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Etiology of human breast cancer is unknown, whereas the Mouse Mammary Tumor Virus (MMTV) is recognized as the etiologic agent of mouse mammary carcinoma. Moreover, this experimental model contributed substantially to our understanding of many biological aspects of the human disease. Several data strongly suggest a causative role of MMTV in humans, such as the presence of viral sequences in a high percentage of infiltrating breast carcinoma and in its preinvasive lesions, the production of viral particles in primary cultures of breast cancer, the ability of the virus to infect cells in culture. This paper demonstrates that MMTV is present in human saliva and salivary glands. MMTV presence was investigated by fluorescent PCR, RT-PCR, FISH, immunohistochemistry, and whole transcriptome analysis. Saliva was obtained from newborns, children, adults, and breast cancer patients. The saliva of newborns is MMTV-free, whereas MMTV is present in saliva of children (26.66%), healthy adults (10.60%), and breast cancer patients (57.14% as DNA and 33.9% as RNA). MMTV is also present in 8.10% of salivary glands. RNA-seq analysis performed on saliva of a breast cancer patient demonstrates a high expression of MMTV RNA in comparison to negative controls. The possibility of a contamination by murine DNA was excluded by murine mtDNA and IAP LTR PCR. These findings confirm the presence of MMTV in humans, strongly suggest saliva as route in inter-human infection, and support the hypothesis of a viral origin for human breast carcinoma.

Papillary thyroid carcinoma is the most frequent histologic type of thyroid tumor. Few studies investigated the role of c-KIT expression in thyroid tumors, suggesting a role for this receptor and its ligand in differentiation and growth control of thyroid epithelium and a receptor loss following malignant transformation. We investigated and correlated c-KIT expression levels and two known markers of thyrocytes differentiation, PAX8 and TTF-1, in malignant and benign cytological thyroid samples. Moreover, we performed functional studies on human papillary thyroid carcinoma cell line to associated c-KIT expression to thyrocytes differentiation and tumor proliferation. c-KIT and PAX8 expression resulted higher in benign samples compared to the malignant ones, and the expression levels of these two genes were significantly correlated to each other. We also observed that c-KIT overexpression led to an increase of PAX8 expression level together with a decrease of proliferation. Furthermore, c-KIT overexpressing cells showed a regression of typical morphological features of malignancy. Taken together these results suggest that c-KIT could be involved in the differentiation of thyroid cells and in tumor progression.

ObjectiveAnaplastic thyroid cancer (ATC) is often unoperable and chemotherapy and radiotherapy are the main treatments. Until now ‘primary ATC cell cultures’ (ANA) have been developed from surgical biopsies. The possibility to obtain ANA from fine-needle aspiration (FNA-ANA) and to test their sensitivity to different drugs could increase the effectiveness of treatments and avoid unnecessary surgical procedures.DesignTo obtain FNA-ANA from six ATC patients before undergoing surgery and to evaluate the chemosensitivity of FNA-ANA to chemotherapeutic agents and thiazolidinediones (TZD).Methods and resultsFNA-ANA from the six ATC patients were cultured in RPMI 1640 and propagated in DMEM. Chemosensitivity was evaluated by inhibiting the proliferation with increasing concentrations of five different chemotherapeutic agents (bleomycin, cisplatin, gemcitabine, etoposide, and carboplatin) or TZD (rosiglitazone). Chemotherapeutic agents significantly inhibited (PPV600EBRAF mutation was observed in two ATC patients; the inhibition of proliferation by drugs was similar in tumors with or without V600EBRAF mutation.ConclusionsOur study demonstrates 1) the possibility to obtain FNA-ANA, and opens the way to the use of FNA-ANA to test the chemosensitivity to different drugs (chemotherapeutic agents or TZD; and possibly the radiosensitivity) in each patient, avoiding unnecessary surgical procedures and the administration of inactive chemotherapeutics; and 2) that etoposide is highly effective in reducing ATC cell growth in vitro.

