Paolo Viacava

Should parafibromin staining replace HRTP2 gene analysis as an additional tool for histologic diagnosis of parathyroid carcinoma

OBJECTIVE HRPT2 gene mutations are associated with parathyroid carcinomas, and absence of parafibromin immunoreactivity has been suggested as a diagnostic marker of malignancy. The aim of our study was to extend parafibromin studies in a series of benign and malignant parathyroid tumors and cross-validate the results of immunohistochemistry with those of HRPT2 analysis. DESIGN AND PATIENTS We performed parafibromin and cyclin D1 immunostaining and HRPT2 gene analysis using loss of heterozygosity studies and sequencing analysis in parathyroid specimens from 11 patients with carcinoma (eleven primary tumors, one skin, and four lung metastases), 22 with sporadic adenomas, and 4 with atypical adenomas. RESULTS Ten out of eleven parathyroid cancers were negative for parafibromin staining and showed HRPT2 gene abnormalities. The remaining sample was negative for immunostaining and genetic analyses. All but one sporadic adenomas showed parafibromin immunoreactivity and no HRPT2 gene abnormalities. The sample with negative immunostaining carried an HRPT2 mutation. Two atypical adenomas were positive and two negative with parafibromin staining. No HRPT2 abnormalities were found in these samples. Cyclin D1 expression was heterogeneous and there was no relationship between expression/expression level of cyclin D1 and parafibromin expression. CONCLUSIONS We have shown that negative parafibromin staining is almost invariably associated with HRPT2 mutations and confirm that loss of parafibromin staining strongly predicts parathyroid malignancy. In clinical practice, these tests could be particularly useful in the subset of parathyroid tumors with equivocal histological examination. However, their diagnostic value in this setting remains to be proven.

Microvascular density and vascular endothelial growth factor expression in normal pituitary tissue and pituitary adenomas

Microvessel density (MVD) represents a measure of angiogenesis and may be used as an indicator of neoplastic aggressiveness. Vascular endothelial growth factor (VEGF) plays a pivotal role as angiogenic promoter by stimulating endothelial cell proliferation and migration and enhancing vascular permeability. The aim of this study was to investigate MVD and VEGF expression in human pituitary adenomas and normal pituitary gland tissues by immunohistochemistry, and to correlate data with clinical characteristics. Fragments from 46 pituitary adenomas (18 non-functioning, 12 ACTH-secreting, 12 GH-secreting, 4 PRL-secreting) and 19 specimens of normal anterior pituitary gland obtained at surgery were evaluated. MVD in normal anterior pituitary was significantly higher than in tumors (69.2±28.5 vs 29.3±19.7; p<0.0001). Within adenomas, no difference was found in MVD when different histotype, size, sex, age, rate of recurrence or medical pre-surgical treatment were considered. The degree of vascularity was somewhat related only to clinical invasiveness, as evaluated by pre-surgical MRI grading (grade 0 p<0.05 vs grade 1 and vs grade 2). No statistically significant difference in VEGF expression was found between normal tissue and adenomas and among tumors of different histotype (p= 0.3978). Size, sex, age, rate of recurrence and medical pre-surgical treatment did not influence VEGF expression. No correlation was found between MVD and VEGF expression. In conclusion, MVD was reduced in pituitary adenomas with respect to normal gland. VEGF expression is however well preserved in adenomas and this might contribute to adequate tumoral vascular supply with complex mechanisms other than endothelial cells proliferation.

