Germline investigation in male breast cancer of DNA repair genes by next-generation sequencing.

PURPOSE: In order to better define the breast cancer (BC) genetic risk factors in men, a germline investigation was carried out on 81 Male BC cases by screening the 24 genes involved in BC predisposition, genome stability maintenance and DNA repair mechanisms by next-generation sequencing. METHODS: Germline DNAs were tested in a custom multi-gene panel focused on all coding exons and exon-intron boundaries of 24 selected genes using two amplicon-based assays on PGM-Ion Torrent (ThermoFisher Scientific) and MiSeq (Illumina) platforms. All variants were recorded and classified by using a custom pipeline. RESULTS: Clinical pathological data and the family history of 81 Male BC cases were gathered and analysed, revealing the average age of onset to be 61.3 years old and that in 35 cases there was a family history of BC. Our genetic screening allowed us to identify a germline mutation in 22 patients (23%) in 4 genes: BRCA2, BRIP1, MUTYH and PMS2. Moreover, 12 variants of unknown clinical significance (VUS) in 9 genes (BARD1, BRCA1, BRIP1, CHEK2, ERCC1, NBN, PALB2, PMS1, RAD50) were predicted as potentially pathogenic by in silico analysis bringing the mutation detection rate up to 40%. CONCLUSION: As expected, a positive family history is a strong predictor of germline BRCA2 mutations in male BC. Understanding the potential pathogenicity of VUS represents an extremely urgent need for the management of BC risk in Male BC cases and their own families.

Molecular profiling of microinvasive breast cancer microenvironment progression.

BACKGROUND: Tumors develop by progression through a series of stages. Every cell of the tumor microenvironment is constantly changing in the flow of the cancer progression. It has become clear in recent years that stroma is essential for tumor maintenance and growth. Here, we aimed to give a chronological order of gene expression changes given in the dynamical framework of microinvasive breast cancer microenvironment. METHODS: RNA-seq was performed on seven microinvasive breast cancers. For each of them we microdissected seven different portions of the tumor, four related to the breast epithelium and three to the stroma. Breast epithelium was chronologically subdivided in normal breast epithelium (NBE), carcinoma in situ (CIS), emerging invasive fingers (EIF) and invasive breast cancer (IBC). For each of the breast epithelium subdivisions we collected the adjacent stroma (S): S-NBE, S-EIF and S-IBC. RESULTS: The overall differentially expressed genes (DEGs) in all the compartments were analysed and evaluated to understand the pathways involved in tumor progression. Then we analysed the DEGs of the epithelial and stromal portions in comparison with the normal portions. We observed that the stromal cells are necessary for the development and the maintenance of the tumor, especially in tumor progression. Moreover the most important genes involved in the main metabolic pathways were analysed and the communications within the different cell compartments were highlighted. CONCLUSIONS: As a future perspective, a deeply study of the identified key genes, particularly in the stromal cells, will be crucial to develop an anticancer therapy that is undergoing a conversion from a cancer cell-centric strategy to a stroma-centric strategy, more genomically stable.

Male secretory breast cancer: case in a 6-year-old boy with a peculiar gene duplication and review of the literature.

PURPOSE: Secretory breast cancer (SBC) is one of the rarest breast cancer (BC), representing the majority of BC in childhood. Nevertheless, it elicits a lot of interest both for the peculiar morphology and the characteristic genetic features. Currently, there is no consensus on optimal treatment strategy. Therefore, it is useful to report every case in order to establish treatment algorithms. METHODS: We describe the case of a 6-year-old boy diagnosed with a SBC, with peculiar genomic and immunohistochemical features. Moreover, we carried out a review of the literature in order to analyze the present state of knowledge about this rare entity. RESULTS: To the best of our knowledge, there are only 120 cases published in literature, only 32 in males and only 2 younger than 6 years. Furthermore, this one had peculiar genomic and immunohistochemical features. Indeed, even if SBC expresses basal-cell markers, our patient had a triple-negative tumor expressing both basal and luminal cell markers. Furthermore, the boy's genomic profile revealed not only positivity for the typical SBC's translocation t(12;15), but also for a 3q28 duplication, found in his father (healthy) and paternal grandfather (with a previous BC). None were positive for BRCA mutation. This locus includes only one gene encoding for a growth factor recently linked to Early Infantile Epileptic Encephalopathy-47 and Idiopathic ventricular tachycardia. Even if the literature does not provide evidence of a pathogenic role it is not possible to exclude a cancer-predisposing activity. CONCLUSIONS: SBC is a rare type of BC, characterized by triple-negative features with an unexpectedly good prognosis. More data are needed to fully understand the behavior of this cancer and genomic profiling could be helpful in improving its diagnosis and management.

