Carcinoma Effusions Research Articles

Comparison of Claudin-4, BerEP4, Carcinoembryonic Antigen and MOC31 in Serous Fluids Metastases Demonstrate High Sensitivity of Claudin-4 at Low Cellularity.

Claudin-4 has been described as a highly sensitive immunocytochemical marker for detection of metastatic carcinoma cells in effusion cytology specimens. This study aims to challenge the performance of claudin-4 in different types of malignancies and low cellularity specimens, by comparison with other markers in a large cohort of carcinomatous effusion specimens. Cell block preparations from peritoneal and pleural fluid specimens were retrieved, with malignant (carcinoma) diagnoses confirmed by review of hospital diagnosis code and pathology reports. Claudin-4, BerEP4, CEA, and MOC31 immunocytochemistry were performed and scored by expression proportion and intensity. Tumor cellularity was assessed for subgroup analysis of low cellularity specimens. Totally 147 specimens (70 pleural, 77 peritoneal) of 68 lung, 62 breast, 9 gynecological, and 7 gastrointestinal carcinomas were retrieved. The average proportion expression of claudin-4 was highest (89.6%, vs. CEA 40.5%, BerEp4 18.6%, MOC31 16.8%) and the percentage of strong expression was highest for claudin-4 (72.1%). Expression levels of claudin-4 were consistently higher than other markers in subgroups of all primary sites. The difference was more significant for low cellularity specimens. High (≥50%) proportion expression was seen for 96.61% of cases for claudin-4 (vs. BerEp4 8.77%, CEA 46.55%, MOC31 8.77%, p < 0.001). These factors contributed to a low concordance between claudin-4 and BerEp4, CEA and MOC31 (K = 0.010-0.043). Claudin-4 is more sensitive than CEA, BerEp4 and MOC31, suitable for low cellularity specimens of most types of metastatic carcinoma and is a robust immunocytochemical marker for carcinoma that can be used solitarily.

Claudin-4 immunocytochemistry is specific and sensitive for the diagnosis of malignant carcinomatous effusions: Results from a pilot study.

Claudin-4, a tight junction associated protein expressed in epithelial cells, is purported as a highly specific and sensitive marker for epithelial malignancies. Our aim was to assess the sensitivity, specificity and real-time utility of claudin-4 immunocytochemistry (ICC) in the diagnostic work-up of suspected malignant effusions. Claudin-4 (3E2C1 clone) ICC was performed prospectively in effusion cell blocks where other ICC markers were being performed as part of reporting over 3 months. Based on claudin-4 staining in unequivocal malignant and reactive effusions, the sensitivity and specificity was calculated. In cases signed out as inconclusive encompassing atypia of undetermined significance (AUS) and suspicious for malignancy (SFM) and negative for malignancy, change in diagnostic category based on addition of claudin-4 ICC was assessed. Study included 107 effusions. Claudin-4 stained 100% of metastatic adenocarcinomas including those with primaries in lung, breast, ovary, female genital tract, gastrointestinal tract and pancreatic-biliary tract, and was negative in all reactive mesothelial and mesothelioma effusions with sensitivity of 100% (48/48) and specificity of 95% (20/21) for adenocarcinoma. Claudin-4 upgraded the diagnostic category to positive in 70% (16/23) of SFM, 20% (1/5) of AUS, and in 50% (5/10) of negative effusions. Among cases with confirmed serosal involvement status on follow-up, claudin-4 showed sensitivity, specificity, positive predictive value and negative predictive values of 85% (11/13), 100% (3/3), 100% (10/10) and 75% (3/4), respectively, for metastatic adenocarcinoma. Claudin-4 as a single marker is sensitive and specific for adenocarcinoma and is a valuable addition to the ICC armamentarium.

Molecular characteristics of low-grade serous carcinoma in effusions.

