Genomic-based ancillary assays offer improved diagnostic yield of effusion cytology with potential challenges in malignant pleural mesothelioma.

BRCA1-associated protein 1 (BAP1) or methylthioadenosine phosphorylase (MTAP) immunohistochemistry (IHC) or 9p21 fluorescence in situ hybridization (FISH) are useful for the diagnosis of malignant pleural mesothelioma (MPM). However, the effect of these assays on the diagnostic yield of effusion cytology in MPM cases with suspicious cytomorphology or the diagnostic challenges in BAP1 or MTAP IHC have not been fully elucidated. Two cohorts of cytologic preparations obtained from pleural effusions were examined: MPM cases in cohort 1 were used to evaluate whether BAP1 or MTAP IHC or 9p21 FISH increase the diagnostic yield of effusion cytology; cohort 2 included cases suspicious for MPM, to which BAP1 or MTAP IHC was applied to clarify the challenges in the clinical assessment of these assays. In cohort 1 (n = 28), either assay elevated 62.5% of class II or III cases to class V. In cohort 2 (n = 139), 21.7% of BAP1 immunocytochemistry in smears and 10.6% of BAP1 IHC and 9.4% of MTAP IHC in cell blocks, were identified to be challenging. The application of genomic-based assays increased the diagnostic yield of effusion cytology in the diagnosis of MPM. However, diagnostic challenges limit the application of these assays in some cases.

Morphological difference between pleural mesothelioma cells in effusion smears with either BAP1 loss or 9p21 homozygous deletion and reactive mesothelial cells without the gene alterations.

We previously characterized the morphological characteristics of malignant pleural mesothelioma (MPM) cells with 9p21 homozygous deletion (HD) using a combination of the virtual microscopy and fluorescence in situ hybridization (FISH). In this study, we investigated whether MPM cells with BRCA1-associated protein 1 (BAP1) loss show the same morphological characteristics identified in MPM cells with 9p21 HD. MPM cells with either BAP1 loss detected by immunocytochemistry (ICC) or 9p21 HD detected by FISH were identified via virtual microscopy prior to ICC or FISH, followed by analysis and quantification of their morphological characteristics. MPM cells with BAP1 loss or 9p21 HD exhibited significantly more frequent cell-in-cell engulfment, multinucleation, and larger multicellular clusters composed of more than 10 cells than reactive mesothelial cells. In conclusion, MPM cells with BAP1 loss or 9p21 HD share similar cytological features, indicating that the same morphological criteria can be used to detect MPM cells harboring such genetic aberrations.

Deletion status of p16 in effusion smear preparation correlates with that of underlying malignant pleural mesothelioma tissue.

Differentiating malignant pleural mesothelioma (MPM) cells morphologically from reactive mesothelial hyperplasia cells is problematic. Homozygous deletion (HD) of p16 (CDKN2A), detected by FISH, is a good marker of malignancy and is useful to differentiate between these cells. However, the correlation between the p16 status of effusion smears and that of the underlying MPM tissues has not been investigated. We used p16-specific FISH to investigate 20 cases of MPM from which both effusion cytologic smears and histologic specimens were available. In five cases, histologic specimens included both an invasive component and surface mesothelial proliferation. In 14 cases (70%), MPM cells in both tissue sections and effusion smears were p16 HD-positive. Conversely, MPM cells in the remaining six tumors (30%) were p16 HD-negative in both tissue sections and effusion smears. For all five MPM cases with surface mesothelial proliferations and invasive components, the effusion smears, surface mesothelial proliferations, and invasive MPM components all displayed p16 deletion. Moreover, the extent to which p16 was deleted in smears highly correlated with the extent of p16 deletion in tissues. The p16 deletion percentages were also similar among smears, tissue surface proliferations, and invasive components. In cases with clinical and radiologic evidence of a diffuse pleural tumor, detection of p16 deletion in cytologic smear samples may permit MPM diagnosis without additional tissue examination. However, the absence of p16 deletion in cytologic smear samples does not preclude MPM.

Case of pure ovarian squamous cell carcinoma resistant to combination chemotherapy with paclitaxel and carboplatin but responsive to monotherapy with weekly irinotecan.