Association between RAD 51 rs1801320 and susceptibility to glioblastoma.

Glioblastoma is the most common and aggressive malignant primary brain tumor. Despite decades of research and the advent of new therapies, patients with glioblastoma continue to have a very poor prognosis. Radiation therapy has a major role as adjuvant treatment for glioblastoma following surgical resection. Many studies have shown that polymorphisms of genes involved in pathways of DNA repair may affect the sensitivity of the cells to treatment. Although the role of these polymorphisms has been investigated in relation to response to radiotherapy, their role as predisposing factors to glioblastoma has not been clarified yet. In the present study, we evaluated the association between polymorphisms in DNA repair genes, namely: XRCC1 rs25487, XRCC3 rs861539 and RAD51 rs1801320, with the susceptibility to develop glioblastoma. Eighty-five glioblastoma patients and 70 matched controls were recruited for this study. Data from the 1000 Genomes Project (98 Tuscans) were also downloaded and used for the association analysis. Subjects carrying RAD51 rs1801320 GC genotype showed an increased risk of glioblastoma (GC vs GG, χ(2) = 10.75; OR 3.0087; p = 0.0010). The C allele was also significantly associated to glioblastoma (χ(2) = 8.66; OR 2.5674; p = 0.0032). Moreover, RAD51 rs1801320 C allele increased the risk to develop glioblastoma also when combined to XRCC1 rs25487 G allele and XRCC3 rs861539 C allele (χ(2) = 6.558; p = 0.0053).

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.

Role of circulating endothelial progenitor cells in the reparative mechanisms of stable ischemic myocardium.

BACKGROUND: Mobilization of endothelial progenitor cells (EPCs) into circulation from bone marrow in patients with acute myocardial infarction has strong scientific evidence; less is known about EPC mobilization in patients with stable coronary artery disease (CAD). The aim of this study was to investigate the association of stable ischemic heart disease with EPC levels in tissue and blood. METHODS: Fifty-five consecutive patients admitted to a single treatment center for valve or coronary artery bypass grafting (CABG) surgeries were included in the study. Blood samples were collected in the morning before surgery and analyzed by flow-cytometry to determine peripheral EPC levels (EPC/ml). Tissue EPC (CD34+VEGFR2+) levels were assessed on a right atrial appendage segment. RESULTS: Mean age was 76±5years, 48% were men, and 53% had CAD The number of CD34+ VEGFR2+ cells in the tissue of patients with CAD was significantly higher (p<0.005) and circulating EPC showed a tendency to be reduced by approximately 20% in peripheral blood of patients with CAD when compared to those without CAD. CONCLUSION: Patients with stable CAD had higher EPC density values (EPC/mm2) and were more likely to have lower EPC blood levels when compare with normal controls.

Immunohistochemical markers of neural progenitor cells in the early embryonic human cerebral cortex.

The development of the human central nervous system represents a delicate moment of embryogenesis. The purpose of this study was to analyze the expression of multiple immunohistochemical markers in the stem/progenitor cells in the human cerebral cortex during the early phases of development.  To this end, samples from cerebral cortex were obtained from 4 human embryos of 11 weeks of gestation. Each sample was formalin-fixed, paraffin embedded and immunostained with several markers including GFAP, WT1, Nestin, Vimentin, CD117, S100B, Sox2, PAX2, PAX5, Tß4, Neurofilament, CD44, CD133, Synaptophysin and Cyclin D1. Our study shows the ability of the different immunohistochemical markers to evidence different zones of the developing human cerebral cortex, allowing the identification of the multiple stages of differentiation of neuronal and glial precursors. Three important markers of radial glial cells are evidenced in this early gestational age: Vimentin, Nestin and WT1. Sox2 was expressed by the stem/progenitor cells of the ventricular zone, whereas the postmitotic neurons of the cortical plate were immunostained by PAX2 and NSE. Future studies are needed to test other important stem/progenitor cells markers and to better analyze differences in the immunohistochemical expression of these markers during gestation.

