The Effect of Black Cohosh on Ki67 expression and Tumor Volume: A Pilot Study of Ductal Carcinoma in Situ Patients.

BACKGROUND: Black cohosh (BC) (Cimicifuga racemosa) may prevent and treat breast cancer through anti-proliferative, pro-apoptotic, anti-estrogenic, and anti-inflammatory effects. This study sought to evaluate the effect of BC on tumor cellular proliferation, measured by Ki67 expression, in a pre-operative window trial of ductal carcinoma in situ (DCIS) patients. METHODS: Patients were treated pre-operatively for 2 to 6 weeks with BC extract. Eligible subjects were those who had DCIS on core biopsy. Ki67 was measured using automated quantitative immunofluorescence (AQUA) pre/post-operatively. Ki67, tumor volume, and hormone changes were assessed with 2-sided Wilcoxon signed-rank tests, α = .05. RESULTS: Thirty-one patients were treated for an average of 24.5 days (median 25; range 15-36). Ki67 decreased non-significantly (n = 26; P = .20; median pre-treatment 1280, post-treatment 859; range pre-treatment 175-7438, post-treatment 162-3370). Tumor volume, estradiol, and FSH did not change significantly. No grade 3 or 4 adverse events were reported. CONCLUSIONS: BC use showed no significant impact on cellular proliferation, tumor volume, or invasive disease upgrade rates in DCIS patients. It was well-tolerated, with no observed significant toxicities. Further study is needed to elucidate BC's role in breast cancer treatment and prevention.ClinicalTrials.gov Identifier: NCT01628536https://clinicaltrials.gov/ct2/show/NCT01628536.

Nuclear shape and orientation features from H&E images predict survival in early-stage estrogen receptor-positive breast cancers.

Early-stage estrogen receptor-positive (ER+) breast cancer (BCa) is the most common type of BCa in the United States. One critical question with these tumors is identifying which patients will receive added benefit from adjuvant chemotherapy. Nuclear pleomorphism (variance in nuclear shape and morphology) is an important constituent of breast grading schemes, and in ER+ cases, the grade is highly correlated with disease outcome. This study aimed to investigate whether quantitative computer-extracted image features of nuclear shape and orientation on digitized images of hematoxylin-stained and eosin-stained tissue of lymph node-negative (LN-), ER+ BCa could help stratify patients into discrete (<10 years short-term vs. >10 years long-term survival) outcome groups independent of standard clinical and pathological parameters. We considered a tissue microarray (TMA) cohort of 276 ER+, LN- patients comprising 150 patients with long-term and 126 patients with short-term overall survival, wherein 177 randomly chosen cases formed the modeling set, and 99 remaining cases the test set. Segmentation of individual nuclei was performed using multiresolution watershed; subsequently, 615 features relating to nuclear shape/texture and orientation disorder were extracted from each TMA spot. The Wilcoxon's rank-sum test identified the 15 most prognostic quantitative histomorphometric features within the modeling set. These features were then subsequently combined via a linear discriminant analysis classifier and evaluated on the test set to assign a probability of long-term vs. short-term disease-specific survival. In univariate survival analysis, patients identified by the image classifier as high risk had significantly poorer survival outcome: hazard ratio (95% confident interval) = 2.91(1.23-6.92), p = 0.02786. Multivariate analysis controlling for T-stage, histology grade, and nuclear grade showed the classifier to be independently predictive of poorer survival: hazard ratio (95% confident interval) = 3.17(0.33-30.46), p = 0.01039. Our results suggest that quantitative histomorphometric features of nuclear shape and orientation are strongly and independently predictive of patient survival in ER+, LN- BCa.

Copy Number Changes Are Associated with Response to Treatment with Carboplatin, Paclitaxel, and Sorafenib in Melanoma.

PURPOSE: Copy number alterations have been shown to be involved in melanoma pathogenesis. The randomized phase III clinical trial E2603: carboplatin, paclitaxel, ± sorafenib (CP vs. CPS) offers a large collection of tumor samples to evaluate association of somatic mutations, genomic alterations, and clinical outcomes, prior to current FDA-approved therapies. EXPERIMENTAL DESIGN: Copy number and mutational analysis on 119 pretreatment samples was performed. RESULTS: CPS therapy was associated with improved progression-free survival (PFS) compared with CP in patients with tumors with RAF1 (cRAF) gene copy gains (HR, 0.372; P = 0.025) or CCND1 gene copy gains (HR, 0.45; P = 0.035). CPS therapy was associated with improved overall survival (OS) compared with CP in patients with tumors with KRAS gene copy gains (HR, 0.25; P = 0.035). BRAF gene copy gain and MET amplification were more common in samples with V600K versus V600E mutations (P < 0.001), which was validated in The Cancer Genome Atlas (TCGA) dataset. CONCLUSIONS: We observed improved treatment response with CPS in patients with melanoma whose tumors have RAF1 (cRAF), KRAS, or CCND1 amplification, all of which can be attributed to sorafenib targeting CRAF. These genomic alterations should be incorporated in future studies for evaluation as biomarkers.