Primary ovarian squamous cell carcinoma is uncommon, and the optimal treatment strategy for this disease has not yet been established. A 71-year-old woman diagnosed with FIGO stage IIb pure ovarian squamous cell carcinoma underwent cytoreductive surgery followed by combination chemotherapy with paclitaxel and carboplatin. After the second treatment course, a recurrent mass grew rapidly, and serum tumor maker levels increased. Monotherapy with weekly irinotecan was then instituted. This second-line chemotherapy was remarkably effective, and the patient subsequently underwent complete interval debulking surgery with a pathological complete response after the third treatment course. Weekly irinotecan is an effective choice for primary ovarian squamous cell carcinoma resistant to combination chemotherapy with paclitaxel and carboplatin.

Guidelines for the cytopathologic diagnosis of epithelioid and mixed-type malignant mesothelioma. Complementary statement from the International Mesothelioma Interest Group, also endorsed by the International Academy of Cytology and the Papanicolaou Society of Cytopathology.

OBJECTIVE: To provide practical guidelines for the cytopathologic diagnosis of malignant mesothelioma. DATA SOURCES: Cytopathologists with an interest in the field involved in the International Mesothelioma Interest Group (IMIG) and the International Academy of Cytology (IAC) contributed to this update. Reference material includes peer-reviewed publications and textbooks. RATIONALE: This article is the result of discussions during and after the IMIG 2012 conference in Boston, followed by thorough discussions during the 2013 IAC meeting in Paris. Additional contributions have been obtained from cytopathologists and scientists who could not attend these meetings, with final discussions and input during the IMIG 2014 conference in Cape Town.

The first identification and retrospective study of Severe Fever with Thrombocytopenia Syndrome in Japan.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is caused by SFTS virus (SFTSV), a novel bunyavirus reported to be endemic in central and northeastern China. This article describes the first identified patient with SFTS and a retrospective study on SFTS in Japan. METHODS: Virologic and pathologic examinations were performed on the patient's samples. Laboratory diagnosis of SFTS was made by isolation/genome amplification and/or the detection of anti-SFTSV immunoglobulin G antibody in sera. Physicians were alerted to the initial diagnosis and asked whether they had previously treated patients with symptoms similar to those of SFTS. RESULTS: A female patient who died in 2012 received a diagnosis of SFTS. Ten additional patients with SFTS were then retrospectively identified. All patients were aged ≥50 years and lived in western Japan. Six cases were fatal. The ratio of males to females was 8:3. SFTSV was isolated from 8 patients. Phylogenetic analyses indicated that all of the Japanese SFTSV isolates formed a genotype independent to those from China. Most patients showed symptoms due to hemorrhage, possibly because of disseminated intravascular coagulation and/or hemophagocytosis. CONCLUSIONS: SFTS has been endemic to Japan, and SFTSV has been circulating naturally within the country.

Association of apixaban therapy and prothrombin time in patients with atrial fibrillation.

BACKGROUND: This study evaluated whether measuring prothrombin time (PT) using particular reagents of interest predicted apixaban-associated anticoagulant activity in Japanese patients with non-valvular atrial fibrillation (NVAF). METHODS AND RESULTS: Two reagents, Shinplastin Excel S and Coagpia PT-N, were used to evaluate PT under apixaban therapy. From June 2013 to February 2014, 103 NVAF patients were recruited, and PT was measured at 3 time points: (1) anytime in the outpatient clinic, (2) at peak, and (3) at trough. In spike-in experiments using pooled citrated normal human platelet-poor plasma with these PT reagents, apixaban prolonged PT values in a concentration-dependent manner. PT values significantly correlated between both reagents (r=0.97) in outpatients. PT values in outpatients taking 5-mg apixaban bid were significantly prolonged and had wide inter- and intraindividual variability. Peak values were significantly higher than trough values, with both values higher than normal. The dose change of apixaban from 5 mg bid to 2.5 mg bid in outpatients halved the degree of PT prolongation in each NVAF patient. CONCLUSIONS: The PT value measured by these specific reagents can predict apixaban-associated anticoagulant activity, although there is significant interpatient variability.

