Adverse childhood experiences and global mental health: avenues to reduce the burden of child and adolescent mental disorders.

Mental disorders are one of the largest contributors to the burden of disease globally, this holds also for children and adolescents, especially in low- and middle-income countries. The prevalence and severity of these disorders are influenced by social determinants, including exposure to adversity. When occurring early in life, these latter events are referred to as adverse childhood experiences (ACEs).In this editorial, we provide an overview of the literature on the role of ACEs as social determinants of mental health through the lenses of global mental health. While the relation between ACEs and mental health has been extensively explored, most research was centred in higher income contexts. We argue that findings from the realm of global mental health should be integrated into that of ACEs, e.g. through preventative and responsive psychosocial interventions for children, adolescents and their caregivers. The field of global mental health should also undertake active efforts to better address ACEs in its initiatives, all with the goal of reducing the burden of mental disorders among children and adolescents globally.

Radiologically defined lipid-poor adrenal adenomas: histopathological characteristics.

BACKGROUND: Adrenal lipid-poor adenomas (LPA) are defined by high unenhanced density (≥ 10 HU), and absolute and relative contrast medium washout > 60% and > 40%, respectively, at computerized tomography (CT). To date, no thorough histopathological characterization has been performed in those frequent lesions (one-third of adrenal adenomas). Our aim was to analyze the histopathological characteristics of adrenal LPA. METHODS: Patients with LPA (n = 57) were selected among consecutive subjects referred for an adrenal incidentaloma or ACTH-independent Cushing syndrome. FluoroDeoxyGlucose-Positron Emission Tomography (FDG-PET) was performed in 37 patients. In patients treated by adrenalectomy (n = 17), Weiss score and Lin-Weiss-Bisceglia score (in tumors composed entirely or predominantly of oncocytes) were calculated. RESULTS: Radiological parameters did not differ among patients with ACTH-independent Cushing syndrome (n = 6) and those with adrenal incidentalomas associated with primary aldosteronism (n = 2), autonomous cortisol secretion (n = 14), or non-functioning (n = 35). Patients treated by adrenalectomy had larger tumors (28.9 ± 11.2 vs 17.3 ± 8.4 mm, P < 0.001), higher CT unenhanced density (29.1 ± 11.0 vs 23.1 ± 9.0 HU, P = 0.043), and FDG-PET adrenal uptake (9.0 ± 6.4 vs 4.4 ± 2.3 SUV, P = 0.003) than non-operated ones. Oncocytic features > 75% of the tumor were detected in 12/17 cases (70.6%). Five of those showed borderline-malignant histopathological characteristics by Lin-Weiss-Bisceglia score. Among remaining non-oncocytic tumors, 1/5 had a Weiss score ≥ 3. Overall, 6/17 tumors (35.3%) had borderline-malignant potential. Radiological parameters were similar between patients with benign and borderline-malignant tumors. CONCLUSIONS: Adrenal LPA are a heterogeneous group of tumors, mostly composed of oncocytomas. Up to 1/3 of those tumors may have a borderline-malignant potential at histopathology.

Astrochemical evolution along star formation: Overview of the IRAM Large Program ASAI.

Evidence is mounting that the small bodies of our Solar System, such as comets and asteroids, have at least partially inherited their chemical composition from the first phases of the Solar System formation. It then appears that the molecular complexity of these small bodies is most likely related to the earliest stages of star formation. It is therefore important to characterize and to understand how the chemical evolution changes with solar-type protostellar evolution. We present here the Large Program "Astrochemical Surveys At IRAM" (ASAI). Its goal is to carry out unbiased millimeter line surveys between 80 and 272 GHz of a sample of ten template sources, which fully cover the first stages of the formation process of solar-type stars, from prestellar cores to the late protostellar phase. In this article, we present an overview of the surveys and results obtained from the analysis of the 3 mm band observations. The number of detected main isotopic species barely varies with the evolutionary stage and is found to be very similar to that of massive star-forming regions. The molecular content in O- and C- bearing species allows us to define two chemical classes of envelopes, whose composition is dominated by either a) a rich content in O-rich complex organic molecules, associated with hot corino sources, or b) a rich content in hydrocarbons, typical of Warm Carbon Chain Chemistry sources. Overall, a high chemical richness is found to be present already in the initial phases of solar-type star formation.