The molecular characteristics of low-grade serous carcinoma (LGSC) in serous effusions have not been studied previously. The present study analysed the molecular profile of LGSC at this anatomical site. Specimens consisted of a series of 17 serous effusions (15 peritoneal, 2 pleural) from 16 patients, of which 15 were LGSC and 2 serous borderline tumour (SBT) who later progressed to LGSC. For comparative purposes, 9 surgical specimens from 6 patients with LGSC were analysed. Fresh-frozen cell pellets and surgical specimens underwent targeted next-generation sequencing covering 50 unique genes. Mutations were found in tumours from 14 of the 22 patients, of whom 4 had 2 different mutations and 10 had a single mutation. Overall, the most common mutations were in KRAS (n= 3) and BRAF (n= 3), followed by NRAS (n= 2), CDK2NA (n= 2), TP53 (n= 2), ATM (n= 2). Mutations in MET, STK11, ERBB2 and FLT3 were found in one case each. Patient-matched specimens had the same molecular profile. Both effusions with TP53 mutation had concomitant ATM mutation, and both stained immunohistochemically with a wild-type pattern. The absence of mutations was associated with a trend for shorter overall survival in univariate analysis (p= 0.072). The molecular alterations in LGSCs in serous effusions are consistent with those found in solid tumours, with frequent alterations in the mitogen-activated protein kinase pathway. Mutations in LGSC may be a marker of better outcomes.

Identifying the primary site of malignancy in carcinomatous serous effusions using immunocytochemistry.

Malignant effusions are commonly encountered in day-to-day cytology practice. Determining the primary site of malignancy in carcinomatous effusions is a Herculean task. Cytology coupled with immunocytochemistry (ICC) is often found to be helpful in this context. This study was conducted to evaluate the diagnostic utility of ICC on sections from cell blocks (CBs) in the detection of the primary site of origin in cases of metastatic carcinomatous effusions. To determine the origin of the primary tumour, TTF1 (lung), PAX-8 (ovary), CDX2 (colorectal), GATA3 (breast), and CK19 (pancreaticobiliary) were employed, depending on the clinical and radiological findings, and serum tumour markers. A total of 13,459 serous effusion samples were received for cytological evaluation from January 2017 to December 2021, of which 2708 (20.1%) were carcinomatous effusions. Out of these, 1044 (38.5%), 1611 (59.5%), and 53 (2.0%) were from pleural, peritoneal and pericardial cavities, respectively. Of these, the majority were adenocarcinoma. ICC was performed in 309 (11.4%) cases. The ovary was the most common primary site in 179 cases (57.9%), followed by the lung (75, 24.3%), pancreaticobiliary system (12, 3.9%), colon/rectum (8, 2.6%), breast (6, 1.9%), prostate (2, 0.6%) and kidney (1, 0.3). The lung was the most common primary site in pleural (67/113, 59.3%) and pericardial (6/8, 75%) effusions. The ovary (168/188, 89.4%) was the most common primary site for carcinomatous effusions in the peritoneal cavity. However, in 17 (5.5%) cases, the exact primary site could not be established. Judicious and methodical use of ICC on CBs helps to identify the primary site of the tumour in most carcinomatous effusions. This is of immense help to the treating clinician in directing appropriate therapy.

Metastatic Carcinoma in Effusions.

Serous cavity may be involved by any neoplasm, including very rare examples of involvement by central nervous system tumors leading to a malignant effusion. The serous cavity lining is rich in lymphatics with lymphatic lacunae opening directly through narrow gaps (stoma) in the lining. Carcinomas mainly metastasize to serosa via the lymphatic vessels, which may be blocked leading to effusion. Primary carcinomas of organs such as lung, intestines, liver, ovary, etc., lined by serosal membranes may spread by direct extension, resulting in malignant effusions. As standard of practice, unless specified, cytopathologic examination of serous effusions implies detection of malignant cells.As compared to a surgical biopsy from a small focal area of an extensive serosal surface, effusion fluid from respective cavity exfoliates the cells from the entire serosal surface with minimal chance of sampling artifact. Because of this, effusion fluid cytology generally provides a higher diagnostic yield as compared to biopsy of the serous lining, as demonstrated by some studies. However, various challenges related to effusion fluid cytology makes the interpretation of effusion fluid cytology a field with potential misinterpretations, especially for those without proper experience or training.Developing and following a methodical approach is important for appropriate cytologic examination of effusion fluids. Proper approach may achieve definitive interpretation even without ancillary tests. However, lack of appropriate approach and processing may introduce a significant variation in interpretation due to combination of well-recognized diagnostic pitfalls, which may lead to lower reproducibility and even serious misinterpretations.Current review discusses in brief appropriate approach to processing and evaluating effusion fluid cytology for metastatic carcinoma. At general level, this is comparable to that of other specimens; however, it is critical to modify with reference to the limitations associated with effusion cytology.

