Breast cancer organoids from malignant pleural effusion-derived tumor cells as an individualized medicine platform.

Malignant pleural effusion (MPE) presents a severe medical condition in patients with advanced breast cancer (BC). We applied organoid culture technology to culture preoperative puncture specimen and corresponding surgical specimen-derived tumor cells from early BC patients and pleural effusion-derived tumor cells from advanced BC patients with MPE to study whether in vitro models could predict therapies of clinical patients. We successfully expanded pleural effusion-derived tumor organoids from 1 advanced triple-negative breast cancer (TNBC) patient with MPE which had been continuously propagated for more than 3 months. The organoids matched the histological characteristics of primary BC and metastatic supraclavicular lymph nodes by H&E staining and retained negative expression of TNBC biomarkers: estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2, and positive expression of antigen Ki-67. Multiple mutations were detected from this advanced TNBC patient with MPE by high-throughput sequencing of metastatic supraclavicular lymph node and the plasma sample. We performed the 3D drug screening tests combined with the clinical medication situation of this patient. The pleural effusion-derived tumor organoids were sensitive to capecitabine (IC50 1.580 µmol) and everolimus (IC50 4.008 µmol) single-agent treatments. The sensitivity to capecitabine was consistent with the clinical treatment response of this patient for capecitabine and with the sequencing results that reported MTHFR gene polymorphism mutation and TYMS -6bp/-6bp polymorphism mutation indicating effectiveness to fluorouracil. Our results suggested that an effective platform for ex vivo pleural effusion-derived tumor organoids from advanced TNBC patients with MPE could be used to identify treatment options and explore the clinicopathological characteristics of these patients.

Yield of Malignant Pleural Effusion for Detection of Oncogenic Driver Mutations in Lung Adenocarcinoma.

BACKGROUND: Pleural fluid can be used to assess targetable mutations in patients with lung adenocarcinoma. The primary objective of this study was to assess the yield of pleural fluid cytology for targetable oncogenic mutations (EGFR, KRAS, BRAF, ALK, and ROS1 gene rearrangements). We also assessed pleural fluid volume necessary for molecular testing. METHODS: Retrospective review was performed of 134 consecutive patients with lung adenocarcinoma associated malignant pleural effusions. EGFR and KRAS testing was done using PCR amplification followed by DNA sequencing, or next generation sequencing in more recent cases that included BRAF assessment. Fluorescence in situ hybridization employing break-apart probes was used to test for ALK and ROS1 rearrangements. RESULTS: Mutation analysis on pleural fluid cell-block was performed on 56 patients. It was adequate for complete analysis ordered including EGFR, KRAS, BRAF, ALK, and ROS1 rearrangements on 40 (71.4%) samples. For individual mutations, EGFR testing was possible in 38 of 49 (77.6%); KRAS 22 of 28 (78.6%); BRAF 10 of 13 (76.9%), ALK gene rearrangement 42 of 51 (82.4%) and ROS1 gene rearrangement in 21 of 28 (75%) pleural fluid specimens. The analysis was satisfactory in 13 of 19 (68.4%) samples with ≤100 mL versus 27 of 37 (72.9%) with >100 mL of fluid tested (P-value=0.7). CONCLUSION: Genetic mutation analysis can be performed on malignant pleural effusions secondary to lung adenocarcinoma, independent of fluid volume.

A Prospective Study to Evaluate a Diagnostic Algorithm for the Use of Fluid Lymphocyte Subset Analysis in Undiagnosed Unilateral Pleural Effusions.

