CMA mediates resistance in breast cancer models.

BACKGROUND: Breast cancer (BC) is the most common malignancy in women and the second leading cause of cancer-related death; chemoresistance is still a clinical challenge mainly because of the different molecular features of this kind of tumour. Doxorubicin (Doxo) is widely used despite its adverse effects and the common onset of resistance. Chaperone-Mediated Autophagy (CMA) has been identified as an important mechanism through which chemotherapeutics can exert their cytotoxic effects and, in this context, LAMP-2A, the key player of CMA, can be a useful biomarker. METHODS: A cohort of patients and breast cancer cells have been screened for Doxo effect and CMA activation by analysing the LAMP-2A level. Molecular silencing has been used to clarify CMA role in BC responsiveness to treatments. Low Doxo doses were combined with other drugs (TMZ or PX-478, a HIF-1α inhibitor) to evaluate their cytotoxic ability and their role in modulating CMA. RESULTS: In this paper, we showed that CMA is an important mechanism mediating the responsiveness of breast cancer cell to different treatments (Doxo and TMZ, as suggested by triple negative cells that are TMZ-resistant and fails to activate CMA). The LAMP-2A expression level was specific for different cell lines and patient-derived tumour subtypes, and was also useful in discriminating patients for their survival rates. Moreover, molecular silencing or pharmacological blockage of HIF-1α activity reverted BC resistance to TMZ. The combination of low-dose Doxo with TMZ or PX-478 showed that the drug associations have synergistic behaviours. CONCLUSION: Here, we demonstrated that CMA activity exerts a fundamental role in the responsiveness to different treatments, and LAMP-2A can be proposed as a reliable prognostic biomarker in breast cancer. In this context, HIF-1α, a potential target of CMA, can also be assessed as a valuable therapeutic target in BC in view of identifying new, more efficient and less toxic therapeutic drug combinations. Moreover, the possibility to combine Doxo with other drugs acting on different but coherent molecular targets could help overcome resistance and open the way to a decrease in the dose of the single drugs.

Diagnostic Circulating miRNAs in Sporadic Amyotrophic Lateral Sclerosis.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by the neurodegeneration of motoneurons. About 10% of ALS is hereditary and involves mutation in 25 different genes, while 90% of the cases are sporadic forms of ALS (sALS). The diagnosis of ALS includes the detection of early symptoms and, as disease progresses, muscle twitching and then atrophy spreads from hands to other parts of the body. The disease causes high disability and has a high mortality rate; moreover, the therapeutic approaches for the pathology are not effective. miRNAs are small non-coding RNAs, whose activity has a major impact on the expression levels of coding mRNA. The literature identifies several miRNAs with diagnostic abilities on sALS, but a unique diagnostic profile is not defined. As miRNAs could be secreted, the identification of specific blood miRNAs with diagnostic ability for sALS could be helpful in the identification of the patients. In the view of personalized medicine, we performed a meta-analysis of the literature in order to select specific circulating miRNAs with diagnostic properties and, by bioinformatics approaches, we identified a panel of 10 miRNAs (miR-193b, miR-3911, miR-139-5p, miR-193b-1, miR-338-5p, miR-3911-1, miR-455-3p, miR-4687-5p, miR-4745-5p, and miR-4763-3p) able to classify sALS patients by blood analysis. Among them, the analysis of expression levels of the couple of blood miR-193b/miR-4745-5p could be translated in clinical practice for the diagnosis of sALS.

Finite-Temperature Disordered Bosons in Two Dimensions.

We study phase transitions in a two dimensional weakly interacting Bose gas in a random potential at finite temperatures. We identify superfluid, normal fluid, and insulator phases and construct the phase diagram. At T=0 one has a tricritical point where the three phases coexist. The truncation of the energy distribution at the trap barrier, which is a generic phenomenon in cold atom systems, limits the growth of the localization length and in contrast to the thermodynamic limit the insulator phase is present at any temperature.

Pathway-based expression profile for breast cancer diagnoses.

Microarray experiments have made possible to identify breast cancer marker gene signatures. However, gene expression-based signatures present limitations because they do not consider metabolic role of the genes and are affected by genetic heterogeneity across patient cohorts. Considering the activity of entire pathways rather than the expression levels of individual genes can be a way to exceed these limits. We evaluated and compared five methods of pathway-level aggregation of gene expression data. Our results confirmed the important role of pathway expression profile in breast cancer diagnostic classification (accuracy >90%). However, although assessed on a limited number of samples and datasets, this study shows that using dissimilarity representation among patients does not improve the classification of pathway-based expression profiles.