Application of multicolour flow cytometry in the detection of metastatic carcinoma in serous effusions: Special emphasis in atypical cytology.

We evaluated the role of simultaneous use of multiple antibodies in flow cytometry (FCM) to detect metastatic carcinomas in effusion samples. Cytological examination of 75 successive cases of effusion samples was performed. There were 48 peritoneal, 26 pleural and one pericardial fluid. Multi-coloured FCM examination was undertaken using a cocktail of CD45, CD14 and epithelial cell adhesion molecule (EpCAM), antibodies tagged with different fluorochromes. The percentage of EpCAM positivity was calculated in the CD45 and CD14 dual negative population by selective gating. The EpCAM value was correlated with the cytological findings, follow-up data and MOC-31 immunostaining. There were 20 benign, 35 malignant and 20 atypical cases diagnosed on cytomorphology. The primary sources of carcinomas were mainly from the ovary, followed by lung, gall bladder, intestine and other areas. Out of 20 cytologically benign cases, there were two malignant cases on the final follow-up, and EpCAM on FCM picked up all 18 benign cases and one malignant case. Out of 35 cytologically detected malignant cases, EpCAM picked up 32 malignant cases. The EpCAM detected 15/18 malignant and both benign cases out of 20 cytological atypical cases. EpCAM antibody by FCM showed 87% sensitivity, 100% specificity, 100% positive predictive value and 74% negative predictive value. This comprehensive study highlights the potential use of multi-coloured FCM along with cytological examination to diagnose metastatic carcinoma in effusion samples. Multi-coloured FCM is rapid and quantitative and is helpful in atypical cases.

P16 as a marker of carcinoma in effusions and peritoneal washing

BackgroundConsidering the potential of p16 as a marker for diagnosis, prognosis and therapeutic response, the aim of this study was to assess its presence, via immunocytochemistry, in metastatic carcinoma of different primary sites and histological types obtained from effusions and peritoneal washings. A total of 118 samples including 85 of metastatic carcinoma and 33 samples of benign effusion/peritoneal washing were prepared by the plasma/thromboplastin method. Immunocytochemistry reactions were performed on cell block sections using antibodies against p16, claudin-4, MOC-31, calretinin, HBME and CD68.ResultsP16 overexpression was observed in 88.23% of all carcinoma samples. All cervix adenocarcinoma samples showed p16 overexpression. Overexpression in adenocarcinomas of ovary, lung and breast was observed in 93.75, 93.10 and 75% of the samples, respectively. Overexpression was observed in all different histological types analyzed: small cell carcinoma (lung), squamous cell carcinoma (cervical) and urothelial carcinoma (bladder). The specificity of p16 for carcinoma detection was of 96.96%.ConclusionOverexpression of p16 was observed in most metastatic carcinoma, from different primary sites and histological types, obtained from effusions and peritoneal washings. Due to its high frequency of overexpression in metastatic carcinoma, p16 may play a possible role in tumor progression and it may be considered as a complementary diagnostic marker depending on histological type and primary site of carcinoma.

Anti-EpCAM antibodies for detection of metastatic carcinoma in effusions and peritoneal wash.