Development and clinical validation of an in situ biopsy-based multimarker assay for risk stratification in prostate cancer.

PURPOSE: Prostate cancer aggressiveness and appropriate therapy are routinely determined following biopsy sampling. Current clinical and pathologic parameters are insufficient for accurate risk prediction leading primarily to overtreatment and also missed opportunities for curative therapy. EXPERIMENTAL DESIGN: An 8-biomarker proteomic assay for intact tissue biopsies predictive of prostate pathology was defined in a study of 381 patient biopsies with matched prostatectomy specimens. A second blinded study of 276 cases validated this assay's ability to distinguish "favorable" versus "nonfavorable" pathology independently and relative to current risk classification systems National Comprehensive Cancer Network (NCCN and D'Amico). RESULTS: A favorable biomarker risk score of ≤0.33, and a nonfavorable risk score of >0.80 (possible range between 0 and 1) were defined on "false-negative" and "false-positive" rates of 10% and 5%, respectively. At a risk score ≤0.33, predictive values for favorable pathology in very low-risk and low-risk NCCN and low-risk D'Amico groups were 95%, 81.5%, and 87.2%, respectively, higher than for these current risk classification groups themselves (80.3%, 63.8%, and 70.6%, respectively). The predictive value for nonfavorable pathology was 76.9% at biomarker risk scores >0.8 across all risk groups. Increased biomarker risk scores correlated with decreased frequency of favorable cases across all risk groups. The validation study met its two coprimary endpoints, separating favorable from nonfavorable pathology (AUC, 0.68; P < 0.0001; OR, 20.9) and GS-6 versus non-GS-6 pathology (AUC, 0.65; P < 0.0001; OR, 12.95). CONCLUSIONS: The 8-biomarker assay provided individualized, independent prognostic information relative to current risk stratification systems, and may improve the precision of clinical decision making following prostate biopsy.

Correlation of somatic mutations and clinical outcome in melanoma patients treated with Carboplatin, Paclitaxel, and sorafenib.

PURPOSE: Sorafenib is an inhibitor of VEGF receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and RAF kinases, amongst others. We assessed the association of somatic mutations with clinicopathologic features and clinical outcomes in patients with metastatic melanoma treated on E2603, comparing treatment with carboplatin, paclitaxel ± sorafenib (CP vs. CPS) EXPERIMENTAL DESIGN: Pretreatment tumor samples from 179 unique individuals enrolled on E2603 were analyzed. Genotyping was performed using a custom iPlex panel interrogating 74 mutations in 13 genes. Statistical analysis was performed using Fisher exact test, logistic regression, and Cox proportional hazards models. Progression-free survival (PFS) and overall survival were estimated using Kaplan-Meier methods. RESULTS: BRAF and NRAS mutations were found at frequencies consistent with other metastatic melanoma cohorts. BRAF-mutant melanoma was associated with worse performance status, increased number of disease sites, and younger age at diagnosis. NRAS-mutant melanoma was associated with better performance status, fewer sites of disease, and female gender. BRAF and NRAS mutations were not significantly predictive of response or survival when treated with CPS versus CP. However, patients with NRAS-mutant melanoma trended toward a worse response and PFS on CP than those with BRAF-mutant or WT/WT melanoma, an association that was reversed for this group on the CPS arm. CONCLUSIONS: This study of somatic mutations in melanoma is the last prospectively collected phase III clinical trial population before the era of BRAF-targeted therapy. A trend toward improved clinical response in patients with NRAS-mutant melanoma treated with CPS was observed, possibly due to the effect of sorafenib on CRAF.

Expression of drug targets in patients treated with sorafenib, carboplatin and paclitaxel.

