Detection of the BRAF(V600E) Mutation in Fine Needle Aspiration Cytology of Thyroid Papillary Microcarcinoma Cells Selected by Manual Macrodissection: An Easy Tool to Improve the Preoperative Diagnosis.

Background: 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. Methods: 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. Results: 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 by >70%. All of the goiters had a wild-type BRAF status. Conclusions: 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

Whole-exome analysis in osteosarcoma to identify a personalized therapy

Osteosarcoma is the most common pediatric primary non-hematopoietic bone tumor. Survival of these young patients is related to the response to chemotherapy and development of metastases. Despite many advances in cancer research, chemotherapy regimens for osteosarcoma are still based on non-selective cytotoxic drugs. It is essential to investigate new specific molecular therapies for osteosarcoma to increase the survival rate of these patients. We performed exomic sequence analyses of 8 diagnostic biopsies of patients with conventional high grade osteosarcoma to advance our understanding of their genetic underpinnings and to correlate the genetic alteration with the clinical and pathological features of each patient to identify a personalized therapy. We identified 18,275 somatic variations in 8,247 genes and we found three mutated genes in 7/8 (87%) samples (KIF1B, NEB and KMT2C). KMT2C showed the highest number of variations; it is an important component of a histone H3 lysine 4 methyltransferase complex and it is one of the histone modifiers previously implicated in carcinogenesis, never studied in osteosarcoma. Moreover, we found a group of 15 genes that showed variations only in patients that did not respond to therapy and developed metastasis and some of these genes are involved in carcinogenesis and tumor progression in other tumors. These data could offer the opportunity to get a key molecular target to identify possible new strategies for early diagnosis and new therapeutic approaches for osteosarcoma and to provide a tailored treatment for each patient based on their genetic profile

A mouse mammary tumor virus env-like exogenous sequence is strictly related to progression of human sporadic breast carcinoma

A viral etiology of human breast cancer (HBC) has been postulated for decades since the identification of mouse mammary tumor virus (MMTV). The detection of MMTV env-like exogenous sequences (MMTVels) in 30% to 40% of invasive HBCs increased attention to this hypothesis. Looking for MMTVels during cancer progression may contribute to a better understanding of their role in HBC. Herein, we analyzed HBC preinvasive lesions for the presence of MMTVels. Samples were obtained by laser microdissection of FFPE tissues: 20 usual-type ductal hyperplasias, 22 atypical ductal hyperplasias (ADHs), 49 ductal carcinomas in situ (DCISs), 20 infiltrating ductal carcinomas (IDCs), and 26 normal epithelial cells collateral to a DCIS or an IDC. Controls included reductive mammoplastic tissue, thyroid and colon carcinoma, and blood samples from healthy donors. MMTVels were detected by fluorescence-nested PCR. DNA samples from the tissues of nine patients were analyzed by real-time quantitative PCR, revealing a different viral load correlated with stage of progression. Furthermore, as never previously described, the presence of MMTVels was investigated by chromogenic in situ hybridization. MMTVels were found in 19% of normal epithelial cells collateral to a DCIS or an IDC, 27% of ADHs, 82% of DCISs, and 35% of IDCs. No MMTVels were found in the control samples. Quantitative PCR and chromogenic in situ hybridization confirmed these results. These data could contribute to our understanding of the role of MMTVels in HBC. (Am J Pathol 2011, 179:2083-2090; DOI: 10.1016/j.ajpath.2011.06.046

Investigating molecular alterations to profile short- and long-term recurrence-free survival in patients with primary glioblastoma

Glioblastoma (GB) is the most aggressive type of primary brain tumor. Despite the progress in recent years regarding the diagnosis and treatment of GB, the recurrence rate remains high, due to the infiltrative and dispersive nature of the tumor, which typically results in poor patient prognosis. In the present study, 19 formalin-fixed, paraffin-embedded GB samples were selected from patients with GB tumors. The samples were classified into a short or long recurrence-free survival (RFS) group, based on the time of first recurrence of the disease in the patients. The 19 samples were molecularly characterized for mutations in the isocitrate dehydrogenase 1 (IDH1) gene, amplification of the epidermal growth factor receptor (EGFR) gene, presence of the EGFR variant III, and methylation of the promoter region of the O6-methylguanine-DNA methyltransferase (MGMT) gene. Then, the expression of 84 genes involved in cell-cell and cell-matrix interactions, and that of 84 microRNAs (miRNAs) associated with brain cancer, was profiled. In addition, a copy number variation analysis of 23 genes reported to undergo frequent genomic alterations in human glioma was also performed. Differences in the expression levels of a number of genes were detected across the short and long RFS groups. Among these genes, 5 in particular were selected, and a 5-genes combination approach was developed, which was able to differentiate between patients with short and long RFS outcome. The high levels of sensitivity and precision displayed by this 5-genes combination approach, which were confirmed with a cross-validation method, provide a strong foundation for further validation of the involvement of the aforementioned genes in GB in a larger patient population. In conclusion, the present study has demonstrated how the expression pattern of miRNAs and mRNAs in patients with GB defines a particular molecular hallmark that may increase or reduce the aggressive behavior of GB tumors, thus influencing the survival rates of patients with GB, their response to therapy and their tendency to suffer a relapse

