XRCC1 and ERCC1 variants modify malignant mesothelioma risk: a case-control study.

Malignant pleural mesothelioma (MPM) is a rare aggressive tumor associated with asbestos exposure. The possible role of genetic factors has also been suggested and MPM has been associated with single nucleotide polymorphisms (SNPs) of xenobiotic and oxidative metabolism enzymes. We have identified an association of the DNA repair gene XRCC1 with MPM in the population of Casale Monferrato, a town exposed to high asbestos pollution. To extend this observation we examined 35 SNPs in 15 genes that could be involved in MPM carcinogenicity in 220 MPM patients and 296 controls from two case-control studies conducted in Casale (151 patients, 252 controls) and Turin (69 patients, 44 controls), respectively. Unconditional multivariate logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (95% CIs). Two DNA repair genes were associated with MPM, i.e. XRCC1 and ERCC1. Considering asbestos-exposed only, the risk increased with the increasing number of XRCC1-399Q alleles (Casale: OR=1.44, 95%CI 1.02-2.03; Casale+Turin: OR=1.34, 95%CI 0.98-1.84) or XRCC1 -77T alleles (Casale+Turin: OR=1.33, 95%CI 0.97-1.81). The XRCC1-TGGGGGAACAGA haplotype was significantly associated with MPM (Casale: OR=1.76, 95%CI 1.04-2.96). Patients heterozygotes for ERCC1 N118N showed an increased OR in all subjects (OR=1.66, 95%CI 1.06-2.60) and in asbestos-exposed only (OR=1.59, 95%CI 1.01-2.50). When the dominant model was considered (i.e. ERCC1 heterozygotes CT plus homozygotes CC versus homozygotes TT) the risk was statistically significant both in all subjects (OR=1.61, 95%CI 1.06-2.47) and in asbestos-exposed only (OR=1.56, 95%CI 1.02-2.40). The combination of ERCC1 N118N and XRCC1 R399Q was statistically significant (Casale: OR=2.02, 95%CI 1.01-4.05; Casale+Turin: OR=2.39, 95%CI 1.29-4.43). The association of MPM with DNA repair genes support the hypothesis that an increased susceptibility to DNA damage may favour asbestos carcinogenicity.

Asbestos induces doxorubicin resistance in MM98 mesothelioma cells via HIF-1alpha.

Human malignant mesothelioma (HMM), which is strongly related to asbestos exposure, exhibits high resistance to many anticancer drugs. Asbestos fibre deposition in the lung may cause hypoxia and iron chelation at the fibre surface. Hypoxia-inducible factor (HIF)-1alpha, which is upregulated by a decreased availability of oxygen and iron, controls the expression of membrane transporters, such as P-glycoprotein (Pgp), which actively extrude the anticancer drugs. The present study aimed to assess whether asbestos may play a role in the induction of doxorubicin resistance in HMM cells through the activation of HIF-1alpha and an increased expression of Pgp. After 24-h incubation with crocidolite asbestos or with the iron chelator dexrazoxane, or under hypoxia, HMM cells were tested for HIF-1alpha activation, Pgp expression, accumulation of doxorubicin and sensitivity to its toxic effect. Crocidolite, dexrazoxane and hypoxia caused HIF-1alpha activation, Pgp overexpression and increased resistance to doxorubicin accumulation and toxicity. These effects were prevented by the co-incubation with the cell-permeating iron salt ferric nitrilotriacetate, which caused an increase of intracellular iron bioavailability, measured as increased activity of the iron regulatory protein-1. Crocidolite, dexrazoxane and hypoxia induce doxorubicin resistance in human malignant mesothelioma cells by increasing hypoxia-inducible factor-1alpha activity, through an iron-sensitive mechanism.

Testis sparing enucleation of a Leydig-cell tumour in a boy.