Angiogenesis in human normal and pathologic adrenal cortex.

The angiogenic phenotype of 13 normal adrenal glands (N), 13 aldosterone-producing adenomas (APA), 12 cortisol-producing adenomas (CPA), 13 nonfunctioning adrenal cortical adenomas (NFA), and 13 adrenal cortical carcinomas (CA) was investigated. Intratumoral vascular density was explored by CD34, a marker of endothelial cells, and the angiogenic status was investigated by vascular endothelial growth factor (VEGF) expression, an important angiogenic factor expressed by tumoral cells. Vascular density, quantified as the number of vessels per square millimeter, was significantly lower (P < 0.0001) in CA (110.3 +/- 27.8) than in N (336.6 +/- 14.5), APA (322.8 +/- 19.1), CPA (288.5 +/- 14.3), and NFA (274.2 +/- 19.8). VEGF expression, calculated as the percentage of positive cells, was significantly greater (P < 0.0001) in CA (85.3 +/- 2.1) than in APA (56.5 +/- 7.5), CPA (38.5 +/- 7.0), N (33.1 +/- 6.1), and NFA (0.76 +/- 0.6). In APA, a negative relation between CD34 and plasma renin activity (P < 0.0002) and a positive association between CD34 and aldosterone levels (P < 0.05) was found. In conclusion, the angiogenic phenotype of CA is characterized by VEGF overexpression but low vascularization, a finding suggesting a dissociation between angiogenic potential and neoangiogenic capabilities of these tumors. The lack of VEGF expression in NFA and the close association between angiogenesis and functional status in APA also suggest a possible influence of the angiogenic phenotype on hormonal secretion of these endocrine tumors.

Activating thyrotropin receptor mutations are present in nonadenomatous hyperfunctioning nodules of toxic or autonomous multinodular goiter.

Toxic multinodular goiter, a heterogeneous disease producing hyperthyroidism, is frequently found in iodine-deficient areas. The pathogenesis of this common clinical entity is still unclear. The aim of the present study was to search for activating TSH receptor (TSHr) or Gs alpha mutations in areas of toxic or functionally autonomous multinodular goiters that appeared hyperfunctioning at thyroid scintiscan but did not clearly correspond to definite nodules at physical or ultrasonographic examination. Surgical tissue specimens from nine patients were carefully dissected, matching thyroid scintiscan and thyroid ultrasonography, to isolate hyperfunctioning and nonfunctioning areas even if they did not correspond to well-defined nodules. TSHr and Gs alpha mutations were searched for by direct sequencing after PCR amplification of genomic DNA. Only 2 adenomas were identified at microscopic examination, whereas the remaining 18 hyperfunctioning areas corresponded to hyperplastic nodules containing multiple aggregates of micromacrofollicules not surrounded by a capsule. Activating TSHr mutations were detected in 14 of these 20 hyperfunctioning areas, whereas no mutation was identified in nonfunctioning nodules or areas contained in the same gland. No Gs alpha mutation was found. In conclusion, activating TSHr mutations are present in the majority of nonadenomatous hyperfunctioning nodules scattered throughout the gland in patients with toxic or functionally autonomous multinodular goiter.

Functioning and nonfunctioning thyroid adenomas involve different molecular pathogenetic mechanisms.

