Transcriptional Landscape of Human Tissue Lymphocytes Unveils Uniqueness of Tumor-Infiltrating T Regulatory Cells.

Tumor-infiltrating regulatory T lymphocytes (Treg) can suppress effector T cells specific for tumor antigens. Deeper molecular definitions of tumor-infiltrating-lymphocytes could thus offer therapeutic opportunities. Transcriptomes of T helper 1 (Th1), Th17, and Treg cells infiltrating colorectal or non-small-cell lung cancers were compared to transcriptomes of the same subsets from normal tissues and validated at the single-cell level. We found that tumor-infiltrating Treg cells were highly suppressive, upregulated several immune-checkpoints, and expressed on the cell surfaces specific signature molecules such as interleukin-1 receptor 2 (IL1R2), programmed death (PD)-1 Ligand1, PD-1 Ligand2, and CCR8 chemokine, which were not previously described on Treg cells. Remarkably, high expression in whole-tumor samples of Treg cell signature genes, such as LAYN, MAGEH1, or CCR8, correlated with poor prognosis. Our findings provide insights into the molecular identity and functions of human tumor-infiltrating Treg cells and define potential targets for tumor immunotherapy.

Assessment of pregnancy dietary intake and association with maternal and neonatal outcomes.

BACKGROUND: Maternal dietary habits are contributors of maternal and fetal health; however, available data are heterogeneous and not conclusive. METHODS: Nutrient intake during pregnancy was assessed in 503 women with uncomplicated pregnancies, using the validated Food Frequency Questionnaire developed by the European Prospective Investigation into Cancer and Nutrition (EPIC-FFQ). RESULTS: In all, 68% of women had a normal body mass index at the beginning of pregnancy, and 83% of newborns had an appropriate weight for gestational age. Maternal pre-pregnancy body mass index (BMI), gestational weight gain (GWG), and placental weight were independently correlated with birth weight. GWG was not related to the pre-pregnancy BMI. EPIC-FFQ evaluation showed that 30% of women adhered to the European Food Safety Authority (EFSA) ranges for macronutrient intake. In most pregnant women (98.1%), consumption of water was below recommendations. Comparing women with intakes within EFSA ranges for macronutrients with those who did not, no differences were found in BMI, GWG, and neonatal or placental weight. Neither maternal nor neonatal parameters were associated with the maternal dietary profiles. CONCLUSIONS: In our population, maternal pre-pregnancy BMI, GWG, and placental weight are determinants of birth weight percentile, while no association was found with maternal nutrition. Future studies should explore associations through all infancy. IMPACT: Maternal anthropometrics and nutrition status may affect offspring birth weight. In 503 healthy women, maternal pre-pregnancy body mass index (BMI), gestational weight gain (GWG), and placental weight were independently correlated to neonatal birth weight. GWG was not related to the pre-pregnancy BMI. In all, 30% of women respected the EFSA ranges for macronutrients. Neither maternal nor neonatal parameters were associated with maternal dietary profiles considered in this study. Maternal pre-pregnancy BMI, GWG, and placental weight are determinants of neonatal birth weight percentile, while a connection with maternal nutrition profiles was not found.

Inhibition of de novo lipogenesis targets androgen receptor signaling in castration-resistant prostate cancer.

A hallmark of prostate cancer progression is dysregulation of lipid metabolism via overexpression of fatty acid synthase (FASN), a key enzyme in de novo fatty acid synthesis. Metastatic castration-resistant prostate cancer (mCRPC) develops resistance to inhibitors of androgen receptor (AR) signaling through a variety of mechanisms, including the emergence of the constitutively active AR variant V7 (AR-V7). Here, we developed an FASN inhibitor (IPI-9119) and demonstrated that selective FASN inhibition antagonizes CRPC growth through metabolic reprogramming and results in reduced protein expression and transcriptional activity of both full-length AR (AR-FL) and AR-V7. Activation of the reticulum endoplasmic stress response resulting in reduced protein synthesis was involved in IPI-9119-mediated inhibition of the AR pathway. In vivo, IPI-9119 reduced growth of AR-V7-driven CRPC xenografts and human mCRPC-derived organoids and enhanced the efficacy of enzalutamide in CRPC cells. In human mCRPC, both FASN and AR-FL were detected in 87% of metastases. AR-V7 was found in 39% of bone metastases and consistently coexpressed with FASN. In patients treated with enzalutamide and/or abiraterone FASN/AR-V7 double-positive metastases were found in 77% of cases. These findings provide a compelling rationale for the use of FASN inhibitors in mCRPCs, including those overexpressing AR-V7.