[Latent malignant lymphoma diagnosed at autopsy in a patient with cold agglutinin disease coexisting thrombotic thrombocytopenic purpura].

An 89-year-old woman presented to our hospital with hemolytic anemia and a high titer of cold agglutinins. Red cell agglutination was observed on a blood smear. Agglutination visibly decreased after warming the blood; therefore, the patient was diagnosed with cold agglutinin disease (CAD). Bone marrow aspiration revealed no infiltration of malignant cells. Computed tomography indicated moderate splenomegaly. The patient had neither an infection nor autoimmune disease. Initial steroid therapy was ineffective and hemolysis worsened. Meanwhile, thrombocytopenia, delirium, fever, and schistocytes in the blood were observed. The progression of hemolysis was attributed not only to CAD but also to coexisting thrombotic thrombocytopenic purpura (TTP) because of the decreased ADAMTS 13 level. Autopsy revealed mild paraaortic lymphadenopathy and splenomegaly. Microscopic examination revealed lymphoma cell infiltration in the spleen, liver, bone marrow, and paraaortic lymph nodes. These observations suggested that TTP and CAD were both secondary complications. This case highlights the importance of an autopsy for the detection of latent lymphoma, which can be difficult to diagnose before the patient's death. Careful examination to exclude lymphomas is important in patients with CAD at the time of diagnosis.

[Long-term survival after complete resection of primary cardiac undifferentiated sarcoma;report of a case].

A 34-year-old man experienced lower limb ischemia due to tumor emboli as an initial symptom. Histopathologic examination of the embolic material revealed undifferentiated sarcoma. By echocardiography the original tumor was arising from the posterior mitral leaflet, and therefore, excision of the mitral valve resulted in complete resection of the sarcoma. Mitral valve replacement was performed, and his postoperative course was uneventful. He did not receive postoperative adjuvant therapy. The patient has been undergoing positron emission tomography and electrocardiography on an outpatient basis to check for signs of recurrence. However, no signs of recurrence have been detected for 7 years postoperatively, and the patient leads an active life.

Fluorescence in situ hybridization detection of chromosome 22 monosomy in pleural effusion cytology for the diagnosis of mesothelioma.

BACKGROUND: Malignant pleural mesothelioma (MPM) is characterized by mutations in several genes, including cyclin-dependent kinase-inhibitor 2A/p16 in the 9p21 locus, BRCA1-associated protein 1 (BAP1), and neurofibromatosis type 2 (NF2) in the 22q12 locus. Recent studies indicate that fluorescence in situ hybridization (FISH) detects hemizygous loss of NF2 in tissue specimens of MPM. The authors investigated whether NF2 FISH, either alone or in combination with other diagnostic assays (9p21 FISH, methylthioadenosine phosphorylase [MTAP] immunohistochemistry [IHC], and BAP1 IHC), effectively distinguishes MPM cells from reactive mesothelial cells (RMCs) in cell blocks prepared from pleural effusions. METHODS: FISH assays were used to examine the deletion status of NF2 and 9p21, and IHC was used to determine the expression of MTAP and BAP1 in cell blocks from 54 cases with MPM and 18 cases with RMCs. RESULTS: Hemizygous NF2 loss (chromosome 22 monosomy or hemizygous deletion) showed 51.9% sensitivity (48.1% for chromosome 22 monosomy and 3.7% for hemizygous deletion) and 100% specificity in differentiating MPM cells from RMCs. Combinations of NF2 FISH, 9p21 FISH, and BAP1 IHC assays yielded greater sensitivity (98.1%) than any assay alone (9p21 FISH, 61.1%; MTAP IHC, 52.8%; or BAP1 IHC, 60.4%). The level of hemizygous NF2 loss in cell blocks positively correlated with that in corresponding tissues. Furthermore, to overcome cytologic specimen-specific challenges, FISH combined with cytokeratin AE1/AE3 immunofluorescence was necessary in 25.9% of MPM cases for FISH assessment of predominantly scattered MPM cells. CONCLUSIONS: NF2 FISH alone or in combination with other diagnostic assays effectively differentiates MPM cells from RMCs in cell blocks prepared from pleural effusions.

