LAG3 is an independent prognostic biomarker and potential target for immune checkpoint inhibitors in malignant pleural mesothelioma: a retrospective study.

BACKGROUND: Lymphocyte-activation gene 3 (LAG3) is an immune checkpoint receptor; novel LAG3 immune checkpoint inhibitors (ICIs) exhibit therapeutic activity in melanoma. The role of LAG3and ICIs of LAG3 are unknown in malignant pleural mesothelioma (MPM). This study aimed to uncover the prognostic landscape of LAG3 in multiple cancers and investigate the potential of using LAG3 as an ICIs target in patients with MPM. METHODS: We used The Cancer Genome Atlas (TCGA) cohort for assessing mRNA expression and our cohort for immunohistochemical expression. TCGA cohort were analyzed using the Wilcoxon rank-sum test to compare mRNA expression between normal and tumor tissues in multiple cancers. We used 86 MPM cases from TCGA and 38 MPM cases from our cohort to analyze the expression of LAG3 in tumor-infiltrating lymphocytes. The mean LAG3 mRNA expression was set as the cut-off and samples were classified as positive/negative for immunohistochemical expression. Overall survival (OS) of patients with MPM was determined using the Kaplan-Meier method based on LAG3 mRNA and immunohistochemical expression. OS analysis was performed using the multivariate Cox proportional hazards model. The correlation of LAG3 expression and mRNA expression of tumor immune infiltration cells (TIICs) gene markers were estimated using Spearman correlation. To identify factors affecting the correlation of LAG3 mRNA expression, a multivariate linear regression model was performed. RESULTS: LAG3 mRNA was associated with prognosis in multiple cancers. Elevated LAG3 mRNA expression was correlated with a better prognosis in MPM. LAG3 expression was detected immunohistochemically in the membrane of infiltrating lymphocytes in MPM. LAG3 immunohistochemical expression was correlated with a better prognosis in MPM. The multivariate Cox proportional hazards model revealed that elevated LAG3 immunohistochemical expression indicated a better prognosis. In addition, LAG3 mRNA expression was correlated with the expression of various gene markers of TIICs, the most relevant to programmed cell death 1 (PD-1) with the multivariate linear regression model in MPM. CONCLUSIONS: LAG3 expression was correlated with prognosis in multiple cancers, particularly MPM; LAG3 is an independent prognostic biomarker of MPM. LAG3 regulates cancer immunity and is a potential target for ICIs therapy. PD-1 and LAG3 inhibitors may contribute to a better prognosis in MPM. TRIAL REGISTRATION: This study was registered with UMIN000049240 (registration day: August 19, 2022) and approved by the Institutional Review Board (approval date: August 22, 2022; approval number: 2022-0048) at Tokyo Women's Medical University.

Expression of T-cell immunoreceptor with immunoglobulin and tyrosine-based inhibitory motif domains (TIGIT) in anaplastic thyroid carcinoma.

BACKGROUND: Immune checkpoint proteins have not been fully examined in follicular cell-derived thyroid carcinoma and medullary thyroid carcinoma (MTC). Anaplastic thyroid carcinoma (ATC) is one of the most aggressive carcinomas. Even multimodal treatment does not result in favorable clinical outcomes for patients with ATC. Anti-tumor immunity has therefore been highlighted as having therapeutic promise for ATC. METHODS: We examined a novel immune checkpoint receptor, T-cell immunoreceptor with immunoglobulin and tyrosine-based inhibitory motif domains (TIGIT), in variable thyroid lesions: adenomatous goiter, follicular adenoma, and thyroid carcinoma (TC) using immunohistochemistry (IHC). RESULTS: Our IHC results showed that TIGIT expression was detected in cancer cells of MTC and high-grade TC: poorly differentiated thyroid carcinoma (PDTC) and ATC. Neoplastic cells were positive for TIGIT in four of five MTCs (80.0%), 17 of 31 ATCs (54.8%) and in 3 of 12 PDTCs (25.0%). TIGIT was not detected in any adenomatous goiters, thyroid benign tumors, or differentiated thyroid carcinoma (DTCs). Intriguingly, ATC cells showing pleomorphic/giant cell features were positive for TIGIT, while ATC cells with other cell morphologies lacked the immunoreactivity. Intra-tumoral immune cell was inclined to be enriched in TIGI-positive ATC. Although coexisting papillary thyroid carcinoma (PTC) components demonstrated high-grade microscopic features, neither the PTC nor follicular thyroid carcinoma (FTC) components expressed TIGT in any composite ATCs. CONCLUSION: TIGIT was immunohistochemically found in MTC with high frequency and partially in high-grade TC. TIGIT expression in cancer cells may be beneficial for a potential utility in MTC and a subset of high-grade TC, especially ATC therapy.