Laboratory measurements and astronomical search for cyanomethanimine.

CONTEXT: C-cyanomethanimine (HNCHCN), existing in the two Z and E isomeric forms, is a key prebiotic molecule, but, so far, only the E isomer has been detected toward the massive star-forming region. Sagittarius B2(N) using transitions in the radio wavelength domain. AIMS: With the aim of detecting HNCHCN in Sun-like-star forming regions, the laboratory investigation of its rotational spectrum has been extended to the millimeter-/submillimeter-wave (mm-/submm-) spectral window in which several unbiased spectral surveys have been already carried out. METHODS: High-resolution laboratory measurements of the rotational spectrum of C-cyanomethanimine were carried out in the 100-420 GHz range using a frequency-modulation absorption spectrometer. We then searched for the C-cyanomethanimine spectral features in the mm-wave range using the high-sensitivity and unbiased spectral surveys obtained with the IRAM 30-m antenna in the ASAI context, the earliest stages of star formation from starless to evolved Class I objects being sampled. RESULTS: For both the Z and E isomers, the spectroscopic work has led to an improved and extended knowledge of the spectroscopic parameters, thus providing accurate predictions of the rotational signatures up to ~700 GHz. So far, no C-cyanomethanimine emission has been detected toward the ASAI targets, and upper limits of the column density of ~ 1011-1012 cm-2 could only be derived. Consequently, the C-cyanomethanimine abundances have to be less than a few 10-10 for starless and hot-corinos. A less stringent constraint, ≤ 10-9, is obtained for shocks sites. CONCLUSIONS: The combination of the upper limits of the abundances of C-cyanomethanimine together with accurate laboratory frequencies up to ~ 700 GHz poses the basis for future higher sensitivity searches around Sun-like-star forming regions. For compact (typically less than 1″) and chemically enriched sources such as hot-corinos, the use of interferometers as NOEMA and ALMA in their extended configurations are clearly needed.

Determination of Mammalian Target of Rapamycin Hyperactivation as Prognostic Factor in Well-Differentiated Neuroendocrine Tumors.

PURPOSE: To evaluate the role of the activation of mTOR (phosphorylated mTOR, p-mTOR) and the expression SSTR2A and IGF-1R as prognostic factor in well-differentiated neuroendocrine tumors. METHODS: A retrospective study was conducted on data from patients with diagnosis of neuroendocrine tumor originated from pancreas (pNET) or gastrointestinal tract (stomach, appendix, and ileus; GI-NET) made between January 2003 and December 2004 and followed up at our institution. Archival material should be available for revision according to WHO 2010 neuroendocrine tumor classification and for p-mTOR, SSTR2A, and IGF-1R immunostaining, calculating a quantitative score (QS). We evaluated clinical, pathological, and immunohistochemistry features for association with the presence of advanced disease at diagnosis and disease relapse in patients who have undergone radical surgery. RESULTS: Archival material from 64 patients was analyzed (37 pNETs and 27 GI-NETs). In these patients, G2 grading, low SSTR2A QS, and high p-mTOR QS were associated with advanced disease at diagnosis at multivariate analysis. Risk of recurrence in 49 patients with R0-resected tumors was higher for G2 grading, stage IIIB-IV, low IGF-1R QS, and high p-mTOR QS at univariate analysis. CONCLUSIONS: With the limits of retrospective data, activation of m-TOR is correlated with advanced disease at diagnosis and with shorter disease-free survival after R0 resection. Validation through prospective studies is needed.

