Metformin Promotes Antitumor Immunity via Endoplasmic-Reticulum-Associated Degradation of PD-L1.

Metformin has been reported to possess antitumor activity and maintain high cytotoxic T lymphocyte (CTL) immune surveillance. However, the functions and detailed mechanisms of metformin's role in cancer immunity are not fully understood. Here, we show that metformin increases CTL activity by reducing the stability and membrane localization of programmed death ligand-1 (PD-L1). Furthermore, we discover that AMP-activated protein kinase (AMPK) activated by metformin directly phosphorylates S195 of PD-L1. S195 phosphorylation induces abnormal PD-L1 glycosylation, resulting in its ER accumulation and ER-associated protein degradation (ERAD). Consistently, tumor tissues from metformin-treated breast cancer patients exhibit reduced PD-L1 levels with AMPK activation. Blocking the inhibitory signal of PD-L1 by metformin enhances CTL activity against cancer cells. Our findings identify a new regulatory mechanism of PD-L1 expression through the ERAD pathway and suggest that the metformin-CTLA4 blockade combination has the potential to increase the efficacy of immunotherapy.

A prospective comparison of ER, PR, Ki67 and gene expression in paired sequential core biopsies of primary, untreated breast cancer.

BACKGROUND: Sequential biopsy of breast cancer is used to assess biomarker effects and drug efficacy. The preoperative "window of opportunity" setting is advantageous to test biomarker changes in response to therapeutic agents in previously untreated primary cancers. This study tested the consistency over time of paired, sequential biomarker measurements on primary, operable breast cancer in the absence of drug therapy. METHODS: Immunohistochemistry was performed for ER, PR and Ki67 on paired preoperative/operative tumor samples taken from untreated patients within 2 weeks of each other. Microarray analysis on mRNA extracted from formalin fixed paraffin embedded cores was performed using Affymetrix based arrays on paired core biopsies analysed using Ingenuity Pathway Analysis (IPA) and Gene Set Analysis (GSA). RESULTS: In 41 core/resection pairs, the recognised trend to lower ER, PR and Ki67 score on resected material was confirmed. Concordance for ER, PR and Ki67 without changing biomarker status (e.g. ER+ to ER-) was 90, 74 and 80 % respectively. However, in 23 paired core samples (diagnostic core v on table core), Ki67 using a cut off of 13.25 % was concordant in 22/23 (96 %) and differences in ER and PR immunohistochemistry by Allred or Quickscore between the pairs did not impact hormone receptor status. IPA and GSA demonstrated substantial gene expression changes between paired cores at the mRNA level, including reduced expression of ER pathway analysis on the second core, despite the absence of drug intervention. CONCLUSIONS: Sequential core biopsies of primary breast cancer (but not core versus resection) was consistent and is appropriate to assess the effects of drug therapy in vivo on ER, PR and Ki67 using immunohistochemistry. Conversely, studies utilising mRNA expression may require non-treatment controls to distinguish therapeutic from biopsy differences.

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

Metformin has therapeutic potential against breast cancer, but the mechanisms of action in vivo remain uncertain. This study examined biomarker effects of metformin in primary breast cancer in a preoperative window of opportunity trial. Non-diabetic women with operable invasive breast cancer were randomized to receive open label pre-operative metformin (500 mg daily for 1 week then 1 g twice daily for a further week) or as controls, not receiving metformin. Patients in both arms had a core biopsy pre-randomisation and again at the time of surgery. Immunohistochemistry for phospho-AMPK (pAMPK), phospho-Akt (pAkt), insulin receptor, cleaved caspase-3, and Ki67 was performed on formalin-fixed paraffin-embedded cores, scored blinded to treatment and analysed by paired t test. In metformin-treated patients, significant up-regulation of pAMPK (paired t test, p = 0.04) and down-regulation of pAkt (paired t test, p = 0.043) were demonstrated compared to the control group. Insulin receptor and serum insulin remained similar following metformin treatment compared with a rise in insulin receptor and insulin in controls. Significant falls in Ki67 and cleaved caspase-3 (paired t test, p = 0.044) were seen in the metformin-treated patients but not in the control group. Changes were independent of body mass index. These biomarker data suggest mechanisms for metformin action in vivo in breast cancer patients via up-regulation of tumor pAMPK, down-regulation of pAkt, and suppression of insulin responses reflecting cytostatic rather than cytotoxic mechanisms.

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.

Determination of Chemical Irritation Potential Using a Defined Gene Signature Set on Tissue-Engineered Human Skin Equivalents.