ANKRd44 gene silencing: a putative role in trastuzumab resistance in HER2-like breast cancer

Trastuzumab is an effective therapeutic treatment for Her2-like breast cancer; despite this most of these tumors develop resistance to therapy due to specific gene mutations or alterations in gene expression. Understanding the mechanisms of resistance to Trastuzumab could be a useful tool in order to identify combinations of drugs that elude resistance and allow a better response for the treated patients. Twelve primary biopsies of Her2+/hormone receptor negative (ER-/PgR-) breast cancer patients were selected based on the specific response to neoadjuvant therapy with Trastuzumab and their whole exome was sequenced leading to the identification of 18 informative gene mutations that discriminate patients selectively based on response to treatment. Among these genes, we focused on the study of the ANKRD44 gene to understand its role in the mechanism of resistance to Trastuzumab. The ANKRD44 gene was silenced in Her2-like breast cancer cell line (BT474), obtaining a partially Trastuzumab-resistant breast cancer cell line that constitutively activates the NF-kb protein via the TAK1/AKT pathway. Following this activation an increase in the level of glycolysis in resistant cells is promoted, also confirmed by the up-regulation of the LDHB protein and by an increased TROP2 protein expression, found generally associated with aggressive tumors. These results allow us to consider the ANKRD44 gene as a potential gene involved in Trastuzumab resistance

Is maximal acid output useful in identifying relapsing duodenal ulcer patients?

In a group of 94 duodenal ulcer patients consecutively studied and followed-up for 1 year, subjects with maximal acid output (M.A.O.) above 60 mmol/hour (mostly heavy-smoking men), were found to relapse more frequently than those with lower M.A.O. values (72.2% versus 27.6%; p less than 0.0005). A significantly higher relapse rate was also detected in patients treated with maintenance dosages of effective antisecretory drugs (cimetidine, ranitidine, pirenzepine) and presenting M.A.O. over 60 mmol/hour. It is yet to be established whether an alternative therapeutic regime may prove effective in preventing ulcer recurrence in patients with gastric hypersecretion

Recurrence of Graves' disease in thyroglossal duct remnants: relapse after total thyroidectomy.

BACKGROUND: Ectopic thyroid tissue can be found anywhere between the foramen cecum and the normal position of the thyroid gland, most commonly located in the anterior cervical area, the region of the thyroglossal duct. Although thyroid cancer has been described frequently in thyroglossal duct remnants, thyroid dysfunction related to this tissue is rare. We report a patient with recurrent Graves' disease arising in a thyroglossal duct remnant. SUMMARY: A 40-year-old woman with a history of total thyroidectomy for Graves' disease, presented with a slowly enlarging midline neck mass in association with clinical signs of hyperthyroidism. Serum-free triiodothyronine (6.6 pg/mL) and serum-free thyroxine (2.2 ng/dL) were elevated (normal range, 2.3-4.2 pg/mL and 0.9-1.8 ng/dL, respectively), and thyroid-stimulating hormone was suppressed (<0.01 mIU/mL; normal range, 0.35-5.50 mIU/mL). Neck ultrasonography showed a solid mass, localized at the infrahyoid area; radionuclide scanning confirmed an increased uptake at the same level. A 4 cm solid mass was removed by the Sistrunk technique. Microscopic examination revealed marked follicular hyperplasia, with tall cells, small follicles, scant, and scalloped colloid, in association with patchy lymphocytic infiltrate consistent with Graves' disease. CONCLUSIONS: There appears to be no reason why thyroid cells within thyroglossal duct remnants should not be influenced by the thyroid-stimulating immunoglobulins of Graves' disease. Thyrotoxicosis resulting from this must be very rare, however, as were unable to find reports of patients with thyrotoxicosis due to Graves' disease in thyroglossal duct remnants. Although some thyroid tissue can be found within the thyroglossal duct in 1.6% to 40% of normal adults, the risk of thyroid dysfunction from this is far too low to justify new therapeutic approaches