BACKGROUND: Haematological malignancy is an important cause of pleural effusion. Pleural effusions secondary to haematological malignancy are usually lymphocyte predominant. However, several other conditions such as carcinoma, tuberculosis, and chronic heart failure also cause lymphocytic effusions. Lymphocyte subset (LS) analysis may be a useful test to identify haematological malignancy in patients with lymphocytic effusions. However, research into their utility in pleural effusion diagnostic algorithms has not yet been published. OBJECTIVES: We aimed to determine the clinical utility of pleural fluid LS analysis and whether it can be applied to a diagnostic algorithm to identify effusions secondary to haematological malignancy. The secondary aim was to evaluate the diagnostic value of pleural fluid differential cell count. METHODS: Consecutive consenting patients presenting to our pleural service between 2008 and 2013 underwent thoracentesis and differential cell count analysis. We proposed an algorithm which selected patients with lymphocytic effusions (>50%) to have further fluid sent for LS analysis. Two independent consultants agreed on the cause of the original effusion after a 12-month follow-up period. RESULTS: A total of 60 patients had samples sent for LS analysis. LS analysis had an 80% sensitivity (8/10) and a 100% specificity for the diagnosis of haematological malignancy. The positive and negative predictive values were 100 and 96.1%, respectively. Overall 344 differential cell counts were analysed; 16% of pleural effusions with a malignant aetiology were neutrophilic or eosinophilic at presentation. A higher neutrophil and eosinophil count was associated with benign diagnoses, whereas a higher lymphocyte count was associated with malignant diagnoses. CONCLUSIONS: LS analysis may identify haematological malignancy in a specific cohort of patients with undiagnosed pleural effusions. A pleural fluid differential cell count provides useful additional information to streamline patient pathway decisions.

A pilot study of an autologous tumor-derived autophagosome vaccine with docetaxel in patients with stage IV non-small cell lung cancer.

BACKGROUND: Tumor-derived autophagosome vaccines (DRibbles) have the potential to broaden immune response to poorly immunogenic tumors. METHODS: Autologous vaccine generated from tumor cells harvested from pleural effusions was administered to patients with advanced NSCLC with the objectives of assessing safety and immune response. Four patients were vaccinated and evaluable for immune response; each received two to four doses of vaccine. Study therapy included two cycles of docetaxel 75 mg/m2 on days 1 and 29 to treat the tumor, release hidden antigens and produce lymphopenia. DRibbles were to be administered intradermally on days 14, 43, 57, 71, and 85, together with GM-CSF (50 µg/d x 6d, administered via SQ mini pump). Peripheral blood was tested for immune parameters at baseline and at each vaccination. RESULTS: Three of four patients had tumor cells available for testing. Autologous tumor-specific immune response was seen in two of the three, manifested by IL-5 (1 patient after 3 doses), and IFN-γ, TNF-α, IL-5, IL-10 (after 4 doses in one patient). All 4 patients had evidence of specific antibody responses against potential tumor antigens. All patients came off study after 4 or fewer vaccine treatments due to progression of disease. No significant immune toxicities were seen during the course of the study. CONCLUSIONS: DRibble vaccine given with GM-CSF appeared safe and capable of inducing an immune response against tumor cells in this small, pilot study. There was no evidence of efficacy in this small poor-prognosis patient population, with treatment not feasible. Trial registration NCT00850785, initial registration date February 23, 2009.

[Clinical Assessment of Chemosensitivity Test in Xeno-free Culture of Autologous 
Malignant Effusion Cells from Patients with Advanced Lung Adenocarcinoma].