Inflammatory cell burden and phenotype in endomyocardial biopsies with antibody-mediated rejection (AMR): a multicenter pilot study from the AECVP.

This multicenter case-controlled pilot study evaluated myocardial inflammatory burden (IB) and phenotype in endomyocardial biopsies (EMBs) with and without pathologic antibody-mediated rejection (pAMR). Sixty-five EMBs from five European heart transplant centers were centrally reviewed as positive (grade 2, n = 28), suspicious (grade 1, n = 7) or negative (n = 30) for pAMR. Absolute counts of total, intravascular (IV) and extravascular (EV) immunophenotyped mononuclear cells were correlated with pAMR grade, capillary C4d deposition, donor specific antibody (DSA) status and acute cellular rejection (ACR). In pAMR+ biopsies, equivalent number of IV CD3+ T lymphocytes (23 ± 4/0.225 mm(2) ) and CD68+ macrophages (21 ± 4/0.225 mm(2) ) were seen. IB and cell phenotype correlated with pAMR grade, C4d positivity and DSA positivity (p < 0.0001). High numbers of IV T lymphocytes were associated with low grade ACR (p = 0.002). In late-occurring AMR EV plasma cells occurring in 34% of pAMR+ EMBs were associated with higher IB. The IB in AMR correlated with pAMR+, C4d positivity and DSA positivity. In pAMR+ equivalent numbers of IV T lymphocytes and macrophages were found. The presence of plasma cells was associated with a higher IB and occurrence of pAMR late after transplantation.

Detection of metastases from differentiated thyroid cancer by different imaging techniques (neck ultrasound, computed tomography and [18F]-FDG positron emission tomography) in patients with negative post-therapeutic ¹³¹I whole-body scan and detectable serum thyroglobulin levels.

INTRODUCTION: DTC patients having detectable Tg and negative post-therapeutic (131)I-WBS have to be investigated by different imaging techniques to detect metastases. PURPOSE: Comparison of neck US, CT and [18F]-FDG PET scan. METHODS: In 49 DTC patients with biochemical disease, neck was examined by US, CT and [18F]-FDG PET. FNA was performed and Tg was determined by FNA-Tg in selected cases of suspicious lymph nodes. Thorax was examined by CT and PET. Serum Tg was measured on LT4 therapy (basal Tg) and after the stimulation with recombinant human TSH (peak Tg). RESULTS: A thyroid remnant was seen by US, CT and PET in eight patients; recurrences were seen by US, CT and PET in six, five and five patients, respectively. Two metastatic nodes were identified by US and CT but not by PET. Lung micronodules were detected by CT in 7/49 (14.3 %) patients and by FDG PET in three of them. Basal Tg ranged from 0.5-1,725 ng/ml while peak Tg ranged from 0.5 to 2,135 ng/ml: the distribution between positive and negative patients was similar. Bone scan was negative in all cases. CONCLUSIONS: In DTC patients with detectable Tg and negative I-131 post-therapy WBS, imaging examination revealed remnant or metastases in 43 % of cases. Remnant and recurrences were equally detected by the three techniques; US was better than [18F]-FDG PET for lymph node metastases since this latter method can give false both positive and negative results; chest examination is best made by CT versus FDG PET due to its higher spatial resolution.

Van-Gogh-like 2 antagonises the canonical WNT pathway and is methylated in colorectal cancers.