No Evidence for Mutations in the Calcium‐Sensing Receptor Gene in Sporadic Parathyroid Adenomas

Inactivating mutations of the calcium‐sensing receptor gene (CaR) might explain abnormalities in the regulation of both parathyroid cell proliferation and parathyroid hormone secretion. In a previous study, using RNAse A protection assay, no mutations were identified in a series of parathyroid specimens from patients with primary and secondary hyperparathyroidism, but the analysis was incomplete, since part of exon 6 could not be analyzed. In the present study, we examined the presence of mutations in the CaR gene in 20 parathyroid adenomas using direct sequencing. The entire coding region of the CaR gene was successfully amplified by polymerase chain reaction and directly sequenced. This analysis did not identify CaR gene mutations in any tumors studied. A polymorphism that encoded a single amino acid change (Ala826Thr) was identified in 4 parathyroid adenomas and in 8 of 50 normal unrelated subjects. Loss of heterozygosity studies were also performed on adenomas using markers for the locus of the CaR gene on chromosome 3q. No allelic loss was demonstrated. In conclusion, our results extend previous observation and suggest that clonal somatic mutations of the CaR gene and allelic loss at the CaR locus on chromosome 3q do not play a major role in the pathogenesis of sporadic parathyroid tumors.

Antiangiogenic and anticolorectal cancer effects of metronomic irinotecan chemotherapy alone and in combination with semaxinib

Metronomic chemotherapy refers to the administration of chemotherapy at low, nontoxic doses on a frequent schedule with no prolonged breaks. The aim of the study is to rationally develop a CPT-11 metronomic regimen in preclinical settings of colon cancer. In vitro cell proliferation, apoptosis and thrombospondin-1/vascular endothelial growth factor (TSP-1/VEGF) expression analyses were performed on endothelial (HUVEC, HMVEC-d) and colorectal cancer (HT-29, SW620) cells exposed for 144 h to metronomic concentrations of SN-38, the active metabolite of CPT-11. HT-29 human colorectal cancer xenograft model was used, and tumour growth, microvessel density and VEGF/TSP-1 quantification was performed in tumours. In vitro and in vivo combination studies with the tyrosine inhibitor semaxinib were also performed. SN-38 preferentially inhibited endothelial cell proliferation alone and interacted synergistically with semaxinib; it induced apoptosis and increased the expression and secretion of TSP-1. Metronomic CPT-11 alone and combined with semaxinib significantly inhibits tumour growth in the absence of toxicity, which was accompanied by decreases in microvessel density and increases in TSP-1 gene expression in tumour tissues. In vitro results show the antiangiogenic properties of low-concentration SN-38, suggesting a key role of TSP-1 in this effect. In vivo, the CPT-11 metronomic schedule is effective against tumour and microvessel growth without toxic effect on mice.

Angiogenesis and VEGF expression in pre-invasive lesions of the human breast.

Angiogenesis (as microvascular density—MVD) and vascular endothelial growth factor (VEGF) expression were evaluated by immunohistochemistry in all types of human pre‐invasive breast lesion, un‐associated with invasive carcinoma, including florid ductal hyperplasia of usual type (FDHUT, 40 cases), atypical ductal hyperplasia (ADH, 10), well‐differentiated intraductal carcinoma (WDIC, 16), intermediately differentiated intraductal carcinoma (IDIC, 25), poorly differentiated intraductal carcinoma (PDIC, 20), atypical lobular hyperplasia (ALH, 13), and lobular carcinoma in situ (LCIS, 12). Both parameters were also studied in normal glandular structures obtained from normal breasts or from breasts containing pre‐invasive lesions. Increased vascularization was present in all lesion types (MVD mean values (expressed as vessel number/mm2): 115 ± 8 in normal lobules, 146 ± 26 in lesions; p < 0.05) and increased with lesion severity. In ductal lesions, MVDs were significantly higher in PDIC (190 ± 65) and IDIC (167 ± 61) than in FDHUT (123 ± 40) and ADH (122 ± 47); MVD was much higher in PDIC than in WDIC (p < 0.001). WDIC showed peculiar features, with a degree of vascularization closer to hyperplasia than to the other histological types of in situ ductal cancer; this observation is in line with the hypothesis that IDIC and PDIC may originate ‘de novo’, without a mandatory transition through WDIC. LCIS was more vascularized than ALH (168 ± 50 and 125 ± 40, respectively; p < 0.05), showing MVD values similar to those of PDIC and IDIC. The vascularization of normal lobules was constant, regardless of their association with lesions. VEGF expression in normal glandular structures was lower than in lesions, with the highest levels found in ductal lesions when compared with lobular lesions. No correlation was found between VEGF expression and the degree and/or type of vascularization. Copyright

Large needle aspiration biopsy and galectin-3 determination in selected thyroid nodules with indeterminate FNA-cytology.