Epithelial cell adhesion molecule (EpCAM) has been used as diagnostic/prognostic marker and therapeutic target. The aim of the present study was to compare immunoreactivity of antibodies against distinct epitopes in the ectodomain of EpCAM for detection of carcinoma from different primary sites and of different histological types in effusions and peritoneal wash. Two antibodies against epitopes in the EGF-like domain I (clones Moc-31 and Ber-EP4) and one antibody against the epitope in the cysteine-poor region (158210) of EpCAM were used (all commercially available). Independently of the clone used, EpCAM overexpression was observed in almost all samples when all the adenocarcinoma samples were analyzed together. By using Moc-31, EpCAM overexpression was observed in all samples of adenocarcinoma. Absence of EpCAM overexpression was observed in a few adenocarcinoma samples at some sites of tumor origin, including ovary, breast and stomach, when Ber-EP4 and 158210 were used. Regarding carcinomas aside from adenocarcinomas, histological types, such as squamous cell, urothelial and small cell carcinoma showed different degrees of EpCAM expression according to the antibody used. In squamous cell carcinoma, overexpression was observed only with the clone 158210. It was concluded that, overall, most samples of metastatic carcinoma from effusions showed overexpression of EpCAM. However, there are significant variations in its detection according to the primary site, histological type of the carcinoma and depending on the antibody used. Thus, the use of more than one type of anti-EpCAM antibody would increase the chance of its detection in metastatic carcinoma effusion.

The cell tube block technique and an immunohistochemistry panel including Wilms tumor 1 to assist in diagnosing cavitary effusions in dogs and cats.

Cell blocks and immunohistochemistry (IHC) are increasingly recognized as being complementary tools for cytologic diagnostics, especially for neoplastic diseases. The study aimed to evaluate the utility of cell tube block (CTB) IHC for refining the diagnosis of effusions in dogs and cats. Cavitary effusions (n=25) from dogs and cats classified by cytology as reactive, neoplastic, borderline (suspicious of neoplasia), and chylous were studied. CTB sections were stained with H&E, and immunostained with PAX-5, CD3, pancytokeratin (CK), vimentin, and Wilms tumor 1 protein (WT1) antibodies, according to the cytologic diagnoses. A histologic case series of confirmed normal, reactive, and neoplastic mesothelium and several different carcinomas were included to test the utility of WT1 as a marker of mesothelial cells. CTBs had a layered appearance with reduced background staining. CD3 and PAX5 immunolabeling allowed immunophenotype assessment in all of the lymphoma cases. In carcinomatous effusions, neoplastic cells were CK-positive, WT1-negative, and vimentin-negative (except for two cases). Wilms tumor 1 protein was positive in the nuclei of normal, reactive, and neoplastic mesothelial cells, and ovarian carcinomatous cells. Other carcinomas and lymphomas were negative. CTBs are valuable tools to assist in making a diagnosis of cavitary effusions in dogs and cats, and WT1 is a promising marker to differentiate mesothelial from carcinomatous cells.

Comparative study of paired box gene 8, cytokeratin 7, and cytokeratin 20 in differentiating the ovarian from nonovarian carcinoma cells in peritoneal effusions

Background In female patients, ovarian carcinoma is considered a common cause of malignant peritoneal effusions that shows a good response to chemotherapy, an issue that mandates quick and accurate diagnosis of those cases. PAX8 is a cell lineage factor that plays a crucial role in the morphogenesis of the kidney, thyroid gland, and Mullerian system. It is expressed in 95–100% of ovarian carcinomas in tissue sections. Recently, it was suggested as a potential marker for the diagnosis of ovarian carcinomas in effusions. Aim This study aimed at assessing the role of CK7, CK20, and PAX8 in differentiating ovarian from nonovarian carcinomas in peritoneal effusions. Patients and methods Immunocytochemical evaluation of CK7, CK20, and PAX8 expression in 40 cell blocks of malignant peritoneal effusion samples was carried out. In addition, the assessment of PAX8 level using double-antibody sandwich enzyme-linked immunosorbent assay technique in the serum of the included cases was also carried out. Results Of the studied 40 cases, 21 (52.5%) proved to be ovarian carcinomas, whereas 19 (47.5%) were nonovarian. Diffuse CK7 expression (scores 3, 4) was significantly detected more frequently in effusions from ovarian than from nonovarian carcinomas. In contrast, CK20 expression tended to be more prevalent in effusions of nonovarian than in those of ovarian origin; however, this was statistically insignificant. PAX8 expression was significantly more frequent in effusions from ovarian carcinomas than from nonovarian carcinomas. Sensitivity and specificity of PAX8 in the diagnosis of ovarian carcinoma was higher than that of CK7, while a combination of both CK7 and PAX8 immune scores using a generated regression equation was the most specific. In contrast, serum PAX8 level was statistically higher in ovarian more than in nonovarian carcinoma cases. Conclusion PAX8 is a useful immune marker for the diagnosis of ovarian carcinoma in peritoneal effusions. However, the combination of both CK7 and PAX8 using a generated regression equation increases the specificity of diagnosis. PAX8 serum level is a potential serum marker in diagnosing ovarian carcinomas.