There are no physical or visual manifestations that define skin sensitivity or irritation; a subjective diagnosis is made on the basis of the evaluation of clinical presentations, including burning, prickling, erythema, and itching. Adverse skin reaction in response to topically applied products is common and can limit the use of dermatological or cosmetic products. The purpose of this study was to evaluate the use of human skin equivalents based on immortalized skin keratinocytes and evaluate the potential of a 22-gene panel in combination with multivariate analysis to discriminate between chemicals known to act as irritants and those that do not. Test compounds were applied topically to full-thickness human skin equivalent or human ex vivo skin and gene signatures determined for known irritants and nonirritants. Principle component analysis showed the discriminatory potential of the 22-gene panel. Linear discrimination analysis, performed to further refine the gene set for a more high-throughput analysis, identified a putative seven-gene panel (IL-6, PTGS2, ATF3, TRPV3, MAP3K8, HMGB2, and matrix metalloproteinase gene MMP-3) that could distinguish potential irritants from nonirritants. These data offer promise as an in vitro prediction tool, although analysis of a large chemical test set is required to further evaluate the system.

Myiasis.

Late presentation of breast cancer is more likely to be complicated and fatal. Local invasion, tissue destruction, skin lose, and superadded infection/infestation make surgical intervention very challenging.

Predicting Patients Found to Have Malignancy at Nipple Duct Surgery.

INTRODUCTION: The unexpected diagnosis of breast cancer following total duct excision is distressing for patients. Despite advances in radiology and the description of suspicious nipple discharge, pre-operative diagnosis of malignancy still evades us. The aim of this study was to review the pathological findings of total duct excision and microdochectomy with reference to pre-operative symptoms, ultrasound, or mammographic findings and identify features associated with increased likelihood of malignant disease. METHODS: Data were collected retrospectively of all patients who underwent total duct excision surgery in a single centre (2011-2017). Pre-operative demographics, symptoms, and imaging findings were recorded and correlated with subsequent pathology. RESULTS: 214 patients underwent total duct excision; data were available for 211. Median age was 53 years. 175/211 (82.9%) patients had benign pathology (duct ectasia, papilloma without atypia, fibrocystic change) on final histological examination, 21/211 (10%) had "risk" lesions (papilloma with atypia, atypical ductal hyperplasia), and 15/211 (7.1%) had malignancy (ductal carcinoma in situ). Of the 15 patients with malignant lesions, 6/15 (40%) had normal imaging (M1, U1). 71/211 (33.6%) had normal imaging (M1, U1): 60/71 (84.5%) had benign disease, 5/71 (7%) had "risk" lesions, and 6/71 (8.5%) had malignant lesions. 83/211 (39.3%) patients presented with bloody discharge: 64/83 (77.1%) had benign pathology, 9/83 (10.8%) risk, and 10/83 (12%) malignancy. 38/211 (18%) patients presented with non-bloody discharge: 32/38 (84.2%) had benign disease, 4/38 (10.5%) risk, and 2/38 (5.3%) malignant lesions. CONCLUSION: Neither imaging nor presenting symptoms correlate with the likelihood of malignant disease being present at final pathology. Even with advances in pre-operative diagnosis, total duct excision remains an essential diagnostic and therapeutic procedure.

Histological evaluation of AMPK signalling in primary breast cancer.

BACKGROUND: AMP-activated protein kinase (AMPK) acts as a cellular fuel gauge that responds to energy stress by suppressing cell growth and biosynthetic processes, thus ensuring that energy-consuming processes proceed only if there are sufficient metabolic resources. Malfunction of the AMPK pathway may allow cancer cells to undergo uncontrolled proliferation irrespective of their molecular energy levels. The aim of this study was to examine the state of AMPK phosphorylation histologically in primary breast cancer in relation to clinical and pathological parameters. METHODS: Immunohistochemistry was performed using antibodies to phospho-AMPK (pAMPK), phospho-Acetyl Co-A Carboxylase (pACC) an established target for AMPK, HER2, ERalpha, and Ki67 on Tissue Micro-Array (TMA) slides of two cohorts of 117 and 237 primary breast cancers. The quick score method was used for scoring and patterns of protein expression were compared with clinical and pathological data, including a minimum 5 years follow up. RESULTS: Reduced signal, compared with the strong expression in normal breast epithelium, using a pAMPK antibody was demonstrated in 101/113 (89.4%) and 217/236 (91.9%) of two cohorts of patients. pACC was significantly associated with pAMPK expression (p = 0.007 & p = 0.014 respectively). For both cohorts, reduced pAMPK signal was significantly associated with higher histological grade (p = 0.010 & p = 0.021 respectively) and axillary node metastasis (p = 0.061 & p = 0.039 respectively). No significant association was found between pAMPK and any of HER2, ERalpha, or Ki67 expression, disease-free survival or overall survival. CONCLUSION: This study extends in vitro evidence through immunohistochemistry to confirm that AMPK is dysfunctional in primary breast cancer. Reduced signalling via the AMPK pathway, and the inverse relationship with histological grade and axillary node metastasis, suggests that AMPK re-activation could have therapeutic potential in breast cancer.

