Sofia Asioli

Teratocarcinoma-Derived Growth Factor-1 Is Upregulated in Aldosterone-Producing Adenomas and Increases Aldosterone Secretion and Inhibits Apoptosis In Vitro

Aldosterone-producing adenomas (APA) are a frequent cause of secondary hypertension characterized by autonomous hypersecretion of aldosterone. However, the molecular mechanisms involved in adrenal tumorigenesis and deregulated aldosterone secretion are currently unknown. To identify putative functional genes, a transcriptional screening was performed on 8 APA and 3 normal adrenals (NA) using oligonucleotide microarrays. Data were next validated on an expanded set of samples by real-time PCR (APA, n=19; NA, n=10). The epidermal growth factor–like teratocarcinoma-derived growth factor-1 (TDGF-1) was upregulated in APA compared with NA (14.7-fold and 21.4-fold by microarray and real-time PCR, respectively). In vitro studies and Western blot analysis using the NCI H295R adrenocortical cell line showed that TDGF-1 increased Akt phosphorylation on Thr308 and Ser473, consistent with activation of phosphatidylinositol 3-kinase/Akt signaling, and also demonstrated a concomitant inactivation of the Akt substrate glycogen synthesis kinase-3&bgr; via Ser9 phosphorylation. Furthermore, TDGF-1 mediated a 3.8±0.4-fold increase in aldosterone secretion (n=4) that was specifically blocked by the phosphatidylinositol 3-kinase inhibitors wortmannin (50 nmol/L) and LY294002 (20 &mgr;mol/L). Finally, TDGF-1 protected H295R cells from apoptosis induced by staurosporine, causing a decrease in caspase-3 activity, a reduction in the inactivation of poly(ADP-ribose) polymerase, and an inhibition of DNA fragmentation, detected by the TUNEL reaction and fluorescence microscopy that was blocked by LY294002. Taken together, our data suggest that TDGF-1, which is significantly upregulated in APA and mediates aldosterone hypersecretion and deregulated growth in adrenocortical cells in vitro, may represent a key player in the development and pathophysiology of primary aldosteronism.

Tumor staging but not grading is associated with adverse clinical outcome in neuroendocrine tumors of the appendix: a retrospective clinical pathologic analysis of 138 cases.

Appendiceal neuroendocrine neoplasms (NENs) are rare and usually incidentally discovered. Most cases are clinically indolent, although the rare aggressive ones are poorly predictable. The aim of this study was to test the applicability and prognostic significance of the new World Health Organization (WHO) classification and to test the several pathologic features and TNM staging systems (American Joint Committee on Cancer and European Neuroendocrine Tumor Society) in these tumors. A multi-institutional retrospective series of 138 appendiceal NENs was selected on the basis of the availability of both pathologic material and clinical information, including follow-up data. All cases were reviewed to record pathologic features and to apply year 2000 and 2010 WHO classifications, as well as European Neuroendocrine Tumor Society and American Joint Committee on Cancer TNM stages. Clinical and pathologic characteristics were compared with disease outcome by contingency, univariate, and multivariate survival analyses. Although up to one third of cases presented several malignancy-associated pathologic features, only 4 patients died of the disease. Adverse outcome was significantly associated with extramural extension (including mesoappendix), well-differentiated carcinoma diagnosis (2000 WHO classification), pT3-4 stage, older age, and presence of positive resection margins, but not with tumor size, mitotic or proliferative indexes, and, consequently, 2010 WHO grading. In the appendix, at variance with midgut/hindgut NENs, the 2000 WHO classification performs better than the grading-based 2010 WHO scheme and, together with tumor stage, is the most relevant parameter associated with clinical aggressiveness.

In Situ Hybridization Analysis of miR-146b-5p and miR-21 in Thyroid Nodules: Diagnostic Implications

Some thyroid nodules such as follicular adenomas (FAs), follicular variant of papillary thyroid carcinomas (FVPTCs), and follicular thyroid carcinomas (FTCs) exhibit similar clinical presentations and gross morphologic appearances. The differential diagnosis of these lesions is sometimes difficult based on morphologic, cytologic, or clinical features alone. miR-146b-5p and miR-21 deregulation has been associated with progression and metastasis of thyroid cancers. However, the utility of in situ hybridization (ISH) to determine the cellular localization, diagnostic, and prognostic significance of miR-146b-5p and miR-21 expression in thyroid tumors has not been extensively analyzed. In order to examine the expression of miR-146b-5p and miR-21 in benign and malignant thyroid tissues and to determine if these microRNAs could be assigned to distinct histomorphological types of thyroid nodules, we analyzed miR-146b-5p and miR-21 expression in thyroid nodules on tissue microarrays (TMAs) with 193 thyroid specimens by ISH. miR-146b-5p and miR-21 expression in thyroid tissues was also analyzed by quantitative reverse transcription–polymerase chain reaction (qRT-PCR). miR-146b-5p was highly expressed (89xa0%) in papillary thyroid carcinomas (PTCs) and 41xa0% of FVPTC. The expression of miR-146b-5p was not expressed in most FTCs, anaplastic thyroid carcinomas (ATCs), poorly differentiated thyroid carcinomas (PDTCs), or FAs (7, 8, 0, and 0xa0%, respectively). MiR-21 was overexpressed in 83xa0% of ATCs, 79xa0% of PTCs, 34xa0% of FVPTCs, and 19xa0% of PDTCs. The expression of miR-21 was not expressed in most FAs (9xa0%) or FTCs (4xa0%). Normal thyroid tissues and most benign goiters were negative for miR-146b-5p and miR-21. qRT-PCR analysis supported the ISH findings. PTC cases with positive expression of miR-146b-5p and miR-21 had significantly poorer disease-free survival rates. Immunohistochemical staining for HBME-1 showed positive staining in PTCs (100xa0%) and FVPTCs (92xa0%) with a subset of FTC (40xa0%) staining positive, while all FAs were negative. Since miR-146b-5p was mainly expressed in PTC including FVPTC and was not expressed in most FTC, PDTC, or ATC, it may serve as a useful diagnostic marker for PTC. ISH is a useful method to analyze microRNA expression in formalin-fixed paraffin-embedded thyroid tissues.