BACKGROUND: Aberrant activation of the canonical WNT signaling is a feature of colorectal cancer (CRC). Van-Gogh-like 2 (VANGL2) belongs to the non-canonical WNT pathway whose activation inhibits canonical WNT signaling. In this study, we investigated the role of VANGL2 and its epigenetic regulation in CRC. METHODS: Van-Gogh-like 2 expression and promoter methylation after 5-aza-2'-deoxycytidine (5-aza) treatment were evaluated in CRC cells. DNA samples from 418 sporadic CRCs were tested for VANGL2 promoter methylation and microsatellite instability (MSI). Proliferation, colony formation and activation of the WNT pathway were tested in cells after VANGL2 overexpression. RESULTS: Van-Gogh-like 2 mRNA was significantly higher in 5-aza-treated RKO, LOVO and SW48, whereas no differences were found in SW480. Van-Gogh-like 2 was fully methylated in RKO, SW48, HCT116, DLD1 and Caco2; partially methylated in LOVO, LS174T and SW837; and unmethylated in SW480, SW620 and HT29. Higher expression of VANGL2 mRNA was found in the unmethylated cell lines. In CRC specimens (8.93% MSI), methylated VANGL2 was associated with MSI, higher grade, proximal colon location and BRAF mutation. Van-Gogh-like 2 overexpression in SW480 significantly decreased proliferation, colony formation and ß-catenin levels. CONCLUSION: Van-Gogh-like 2 is frequently methylated in MSI-CRCs with BRAF mutation and may act as a tumour suppressor gene, counteracting WNT/ß-catenin signaling.

Salivary glands ultrasound examination after radioiodine-131 treatment for differentiated thyroid cancer.

BACKGROUND: The most important side effect of radioiodine ((131)I) therapy is sialoadenitis and xerostomy. AIM: To evaluate by ultrasound (US) parotid and submandibular glands after (131)I therapy for differentiated thyroid cancer (DTC). PATIENTS: Seventy-six subjects thyroidectomized for DTC submitted to salivary glands US examination. Forty-three of them had been previously treated with (131)I: 22 with 1.11 GBq (30 mCi) for remnant ablation, and 21 with higher doses [up to 44.4 GBq (1200 mCi)] for metastases. Thirty-three subjects studied before (131)I therapy served as controls. Parotid and submandibular volume, homogeneity, and echogenicity were determined. (131)I-treated patients filled a questionnaire about sialoadenitis symptoms. RESULTS: Parotid gland volume was significantly higher in treated patients (28.3±16.2 ml) than in untreated patients (20.7±10.4 ml, p=0.0154) and related to the time from last (131)I therapy. Three had parotid volume <1.5 ml and complained severe xerostomy. Submandibular gland volume was similar in treated (11.2±7.6 ml) and untreated patients (8.6±4.2 ml, p=0.0602). Homogeneity and echogenicity were similar in treated and untreated patients. Sialoadenitis symptoms were reported in 26% and were related to the (131)I cumulative dose. Symptoms were not related to gland volume. Hypoechogenicity and inhomogeneity of the parotids were more frequent in patients with salivary stickiness. CONCLUSION: Parotid, but not submandibular, volume is increased after (131)I treatment depending on the received activity and the time from irradiation but not on sialoadenitis symptoms. Xerostomy is associated to gland atrophy at US.

Nuclear receptors agonists exert opposing effects on the inflammation dependent survival of breast cancer stem cells.

Recent literature highlights the importance of pro-inflammatory cytokines in the biology of breast cancer stem cells (CSCs), unraveling differences with respect to their normal counterparts. Expansion of mammospheres (MS) is a valuable tool for the in vitro study of normal and cancer mammary gland stem cells. Here, we expanded MSs from human breast cancer and normal mammary gland tissues, as well from tumorigenic (MCF7) and non-tumorigenic (MCF10) breast cell lines. We observed that agonists for the retinoid X receptor (6-OH-11-O-hydroxyphenanthrene), retinoic acid receptor (all-trans retinoic acid (RA)) and peroxisome proliferator-activated receptor (PPAR)-γ (pioglitazone (PGZ)), reduce the survival of MS generated from breast cancer tissues and MCF7 cells, but not from normal mammary gland or MCF10 cells. This phenomenon is paralleled by the hampering of pro-inflammatory Nuclear Factor-κB (NF-κB)/Interleukin-6 (IL6) axis that is hyperactive in breast cancer-derived MS. The hindrance of such pathway associates with the downregulation of MS regulatory genes (SLUG, Notch3, Jagged1) and with the upregulation of the differentiation markers estrogen receptor-α and keratin18. At variance, the PPARα agonist Wy14643 promotes MS formation, upregulating NF-κB/IL6 axis and MS regulatory genes. These data reveal that nuclear receptors agonists (6-OH-11-O-hydroxyphenanthrene, RA, PGZ) reduce the inflammation dependent survival of breast CSCs and that PPARα agonist Wy14643 exerts opposite effects on this phenotype.

