Immunohistochemical analysis of nuclear versus cytoplasmic staining of WT1 in malignant mesotheliomas and primary pulmonary adenocarcinomas

CONTEXT: Previous studies have indicated certain immunohistochemical markers, including WT1, may be helpful in distinguishing adenocarcinomas from mesotheliomas, but to date there are no reliable, widely accepted, commercially available antibodies positive in mesotheliomas and negative in adenocarcinomas. We compared the nuclear and cytoplasmic staining patterns of WT1 in these 2 malignancies using a commercially available antibody and examined the expression of 2 other previously reported positive markers, calretinin and thrombomodulin. METHODS: Sixty-seven mesotheliomas and 51 adenocarcinomas, all paraffin embedded, were retrieved from recent case files. The diagnosis of mesothelioma was based on typical clinical and morphologic features, as well as immunohistochemistry; electron microscopy had been performed on 16 cases. The diagnosis of adenocarcinoma was based on typical light microscopic findings and a positive stain for mucin. Commercially available antibodies to WT1, thrombomodulin, and calretinin were applied. Because of the conflict surrounding calretinin, 2 anticalretinin antibodies (from Chemicon Inc and Zymed Laboratories) were utilized. RESULTS: Fifty of 67 mesotheliomas showed strong nuclear staining with WT1. No adenocarcinomas (0/51) showed nuclear staining. Twenty-three of 67 mesotheliomas were positive for thrombomodulin, and 35 of 67 mesotheliomas were positive for calretinin with the Chemicon antibody. Nine of 15 mesotheliomas were positive for calretinin with the Zymed antibody. CONCLUSIONS: Thrombomodulin and calretinin did not prove useful in discriminating between mesotheliomas and adenocarcinomas. The degree of positivity with calretinin may be dependent on the specific antibody utilized. Nuclear staining for WT1 is highly specific for mesothelioma and, in the appropriate clinical setting, can be a helpful adjunct in the distinction between adenocarcinomas and mesotheliomas.     

Ultrastructural histochemistry of mesotheliomas and adenocarcinomas in malignant effusions

The results of electron microscopic examination of cytologic specimens from six cases of mesothelioma and 10 cases of metastatic carcinoma of different origins are presented. The formation of cell clusters in malignant effusions from the two neoplasms has been thoroughly investigated: in mesotheliomas, cells had longer, more slender microvilli than in carcinomas and more abundant bundles of intermediate filaments; the central cavity often seen in the clusters frequently contained collagen and showed basement membrane production. The application of periodic acid-silver methenamine (PASM) and phosphotungstic acid (PTA) demonstrated a peculiar ultrastructural difference in cell coat staining in the two tumor types: in mesotheliomas, PTA and PASM were consistently negative along the outer surface of the cell aggregates, while carcinomas displayed a positive reaction either on the outer surface or on both inner and outer surfaces of the clusters. The diagnostic significance of the above-mentioned difference between the two neoplasms will require further investigation in a larger number of cases.     

Keratins in malignant mesotheliomas and pleural adenocarcinomas: comparative immunohistochemical analysis with polyclonal and monoclonal antibodies

The distribution of intra-cellular keratins was studied in normal pleural mesothelium, malignant mesotheliomas and adenocarcinomas. This study was performed on deparaffinized sections of tissue fixed in Bouin's solution by indirect immunofluorescence with a monoclonal antibody (KL1) and a conventional keratin antiserum (AKS). Discrepancies were detected using one antibody or the other. Cells from normal mesothelium and 18 cases of malignant mesotheliomas (papillary, tubulary, solid epithelial type) were strongly labelled only by KL1. The 2 cases of sarcomatoid type were negative with both antibodies. In contrast 5 metastatic adenocarcinomas and 5 lung adenocarcinomas were weakly positive or negative with both antibodies. These data confirm the presence of cytokeratins in epithelial differentiation process. Although a clear-cut distinction between mesotheliomas and adenocarcinomas was not possible using these keratin antibodies. Our data point out the importance of reactivity pattern of the antibody used in such investigations.     