The molecular biology of follicular cell growth in thyroid nodules is still poorly understood. Because gain-of-function (activating) mutations of the thyroid-stimulating hormone receptor (TShR) and/or Gs alpha genes may confer TSh-independent growth advantage to neoplastic thyroid cells, we searched for somatic mutations of these genes in a series of hyperfunctioning and nonfunctioning follicular thyroid adenomas specifically selected for their homogeneous gross anatomy (single nodule in an otherwise normal thyroid gland). TShR gene mutations were identified by direct sequencing of exons 9 and 10 of the TShR gene in genomic DNA obtained from surgical specimens. Codons 201 and 227 of the Gs alpha gene were also analyzed. At histology, all hyperfunctioning nodules and 13 of 15 nonfunctioning nodules were diagnosed as follicular adenomas. Two nonfunctioning thyroid nodules, although showing a prevalent microfollicular pattern of growth, had histological features indicating malignant transformation (a minimally invasive follicular carcinoma and a focal papillary carcinoma). Activating mutations of the TShR gene were found in 12 of 15 hyperfunctioning follicular thyroid adenomas. In one hyperfunctioning adenoma, which was negative for TShR mutations, a mutation in codon 227 of the Gs alpha gene was identified. At variance with hyperfunctioning thyroid adenomas, no mutation of the TShR or Gs alpha genes was detected in nonfunctioning thyroid nodules. In conclusion, our findings clearly define a different molecular pathogenetic mechanism in hyperfunctioning and nonfunctioning follicular thyroid adenomas. Activation of the cAMP cascade, which leads to proliferation but maintains differentiation of follicular thyroid cells, typically occurs in hyperfunctioning thyroid adenomas. Oncogenes other than the TShR and Gs alpha genes are probably involved in nonfunctioning follicular adenomas.

Parathyroid expression of calcium-sensing receptor protein and in vivo parathyroid hormone-Ca(2+) set-point in patients with primary hyperparathyroidism.

A reduced expression of calcium-sensing receptor (CaR) messenger ribonucleic acid and protein accompanied by abnormalities in parathyroid cell proliferation and PTH secretion are present in primary hyperparathyroidism. We studied the expression of CaR protein by immunohistochemistry in 36 sporadic parathyroid adenomas and investigated the relationship between CaR expression and several preoperative clinical parameters, including the set-point of Ca(2+)-regulated PTH secretion (measured in vivo). The adenomas were classified in 4 categories according to the intensity of immunohistochemical staining: 5 (14%) showed a CaR staining intensity similar to that of normal parathyroid ( ), 10 (27%) showed moderate staining (++), 16 (45%) showed weak staining (+), and 5 (14%) were negative (-). The intensity of CaR staining was not related to preoperative serum Ca(2+), PTH levels or adenoma volume. Twenty-nine patients underwent preoperatively the calcium infusion test to evaluate the PTH-Ca(2+) set-point. Individual values of PTH-Ca(2+) set-point ranged from 1.38-1.93 mmol/L and were significantly correlated with basal Ca(2+) levels (r = 0.96; P: = 0. 0001) and adenoma volume (r = 0.5; P: = 0.01). The mean PTH-Ca(2+) set-point values were significantly different in the 4 groups of patients classified according to immunohistochemical staining intensity of their adenoma (P: = 0.025; F = 3.78); the mean PTH-Ca(2+) set-point was significantly higher in the groups classified as negative than in those classified as weak or moderate. No correlation was observed between the PTH-Ca(2+) set-point and basal PTH levels or between the percent maximal PTH inhibition and adenoma volume and basal PTH or Ca(2+) levels. In summary, our data suggest that there is a relationship between apparent CaR protein expression and PTH-Ca(2+) set-point abnormality, suggesting that a reduced receptor content might have an important role in the pathogenesis of primary hyperparathyroidism.

Primary chemotherapy in locally advanced breast cancer (LABC): effects on tumour proliferative activity, bcl-2 expression and the relationship between tumour regression and biological markers.

