Cytologic re-evaluation of negative effusions from patients with malignant mesothelioma.

BACKGROUND: Cytology is a controversial means of diagnosing malignant mesothelioma due to the high rates of negative samples. The aim of the present study was to review effusions originally reported as "negative" in patients with histologically-proven mesothelioma to evaluate possible pitfalls. METHODS: We reviewed the cytologic slides of 25 specimens that refer to 15 epithelioid, 5 biphasic, 4 sarcomatoid and 1 well-differentiated papillary mesotheliomas. For comparison, we also reviewed 23 specimens from non-neoplastic conditions. For each effusion, we evaluated the background and calculated a score considering the following items: amount of mesothelial cells, architectural pattern and atypical features, and a revised diagnosis was rendered. RESULTS: More than half of the effusions initially called "negative" (but mesothelioma by histology) were considered atypical/ suspicious (false-negative diagnosis); the remaining cases were true-negative or inadequate. Almost all effusions initially called "negative" (but non-neoplastic by histology) were considered negative. The only item that seems to discriminate between the two groups is atypia of mesothelial cells. CONCLUSIONS: The present study has highlighted the following pitfalls: (i) to report effusions devoid of mesothelial cells as negative that instead should be reported as inadequate/non-diagnostic; (ii) to underestimate low cellular effusions containing atypical mesothelial cells or high cellular effusions containing bland mesothelial cells with a morular pattern; (iii) to consider that an inflammatory background may obscure a scant number of mesothelial cells. A categorized system (inadequate (M1), negative (M2), atypical (M3) and suspicious (M4)) for reporting effusion cytology may be of help in the diagnostic work-up of patients with effusions suspicious for mesothelioma.

A one-generation cluster of malignant mesothelioma within a family reveals exposure to asbestos-contaminated jute bags in Naples, Italy.

Substantial evidence supports the role of asbestos in malignant mesothelioma. Clustering for this malignancy among relatives not only suggests genetic susceptibility as a relevant component but also provides a clue to investigate non-occupational sources of exposure. We identified five cases of malignant mesothelioma within one family with exposure to asbestos experienced during childhood, as 'next door' residents of a workshop recycling asbestos-contaminated jute sacks in Naples, Italy. This cluster discloses the health risk in the reuse of bags that previously had contained asbestos. Furthermore, it emphasizes the role of asbestos in the genetic-environmental interaction issue of malignant mesothelioma.

Malignant pleural mesothelioma in bakers and pastry cooks.

BACKGROUND: The occurrence of malignant pleural mesothelioma (MPM) among bakers and pastry cooks has never been documented. CASE REPORTS: We detected eight cases of MPM in bakers, pastry cooks, and biscuit cooks engaged in making, baking/cooking, and selling pastry/bread in two hospital-based series (Rome and Orbassano/Turin, Italy; period 1990-1997; 222 cases). Field-investigations revealed asbestos-containing material (ACM) in ovens for baking bread, that were manufactured prior to the 1980s. CONCLUSIONS: It is suggested that there is a possible new association of the risk of having worked as a baker or pastry cook and MPM. Presumptive source of exposure to asbestos was the use of asbestos-insulated ovens.

DNA copy number changes in familial malignant mesothelioma.

Malignant mesothelioma (MM) is predominantly a sporadic malignancy linked to exposure to asbestos. Clustering of MM in families suggests genetic susceptibility as a contributing factor. We performed comparative genomic hybridization (CGH) analysis on tumor samples from members of a family with MM of the pleura and a history of parental cancer. Our specific aim was to find a recurrent copy number loss indicating the chromosomal area to which a gene underlying the development of MM could be assigned according to the Knudson two-hit hypothesis. We found losses at 1p, 6q, 9p, 13q, and 14q. The copy number changes were very similar to those reported in sporadic cases. Our findings and results from sporadic cases highlight the importance of cloning the genes in the loss sites at 1p, 6q, 14q, and 22q.