Testicular tumours are very rare in paediatric age, accounting only for 1% of all paediatric tumours. Testicular tumours can originate either from germ cells (77.4%) or from stromal cells (7.1%) or from other cells. Leydig-cell tumours account for 1% of all testicular tumours and 39% of gonadal stromal tumours and in the prepubertal male are responsible for causing precocious pseudopuberty. In the past, orchiectomy has been considered the treatment of choice, but in consideration of the fact that Leydig Cell tumours in children invariably show a benign behaviour, in recent years some authors have suggested a more conservative approach. In the herein reported case, a decision was made to simply enucleate the tumour leaving the testis. After one year, imaging shows a normal testis with no sign of recurrence.

Interleukin-2 induces cell cycle perturbations leading to cell growth inhibition and death in malignant mesothelioma cells in vitro.

Previous report indicated that Interleukin-2 (IL-2) is able to inhibit the growth of IL-2-receptor-positive cancer cell lines without any involvement of the immune system, through IL-2-induced alterations of the cell cycle kinetics. In this study we provide evidence that IL-2 exerts anti-proliferative effect on three human malignant mesothelioma (MMe) cells in vitro, while no effects were observed on normal human mesothelial cell (HMC) primary cultures. The growth inhibitory effect of IL-2 on neoplastic cells appeared to depend on the baseline proliferative status of these cells. Indeed, in highly proliferating MMe cells, we observed a reduction of malignant cells in the S-phase of the cell cycle, with an accumulation in G0/G1, followed by apotosis for longer incubations or exposure to higher doses. On the contrary, in MMe cells proliferating at lower rate, IL-2 induces only a late cytotoxic effect, leading to apoptosis, without significantly affecting the cell cycle. IL-2Rbeta mRNA was detectable by RT-PCR in all MMe cells, IL-2Ralpha mRNA in one only out the three assayed and IL-2Rgamma mRNA in none. In addition, mRNA specific for the IL-2Rbeta-associated Jak-1 tyrosine kinase was expressed in all MMe cell lines, further suggesting that IL-2Rbeta may play a role in the observed effects. Very low, albeit detectable, levels of IL-2Rbeta chain appeared to be expressed at the cell surface of MMe cells by indirect immunofluorescence and FACS analyses. Finally, Ca(++) fluxes were rapidly induced when MMe cells were exposed to exogenous IL-2.

Establishment of four new mesothelioma cell lines: characterization by ultrastructural and immunophenotypic analysis.

The aim of this study was to assess the biological characteristics of four new malignant mesothelioma (MM) cell lines. Since simian virus (SV)40 sequences have been recently detected in MM, SV40 large T antigen (Tag) expression was also analysed. MM cell lines were characterized by morphological, ultrastructural and cytogenetic analysis. Expression of Tag and of relevant MM markers was studied by immunocytochemistry, surface antigens by indirect immunofluorescence and immunomodulating cytokines by enzyme-linked immunosorbent assay (ELISA). The four MM cell lines, established from pleural effusions, showed a slow proliferation rate and pleomorphic changes during culture. Cell lines expressed vimentin, cytokeratins 8 and 18, and the mesothelial antigen recognized by HBME-1 monoclonal antibody, but not carcinoembryonic antigen. Surface human leukocyte antigen (HLA)-class I and intercellular adhesion molecule (ICAM)-1 molecules were present on all the cell lines. While HLA class II and CD86 were constitutively undetectable, HLA-class II was present after interferon (IFN)-gamma stimulation. All cell lines displayed abnormal karyotypes with chromosome 6 abnormalities. Transforming growth factor (TGF)-beta2 and interleukin (IL)-6 were constitutively secreted, while tumour necrosis factor (TNF)-alpha was secreted only in response to lipopolysaccharide. Intranuclear Tag was expressed in two cell lines. The persistence of large T antigen with human leukocyte antigen class I and intercellular adhesion molecule-1 positivity may point to large T antigen as a target for cytotoxic T-lymphocyte-based immunotherapy in some malignant mesothelioma patients.

