Highly Sensitive Microsatellite Instability and Immunohistochemistry Assessment in Endometrial Aspirates as a Tool for Cancer Risk Individualization in Lynch Syndrome.

Women with Lynch syndrome (LS) are at increased risk of endometrial cancer (EC), among other tumors, and are characterized by mismatch repair (MMR) deficiency and microsatellite instability (MSI). While risk-reducing gynecologic surgeries effectively decrease EC incidence, doubts arise regarding the appropriate timing of the surgery. We explored the usefulness of highly sensitive MSI (hs-MSI) assessment in endometrial aspirates for individualizing gynecologic surveillance in LS carriers. Ninety-three women with LS, 25 sporadic EC patients (9 MMR-proficient and 16 MMR-deficient), and 30 women with benign gynecologic disease were included in this study. hs-MSI was assessed in prospectively collected endometrial aspirates in 67 LS carriers, EC cases, and controls. MMR, PTEN, ARID1A, and PAX2 protein expression patterns were evaluated in the LS samples. Follow-up aspirates from 8 LS carriers were also analyzed. Elevated hs-MSI scores were detected in all aspirates from MMR-deficient EC cases (3 LS and 16 sporadic) and negative in aspirates from controls and MMR-proficient EC cases. Positive hs-MSI scores were also detected in all 4 LS aspirates reported as complex hyperplasia. High hs-MSI was also present in 10 of 49 aspirates (20%) from LS carriers presenting a morphologically normal endometrium, where MMR protein expression loss was detected in 69% of the samples. Interestingly, the hs-MSI score was positively correlated with MMR-deficient gland density and the presence of MMR-deficient clusters, colocalizing PTEN and ARID1A expression loss. High hs-MSI scores and clonality were evidenced in 2 samples collected up to 4 months before EC diagnosis; hs-MSI scores increased over time in 5 LS carriers, whereas they decreased in a patient with endometrial hyperplasia after progestin therapy. In LS carriers, elevated hs-MSI scores were detected in aspirates from premalignant and malignant lesions and normal endometrium, correlating with MMR protein loss. hs-MSI assessment and MMR immunohistochemistry may help individualize EC risk assessment in women with LS.

Silibinin is a suppressor of the metastasis-promoting transcription factor ID3.

BACKGROUND: ID3 (inhibitor of DNA binding/differentiation-3) is a transcription factor that enables metastasis by promoting stem cell-like properties in endothelial and tumor cells. The milk thistle flavonolignan silibinin is a phytochemical with anti-metastatic potential through largely unknown mechanisms. HYPOTHESIS/PURPOSE: We have mechanistically investigated the ability of silibinin to inhibit the aberrant activation of ID3 in brain endothelium and non-small cell lung cancer (NSCLC) models. METHODS: Bioinformatic analyses were performed to investigate the co-expression correlation between ID3 and bone morphogenic protein (BMP) ligands/BMP receptors (BMPRs) genes in NSCLC patient datasets. ID3 expression was assessed by immunoblotting and qRT-PCR. Luciferase reporter assays were used to evaluate the gene sequences targeted by silibinin to regulate ID3 transcription. In silico computational modeling and LanthaScreen TR-FRET kinase assays were used to characterize and validate the BMPR inhibitory activity of silibinin. Tumor tissues from NSCLC xenograft models treated with oral silibinin were used to evaluate the in vivo anti-ID3 effects of silibinin. RESULTS: Analysis of lung cancer patient datasets revealed a top-ranked positive association of ID3 with the BMP9 endothelial receptor ACVRL1/ALK1 and the BMP ligand BMP6. Silibinin treatment blocked the BMP9-induced activation of the ALK1-phospho-SMAD1/5-ID3 axis in brain endothelial cells. Constitutive, acquired, and adaptive expression of ID3 in NSCLC cells were all significantly downregulated in response to silibinin. Silibinin blocked ID3 transcription via BMP-responsive elements in ID3 gene enhancers. Silibinin inhibited the kinase activities of BMPRs in the micromolar range, with the lower IC50 values occurring against ACVRL1/ALK1 and BMPR2. In an in vivo NSCLC xenograft model, tumoral overexpression of ID3 was completely suppressed by systematically achievable oral doses of silibinin. CONCLUSIONS: ID3 is a largely undruggable metastasis-promoting transcription factor. Silibinin is a novel suppressor of ID3 that may be explored as a novel therapeutic approach to interfere with the metastatic dissemination capacity of NSCLC.