Epithelioid pleural mesotheliomas and pulmonary adenocarcinomas: a comparative DNA flow cytometric study.

The DNA flow cytometric characteristics of 23 immunohistochemically and ultrastructurally proven pleural epithelioid mesotheliomas were compared with those of 41 primary pulmonary adenocarcinomas. Multiple, separate tissue blocks were analyzed from each neoplasm to assess DNA heterogeneity. All of the pleural mesotheliomas and 80% of the pulmonary adenocarcinomas manifested a homogeneous DNA ploidy (DNA stability). A significant statistical difference in the ploidy pattern between pleural mesotheliomas and pulmonary adenocarcinomas was noted (P < .001). Mesotheliomas were mostly diploid (78%) and adenocarcinomas were preponderantly aneuploid (88%). The proliferative rate and DNA indexes of the aneuploid adenocarcinomas were significantly higher than those of pleural mesotheliomas (P < .001). There was no statistical significance between the proliferative rates of diploid mesotheliomas and those of adenocarcinomas. We conclude that the DNA flow cytometric characteristics of mesotheliomas are significantly different from those of pulmonary adenocarcinomas. The clinical implications of these findings are discussed.     

An ultrastructural comparison of mesotheliomas with adenocarcinomas of the lung and breast

The ultrastructural features of 15 mesotheliomas were compared with those of equal numbers of adenocarcinomas of the lung and of the breast in a double-blind study. Combined quantitative and qualitative features were evaluated to provide criteria for distinguishing among these three tumors, which may present as either primary or metastatic pleural tumors. mesotheliomas could be distinguished from adenocarcinomas of the lung by length of microvilli (mean ratios of length to diameter [LDR], 15.7 and 8.7, respectively; P less than 0.01) and content of tonofilaments. Length of microvilli was also useful in distinguishing mesotheliomas from breast adenocarcinomas (mean LDR, 15.7 and 6.9, respectively; P less than 0.001). Adenocarcinomas of the lung could be distinguished from adenocarcinomas of the breast by tonofilament content and the presence of intracytoplasmic lumina. Combined quantitative and qualitative criteria are essential for maximal ultrastructural discrimination among these tumors.     

The ultrastructural localization of keratin proteins and carcinoembryonic antigen in malignant mesotheliomas.

The immunoultrastructural localization of keratin proteins and carcinoembryonic antigen (CEA) in mesothelioma cells was accomplished with a low-temperature embedding colloidal gold technique. The keratin antiserum labeled only intermediate filaments. These filaments surrounded the cytoplasmic organelles and were inserted into desmosomes. The CEA antiserum labeled cytoplasmic vesicles and droplets. No definite labeling of microvilli was observed.     

L&H variants of Reed-Sternberg cells express sialylated Leu M1 antigen.

Anti-Leu M1 generally does not stain the lymphocytic and histiocytic (L&H) variants of Reed-Sternberg cells in the lymphocyte predominant type of Hodgkin's disease. However, the authors found that after neuraminidase treatment for removal of sialic acid, the L&H cells in more than half of the cases studied could be stained by anti-Leu M1. This result strongly suggests that L&H cells differ from the Reed-Sternberg cells in other types of Hodgkin's disease in their unique capacity to sialylate the 150-kd Leu M1 antigen.     

Antigenic phenotype of Langerhans cell histiocytosis: an immunohistochemical study demonstrating the value of LN-2, LN-3, and vimentin

In order to determine the antigenic phenotype of the proliferating cells in Langerhans cell histiocytosis (LCH), we studied 15 such examples by using formalin- and B5-fixed, paraffin-embedded tissues. We used a panel of antibodies that are known to react with lymphocyte- and histiocyte-associated antigens. These included LN-1, LN-2, and LN-3 monoclonal antibodies (MoAbs), MoAbs to leukocyte common antigen (LCA), Leu-M1 antigen, vimentin, and epithelial membrane antigen (EMA), as well as polyclonal antibodies to lysozyme and S100 protein. The antigens encountered most frequently in LCH cells were S100 protein (93% of cases), vimentin (86%), and those detected by LN-2 (80%) and LN-3 (82%). Lysozyme was detected focally in two cases and diffusely in one case. The LCH cells were negative for LN-1, LCA, Leu-M1, and EMA. There was only one specimen in which S100 protein was not demonstrated; in this case, LN-3, vimentin, and T6 on frozen section were positive. The phenotype of LCH cells is similar to that of Langerhans' cells and interdigitating histiocytes. Our results demonstrate the value of using a panel of antibodies, including anti-vimentin MoAb, LN-2, and LN-3 for the immunophenotypic diagnosis of LCH in addition to an antibody to S100 protein.     