Thyroid fine-needle aspiration biopsy (FNA)-cytology is widely used for the preoperative characterisation of thyroid nodules but this task is difficult for follicular lesions, which often remain undefined. We propose a strategy for improving the preoperative characterisation of selected follicular thyroid proliferations, which is based on large needle aspiration biopsy (LNAB) and galectin-3 expression analysis. Eighty-five thyroid specimens were obtained by LNAB (20-gauge needles) from thyroid nodules with indeterminate follicular FNA-cytology. Aspirated material was processed as a tissue microbiopsy to obtain cell blocks for both cyto/histo-morphological evaluation and galectin-3 expression analysis, by using a purified monoclonal antibody to galectin-3 and a biotin-free immunoperoxidase staining method. Preoperative diagnosis was compared to the final histology. LNAB and cell-block technique allow a preliminary distinction between nodules with a homogeneous microfollicular/trabecular structure, as frequently observed in tumours, and lesions with mixed normo–micro–macrofollicular architecture, as observed in goitre. Furthermore, LNAB provides optimal substrates for galectin-3 expression analysis. Among 85 cases tested, 14 galectin-3-positive cases were discovered preoperatively (11 thyroid cancers and three adenomas confirmed at the final histology), whereas galectin-3-negative cases were 71 (one carcinoma and 70 benign proliferations at the final histology). Sensitivity, specificity and diagnostic accuracy of this integrated morphologic and phenotypic diagnostic approach were 91.6, 97.2 and 95.3%, respectively. In conclusion, LNAB plus galectin-3 expression analysis when applied preoperatively to selected thyroid nodules candidate to surgery can potentially reduce unnecessary thyroid resections.

MASSIVE ISOLATION, MORPHOLOGICAL AND FUNCTIONAL-CHARACTERIZATION, AND XENOTRANSPLANTATION OF BOVINE PANCREATIC-ISLETS

The limited availability of human donors makes the search for alternative islet sources mandatory for future developments in pancreatic islet transplantation. In this study, we report on the massive isolation of bovine islets of proven in vitro and in vivo viability. The islets were prepared by collagenase digestion, sequential filtrations, and density-gradient purification by modifying a technique previously developed in our laboratory for the porcine pancreas. The prepurification yield was 2,743 +/- 78 islet equivalents (IE)/g pancreas (mean +/- SE), with a postpurification recovery of 78.7 +/- 2.2%. Purity ranged from 80 to 90%. The histological and immunocytochemical studies demonstrated the identity and integrity of the islets with well-preserved insulin-, glucagon-, and somatostatin-containing cells. The morphological integrity of cultured bovine islets was demonstrated for up to 4 weeks from isolation. Insulin secretion from freshly isolated islets was similar at 3.3 mmol/l glucose (0.36 +/- 0.06 pmol.IE-1.min-1) and at 14 mmol/l glucose (0.42 +/- 0.00 pmol.IE-1.min-1), and it increased significantly (P < 0.01) at 25 mmol/l glucose (1.44 +/- 0.12 pmol.IE-1.min-1). Arginine, theophylline, and propionic acid increased insulin secretion from freshly isolated islets at 3.3 and 14 mmol/l glucose, but not at 25 mmol/l glucose. Islets cultured at 37 degrees C in CMRL 1066 culture medium for at least 10 days were shown to become responsive to a lower glucose concentration, as demonstrated by the significant increase of insulin release in response to 14 mmol/l glucose, when compared with basal secretion. This recovered responsivity to glucose was maintained after 4 weeks of culture.(ABSTRACT TRUNCATED AT 250 WORDS)

mRNA markers of breast cancer nodal metastases: comparison between mammaglobin and carcinoembryonic antigen in 248 patients