DigiPatICS: Digital Pathology Transformation of the Catalan Health Institute Network of 8 Hospitals-Planification, Implementation, and Preliminary Results.

Complete digital pathology transformation for primary histopathological diagnosis is a challenging yet rewarding endeavor. Its advantages are clear with more efficient workflows, but there are many technical and functional difficulties to be faced. The Catalan Health Institute (ICS) has started its DigiPatICS project, aiming to deploy digital pathology in an integrative, holistic, and comprehensive way within a network of 8 hospitals, over 168 pathologists, and over 1 million slides each year. We describe the bidding process and the careful planning that was required, followed by swift implementation in stages. The purpose of the DigiPatICS project is to increase patient safety and quality of care, improving diagnosis and the efficiency of processes in the pathological anatomy departments of the ICS through process improvement, digital pathology, and artificial intelligence tools.

Inhibitor of Apoptosis (IAP) survivin is indispensable for survival of HER2 gene-amplified breast cancer cells with primary resistance to HER1/2-targeted therapies.

Primary resistance of HER2 gene-amplified breast carcinomas (BC) to HER-targeted therapies can be explained in terms of overactive HER2-independent downstream pro-survival pathways. We here confirm that constitutive overexpression of Inhibitor of Apoptosis (IAP) survivin is indispensable for survival of HER2-positive BC cells with intrinsic cross-resistance to multiple HER1/2 inhibitors. The IC50 values for the HER1/2 Tyrosine Kinase Inhibitors (TKIs) gefitinib, erlotinib and lapatinib were up to 40-fold higher in trastuzumab-unresponsive JIMT-1 cells than in trastuzumab-naïve SKBR3 cells. ELISA-based and immunoblotting assays demonstrated that trastuzumab-refractory JIMT-1 cells constitutively expressed ~ 4 times more survivin protein than trastuzumab-responsive SKBR3 cells. In response to trastuzumab, JIMT-1 cells accumulated ~10 times more survivin than SKBR3 cells. HER1/2 TKIs failed to down-regulate survivin expression in JIMT-1 cells whereas equimolar doses of HER1/HER2 TKIs drastically depleted survivin protein in SKBR3 cells. ELISA-based detection of histone-associated DNA fragments confirmed that trastuzumab-refractory JIMT-1 cells were intrinsically protected against the apoptotic effects of HER1/2 TKIs. Of note, when we knocked-down survivin expression using siRNA and then added trastuzumab, cell proliferation and colony formation were completely suppressed in JIMT-1 cells. Our current findings may be extremely helpful to design successful combinatorial strategies aimed to circumvent the occurrence of de novo resistance to HER2-directed drugs using survivin antagonists.

Differences in the Impact of COVID-19 on Pathology Laboratories and Cancer Diagnosis in Girona.

INTRODUCTION: The recent COVID-19 pandemic has compromised socio-health care, with consequences for the diagnosis and follow-up of other pathologies. The aim of this study was to evaluate the impact of COVID-19 on cancer diagnosis in Girona, Spain. METHODOLOGY: Observational study of samples received in two pathology laboratories during 2019-2020 (tertiary hospital in Girona and county hospital in Figueres). Date, sample type, and location and morphology were available. Samples were recoded to determine malignancy and grouped by location. Comparisons were made by calendar year and period of exposure to COVID-19. RESULTS: 102,360 samples were included: 80,517 from Girona and 21,843 from Figueres. The reduction in activity in the pathology laboratories in 2020 compared to the previous year was 25.4% in Girona and 27.5% in Figueres. The reduction in cancer diagnoses in 2020 compared to 2019 was 6.8% in Girona and 21% in Figueres. In both laboratories, a decrease was observed in the diagnoses of neoplasms of the lip, oral cavity and pharynx, larynx, colon, rectum and anus, kidney and urinary system, melanoma, and central nervous system. A statistically significant higher probability of a sample received in the pathology laboratory displaying malignancy during COVID-19 was found (Girona: OR = 1.28, 95% CI: 1.23-1.34; Figueres: OR = 1.10, 95% CI: 1.01-1.20) with respect to the COVID-19-free period. CONCLUSIONS: The COVID-19 pandemic has resulted in a reduction in cancer diagnoses by pathology departments that varies according to tumor location and type of hospital. Despite this, the optimization of care resources and the recovery effort have partially reduced the impact of the pandemic in certain neoplasms.