Correction: Rondinone et al. Extensive Placental Methylation Profiling in Normal Pregnancies. Int. J. Mol. Sci. 2021, 22, 2136.

In the original publication [...].

Extensive Placental Methylation Profiling in Normal Pregnancies.

The placental methylation pattern is crucial for the regulation of genes involved in trophoblast invasion and placental development, both key events for fetal growth. We investigated LINE-1 methylation and methylome profiling using a methylation EPIC array and the targeted methylation sequencing of 154 normal, full-term pregnancies, stratified by birth weight percentiles. LINE-1 methylation showed evidence of a more pronounced hypomethylation in small neonates compared with normal and large for gestational age. Genome-wide methylation, performed in two subsets of pregnancies, showed very similar methylation profiles among cord blood samples while placentae from different pregnancies appeared very variable. A unique methylation profile emerged in each placenta, which could represent the sum of adjustments that the placenta made during the pregnancy to preserve the epigenetic homeostasis of the fetus. Investigations into the 1000 most variable sites between cord blood and the placenta showed that promoters and gene bodies that are hypermethylated in the placenta are associated with blood-specific functions, whereas those that are hypomethylated belong mainly to pathways involved in cancer. These features support the functional analogies between a placenta and cancer. Our results, which provide a comprehensive analysis of DNA methylation profiling in the human placenta, suggest that its peculiar dynamicity can be relevant for understanding placental plasticity in response to the environment.

Deregulation of miRNAs-cMYC circuits is a key event in refractory celiac disease type-2 lymphomagenesis.

A percentage of celiac disease (CD) patients develop refractory type-2 disease (RCD2), a condition associated with increased risk of enteropathy-associated T-cell-lymphoma (EATL) and without therapeutic option. Therefore, we profiled the miRNome in series of peripheral T-cell lymphomas (PTCLs), CD, RCD1 or 2 and in the murine interleukin-15 (IL15)-transgenic (TG) model of RCD. The transcriptome was analyzed in 18 intestinal T-cell lymphomas (ITLs). Bioinformatics pipelines provided significant microRNA (miRNA) lists and predicted targets that were confirmed in a second set of patients. Our data show that ITLs have a unique miRNA profile with respect to other PTCLs. The c-MYC regulated miR-17/92 cluster distinguishes monomorphic epitheliotropic ITL (MEITL) from EATL and prognosticates EATL outcome. These miRNAs are decreased in IL15-TG mice upon Janus kinase (JAK) inhibition. The random forest algorithm identified a signature of 38 classifier miRNAs, among which, the miR-200 and miR-192/215 families were progressively lost in RCD2 and ITL-CD, whereas miR-17/92 and C19MC miRNAs were up-regulated. Accordingly, SMAD3, MDM2, c-Myc and activated-STAT3 were increased in RCD2 and EATL tissues while JAK inhibition in IL15-TG mice restored their levels to baseline. Our data suggest that miRNAs circuit supports activation of STAT3 and c-Myc oncogenic signaling in RCD2, thus contributing to lymphomagenesis. This novel understanding might pave the way to personalized medicine approaches for RCD and EATL.

PTEN Expression as a Complementary Biomarker for Mismatch Repair Testing in Breast Cancer.