Gene expression profile of human colon cancer cells treated with cross-reacting material 197, a diphtheria toxin non-toxic mutant.

Cross-Reacting Material 197 (CRM197) is a diphtheria toxin non-toxic mutant that has shown antitumor activity in mice and humans. It is still unclear whether this anti-tumorigenic effect depends on its strong inflammatory-immunological property, its ability to inhibit heparin-binding epidermal growth factor (HB-EGF), or even its possible weak toxicity. CRM197 is utilized as a specific inhibitor of HB-EGF that competes for the epidermal growth factor receptor (EGFR), overexpressed in colorectal cancer and implicated in its progression. In this study we evaluate the effects of CRM197 on HT-29 human colon cancer cell line behaviour and, for CRM197 recognized ability to inhibit HB-EGF, its possible influence on EGFR activation. In particular, while HT-29 does not show any reduction of viability after CRM197 treatment (MTT modified assay), or changes in cell cycle distribution (flow cytometry), in EGFR localization, phospho-EGFR detected signals (immunohistochemistry) or in morphology (scanning electron microscopy, SEM) they show a change in the gene expression profile by microarray analysis (cDNA microarray SS-H19k8). The overexpression of genes like protein phosphatase 2, catalytic subunit, alpha isozyme (PPP2CA), guanine nucleotide-binding protein G subunit alpha-1(GNAI1) and butyrophilin, subfamily 2, member A1 (BTN2A1) has been confirmed with real-time-qPCR. This is the first study where the CRM197 treatment on HT-29 shows a possible scarce implication of endogenous HB-EGF on EGFR expression and cancer cell development. At the same time, our results show the alteration of a specific and selected number of genes.

Prospective study on the FDG-PET/CT predictive and prognostic values in patients treated with neoadjuvant chemoradiation therapy and radical surgery for locally advanced rectal cancer.

BACKGROUND: 2-[fluorine-18]fluoro-2-deoxy-D-glucose-positron emission tomography/computed tomography (FDG-PET/CT) was carried out before and after neoadjuvant chemoradiotherapy (NCRT) followed by radical surgery for locally advanced rectal cancer (LARC). The aim of this study was to define its predictive and prognostic values. PATIENTS AND METHODS: Patients with cT3-T4 N-/+ carcinoma of medium/low rectum received daily 5-fluorouracil-based chemotherapy infusion and radiation therapy on 6-week period followed by surgery 7-8 weeks later. Tumour metabolic activity, expressed as maximum standardised uptake value (SUV-1 = at baseline and SUV-2 = pre-surgery), was calculated in the most active tumour site. Predictive and prognostic values of SUV-1, SUV-2 and Δ-SUV (percentage change of SUV-1 - SUV-2) were analysed towards pathological response (pR) in the surgical specimen and disease recurrence, respectively. RESULTS: Eighty consecutive patients entered the study. SUV-1, SUV-2 and Δ-SUV appeared singly correlated with pR, but not one of them resulted an independent predictive factor at multivariate analysis. After a median follow-up of 44 months, 13 patients (16.2%) presented local and/or distant recurrence. SUV-2 ≤5 was associated with lower incidence of disease recurrence and resulted prognostic factor at multivariate analysis. CONCLUSIONS: Dual-time FDG-PET/CT in patients with LARC treated with NCRT and radical surgery supplies limited predictive information. However, an optimal metabolic response appears associated with a favourable patient outcome.