Silibinin Suppresses Tumor Cell-Intrinsic Resistance to Nintedanib and Enhances Its Clinical Activity in Lung Cancer.

The anti-angiogenic agent nintedanib has been shown to prolong overall and progression-free survival in patients with advanced non-small-cell lung cancer (NSCLC) who progress after first-line platinum-based chemotherapy and second-line immunotherapy. Here, we explored the molecular basis and the clinical benefit of incorporating the STAT3 inhibitor silibinin-a flavonolignan extracted from milk thistle-into nintedanib-based schedules in advanced NSCLC. First, we assessed the nature of the tumoricidal interaction between nintedanib and silibinin and the underlying relevance of STAT3 activation in a panel of human NSCLC cell lines. NSCLC cells with poorer cytotoxic responses to nintedanib exhibited a persistent, nintedanib-unresponsive activated STAT3 state, and deactivation by co-treatment with silibinin promoted synergistic cytotoxicity. Second, we tested whether silibinin could impact the lysosomal sequestration of nintedanib, a lung cancer cell-intrinsic mechanism of nintedanib resistance. Silibinin partially, but significantly, reduced the massive lysosomal entrapment of nintedanib occurring in nintedanib-refractory NSCLC cells, augmenting the ability of nintedanib to reach its intracellular targets. Third, we conducted a retrospective, observational multicenter study to determine the efficacy of incorporating an oral nutraceutical product containing silibinin in patients with NSCLC receiving a nintedanib/docetaxel combination in second- and further-line settings (n = 59). Overall response rate, defined as the combined rates of complete and partial responses, was significantly higher in the study cohort receiving silibinin supplementation (55%) than in the control cohort (22%, p = 0.011). Silibinin therapy was associated with a significantly longer time to treatment failure in multivariate analysis (hazard ratio 0.43, p = 0.013), despite the lack of overall survival benefit (hazard ratio 0.63, p = 0.190). Molecular mechanisms dictating the cancer cell-intrinsic responsiveness to nintedanib, such as STAT3 activation and lysosomal trapping, are amenable to pharmacological intervention with silibinin. A prospective, powered clinical trial is warranted to confirm the clinical relevance of these findings in patients with advanced NSCLC.

Dynamic emergence of the mesenchymal CD44(pos)CD24(neg/low) phenotype in HER2-gene amplified breast cancer cells with de novo resistance to trastuzumab (Herceptin).

Evidence is mounting that the occurrence of the CD44(pos)/CD24(neg/low) cell population, which contains potential breast cancer (BC) stem cells, could explain BC clinical resistance to HER2-targeted therapies. We investigated whether de novo refractoriness to the anti-HER2 monoclonal antibody trastuzumab (Tzb; Herceptin) may relate to the dynamic regulation of the mesenchymal CD44(pos)/CD24(neg/low) phenotype in HER2-positive BC. We observed that the subpopulation of Tzb-refractory JIMT-1 BC cells exhibiting CD44(pos)/CD24(neg/low)-surface markers switched with time. Low-passage JIMT-1 cell cultures were found to spontaneously contain approximately 10% of cells bearing the CD44(pos)/CD24(neg/low) immunophenotype. Late-passage (>60) JIMT-1 cultures accumulated approximately 80% of CD44(pos)/CD24(neg/low) cells and closely resembled the CD44(pos)/CD24(neg/low)-enriched ( approximately 85%) cell population constitutively occurring in HER2-negative MDA-MB-231 mesenchymal BC cells. Dynamic expression of mesenchymal markers was not limited to CD44/CD24 because high-passages of JIMT-1 cells exhibited also reduced expression of the HER2 protein and over-secretion of pro-invasive/metastatic chemokines and metalloproteases. Accordingly, late-passage JIMT-1 cells displayed an exacerbated migratogenic phenotype in plastic, collagen, and fibronectin substrates. Intrinsic genetic plasticity to efficiently drive the emergence of the CD44(pos)/CD24(neg/low) mesenchymal phenotype may account for de novo resistance to HER2 targeting therapies in basal-like BC carrying HER2 gene amplification.

