Evidence for biological effects of metformin in operable breast cancer: a pre-operative, window-of-opportunity, randomized trial.

Metformin may reduce the incidence of breast cancer and enhance response to neoadjuvant chemotherapy in diabetic women. This trial examined the effects of metformin on Ki67 and gene expression in primary breast cancer. Non-diabetic women with operable invasive breast cancer received pre-operative metformin. A pilot cohort of eight patients had core biopsy of the cancer at presentation, a week later (without treatment; internal control), then following metformin 500-mg o.d. for 1 week increased to 1-g b.d. for a further week continued to surgery. A further 47 patients had core biopsy at diagnosis were randomized to metformin (the same dose regimen) or no drug, and 2 weeks later had core biopsy at surgery. Ki67 immunohistochemistry, transcriptome analysis on formalin-fixed paraffin-embedded cores and serum insulin determination were performed blinded to treatment. Seven patients (7/32, 21.9%) receiving metformin withdrew because of gastrointestinal upset. The mean percentage of cells staining for Ki67 fell significantly following metformin treatment in both the pilot cohort (P = 0.041, paired t-test) and in the metformin arm (P = 0.027, Wilcoxon rank test) but was unchanged in the internal control or metformin control arms. Messenger RNA expression was significantly downregulated by metformin for PDE3B (phosphodiesterase 3B, cGMP-inhibited; a critical regulator of cAMP levels that affect activation of AMP-activated protein kinase, AMPK), confirmed by immunohistochemistry, SSR3, TP53 and CCDC14. By ingenuity pathway analysis, the tumour necrosis factor receptor 1 (TNFR1) signaling pathway was most affected by metformin: TGFB and MEKK were upregulated and cdc42 downregulated; mTOR and AMPK pathways were also affected. Gene set analysis additionally revealed that p53, BRCA1 and cell cycle pathways also had reduced expression following metformin. Mean serum insulin remained stable in patients receiving metformin but rose in control patients. This trial presents biomarker evidence for anti-proliferative effects of metformin in women with breast cancer and provides support for therapeutic trials of metformin.

Identification and validation of an anthracycline/cyclophosphamide-based chemotherapy response assay in breast cancer.

BACKGROUND: There is no method routinely used to predict response to anthracycline and cyclophosphamide-based chemotherapy in the clinic; therefore patients often receive treatment for breast cancer with no benefit. Loss of the Fanconi anemia/BRCA (FA/BRCA) DNA damage response (DDR) pathway occurs in approximately 25% of breast cancer patients through several mechanisms and results in sensitization to DNA-damaging agents. The aim of this study was to develop an assay to detect DDR-deficient tumors associated with loss of the FA/BRCA pathway, for the purpose of treatment selection. METHODS: DNA microarray data from 21 FA patients and 11 control subjects were analyzed to identify genetic processes associated with a deficiency in DDR. Unsupervised hierarchical clustering was then performed using 60 BRCA1/2 mutant and 47 sporadic tumor samples, and a molecular subgroup was identified that was defined by the molecular processes represented within FA patients. A 44-gene microarray-based assay (the DDR deficiency assay) was developed to prospectively identify this subgroup from formalin-fixed, paraffin-embedded samples. All statistical tests were two-sided. RESULTS: In a publicly available independent cohort of 203 patients, the assay predicted complete pathologic response vs residual disease after neoadjuvant DNA-damaging chemotherapy (5-fluorouracil, anthracycline, and cyclophosphamide) with an odds ratio of 3.96 (95% confidence interval [Cl] =1.67 to 9.41; P = .002). In a new independent cohort of 191 breast cancer patients treated with adjuvant 5-fluorouracil, epirubicin, and cyclophosphamide, a positive assay result predicted 5-year relapse-free survival with a hazard ratio of 0.37 (95% Cl = 0.15 to 0.88; P = .03) compared with the assay negative population. CONCLUSIONS: A formalin-fixed, paraffin-embedded tissue-based assay has been developed and independently validated as a predictor of response and prognosis after anthracycline/cyclophosphamide-based chemotherapy in the neoadjuvant and adjuvant settings. These findings warrant further validation in a prospective clinical study.

A Predictive Genetic Signature for Response to Fluoropyrimidine-Based Neoadjuvant Chemoradiation in Clinical Stage II and III Rectal Cancer.

PURPOSE: Pre-operative chemoradiation (CRT) is currently the standard of care for patients with clinical stage II and III rectal cancer but only about 45% of patients achieve tumor downstaging and <20% of patients achieve a pathologic complete response. Better methods to stratify patients according to potential neoadjuvant treatment response are needed. We used microarray analysis to identify a genetic signature that correlates with a pathological complete response (pCR) to neoadjuvant CRT. We performed a gene network analysis to identify potential signaling pathways involved in determining response to neoadjuvant treatment. PATIENTS AND METHODS: We identified 31 T3-4 N0-1 rectal cancer patients who were treated with neoadjuvant fluorouracil-based CRT. Eight patients were identified to have achieved a pCR to treatment while 23 patients did not. mRNA expression was analyzed using cDNA microarrays. The correlation between mRNA expression and pCR from pre-treatment tumor biopsies was determined. Gene network analysis was performed for the genes represented by the predictive signature. RESULTS: A genetic signature represented by expression levels of the three genes EHBP1, STAT1, and GAPDH was found to correlate with a pCR to neoadjuvant treatment. The difference in expression levels between patients who achieved a pCR and those who did not was greatest for EHBP1. Gene network analysis showed that the three genes can be connected by the gene ubiquitin C (UBC). CONCLUSION: This study identifies a 3-gene signature expressed in pre-treatment tumor biopsies that correlates with a pCR to neoadjuvant CRT in patients with clinical stage II and III rectal cancer. These three genes can be connected by the gene UBC, suggesting that ubiquitination is a molecular mechanism involved in determining response to treatment. Validating this genetic signature in a larger number of patients is proposed.

The micro RNA target paradigm: a fundamental and polymorphic control layer of cellular expression.

Evidence is emerging that micro RNA (miRNA) is an important and potentially polymorphic regulatory layer for silencing gene expression in vivo. Knowledge of miRNA targeting may help to elucidate the function of many human genes in common diseases, providing a powerful target validation technology. Accurate in silico prediction of miRNA targets in mRNA is a critical capability, allowing effective evaluation of the impact of variation on the creation, strengthening, weakening and destruction of miRNA binding sites. Application of such analyses identifies thousands of single-nucleotide polymorphisms, which may potentially impact miRNA regulation of mRNA. The authors believe this information may offer a real opportunity to study miRNA function at a number of levels. First, sequence-focused analysis will help to define the functional boundaries of miRNA target binding. Second, one may be able to identify miRNA target variants in mRNA with a direct role in human disease, which may be valuable therapeutic targets.