Primary human mesothelioma cells express class II MHC, ICAM-1 and B7-2 and can present recall antigens to autologous blood lymphocytes.

Mesothelioma cells (MMc) are considered to be weakly immunogenic and the experimental approaches attempting to induce an immune response against these cells have been disappointing. Our aim was to investigate whether MMc possess the surface accessory molecules involved in antigen presentation and whether these cells are capable of presenting recall antigens to autologous blood lymphocytes. Four primary MMc cultures were generated from malignant effusions and examined to assess whether the accessory molecules required for antigen presentation were present on their surfaces. Intercellular adhesion molecule-I (ICAM-I; CD54); class I and class II major histocompatibility complex-DR (MHCI and MHCII-DR); B7-1 (CD80.3); and B7-2 (CD86) expression by MMc was studied by immunocytochemical and/or FACScan analysis. MMc were pulsed with purified protein derivative (PPD), Tetanus toxoid (TT) and Candida albicans (CA) bodies, and incubated with autologous lymphocytes. Lymphocyte proliferation was estimated by radionucleotide incorporation. Phenotypic analysis showed the presence of MHCII-DR, ICAM-I and B7-2 on primary MMc cultures, whereas the phenotypic evaluation of 2 established MMc lines did not show the presence of the B7-1 and B7-2 molecules. In addition, MHCII-DR was detectable only after interferon gamma (IFN-gamma) stimulation. Primary MMc cultures acquired the capability to induce lymphocyte proliferation after pulse with the recall antigens. To achieve characterization of these lymphocytes, we generated a PPD-specific CD4+ T-cell clone. PPD-pulsed MMc were shown to specifically induce T-cell clone proliferation through a MHCII-DR-mediated process. We conclude that primary MMc possess the surface molecules required for antigen presentation and can present recall antigens to CD4+ lymphocytes.

The role of immunohistochemistry in the diagnosis of malignant mesothelioma.

Malignant mesothelioma (MM) can place the pathologist in a diagnostic dilemma because of its morphological variability. The contribution of immunohistochemistry to a more accurate recognition of the mesothelial histogenesis of serosal tumours is widely acknowledged. The immunohistochemical diagnosis of epithelial MM is currently mainly one of exclusion based on prevailing negative staining with several tissue markers of adenocarcinomatous differentiation. The contribution of immunohistochemistry to a definite diagnosis of MM has only been established for the sarcomatous forms, which are positively labelled by anticytokeratin antibodies. A few antimesothelial antibodies have been generated in recent years and two of them have recently been marketed although they have yet to be validated on a wide scale in routine laboratory practice. However, none of them appears a priori as the ideal single positive marker for an unequivocal diagnosis of MM.

Newly marketed tissue markers for malignant mesothelioma: immunoreactivity of rabbit AMAD-2 antiserum compared with monoclonal antibody HBME-1 and a review of the literature on so-called antimesothelioma antibodies.

A complementary DNA (cDNA) library was constructed from a human malignant mesothelioma (MM) cell line and a cDNA fragment encoding for a cytoplasmic mesothelial protein recognized by the polyclonal antibody AMAD-1 was then cloned and expressed in Escherichia coli. The purified recombinant protein was used to raise a novel antibody, named AMAD-2, in rabbits. This antibody reacted with normal mesothelium and most MM (15 of 17) on paraffin sections and featured a cytoplasmic labeling. Conversely, AMAD-2 immunostaining of normal and tumor tissues from body sites other than serosal membranes was limited with respect to the proportion of positive specimens and usually less conspicuous than in MM. AMAD-2 immunoreactivity was subsequently compared with staining for HBME-1, another newly marketed antimesothelial monoclonal antibody, concerning the ability to distinguish pleural MM from metastatic pleural tumors of epithelial type. A granular cytoplasmic immunoreactivity for AMAD-2 was present in 50% or more of tumor cells in all 84 MM, regardless of histological type, but also in 3 (7%) of 42 pleural metastases, albeit only focally. HBME-1 was shown in 63 of 66 epithelial MM and in the epithelial component of all 8 mixed MM, with a prevailingly membranous pattern, usually homogeneous and strong, whereas none of the 10 sarcomatous MM was positive. HBME-1 was also expressed in 6 (14%) of 42 pleural metastases in a cytoplasmic or membranous pattern. Compared with HBME-1, AMAD-2 showed a higher degree of specificity and sensitivity for MM. AMAD-2 still proved to be superior to HBME-1, also when sarcomatoid MM were excluded from the assessment. This finding supports the view that AMAD-2 is an antibody highly, although not entirely, specific for the mesothelial lineage, whereas HBME-1 is probably a cell marker more closely related to the epithelial differentiation of MM. Therefore, AMAD-2 is preferable as a positive tissue marker to be incorporated in the optimal immunohistochemical panel for the diagnosis of MM.

