival (hazard ratio [HR] ¼ 1.48; 95% confidence interval [CI] ¼ 1.119-1.966; P ¼ .0061). Survival analysis showed 10-year survival of 53.1% for patients with high stathmin expression versus 67% for low expressers (log-rank, P < .003). Cox multivariate analysis showed high stathmin expression was independent of age, menopausal status, nodal status, nuclear grade, tumor size, and estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression (HR ¼ 1.19; 95% CI ¼ 1.03-1.37; P ¼ .01). The ratio of MAP-tau to stathmin expression showed a positive correlation to disease-free survival (HR ¼ 0.679; 95% CI ¼ 0.517-0.891; P ¼ .0053) with a 10-year survival of 65.4% for patients who had a high ratio of MAP-tau to stathmin versus 52.5% 10-year survival rate for those with a low ratio (log-rank, P ¼ .0009). Cox multivariate analysis showed the ratio of MAP-tau to stathmin was an independent predictor of overall survival (HR ¼ 0.609; 95% CI ¼ 0.422-0.879; P ¼ .008). CONCLUSIONS: Low stathmin and high MAP-tau are associated with increased microtubule stability and better prognosis in breast cancer. Cancer 2012;118:4660-9. V C 2012 American Cancer Society.

Introduction: Biomarkers, such as Estrogen Receptor, are used to determine therapy and prognosis in breast carcinoma. Immunostaining assays of biomarker expression have a high rate of inaccuracy; for example, estimates are as high as 20% for Estrogen Receptor. Biomarkers have been shown to be heterogeneously expressed in breast tumors and this heterogeneity may contribute to the inaccuracy of immunostaining assays. Currently, no evidencebased standards exist for the amount of tumor that must be sampled in order to correct for biomarker heterogeneity. The aim of this study was to determine the optimal number of 20X fields that are necessary to estimate a representative measurement of expression in a whole tissue section for selected biomarkers: ER, HER-2, AKT, ERK, S6K1, GAPDH, Cytokeratin, and MAP-Tau. Methods: Two collections of whole tissue sections of breast carcinoma were immunostained for biomarkers. Expression was quantified using the Automated Quantitative Analysis (AQUA) method of quantitative immunofluorescence. Simulated sampling of various numbers of fields (ranging from one to thirty five) was performed for each marker. The optimal number was selected for each marker via resampling techniques and minimization of prediction error over an independent test set. Results: The optimal number of 20X fields varied by biomarker, ranging between three to fourteen fields. More heterogeneous markers, such as MAP-Tau protein, required a larger sample of 20X fields to produce representative measurement. Conclusions: The optimal number of 20X fields that must be sampled to produce a representative measurement of biomarker expression varies by marker with more heterogeneous markers requiring a larger number. The clinical implication of these findings is that breast biopsies consisting of a small number of fields may be inadequate to represent whole tumor biomarker expression for many markers. Additionally, for biomarkers newly introduced into clinical use, especially if therapeutic response is dictated by level of expression, the optimal size of tissue sample must be determined on a marker-by-marker basis.

Antibodies are among the most frequently used tools in basic science research and in clinical assays, but there are no universally accepted guidelines or standardized methods for determining the validity of these reagents. Furthermore, for commercially available antibodies, it is clear that what is on the label does not necessarily correspond to what is in the tube. To validate an antibody, it must be shown to be specific, selective, and reproducible in the context for which it is to be used. In this review, we highlight the common pitfalls when working with antibodies, common practices for validating antibodies, and levels of commercial antibody validation for seven vendors. Finally, we share our algorithm for antibody validation for immunohistochemistry and quantitative immunofluorescence.

Taxanes are microtubule targeting agents (MTAs) and potent cytotoxic molecules recognized as highly effective chemotherapeutic agents. In large randomized clinical trials, taxane-based chemotherapies provided benefits in overall and disease-free survival, but they are accompanied by significant adverse effects. Thus the clinical utility of taxane therapy would be enhanced if there were companion diagnostic tests so that only the taxane-sensitive patients (about half of breast cancers) could be treated with this drug. Accumulating evidence indicates that microtubule associated proteins (MAPs) may be responsible for tumor cell resistance to taxanes. Our results indicate that MAP-tau functions as a prognostic factor in both the Yale cohort and the TAX 307 cohort with high MAP-tau expression associated with longer overall survival and TTP. Tau does NOT behave as a predictor of response to taxane-based chemotherapy since differences between low and high MAP-tau groups by treatment arm and response rate were not observed in the TAX 307 clinical trial cohort. Our data supports the use of MAP-tau as a prognostic marker, but does not support its use as a singular predictive factor for response to docetaxel. We have also evaluated the microtubule destabilizer, stathmin, and found high expression is associated with worse outcome. Since stathmin expression is useful in predicting survival, it may serve as marker to accurately select patients for current taxane-based or other anti-microtubule therapies in combination with a microtubule stabilizer such as MAP-tau.