BACKGROUND: A great individual differences to chemotherapeutic effects existed in the patient with advanced lung cancer. How to choose the optimum regimens to achieve the individuation and maximum effect of chemotherapy for lung cancer is worth exploring. The study was designed to examine the effect of ex vitro chemo-sensitivity assay in xeno-free culture of autologous malignant effusion cells from patients with advanced lung adenocarcinoma. METHODS: The 50 treatment-naive patients with lung adenocarcinoma complicated with malignant pleural or pericardial effusions were enrolled. Effusions of all cases had been controlled by closed drainage and 300 mL-500 mL of which were retained under sterile condition from 25 cases (Chemo-sensitivity group). Primary malignant effusion cells were isolated from autologous effusions of the patients. Then, xeno-free culture (average 11 days) were intervened with 8 chemotherapeutic drugs commonly used in clinical practice and were determined by CCK-8 assay. Optimum regimens were selected for chemotherapy based on the results of chemosensitivity test. As a contrast, chemotherapy regimens for the other 25 patients (Control group) were on the basis of physician's clinical experience. RESULTS: After four cycles of chemotherapy, in Chemo-sensitivity group, 17 (68.0%) cases were determined for partial response (PR), 5 (20.0%) cases for stable disease (SD), and the objective response rate (ORR) was 68.0%, the disease control rate (DCR) was 88.0%. Meanwhile, in Control group, 9 (36.0%) cases were determined for PR, 7 (28.0%) cases for SD, and, the ORR was 36.0%, the DCR was 64.0%. There were significant differences between the two groups in ORR and DCR (P<0.05). To the end of follow-up, there were 21 cases of death in Chemo-sensitivity group as well as 22 cases in Control group. The mean progression-free survival (PFS) in Chemo-sensitivity group and Control group respectively were 10.0 months and 5.8 months, and the mean overall survival (OS) in the two groups were 30.2 months and 21.2 months respectively. There were also significant differences between the two groups in PFS and OS (P<0.05). Furthermore, the adverse reactions in both groups were mild and controllable. CONCLUSIONS: Xeno-free culture of autologous malignant effusion cells from patients with advanced lung adenocarcinoma and ex vitro chemo-sensitivity assay are beneficial to the rational choices of chemotherapeutic agents used in patients with lung adenocarcinoma complicated with malignant effusions, which is a worthy trial in personalized cell culture for individualized cancer therapy and further studies.

Pleural effusion of malignant aetiology: cell block technique to establish the diagnosis.

We describe cases of two previously healthy women presenting with progressively worsening breathlessness for 1-2 months. In both cases, physical examination was suggestive of a left-sided pleural effusion, confirmed by chest X-ray. Analysis of aspirated fluid showed a lymphocytic exudate, but cytological analysis was negative for malignancy in both patients. CT scan revealed malignancies as the underlying cause of the effusions. Both patients were managed with intercostal drainage in order to collect a sufficient amount of pleural fluid to perform a new technique in our hospital: cell block. This proved to be extremely useful in assessing the definitive diagnosis and management of both women. We briefly discuss the approach to a malignant pleural effusion and the aid of this not-so-new technique.

Young investigator challenge: MicroRNA-21/MicroRNA-126 profiling as a novel tool for the diagnosis of malignant mesothelioma in pleural effusion cytology.

BACKGROUND: In pleural effusion cytology, the distinction of malignant mesothelioma (MM) from reactive mesothelial cells (RMCs) may be challenging, even with the aid of immunocytochemistry or fluorescence in situ hybridization. It has been demonstrated that several microRNAs (miRNAs) are useful for this purpose in cell lines and histologic samples. In the current study, the authors evaluated the utility of an miRNA-based classifier as a complement to cytology. METHODS: Quantitative reverse transcriptase-polymerase chain reaction analysis of 15 miRNAs was performed in mesothelial (MET-5A) and MM (H28 and H2052) cell lines. Significant miRNAs were validated in 51 MM and 40 nonneoplastic pleural histologic samples and then were tested in 29 MM and 24 RMC cytologic specimens. The performance of individual and combined miRNAs was assessed for their ability to differentiate between MM and RMCs. RESULTS: MiRNA-19a (MiR-19a), miR-19b, miR-21, miR-25, and miR-126 were differentially expressed in cell lines and histologic samples. MiR-126 was down-regulated in MM surgical specimens compared with nonneoplastic specimens, whereas all of the other miRNAs were overexpressed. In cytologic specimens, all miRNAs except miR-25 were confirmed, exhibiting a sensitivity or a specificity higher than the threshold of 0.80, as recommended by the International Mesothelioma Interest Group. The best classifier resulted from the combination of miR-21 and miR-126, which achieved 0.86 sensitivity and 0.87 specificity. CONCLUSIONS: Subject to validation of the current results in further larger studies, miR-21 and miR-126 profiling could be an effective and reliable tool for the diagnosis of MM in pleural effusions complementary to cytology evaluation.