Resveratrol targets PD-L1 glycosylation and dimerization to enhance antitumor T-cell immunity.

New strategies to block the immune evasion activity of programmed death ligand-1 (PD-L1) are urgently needed. When exploring the PD-L1-targeted effects of mechanistically diverse metabolism-targeting drugs, exposure to the dietary polyphenol resveratrol (RSV) revealed its differential capacity to generate a distinct PD-L1 electrophoretic migration pattern. Using biochemical assays, computer-aided docking/molecular dynamics simulations, and fluorescence microscopy, we found that RSV can operate as a direct inhibitor of glyco-PD-L1-processing enzymes (α-glucosidase/α-mannosidase) that modulate N-linked glycan decoration of PD-L1, thereby promoting the endoplasmic reticulum retention of a mannose-rich, abnormally glycosylated form of PD-L1. RSV was also predicted to interact with the inner surface of PD-L1 involved in the interaction with PD-1, almost perfectly occupying the target space of the small compound BMS-202 that binds to and induces dimerization of PD-L1. The ability of RSV to directly target PD-L1 interferes with its stability and trafficking, ultimately impeding its targeting to the cancer cell plasma membrane. Impedance-based real-time cell analysis (xCELLigence) showed that cytotoxic T-lymphocyte activity was notably exacerbated when cancer cells were previously exposed to RSV. This unforeseen immunomodulating mechanism of RSV might illuminate new approaches to restore T-cell function by targeting the PD-1/PD-L1 immunologic checkpoint with natural polyphenols.

The serine 2481-autophosphorylated form of mammalian Target Of Rapamycin (mTOR) is localized to midzone and midbody in dividing cancer cells.

Using a high-resolution, automated confocal high-content imaging system, we investigated the sub-cellular localization of the Serine 2481-autophosphorylated form of mTOR (PP-mTOR(Ser2481)) during mitosis and cytokinesis in human cancer cells. PP-mTOR(Ser2481) exhibited a punctate nuclear distribution in interphase cancer cells, with the number of PP-mTOR(Ser2481) nuclear speckles positively relating with the proliferative capacity of cancer cells. PP-mTOR(Ser2481) expression dynamically rearranged within the cytoplasm in a close association near and between separating chromosomes during early stages of mitosis. Towards the end of anaphase and in telophase, PP-mTOR(Ser2481) drastically focused on the midzone and ultimately in the centre of the midbody at the presumptive cleavage furrow. In cells at cytokinesis, PP-mTOR(Ser2481) appeared as a doublet facing each other at the apical ends of two daughter cells. Three-dimensional analysis confirmed that PP-mTOR(Ser2481) positioned at a ring structure wrapped round by microtubule bundles to connect daughter cells. These results reveal for the first time that PP-mTOR(Ser2481) may be unexpectedly involved in the terminal stages of cytokinesis.

Peripheral zone prostate cancer in patients with elevated PSA levels and low free-to-total PSA ratio: detection with MR imaging and MR spectroscopy.