INTRODUCTION: Sorafenib, a multitarget kinase inhibitor, targets members of the mitogen-activated protein kinase (MAPK) pathway and VEGFR kinases. Here we assessed the association between expression of sorafenib targets and biomarkers of taxane sensitivity and response to therapy in pre-treatment tumors from patients enrolled in ECOG 2603, a phase III comparing sorafenib, carboplatin and paclitaxel (SCP) to carboplatin, paclitaxel and placebo (CP). METHODS: Using a method of automated quantitative analysis (AQUA) of in situ protein expression, we quantified expression of VEGF-R2, VEGF-R1, VEGF-R3, FGF-R1, PDGF-Rß, c-Kit, B-Raf, C-Raf, MEK1, ERK1/2, STMN1, MAP2, EB1 and Bcl-2 in pretreatment specimens from 263 patients. RESULTS: An association was found between high FGF-R1 and VEGF-R1 and increased progression-free survival (PFS) and overall survival (OS) in our combined cohort (SCP and CP arms). Expression of FGF-R1 and VEGF-R1 was higher in patients who responded to therapy ((CR+PR) vs. (SD+PD+ un-evaluable)). CONCLUSIONS: In light of the absence of treatment effect associated with sorafenib, the association found between FGF-R1 and VEGF-R1 expression and OS, PFS and response might reflect a predictive biomarker signature for carboplatin/paclitaxel-based therapy. Seeing that carboplatin and pacitaxel are now widely used for this disease, corroboration in another cohort might enable us to improve the therapeutic ratio of this regimen.

Loss of nuclear localized and tyrosine phosphorylated Stat5 in breast cancer predicts poor clinical outcome and increased risk of antiestrogen therapy failure.

PURPOSE: To investigate nuclear localized and tyrosine phosphorylated Stat5 (Nuc-pYStat5) as a marker of prognosis in node-negative breast cancer and as a predictor of response to antiestrogen therapy. PATIENTS AND METHODS: Levels of Nuc-pYStat5 were analyzed in five archival cohorts of breast cancer by traditional diaminobenzidine-chromogen immunostaining and pathologist scoring of whole tissue sections or by immunofluorescence and automated quantitative analysis (AQUA) of tissue microarrays. RESULTS: Nuc-pYStat5 was an independent prognostic marker as measured by cancer-specific survival (CSS) in patients with node-negative breast cancer who did not receive systemic adjuvant therapy, when adjusted for common pathology parameters in multivariate analyses both by standard chromogen detection with pathologist scoring of whole tissue sections (cohort I; n = 233) and quantitative immunofluorescence of a tissue microarray (cohort II; n = 291). Two distinct monoclonal antibodies gave concordant results. A progression array (cohort III; n = 180) revealed frequent loss of Nuc-pYStat5 in invasive carcinoma compared to normal breast epithelia or ductal carcinoma in situ, and general loss of Nuc-pYStat5 in lymph node metastases. In cohort IV (n = 221), loss of Nuc-pYStat5 was associated with increased risk of antiestrogen therapy failure as measured by univariate CSS and time to recurrence (TTR). More sensitive AQUA quantification of Nuc-pYStat5 in antiestrogen-treated patients (cohort V; n = 97) identified by multivariate analysis patients with low Nuc-pYStat5 at elevated risk for therapy failure (CSS hazard ratio [HR], 21.55; 95% CI, 5.61 to 82.77; P < .001; TTR HR, 7.30; 95% CI, 2.34 to 22.78; P = .001). CONCLUSION Nuc-pYStat5 is an independent prognostic marker in node-negative breast cancer. If confirmed in prospective studies, Nuc-pYStat5 may become a useful predictive marker of response to adjuvant hormone therapy.

Vertical targeting of the phosphatidylinositol-3 kinase pathway as a strategy for treating melanoma.

PURPOSE: Melanoma is relatively resistant to chemotherapy; improved targeting of molecules critical for cell proliferation and survival are needed. Phosphatidylinositol-3 kinase (PI3K) is an important target in melanoma; however, activity of PI3K inhibitors (PI3KI) is limited. Our purpose was to assess mTOR as a cotarget for PI3K. METHODS: Using a method of quantitative immunofluorescence to measure mTOR expression in a large melanoma cohort, we studied associations with PI3K subunits, p85 and p110α. We assessed addition of the mTOR inhibitor rapamycin to 2 PI3KIs, NVP-BKM120 and LY294002. We studied in vitro activity of a novel dual PI3K/mTOR inhibitor NVP-BEZ235 and activity of the combination of NVP-BEZ235 and the MAP/ERK kinase (MEK) inhibitor AZD6244. RESULTS: Strong coexpression of mTOR and p110α was observed (ρ = 0.658; P < 0.0001). Less coexpression was seen with p85 (ρ = 0.239; P < 0.0001). Strong synergism was shown between rapamycin and both PI3KIs. Activity of both PI3KIs was similarly enhanced with all rapamycin concentrations used. The dual PI3K/mTOR inhibitor effectively inhibited viability in 23 melanoma cell lines (IC(50) values in the nanomolar range), regardless of B-Raf mutation status, with resultant reduction in clonogenicity and downregulation of pAkt and pP70S6K. Synergism was seen when combining NVP-BEZ235 and AZD6244, with resultant increases in poly(ADP-ribose) polymerase and caspase-2 cleavage. CONCLUSIONS: mTOR and p110α are coexpressed in melanoma. Rapamycin concentrations as low as 1 nmol/L enhance activity of PI3KIs. The dual PI3K/mTOR inhibitor NVP-BEZ235 is highly active in melanoma cells in vitro, suggesting that concurrent PI3K and mTOR targeting in melanoma warrants further investigation, both alone and in combination with MEK inhibitors.