Mismatch repair (MMR) analysis in breast cancer may help to inform immunotherapy decisions but it lacks breast-specific guidelines. Unlike in other neoplasms, MMR protein loss shows intra-tumor heterogeneity and it is not mirrored by microsatellite instability in the breast. Additional biomarkers can improve MMR clinical testing. Phosphatase and tensin homolog (PTEN) inactivation is an early oncogenic event that is associated with MMR deficiency (dMMR) in several tumors. Here, we sought to characterize the diagnostic utility of PTEN expression analysis for MMR status assessment in breast cancer. A total of 608 breast cancers were profiled for their MMR and PTEN status. Proteins expression and distribution were analyzed by immunohistochemistry (IHC) on tissue microarrays and confirmed on full sections; PTEN copy number alterations were detected using a real-time PCR assay. Overall, 78 (12.8%) cases were MMR-heterogeneous (hMMR), while all patterns of PTEN expression showed no intra-tumor heterogeneity. Wild-type PTEN expression was observed in 15 (18.5%) dMMR tumors (p < 0.0001). Survival analyses revealed significant correlations between MMR-proficient (pMMR), PTEN expression, and a better outcome. The positive predictive value of PTEN-retained status for pMMR ranged from 94.6% in estrogen receptor (ER)+/HER2- tumors to 100% in HER2-amplified and ER-/HER2- cases. We propose a novel diagnostic algorithm where PTEN expression analysis can be employed to identify pMMR breast cancers.

The Mitochondrial Unfoldase-Peptidase Complex ClpXP Controls Bioenergetics Stress and Metastasis.

Mitochondria must buffer the risk of proteotoxic stress to preserve bioenergetics, but the role of these mechanisms in disease is poorly understood. Using a proteomics screen, we now show that the mitochondrial unfoldase-peptidase complex ClpXP associates with the oncoprotein survivin and the respiratory chain Complex II subunit succinate dehydrogenase B (SDHB) in mitochondria of tumor cells. Knockdown of ClpXP subunits ClpP or ClpX induces the accumulation of misfolded SDHB, impairing oxidative phosphorylation and ATP production while activating "stress" signals of 5' adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and autophagy. Deregulated mitochondrial respiration induced by ClpXP targeting causes oxidative stress, which in turn reduces tumor cell proliferation, suppresses cell motility, and abolishes metastatic dissemination in vivo. ClpP is universally overexpressed in primary and metastatic human cancer, correlating with shortened patient survival. Therefore, tumors exploit ClpXP-directed proteostasis to maintain mitochondrial bioenergetics, buffer oxidative stress, and enable metastatic competence. This pathway may provide a "drugable" therapeutic target in cancer.

OATPB1/B3 and MRP3 expression in hepatocellular adenoma predicts Gd-EOB-DTPA uptake and correlates with risk of malignancy.

BACKGROUND AND AIMS: Hepatobiliary phase (HBP) Gd-EOB-DTPA-enhanced magnetic resonance imaging (MRI) has increased the accuracy in differentiating focal nodular hyperplasia (FNH) and hepatocellular adenoma (HCA). However, the ability of this technique to distinguish HCA subtypes remains controversial. The aim of this study was to investigate the expression of hepatocyte transporters (OATPB1/B3, MRP2, MRP3) in HCA subtypes, hence to understand their MRI signal intensity on HBP Gd-EOB-DTPA-enhanced MRI. METHODS: By means of immunohistochemistry (IHC), we scored the expression of OATPB1/B3, MRP2 and MRP3, in resected specimens of FNH (n = 40), subtyped HCA (n = 58) and HCA with focal malignant transformation (HCA-HCC, n = 4). Results were validated on a supplementary set of FNH (n = 6), subtyped HCA (n = 17) and HCA-HCC (n = 1) with Gd-EOB-DTPA MR images. RESULTS: All FNH showed a preserved expression of hepatocytes transporters. Beta-catenin-activated HCA (at highest risk of malignant transformation) and HCA-HCC were characterized by preserved/increased OATPB1/B3 expression (predictor of hyperintensity on HBP), as opposed to other HCA subtypes (P < 0.01) that mostly showed OATPB1/B3 absence (predictor of hypointensity on HBP). HCA-HCC showed an additional MRP3 overexpressed profile (P < 0.01). On HBP Gd-EOB-DTPA-enhanced MRI, FNH and HCA signal intensity reflected the profile predicted by their specific OATPB1/B3 tissue expression. The hyperintense vs hypointense HBP signal criterion was able to distinguish all higher risk HCA and HCA-HCC (100% accuracy). CONCLUSIONS: OATPB1/B3 and MRP3 IHC and signal intensity on HBP Gd-EOB-DTPA-enhanced MRI can help to stratify HCA according to their risk of malignant transformation.

A novel tool for predicting extracapsular extension during graded partial nerve sparing in radical prostatectomy.