[Value of Cell Block in the Diagnosis of Malignant Pleural Effusion].

Malignant pleural effusion (MPE ) is due tumor which arises from the mesothelium or metastases from tumor origniating other sites. In large, for undiagnosed unilateral pleural effusions, the most frequent and important diagnosis to be established or excluded is malignancy. Cell block is prepared from residual fluid which is centrifuged or is naturally sedimenting to obtain clots at the bottom of the container. The cell block technique is simple, relatively non-invasive, reproducible and has a high yield for malignant plerual effusion. It plays an important role in the diagnosis, guiding the treatment of maligant pleural effusion. Herein, we summarize the technologys which make the cell block, the differential diagnostic value when multiple sections of the cell block are processed for immunhistochemistry, advantages in the diagnosis of malignant pleural effusion, the clinical value of gene screening in cell block. The aim of this article is to discuss the value of cell block in diagnosis of maligant pleural effusion.

Detection of malignant mesothelioma using nuclear structure of mesothelial cells in effusion cytology specimens.

Mesothelioma is a form of cancer generally caused from previous exposure to asbestos. Although it was considered a rare neoplasm in the past, its incidence is increasing worldwide due to extensive use of asbestos. In the current practice of medicine, the gold standard for diagnosing mesothelioma is through a pleural biopsy with subsequent histologic examination of the tissue. The diagnostic tissue should demonstrate the invasion by the tumor and is obtained through thoracoscopy or open thoracotomy, both being highly invasive surgical operations. On the other hand, thoracocentesis, which is removal of effusion fluid from the pleural space, is a far less invasive procedure that can provide material for cytological examination. In this study, we aim at detecting and classifying malignant mesothelioma based on the nuclear chromatin distribution from digital images of mesothelial cells in effusion cytology specimens. Accordingly, a computerized method is developed to determine whether a set of nuclei belonging to a patient is benign or malignant. The quantification of chromatin distribution is performed by using the optimal transport-based linear embedding for segmented nuclei in combination with the modified Fisher discriminant analysis. Classification is then performed through a k-nearest neighborhood approach and a basic voting strategy. Our experiments on 34 different human cases result in 100% accurate predictions computed with blind cross validation. Experimental comparisons also show that the new method can significantly outperform standard numerical feature-type methods in terms of agreement with the clinical diagnosis gold standard. According to our results, we conclude that nuclear structure of mesothelial cells alone may contain enough information to separate malignant mesothelioma from benign mesothelial proliferations.

Utility of nuclear morphometry in effusion cytology.

BACKGROUND: The cytological analysis of serous effusions is a common investigation and yields important diagnostic information. However, the distinction of reactive mesothelial cells from malignant cells can sometimes be difficult for the cytopathologist. Hence cost-effective ancillary methods are essential to enhance the accuracy of cytological diagnosis. The aim of this study was to examine the utility of nuclear morphometry in differentiating reactive mesothelial cells from malignant cells in effusion smears. MATERIALS AND METHODS: Sixty effusion smears consisting of 30 effusions cytologically classified as malignant (adenocarcinomas) and 30 benign effusions showing reactive mesothelial cells were included in the study. ImageJ was used to measure the nuclear area, perimeter, maximal feret diameter, minimal feret diameter and the circularity. A total of ten representative cells were studied in each case. RESULTS: Significant differences were found between benign and malignant effusions for the nuclear area, perimeter, maximal feret diameter and minimal feret diameter. No significant difference was found for circularity, a shape descriptor. Receiver operating characteristic (ROC) curve analysis revealed that nuclear area, perimeter, maximal feret diameter, and minimal feret diameter are helpful in discriminating benign and malignant effusions. CONCLUSIONS: Computerised nuclear morphometry is a helpful ancillary technique to distinguish benign and malignant effusions. ImageJ is an excellent cost effective tool with potential diagnostic utility in effusion cytology.