The rate of tumour cell proliferation evaluated by the [3H]-thymidine labelling index ([3H]-dT-LI) is known to be an independent prognostic factor in patients with operable breast cancer and significantly predicts the response to chemotherapy in patients with advanced disease. In locally advanced breast cancer (LABG), we examined whether chemotherapy induced modifications in [3H]-dt-LI, and bcl-2 expression and their relationship with tumour regression and prognosis. 70 LABC patients received three courses of primary chemotherapy (FEC: 5-fluorouracil 600 mg/m2, epidoxorubicin 60 mg/m2, cyclophosphamide 600 mg/m2, followed by surgery and subsequent adjuvant chemotherapy consisting of three courses of FEC alternated with three courses of CMF (cyclophosphamide 600 mg/m2, methotrexate 40 mg/m2, 5-fluorouracil 600 mg/m2). Tumour biological markers were evaluated on diagnostic biopsy, before primary chemotherapy and at surgery. Tumour cell proliferation was determined by [3H]-dT-LI, whilst bcl-2 expression was examined by immunohistochemical staining. The overall response rate to primary FEC was 74.3% (95% confidence interval 57.6-83.2%). The response rate correlated with high [3H]-dT-LI: 88% (29/33) of patients with high [3H]-dT-LI achieved an objective response compared with 62% (23/37) of patients with low [3H]-dT-LI (P = 0.014). The 3 patients achieving a pathological complete response after induction treatment had high proliferative tumours. The highest 2-year relapse free survival (66.6%) was observed in patients with low [3H]-dT-LI after primary chemotherapy. The median bcl-2 expression values before and after primary chemotherapy were 0% (range 0-80) and 30% (range 0-90), respectively (P = 0.03). Our data indicate that primary chemotherapy can modulate tumour cell kinetics and apoptosis-related genes. Pretreatment proliferative activity correlated with tumour response, whilst post-treatment [3H]-dT-LI correlated with relapse free survival.

Cryopreservation of purified bovine islets of Langerhans.

In this study, bovine islets were isolated by collagenase digestion and density gradient purification, equilibrated stepwise with 3 M dimethylsulfoxide at 24 degrees C, nucleated at -150 degrees C, slow cooled at 0.25 degrees C/min down to -40 degrees C, and finally stored at -150 degrees C. After variable periods of time, the islets were quickly thawed at 37 degrees C, and dimethylsulfoxide was removed by 0.75 M sucrose. Postthawing recovery was 86 +/- 6% islet equivalents. Histology confirmed the identity and morphological integrity of the islets. Insulin release from the frozen-thawed islets was 0.13 +/- 0.03 microU/is/min at 3.3 mmol/L glucose and increased significantly (0.27 +/- 0.04 microU/is/min, P < 0.05) at 25 mmol/L glucose. Encapsulated, cryopreserved islets reversed hyperglycemia in diabetic mice after 6-8 days following implantation. Therefore, the method described in this paper permitted successful cryopreservation of bovine islets of proven in vitro and in vivo viability.

Preoperative localization of suspicious parathyroid adenomas by assay of parathyroid hormone in needle aspirates.

OBJECTIVE: To determine the usefulness of parathyroid hormone (PTH) measurement in needle aspirates of a suspicious neck mass to confirm its parathyroid nature in patients with primary hyperparathyroidism. METHODS: Thirty-three patients with surgically proved primary hyperparathyroidism were submitted to neck ultrasound (US), parathyroid scintigraphy, and assay of PTH in the aspirate (PTHa) of the suspicious cervical mass. RESULTS: Based on the results of neck US and parathyroid scintigraphy, patients were divided into two groups. Group 1: 16 patients (seven with nodular goiter) with concordant positive US and scintigraphic results. In all but one patient, PTHa was detectable and often markedly elevated (> 1000 pg in 12 patients, between 292 pg and 803 pg in three patients and 53 pg in one patient). The patient with undetectable PTHa had a small lower left parathyroid adenoma (8x8x10 mm). Group 2: 17 patients (12 with nodular goiter) with discordant US and scintigraphic results. PTHa established the parathyroid nature of the mass in 13 cases (> 1000 pg in 8 patients, between 501 pg and 953 pg in three patients and 90 and 79 pg in two patients): 11 of these had a suspected lesion by US examination but the scintigraphy results were negative; two had a mass that gave positive scintigraphy results but was of uncertain origin according to US: in both cases an intrathyroidal parathyroid adenoma was found. PTHa was undetectable in four cases (three with nodular goiter): all of these had equivocal US results, and three had positive scans and one a negative scan. CONCLUSIONS: Assay of PTHa is a simple method and should be useful for confirming the parathyroid nature of a cervical mass in patients with discordant or non-diagnostic US and scintigraphic results.