Isolation of canine mammary cells with stem cell properties and tumour-initiating potential.

Recent data suggest that mammary carcinogenesis may be driven by cancer stem cells (CSCs) derived from mutated adult stem cells, which have acquired aberrant cell self-renewal or by progenitor cells that have acquired the capacity for cell self-renewal. Spontaneous mammary cancers in cats and dogs are important models for the understanding of human breast cancer and may represent alternative species model systems that can significantly contribute to the study of human oncogenesis. With the goal of identifying markers for isolating human breast CSCs, we have generated a canine model system to isolate and characterize normal and CSCs from dog mammary gland. Insight into the hierarchical organization of canine tumours may contribute to the development of universal concepts in oncogenesis by CSCs. Cells with stem cell properties were isolated from normal and tumoural canine breast tissue and propagated as mammospheres and tumourspheres in long-term non-adherent culture conditions. We showed that cells obtained from spheres that display self-renewing properties, have multi-lineage differentiation potential, could generate complex branched tubular structures in vitro and form tumours in NOD/SCID mice. We analysed these cells for the expression of human stem and CSC markers and are currently investigating the tumour-initiating properties of these cells and the hierarchical organization of normal and neoplastic canine mammary tissue.

[An unusual localization of thryoglossal duct cyst]. / Localizzazione inusuale di cisti del dotto tireoglosso.

Thyroglossal duct cysts are most common neck masses after benign lymphonodes. They originate from primitive thiroglossal duct, so they could be locate along its course. Every mass in the middle line of the neck can be considered as a thyroglossal cyst. Best treatment is surgery (Sistrunk procedure). We present a case of unusual localization at floor of the mouth of thyroglossal in a 34 years old woman. To our knowledge in literature, only two cases, have been reported both.

Distinct populations of tumor-initiating cells derived from a tumor generated by rat mammary cancer stem cells.

Tumors derived from rat LA7 cancer stem cells (CSCs) contain a hierarchy of cells with different capacities to generate self-renewing spheres and tubules serially ex vivo and to evoke tumors in vivo. We isolated two morphologically distinct cell types with distinct tumorigenic potential from LA7-evoked tumors: cells with polygonal morphology that are characterized by expression of p21/(WAF1) and p63 and display hallmarks of CSCs and elongated epithelial cells, which generate tumors with far less heterogeneity than LA7 CSCs. Serial transplantation of elongated epithelial cells results in progressive loss of tumorigenic potential; tumor heterogeneity; CD44, E-cadherin, and epithelial cytokeratin expression and increased alpha-smooth muscle actin I and vimentin expression. In contrast, serial transplantation of LA7 CSCs can be performed indefinitely and results in tumors that maintain their heterogeneity, consistent with self-renewal and multilineage differentiation potential. Collectively, our data show that polygonal cells are CSCs, whereas epithelial elongated cells are lineage-committed progenitors with tumorigenic potential, and suggest that tumor progenitors, although lacking indefinite self-renewal potential, nevertheless may make a substantial contribution to tumor development. Because LA7 cells can switch between conditions that favor maintenance of pure CSCs vs. differentiation into other tumor cell types, this cell system provides the opportunity to study factors that influence CSC self-renewal and differentiation. One factor, p63, was identified as a key gene regulating the transition between CSCs and early progenitor cells.

The properties of a mammary gland cancer stem cell.

The cancer stem cell hypothesis posits that tumors are derived from a single cancer-initiating cell with stem cell properties. The task of identifying and characterizing a single cancer-initiating cell with stem cell properties has proven technically difficult because of the scarcity of the cancer stem cells in the tissue of origin and the lack of specific markers for cancer stem cells. Here we show that a single LA7 cell derived from rat mammary adenocarcinoma has the following properties: the differentiation potential to generate all of the cell lineages of the mammary gland; the ability to generate branched duct-like structures that recapitulate morphologically and functionally the ductal-alveolar-like architecture of the mammary tree; and the capacity to initiate heterogeneous tumors in nonobese diabetic-SCID mice. In addition, we show that cultured cells derived from tumors generated by a single LA7 cell-injection have properties similar to LA7 cells, can generate all of the cell lineages of the mammary gland, and recapitulate the ductal-alveolar-like architecture of the mammary tree. The properties of self-renewal, extensive capacity for proliferation, multilineage differentiation potential, and single-cell tumor-initiation potential suggest that LA7 cells are cancer stem cells and can be used as a model system to study the dynamics of tumor formation at the single-cell level.

Pretreatment haemoglobin levels significantly predict the tumour response to primary chemotherapy in human breast cancer.