PURPOSE: To retrospectively assess the value of endorectal magnetic resonance (MR) imaging and MR spectroscopy combined with the free-to-total prostate-specific antigen (PSA) ratio for detecting prostate cancer in men with elevated PSA levels. MATERIALS AND METHODS: The institutional review board approved the study, and all patients provided informed written consent. Endorectal MR imaging and MR spectroscopy were performed in 54 patients with PSA levels greater than 3 ng/mL but less than 15 ng/mL and free-to-total PSA ratio of less than 20%, followed by sextant biopsy in the peripheral zone. For each patient, MR imaging and MR spectroscopic findings, PSA level, and free-to-total PSA ratio were analyzed and compared with biopsy results and/or histopathologic tumor maps with regard to a sextant-modified distribution. The likelihood of cancer in each sextant according to MR and MR spectroscopic findings was graded independently on a scale of 1 (benign) to 5 (malignant). Detection accuracy and a multivariate logistic regression analysis were used to determine the most accurate combination of imaging, and clinical tests were used to detect prostate cancer according to the area under the receiver operating characteristic curve (AUC). RESULTS: The model incorporating MR imaging, MR spectroscopy, and free-to-total PSA ratio (AUC = 97.5%) was significantly more accurate in predicting prostate cancer than models using MR imaging alone (AUC = 85.1%; P = .007), MR spectroscopy alone (AUC = 87.2%; P = .041), or MR imaging and free-to-total PSA ratio combined (AUC = 90.8%; P = .038). CONCLUSION: MR and MR spectroscopy combined with free-to-total PSA ratio improves the predictive value for prostate cancer detection.

Neoadjuvant Metformin Added to Systemic Therapy Decreases the Proliferative Capacity of Residual Breast Cancer.

The proliferative capacity of residual breast cancer (BC) disease indicates the existence of partial treatment resistance and higher probability of tumor recurrence. We explored the therapeutic potential of adding neoadjuvant metformin as an innovative strategy to decrease the proliferative potential of residual BC cells in patients failing to achieve pathological complete response (pCR) after pre-operative therapy. We performed a prospective analysis involving the intention-to-treat population of the (Metformin and Trastuzumab in Neoadjuvancy) METTEN study, a randomized multicenter phase II trial of women with primary, non-metastatic (human epidermal growth factor receptor 2) HER2-positive BC evaluating the efficacy, tolerability, and safety of oral metformin (850 mg twice-daily) for 24 weeks combined with anthracycline/taxane-based chemotherapy and trastuzumab (arm A) or equivalent regimen without metformin (arm B), before surgery. We centrally evaluated the proliferation marker Ki67 on sequential core biopsies using visual assessment (VA) and an (Food and Drug Administration) FDA-cleared automated digital image analysis (ADIA) algorithm. ADIA-based pre-operative values of high Ki67 (≥20%), but not those from VA, significantly predicted the occurrence of pCR in both arms irrespective of the hormone receptor status (p = 0.024 and 0.120, respectively). Changes in Ki67 in residual tumors of non-pCR patients were significantly higher in the metformin-containing arm (p = 0.025), with half of all patients exhibiting high Ki67 at baseline moving into the low-Ki67 (<20%) category after neoadjuvant treatment. By contrast, no statistically significant changes in Ki67 occurred in residual tumors of the control treatment arm (p = 0.293). There is an urgent need for innovative therapeutic strategies aiming to provide the protective effects of decreasing Ki67 after neoadjuvant treatment even if pCR is not achieved. Metformin would be evaluated as a safe candidate to decrease the aggressiveness of residual disease after neoadjuvant (pre-operative) systemic therapy of BC patients.

The C Allele of ATM rs11212617 Associates With Higher Pathological Complete Remission Rate in Breast Cancer Patients Treated With Neoadjuvant Metformin.