[Malignant mesothelioma in the industrial area of Colleferro]. / Mesotelioma maligno nel comprensorio industriale di Colleferro.

The study describes the occurrence of pleural and peritoneal malignant mesothelioma in the Colleferro industrial area (Province of Rome, 9 municipalities, population 63,000, period 1993-98) which is the site of a large chemical plant (BPD) producing organic chemicals, acid mixtures, insecticides, explosives and dynamite, and was involved in manufacturing/maintenance of railroad rolling stock. Asbestos was extensively used in these plants in the past. Mesothelioma cases were actively searched from data in files of pathology archives, hospital admission and discharge (records), and death certificates recorded at local health authority register. 23 potential cases were identified for whom clinical charts and pathological slides were reviewed. A multidisciplinary evaluation of all collected information confirmed 18 cases of cyto-histologically proven malignant mesothelioma (pleural/peritoneal ratio of 2.75:1) among residents and/or workers at BPD. The remaining 5 cases were defined as not mesothelioma; however, two were cases of lung cancer (both occupationally exposed to asbestos). All subjects with malignant mesothelioma had been occupationally exposed to asbestos (14 males and 3 females), except one (1 female with domestic exposure). No mesothelioma case was attributable to environmental exposure. Of the 17 cases with occupational asbestos exposure, 15 occurred in BPD workers employed in manufacturing/maintenance of railroad rolling stock (3 cases), general maintenance services (5 cases), or in the armaments sector (7 cases) and 2 in residents but not BPD workers (1 baker, 1 pipefitter). The incidence rate in residents of the 9 municipalities was 5.5 in males and 1.3 in females (standardized on the Italian population x100,000, census 1981). For Colleferro municipality only, the incidence was 10.1 in males and 4.1 in females, which are the highest rates reported so far in Italy. Besides confirming the risk of mesothelioma risk in railroad rolling stock manufacturing and asbestos-insulated pipe maintenance workers, this study identifies a cluster of malignant mesothelioma in explosives production workers.

Human herpesvirus-8 in lymphomatous and nonlymphomatous body cavity effusions developing in Kaposi's sarcoma and multicentric Castleman's disease.

Human herpesvirus-8 (HHV-8) has been associated with Kaposi's sarcoma, multicentric Castleman's disease and primary effusion lymphoma. Kaposi's sarcoma and multicentric Castleman's disease patients may develop body cavity effusions that, unlike primary effusion lymphoma, are poorly characterized. To better define these effusions, pleural and peritoneal fluids derived from 12 human immunodeficiency virus-seropositive and one seronegative patients affected by Kaposi's sarcoma or multicentric Castleman's disease were analyzed by a combination of morphologic, immunophenotypic, and DNA analyses, including polymerase chain reaction amplification of HHV-8, Epstein-Barr virus, and immunoglobulin heavy-chain (IgH) gene sequences. In addition, HHV-8 serologic status was assessed by using an immunofluorescence assay. All patients were adult men with high antibody titers to HHV-8; 11 of the 13 patients were homosexual/bisexual. Effusions revealed monocyte/macrophage-rich infiltration (10 patients) or large-cell lymphoma with CD45(+)/non-T/non-B phenotype (three of 13 patients); polymerase chain reaction analysis showed the presence of HHV-8 sequences (nine of 13 patients), germline IgH (seven of 12 patients) or clonal IgH rearrangements (four of 12 patients), and rarely Epstein-Barr virus sequences (two of 12 patients). In the setting of HHV-8 infection, two effusion types may occur. One fulfills the criteria for HHV-8-positive PEL (lymphoma-morphology, HHV-8-DNA(+), IgH rearrangement). The other seems more reminiscent of an HHV-8-associated nonneoplastic process (monocyte-macrophage morphology, HHV-8-DNA(+/-), germline IgH). Interestingly, a single case of the latter effusion type harbored a B-cell monoclonal proliferation, which suggests the hypothesis that a prelymphomatous effusion may precede overt body cavity lymphoma.