Background Papillary thyroid cancer is the most common endocrine malignancy. The most sensitive and specific diagnostic tool for thyroid nodule diagnosis is fine-needle aspiration (FNA) biopsy with cytological evaluation. Nevertheless, FNA biopsy is not always decisive leading to “indeterminate” or “suspicious” diagnoses in 10 %–30 % of cases. BRAF V600E detection is currently used as molecular test to improve the diagnosis of thyroid nodules, yet it lacks sensitivity. The aim of the present study was to identify novel molecular markers/computational models to improve the discrimination between benign and malignant thyroid lesions. Methods We collected 118 pre-operative thyroid FNA samples. All 118 FNA samples were characterized for the presence of the BRAF V600E mutation (exon15) by pyrosequencing and further assessed for mRNA expression of four genes (KIT, TC1, miR-222, miR-146b) by quantitative polymerase chain reaction. Computational models (Bayesian Neural Network Classifier, discriminant analysis) were built, and their ability to discriminate benign and malignant tumors were tested. Receiver operating characteristic (ROC) analysis was performed and principal component analysis was used for visualization purposes. Results In total, 36/70 malignant samples carried the V600E mutation, while all 48 benign samples were wild type for BRAF exon15. The Bayesian neural network (BNN) and discriminant analysis, including the mRNA expression of the four genes (KIT, TC1, miR-222, miR-146b) showed a very strong predictive value (94.12 % and 92.16 %, respectively) in discriminating malignant from benign patients. The discriminant analysis showed a correct classification of 100 % of the samples in the malignant group, and 95 % by BNN. KIT and miR-146b showed the highest diagnostic accuracy of the ROC curve, with area under the curve values of 0.973 for KIT and 0.931 for miR-146b. Conclusions The four genes model proposed in this study proved to be highly discriminative of the malignant status compared with BRAF assessment alone. Its implementation in clinical practice can help in identifying malignant/benign nodules that would otherwise remain suspicious. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1917-2) contains supplementary material, which is available to authorized users.

ObjectiveChromosomal rearrangements of theRETproto-oncogene is one of the most common molecular events in papillary thyroid carcinoma (PTC). However, their pathogenic role and clinical significance are still debated. This study aimed to investigate the prevalence of RET/PTC rearrangement in a cohort ofBRAFWT PTCs by fluorescencein situhybridization (FISH) and to search a reliable cut-off level in order to distinguish clonal or non-clonal RET changes.DesignFortyBRAFWT PTCs were analyzed by FISH for RET rearrangements. As controls, sixBRAFV600E mutated PTCs, 13 follicular adenomas (FA), and ten normal thyroid parenchyma were also analyzed.MethodsWe performed FISH analysis on formalin-fixed, paraffin-embedded tissue using a commercially available RET break–apart probe. A cut-off level equivalent to 10.2% of aberrant cells was accepted as significant. To validate FISH results, we analyzed the study cohort by qRT-PCR.ResultsSplit RET signals above the cut-off level were observed in 25% (10/40) of PTCs, harboring a percentage of positive cells ranging from 12 to 50%, and in one spontaneous FA (1/13, 7.7%). Overall, the data obtained by FISH matched well with qRT-PCR results. Challenging findings were observed in five cases showing a frequency of rearrangement very close to the cut-off.ConclusionsFISH approach represents a powerful tool to estimate the ratio between broken and non-broken RET tumor cells. Establishing a precise FISH cut-off may be useful in the interpretation of the presence of RET rearrangement, primarily when this strategy is used for cytological evaluation or for targeted therapy.

Clinical management of patients with thyroid nodules indeterminate at fine-needle aspiration (FNA) cytology is still unsettled.Our objective was to establish the clinical outcome of patients with thyroid nodules indeterminate at cytology and to identify the features associated with malignancy.This was a retrospective evaluation of 1520 consecutive patients with indeterminate cytology among 100 065 patients who underwent FNA between January 2000 and December 2010.Of 1520 patients, 371 (24.4 %) had thyroid cancer at histology, the follicular variant of papillary cancer being the most frequent histotype, and 342 patients with cancer were free of disease after thyroidectomy and (131)I remnant ablation, whereas 29 needed further treatment because of persistent disease. Among them, only 12 had persistence of disease at the end of follow-up. Atypias at cytology (P = .001), blurred nodule margins (P = .005), and spot microcalcifications (P = .003) at thyroid ultrasound (US) were significantly associated with malignancy. A clinical score including cytology and US characteristics was calculated; the lowest value showed a high negative predictive value (83.9%) for the presence of malignancy and even higher (99.5%) for the presence of a more cumbersome cancer disease, and only 4 of the 29 patients who needed further treatment were included in the group with the lowest risk score.Patients with Thy 3 cytology and histology of thyroid cancer had an overall good prognosis. A clinical risk score including the results of cytology and US features is helpful in the management of patients with indeterminate thyroid nodules.