Challenges and limitation of MTAP immunohistochemistry in diagnosing desmoplastic mesothelioma/sarcomatoid pleural mesothelioma with desmoplastic features.

AIM: Genomic-based ancillary assays including immunohistochemistry (IHC) for BRCA-1 associated protein-1 (BAP1) and methylthioadenosine phosphorylase (MTAP), and fluorescence in situ hybridization (FISH) for CDKN2A are effective for differentiating pleural mesothelioma (PM) from reactive mesothelial proliferations. We previously reported a combination of MTAP and BAP1 IHC effectively distinguishes sarcomatoid PM from fibrous pleuritis (FP). Nevertheless, cases of sarcomatoid PM with desmoplastic features (desmoPM) are encountered where the IHC assessment is unclear. METHODS AND RESULTS: We evaluated assessment of MTAP IHC, BAP1 IHC, and CDKN2A FISH in 20 desmoPM compared to 24 FP. MTAP and BAP1 IHC could not be assessed in 11 (55 %) and 10 (50 %) cases, respectively, due to loss or faint immunoreactivity of internal positive control cells, while CDKN2A FISH could be evaluated in all cases. The sensitivities for MTAP loss, BAP1 loss, and CDKN2A homozygous deletion in desmoPM were 40 %, 10 %, and 100 %. A combination of MTAP loss and BAP1 loss yielded 45 % of sensitivity. CONCLUSIONS: MTAP IHC is a useful surrogate diagnostic marker in differentiating ordinary sarcomatoid PM from FP, but its effectiveness is limited in desmoPM. CDKN2A FISH is the most effective diagnostic assays with 100 % sensitivity and specificity in discriminating desmoPM from FP in the facilities where the FISH assay is available.

Malignant peritoneal mesothelioma: prognostic significance of clinical and pathologic parameters and validation of a nuclear-grading system in a multi-institutional series of 225 cases.

Malignant peritoneal mesothelioma historically carried a grim prognosis, but outcomes have improved substantially in recent decades. The prognostic significance of clinical, morphologic, and immunophenotypic features remains ill-defined. This multi-institutional cohort comprises 225 malignant peritoneal mesotheliomas, which were assessed for 21 clinical, morphologic, and immunohistochemical parameters. For epithelioid mesotheliomas, combining nuclear pleomorphism and mitotic index yielded a composite nuclear grade, using a previously standardized grading system. Correlation of clinical, morphologic, and immunohistochemical parameters with overall and disease-free survival was examined by univariate and multivariate analyses. On univariate analysis, longer overall survival was significantly associated with diagnosis after 2000 (P = 0.0001), age <60 years (P = 0.0001), ECOG performance status 0 or 1 (P = 0.01), absence of radiographic lymph-node metastasis (P = 0.04), cytoreduction surgery (P < 0.0001), hyperthermic intraperitoneal chemotherapy (P = 0.0001), peritoneal carcinomatosis index <27 (P = 0.01), absence of necrosis (P = 0.007), and epithelioid histotype (P < 0.0001). Among epithelioid malignant mesotheliomas only, longer overall survival was further associated with female sex (P = 0.03), tubulopapillary architecture (P = 0.005), low nuclear pleomorphism (P < 0.0001), low mitotic index (P = 0.0007), and low composite nuclear grade (P < 0.0001). On multivariate analyses, the low composite nuclear grade was independently associated with longer overall and disease-free survival (P < 0.0001). Our data further clarify the interactions of clinical and pathologic features in peritoneal mesothelioma prognosis and validate the prognostic significance of a standardized nuclear-grading system in epithelioid malignant mesothelioma of the peritoneum.

Membranous HEG1 expression is a useful marker in the differential diagnosis of epithelioid and biphasic malignant mesothelioma versus carcinomas.