Expression of Das-1 in primary lung adenocarcinomas represents reactivation of an oncofetal pulmonary antigen.

OBJECTIVE: The monoclonal antibody (mAb) designated as mAb Das-1 was generated against a colonic epithelial protein. It reacts with bronchiolar epithelium and submucosal glands and weakly with alveolar lining cells of fetal lung. Adult alveolar epithelium is, however, nonreactive for mAb Das-1. The present study was designed to evaluate the immunoexpression of Das-1 and to correlate it with the histomorphology of primary lung adenocarcinomas and intestinal metaplasia. METHODS: Eighty-four cases of primary lung adenocarcinomas were reviewed and categorized according to histologic grade and subtype. Immunohistochemical staining with mAb Das-1 was performed with normal colon as control. RESULTS: Expression of Das-1 in the submucosal bronchial glands also served as internal control. Strong and diffuse staining was seen in 33 of the 84 cases of adenocarcinomas (39%). CONCLUSIONS: Expression of Das-1 was seen in primary lung adenocarcinomas even though adult alveolar epithelium is negative. Staining with mAb Das-1 was seen regardless of the grade or histological subtype of the lung adenocarcinomas. As most primary lung adenocarcinomas are not of bronchial gland origin, expression of Das-1 represents activation of oncofetal antigens.     

Use of Leu M1 and antiepithelial membrane antigen monoclonal antibodies for diagnosing Hodgkin's disease

Biopsies of 82 patients diagnosed as having Hodgkin's disease were reviewed. Seventeen were reclassified histologically as non-Hodgkin's lymphoma or reactive lymphoid hyperplasia. A substantial number of cases of Hodgkin's disease were negative when stained with Leu M1. Staining for Leu M1 was not found in the cases of non-Hodgkin's lymphoma or reactive lymphoid hyperplasia. With the exception of the lymphocyte predominant nodular subtype of Hodgkin's disease, epithelial membrane antigen staining was seen in a few cases of Hodgkin's disease and non-Hodgkin's lymphomas. This was not a useful discriminating feature.     

DNA ploidy analysis of pleural mesotheliomas: its usefulness for their distinction from lung adenocarcinomas.

The distinction of malignant mesotheliomas from adenocarcinomas with pleural involvement is often difficult, even with electron microscopic and state-of-the-art histochemical and immunologic studies. We evaluated the DNA ploidy and cell cycle of 45 clinically, morphologically, and immunohistochemically well-characterized malignant mesotheliomas to establish their ploidy profile and compared it with that of 41 pulmonary adenocarcinomas. All the cases were mucin negative and had been immunophenotyped with the following monoclonal antibodies: anti-keratin, anti-CEA, anti-Vimentin, anti-HMFG2, Leu M1 (CD15) and B72.3. Single cell suspensions from the paraffin blocks were prepared following Hedley's technique and were analyzed with a Coulter EPICS V flow cytometer. The resulting histograms were interpreted with the Multicycle software program. Five cases were excluded due to their high coefficients of variation. DNA aneuploidy was defined by the presence of more than one G0/G1 peak on the histograms obtained exclusively from the tumor sample. With this criterion, there is a possibility of missing aneuploid cases with a single aneuploid cycling population; however, fixatives and time of fixation produce such a remarkable variation in the fluorochrome uptake that any control, other than normal tissue present in the sample, was rendered unreliable. Five (14%) cases were DNA aneuploid with DNA indexes ranging from 1.2 to 1.9 (mean = 1.5). Three cases had increased S + G2/M values. Of the aneuploid cases, four were epithelial and one sarcomatous. In comparison, aneuploidy was found in 31 (75%) of the lung adenocarcinomas studied (p < 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of "myelomonocytic" antigens in mesotheliomas and adenocarcinomas involving the serosal surfaces.