Somatostatin analogues do not affect calcium metabolism in patients with acromegaly and primary hyperparathyroidism [corrected] due to MEN 1-like syndrome.

Patients with clinical features of MEN 1 without mutations in the menin gene fulfill the criteria of MEN1-like syndrome. Primary hyperparathyroidism (PHP) is the most frequent clinical finding in both syndromes and is usually treated by surgery. However, PHP has been reported to respond to somatostatin analogues (SSA) in MEN 1 patients. 7 patients with PHP in the context of MEN 1-like syndrome (and absence of mutations in the menin gene) were enrolled in the study and treated with SSA for 6 months for the non-PHP disease before parathyroidectomy. Serum ionized calcium, phosphorus, and PTH concentrations, and 24-h urinary calcium and phosphorus excretion were measured before and after SSA therapy. Mean serum ionized calcium, phosphorus, and PTH concentrations did not significantly change after a 6-month course with SSA. SSA scintigraphy did not reveal uptake in the neck region corresponding to the parathyroid adenoma identified at surgery and confirmed at histology. However, immunohistochemistry revealed SS-type 2A receptor in parathyroid tissue samples of 6 out of 7 patients. SSA therapy does not affect calcium-phosphorus metabolism in patients with MEN 1-like syndrome, suggesting that the drug has no role in controlling PHP in these subset of patients.

Molecular detection of epidermal growth factor receptor in colorectal cancer: does it still make sense?

AIM: The aim of the study was to detect and compare the epidermal growth factor receptor (EGFr) content using different methods, to establish whether the quantitative detection and functional study of EGFr in colorectal cancer, using methods other than immunohistochemistry (IHC), are appropriate. METHOD: Analysis of EGFr by IHC was performed in 230 colorectal cancer patients using monoclonal anti-EGFr. Total and activated EGFr (pY1068) contents were determined in 92 patients and real-time PCR, to determine the level of EGFr messenger RNA, was carried out in 60 patients. RESULTS: There was no association between EGFr IHC groups and the mean total EGFr levels measured using ELISA. CONCLUSION: The study shows that the results of different EGFr detection methods do not correlate with each other. Hence, the real role of EGFr in colorectal cancer remains unsettled. Clinically, the receptor itself does not seem to be important and it would be better to focus on EGFr signalling in downstream pathways.

Role of recombinant human TSH in the management of large euthyroid multinodular goitre: a new therapeutic option? Pros and cons.

Conventional 131I treatment has been used in the last 20 years for large nodular goitres when patients present high surgical risk or simply refuse surgery. 131I therapy causes a mean goitre volume reduction of about 40% after one year. However, the individual response is variable and for low radioiodine uptake and very large goitres, high 131I activities are needed in order to have a adequate 131I accumulation in the thyroid. rhTSH is approved for thyroid cancer management and has been tested off label in large goitres, in whom increases 131I uptake, thus reducing the 131I amount to be administered. The use of lower 131I activities allows to reduce the radiation burden to body and the time of social life restriction. Moreover, depending on the radiation regulations of the different countries, the 131I therapy could be carried out either as outpatients or in a shorter hospitalization period, implying a decrease of costs. The effects of rhTSH on goitre may be due not only to the 131I uptake increase, but also to a more homogeneous distribution of 131I in the gland, and to the thyroid cell activation that makes them more radiosensitive. Acute adverse effects are due to the surge of thyroid hormone in blood and to the goitre volume increase, that cause cardiac symptoms and tracheal compression, respectively. These effects are probably dose dependent and are negligible for rhTSH lower doses.

Recombinant human TSH (rhTSH) in 2009: new perspectives in diagnosis and therapy.