Human herpes virus-8 associated primary effusion lymphoma of the pleural cavity in HIV-negative elderly men.

Human herpes virus-8 (HHV-8)-associated primary effusion lymphoma (PEL) is an unusual lymphoma confined to the body cavities, which primarily affects human immunodeficiency virus (HIV)-positive men at high risk for Kaposi's sarcoma (KS). We describe two HIV-negative elderly Italian men, who developed pleural HHV-8-positive PEL in association with other diseases (systemic hypertension, colonic carcinoma, chronic obstructive airways disease, dilated cardiomyopathy), but without KS. Thoracic computed tomography revealed unilateral pleural effusion and pleural thickening. Thoracentesis disclosed large lymphoma cells, with no T- or B-cell associated antigens, clonal rearrangement of the immunoglobulin heavy chain gene and the presence of HHV-8 but not Epstein-Barr virus deoxyribonucleic acid sequences. Our cases differ from most pleural effusion lymphomas, in that they are non-acquired immunodeficiency syndrome-related. This highlights the possible human herpes virus-8-associated primary effusion lymphoma risk among elderly human immunodeficiency virus-negative patients, particularly Italians, in whom human herpes virus-8 seroprevalence rates and incidence of classic Kaposi's sarcoma are high.

Kaposi's sarcoma following malignant mesothelioma.

We report the unusual occurrence of Kaposi's sarcoma following asbestos-related malignant mesothelioma, in a human deficiency virus (HIV)-negative Italian man. Seropositivity to human herpes virus 8 (HHV8) was documented at the time of mesothelioma diagnosis and preceded the onset of Kaposi' sarcoma with a time lapse of 13 months. HHV8 DNA was detected by polymerase chain reaction in lesional Kaposi's sarcoma but not within mesothelioma. By immunostaining, mesothelioma cells expressed interleukin-6 and platelet-derived growth factor, which are important for survival of Kaposi's sarcoma cells. Besides the possibility of a casual association, we hypothesize that mesothelioma-linked factors may have contributed to the development of Kaposi's sarcoma in the presence of HHV8 infection.

Familial pleural malignant mesothelioma: clustering in three sisters and one cousin.

Malignant mesothelioma is associated with asbestos, yet its occurrence in genetically-related individuals suggests a role of host predisposition. We have identified a cluster of pleural malignant mesothelioma in three sisters and one cousin (male). The women had worked in the same confectionery shop as pastry cooks and/or pastry shop assistants; the use of an asbestos-insulated oven was the putative source of exposure. The man had occupational exposure as a heating system installation worker. The family history reported malignant cancers in first-degree (larynx, brother; pleura and lung, mother), second-degree (lung, aunt and uncle) and third-degree (lung, cousin) relatives. The study reveals the potential existence of the mesothelioma risk among pastry cooks and highlights the fact that inherited factors may be involved in the development of this tumour.

Absence of HHV-8 DNA sequences in malignant mesothelioma.

Human herpesvirus 8 (HHV-8) associated primary effusion lymphomas arise and grow in the body cavities as effusions, but it is not known whether the lining of body cavities and mesothelium derived malignancies are potential targets of HHV-8 infection. We examined a series of 13 diffuse malignant mesotheliomas and four mesothelial cell rich effusion samples of non-neoplastic aetiology from non-immunodepressed patients using the polymerase chain reaction to detect HHV-8 specific sequences. HHV-8 amplification products were absent in diffuse malignant mesotheliomas and in non-neoplastic effusions samples. These results suggest that HHV-8 has a selective tropism among body cavity based tumours, being confined to primary effusion lymphomas.

Cyclin D1 and Cyclin E expression in malignant thyroid cells and in human thyroid carcinomas.