Background: The minor allele (C) of the single-nucleotide polymorphism (SNP) rs11212617, located near the ataxia telangiectasia mutated (ATM) gene, has been associated with an increased likelihood of treatment success with metformin in type 2 diabetes. We herein investigated whether the same SNP would predict clinical response to neoadjuvant metformin in women with early breast cancer (BC). Methods: DNA was collected from 79 patients included in the intention-to-treat population of the METTEN study, a phase 2 clinical trial of HER2-positive BC patients randomized to receive either metformin combined with anthracycline/taxane-based chemotherapy and trastuzumab or equivalent regimen without metformin, before surgery. SNP rs11212617 genotyping was assessed using allelic discrimination by quantitative polymerase chain reaction. Results: Logistic regression analyses revealed a significant relationship between the rs11212617 genotype and the ability of treatment arms to achieve a pathological complete response (pCR) in patients (odds ratio [OR]genotype×arm = 10.33, 95% confidence interval [CI]: 1.29-82.89, p = 0.028). In the metformin-containing arm, patients bearing the rs11212617 C allele had a significantly higher probability of pCR (OR A/C,C/C = 7.94, 95%CI: 1.60-39.42, p = 0.011). Conversely, no association was found between rs11212617 and clinical response in the reference arm (OR A/C,C/C = 0.77, 95%CI: 0.20-2.92, p = 0.700). After controlling for tumor size and hormone receptor status, the rs11212617 C allele remained a significant predictor of pCR solely in the metformin-containing arm. Conclusions: If reproducible, the rs11212617 C allele might warrant consideration as a predictive clinical biomarker to inform the personalized use of metformin in BC patients. Trial Registration: EU Clinical Trials Register, EudraCT number 2011-000490-30. Registered 28 February 2011, https://www.clinicaltrialsregister.eu/ctr-search/trial/2011-000490-30/ES.

Metformin induces a fasting- and antifolate-mimicking modification of systemic host metabolism in breast cancer patients.

Certain dietary interventions might improve the therapeutic index of cancer treatments. An alternative to the "drug plus diet" approach is the pharmacological reproduction of the metabolic traits of such diets. Here we explored the impact of adding metformin to an established therapeutic regimen on the systemic host metabolism of cancer patients. A panel of 11 serum metabolites including markers of mitochondrial function and intermediates/products of folate-dependent one-carbon metabolism were measured in paired baseline and post-treatment sera obtained from HER2-positive breast cancer patients randomized to receive either metformin combined with neoadjuvant chemotherapy and trastuzumab or an equivalent regimen without metformin. Metabolite profiles revealed a significant increase of the ketone body ß-hydroxybutyrate and of the TCA intermediate α-ketoglutarate in the metformin-containing arm. A significant relationship was found between the follow-up levels of homocysteine and the ability of treatment arms to achieve a pathological complete response (pCR). In the metformin-containing arm, patients with significant elevations of homocysteine tended to have a higher probability of pCR. The addition of metformin to an established anti-cancer therapeutic regimen causes a fasting-mimicking modification of systemic host metabolism. Circulating homocysteine could be explored as a clinical pharmacodynamic biomarker linking the antifolate-like activity of metformin and biological tumor response.

Growth and molecular interactions of the anti-EGFR antibody cetuximab and the DNA cross-linking agent cisplatin in gefitinib-resistant MDA-MB-468 cells: new prospects in the treatment of triple-negative/basal-like breast cancer.