Because of the demonstrated efficacy of Leu-M1 as a discriminant between malignant epithelioid mesothelioma (MEM) and adenocarcinomas involving the serosal surfaces (ACSs), the authors assessed the reactivities of related "myelomonocytic" antigens in this context. Paraffin sections from 41 MEMs and 43 ACSs (pulmonary, "serous surface papillary," and metastatic mammary adenocarcinomas) were evaluated for their expression of Leu-M1, LN1, LN2, and the Mac 387 antigen. Diagnoses were based in each case on the results of conventional histologic and electron microscopic examinations. Leu-M1 was detected only in minute foci of three peritoneal MEMs and was absent entirely in pleural mesotheliomas. Conversely, 38 of 43 ACSs expressed this marker. Three cases of peritoneal MEM and one pleural mesothelioma were multifocally LN2 positive, as were 39 of 43 ACSs. LN1 was the most frequently expressed antigen in MEM, being observed in 18 such tumors (10 pleural; 8 peritoneal); it was also detected in 37 of 43 ACSs. Mac 387 failed to label any of the neoplasms assessed in this series. These results demonstrate similar patterns of "myelomonocytic" antigen expression by diverse ACS and a general absence of Leu-M1 and LN2 in MEM. LN1 and the Mac 387 antigen appear to have no additional value when compared with Leu-M1 and LN2 in the immunohistochemical evaluation of serosal neoplasms.     

Nonspecificity of colon-specific antigen in adenocarcinomas. An immunohistochemical study of 422 cases.

To determine the specificity of colon-specific antigen in adenocarcinomas, routinely prepared paraffin-embedded tissue from 422 cases of adenocarcinoma were studied using a commercially available monoclonal antibody to colon-specific antigen and a standard avidin-biotin immunohistochemical technique. Positive reactivity for colon-specific antigen was very common (80% to 100%) in adenocarcinomas of the colon, distal esophagus/stomach, ovary, endocervix, endometrium, lung, pancreas, prostate, and bile ducts. Positive reactions were infrequent in adenocarcinomas of the breast (16%) and in hepatocellular carcinomas (23%). No immunoreactivity was seen in adenocarcinomas of the thyroid or in renal cell carcinomas. It is concluded that colon-specific antigen is not a colon-specific marker in adenocarcinomas. However, it may be useful in ruling out adenocarcinomas of renal or thyroid origin in certain clinical settings.     

Cilia and ciliogenesis in endometrial adenocarcinomas. An ultrastructural analysis

Cilia in neoplastic cells were observed by electron microscopy in specimens from five of six consecutive patients with endometrial adenocarcinoma. Most cilia showed a range of defects, from misalignment and displacement of individual doublets to the absence of up to three peripheral doublets, the pattern varying from 9+2 to 6+2; the central pair of microtubules also was frequently missing. Single peripheral microtubules and displacement of the dynein arms were also observed. The high proportion of cilial defects in neoplastic cells (72%) compared with those in normal endometrium (26%), together with a broader spectrum of cilial abnormalities, suggests that the neoplastic state increases the number and range of cilial lesions.     

Immunohistochemical phenotype of malignant mesothelioma: predictive value of CA125 and HBME-1 expression