Evidence of the involvement of cyclin gene alterations in human cancer is growing. In this study, we sought to determine the pattern of expression of cyclin D1 and cyclin E in normal and malignant thyroid cells. Quiescent rat thyroid cells in culture, induced to synthesize DNA by thyrotropin (TSH), expressed cyclin D1 gene after 6 hr and cyclin E gene with a peak at 18 hr from the stimulus; K-ras-transformed rat thyroid cells, which grew without addition of hormones necessary for normal cell proliferation, expressed elevated levels of cyclin D1 and cyclin E, compared with normal differentiated thyroid cells. Human benign and malignant thyroid tumors and their relative normal tissues were then analyzed. Neither major genetic alterations nor amplifications for cyclin D1 and cyclin E genes were found by Southern blot analysis in genomic DNAs extracted from all types of thyroid tumors. Moreover, statistical analyses of densitometric values from Northern blots did not show increased levels of cyclin D1 and E mRNAs in the tumor samples, compared with normal thyroid. Immunohistochemical analyses of formalin-fixed paraffin-embedded sections of tissues with specific antibodies revealed a prevalent cytoplasmic cyclin E staining in the thyroid tissues analyzed. Cyclin D1, instead, was present in the cytoplasm of normal thyroids and adenomas, but in 31% of thyroid papillary carcinomas analysed, it was overexpressed, with a localization in the nucleus. Our in vivo observations suggest that unlike cyclin E, elevated nuclear cyclin D1 expression defines a subset of thyroid papillary carcinomas, and might be a contributory factor to thyroid tumorigenesis.

Utility of HBME-1 immunostaining in serous effusions.

HBME-1 is an anti-mesothelial cell monoclonal antibody derived from human mesothelioma cells. We investigated 227 body cavity effusions to test its utility in differentiating mesothelioma from adenocarcinoma. HBME-1 outlined cell membranes in non-neoplastic mesothelial cells. Thick surface staining was observed on all mesotheliomas. HBME-1 reactivity was also detected in 24% of metastatic carcinomatous effusions. Most ovarian carcinomas (83%) reacted with this antibody, showing surface staining. Cytoplasmic HBME-1 immunoreactivity was observed in a small proportion of non-ovarian adenocarcinomas (14%). Despite its limited specificity, HBME-1 might be added to the battery of other markers of epithelial and/or mesothelial differentiation to be used in cases of suspected mesothelioma. Evaluation of suspicious cells should include careful study of the pattern of immunostaining.

Malignant mesothelioma in Rome, Italy 1980-1995. A retrospective study of 79 patients.

AIM AND BACKGROUND: To evaluate the characteristics of a case-series of 79 malignant mesothelioma patients collected from the main teaching hospital of Rome, Italy, and other local clinics of Latium Region and to assess the role of asbestos exposure, since previous studies on the occurrence of the disease in this area were lacking. METHODS: The study included cytohistologically diagnosed malignant mesothelioma (71 pleural, 7 peritoneal, and 1 testicular tunica vaginalis) detected or referred for consultation during the period 1980-1995. Information regarding occupational and/or nonoccupational exposures was derived from clinical records and interviews, when available. RESULTS: Patients were resident in Rome and other towns of Latium; a few were from other parts of central and southern Italy. Exposure to asbestos was assessed for 45.5% of patients, another 45.5% had unknown exposure, and for the remaining 9% such information was lacking. Occupational exposure occurred in 53% of men for whom information was available and nonoccupational exposure occurred in 20% of women. The study identified two clusters of cases from an asbestos-cement plant and a facility where asbestos was ubiquitous. Furthermore, most exposed subjects reported occupations in the construction industry, which is particularly active in the Latium Region; others were railroad workers, naval mechanics and navy personnel, bakers, explosive workers and car mechanics. A few patients reported indoor exposure to asbestos at home and/or in the workplace. CONCLUSIONS: The study confirmed that mesothelioma risk is present in several job titles of the construction industry, and it is no longer confined to workers employed in the manufacture or application of asbestos products. The occurrence of malignant mesothelioma in patients with unexpected occupational and nonoccupational exposures indicates the need for further investigation on previously underestimated exposures.