Three prominent hallmarks of triple-negative/basal-like breast carcinomas, a subtype of breast cancer gene phenotype associated with poor relapse-free and overall survival, are overexpression of the epidermal growth factor receptor (EGFR), hyperactivation of the MEK/ERK transduction pathway and high sensitivity to DNA-damaging agents. The cytotoxic interaction between EGFR inhibitors (monoclonal antibodies such as cetuximab and small molecule tyrosine kinase inhibitors such as gefitinib) and DNA cross-linking agents (e.g. platinum derivatives) might represent a promising combination for the treatment of triple-negative/basal-like breast tumors that are dependent upon EGFR/MEK/ERK signaling. We evaluated the growth and molecular interactions of the anti-EGFR antibody cetuximab (erbitux) and the DNA cross-linking agent cisplatin (cis-diammedichloroplatinum; CDDP) in the gefitinib-resistant MDA-MB-468 breast cancer cell line, an in vitro model system that shows many of the recurrent basal-like molecular abnormalities including ER-PR-HER2-negative status, TP53 deficiency, EGFR overexpression, PTEN loss and constitutive activation of the MEK/ERK pathway. Unlike other basal-like breast cancer models, MDA-MB-468 cells do not carry mutations of the key DNA repair gene BRCA1. Concurrent treatment with sub-optimal doses of cetuximab significantly enhanced CDDP-induced apoptotic cell death. However, an isobologram-based mathematical assessment of the nature of the interaction revealed a loss of synergism when employing a high-dose of cetuximab. Since BRCA1 depletion has been found to decrease DNA damage repair and cell survival in MDA-MB-468 cells when treated with DNA-damaging drugs, we employed ELISA-based quantitative analyses to measure BRCA1 protein levels in CDDP+/- cetuximab-treated cells. Cetuximab as single agent was as efficient as CDDP at increasing BRCA1 protein expression. Interestingly, cetuximab co-exposure significantly antagonized the ability of CDDP to up-regulate BRCA1 expression. Low-scale phosphor-proteomic approaches [i.e. phospho-receptor tyrosine kinase (RTK) and phospho-mitogen-activated protein kinases (MAPKs) Array Proteome Profiler capable of simultaneously identifying the relative levels of phosphorylation of 42 different RTKs and 23 different MAPKs and other serine/threonine kinases, respectively] revealed the ability of Cetuximab, as single agent, to paradoxically induce hyper-phosphorylation of EGFR while concomitantly deactivating p42/44 (ERK1/ERK2) MAPK. Unexpectedly, ELISA-based quantitative analyses of EGFR protein content demonstrated that simultaneous exposure to cetuximab and optimal doses of CDDP completely depleted EGFR protein in MDA-MB-468 cells. Although these findings preclinically support, at least in part, ongoing clinical trials for 'triple-negative/basal-like' metastatic breast cancer patients who are receiving either cetuximab alone versus cetuximab plus carboplatin (http://www.clinicaltrials.gov/ct/show/NCT00232505), the unexpected ability of CDDP to promote a complete depletion of the cetuximab target EGFR further suggests that treatment schedules, cetuximab/CDDP doses and BRCA1 status should be carefully considered when combining anti-EGFR antibodies and platinum derivatives in triple-negative/basal-like breast carcinomas.

Solid neuroendocrine breast carcinomas: incidence, clinico-pathological features and immunohistochemical profiling.

Primary pure neuroendocrine breast carcinomas (NEBC) have been considered special features within conventional breast carcinomas until recently. Indeed, the actual incidence of NEBC in BC populations has remained largely unknown due to the lack of unambiguous diagnostic criteria. In 2003, the World Health Organization (WHO) classification of breast tumors definitely established that the immunohistochemical expression of NE markers in more than 50% of the tumor cell population is the unique requisite for NEBC diagnosis. Herein, we sought to determine the incidence, the clinico-pathological features and the immunohistochemical profile of NEBC in a large series of 1368 infiltrating breast tumors collected from 1989 to 2008 in our institution (Dr Josep Trueta University Hospital, Girona, Catalonia). Twelve cases were initially selected to fulfil histopathological patterns compatible with NEBC. Clinical data along with histological and immunohistochemical profiles were collected in all cases. The criterion inclusion was the presence of more than 50% tumor immunoreactivity for one of NE markers including chromogranin, synaptophysin and CD56. Only 7 tumors fully satisfied the NEBC criteria established by the WHO (0.5% prevalence). All the NECB were grade 2 ductal carcinoma infiltrating (DCI) with tumor sizes ranging from 7 to 55 mm. Lymphovascular tumoral emboli was present in 4 cases (57.1% of NEBC) and mucinous features occurred in 2 cases (28.5% of NEBC). Axillary lymph nodes were metastatic in 3 cases (42.8% of NEBC). A positive status for estrogen receptor (ER), progesterone receptor (PR) and synaptophysin was observed in 7 cases (100% of NEBC). None of the NEBC displayed HER2 overexpression. All the patients bearing NECB received hormone therapy and 4 of them underwent radiotherapy and/or chemotherapy. Of note, none of the NEBC patients died from BC-related causes after a median follow-up of 51 months. These findings revealed that: a) Pure solid NEBC do not significantly differ from other breast carcinomas in terms of general clinical features; b) NEBC do not exhibit an aggressive behavior despite the presence of adverse prognostic factors; and c) NEBC immunohistochemical profile mainly corresponds to that of the Luminal A BC subtype. Although it remains to be elucidated whether the good prognosis of NEBC relates to the intrinsic nature of the tumor and/or to a high rate of treatment responses, their immunohistochemical profile strongly suggest that NEBC belong to the Luminal A BC subtype. Forthcoming studies should definitely determine if the clinico-pathological features of NEBC indeed represent an independent good-prognosis subgroup of BC gene signature.