OBJECTIVES: To create a statistical tool for the estimation of extracapsular extension (ECE) level of prostate cancer and determine the nerve-sparing (NS) approach that can be safely performed during radical prostatectomy (RP). PATIENTS AND METHODS: A total of 11 794 lobes, from 6 360 patients who underwent robot-assisted RP between 2008 and 2016 were evaluated. Clinicopathological features were included in a statistical algorithm for the prediction of the maximum ECE width. Five multivariable logistic models were estimated for: presence of ECE and ECE width of >1, >2, >3, and >4 mm. A five-zone decision rule based on a lower and upper threshold is proposed. Using a graphical interface, surgeons can view patient's pre-treatment characteristics and a curve showing the estimated probabilities for ECE amount together with the areas identified by the decision rule. RESULTS: Of the 6 360 patients, 1 803 (28.4%) were affected by non-organ-confined disease. ECE was present in 1 351 lobes (11.4%) and extended beyond the capsule for >1, >2, >3, and >4 mm in 498 (4.2%), 261 (2.2%), 148 (1.3%), 99 (0.8%) cases, respectively. ECE width was up to 15 mm (interquartile range 1.00-2.00). The five logistic models showed good predictive performance, the area under the receiver operating characteristic curve was: 0.81 for ECE, and 0.84, 0.85, 0.88, and 0.90 for ECE width of >1, >2, >3, and >4 mm, respectively. CONCLUSION: This novel tool predicts with good accuracy the presence and amount of ECE. Furthermore, the graphical interface available at www.prece.it can support surgeons in patient counselling and preoperative planning.

PI3K therapy reprograms mitochondrial trafficking to fuel tumor cell invasion.

Molecular therapies are hallmarks of "personalized" medicine, but how tumors adapt to these agents is not well-understood. Here we show that small-molecule inhibitors of phosphatidylinositol 3-kinase (PI3K) currently in the clinic induce global transcriptional reprogramming in tumors, with activation of growth factor receptors, (re)phosphorylation of Akt and mammalian target of rapamycin (mTOR), and increased tumor cell motility and invasion. This response involves redistribution of energetically active mitochondria to the cortical cytoskeleton, where they support membrane dynamics, turnover of focal adhesion complexes, and random cell motility. Blocking oxidative phosphorylation prevents adaptive mitochondrial trafficking, impairs membrane dynamics, and suppresses tumor cell invasion. Therefore, "spatiotemporal" mitochondrial respiration adaptively induced by PI3K therapy fuels tumor cell invasion, and may provide an important antimetastatic target.

Recurrent NAB2-STAT6 gene fusions and oestrogen receptor-α expression in pulmonary adenofibromas.

AIMS: Pulmonary adenofibromas are rare benign fibroepithelial tumours of the lung with unknown histogenesis and an indolent clinical behaviour. Their stroma resembles that of solitary fibrous tumours, whereas the glands are composed of respiratory epithelium organized in a phyllodes-like architecture. Differentiation of pulmonary adenofibromas from other more aggressive intrathoracic tumours is clinically relevant. However, their biology is unknown. Here, we sought to characterize pulmonary adenofibromas at a clinicopathological level and to define whether they could be underpinned by a highly recurrent somatic genetic alteration akin to tumours with similar morphology. METHODS AND RESULTS: Seven pulmonary adenofibromas were subjected to immunohistochemical analysis for thyroid transcription factor 1 (TTF1), napsin A, cytokeratin 7, E-cadherin, CD99, CD34, CD31, STAT6, oestrogen receptor (ER), progesterone receptor, androgen receptor, bcl-2, and vimentin, as well as electron microscopy and capillary sequencing on microdissected samples to evaluate the presence of NAB2-STAT6 fusion genes and MED12 exon 2 mutations in their discrete components. A control group comprising pulmonary solitary fibrous tumours, pulmonary hamartomas and breast fibroadenomas was also analysed. We confirmed that the stromal elements of pulmonary adenofibromas pertain to the fibroblastic lineage, and show ER overexpression in 71% of cases, whereas the epithelium consists of TTF1-positive, E-cadherin positive bronchiolar elements. A highly recurrent NAB2-STAT6 fusion variant (exon 4-exon 2) was detected in the stroma but not in the epithelium. No MED12 mutations were identified. CONCLUSIONS: Here, we demonstrate that pulmonary adenofibromas are neoplastic lesions harbouring the molecular hallmark of solitary fibrous tumours.