Concomitant malignant mesothelioma of the pleura, peritoneum, and tunica vaginalis testis.

We describe the cytohistological, immunohistochemical and ultrastructural findings in a 55-yr-old-man with history of asbestos exposure and diffuse malignant mesothelioma (DMM) of the pleura, peritoneum, and tunica vaginalis presenting with chest pain and scrotal swelling. Pleural fine-needle aspiration (FNA) revealed mesenchymal elements and spindle-shaped epithelial-like cells, while biopsy showed pure sarcomatous tumor invading lung parenchymal. In both samples tumor cells coexpressed cytokeratin and vimentin. Peritoneal and hydrocele effusions contained aggregates of malignant mesothelial cells. Electron microscopy showed intermediate filaments, rare desmosomes and sparse microvilli. Morphological findings were consistent with a DMM, with a biphasic pattern in the pleura and an epithelial one in the peritoneum and tunica vaginalis. Although the possibility of a multicentric origin cannot be ruled out, clinical chronologic sequence suggests that the pleura was the primary involved site, followed by spread to peritoneum and tunica vaginalis.

The diagnostic value of thrombomodulin immunolocalization in serous effusions.

OBJECTIVE: To test the value of an antithrombomodulin monoclonal antibody as a diagnostic tool in detecting mesothelial cells and in differentiating mesothelioma from other malignant effusions. DESIGN: Thrombomodulin is a thrombin receptor that is distributed on surfaces where an anticoagulant activity is expected, including mesothelium. Antigen expression was studied by immunocytochemistry in 226 peritoneal and pleural exudates. RESULTS: The antigen was found in 33 of 33 mesotheliomas, 35 of 35 reactive effusions, 57 of 145 carcinomatous fluids, and in one case of angiosarcoma among seven metastatic nonepithelial tumors. Three distinct staining patterns were demonstrated: (1) thin cell membranes in benign mesothelial cells; (2) thick cell membranes in mesotheliomas; and (3) cytoplasm in carcinomas. All squamous cell carcinomas had demonstrable thrombomodulin, suggesting that antigen expression likely correlates with squamous differentiation. CONCLUSIONS: Thrombomodulin is of diagnostic utility in distinguishing mesothelioma from adenocarcinoma, provided the characteristic "thick membrane" pattern is present, but it should be used in panels with other markers of mesothelial and/or epithelial differentiation.

Platelet-derived growth factor receptor immunoreactivity in mesothelioma and nonneoplastic mesothelial cells in serous effusions.

Expression of the platelet-derived growth factor receptor (PDGFR) was detected by immunocytochemistry in normal and malignant mesothelial cells from 14 benign effusions and 22 mesotheliomas. Two well-characterized antireceptor monoclonal antibodies to the PDGFR alpha-subunit (PR292) and beta-subunit (PR7212) were used. PDGFR alpha-subunit outlined cell membranes intensely in nonneoplastic mesothelial cells, whereas it was focal in mesothelioma and limited to a few cases only. PDGFR beta-subunit was weakly expressed in the cytoplasm of normal mesothelium; in contrast, malignant mesothelial cells showed strong cytoplasmic staining. Our results show that normal mesothelium may be responsive to PDGF by the predominant expression of PDGFR-alpha and less by PDGFR-beta and indicate the presence of growth-stimulation loops in mesothelioma through PDGF/PDGFR-beta interaction. Also, scattered staining for the PDGFR alpha-subunit suggests that a PDGF-A chain/PDGFR-alpha interaction may also be active in mesothelioma cells growing in suspension.

Utility of cytokeratin 20 in identifying the origin of metastatic carcinomas in effusions.