A phase 2 trial of neoadjuvant metformin in combination with trastuzumab and chemotherapy in women with early HER2-positive breast cancer: the METTEN study.

The METTEN study assessed the efficacy, tolerability, and safety of adding metformin to neoadjuvant chemotherapy plus trastuzumab in early HER2-positive breast cancer (BC). Women with primary, non-metastatic HER2-positive BC were randomized (1:1) to receive metformin (850 mg twice-daily) for 24 weeks concurrently with 12 cycles of weekly paclitaxel plus trastuzumab, followed by four cycles of 3-weekly FE75C plus trastuzumab (arm A), or equivalent regimen without metformin (arm B), followed by surgery. Primary endpoint was the rate of pathological complete response (pCR) in the per-protocol efficacy population. pCR rate was numerically higher in the metformin-containing arm A (19 of 29 patients [65.5%, 95% CI: 47.3-80.1]) than in arm B (17 of 29 patients [58.6%, 95% CI: 40.7-74.5]; OR 1.34 [95% CI: 0.46-3.89], P = 0.589). The rate of breast-conserving surgery was 79.3% and 58.6% in arm A and B (P = 0.089), respectively. Blood metformin concentrations (6.2 µmol/L, 95% CI: 3.6-8.8) were within the therapeutic range. Seventy-six percent of patients completed the metformin-containing regimen; 13% of patients in arm A dropped out because of metformin-related gastrointestinal symptoms. The most common adverse events (AEs) of grade ≥3 were neutropenia in both arms and diarrhea in arm A. None of the serious AEs was deemed to be metformin-related. Addition of anti-diabetic doses of metformin to a complex neoadjuvant regimen was well tolerated and safe. Because the study was underpowered relative to its primary endpoint, the efficacy data should be interpreted with caution.

Mitophagy-driven mitochondrial rejuvenation regulates stem cell fate.

Our understanding on how selective mitochondrial autophagy, or mitophagy, can sustain the archetypal properties of stem cells is incomplete. PTEN-induced putative kinase 1 (PINK1) plays a key role in the maintenance of mitochondrial morphology and function and in the selective degradation of damaged mitochondria by mitophagy. Here, using embryonic fibroblasts fromPINK1 gene-knockout (KO) mice, we evaluated whether mitophagy is a causal mechanism for the control of cell-fate plasticity and maintenance of pluripotency. Loss of PINK1-dependent mitophagy was sufficient to dramatically decrease the speed and efficiency of induced pluripotent stem cell (iPSC) reprogramming. Mitophagy-deficient iPSC colonies, which were characterized by a mixture of mature and immature mitochondria, seemed unstable, with a strong tendency to spontaneously differentiate and form heterogeneous populations of cells. Although mitophagy-deficient iPSC colonies normally expressed pluripotent markers, functional monitoring of cellular bioenergetics revealed an attenuated glycolysis in mitophagy-deficient iPSC cells. Targeted metabolomics showed a notable alteration in numerous glycolysis- and TCA-related metabolites in mitophagy-deficient iPSC cells, including a significant decrease in the intracellular levels of α-ketoglutarate -a key suppressor of the differentiation path in stem cells. Mitophagy-deficient iPSC colonies exhibited a notably reduced teratoma-initiating capacity, but fully retained their pluripotency and multi-germ layer differentiation capacity in vivo. PINK1-dependent mitophagy pathway is an important mitochondrial switch that determines the efficiency and quality of somatic reprogramming. Mitophagy-driven mitochondrial rejuvenation might contribute to the ability of iPSCs to suppress differentiation by directing bioenergetic transition and metabolome remodeling traits. These findings provide new insights into how mitophagy might influence the stem cell decisions to retain pluripotency or differentiate in tissue regeneration and aging, tumor growth, and regenerative medicine.