Outcomes of an Australian testing programme for epidermal growth factor receptor mutations in non-small cell lung cancer.

BACKGROUND: Molecular characterisation of non-squamous non-small-cell lung cancer (NSCLC) is required to direct optimal treatment. Treatment of NSCLC with inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase (EGFR-TKI) should be guided by the presence of activating mutations of the EGFR gene. AIM: To gain insight into the rate of testing, the range of tissues samples, test utility and outcome when cost of testing as a barrier to access is removed in the Australian setting. METHODS: In October 2010, a sponsored programme was commenced to gather data on EGFR gene mutation testing in Australia. Partnering laboratories were funded for provision of de-identified results. For participating patients, the programme supported the test charge. Mutation testing was performed using Sanger sequencing of exons 18-21 of the EGFR. RESULTS: Samples 2013 were submitted from 2012 patients. Full sequencing was achieved in 1717 (85%). Failure of full sequencing was more likely in samples derived from fine needle aspiration(FNA) biopsy than tissue biopsy or pleural/pericardial fluid cell blocks OR 3.1 (95% CI 1.9-5.2). There were 359 mutations seen in 337 patients. 14.5% of cases had a classical mutation conferring sensitivity to EGFR-TKI. In addition there was a range of less common mutations - some predicting responses and others of uncertain significance. 1.4% of cases had mutations associated with non-responsiveness to EGFR-TKI. CONCLUSIONS: EGFR gene mutation testing is feasible on local and interstate lung cancer samples. The rate of valid test outcomes is high, but FNA samples are associated with more frequent test failure.

Differentiation and immune regulation of IL-9-producing CD4+ T cells in malignant pleural effusion.

RATIONALE: IL-9-producing CD4(+) T cells (Th9 cells) have been reported to be involved in inflammation and immune diseases. However, the involvement of Th9 cells in malignancy has not been investigated. OBJECTIVES: To elucidate the mechanism by which Th9 cells differentiate in malignant pleural effusion (MPE) and to explore the immune regulation of Th9 cells on lung cancer cells. METHODS: Distribution of Th9 cells in relation to Th17 and Th1 cells in both MPE and blood were determined. The effects and mechanisms of proinflammatory cytokines and regulatory T cells on differentiation of Th9 cells in vitro were explored. The impacts and signal transductions of IL-9, IL-17, and IFN-γ on lung cancer cell lines were also investigated. MEASUREMENTS AND MAIN RESULTS: The numbers of Th9, Th17, and Th1 cells were all increased in MPE when compared with blood. The increase in Th9 cells in MPE was due to the promotion by cytokines and regulatory T cells. By activating STAT3 signaling, both IL-9 and IL-17 substantially promoted the proliferation and migratory activity of lung cancer cells, whereas IFN-γ, which activated STAT1 signaling, was noted to suppress lung cancer cell proliferation and migration. IFN-γ could induce lung cancer cell apoptosis. Moreover, IL-9 and IFN-γ, but not IL-17, could strongly facilitate intercellular adhesion of lung cancer cells to pleural mesothelial cell monolayers. CONCLUSIONS: Our data revealed that Th9 cells were increased in MPE and that Th9 cells exerted an important immune regulation on lung cancer cells in human tumor environment.

Generation and differentiation of IL-17-producing CD4+ T cells in malignant pleural effusion.