The purpose of this study was to evaluate whether tumour response to primary chemotherapy in human breast cancer is influenced by baseline haemoglobin (Hb) status. A total of 157 patients with T2-4, N0-1 M0 breast cancer were treated with chemotherapy consisting of either the CMF regimen + tamoxifen (the first 76 cases) or the single-agent epirubicin (the subsequent 81) before definitive surgery. In total, 144 patients were fully assessable. Ki67, p53, bcl-2, c-erbB2, steroid hormone receptor, and microvessel density were evaluated immunohistochemically in tumour specimens obtained before chemotherapy and at surgery. Tumour shrinkage >50% occurred in 72.1% of patients. Responding patients had higher baseline Hb levels and red blood cell counts than nonresponders (P<0.01 and <0.003, respectively). The distribution of disease response according to increasing cutoffs of baseline Hb status showed that from 12.5 mg l(-1) onwards, patients with Hb levels above the cutoff obtained a greater response rate than those with lower Hb values. The difference attained the statistical significance at 12.5 (76.1 vs 59.5%, P<0.05) and 13.0 g/dl(-1) (81.0 vs 57.6%, P<0.002) cutoffs, respectively. The predictive role of Hb levels was maintained in multivariate analysis after adjustment for clinical and biological characteristics and treatment regimen. Patients with baseline Hb levels

Prognostic value of Goseki histological classification in adenocarcinoma of the cardia.

Various histologic classification systems have been proposed as prognostic factors for gastric cancer. We assessed the prognostic value of Goseki classification as well as the TNM staging system, histological tumour grading, Lauren, WHO, Goseki and Siewert classifications in 100 patients with cardia carcinoma undergoing curative surgery. Two patients were lost at follow-up. The median time of follow-up in the remaining patients was 32.9 months after surgery (range: 0.1-142.1 months). No differences in survival rates were observed according to tumour grading, Lauren or WHO histologic or Siewert topographical classification. No differences were found according to Goseki classes, when considering either the mucin content of the carcinoma (types I and III vs II and IV) or the differentiation grade (types I and II vs III and IV). Multivariate analysis showed that the only lymph node positivity was a significant predictor of survival: 7.2% of patients with, but 41.5% of those without nodal involvement were alive after five years (P=0.0001). In conclusion, we found no prognostic role for Goseki or the traditional histological indexes, while the TNM staging system and particularly lymph node positivity were the main predictors of survival in patients with cardia adenocarcinoma.

Sudden improvement of respiratory failure in a woman with carcinoma of the colon.

We present a case of bronchial and endotracheal metastases completely blocking the left main bronchus and partially occluding the middle lobe bronchus contributing to severe respiratory failure. The patient's lack of consent to laser resection of the mass led to the use of chemotherapy; after the first cycle of treatment a neoplastic mass about 3 cm long was spontaneously expelled with a cough. The expulsion of the metastasis caused rapid improvement of the dyspnea and gas exchange; however, the continuation of the chemotherapy did not bring any further benefit to the patient, who died 115 days after diagnosis.

Video-based system for intraoperative facial nerve monitoring: comparison with electromyography.

OBJECTIVE: To validate a recently developed intraoperative facial nerve monitoring system that is based on video control of facial movements. STUDY DESIGN: In a single-subject design study, involving 15 otoneurosurgical patients, the relationship between intensity of neural stimulation, facial movements, and electrophysiologic voltage were measured. The analysis was performed by measuring the ipsilateral oral commissure displacement in relation to different levels of current administered to the nerve during surgical procedures. SETTING: Electromyography and video system intraoperative facial nerve monitoring. PATIENTS: 15 patients (9 men, 6 women; mean age, 61 yr) undergoing a translabyrinthine approach for removal of acoustic neuroma. RESULTS: Electromyography showed slightly greater sensitivity. With regard to the stimulation-response ratio, facial movement and electromyographic amplitude showed very similar responses. CONCLUSIONS: The video system was considered useful in terms of validity and reliability. Furthermore, the authors' surgical experience showed some limitations of electromyography, especially in terms of electrical artifact during cauterization, totally masking the electrophysiologic monitoring.

[Single-slice and multi-slice computerized tomography: dosimetric comparison with diagnostic reference dose levels]. / Tomografia computerizzata a strato singolo e a strato multiplo: confronto dosimetrico con i livelli diagnostici di riferimento.