Measurement of telomere length in cells from pleural effusion: Asbestos exposure causes telomere shortening in pleural mesothelial cells.

We estimated the telomere lengths of neoplastic and non-neoplastic mesothelial cells and examined their correlation with asbestos exposure and the expression of markers of mesothelial malignancy. Cell blocks of pleural effusion obtained from 35 cases of non-neoplastic disease (NN), 12 cases of malignant mesothelioma (MM) and 12 cases of carcinomatous effusion due to lung adenocarcinoma (LA) were examined. Fifteen of the 35 NN cases had pleural plaques (NNpp+) suggestive of asbestos exposure, and the other 20 cases had no pleural plaques (NNpp-). Telomere length was measured using the tissue quantitative fluorescence in situ hybridization method, and expressed as normalized telomere-to-centromere ratio. NN cases had significantly longer telomeres than MM (P < 0.001) and LA (P < 0.001) cases. Telomeres in NNpp+ cases were slightly shorter than those of NNpp- cases (P = 0.047). MM and LA showed almost the same telomere length. NN cases with shorter telomeres tended to show aberrant expression of epithelial membrane antigen (EMA), CD146, glucose transporter 1 (GLUT1) and IGF-II messenger RNA-binding protein 3 (IMP3). These results suggest that telomere shortening and subsequent genetic instability play an important role in the development of MM. Measurement of telomere length of cells in pleural effusion might be helpful for earlier detection of MM.

Características citológicas de cistoadenocarcinoma papilar ovariano em um cão

Background: Ovarian papillary cystadenocarcinoma is a rare neoplasm associated with peritoneal implantation and malignant effusion. Most dogs are asymptomatic until the nodules become large and the abdominal volume is increased. From the clinical suspicion, the diagnosis can be obtained through imaging and histopathology, however, cytological analysis has become an alternative method for the early detection of this neoplasm. In order to demonstrate the importance of cytology in the diagnosis of ovarian neoplasms and its metastasis, it is reported a case of metastatic ovarian papillary cystadenocarcinoma in a dog.Case: Female, intact, teckel, 5-year-old, with increased abdominal volume. Physical examination revealed ascites and intracavitary mass, abdominocentesis and fine needle puncture of the mass were performed for cytological evaluation. In the cavity fluid it was observed: dark red color, cloudy appearance, hematocrit of 35%; (7.6 g / dL), pH (8.0), 22,000 nucleated cells / μL, marked cellularity of pleomorphic epithelial cells arranged in three-dimensional cohesive groups, sometimes in acinar or tubular pattern, nucleus with loose chromatin coarse, scarse to moderate cytoplasm, perinuclear halo, multiple and evident nucleoli compatible with carcinomatous neoplastic effusion. In the cytological evaluation of the tumor, epithelial cells were observed, with the same microscopic characteristics of the abdominal fluid. A laparotomy that did not show metastasis was performed, multiple nodes interspersed with cystic regions containing yellow-red fluid in the right ovary were visualized. Histopathology showed: neoplastic cells proliferation of ovarian glandular tissue, scarce cytoplasm, poorly delimited, nucleus ranging from oval to cylindrical with marked pleomorphism, evident nucleoli and loose chromatin, mitotic figures and papillary growth. Neoplastic cells forming irregular cavities with proteinaceous fluid, scarce connective tissue intermingling the cellular nest and areas of hemorrhage. Cytological and histopathological analyzes were compatible with ovarian papillary cystadenocarcinoma. After three months of excision, the dog returned with thoracic effusion that presented the same characteristics of the abdominal fluid, indicating metastasis.Discussion: Metastasis and effusion were observed in 48% and 86% of dogs with this tumor, respectively. Cell exfoliation, release of fluid through the tumor capsule or rupture of cysts can result in transcelomic metastatic implants that exert pressure and obstruct peritoneal and diaphragmatic lymphatic vessels causing effusion. The macro and microscopic characteristics of the abdominal effusion reinforced the suspicion of neoplasia, and the cytomorphological evaluation of the tumor, which identified carcinomatous cells similar to that of the effusion, allowed the presumptive diagnosis of the neoplasia. The macroscopic presence of multiple nodes interspersed with cystic regions containing red fluid in the right ovary, identified after surgical excision, reinforced the cytologic diagnosis. Histopathological examination identified wellestablished microscopic features that allowed the definitive and confirmatory diagnosis of neoplasia. Radiological analysis of the chest was not enough to detect the metastasis diagnosed by effusion analysis, however, small nodules (less than 6 mm) are difficult to identify by imaging. Thus, it is important to emphasize the importance of cytological evaluation of tumor and effusions for detection of neoplastic cells for the diagnosis of intracavitary neoplasia and metastasis.