Expression of sorafenib targets in melanoma patients treated with carboplatin, paclitaxel and sorafenib.

BACKGROUND: Sorafenib, a multitarget kinase inhibitor, inhibits members of the mitogen-activated protein kinase (MAPK) pathway and receptor tyrosine kinases, including vascular endothelial growth factor receptor 2 (VEGF-R2). Sorafenib, carboplatin, and paclitaxel (SCP) has antitumor activity in melanoma patients, but no association was found between response and activating B-Raf V600E mutations. We assessed the expression of sorafenib targets in SCP-treated patient specimens and evaluated the association with response and progression-free survival. EXPERIMENTAL DESIGN: Using automated quantitative analysis, we quantified the expression of VEGF-R1, VEGF-R2, VEGF-R3, fibroblast growth factor receptor 1, platelet-derived growth factor receptor beta, c-Kit, B-Raf, C-Raf, meiosis-specific serine/threonine protein kinase 1, and extracellular regulated kinase 1/2 (ERK1/2) in pretreatment specimens from 46 patients. Furthermore, we assessed ERK1/2 expression in 429 archival melanomas. RESULTS: VEGF-R2 expression was significantly higher in patients with a complete or partial response (P = 0.0435), whereas ERK1/2 was higher in patients who did not respond (P = 0.0417). High ERK1/2 was an independent predictor of poor survival. High ERK1/2 was associated with decreased survival in the archival melanoma cohort, suggesting that high ERK1/2-expressing tumors are biologically more aggressive. All of the six patients with both high VEGF-R2 and low ERK1/2 responded to SCP. CONCLUSIONS: High VEGF-R2 expression is associated with response to SCP in melanoma, whereas high ERK1/2 is associated with resistance. Collection of specimens from SCP-treated melanoma patients in a cooperative group phase III trial comparing this regimen with the chemotherapy alone is ongoing, and confirmation of these findings is necessary. These markers might be useful for predicting response to sorafenib when given with other chemotherapies and in other diseases, resulting in the possible elimination of unnecessary treatment of patients unlikely to respond.

Prognostic significance of cadherin-based adhesion molecules in cutaneous malignant melanoma.

BACKGROUND: The need for novel molecular prognostic markers that can supplement validated clinicopathologic correlates for cutaneous malignant melanoma is well recognized. Proteins that mediate the epithelial-mesenchymal transition, the process by which a cancer cell disengages from its parent tumor, are important candidates. METHODS: The prognostic relevance of E-cadherin, N-cadherin, and P-cadherin, calcium-dependent transmembrane glycoproteins that regulate cell-cell adhesion, and their adaptors, alpha-catenin, beta-catenin, and p120-catenin, was evaluated on a cohort of 201 primary and 274 metastatic melanoma tumors using fluorescence-based immunohistochemical methods and Automated Quantitative Analysis of protein expression on digitally captured photomicrographs. RESULTS: Increasing levels of N-cadherin expression improved overall survival (log-rank = 7.31; P = 0.03) but did not retain significance following adjustment for established clinicopathologic correlates (P = 0.50). Higher levels of E-cadherin approached significance for favorable prognosis on both univariate (P = 0.13) and multivariable (P = 0.10) analyses. Hierarchical clustering of the composite profiles for all six markers identified four unique clusters that yielded differential overall survival (log-rank = 10.54; P = 0.01). Cluster 4, expressing high E-cadherin and N-cadherin levels, possessed the most favorable outcome and cluster 2, featuring low E-cadherin and alpha-catenin but modest N-cadherin, showed least favorable outcomes. Cluster 2 remained significant on multivariable analysis (hazard ratio, 3.29; 95% confidence interval, 1.50-7.19; P = 0.003). CONCLUSIONS: Although none of the cadherin-based adhesion molecules were independently prognostic, multimarker profiles were significant. Similar to epithelial-derived tumors, loss of E-cadherin correlates with poor outcome. In contrast, for neural crest-derived cutaneous malignant melanoma, N-cadherin overexpression can be associated with either a successful epithelial-mesenchymal transition or a favorably differentiated tumor. Additional cadherin profiles are needed to discriminate these distinctive phenotypes.