Therapeutic Potential of Immunoproteasome Inhibition in Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy is an inherited fatal genetic disease characterized by mutations in dystrophin gene, causing membrane fragility leading to myofiber necrosis and inflammatory cell recruitment in dystrophic muscles. The resulting environment enriched in proinflammatory cytokines, like IFN-γ and TNF-α, determines the transformation of myofiber constitutive proteasome into the immunoproteasome, a multisubunit complex involved in the activation of cell-mediate immunity. This event has a fundamental role in producing peptides for antigen presentation by MHC class I, for the immune response and also for cytokine production and T-cell differentiation. Here, we characterized for the first time the presence of T-lymphocytes activated against revertant dystrophin epitopes, in the animal model of Duchenne muscular dystrophy, the mdx mice. Moreover, we specifically blocked i-proteasome subunit LMP7, which was up-regulated in dystrophic skeletal muscles, and we demonstrated the rescue of the dystrophin expression and the amelioration of the dystrophic phenotype. The i-proteasome blocking lowered myofiber MHC class I expression and self-antigen presentation to T cells, thus reducing the specific antidystrophin T cell response, the muscular cell infiltrate, and proinflammatory cytokine production, together with muscle force recovery. We suggest that i-proteasome inhibition should be considered as new promising therapeutic approach for Duchenne muscular dystrophy pathology.

Morphophenotypic changes in human multistep hepatocarcinogenesis with translational implications.

BACKGROUND & AIMS: Human hepatocarcinogenesis in cirrhosis is thought to be multistep and characterized by a spectrum of nodular lesions, ranging from low to high grade dysplastic nodules (LGDN and HGDN) to early and progressed hepatocellular carcinoma (eHCC and pHCC). The aim of this study was to investigate the morphophenotypical changes of this sequence and their potential translational significance. METHODS: We scored the vascular profile, ductular reaction/stromal invasion and overexpression of five biomarkers (GPC3, HSP70, GS, CHC, and EZH2), in a series of 100 resected nodules (13 LGDN, 16 HGDN, 42 eHCC and 29 small pHCC). RESULTS: The score separated the four groups of nodules as individual entities (p<0.01). In the sequence, biomarker's overexpression progressively increased with parallel decrease of ductular reaction; the vascular remodeling started very early (LGDN) but did not further develop in a proportion of HCC. eHCC was the most heterogeneous entity, with marginal overlap with HGDN and pHCC. Liver environment (fibrosis, etiology) did not impact on the phenotype of the different nodules. A subclass of eHCC (16/42) without evidence of stromal invasion was identified, suggesting a "preinvasive stage" (p<0.05). For diagnosis, the application of four and five biomarkers (rather than the usual three) improved the sensitivity of the assay for the detection of eHCC (76% and 93% vs. 52%); biomarkers in alternative combinations, and also increased the sensitivity of the assay (GS+CHC+EZH2: 76%; GS+CHC+EZH2+HSP70: 90%). CONCLUSIONS: This study supports the multistep nature of human hepatocarcinogenesis, and suggests that eHCC is more heterogeneous than previously thought. This provides further information of the potential translational significance into clinical practice.

Analysis of NSCLC tumour heterogeneity, proliferative and 18F-FDG PET indices reveals Ki67 prognostic role in adenocarcinomas.

AIMS: The role of tumour metabolic and proliferative indices in predicting non-small-cell lung cancer (NSCLC) patients' prognosis is unclear. We correlated fluorine 18 ((18) F)-fluorodeoxyglucose (FDG)-positron emission tomography (PET) value and Ki67 index to patients' survival, taking into account tumour heterogeneity, disease characteristics and genetic aberrations. METHODS AND RESULTS: A series of 383 NSCLCs was arranged into tissue microarrays and Ki67 staining was analysed by immunohistochemistry. The maximum standardized uptake (SUV(MAX) ) value detected by (18) F-FDG-PET analysis was calculated over a region of interest. Large-cell and squamous cell carcinomas had higher proliferative and metabolic activities than adenocarcinomas, and the two measures were correlated significantly. The hot-spot Ki67 value was correlated with patients' survival and the cut-off to discriminate patients in the survival risk groups was 20%. Ki67 hot-spot values were greater in anaplastic lymphoma kinase (ALK) rearranged tumours. Adenocarcinomas showed the highest intratumour heterogeneity in proliferative activity and the hot-spot Ki67 value predicted only the prognosis of patients in this group. Although tumour metabolic activity was not associated with patients' prognosis, a SUV(MAX) > 2 was related to nodal metastases, tumour size and grade. CONCLUSIONS: Our results highlight how tumour heterogeneity influences evaluation of prognostic biomarkers. Our data support Ki67 evaluation to estimate NSCLC patients' prognosis, particularly for adenocarcinoma.