A candidate molecular signature associated with tamoxifen failure in primary breast cancer.

INTRODUCTION: Few markers are available that can predict response to tamoxifen treatment in estrogen receptor (ER)-positive breast cancers. Identification of such markers would be clinically useful. We attempted to identify molecular markers associated with tamoxifen failure in breast cancer. METHODS: Eighteen initially ER-positive patients treated with tamoxifen requiring salvage surgery (tamoxifen failure [TF] patients) were compared with 17 patients who were disease free 5 years after surgery plus tamoxifen adjuvant therapy (control patients). cDNA microarray, real-time quantitative PCR, and immunohistochemistry on tissue microarrays were used to generate and confirm a gene signature associated with tamoxifen failure. An independent series of 33 breast tumor samples from patients who relapsed (n = 14) or did not relapse (n = 19) under tamoxifen treatment from a different geographic location was subsequently used to explore the gene expression signature identified. RESULTS: Using a screening set of 18 tumor samples (from eight control patients and 10 TF patients), a 47-gene signature discriminating between TF and control samples was identified using cDNA arrays. In addition to ESR1/ERalpha, the top-ranked genes selected by statistical cross-analyses were MET, FOS, SNCG, IGFBP4, and BCL2, which were subsequently validated in a larger set of tumor samples (from 17 control patients and 18 TF patients). Confirmation at the protein level by tissue microarray immunohistochemistry was observed for ER-alpha, gamma-synuclein, and insulin-like growth factor binding protein 4 proteins in the 35 original samples. In an independent series of breast tumor samples (19 nonrelapsing and 14 relapsing), reduced expression of ESR1/ERalpha, IGFBP4, SNCG, BCL2, and FOS was observed in the relapsing group and was associated with a shorter overall survival. Low mRNA expression levels of ESR1/ERalpha, BCL2, and FOS were also associated with a shorter relapse-free survival (RFS). Using a Cox multivariate regression analysis, we identified BCL2 and FOS as independent prognostic markers associated with RFS. Finally, the BCL2/FOS signature was demonstrated to have more accurate prognostic value for RFS than ESR1/ERalpha alone (likelihood ratio test). CONCLUSIONS: We identified molecular markers including a BCL2/FOS signature associated with tamoxifen failure; these markers may have clinical potential in the management of ER-positive breast cancer.

Targeting AMPK: a new therapeutic opportunity in breast cancer.

BACKGROUND: This paper reviews the mammalian Target Of Rapamycin (mTOR) pathway dysregulation in breast cancer, and the current evidence targeting this pathway directly or through activation of AMP-activated protein kinase (AMPK) as an additional therapeutic opportunity for intervention in breast cancer. METHODS: Relevant articles were identified through computerised searches of Medline and Pubmed. Secondary articles were identified from the reference lists of key papers and by hand searching. RESULTS AND CONCLUSION: The current consensus to target the AMPK/mTOR pathway in breast cancer is based on in vitro and epidemiological evidences. A low incidence of cancer in diabetic patients on metformin has been explained in vitro by the drug's anti-proliferative effect through activation of AMPK. There is a need to explore the anticancer effects of metformin and the potential to develop the therapeutic avenues offered by targeting the AMPK/mTOR pathway.

Effects of prolonged exposure to low dose metformin in thyroid cancer cell lines.