Sialylated HEG1 has been reported as a highly specific and sensitive mesothelioma marker but a comprehensive evaluation of its expression in carcinomas in different organs, various sarcomas and reactive mesothelial proliferations has not been reported. The aim of this study was to evaluate the clinical applicability of HEG1 as a marker in the diagnosis of mesothelioma. HEG1 immunoreactivity was evaluated in whole sections of 122 mesotheliomas, 75 pulmonary carcinomas, 55 other carcinomas, 16 mesenchymal tumors, and 24 reactive mesothelial proliferations and in tissue microarrays containing 70 epithelioid (EM), 36 biphasic (BM), and 2 sarcomatoid mesotheliomas (SM). In whole sections and tissue microarrays, respectively, membranous HEG1 was expressed in 93.0% and 85.5% of EM, 81.3% and 69.4% of BM, 0% and 0% of SM. HEG1 was not expressed in pulmonary adenocarcinomas. HEG1 was expressed as cytoplasmic immunoreactivity in pulmonary squamous cell carcinomas (21.7%). Membranous HEG1 staining was seen in ovarian carcinomas (66.7%), thyroid carcinomas (100%), reactive conditions (16.7%), and mesenchymal tumors (18.8%). The sensitivity of membranous HEG1 expression to distinguish EM/BM from all carcinomas was 88.8%. The specificity for the differential diagnosis between EM/BM and all carcinomas and pulmonary carcinomas was 92.3% and 98.7%, respectively.

Combination of a p53-activating CP-31398 and an MDM2 or a FAK inhibitor produces growth suppressive effects in mesothelioma with wild-type p53 genotype.

A majority of mesothelioma had the wild-type p53 genotype but was defective of p53 functions primarily due to a genetic defect in INK4A/ARF region. We examined a growth suppressive activity of CP-31398 which was developed to restore the p53 functions irrespective of the genotype in mesothelioma with wild-type or mutated p53. CP-31398 up-regulated p53 levels in cells with wild-type p53 genotype but induced cell growth suppression in a p53-independent manner. In contrasts, nutlin-3a, an MDM2 inhibitor, increased p53 and p21 levels in mesothelioma with the wild-type p53 genotype and produced growth suppressive effects. We investigated a combinatory effect of CP-31398 and nutlin-2a and found the combination produced synergistic growth inhibition in mesothelioma with the wild-type p53 but not with mutated p53. Western blot analysis showed that the combination increased p53 and the phosphorylation levels greater than treatments with the single agent, augmented cleavages of PARP and caspase-3, and decreased phosphorylated FAK levels. Combination of CP-31398 and defactinib, a FAK inhibitor, also achieved synergistic inhibitory effects and increased p53 with FAK dephosphorylation levels greater than the single treatment. These data indicated that a p53-activating CP-31398 achieved growth inhibitory effects in combination with a MDM2 or a FAK inhibitor and suggested a possible reciprocal pathway between p53 elevation and FAK inactivation.

MTAP immunohistochemistry is an accurate and reproducible surrogate for CDKN2A fluorescence in situ hybridization in diagnosis of malignant pleural mesothelioma.

Ancillary studies facilitate accurate diagnosis of morphologically challenging mesothelial proliferations. The current diagnostic algorithm proceeds from BAP1 immunohistochemistry to CDKN2A fluorescence in situ hybridization. While MTAP immunohistochemistry has recently shown promise as a surrogate for CDKN2A fluorescence in situ hybridization, it has been examined in only a few single-institution studies. Furthermore, there are no published reports on interobserver agreement or interlaboratory reproducibility for MTAP immunohistochemistry. We performed MTAP immunohistochemistry on 20 benign mesothelial lesions and 99 malignant mesotheliomas from five mesothelioma centers in four countries, and each MTAP stain was independently interpreted by four pathologists. CDKN2A fluorescence in situ hybridization data were available for a subset of cases, and a subset of cases was subjected in MTAP immunohistochemistry in multiple laboratories to assess interlaboratory reproducibility. Interobserver agreement in MTAP immunostain interpretation was excellent for all mesothelial lesions (kappa: 0.85) and for malignant mesothelioma cases only (kappa: 0.82). Interlaboratory reproducibility was also excellent (kappa values for paired protocols: 0.77-0.89). MTAP loss by immunohistochemistry was 78% sensitive and 96% specific for CDKN2A homozygous deletion. MTAP immunohistochemistry is a reliable surrogate for CDKN2A fluorescence in situ hybridization in diagnosis of malignant mesothelioma. Interobserver agreement is excellent for interpretation of MTAP staining, and protocols performed in different laboratories yield concordant MTAP staining results. Rare cases with immunohistochemical MTAP loss may retain normal CDKN2A copy number, and the MTAP staining results should be correlated with clinicopathologic findings and other ancillary studies.