IL-17-producing CD4(+) T (Th17) cells have been found to be increased in some human cancers; however, the possible implication of Th17 cells in regulating antitumor responses in malignant pleural effusion (MPE) remains to be elucidated. In the current study, distribution and phenotypic features of Th17 cells in both MPE and peripheral blood from patients with lung cancer were determined by flow cytometry or double immunofluorescence staining. The impacts of cytokines on Th17 cell generation and differentiation were explored. The chemoattractant activity of chemokines CCL20 and CCL22 for Th17 cells in vitro was also observed. It was found that the increased Th17 cells could be found in MPE compared with blood. The in vitro experiments showed that IL-1ß, IL-6, IL-23, or their various combinations could promote Th17 cell generation and differentiation from naive CD4(+) T cells. MPE was chemotactic for Th17 cells, and this activity was partly blocked by anti-CCL20 and/or CCL22 Abs. Our data also showed that the accumulation of Th17 cells in MPE predicted improved patient survival. It could be concluded that the overrepresentation of Th17 cells in MPE might be due to Th17 cell differentiation and expansion stimulated by pleural proinflammatory cytokines and to recruitment of Th17 cells from peripheral blood induced by pleural chemokines CCL20 and CCL22. Furthermore, the accumulation of Th17 cells in MPE predicted improved patient survival. These data provide the basis for developing immune-boosting strategies based on ridding the cancer patient of this cell population.

Establishment of an HS23 stromal cell-dependent myeloma cell line: fibronectin and IL-6 are critical.

A multiple myeloma (MM) cell line, MSG1, which depends on HS23 stromal cells for its survival, was established from the pleural effusion of a patient with MM who expressed the M-protein of IgA-λ in his serum. During the first 2 months of culture, the myeloma cells survived on adhesive cells from the pleural effusion and, subsequently, they continued to proliferate on HS23 stromal cells. The phenotype of the established MSG1 cell line was: CD138(+), CD38(++), CD19⁻, CD56⁻, VLA-4(+), VEGFR1(+) and VEGFR2(+). Immunohistochemical staining also demonstrated expression of the IgA and λ chain in MSG1 cytoplasm. Karyotype analysis indicated complex chromosomal abnormalities; hypertriploidy, including the deletion of chromosomes 13 and 17, and c-myc translocation. MSG1 cells continued to proliferate, not only when co-cultured with HS23 cells, but also when cultured only on fibronectin-coated plates with the supernatant of HS23 cells or with control medium containing IL-6. Tocilizumab, an anti-IL-6 receptor antibody, inhibited MSG1 survival under these conditions. Therefore, MSG1 may be a unique myeloma cell line that is useful for the study of cell adhesion-mediated drug resistance induced by adhesion molecules and IL-6 stimulation of myeloma cells.

Host-derived interleukin-5 promotes adenocarcinoma-induced malignant pleural effusion.

RATIONALE: IL-5 is a T helper 2 cytokine important in the trafficking and survival of eosinophils. Because eosinophils can be found in malignant pleural effusions (MPE) from mice and humans, we asked whether IL-5 is involved in the pathogenesis of MPE. OBJECTIVES: To determine the role of IL-5 in MPE formation. METHODS: The effects of IL-5 on experimental MPE induced in C57BL/6 mice by intrapleural injection of syngeneic lung (Lewis lung cancer [LLC]) or colon (MC38) adenocarcinoma cells were determined using wild-type (il5(+/+)) and IL-5-deficient (il5⁻(/)⁻) mice, exogenous administration of recombinant mouse (rm) IL-5, and in vivo antibody-mediated neutralization of endogenous IL-5. The direct effects of rmIL-5 on LLC cell proliferation and gene expression in vitro were determined by substrate reduction and microarray. MEASUREMENTS AND MAIN RESULTS: Eosinophils and IL-5 were present in human and mouse MPE, but the cytokine was not detected in mouse (LLC) or human (A549) lung and mouse colon (MC38) adenocarcinoma-conditioned medium, suggesting production by host cells in MPE. Compared with il5(+/+) mice, il5⁻(/)⁻ mice showed markedly diminished MPE formation in response to both LLC and MC38 cells. Exogenous IL-5 promoted MPE formation in il5(+/+) and il5⁻(/)⁻ mice, whereas anti-IL-5 antibody treatment limited experimental MPE in il5(+/+) mice. Exogenous IL-5 had no effects on LLC cell proliferation and gene expression; however, IL-5 was found to be responsible for recruitment of eosinophils and tumor-promoting myeloid suppressor cells to MPE in vivo. CONCLUSIONS: Host-derived IL-5 promotes experimental MPE and may be involved in the pathogenesis of human MPE.