Malignant mesothelioma of the pleura. The reproducibility of the immunohistological diagnosis.

The reproducibility of the histopathological diagnosis of pleural malignant mesothelioma (MM), after supplementing routine H&E stain by immunohistochemistry (IH) in 77 cases of original diagnoses of MM, was assessed by examining interobserver variation between five pathologists. A battery of commercial antibodies (cytokeratins, vimentin, HMFG-2, anti Leu-M1 [CD15], BerEP4, B72.3 [TAG-72], carcinoembyonic antigen), considered to be useful in enhancing diagnostic accuracy, was used. The number of definitively classified tumors (accepted MM plus rejected MM) increased from 57 on H&E stain to 60 after IH, with 59 (76.6%) cases being accepted as true MM. Based on IH, the chance-adjusted interobserver agreement was poor (kappa w = 0.29) and lower than that observed on previous H&E alone. The intraobserver agreement for four of the five pathologists was rather good (kappa w = 0.54-0.56). The inter- and intraobserver concordance was higher in accepting than excluding the cases as MM. A larger number of cases were classified by all reviewers as mixed or sarcomatous variants after IH. In the interpretation of each immunostain, kappa values ranged from 0.19 for B72.3 to 0.62 for HMFG-2, which were respectively the least and the most consistently interpreted immunostains. The information additionally contributed by IH did not seem to change the pathologists' diagnoses very much in comparison with those made by routine H&E stain. Until highly specific and sensitive probes for the positive identification of MM become available, a careful scrutiny of routinely stained preparations still remains the most rewarding component of the diagnostic pathway.

[Cellular proliferation, expression of p53, EGFR and apoptosis index of healthy mucosa of the bladder with TCC; pre- and post-intravesical BCG immunohistochemical study]. / Proliferazione cellulare, espressione di p53, EGFR ed indice di apoptosi nella mucosa sana di vesciche con TCC; studio immunoistochimico pre e post BCG endovescicale.

At present, the most efficacious and used immunostimulant agent in the superficial bladder cancer immunotherapy field, is the BCG, even if its mechanism of action is still partly unknown. The therapeutic effects of BCG don't seem to depend exclusively on local immune response, so that according to this assertion, this immunohistochemical study had been conducted on 14 patients affected by superficial bladder cancer (pTa-pT1) which aimed to value both the apoptosis and proliferation indexes and the expression of the genetic product p53 and EGFR before and after the exposition of the vesical mucosa to the BCG. The BCG treatment can reduce the proliferation index of the normal urothelial cells in a statistically significant way whereas it would exclude a cytostatic effect mediate by negative modulation of EGFR from the cytokinins induced by BCG itself. The index of apoptosis of the urothelium does not increase after BCG and decreased expression of p53 associated after the treatment, although statistically not significant, it would seem to bear, the prophylactic efficacy of BCG according to the follow up of the patients included in the study.

Lipoblastic differentiation in a primary localized fibrous mesothelioma of the peritoneum.