PURPOSE: The absorbed dose during clinical examinations of the head, thorax, abdomen and pelvis performed with a single-slice CT scanner and a new multi-slice CT system was measured and compared. Technical parameters, defined at installation and memorized on the two CT machines relate to a standard-sized patient and were considered the reference standard. Our experimental data were also been compared with the Diagnostic Reference Levels (D.L. 26/5/2000 n.187, Annex V). MATERIAL AND METHODS: We compared the performance of a multi-slice GE LightSpeed QX/i ADVANTAGE to that of a single-slice GE ProSpeed SX. The radiation beam profiles were measured at isocenter using a phosphor plate. Dose measurements were performed, according to the EUR 16262 EN Guidelines, with a 10-cm long CT pencil ionisation chamber and two PMMA phantoms (CEI EN 61223-2-6) for head and body respectively. RESULTS: The obtained (normalised and weighted) computed tomographic dose index (nCTDIW) values were systematically higher for the multi-slice system (up to 36%) and the dose-length product (DLP) values on the multi-slice scanner exceeded the equivalent single-slice DLP values. The values were, however, always lower than DRLs, except in the case of the head multi-slice protocol, the technical parameters of which need to be improved. Our results allowed moreover to calibrate the automatic dose evaluation system of the multi-slice system, which systematically underestimated DLP values. DISCUSSION AND CONCLUSIONS: The comparison showed that the multi-slice scanner delivers a higher dose compared to the single-slice scanner. This is due to the radiation beam profile which is wider than the total active detector width, to the shorter focal spot-to-isocenter distance and to the effective scan length, which is longer than the nominal irradiated volume because the reconstruction algorithm of a multi-slice helical CT image requires the projection data from all detector rows. Nevertheless, the technology of new CT systems equipped with a multiple row detectors array can improve the protection of the patient thanks to very short irradiation time (less than 1 s) and reduced current values. In order to optimize the dose to the patient some acquisition parameters have been adjusted for head examinations.

The C-terminal domain of yeast Ero1p mediates membrane localization and is essential for function.

In eukaryotes, members of the Ero1 family control oxidative protein folding in the endoplasmic reticulum (ER). Yeast Ero1p is tightly associated with the ER membrane, despite cleavage of the leader peptide, the only hydrophobic sequence that could mediate lipid insertion. In contrast, human Ero1-Lalpha and a yeast mutant (Ero1pDeltaC) lacking the 127 C-terminal amino acids are soluble when expressed in yeast. Neither Ero1-Lalpha nor Ero1pDeltaC complements an ERO1 disrupted strain. Appending the yeast C-terminal tail to human Ero1-Lalpha restores membrane association and allows growth of ERO1 disrupted cells. Therefore, the tail of Ero1p mediates membrane association and is crucial for function.

Relationship between tumour shrinkage and reduction in Ki67 expression after primary chemotherapy in human breast cancer.

The association between tumour shrinkage and reduction in kinetic cell activity after primary chemotherapy in human breast cancer is still a matter of investigation. 157 patients with T2-4, N0-1, M0 breast cancer received primary chemotherapy consisting of either the CMF regimen + tamoxifen (the first consecutive 76 cases) or the single agent epirubicin (the subsequent 81). Ki67, p53, bcl2, c-erbB2 and steroid hormone receptors were evaluated immunohistochemically in tumour specimens obtained before chemotherapy and at surgery. Tumour shrinkage of >50% occurred in 72.4% of patients. Ki67 expression significantly decreased after chemotherapy; the reduction correlated with tumour response in both univariate (P < 0.005) and multivariate analysis (P = 0.02). p53, bcl-2, steroid hormone receptor and c-erbB2 immunostaining were scarcely affected. Baseline bcl2 (P = 0.04) and c-erbB2 (P = 0.02) were directly and inversely associated with the reduction in Ki67 immunostaining, respectively. Baseline p53 expression (P < 0.01) was directly related with Ki67 expression at residual tumour, whereas oestrogen receptor expression (P < 0.001) was inversely related. Ki67 at residual tumour was a better predictor for relapse-free survival (RFS) than baseline Ki67. Clinical response (P < 0.03), but not reduction in Ki67, was a significant independent predictor for disease recurrence. Chemotherapy was found to induce tumour shrinkage and to reduce the number of cells in the cell cycle, but its effect on tumour biology/aggressiveness was minimal. Reduction in Ki67 immunostaining correlated with clinical response but failed to be related to RFS. Ki67 expression at surgery rather than at baseline appears to be a better predictor for disease relapse.

Effects of liposome-entrapped annamycin in human breast cancer cells: interference with cell cycle progression and induction of apoptosis.