Immunocytochemistry of CD146 is useful to discriminate between malignant pleural mesothelioma and reactive mesothelium.

Malignant pleural mesothelioma is a refractory tumor with poor prognosis associated with asbestos exposure. Pleural effusion is frequently observed in patients with malignant pleural mesothelioma, and cytological analysis is effective to detect malignant pleural mesothelioma. However, cytological discrimination between malignant pleural mesothelioma and reactive mesothelium is often difficult. Increased expression of CD146, a cell adhesion molecule, has been reported to be closely associated with an advanced stage of malignant melanoma, prostate cancer, and ovarian cancer. In this study, to evaluate the diagnostic utility of CD146 for discrimination between malignant pleural mesothelioma and reactive mesothelium, we examined immunocytochemical expression of CD146 in malignant pleural mesothelioma and reactive mesothelium using two clones of CD146 antibody, OJ79 and EPR3208, on smear specimens of effusion fluids. Immunocytochemical stains were semiquantitatively scored on the basis of immunostaining intensity (0, negative; 1, weak positive; 2, moderate positive; and 3, strong positive). CD146 expression was detected in 15 of 16 malignant pleural mesothelioma with median immunostaining score of 3 by OJ79, and in 19 of 21 malignant pleural mesothelioma with median immunostaining score of 2 by EPR3208. Strong immunoreactivity of CD146 was observed at the apposing surfaces of cell-cell interactions on the plasma membrane of mesothelioma cells. In addition, one OJ79-negative case of malignant pleural mesothelioma was positive for CD146 by EPR3208 and two EPR3208-negative cases of malignant pleural mesothelioma were CD146 positive by OJ79, showing that all 23 malignant pleural mesothelioma cases were positive for CD146 by either OJ79 or EPR3208. On the other hand, CD146 expression was undetectable in all reactive mesothelium cases by OJ79 and EPR3208. The sensitivity of OJ79 and EPR3208 was 94 and 90%, respectively, and the specificity was 100% for both clones. We propose that CD146 is a sensitive and specific immunocytochemical marker enabling differential diagnosis of malignant pleural mesothelioma from reactive mesothelium.

[A recurrent case of lipid-secreting carcinoma of the breast successfully treated with capecitabine].

We herein report a recurrent case of lipid-secreting carcinoma of the breast which was successfully treated with capecitabine. A 50-year-old female underwent a pectoralis-preserving mastectomy for left breast cancer in December 2002. The clinical staging of the disease was T2N1M0 (stage II B) and ER (-), PgR(-), HER2 (1+). Microscopic examinations revealed solid alveolar proliferation in the majority of the tumor cells, which had an abundant foamy cytoplasm. A variable amount of neutral lipid was also identified in the cytoplasm of the tumor cells by Sudan III staining. After the operation, the patient received two courses of systemic chemotherapy using docetaxel (60 mg/m(2)). In March 2004, she was diagnosed to have a recurrence in the thoracic wall. She received radiotherapy (total 50 Gy radiation), but it proved to be ineffective. In June 2004, treatment using capecitabine (2,400 mg/day) was therefore attempted. Two courses of the treatment resulted in a complete response of the tumor. The above patient has since continued to show a complete response with capecitabine for over 3.5 years.

Expression of vascular endothelial growth factor in malignant mesothelioma.

Malignant mesothelioma is the most common primary pleural neoplasm. Angiogenesis is an important component of a variety of pathological processes, including carcinogenesis and tumor metastases. Vascular endothelial growth factor (VEGF) is the most potent known endothelial, cell specific mitogen. The authors assessed the relation between VEGF expression and clinicopathological variables or overall survival, in malignant mesothelioma. We studied 37 patients with malignant pleural mesothelioma and found that 36 out of 37 (97.3%) malignant mesothelioma samples were stained positively for VEGF. An increased expression of VEGF was observed in the epithelioid type compared with the other histological types of malignant mesothelioma, including the biphasic and sarcomatoid types. No statistically significant association was observed between VEGF expression and gender, age, or clinical stage. Furthermore, the expression of VEGF did not impact on the survival of patients with malignant mesothelioma. Although VEGF expression might be important for tumor development and maintenance, it was not identified as a prognostic factor in malignant mesothelioma.