Lipoblastic differentiation in fibrous mesotheliomas is an extremely rare occurrence. We present the histological and immunohistochemical features of a case of localized peritoneal mesothelioma with lipoblastic differentiation in an 80-year old man and discuss the differential diagnosis with liposarcoma.

Malignant mesothelioma of the pleura: interobserver variability.

AIMS: To assess the consistency of histopathological diagnosis of pleural malignant mesothelioma by estimating interobserver agreement between five pathologists. METHODS: Eighty eight histological sets pertaining to original diagnoses of pleural malignant mesothelioma were reviewed separately by each pathologist. Diagnostic likelihood was graded as definite (A), probable (B), possible (C), improbable (D), and definitely not (E) malignant mesothelioma. The following indexes were estimated: observed proportion of agreement (Po), kappa statistics and proportion of agreement for "positive" (Ppos) and "negative" (Pneg) diagnoses. RESULTS: Sixty cases (68.2%) were rated by at least three reviewers as A or B and by none of the others as D or E. Five (5.7%) were rated by at least two reviewers as D or E and by none of the others as A or B. Nine (10.2%) showed a serious disagreement, rated A or B and D or E. Agreement for sets obtained at necropsy/surgery (median kappa w = 0.57) was similar to that for endoscopic material (median kappa w = 0.54). Agreement was poor on material obtained by needle biopsy (median kappa w = 0.21). The median value of Ppos varied between 0.94 (necropsy/surgery) and 0.67 (needle biopsy) and that of Pneg between 0.78 (necropsy/surgery) and 0.34 (unspecified biopsy). Interobserver agreement on histological typing was good overall (median kappa = 0.59). CONCLUSIONS: Of the original histological diagnoses, 70% were consistently reproduced through panel review. Most indexes of agreement between pathologists ranged from poor (needle biopsy) to moderate (necropsy/surgery). Agreement in confirming malignant mesothelioma was greater than that regarding exclusion of this diagnosis. Of the cases finally considered to have malignant mesothelioma, the reproducibility of histological typing was relatively satisfactory.

Late effects of intravesical bacillus Calmette-Guérin immunotherapy on bladder mucosa infiltrating lymphocytes: an immunohistochemical study.

During intravesical bacillus Calmette-Guérin (BCG) treatment for the prophylaxis of recurrent superficial bladder carcinoma, patients typically show a local inflammatory response involving mainly T lymphocytes, most of which have the helper-induced phenotype (CD4+) (CD4+/CD8+ ratio > 1). To evaluate whether this immunophenotypic profile of the lymphocytes persists also after the completion of this immunotherapy, we examined bladder biopsy specimens during the posttreatment follow-up period of 24 patients, previously submitted to a 2-year BCG administration. The intensity of inflammatory response differed among the patients and in 10 of them even between the scar and the normal mucosa of the bladder. A reversal to the pretreatment CD4+/CD8+ ratio < 1 occurred in the majority of subjects, including the 3 patients with histologically confirmed tumour recurrence. In addition, 11 tumour-free patients showed prevailing CD4+ cells in the scar mucosa and prevailing CD8+ in the normal mucosa of their bladder or vice versa. From these findings it appears that the long-term host response to BCG does not depend exclusively on an intense, long-lasting local mononuclear immune reaction.

Idiopathic granulomatous mastitis. Report of a case clinically and mammographically simulating breast carcinoma.

A case of bilateral idiopathic granulomatous mastitis in a 67-year-old woman is described. The clinical presentation and mammographic findings raised strong suspicions of malignancy, excluded by the histological examination, which made it possible also to rule out possible known causes of granulomatous inflammation of the breast.

Expression of intercellular adhesion molecule-1 (ICAM-1) by reactive mesothelial cells in pleural effusions.

