Antonio Giuseppe Naccarato

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.

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.

Region-dependent effects of flibanserin and buspirone on adenylyl cyclase activity in the human brain

The mode of action of antidepressant drugs may be related to mechanisms of receptor adaptation, involving overall the serotonin 1A (5-HT1A) receptor subtype. However, so far, the clinical effectiveness of selective compounds acting at this level has proved disappointing. This could be explained by the heterogeneity of 5-HT1A receptors within the central nervous system. In animals, two 5-HT1A agonists, flibanserin and buspirone, have shown different pharmacological properties, depending on the brain region. Since no evidence supports this observation in humans, this study sought to investigate whether these two drugs exert different effects on 5-HT1A receptor activation in three different human brain areas: the prefrontal cortex, hippocampus and raphe nuclei. 5-HT1A-mediated inhibition of forskolin-stimulated adenylyl cyclase (AC) was taken as an index of 5-HT1A receptor activation. Flibanserin significantly reduced the activity of AC post-synaptically, i.e. in the prefrontal cortex [EC50 (mean +/- S.E.M.), 28 +/- 10.2 nM; Emax, 18 +/- 2.3%] and in the hippocampus (EC50, 3.5 +/- 3.1 nM; Emax, 20 +/- 4.0%), but had no effect in the raphe nuclei, i.e. at pre-synaptic level. Vice versa, buspirone was only slightly but significantly effective in the raphe (EC50, 3.0 +/- 2.8 nM; Emax, 12 +/- 1.9%). Agonist effects were sensitive to the 5-HT1A antagonists WAY-100135 and pindobind 5-HT1A in the cortex and raphe nuclei, whereas buspirone antagonized flibanserin in the hippocampus. These findings suggest a region-related action of flibanserin and buspirone on forskolin-stimulated AC activity in human brain.

ALK-1-negative anaplastic large cell lymphoma associated with breast implants: a new clinical entity.

Concerns have been raised recently regarding the increasing number of reports of non-Hodgkin lymphoma (NHL) that developed in close proximity to silicone or saline breast implants. In particular, an increased risk of anaplastic large cell lymphoma (ALCL) in patients with breast prostheses has been proposed. We reviewed clinical and pathologic findings in 40 women who received a diagnosis of breast NHL arising in association with breast implants and of 27 patients who had a diagnosis of ALCL with breast involvement reported in the published literature. Among the 40 reported cases of prosthesis-associated breast lymphomas, 28 were anaplastic lymphoma kinase-1-negative (ALK-1(-)) ALCLs, whereas of 27 ALCLs in patients without implants found in the literature, only 10 were ALK-1(-). The finding of 28 cases of breast ALK-1(-) ALCL occurring in patients with implants compared with 10 cases in women without implants is in favor of an association between silicone breast prostheses and ALK-1(-) ALCL. Although the incidence of this type of lymphoma remains remarkably low given that breast prostheses have been widely used for decades, clinical and pathologic evidence for a causative role is becoming dramatically strong. The histologic, phenomenologic, and clinical similarities of the majority of implant-related ALK-1(-) ALCLs suggest a common mechanism, especially when compared with the counterpart of patients without implants in which very few and highly dishomogeneous cases of the same malignancy were detected. There is convincing evidence that primary implant-related ALK-1(-) ALCL represents a distinct clinicopathologic entity that has been inappropriately fitted into the category of systemic ALK-1(-) ALCL. Thus it should be recognized as a separate category and classified on its own.

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

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.

THE SPECTRUM OF 67-kD LAMININ RECEPTOR EXPRESSION IN BREAST CARCINOMA PROGRESSION

Laminin is a glycoprotein of the basement membrane (BM), involved in a variety of normal and pathological cellular events including tumour invasion and metastasis. Cells bind laminin through different types of receptor. The 67‐kD laminin receptor (67LR) is a cell‐surface protein which binds laminin with high affinity. 67LR expression has been shown to increase in neoplastic cells, compared with normal tissues, and 67LR seems to play an important role during the first steps of neoplastic progression. In this study, 67LR expression was analysed during the morphological phases of breast cancer progression from normal tissue to invasive carcinoma. A total of 506 formalin‐fixed, paraffin‐embedded normal breast structures and lesions were stained by immunohistochemistry using the MLuC5 monoclonal antibody, which is specific for 67LR. The results show that in normal breast and in any kind of breast lesion, myoepithelial and endothelial cells express 67LR. While 67LR is not seen in the epithelium of normal breast, cysts, adenosis, and benign tumours, it is expressed in the epithelial cells of several hyperplasias and carcinomas in situ, both ductal and lobular, as well as in all invasive carcinomas. The 67LR‐positive cell subpopulation expands from hyperplastic lesions to invasive carcinoma, suggesting that 67LR could be related to the induction and progression of breast cancer.