Amphiregulin triggered epidermal growth factor receptor activation confers in vivo crizotinib-resistance of EML4-ALK lung cancer and circumvention by epidermal growth factor receptor inhibitors.

Crizotinib, a first‐generation anaplastic lymphoma kinase (ALK) tyrosine‐kinase inhibitor, is known to be effective against echinoderm microtubule‐associated protein‐like 4 (EML4)‐ALK‐positive non‐small cell lung cancers. Nonetheless, the tumors subsequently become resistant to crizotinib and recur in almost every case. The mechanism of the acquired resistance needs to be deciphered. In this study, we established crizotinib‐resistant cells (A925LPE3‐CR) via long‐term administration of crizotinib to a mouse model of pleural carcinomatous effusions; this model involved implantation of the A925LPE3 cell line, which harbors the EML4‐ALK gene rearrangement. The resistant cells did not have the secondary ALK mutations frequently occurring in crizotinib‐resistant cells, and these cells were cross‐resistant to alectinib and ceritinib as well. In cell clone #2, which is one of the clones of A925LPE3‐CR, crizotinib sensitivity was restored via the inhibition of epidermal growth factor receptor (EGFR) by means of an EGFR tyrosine‐kinase inhibitor (erlotinib) or an anti‐EGFR antibody (cetuximab) in vitro and in the murine xenograft model. Cell clone #2 did not have an EGFR mutation, but the expression of amphiregulin (AREG), one of EGFR ligands, was significantly increased. A knockdown of AREG with small interfering RNAs restored the sensitivity to crizotinib. These data suggest that overexpression of EGFR ligands such as AREG can cause resistance to crizotinib, and that inhibition of EGFR signaling may be a promising strategy to overcome crizotinib resistance in EML4‐ALK lung cancer.

Young investigator challenge: The utility of GATA3 immunohistochemistry in the evaluation of metastatic breast carcinomas in malignant effusions.

It is not uncommon to encounter challenges in the immunohistochemical confirmation of metastatic breast cancer given the limited sensitivities of mammaglobin and gross cystic disease fluid protein 15 (GCDFP-15/BRST-2) and the significant proportion of triple-negative breast carcinomas (ie, tumors that are negative for estrogen receptor [ER], and progesterone receptor [PgR], and human epidermal growth factor 2 [HER2]). GATA binding protein 3 (GATA3) has emerged as a potentially useful immunohistochemical adjunct during the evaluation of metastatic breast carcinomas in cytology specimens. The objective of the current study was to examine GATA3 expression in the context of malignant effusions secondary to both mammary and extramammary malignancies. In total, 306 malignant effusions (from 62 metastatic breast carcinomas and 244 extramammary malignancies) were examined using GATA3 immunohistochemistry. Effusions with metastatic breast carcinoma were also examined using immunohistochemistry for additional breast markers (ER, PgR, HER2, mammaglobin, and GCDFP-15/BRST-2). GATA3 immunohistochemistry highlighted the tumor cells in 58 of the 62 samples (93.5%) from patients with metastatic breast carcinoma, which was higher than the observed sensitivity of immunohistochemistry for ER (63.8%), PgR (41.4%), HER2 (15.5%), mammaglobin (22.4%), and GCDFP-15/BRST-2 (5.2%). GATA3 expression also was observed in a subset of malignant effusions secondary to extramammary primaries, specifically, in 28 of 244 specimens (11.5%). GATA3 is a highly sensitive marker for the detection of metastatic breast carcinomas in effusion specimens. However, this marker is not entirely specific for malignancies of breast origin. Thus, GATA3 should be used in conjunction with additional immunohistochemical markers during the cytologic evaluation of malignant effusions.