Background Thyroid nodules with indeterminate cytological features on fine needle aspiration (FNA) cytology have a 20% risk of thyroid cancer. The aim of the current study was to determine the diagnostic utility of an 8-gene assay to distinguish benign from malignant thyroid neoplasm. Methods The mRNA expression level of 9 genes (KIT, SYNGR2, C21orf4, Hs.296031, DDI2, CDH1, LSM7, TC1, NATH) was analysed by quantitative PCR (q-PCR) in 93 FNA cytological samples. To evaluate the diagnostic utility of all the genes analysed, we assessed the area under the curve (AUC) for each gene individually and in combination. BRAF exon 15 status was determined by pyrosequencing. An 8-gene computational model (Neural Network Bayesian Classifier) was built and a multiple-variable analysis was then performed to assess the correlation between the markers. Results The AUC for each significant marker ranged between 0.625 and 0.900, thus all the significant markers, alone and in combination, can be used to distinguish between malignant and benign FNA samples. The classifier made up of KIT, CDH1, LSM7, C21orf4, DDI2, TC1, Hs.296031 and BRAF had a predictive power of 88.8%. It proved to be useful for risk stratification of the most critical cytological group of the indeterminate lesions for which there is the greatest need of accurate diagnostic markers. Conclusion The genetic classification obtained with this model is highly accurate at differentiating malignant from benign thyroid lesions and might be a useful adjunct in the preoperative management of patients with thyroid nodules.

Papillary carcinomas with diameters that are less than or equal to 1 cm (thyroid papillary microcarcinoma [mPTC]) are quite common but can carry more risk than previously thought. The proper treatment and management of these lesions is still being debated. Even though fine needle aspiration cytology (FNAC) is considered the best method for the diagnosis of thyroid nodules, its efficacy is still questioned for mPTC. We investigated the role of BRAF gene status in preoperative cytological samples, using manual macrodissection as an additional tool to improve the diagnostic accuracy of mPTC.DNA was extracted directly from stained FNAC smears of 95 patients including 85 with histological diagnoses of papillary thyroid carcinoma (PTC) ≤1 cm and 10 with goiters. The cytological diagnoses of the 95 cases included the following: 42 samples were suspicious for papillary carcinoma, 38 were PTCs, and 15 were indeterminate lesions. DNA was then extracted from the FNAC slides after performing a "manual macrodissection" procedure. The BRAF(V600E) mutational status was determined by sequence analysis in all the patients.In this study, we showed that the BRAF(V600E) mutation was present with a high frequency in patients with mPTC (74%). The presence of the mutation was independent of the size of the tumor. In our study, the combination of the cytological diagnosis and the molecular analysis was able to identify 82% of all cases of mPTC, with an increase of 37% compared with a morphological diagnosis alone. The morpho-molecular analysis was able to reduce the number of suspicious cases by70%. All of the goiters had a wild-type BRAF status.The analysis of BRAF mutational status in FNAC obtained from papillary microcarcinomas demonstrates that molecular pathology, combined with morphology and molecular biology is a powerful tool for cytological diagnosis of mPTC. Our results also confirm the data supporting the biological relevance of PTCs with diameters that are ≤1 cm and the importance of "manual macrodissection" in the molecular analysis of cytological material.

identified among the 141 HIV-infected women, of which 16 viral types were classified as cancer high-risk or probable high-risk viruses. The most common types identified were HPV types 58 (12.1%, p < 0.001), 16 (10.7%, p = 0.06), 61 (8.4%, p < 0.05), 53 (7.9%, p = 0.55) and 6 (7.6%, p = 0.41). Nineteen distinct HPV genotypes were identified among the 50 HIV-uninfected women. Ten types were of high-risk or probable high-risk. The most common types identified in HIV-uninfected subjects were HPV types 53 (10.4%), 6 (9.1%), 16 (9.1%), 18 (9.1%) and 66 (7.8%). Discussion HPV infection was mainly associated with HIV positive status. HPV multiple infections were high in the population studied, independently of HIV status. HPV Genotypes are different in the two groups. The HPV types identified are partially different from those more commonly identified in Western countries.