We demonstrate an integrated approach to the study of a transcriptional regulatory cascade involved in the progression of breast cancer and we identify a protein associated with disease progression. Using chromatin immunoprecipitation and genome tiling arrays, whole genome mapping of transcription factor-binding sites was combined with gene expression profiling to identify genes involved in the proliferative response to estrogen (E2). Using RNA interference, selected ERalpha and c-MYC gene targets were knocked down to identify mediators of E2-stimulated cell proliferation. Tissue microarray screening revealed that high expression of an epigenetic factor, the E2-inducible histone variant H2A.Z, is significantly associated with lymph node metastasis and decreased breast cancer survival. Detection of H2A.Z levels independently increased the prognostic power of biomarkers currently in clinical use. This integrated approach has accelerated the identification of a molecule linked to breast cancer progression, has implications for diagnostic and therapeutic interventions, and can be applied to a wide range of cancers.

The authors have not changed the content of the original article, but would like to make the following corrections to the acknowledgments section of the published manuscript.

INTRODUCTION: Taxanes are microtubule targeting agents (MTAs) and potent cytotoxic molecules recognized as highly effective chemotherapeutic agents. In large randomized clinical trials, taxane-based chemotherapies provided benefits in overall and disease-free survival, but they are accompanied by significant adverse effects. Thus the clinical utility of taxane therapy would be enhanced if there were companion diagnostic tests so that only the taxane-sensitive patients (about half of breast cancers) could be treated with this drug. Accumulating evidence indicates that microtubule associated proteins (MAPs) may be responsible for tumor cell resistance to taxanes. Here we use a large retrospective cohort to measure expression levels of stathmin, a microtubule destabilizing protein, and to determine prognostic value, as a first step toward development of a companion diagnostic. METHODS: Stathmin expression was measured in a large retrospective breast cancer cohort (n= 645) with 20 year follow-up using tissue microarray technology (TMA) in two-fold redundancy and quantitative immunofluorescence (AQUA). Patient stathmin expression levels were correlated with clinical, pathological, and disease free survival variables. RESULTS: Stathmin expression exhibited nonparametric distribution with high correlation (R= 0.70) between redundant TMA cores. Prognostic evaluation of the cohort using univariate analysis indicated an inverse correlation between high stathmin expression and overall survival (HR = 1.30, 95% confidence interval [CI] = 1.02-1.65; p< 0.028). Kaplan Meier survival analysis showed 10-year survival of 54% for patients with high stathmin expression versus a 65% survival rate for low expressers (log rank, P CONCLUSIONS: Stathmin expression in breast tumor tissue functions as a prognostic factor where high expression is associated with worse outcome. Since stathmin expression is useful in predicting survival, it may serve as marker to accurately select patients for current taxane-based or other anti-microtubule therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1753.

Introduction Microtubule associated proteins (MAPs) endogenously regulate microtubule stabilization and have been reported as prognostic and predictive markers for taxane response. The microtubule stabilizer, MAP-tau, has shown conflicting results. We quantitatively assessed MAP-tau expression in two independent breast cancer cohorts to determine prognostic and predictive value of this biomarker. Methods MAP-tau expression was evaluated in the retrospective Yale University breast cancer cohort (n = 651) using tissue microarrays and also in the TAX 307 cohort, a clinical trial randomized for TAC versus FAC chemotherapy (n = 140), using conventional whole tissue sections. Expression was measured using the AQUA method for quantitative immunofluorescence. Scores were correlated with clinicopathologic variables, survival, and response to therapy. Results Assessment of the Yale cohort using Cox univariate analysis indicated an improved overall survival (OS) in tumors with a positive correlation between high MAP-tau expression and overall survival (OS) (HR = 0.691, 95% CI = 0.489-0.974; P = 0.004). Kaplan Meier analysis showed 10-year survival for 65% of patients with high MAP-tau expression compared to 52% with low expression (P = .006). In TAX 307, high expression was associated with significantly longer median time to tumor progression (TTP) regardless of treatment arm (33.0 versus 23.4 months, P = 0.010) with mean TTP of 31.2 months. Response rates did not differ by MAP-tau expression (P = 0.518) or by treatment arm (P = 0.584). Conclusions Quantitative measurement of MAP-tau expression has prognostic value in both cohorts, with high expression associated with longer TTP and OS. Differences by treatment arm or response rate in low versus high MAP-tau groups were not observed, indicating that MAP-tau is not associated with response to taxanes and is not a useful predictive marker for taxane-based chemotherapy.