Lack of stereoselectivity of 8-hydroxy-2(di-N-propylamino)tetralin-mediated inhibition of forskolin-stimulated adenylyl cyclase activity in human pre- and post-synaptic brain regions.

The stereoselectivity of the serotonin1A (5-HT1A) receptor compound 8-hydroxy-2(di-N-propylamino)tetralin (8-OH-DPAT) on forskolin-stimulated adenylyl cyclase activity was investigated in membranes from human 5-HT pre-synaptic (raphe nuclei) and post-synaptic (hippocampus and prefrontal cortex) regions of autopsy brains. After sample incubation with agonists and antagonists, results showed that both the racemic mixture of 8-OH-DPAT or its (+) and (-) enantiomers behaved as full agonists in the tested brain regions. Enantiomer potency (EC50, nM) and efficacy (percentage of maximal inhibition, %) values were similar in all regions under investigation. However, some inter and intra-region variations in racemic 8-OH-DPAT potency and efficacy have been observed. In particular, the potency of racemic 8-OH-DPAT was higher in the prefrontal cortex and raphe nuclei than in the hippocampus, where it was in fact lower than either single enantiomers. Agonist effects were competitively reversed by 5-HT1A antagonists, although once again a different profile was revealed in the hippocampus. The data underscores the lack of stereospecificity of 8-OH-DPAT-mediated inhibition of adenylyl cyclase activity in either pre- or post-synaptic human brain regions. Moreover, such results have significant implication, as they support the notion that human 5-HT1A receptors might vary from one brain region to the other.

Further characterisation of [3H]8-hydroxy-2-(di-N-propylamino)tetralin binding sites in human brain postmortem.

The saturation parameters and the pharmacological characteristics of the binding of the serotonin 1A (5-HT1A) receptor agonist [3H]8-hydroxy-2-(di-N-propylamino)tetralin ([3H]8-OH-DPAT), as well as the effects of nucleotides and divalent cations (Mg2+, Mn2+) on it, were compared in some human postmortem brain regions: the main cortical areas, hippocampus and striatum. [3H]8-OH-DPAT labelled a single population of recognition sites with the highest maximal capacity (Bmax) in the hippocampus and the lowest affinity in the striatum. Among the various cortical areas, the frontal cortex exhibited the highest Bmax. The pharmacological profile of the [3H]8-OH-DPAT binding sites was consistent with the labelling of the 5-HT1A receptor in the hippocampus and cortex, whereas the striatal site shared strong similarity to the presynaptic serotonin transporter. Modulation of [3H]8-OH-DPAT binding by divalent cations and nucleotides was detectable and stable in autopsy brains. In particular, nucleotide effects were area-dependent: guanosine thiotriphosphate (GTP gamma S) reduced [3H]8-OH-DPAT binding to the same extent in the hippocampus and frontal cortex, while having no effect in the striatum. Divalent cation effects depended also upon the brain area: in the striatum, they inhibited [3H]8-OH-DPAT binding, while stimulating it in the hippocampus and, with less extent, in the frontal cortex. In summary, these findings suggest that the [3H]8-OH-DPAT binding and its modulatory parameters in human brain tissues seem to show similarities but also some differences with respect to those determined in the rat brain. Furthermore, postmortem stability of GTP and divalent cation sensitive 5-HT1A receptors underlines the need for further studies on the regulatory and functional properties of this receptor in the human brain.

Apocrine epithelium of the breast: does it result from metaplasia?