Although most thyroid nodule fine-needle aspiration (FNA) diagnoses are definitive or nearly definitive, about 30% of them are not read as definitively benign or malignant, the so-called indeterminate or suspicious FNA diagnosis. The prevalence of malignancy in FNA samples with these diagnoses varies from 10% to 52%. The first aim of this study was to determine if BRAF V600E analysis of thyroid FNA cytological smears could be performed with a relatively simple protocol. We also sought to determine if assessing the presence of BRAF gene mutations in preoperative FNA cytology slides would provide diagnostic information for FNA samples with a reading of indeterminate or suspicious thyroid lesions.DNA was extracted directly from FNA-stained smears of 111 patients with thyroid lesions having different cytological diagnoses. There was 1 cystic nodule, 20 microfollicular proliferations without atypia, 32 that were suspicious for papillary carcinoma, 56 papillary thyroid carcinomas (PTC), and 2 poorly differentiated carcinomas. The BRAF V600E mutational status was determined by sequencing analysis in all patients. The histopathological diagnosis was obtained in all cases.We observed that 56/90 (62.3%) patients received a definitive diagnosis of PTC when only cytology was used. After molecular analysis, the BRAF V600E mutation was detected in 18/32 (56.2%) cases with a cytology of suspicious for papillary carcinoma and 41/56 (73.2%) with PTC. According to the morpho-molecular analysis (i.e., traditional cytology combined with BRAF V600E analysis) 74/90 (82.2%) patients could be assigned a definitive diagnosis of PTC. Therefore, the addition of molecular analysis yielded an increase of 20% in the sensitivity compared to cytology alone.The method of molecular analysis of thyroid FNA smears described here can be easily performed after the FNA, thereby avoiding inconvenience and additional time during the FNA and permitting later analysis of samples having indeterminate cytology features. The increased sensitivity of this preoperative morpho-molecular analysis should provide information that is useful in deciding the extent of thyroid surgery for thyroid nodules that are indeterminate or suspicious on cytology.

No study has evaluated the antiproliferative effects of thiazolidinediones and antiblastics in 'primary cultured human anaplastic thyroid cancer cells'.Primary anaplastic cells proliferation was evaluated after incubation with increasing concentrations of rosiglitazone or pioglitazone or antiblastics (bleomycin, cisplatin, gemcitabine) by a proliferation assay (WST-1-tetrazolium reaction) and cell counting.A reduction of proliferation by thiazolidinediones at 1 h (from the start of tetrazolium reaction) [of 11% and 25%, with rosiglitazone, 10 or 20 (P = 0.0001) microM, respectively; of 7% and 17%, with pioglitazone, 10 or 20 (P = 0.0125) microM, respectively], and at 2 h [of 14% and 24%, with rosiglitazone, 10 (P = 0.0043) or 20 (P0.0001) microM, respectively; of 9% and 21%, with pioglitazone, 10 (P = 0.0397) or 20 (P = 0.0001) microM, respectively] was shown. No significant thiazolidinediones effect was observed in normal thyroid follicular cells. Bleomycin, cisplatin and gemcitabine significantly (P0.0001) inhibited (50%) anaplastic cells proliferation. Cell counting confirmed the above mentioned results. Inhibition of proliferation was similar in tumours with or without (V600E)BRAF mutation, both for thiazolidinediones and antiblastics.Thiazolidinediones exert an antiproliferative effect in primary cultured human anaplastic carcinoma cells in vitro, such as antiblastics.