MALAT1 Long Non-coding RNA Expression in Thyroid Tissues: Analysis by In Situ Hybridization and Real-Time PCR

Long non-coding RNAs (lncRNAs) are important for transcription and for epigenetic or posttranscriptional regulation of gene expression and may contribute to carcinogenesis. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), an lncRNA involved in the regulation of the cell cycle, cell proliferation, and cell migration, is known to be deregulated in multiple cancers. Here, we analyzed the expression of MALAT1 on 195 cases of benign and malignant thyroid neoplasms by using tissue microarrays for RNA in situ hybridization (ISH) and real-time PCR. MALAT1 is highly expressed in normal thyroid (NT) tissues and thyroid tumors, with increased expression during progression from NT to papillary thyroid carcinomas (PTCs) but is downregulated in poorly differentiated thyroid cancers (PDCs) and anaplastic thyroid carcinomas (ATCs) compared to NT. Induction of epithelial to mesenchymal transition (EMT) by transforming growth factor (TGF)-beta in a PTC cell line (TPC1) led to increased MALAT1 expression, supporting a role for MALAT1 in EMT in thyroid tumors. This is the first ISH study of MALAT1 expression in thyroid tissues. It also provides the first piece of evidence suggesting MALAT1 downregulation in certain thyroid malignancies. Our findings support the notion that ATCs may be molecularly distinct from low-grade thyroid malignancies and suggest that MALAT1 may function both as an oncogene and as a tumor suppressor in different types of thyroid tumors.

Polymorphous adenocarcinoma of the breast. Report of three cases.

We report three cases of polymorphous adenocarcinoma (PLA) of the breast in 37-, 55- and 74-year-old women, respectively. The patients have no evidence of previous malignancy. The tumours consist of monotonous cells showing a wide spectrum of growth patterns: solid nests, trabeculae, tubules, cribriform structures, strands and fascicles reminiscent of polymorphous low-grade adenocarcinoma of salivary glands. To our knowledge, PLA has never been reported in the breast; therefore, this tumour should be added to the list of neoplastic lesions of the breasts that have the same features as those of the salivary glands.

DNA methylation analysis by bisulfite next-generation sequencing for early detection of oral squamous cell carcinoma and high-grade squamous intraepithelial lesion from oral brushing.

PURPOSEnOral squamous cell carcinoma (OSCC) is commonly preceded by oral potentially malignant lesions (OPML). The aim of the present study was to assess, by bisulfite next-generation sequencing (NGS), the methylation status of a list of candidate genes obtained from oral brushings to early detect OPML and OSCC.nnnMATERIAL AND METHODSnOral brushing specimens from 11 OSCC, 11 high-grade squamous intraepithelial lesions (HG-SIL), 9 low-grade SIL (LG-SIL), 9 oral lichen planus (OLP), and 8 healthy donors were included in this study. We investigated, by means of bisulfite NGS, the promoter of GP1BB, ZAP70, KIF1A, p16[CDKN2A], CDH1, miR137, and miR375. Statistical significance between lesions and a pool of healthy donors were evaluated with the Mann-Whitney U test.nnnRESULTSnZAP70 was found to be hypermethylated in 100% of OSCC and HG-SIL and in 28.6% of LG-SIL. GP1BB hypomethylation was detected in 90.9% OSCC and HG-SIL and in 37.5% of LG-SIL. MiR137 was hypermethylated in 100% of OLP, 44.4% of OSCC, 40% HG-SIL, and 25% LG-SIL. KIF1A hypermethylation was found to be associated with TP53 mutations (p < 0.0001).nnnCONCLUSIONnIn the present preliminary cohort of patients, DNA methylation analysis of GP1BB and ZAP70 seems to be a promising noninvasive tool for early detection of OSCC and HG SIL from oral brushing specimens.