Thyrotropin stimulating hormone (TSH) exerts a physiological stimulus to growth, function and ability to take up iodine of both normal and malignant follicular cells. For this reason, the metastases deriving from well differentiated thyroid cancer (DTC) can be effectively treated with radioiodine but this procedure requires a strong TSH stimulus that can be obtained by withdrawing the L-thyroxine (LT4) therapy. However, both the social and personal life of patients while they are withdrawing the LT4 therapy are heavily affected by hypothyroidism. After more than a decade since the development of recombinant human TSH (rhTSH), this molecule has been introduced in the clinical practice (1998 in USA and 2001 in Europe) as a safe and reliable alternative to LT4 withdrawal. For several years the main clinical application of rhTSH was for diagnostic purposes (i.e. thyroglobulin stimulation) but, after the more recent demonstration of its efficacy in preparing DTC patients for radioiodine post surgical thyroid remnant ablation, also this application has been officially recognized worldwide. The validity of rhTSH has been also demonstrated in stimulating metastatic thyroid cancer cells but this employment is not yet officially approved and it can be used only in patients with contraindication to hypothyroidism (i.e. "compassionate use"). Other possible uses of rhTSH stimulation are related to its ability to enhance both 18FDG uptake during PET scan of metastatic DTC patients and the effectiveness of conventional chemotherapy. The aim of this review was to recall how the rhTSH has been developed and progressively introduced in the clinical management of DTC patients.

High prevalence of retinoblastoma protein loss in triple-negative breast cancers and its association with a good prognosis in patients treated with adjuvant chemotherapy.

BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive disease, nevertheless exhibiting a high response rate to chemotherapy. Since the retinoblastoma protein (pRb) loss confers a high sensitivity to chemotherapy regimens, we evaluated the prevalence of pRb loss in TNBCs and its relevance on the clinical outcome of patients treated with adjuvant chemotherapy. PATIENTS AND METHODS: pRb status was prospectively evaluated by immunocytochemistry in 518 consecutive patients with complete receptor information. The predictive value of pRb status in TNBCs was determined according to the adjuvant therapeutic treatments. RESULTS: Fifty-three tumors were identified as TNBCs. The prevalence of pRb loss was significantly higher in TNBCs than in the other cancer subtypes. All patients with TNBCs lacking pRb and treated with systemic chemotherapy (cyclophosphamide, methotrexate and 5-fluorouracil) were disease free at a medium follow-up time of 109 months, whereas the clinical outcome of those expressing pRb was significantly poorer (P = 0.008). Analysis of disease-free survival including the established anatomo-clinical prognostic parameters indicated pRb loss as the only significant predictive factor. CONCLUSIONS: pRb loss is much more frequent in TNBCs than in the other breast cancer subtypes. Patients with TNBCs lacking pRb had a very favorable clinical outcome if treated with conventional adjuvant chemotherapy.

The basal-like breast carcinoma phenotype is regulated by SLUG gene expression.

Basal-like breast carcinoma is an aggressive form of breast cancer, characterized by the absence of oestrogen receptor and HER2 expression, the presence of cytokeratin 5 and epidermal growth factor receptor expression, and by the up-regulation of stem cell regulatory genes. We show here that tumour tissues expressing high levels of SLUG mRNA show a basal-like breast carcinoma phenotype and that such tumours also express high levels of stem cell-regulatory genes, ie CD133, Bmi1. Further, we show that stem/progenitor cells, isolated from ductal breast carcinoma and from normal mammary gland as mammospheres, express SLUG, CD133, and Bmi1 mRNA and show a phenotype similar to that of basal-like breast carcinoma. We also report that SLUG expression in tumour tissues correlates with that of the hypoxia survival gene carbonic anhydrase IX. In this regard, we report that the exposure of SLUG-negative/luminal-like MCF-7 cells to a hypoxic environment promotes the onset of the basal-like breast carcinoma phenotype, together with up-regulation of the SLUG gene, which in turn blunts oestrogen receptor-alpha and boosts carbonic anhydrase IX gene expression. Finally, we show that SLUG expression promotes the invasiveness of MCF-7 cells exposed to hypoxia and sustains the in vivo aggressiveness of hypoxia-selected, MCF-7-derived cells in xenografts. These data indicate that SLUG gene expression is part of a hypoxia-induced genetic programme which sets up a basal/stem cell-like, aggressive phenotype in breast cancer cells.