Tumor size, stage and grade alterations of urinary peptidome in RCC.

BACKGROUND: Several promising biomarkers have been found for RCC, but none of them has been used in clinical practice for predicting tumour progression. The most widely used features for predicting tumour aggressiveness still remain the cancer stage, size and grade. Therefore, the aim of our study is to investigate the urinary peptidome to search and identify peptides whose concentrations in urine are linked to tumour growth measure and clinical data. METHODS: A proteomic approach applied to ccRCC urinary peptidome (n = 117) based on prefractionation with activated magnetic beads followed by MALDI-TOF profiling was used. A systematic correlation study was performed on urinary peptide profiles obtained from MS analysis. Peptide identity was obtained by LC-ESI-MS/MS. RESULTS: Fifteen, twenty-six and five peptides showed a statistically significant alteration of their urinary concentration according to tumour size, pT and grade, respectively. Furthermore, 15 and 9 signals were observed to have urinary levels statistically modified in patients at different pT or grade values, even at very early stages. Among them, C1RL, A1AGx, ZAG2G, PGBM, MMP23, GP162, ADA19, G3P, RSPH3, DREB, NOTC2 SAFB2 and CC168 were identified. CONCLUSIONS: We identified several peptides whose urinary abundance varied according to tumour size, stage and grade. Among them, several play a possible role in tumorigenesis, progression and aggressiveness. These results could be a useful starting point for future studies aimed at verifying their possible use in the managements of RCC patients.

MicroRNA-425-3p predicts response to sorafenib therapy in patients with hepatocellular carcinoma.

BACKGROUND & AIMS: Sorafenib is the standard of care in advanced hepatocellular carcinoma (HCC), however no criteria have been established to select patients likely to benefit from this therapy. In this study, we evaluated the predictive role of microRNAs (miRNAs) in this setting of patients. METHODS: We profiled 522 miRNA in a series of 26 HCC patients treated with sorafenib (training set) and validated the results in an independent series of 58 patients (validation set). Formalin-fixed paraffin-embedded tumour and cirrhotic liver biopsies were used for RNA extraction and miRNAs profiling with TaqMan Arrays technology. Statistical analyses were used to correlate miRNA levels with clinical outcome, including time to progression (TTP), progression free (PFS), and overall survival. Cell viability and cell motility of HuH-7 or HepG2 HCC cells were tested in vitro after transfection with specific miRNA precursor, inhibitor or controls and sorafenib treatment. RESULTS: Six miRNAs were significantly associated with clinical variables in the training set and only miR-425-3p could be further validated. Higher levels of miR-425-3p were associated with longer TTP and PFS (P = 0.0008; HR = 0.4; 95% CI = 0.2-0.7 and P = 0.007; HR = 0.5; 95% CI = 0.3-0.9 respectively). Multivariate analysis confirmed the predictive significance of miR-425-3p. Furthermore, an association between increased miR-425-3p, cell death and reduced cell motility was defined in vitro in HCC cell lines treated with sorafenib. CONCLUSIONS: Assessment of miR-425-3p levels in liver biopsies could help in stratifying patients with advanced HCC for sorafenib treatment. These promising results need to be confirmed in a large prospective study.

Histological evaluation of duodenal biopsies from coeliac patients: the need for different grading criteria during follow-up.