[Primary effusion lymphoma in two kidney transplant recipients]. / Lymphome des séreuses chez le transplanté rénal: deux cas.

BACKGROUND: Primary effusion lymphoma (PEL) is a highly malignant non-Hodgkin lymphoma associated with Kaposi's sarcoma-associated herpesvirus/human herpesvirus-8 infection (KSHV/HHV-8). It is chiefly seen in HIV patients and is rare in transplant recipients, possibly going unrecognized. OBSERVATION: We describe two male kidney transplant recipients, aged 47 and 51 years, followed for Kaposi's sarcoma in skin, lymph nodes, gastrointestinal (GI) tract and lung whose disease was poorly controlled by sirolimus and chemotherapy. Recurrent pleural effusion contrasted with reduction of cutaneous Kaposi lesions. KHSV viral loads were negative or very low in plasma, were negative or very low, whereas those in pleural effusion were high. Lymphoma cells were discovered only seven to nine months after the initial effusion despite repeated needle biopsies. In one patient, tumour cells were co-infected with Epstein-Barr virus. CONCLUSION: The contrast between a very low KHSV viral load in plasma and a very high viral load pleural effusion should alert physicians and prompt suspicion of PEL in Kaposi's sarcoma patients with recurrent serous effusion. The potential inhibitory role of sirolimus on PEL progression is discussed.

Scoring system for differential diagnosis of malignant mesothelioma and reactive mesothelial cells on cytology specimens.

Cytology is the only useful tool in the detection of malignant mesothelioma (MM) at an early stage. No other methods, such as immunocytochemistry or electron microscopy, are available to distinguish MM from reactive mesothelial cells (RMC). Some objective analysis of cytology specimens is necessary. On the basis of our case review and cytological features described in previous articles, we developed a scoring system for malignant mesothelioma (SSMM) of effusion cytology to distinguish MM cells from RMC. Mesothelioma cells in effusions from 22 patients (20 pleural and 2 peritoneal mesotheliomas) were compared with RMC from 20 patients without obvious tumor cells and 50 effusions containing metastatic carcinoma cells. The SSMM is based on characteristic features of mesothelial and malignant cells. The total achievable score is 10 points: one point each is given for variety of cell size, cyanophilic cytoplasm with villosity/windows/bleb, sheet-like arrangement, mirror-ball-like cell clusters, nuclear atypia, and cannibalism, respectively. Further two points each are ascribed for acidophilic large nucleoli and multinucleated cells with more than eight nuclei. The total score for each of the 22 mesotheliomas was more than 5 points. On the other hand, all RMC and the 50 metastatic carcinoma cases scored less than 3 points, aside from two cases that were treated with OK432. No single characteristic feature was observed to be consistent within the 22 mesotheliomas analyzed. Ancillary use of immunocytochemistry, such as podoplanin (D2-40) and calretinin, supported the diagnostic accuracy of the SSMM. SSMM is useful for the differential diagnosis of MM.

Establishment and characterization of HER2-positive cell line derived from pleural effusion of human breast scirrhous carcinoma.

We have established a cell line derived from a pleural effusion of breast scirrhous carcinoma. This cell line presented immunohistochemically negative for estrogen receptor and slightly positive for progesterone receptor, and positive for c-erbB/HER2/neu. However the original tumor was found to be positive for estrogen receptor and progesterone receptor and slightly positive for c-erbB/HER2/neu expression. Enzyme and electrochemiluminescence immunoassays of tumor markers in the conditioned medium by the cell line revealed they secrete CA15-3, NCC-ST-439 and HER2 protein. We believe the cell line will contribute to the therapeutic study of malignant breast cancer.