Use of p16 FISH for differential diagnosis of mesothelioma in smear preparations.

Because most of malignant pleural mesothelioma (MPM) patients first present with pleural effusion, detection of mesothelioma cells on effusion smears is critical for early diagnosis. Recently, accumulating evidence indicating that the cytological diagnosis of MPM supported by ancillary techniques is as reliable as that based on histopathology has led to new guidelines for the cytopathologic diagnosis of MPM. Based on the guidelines, a combination of cytomorphological criteria and verification by ancillary techniques is required for the cytologic diagnosis of MPM. Detection of p16 homozygous deletion by fluorescence in situ hybridization (FISH) is the most reliable ancillary technique for differentiating MPM from reactive mesothelial cells (RMC) because of its relatively high sensitivity and extremely high specificity. We showed that the p16 deletion status of MPM cells in pleural effusions reflected that of the underlying invasive MPM tissues, indicating the usefulness of p16 FISH in effusion smear cytology for MPM diagnosis. Thus, for differentiating MPM from RMC, we propose to perform p16 FISH as often as possible. A positive p16 homozygous deletion supports the diagnosis of MPM. However, a negative result does not rule out the possibility of MPM. In such cases, a morphological assessment is critical. Therefore, we analyzed the morphological characteristics of p16 deletion-positive mesothelioma cells using a combination of virtual microscopy and p16 FISH, and identified three morphological characteristics useful for the differentiation, including cell-in-cell engulfment with or without hump formation, multinucleate cells, and larger berry-like cell aggregates. Diagn. Cytopathol. 2016;44:774-780. © 2016 Wiley Periodicals, Inc.

Low homozygous/high heterozygous deletion status by p16 FISH correlates with a better prognostic group than high homozygous deletion status in malignant pleural mesothelioma.

OBJECTIVES: Homozygous deletion (homo-d) of the p16 (CDKN2A) gene, as determined by fluorescence in situ hybridization (FISH), helps differentiate malignant pleural mesothelioma (MPM) from reactive mesothelial hyperplasia (RMH). Heterozygous deletion (hetero-d) has also been identified variably in p16 FISH. This study aimed to investigate the significance of homo-d and hetero-d of p16 in the diagnosis and prognosis of MPM. MATERIALS AND METHODS: p16 FISH was performed in 93 MPMs and 47 RMHs. Real-time polymerase chain reaction (PCR) and methylation specific PCR (MSP) were also performed for cases in which DNA was available. Overall survival (OS) was assessed via the Kaplan-Meier method and logrank test. RESULTS: Cutoff values for homo-d and hetero-d were set at 10% and 47%, respectively, based on p16 FISH results in RMH. In MPM, 80/93 (86.0%) were homo-d positive, and 15/93 (16.1%) were hetero-d positive. No RMH was homo/hetero-d positive. In nine cases of MPM with the low homo-d (<30%)/high hetero-d (>47%) pattern, FISH with a shorter probe caused a slight increase (from 20.1% to 26.5%) in the mean percentage of homo-d and a decrease in that of hetero-d (from 59.6% to 55.6%). Four cases in which the low homo-d/high hetero-d pattern was maintained with the shorter probe were further analyzed by real-time PCR, which separated them into a two (n=2) or one allele deletion group (n=2). MSP revealed no promoter methylation in the two cases with one allele deletion. The OS was significantly shorter in homo-d positive cases (n=24) than homo-d negative cases (n=5, p=0.0002) in the 29 MPM cases with follow-up data. Also, low homo-d/high hetero-d cases (n=5) had a significantly better prognosis than high homo-d (≥30%) cases (n=17, p=0.011). CONCLUSIONS: Within p16 homo-d positive MPMs with poorer prognosis, the low homo-d/high hetero-d pattern may belong to a better prognostic subgroup in homo-d positive MPMs.