BACKGROUND: Coeliac disease is characterised by villous atrophy, which usually normalises after gluten withdrawal. Sometimes the revaluation of duodenal histology is required during follow-up, even if the methodology for comparing duodenal histology before and after introducing a gluten-free diet is not yet established. Our aim was to evaluate a novel criterion to compare duodenal histology in coeliac disease before and after gluten withdrawal. METHODS: Duodenal biopsies from coeliac patients were retrospectively reviewed to compare duodenal histology at diagnosis and after at least one year on a gluten-free diet. Two different methods were used: the first was represented by the classical Marsh-Oberhuber score, the second compared the areas covered by each Marsh-Oberhuber grade and expressed as percentages, the final grade being calculated from the analysis of ten power fields per duodenal biopsy. RESULTS: Sixty-nine patients (17 males 52 females, age at diagnosis 36 ± 15 years) who underwent duodenal biopsies, were considered. According to the classical Marsh-Oberhuber scale, 32 patients did not present atrophy during follow-up while 37 showed duodenal atrophy, among whom 26 improved the grade of severity and 11 retained the same one. Of these latter, according to the second method, eight patients were considered improved, two showed a worsened duodenal damage and only one remained unchanged; the evaluation changed in 91 % of cases. CONCLUSIONS: The proposed semi-quantitative approach (i.e. the second method) for the evaluation of histology at follow-up provides additional information about the progression/regression of the mucosal damage.

Survivin family proteins as novel molecular determinants of doxorubicin resistance in organotypic human breast tumors.

INTRODUCTION: The molecular determinants of breast cancer resistance to first-line anthracycline-containing chemotherapy are unknown. METHODS: We examined the response to doxorubicin of organotypic cultures of primary human breast tumors ex vivo with respect to cell proliferation, DNA damage and modulation of apoptosis. Samples were analyzed for genome-wide modulation of cell death pathways, differential activation of p53, and the role of survivin family molecules in drug resistance. Rational drug combination regimens were explored by high-throughput screening, and validated in model breast cancer cell types. RESULTS: Doxorubicin treatment segregated organotypic human breast tumors into distinct Responder or Non Responder groups, characterized by differential proliferative index, stabilization of p53, and induction of apoptosis. Conversely, tumor histotype, hormone receptor or human epidermal growth factor receptor-2 (HER2) status did not influence chemotherapy sensitivity. Global analysis of cell death pathways identified survivin and its alternatively spliced form, survivin-ΔEx3 as uniquely overexpressed in Non Responder breast tumors. Forced expression of survivin-ΔEx3 preserved cell viability and prevented doxorubicin-induced apoptosis in breast cancer cell types. High-throughput pharmacologic targeting of survivin family proteins with a small-molecule survivin suppressant currently in the clinic (YM155) selectively potentiated the effect of doxorubicin, but not other chemotherapeutics in breast cancer cell types, and induced tumor cell apoptosis. CONCLUSIONS: Survivin family proteins are novel effectors of doxorubicin resistance in chemotherapy-naive breast cancer. The incorporation of survivin antagonist(s) in anthracycline-containing regimens may have improved clinical activity in these patients.

miR-296/Scribble axis is deregulated in human breast cancer and miR-296 restoration reduces tumour growth in vivo.

miR-296-5p is a central regulator of signalling pathways affecting development, stem cell differentiation and cancer. We hypothesized that miR-296-5p is involved in breast cancer onset and progression possibly through regulation of its target SCRIB (Scribble), a polarity protein recently implicated in the acquisition of cancer stem cell traits and in cell motility. We found that miR-296-5p levels were consistently reduced in human breast cancer tissues compared with non-neoplastic mammary parenchyma, and low expression of this miRNA predicted shorter disease-free survival independently of classic clinicopathological parameters. Further, reduced miR-296-5p levels were significantly correlated with an earlier spread of cancer in the overall series and with distant metastases in the subset. In contrast with its regulator, SCRIB was overexpressed and mislocalized in primary breast cancers or locoregional or distant metastatic lesions compared with normal parenchyma. Notably, SCRIB mislocalization was associated with overall survival, metastatic spread and organ tropism in patients with breast cancer. Finally, direct injection of a precursor miR-296-5p into tumours of a breast cancer xenograft model significantly decreased tumour growth. Our results show that the miR-296-5p/SCRIB axis plays a role in breast carcinogenesis and an miR-296-5p-based therapeutic approach hampers breast cancer tumour growth in vivo. Modulation of miR-296-5p may represent a new therapeutic option for patients with breast cancer.