Carbonic anhydrase IX (CAIX) does not differentiate between benign and malignant mesothelium.

To examine carbonic anhydrase IX (CAIX), a marker of renal cell carcinoma that recently has been described in malignant effusions. Pleural and peritoneal fluids with the following diagnoses-reactive (n = 23), carcinoma (n = 17), and "suspicious for mesothelioma" (n = 4)-were immunostained for CAIX, calretinin, Ber-EP4, and MOC31. A tissue microarray of epithelioid (n = 27) and sarcomatoid (n = 8) mesotheliomas and three cases of benign mesothelium were also immunostained for CAIX. Mesothelial cells in both reactive (18/23) and malignant effusions (18/21) were positive for CAIX (P > .05). In carcinomatous effusions, CAIX expression was restricted to the mesothelial cells. Agreement between CAIX and calretinin expression was present in 89% of cases. In tissues, CAIX was positive in 100% of benign and 91% of malignant mesothelium. CAIX can be a useful ancillary marker for identifying mesothelial cells. There is no difference in CAIX expression between benign and malignant mesothelium. Caution should be exercised while evaluating for metastasis from renal cell carcinoma.

Immunological screening for tumor cells in serous body fluids has added value with the CELL-DYN Sapphire

Conventional cytological examination has limited sensitivity for detecting tumor cells in serous body cavity effusions and therefore, adjuvant techniques are necessary for a reliable diagnosis. Flow cytometry has proven benefit in these circumstances. The aim of our study was to explore the feasibility of CELL-DYN Sapphire, an advanced hematology analyzer with flow cytometric capabilities, for detecting tumor cells in serous body fluids, using CD326 monoclonal antibodies, which are directed against the epithelial marker EpCAM. One hundred and five serous fluids (39 peritoneal and 66 pleural effusions) were analyzed by the CELL-DYN Sapphire using monoclonal antibody combinations CD3/CD19 and CD45/CD326. Of all samples a cytospin preparation was made and microscopically examined; the pathology findings served as a reference. Using a threshold of 1% CD326+ cells, CELL-DYN Sapphire identified nine out of 12 cases with tumor cells in the serous effusions (sensitivity 75%), whereas routine cytology found eight cases (sensitivity 67%). The combination of immunophenotyping and cytology identified all 12 cases with tumor cells in the effusion fluid (sensitivity 100%). The specificities were 92% and 100%, respectively. We demonstrated that it is feasible to run an immunophenotypic assay on CELL-DYN Sapphire for detecting tumor cells in serous body fluids. In addition, this study confirmed that a combination of conventional cytology and flow cytometry had a very high diagnostic yield in cases of carcinomatous effusions.

Incidence of Merkel Cell Carcinoma and Other Neuroendocrine Carcinomas in Effusions: A 5-Year Single Institution Experience

Incidence of Merkel Cell Carcinoma and Other Neuroendocrine Carcinomas in Effusions: A 5-Year Single Institution Experience

Rab25 is overexpressed in Müllerian serous carcinoma compared to malignant mesothelioma