Clinical significance of the expression of tumor-associated antigen, RCAS1, and its soluble protein in pleural fluid in malignant mesothelioma.

RCAS1 is a type II membrane protein which is also secreted as a soluble protein. RCAS1 may play a role in evading immune surveillance by tumor cells and be responsible for the aggressive behavior of tumors. We examined the usefulness of RCAS1 in the follow-up of malignant mesothelioma. In addition, we examined the usefulness of its soluble protein (sRCAS1) in pleural effusion for the diagnosis of malignant mesothelioma. We studied 38 patients with pleural malignant mesothelioma and examined the correlation between RCAS1 expression, clinicopathologic variables and overall survival. We also determined the pleural fluid sRCAS1 concentration with an enzyme-linked immunosorbent assay (ELISA). We found that 34 out of 38 (89.5%) malignant mesothelioma cells stained for RCAS1. The positive rate was 90.9% in biphasic type, 78.6% in epithelioid type, and 100% in sarcomatoid type. No statistically significant correlation was observed between RCAS1 expression and gender, age, histology or clinical stage. Interestingly, survival of the malignant mesothelioma patients with RCAS1 expression was significantly increased compared with those without (median survival time: 13.0 vs. 4.3 months, p=0.011). Multivariate analysis of prognostic factors, using the Cox proportional hazards model, revealed that RCAS1 expression had a significantly positive effect on survival. sRCAS1 concentrations in pleural fluid in malignant mesothelioma were lower than those in lung cancer (2.18+/-2.20 vs. 46.3+/-129 U/ml; p=0.019). RCAS1 expression is informative for the follow-up of malignant mesothelioma patients. In addition, sRCAS1 in pleural fluid may be useful for the diagnosis of malignant mesothelioma.

Guidelines for cytopathologic diagnosis of epithelioid and mixed type malignant mesothelioma. Complementary statement from the International Mesothelioma Interest Group, also endorsed by the International Academy of Cytology and the Papanicolaou Society of Cytopathology.

To provide practical guidelines for the cytopathologic diagnosis of malignant mesothelioma (MM). Cytopathologists involved in the International Mesothelioma Interest Group (IMIG) and the International Academy of Cytology (IAC), who have an interest in the field contributed to this update. Reference material includes peer-reviewed publications and textbooks. This article is the result of discussions during and after the IMIG 2012 conference in Boston, followed by thorough discussions during the 2013 IAC meeting in Paris. Additional contributions have been obtained from cytopathologists and scientists, who could not attend these meetings, with final discussions and input during the IMIG 2014 conference in cape town. During the previous IMIG biennial meetings, thorough discussions have resulted in published guidelines for the pathologic diagnosis of MM. However, previous recommendations have stated that the diagnosis of MM should be based on histological material only.[12] Accumulating evidence now indicates that the cytological diagnosis of MM supported by ancillary techniques is as reliable as that based on histopathology, although the sensitivity with cytology may be somewhat lower.[345] Recognizing that noninvasive diagnostic modalities benefit both the patient and the health system, future recommendations should include cytology as an accepted method for the diagnosis of this malignancy.[67] The article describes the consensus of opinions of the authors on how cytology together with ancillary testing can be used to establish a reliable diagnosis of MM.

Guidelines for the cytopathologic diagnosis of epithelioid and mixed-type malignant mesothelioma: Complementary Statement from the International Mesothelioma Interest Group, Also Endorsed by the International Academy of Cytology and the Papanicolaou Society of Cytopathology.

OBJECTIVE: To provide practical guidelines for the cytopathologic diagnosis of malignant mesothelioma. DATA SOURCES: Cytopathologists with an interest in the field involved in the International Mesothelioma Interest Group (IMIG) and the International Academy of Cytology (IAC) contributed to this update. Reference material includes peer-reviewed publications and textbooks. RATIONALE: This article is the result of discussions during and after the IMIG 2012 conference in Boston, followed by thorough discussions during the 2013 IAC meeting in Paris. Additional contributions have been obtained from cytopathologists and scientists who could not attend these meetings, with final discussions and input during the IMIG 2014 conference in Cape Town.