Background: Thyroid cancer is generally associated with an excellent prognosis, but there is significant long-term morbidity with standard treatment. Some sub-types however have a poor prognosis. Metformin, an oral anti-diabetic drug is shown to have anti-cancer effects in several types of cancer (breast, lung and ovarian cancer). The proposed mechanisms include activation of the Adenosine Mono-phosphate-activated Protein Kinase (AMPK) pathway and inhibition of the mTOR pathway (which promotes growth and proliferation). By inhibiting hepatic gluconeogenesis and increasing glucose uptake by muscles, metformin decreases blood glucose and circulating Insulin levels. Aims: Explore the effect of metformin on the growth and proliferation of thyroid cancer cell lines. Methods: The effects of metformin on thyroid cancer cell lines (FTC-133, K1E7, RO82-W-1, 8305C and TT) and normal thyroid follicular cells (Nthy-ori 3-1) were investigated using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay for cell proliferation; clonogenic assays; FACS analysis for apoptosis and cell cycle, H2A.X phosphorylation (γH2AX) assay for DNA repair and scratch assay for cell migration. Results: Metformin inhibited cell proliferation and colony formation at 0.03 mM and above and inhibited cell migration at 0.3 mM. At concentrations of 0.1 mM and above metformin increased the percentage of apoptotic cells and induced cell cycle arrest in G0/G1 phase at minimum concentration of 0.3 mM. Unlike previous reports, no effect on DNA repair response was demonstrated. Conclusion: Metformin suppressed growth of all thyroid cancer cell lines, at concentrations considered to be within in the therapeutic range for diabetic patients on metformin (<0.3 mM).

Phase II Randomized Preoperative Window-of-Opportunity Study of the PI3K Inhibitor Pictilisib Plus Anastrozole Compared With Anastrozole Alone in Patients With Estrogen Receptor-Positive Breast Cancer.

PURPOSE: Preclinical data support a key role for the PI3K pathway in estrogen receptor-positive breast cancer and suggest that combining PI3K inhibitors with endocrine therapy may overcome resistance. This preoperative window study assessed whether adding the PI3K inhibitor pictilisib (GDC-0941) can increase the antitumor effects of anastrozole in primary breast cancer and aimed to identify the most appropriate patient population for combination therapy. PATIENTS AND METHODS: In this randomized, open-label phase II trial, postmenopausal women with newly diagnosed operable estrogen receptor-positive, human epidermal growth factor receptor 2 (HER2)-negative breast cancers were recruited. Participants were randomly allocated (2:1, favoring the combination) to 2 weeks of preoperative treatment with anastrozole 1 mg once per day (n = 26) or the combination of anastrozole 1 mg with pictilisib 260 mg once per day (n = 49). The primary end point was inhibition of tumor cell proliferation as measured by change in Ki-67 protein expression between tumor samples taken before and at the end of treatment. RESULTS: There was significantly greater geometric mean Ki-67 suppression of 83.8% (one-sided 95% CI, ≥ 79.0%) for the combination and 66.0% (95% CI, ≤ 75.4%) for anastrozole (geometric mean ratio [combination:anastrozole], 0.48; 95% CI, ≤ 0.72; P = .004). PIK3CA mutations were not predictive of response to pictilisib, but there was significant interaction between response to treatment and molecular subtype (P = .03); for patients with luminal B tumors, the combination:anastrozole geometric mean ratio of Ki-67 suppression was 0.37 (95% CI, ≤ 0.67; P = .008), whereas no significant Ki-67 response was observed for pictilisib in luminal A tumors (1.01; P = .98). Multivariable analysis confirmed Ki-67 response to the combination treatment of patients with luminal B tumors irrespective of progesterone receptor status or baseline Ki-67 expression. CONCLUSION: Adding pictilisib to anastrozole significantly increases suppression of tumor cell proliferation in luminal B primary breast cancer.

Delay from symptom onset increases the conversion rate in laparoscopic cholecystectomy for acute cholecystitis.

BACKGROUND: Randomized trials suggest that laparoscopic cholecystectomy should be performed on first admission for acute cholecystitis. However, this is not widely practiced, possibly because of a perceived high conversion rate. We hypothesized that delay from onset of symptoms may increase the conversion rate. METHODS: We performed a retrospective case note review of patients undergoing emergency cholecystectomy in a single institution between January 2002 and December 2005. We analyzed whether delay from onset of symptoms was related to the conversion rate in patients with a histopathological diagnosis of acute cholecystitis. RESULTS: Of patients who underwent emergency laparoscopic cholecystectomy in our institution, 32.4% (197/608) had acute cholecystitis on histopathology. The conversion rate of those with acute cholecystitis was considerably higher (24.4%) than for those with other pathologies (6.3%). For patients with acute cholecystitis, the conversion rates increased with duration of symptoms: 9.5%, 16.1%, 38.9%, and 38.6% for delays of 0-2 days, 3-4 days, 5-6 days, and > 6 days from symptom onset, respectively (chi-square for trend = 14.27, DF = 1, p = 0.00016). Most conversions were due to the presence of acute inflammatory adhesions. CONCLUSIONS: Early intervention for acute cholecystitis (preferably within 2 days of onset of symptoms) is most likely to result in successful laparoscopic cholecystectomy; increasing delay is associated with conversion to open surgery.