Rab25, an epithelial-specific member of the Rab family of small GTPases, was previously shown to be overexpressed in ovarian/primary peritoneal serous carcinoma compared to malignant mesothelioma using gene expression arrays. The objective of this study was to validate this finding at the mRNA and protein level. Quantitative PCR analysis of 112 Müllerian serous carcinomas (84 effusions, 28 primary ovarian carcinomas) and 22 malignant mesotheliomas (19 effusions, 3 solid specimens) showed significantly higher RAB25 mRNA expression in the former tumor (p < 0.001). Immunohistochemical analysis of Rab25 protein expression in 245 effusions showed significantly higher expression of this protein in Müllerian serous carcinoma compared to malignant mesothelioma (189/209 vs. 12/36 positive tumors, respectively; p < 0.001). Immunostaining of 101 patient-matched solid Müllerian carcinoma specimens (34 primary carcinomas, 67 metastases) showed expression levels comparable to effusions (94/101 positive specimens; p > 0.05). Rab25 mRNA and protein expression levels in Müllerian carcinoma effusions did not correlate with overall or progression-free survival. Our data confirm that Rab25 effectively differentiates Müllerian carcinomas from malignant mesothelioma at the mRNA and protein level, suggesting a role in the diagnostic work-up of serosal cancers.

PINCH‐2 expression in cancers involving serosal effusions using quantitative PCR

The PINCH-2 gene was previously shown to be overexpressed in malignant mesothelioma compared with ovarian/peritoneal serous carcinoma in Affymetrix array analysis. The objective of the present study was to validate this finding at the mRNA and protein level. Effusions (n = 91; 71 ovarian and 10 breast carcinomas, 10 malignant mesotheliomas) were assayed for PINCH-2 mRNA expression using quantitative PCR. PINCH-2 protein expression was analysed in 37 effusions using flow cytometry. Quantitative PCR analysis showed significantly higher PINCH-2 mRNA levels in mesotheliomas compared with carcinomas (P = 0.004). Values of <10 copies were found exclusively in carcinoma effusions (25.4% of ovarian and 50% of breast carcinomas). However, PINCH-2 protein expression by flow cytometry did not differ significantly between the three cancer types. No association was observed between PINCH-2 levels and patient survival or expression of previously-studied molecules related to adhesion, metastasis and apoptosis inhibition in ovarian carcinoma. Our data suggest that PINCH-2 mRNA is overexpressed in malignant mesothelioma compared with carcinomas involving serosal cavities, and that low levels of this gene argue against the diagnosis of mesothelioma. The frequent PINCH-2 protein expression in all three studied cancers suggests a role for this molecule in cancer cell biology in effusions and merits further research.

Diagnostic utility of PAX8 and PAX2 immunohistochemistry in the identification of metastatic Müllerian carcinoma in effusions

Morphologic distinction of Müllerian carcinomas from non-Müllerian carcinomas in effusion specimens by cytomorphology alone can be diagnostically challenging. Therefore, immunohistochemical adjuncts can be useful in differentiating Müllerian from non-Müllerian metastases. In this study, we evaluated the expression of PAX8 and PAX2 in malignant effusions collected from patients with known Müllerian and non-Müllerian carcinomas. Sections from cell blocks prepared from 152 effusion specimens (54 and 98 cases representing metastases from Müllerian and non-Müllerian primaries, respectively) were immunostained with rabbit polyclonal antibodies against PAX8 and PAX2. Immunopositivity was defined as the presence of strong nuclear staining in at least 25% of the tumor cells. Fifty-two (96%) and 13 (24%) of the 54 Müllerian carcinomas were positive for PAX8 and PAX2, respectively. PAX8 positivity was seen in only four (4%) of 98 non-Müllerian carcinomas; these represented metastasis from a large cell neuroendocrine lung carcinoma, papillary thyroid carcinoma, renal cell carcinoma, and acinic cell carcinoma of the parotid gland. PAX2 positivity was not seen in any of the non-Müllerian carcinomas. The results demonstrate that both PAX8 and PAX2 are highly specific markers for metastatic Müllerian carcinomas in cell block preparations from effusion specimens (96% and 100%, respectively). PAX8, however, is more sensitive than PAX2 in identifying Müllerian carcinomas in fluids (96% versus 24%). Overall, immunohistochemistry for PAX8 and PAX2 represent diagnostically useful adjuncts in identifying a Müllerian carcinoma as a source of a malignant effusion.