The aim of the present study was to assess recurrence rates and times in patients with squamous intraepithelial lesion (SIL) of the uterine cervix treated with loop electrosurgical excision procedure (LEEP) conization, in order to define categories of patients who have a different risk of recurrence and who need a different surveillance protocol. This study was carried out on 119 consecutive patients who underwent LEEP. All patients were followed up with cervical smear and colposcopy after 3, 6, and 12 months in the first-year posttreatment, and every 6–12 months afterwards. Human papillomavirus (HPV) testing was performed at the time of LEEP and repeated 3–6 months later. The histologic examination of LEEP specimens revealed stage IA1 squamous cell cervical cancer in 4 (3.4%) cases, high-grade SIL in 75 (63%) cases, and low-grade SIL in 40 (33.6%) cases. The four patients with stage IA1 cervical cancer were not included in the further analyses. Disease recurred in none of the 50 patients with negative posttreatment HPV testing, in 4 (9.3%) of the 43 patients with positive posttreatment HPV testing and negative surgical margins, and in 8 (36.4%) of 22 patients with positive posttreatment HPV testing and positive margins. The combined evaluation of surgical margin status and posttreatment HPV testing could allow to subdivide patients treated with LEEP into categories at different risk of recurrence, requiring new tailored surveillance procedures.

Summary Objective Anaplastic thyroid cancer (ATC) is often inoperable and chemotherapy and radiotherapy are the main treatments. Until now, ‘primary ATC cell cultures’ (ANA) have been developed from surgical biopsies. We investigated the possibility of obtaining ANA from fine-needle aspiration (FNA-ANA) and testing their sensitivity to chemotherapeutic agents, which could enable treatments to be more effective and avoid unnecessary surgical procedures. Design and patients The aim of this study was to obtain FNA-ANA from three ATC patients and to evaluate the chemosensitivity of FNA-ANA to chemotherapeutic agents. Measurements and results FNA-ANA from ATC patients were cultured in RPMI 1640 and propagated in Dulbecco's modified Eagle's medium (DMEM). Chemosensitivity was evaluated by inhibiting the proliferation (analysing the number of viable cells by the cleavage of tetrazolium salts), by increasing the concentration of four different chemotherapeutic agents: bleomycin, cisplatin, gemcitabine and etoposide. The chemotherapeutic agents significantly inhibited (> 50%) FNA-ANA proliferation. Another ANA for each patient was obtained from a surgical biopsy specimen; the results for the chemosensitivity tests were similar to those obtained using FNA-ANA. Conclusions Our study demonstrates the possibility of obtaining FNA-ANA, and opens the way to the use of FNA-ANA as a means of testing the chemosensitivity to different chemotherapeutic agents (and possibly the radiosensitivity) in each patient, avoiding unnecessary surgical procedures and the administration of inactive chemotherapeutics.

Background A large amount of information has been collected on the molecular tumorigenesis of thyroid cancer. A low expression of c-KIT gene has been reported during the transformation of normal thyroid epithelium to papillary carcinoma suggesting a possible role of the gene in the differentiation of thyroid tissue rather than in the proliferation. The initial presentation of thyroid carcinoma is through a nodule and the best way nowadays to evaluate it is by fine-needle aspiration (FNA). However many thyroid FNAs are not definitively benign or malignant, yielding an indeterminate or suspicious diagnosis which ranges from 10 to 25% of FNAs. BRAF mutational analysis is commonly used to assess the malignancy of thyroid nodules but unfortunately it still leaves indeterminate diagnoses. The development of molecular initial diagnostic tests for evaluating a thyroid nodule is needed in order to define optimal surgical approach for patients with uncertain diagnosis pre- and intra-operatively. Methods In this study we extracted RNA from 82 FNA smears, 46 malignant and 36 benign at the histology, in order to evaluate by quantitative Real Time PCR the expression levels of c-KIT gene. Results We have found a highly preferential decrease rather than increase in transcript of c-KIT in malignant thyroid lesions compared to the benign ones. To explore the diagnostic utility of c-KIT expression in thyroid nodules, its expression values were divided in four arbitrarily defined classes, with class I characterized by the complete silencing of the gene. Class I and IV represented the two most informative groups, with 100% of the samples found malignant or benign respectively. The molecular analysis was proven by ROC (receiver operating characteristic) analysis to be highly specific and sensitive improving the cytological diagnostic accuracy of 15%. Conclusion We propose the use of BRAF test (after uncertain cytological diagnosis) to assess the malignancy of thyroid nodules at first, then the use of the c-KIT expression to ultimately assess the diagnosis of the nodules that otherwise would remain suspicious. The c-KIT expression-based classification is highly accurate and may provide a tool to overcome the difficulties in today's preoperative diagnosis of thyroid suspicious malignancies.