Benign and malignant breast lesions may show an apocrine epithelium considered to be the result of metaplasia. In an attempt to clarify the histogenesis of the breast apocrine epithelium we searched for the presence of apocrine cells or cells with apocrine differentiation during human breast development. We analysed 10 autopsy specimens of female breasts from fetuses between 28 and 40 weeks of gestational age and tissue from 6 normal breasts, obtained after breast reduction in nulliparous young women between 22 and 28 years of age. Formalin-fixed, paraffin-embedded sections were stained with haematoxylin-eosin, PAS-diastase and a monoclonal antibody (BRST-2) anti-GCDFP-15, which is a specific apocrine marker. A 40-week fetal breast was analysed by electron microscopy. No cells with histological and ultrastructural apocrine features were found in the ducts of fetal breasts. All fetal breasts showed some ducts with sparse GCDFP-15-immunoreactive cells; the number of these cells increased with gestational age. PAS-diastase was negative. No cells with apocrine morphology were found in ducts and lobules of normal adult breasts. Scattered GCDFP-15-positive luminal epithelial cells and rare PAS-diastase-positive cells were observed in some lobules of all adult breasts. Cells with biochemical characteristics (GCDFP-15 expression) of apocrine differentiation are evident during human fetal breast development and persist in adult mammary glands. Unknown stimuli may induce these cells to take on an apocrine morphology. Apocrine epithelium of the breast may be a normal process of differentiation rather than metaplasia. We suggest the term "apocrine differentiation precursor cells" for GCDFP-15-positive breast epithelial cells with no apocrine morphology.

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

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.

Bio-morphological events in the development of the human female mammary gland from fetal age to puberty.

Bio-morphological understanding of the developing human mammary glands may clarify some aspects of breast pathology, including cancer. In particular, some epidemiological data suggests that during fetal growth an altered intrauterine hormonal status, especially a change in estrogen status, could predispose to carcinogenesis. In an attempt to achieve new information on early breast growth, a series of developing human breasts have been analyzed, namely: 4 fetal breasts (28-32 weeks of gestational age), 7 infant breasts (7 h to 2 years) and 1 puberal breast (12 years). In addition to the morphological features, we studied the immunohistochemical expression of some markers involved in morphogenesis, such as MIB-1 for cell proliferation, bcl-2 for apoptosis control, CD34 for vasculogenesis, estrogen (ER) and progesterone (PR) receptors for hormonal profile, and smooth-muscle actin for myoepithelial differentiation. The results were as follows: (a) lobules, absent between 28 weeks and 2 days, were well evident at 2 years of age and at puberty; (b) myoepithelial cells appeared from 28 weeks onward and persisted later with no modification in quantity and distribution; (c) epithelial cell proliferation was constantly low; (d) in all breasts inner epithelial cells showed diffuse bcl-2 positivity, while basal myoepithelial-like cells were generally negative; (e) all breasts were well vascularized with two different patterns: periductal vascularization (PDV) and interductal vascularization (IDV), IDV being always present, whereas PDV was found only in infant breasts; (f) ER and PR were almost absent in fetal and infant breasts, while their expression was high in the epithelial cells of the puberal breast; (g) stromal cells had no hormonal receptors and were heterogeneous for proliferation and bcl-2 expression. Interestingly, two fetal breasts showed high proliferation and high ER expression, respectively, in their epithelial compartment. This could be the expression of an altered hormonal environment in utero, representing a basis for possible subsequent cancer initiation.

Apparent absence of aging and gender effects on serotonin 1A receptors in human neocortex and hippocampus.

The effects of gender, aging and gender x age on the binding of the 5-HT1A receptor high-affinity agonist [3H]8-hydroxy-2(di-N-propylamino)tetralin ([3H]8-OH-DPAT), were evaluated and compared in tissues of human prefrontal, temporal, parietal, occipital cortex and hippocampus obtained from 21 autopsy subjects. The results revealed no variation with age or gender in either the [3H]8-OH-DPAT maximum binding capacity (Bmax) or dissociation constant (Kd) values. On the other hand, when separate correlations to subject ages were performed for men and women, aging effects on [3H]8-OH-DPAT Bmax and Kd were detected: in men, a significant age-dependent decrease in Kd values was observed in the occipital cortex; in women, the Bmax significantly decreased with aging in the parietal cortex and hippocampus, while increasing in occipito-cortical membranes. Overall, the present study reveals that, although neither gender nor aging 'per se' seem to modify the 5-HT1A receptor binding, gender may reveal region-specific aging effects, i.e. on receptor affinity in men and receptor density in women. Such findings should stimulate further investigation on the hypothesized existence of gender x age-related cross-connections between serotonergic system and hypothalamus-pituitary-gonadal circuits.