Reactive mesothelial cells in serous effusions have been assessed for their capacity to express intercellular adhesion molecule-1 (ICAM-1), when pleural cavities are involved in inflammatory and immune reactions. Cytospin smears prepared from 10 benign and 10 malignant effusion specimens were used in an immunocytochemical procedure. Mesothelial cells were positively immunolabeled by a monoclonal antibody to human ICAM-1, whereas lymphocytes clustered around them reacted positively with a monoclonal antibody to human Lymphocyte Function-associated antigen-1 alpha chain (LFA-1-alpha). The ICAM-1/LFA-1 interaction enables mesothelial cells to regulate leukocyte transmigration to and accumulation in serous cavities and therefore they are actively involved in the development of serous effusions.

The in-vitro hematopoietic capacity of the adult human mesothelial cell: a model of cell differentiation induced by the structure of the microenvironment.

Upon exposure to collagen sponges, cultured adult human mesothelial cells were shown to differentiate into hematopoietic cells similar to those of the red bone marrow. This transformation was confirmed by morphological analysis and by cell immunoreactivity toward specific antibodies directed to antigens of the hematopoietic cell lines at various stages of differentiation. Besides demonstrating that the pluri-potentiality of the mesothelium persist into adulthood, this observation suggests that the process of differentiation may also be influenced by the structural organization of the microenvironment hosting the mesothelial cells.

Giant cell fibroblastoma of the scrotum. A case report.

A case of giant cell fibroblastoma occurring in the scrotum of a 21-year-old male is described. It may have been a recurrence of a previous lesion in the same region, which was only clinically diagnosed as fibrolipoma when excised 10 years before. Giant cell fibroblastoma is a benign, locally recurrent tumor of youth and childhood which arises from superficial soft tissue. To our knowledge, only 2 other cases of scrotal involvement by this neoplasm have been reported.

Cell proliferation in breast carcinoma assessed by a PCNA grading system and its relation to other prognostic variables.

This study was undertaken to relate the expression of the proliferating cell nuclear antigen (PCNA), a proliferation marker of putative prognostic significance, to some more established prognostic factors in a series of 60 consecutive breast cancer surgical specimens. PCNA was detected by the PC10 monoclonal antibody (MAb) using an immunohistochemical method and PCNA immunostaining was estimated on a semiquantitative basis, a cut-off value of 50% of positively stained tumour cells discriminating between the high (> 50%) and low (< 50%) PCNA grade. The PCNA grade did not correlate with tumour size and axillary node status. However, a high PCNA grade tended to be associated with a poor histological grade and there was an inverse relationship with oestrogen-receptor status, as determined by means of the immuno-histochemical staining for the oestrogen-induced pS2 protein. These conflicting results suggest that the possible prognostic usefulness of PCNA immunostaining, as a measure of cell proliferation rate, in breast cancer is yet to be demonstrated and can be validated only by direct relation to survival data.

Morphometric assessment of nucleolar activity in liver cells during the evolutive pathway of chronic hepatitis C to cirrhosis.

Using a computer-assisted image analysis system, we performed a morphometric study of silver-stained nucleoli of hepatocytes in liver biopsy specimens from hepatitis C virus-positive patients with chronic persistent hepatitis (3 cases), chronic active hepatitis (4 cases), and cirrhosis (4 cases). The number and the total area of nucleoli, the average area of each nucleolus and the nuclear area were determined for each of 100 hepatocytes per case. A continuing increase in the area of both nucleoli and nuclei paralleled a progressive decrease in the number of nucleoli during the evolution of chronic hepatitis C to liver cirrhosis. These findings would indicate that the hepatitis C virus-induced liver damage causes reactive changes in surviving hepatocytes resulting in an increased nucleolar biosynthetic activity rather than in an increment of cell proliferation rate. Therefore, the liver response to hepatitis C virus injury seems to be mainly based on a condition of cell "hypertrophy", whereas we previously showed that a process also of compensatory hyperplasia occurs in chronic hepatitis B, possibly resulting from a different pathogenesis of the viral damage.