Using a commercially available monoclonal antibody (Ks20.1) and the avidin-biotin peroxidase method on cytospins and cell blocks, we analyzed cytokeratin (CK) 20 expression in 169 serous effusions. Cytoplasmic staining was observed in 44/151 malignant fluids. Colon, gastric, and pancreatic adenocarcinomas and mucinous ovarian tumors were most frequently positive. Single cases of transitional-cell and squamous cell carcinomas were reactive as well. Lung and breast cancers were mostly negative. Nonmucinous ovarian tumors were invariably unlabeled as were mesotheliomas and normal mesothelial cells. The study shows that CK 20 is valuable in distinguishing tumor cell origin in effusions. In particular, it identifies a set of carcinomas with the majority arising from the gastrointestinal tract, and represents a highly characteristic marker for colorectal cancer.

C-erbB-2 oncoprotein immunostaining in serous effusions.

The product of c-erbB-2 gene is detected in a proportion of carcinomas from various sites and is generally associated with a high degree of malignancy. A series of 58 effusions containing malignant cells and 16 cytologically negative serous effusions was assessed by immunocytochemical methods for c-erbB-2 expression using the monoclonal antibody NCL-B11, which recognizes the internal domain of the c-erbB-2 oncoprotein. Both alcohol-fixed smears and cell blocks from formalin-fixed specimens were used. A crisp, clear cut membrane-associated positive staining was evident in 51% (30/58) malignant effusions and was restricted to metastatic adenocarcinomas. Breast and ovarian cancers showed the highest incidence of positivity. Mesotheliomas as well as non-neoplastic effusions were consistently negative. Paraffin blocks from formalin-fixed cells displayed a weak immunoreactivity when compared with their alcohol-fixed counterparts. The study shows that the c-erbB-2 oncoprotein can be easily identified in standard cytological smears: it can be of assistance in differentiating adenocarcinomas from mesotheliomas, and in selected cases it can provide a further prognostic indicator, replacing tissue immunohistochemistry.

Immunological detection of cat uterine proteins in ferrets, dogs and baboons.

In the cat, a high molecular weight (greater than 320 kDa) estrogen (E2)-dependent protein (CUPED) and a group of low molecular weight (28 kDa, 36 kDa, 41 kDa) progesterone (P)-dependent proteins (PDP) are synthesized by the epithelial cells of the deep uterine glands and secreted into the lumen. The present study was designed to investigate the possibility that other species synthesize proteins similar to CUPED and PDP in response to steroids. Uterine flushings and media from cultured endometrial explants obtained from E2 or P dominated animals were analyzed on Western blots for the presence of proteins which cross-react with antibodies raised against CUPED and PDP. CUPED antibody immunoreacted with a protein band at approximately the same molecular weight as CUPED in the media and flushings from E2-treated ferret, dog and baboon, but not from rat, mouse or hamster. In the cat, ferret and dog, specific immunocytochemical staining was observed in the apical portion of the secretory cells. In the baboon, staining was observed in both the basal and apical areas of the epithelial cells. The presence of CUPED in the uterine lumen during the E2-dominated phase of the reproductive cycle in several species suggests that CUPED may play a general role in providing the necessary biochemical milieu for reproductive success. The PDP antibody did not cross-react with proteins in the media of species other than cat, and therefore may be unique to the cat.

Long-term treatment with thymomodulin reduces airway hyperresponsiveness to methacholine.

We investigated the effect of thymomodulin, a calf thymus acid lysate with immunomodulating activity, on bronchial hyperresponsiveness to methacholine of atopic subjects with asthma. In 16 subjects we measured airway responsiveness at 30, 60, and 90 days after treatment with placebo (eight subjects) or thymomodulin (eight subjects; 80 mg daily orally). The degree of bronchial responsiveness to methacholine was significantly reduced at 90 days during treatment with thymomodulin and remained reduced, even if not significantly, 60 days after cessation of treatment.