The effects of liposome-encapsulated annamycin (L-Ann) were investigated in two human breast cancer cell lines, MCF7 and MDA-MB-435. For comparative purposes, doxorubicin (Dx) was used throughout the study. A 4-hour treatment with L-Ann was significantly more active in MDA-MB-435 than in MCF7 cells (IC(50) values of 0.03 and 0.08 microg/ml, respectively), whereas Dx was equally active in the two cell lines (IC(50) 0.12 microg/ml). L-Ann induced an accumulation of cells in G2M phases which was dose-dependent in MDA-MB-435 but not in MCF7 cells. Dx also caused a dose-dependent increase of G2M cell fraction in MDA-MB-435 cells, whereas a G2M cell accumulation was observed only after treatment with the highest Dx concentration in MCF7 cells. G2M phase cell accumulations induced in MCF7 cells by L-Ann or Dx were accompanied by a decrease in cdc2 kinase activity and in cyclin B1 and cdc2 expression. Conversely, in MDA-MB-435 cells exposed to L-Ann or Dx, cdc2 kinase activity, cyclin B1 and cdc2 expression increased in parallel to the increase in the number of cells accumulated in the G2M phase. L-Ann and Dx induced apoptosis in MDA-MB-435 but not in MCF7 cells. In MDA-MB-435 cells exposed to L-Ann or Dx, no change was observed in the expression of bax, but there was a p53-independent increase in p21(waf1) expression. In MCF7 cells, treatment with L-Ann or Dx induced an increase in p53 expression with a consequent transactivation of p21(waf1) and bax. Our results indicate that L-Ann is more cytotoxic than Dx in breast cancer cells and is able to induce apoptosis through p53-independent mechanisms.

p53 but not bcl-2 immunostaining is predictive of poor clinical complete response to primary chemotherapy in breast cancer patients.

Preoperative chemotherapy administered to breast cancer (BC) patients is a model for studying in vivo the interaction between cytotoxic treatment and clinical and biological parameters. Apoptosis induced by anticancer agents is a mechanism of treatment activity; therefore, overexpression of genes inhibiting the apoptotic pathway could produce drug resistant tumors. In the present study, the two most studied inhibitors of apoptosis, the bcl-2 gene and the mutant p53, have been evaluated to assess whether they may play a role in modulating response of BC to primary chemotherapy. From August 1990 to January 1997, 143 patients bearing T(2-4)N(0-1)M0 primary BC were submitted to two different chemotherapeutic regimens before surgery. The first 64 received the cyclophosphamide, methotrexate, 5-fluorouracil (CMF) regimen (on days 1 and 8 and every 28 days thereafter) associated with tamoxifen (30 mg daily) in case of estrogen receptor (ER)-positive BC, and the remaining 79 were submitted to single agent epirubicin (120 mg/m2 every 21 days). The expression of p53, bcl-2, Ki67, ER, progesterone receptor, c-erbB2, and the multidrug resistance P-glycoprotein (gp-170) was evaluated in BC specimens obtained at diagnosis by incision biopsy and at postchemotherapy surgery. At the end of chemotherapy administration (median, 3 cycles; range, 2-6), the clinical complete response (cCR) rate was superimposable in the patient subgroups with bcl-2-positive or -negative primary tumors; conversely, p53 expression, at a cutoff of 10% positive cells, was significantly associated with a lower cCR rate (9.4 versus 27.0%; P < 0.04). p53 was a significant predictor for poor cCR in the subset submitted to epirubicin (3.6 versus 25.5%; P < 0.02; in patients with p53+ and p53- BC, respectively); by contrast, only a trend toward lower cCR has been observed in patients with p53+ tumors receiving CMF +/- tamoxifen with respect to p53- ones. The distribution of cCR according to the gp-170-positive or -negative tumors was 8 versus 22% in patients submitted to epirubicin and 29 versus 30% in those receiving CMF +/- tamoxifen, respectively. In a multivariate regression analysis, after adjusting for treatment administered (epirubicin versus CMF +/- tamoxifen), menopausal status, tumor and node status, histology grade, ER, progesterone receptor, c-erbB2, Ki67, bcl-2, and gp-170 expression, the p53 status maintained an independent predictive role for cCR. Most of the tumors undergoing change in percentage of p53 expression after both treatments originally harbored mutant protein, and only four BC specimens that were p53 negative before chemotherapy became positive afterward. These data confirm in vivo the concept that the responsiveness of tumors to chemotherapy in part derives from the capability of BC cells to undergo apoptosis. The role of mutated p53 in preventing response is more evident in patients submitted to epirubicin, and this may be caused by the up-regulation of multidrug resistance gene expression by p53 inactivation. p53 is a stable phenotype and is not inducible by at least three or four chemotherapy cycles.