Histological detection of axillary lymph node metastases is still the most valuable prognostic parameter for breast cancer, but about 30% of node‐negative patients relapse within five years, suggesting that current methods are inadequate for identifying metastatic disease. More sensitive, PCR‐based methods for the detection of metastatic cells are now available, enabling the amplification of cancer cell‐specific mRNA messages by the RT‐PCR assay. An ideal tumour marker, consistently expressed in tumour samples and not at all in normal lymph nodes, remains to be identified. The present study first investigated the expression of seven mRNA markers, CEA, CK19, c‐Met, mammaglobin, MUC‐1, β1→GalNAc‐T and p97, selected on the basis of their previously reported specificity for breast cancer cells. Eighteen lymph nodes were examined from patients without tumours. Only mammaglobin mRNA and CEA mRNA were not expressed in normal nodes. All of the other markers showed a band of expression in 17%–55% of cases, indicating that they are not breast cancer‐specific. CEA mRNA and mammaglobin mRNA expression could be detected in 15/20 (75%) and 19/20 (95%) primary breast carcinomas, respectively. The expression of mammaglobin mRNA and CEA mRNA was then compared in axillary lymph nodes from 248 consecutive breast cancer patients, 89 with histologically documented lymph node metastasis and 159 without histological evidence of metastatic disease. Ninety‐seven per cent of the patients with histologically involved nodes showed expression of mammaglobin mRNA, whereas CEA mRNA was expressed in 79% of these cases. In the group of patients with histologically negative lymph nodes, 46 (29%) and 32 (20%) were found to be positive for mammaglobin and CEA expression, respectively, indicating the presence of metastases not detected by routine histological examination of one lymph node section. These results show that both mammaglobin RT‐PCR and CEA RT‐PCR are useful tools for the detection of breast cancer metastases in axillary lymph nodes. The detection sensitivity of the mammaglobin RT‐PCR is far superior to that of the CEA RT‐PCR, allowing the diagnosis of occult metastases in nearly one‐third of cases. Copyright

Genetic analyses in familial isolated hyperparathyroidism: implication for clinical assessment and surgical management.

Objective  Familial isolated primary hyperparathyroidism (FIPH) can result from either incomplete expression of a syndromic form of familial primary hyperparathyroidism [multiple endocrine neoplasia type 1 (MEN 1), hyperparathyroidism–jaw tumour syndrome (HPT‐JT) or familial hypocalciuric hypercalcaemia (FHH)] or still unrecognized causes.

Spectrum of GCDFP-15 expression in human fetal and adult normal tissues

Abstract GCDFP-15, a glycoprotein identified in the cyst fluid of cystic breast disease, is considered to be a marker of apocrine differentiation. Studies on GCDFP-15 localization in adult normal tissues are lacking, and no information on GCDFP-15 expression during fetal development has been reported. We investigated GCDFP-15 expression in a large series of formalin-fixed, paraffin-embedded normal human adult and fetal tissues using the monoclonal antibody BRST-2. In normal adult tissues GCDFP-15 expression was found in all apocrine, lacrimal, ceruminous and Moll’s glands and in numerous serous cells of the submandibular, sublingual and minor salivary glands. The serous cells of nasal and bronchial glands were also positive; parotid and laryngeal glands showed rare immunoreactive cells. GCDFP-15-positive cells were observed in all cutaneous eccrine glands from different body sites. In fetal tissues immunoreactivity was observed in numerous acinous cells of all tracheal, bronchial and submandibular salivary glands. GCDFP-15 positivity was identified in numerous cells of all axillary sweat glands and in rare cells of some sweat glands of the thorax, abdomen, back, leg and arm. In both apocrine and nonapocrine glands GCDFP-15 was always localized in the secretory component. These data suggest that GCDFP-15 is a glandular differentiation marker associated with apocrine secretion; that it is expressed in glands that have phylogenetic origins in common with apocrine glands (submandibular salivary and submucosal bronchial glands); and that eccrine cutaneous glands express GCDFP-15 and thus might be referred to as mixed apocrine-eccrine glands. GCDFP-15 is expressed during fetal development and may represent a common marker of embryologically linked glandular structures.