Histological diagnosis of malignant mesothelioma and differentiation from adenocarcinoma is often difficult. Definitive pathological confirmation of malignant mesothelioma requires demonstration of an appropriate immunohistochemical phenotype. Selection of an optimum panel of immunohistochemical antibodies for the reliable identification of malignant mesothelioma is hindered by the absence of a specific immunohistochemical label for mesothelioma cells. Recently, we have found that the ovarian carcinoma cell antibody CA125 labels malignant mesothelioma cells, and the antibody HBME-1 has been developed as a sensitive mesothelial cell marker. We have compared the immunohistochemical staining patterns achieved with CA125 and HBME-1 to those obtained using a panel of eight further antibodies in 17 malignant mesotheliomas and 14 primary and secondary adenocarcinomas within lung and pleura. CA125 labelled malignant mesothelioma cells in 15 of 17 cases (88%), and adenocarcinoma cells in seven of 14 cases (50%). HBME-1 labelled mesothelioma cells in all 17 cases (100%) but also labelled adenocarcinoma cells in 10 of 14 cases (71%). BerEP4 positively labelled one malignant mesothelioma but was negative in the remaining 16 cases and positively labelled nine of 14 adenocarcinomas (64%). Monoclonal anti-CEA, AUA-1, CA19.9 and LeuM1 labelled no malignant mesotheliomas and were positive in 10 (71%), nine (64%), eight (57%) and six (43%) of 14 cases of adenocarcinoma, respectively. Diastase-PAS staining detected neutral mucin in none of the malignant mesotheliomas but in 10 (71%) of the 14 adenocarcinomas. We conclude that CA125 and HBME-1 do not label mesothelial cells with sufficient specificity to be useful for differentiating malignant mesothelioma from adenocarcinoma, although negative staining with HBME-1 makes a diagnosis of malignant mesothelioma unlikely. As there remains an absence of a specific positive mesothelial cell marker this distinction is still most reliably made using a panel of antibodies including at least two of the following: anti-CEA, AUA-1, BerEP4, LeuM1 and CA19.9, in combination with histochemical assessment of neutral mucin production.     

Carcinoembryonic antigen: immunohistologic identification in invasive and intraepithelial carcinomas of the lung.

A total of 58 pulmonary lesions from 48 patients were examined for carcinoembryonic antigen (CEA). The three-layer immunoperoxidase procedure for antigen detection was used with a monospecific anti-CEA antiserum. The control serum was the same antiserum with its specificity removed by affinity chromatography. Normal goat serum was also used as a control. Carcinoembryonic antigen was present in the majority of pulmonary adenocarcinomas and generally absent in the squamous cancers. The major exception was in the well-differentiated squamous lesions where CEA was occasionally found in the keratinizing areas. Of special interest was the finding of CEA in all areas of intraepithelial squamous neoplasia studied.     

Molecular analysis of varicella-zoster virus strains circulating in Tanzania demonstrating the presence of genotype M1

Based on analysis of 16,392 bp encompassing the complete open reading frames (ORFs) 1, 5, 31, 36, 37, 47, 60, 62, 67, and 68 of the genome of genotype M1 varicella-zoster virus (VZV) was found in swab samples originating from eight Tanzanian zoster patients. Moreover, sequence analysis suggests recombination events between different VZV genotypes within ORFs 1, 31, 60, and 67.     

DNA quantitation by image analysis of paraffin-embedded colorectal adenocarcinomas and its prognostic value.

DNA content was measured by image analysis in a retrospective study of formalin-fixed paraffin-embedded colorectal carcinomas from 213 patients who were followed up for at least 5 yr. DNA histograms were classified as diploid, aneuploid, or tetraploid. Diploid tumors comprised 29% of all cases, aneuploid 50%, and tetraploid 21%. Aneuploid tumors were found more often in patients with advanced disease and in carcinomas arising in the rectum. Pathologic stage, histologic grade, and ploidy were individually related to survival and recurrence. However, after stage stratification, histologic grade was no longer a significant prognostic factor. In patients without regional or distant metastases (Dukes' Stage A and Stage B), patients with aneuploid tumors had a statistically worse prognosis than patients with diploid or tetraploid tumors (P less than 0.01). The prognostic value of ploidy in this group of patients was maintained only in tumors arising in the distal colon and rectum (P less than 0.04). In patients with regional or distant metastases, DNA content did not provide additional prognostic information. In conclusion, DNA quantitation can be evaluated reliably by image analysis of archival material and can provide valuable prognostic information, especially in patients with Dukes' Stage A and Stage B disease. It may prove useful in guiding adjuvant therapy in these patients.