Localised pleural malignant mesothelioma. Report of two cases simulating pulmonary carcinoma and review of the literature

AimsTo describe two cases of localised malignant mesothelioma with a predominantly intrapulmonary growth which led to a pre-operative diagnosis of pulmonary carcinoma.Materials and methodsBoth cases presented as intrapulmonary masses, while at computed tomography scan the pleura appeared not significantly thickened. In one patient, the main bronchus was diffusely infiltrated by the tumour. In both cases, a preoperative biopsy showed a proliferation of large cells leading to a diagnosis of non-small-cell lung carcinoma. Histological examination of the surgical specimens revealed features consistent with epithelioid mesothelioma with deciduoid features in one case and with biphasic mesothelioma in the other. Both cases were diffusely positive with anti-calretinin antibody, while anti-TTF1, anti-surfactant and anti-CEA antisera were negative.ConclusionsLocalised malignant mesotheliomas are unusual and predominantly intrapulmonary growth is rare. Pathologists should be aware of this possibility to avoid misdiagnosis, particularly in small biopsies.

Primary neuroendocrine (Merkel cell) carcinoma of the nipple

We report a case of Merkel cell carcinoma of the nipple in an 82-year-old woman. She had swelling and pain in her right nipple for a period of 11 months, and a clinical diagnosis of primary mammary neoplasm was made. The laboratory findings were normal and both mammogram and CT scan ruled out lesions in the mammary parenchyma. A fine needle aspiration of the lump of the nipple yielded a diagnosis of malignant small cell tumor; subsequently she underwent an excisional biopsy of the nipple. A diagnosis of Merkel cell carcinoma was made at histology. Postoperatively, 36 Gy of adjuvant radiotherapy was applied, and 6 months later she had a radical mastectomy and axillary lymphadenectomy. No tumor was found in the breast, but 13 of 17 lymph nodes contained metastatic deposits of the Merkel cell carcinoma. She died 15 months after the initial excisional biopsy with diffuse visceral metastases. S. Asioli · T. Dorji · V. Eusebi (✉) Department of Oncology, Section of Anatomic Pathology, “M. Malpighi,” University of Bologna, Ospedale Bellaria, Via Altura 3, 40139 Bologna, Italy e-mail: vincenzo.eusebi@ausl.bologna.it Tel.: +39-051-6225523, Fax: +39-051-6225759

Crooke’s hyalinization in silent corticotroph adenoma: Report of two cases

Corticotroph adenomas rarely show Crooke’s hyaline change in neoplastic cells, a feature similar to that of normal corticotroph cells exposed to excess cortisol. Crooke’s cell adenomas are usually associated with Cushing’s disease. Nonfunctioning examples are uncommon. We report two clinically silent corticotroph adenomas featuring extensive Crooke’s hyalinization in neoplastic cells. The two patients were 49 and 59 yr of age and neither had Cushing’s disease. Serum and urinary cortisol were normal. One patient had elevated serum adrenocorticotropic hormone. In our experience, the two patients accounted for 0.4% of pituitary adenomas operated on from January 1992 to December 2001 and 3.5% of all corticotroph adenomas. The two lesions had features of the subtype 1 silent corticotroph adenoma. Cytogenetic analysis performed on one lesion showed a normal karyotype (46;XY). Hyalinization in clinically silent Crooke’s cell adenoma indicates that hyaline changes do not always relate to excess cortisol. It is known that neoplastic Crooke’s cells show immunoreactivity for glucocorticoid receptors stronger than nontumorous Crooke’s corticotrophs. This fact suggests that receptor overexpression or lack of receptor downregulation may result in hypersensitivity of neoplastic Crooke’s cells to physiologic cortisol plasma levels.

TP53 alterations in pancreatic acinar cell carcinoma: new insights into the molecular pathology of this rare cancer

The molecular alterations of pancreatic acinar cell carcinomas (ACCs) are poorly understood and have been reported as being different from those in ductal adenocarcinomas. Loss of TP53 gene function in the pathogenesis of ACCs is controversial since contradictory findings have been published. A comprehensive analysis of the different possible genetic and epigenetic mechanisms leading to TP53 alteration in ACC has never been reported and hence the role of TP53 in the pathogenesis and/or progression of ACC remains unclear. We investigated TP53 alterations in 54 tumor samples from 44 patients, including primary and metastatic ACC, using sequencing analysis, methylation-specific multiplex ligation probe amplification, fluorescence in situ hybridization, and immunohistochemistry. TP53 mutations were found in 13xa0% of primary ACCs and in 31xa0% of metastases. Primary ACCs and metastases showed the same mutational profile, with the exception of one case, characterized by a wild-type sequence in the primary carcinoma and a mutation in the corresponding metastasis. FISH analysis revealed deletion of the TP53 region in 53xa0% of primary ACCs and in 50xa0% of metastases. Promoter hypermethylation was found in one case. The molecular alterations correlated well with the immunohistochemical findings. A statistically significant association was found between the combination of mutation of one allele and loss of the other allele of TP53 and worse survival.