Controversial relationship between the expression of the RB pathway components and RB protein phosphorylation in human breast cancer.

Recent data challenge the relevance of the RB pathway to cancer based on RB inactivation, at least in breast tumors. To obtain information on the actual role of the components of the RB pathway in tumor progression we decided to investigate whether their quantitative changes were associated with variations in the level of RB phosphorylation in human breast cancer. A series of 68 human primary breast carcinomas was studied. Five cases were excluded from the study due to their lack of RB expression. In the remaining 63 cases the expression of cyclin D1, cdk4, cyclin E, and INK4a mRNA was assessed by real-time RT-PCR. The level of RB phosphorylated protein (ppRB) and p27 expression was immunohistochemically analyzed by measuring the percentage of stained cells (labeling index, LI). Cell proliferation rate was measured by Ki67 LI evaluation. The ppRB LI ranged from 5.2 to 73.8 and, as expected, was strongly related to the Ki67 LI (r=0.80; p<0.001). The expression of cyclin D1 mRNA, expressed in arbitrary units (a. u.), ranged from 1.15 to 123.0 and was inversely related to the ppRB LI (p=0.021) and Ki67 LI (p<0.001). Neither the cdk4 (range from 0.07 to 1.13 a. u.) nor the cyclin E (range from 0.13 to 9.27 a. u.) mRNA expression was significantly associated with the ppRB LI (p=0.962 and p=0.103, respectively). Cyclin E was related to Ki67 LI (p=0.022). Both INK4a mRNA (range from 0.01 to 0.60 a. u.) and p27 (LI from 0.0 to 73.1) values were inversely related to the ppRB LI (p=0.022 and p=0.014, respectively). Cyclin D1, cdk4, and cyclin E mRNA expressions were not significantly related to one another. In human primary breast cancers, the expression levels of the factors known to facilitate the cell cycle progression by RB protein phosphorylation were not positively related to ppRB-LI. Pathological increases of cyclin D, cdk4, and cyclin E are very likely associated with other biological functions other than their well-established action on cell cycle progression.

Relationship between the RB1 mRNA level and the expression of phosphorylated RB protein in human breast cancers: their relevance in cell proliferation activity and patient clinical outcome.

The aim of the present study was to ascertain the relationship between the level of RB1 mRNA and the expression of phosphorylated RB protein and the relevance of these two parameters in cancer cell proliferation and clinical outcome in human breast cancer. Sixty-eight primary human breast cancers were considered. The amount of RB1 mRNA was evaluated by quantitative RT-PCR analysis. The level of RB phosphorylation was immunohistochemical defined by measuring the phosphorylated (pp) RB labelling index (LI). Cell proliferation rate was measured by calculating the Ki67 LI. No relation was found between the RB1 mRNA level and the ppRB LI (p=0.565). Both RB1 mRNA value and ppRB LI were related (in an inverse and direct manner, respectively) to Ki67 LI. RB1 mRNA expression was more strictly associated with KI67 LI (p=0.001) than the ppRB LI (p=0.013). Regarding the patient clinical outcome, the separately considered RB parameters did not reach the prognostic significance. However, patients with low RB1 mRNA quantity and patients with high ppRB LI, taken together, had a significantly shorter disease free and overall survival than the group comprehending patients with high RB1 mRNA value and low ppRB LI, and this despite the low number of patients considered. Our results demonstrated that the ppRB LI was independent of the RB1 mRNA level; that both RB parameters are related to the cell proliferation rate and, if collectively considered, have a high informative value on breast tumour prognosis.