Adenomatoid Tumor: A Review of Pathology With Focus on Unusual Presentations and Sites, Histogenesis, Differential Diagnosis, and Molecular and Clinical Aspects With a Historic Overview of Its Description.

Adenomatoid tumors have been described almost a century ago, and their nature has been the subject of debate for decades. They are tumors of mesothelial origin usually involving the uterus, the Fallopian tubes, and the paratesticular region. Adenomatoid tumors of the adrenal gland, the liver, the extragenital peritoneum, the pleura, and the mediastinum have been rarely reported. They are usually small incidental findings, but large, multicystic and papillary tumors, as well as multiple tumors have been described. Their pathogenesis is related to immunosuppression and to TRAF7 mutations. Despite being benign tumors, there are several macroscopic or clinical aspects that could raise diagnostic difficulties. The aim of this review was to describe the microscopic and macroscopic aspects of adenomatoid tumor with a special focus on its differential diagnosis and pathogenesis and the possible link of adenomatoid tumor with other mesothelial lesions, such as the well-differentiated papillary mesothelioma and the benign multicystic mesothelioma, also known as multilocular peritoneal cysts.

PD-L1, FGFR1, PIK3CA, PTEN, and p16 expression in pulmonary emphysema and chronic obstructive pulmonary disease with resected lung squamous cell carcinoma.

BACKGROUND: Emphysema and chronic obstructive pulmonary disease (COPD) are well known independent risk factors for lung cancer. However, the developmental mechanisms between emphysema/COPD and lung cancer remain unknown. The purpose of this study was to evaluate PD-L1, FGFR1, PIK3CA, PTEN, and p16 expression in squamous cell carcinoma (SCC) associated with emphysema/COPD. METHODS: A total of 59 patients with squamous cell lung carcinoma (SCC) resected between 2008 and 2012 were retrospectively reviewed. Emphysema was assessed according to the Goddard score. Total severity was divided into none-mild (0-7), moderate (8-15), and severe (≥ 16). Local severity around the existing tumor was divided into no emphysema (0) and presence of emphysema (1-4). COPD severity was based on the Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria. PD-L1, FGFR1, PIK3CA, PTEN, and p16 expression were evaluated by immunohistochemistry (IHC). Expression level was classified as tumor cells (TC) 3 (≥ 50%), TC2 (5-49%), TC1 (1-4%), or TC0 (< 1%), and as tumor-infiltrating immune cells (IC) 3 (≥ 50%), IC2 (5-49%), IC1 (1-4%), or IC0 (< 1%) for PD-L1. Expression level was compared between none-mild/moderate-severe total emphysema, no/presence of local emphysema, no COPD/COPD, and GOLD 1/GOLD 2, 3. RESULTS: PD-L1 expression was significantly correlated with severity of emphysema in TC0, 1, 2 vs. TC3 (P = 0.012). PD-L1 was significantly higher inversely in none-mild emphysema compared to moderate-severe (95% CI, 0.061-5.852, P = 0.045). There were no other significant associations between PD-L1, FGFR1, PIK3CA, PTEN, and p16 expression and total/local severity of emphysema or presence of COPD/GOLD stage. CONCLUSIONS: PD-L1 expression in SCC was correlated with severity of emphysema in TC0, 1, 2 vs. TC3 and more frequent in none-mild emphysema than moderate-severe emphysema.

Nuclear grade and necrosis predict prognosis in malignant epithelioid pleural mesothelioma: a multi-institutional study.

A recently described nuclear grading system predicted survival in patients with epithelioid malignant pleural mesothelioma. The current study was undertaken to validate the grading system and to identify additional prognostic factors. We analyzed cases of epithelioid malignant pleural mesothelioma from 17 institutions across the globe from 1998 to 2014. Nuclear grade was computed combining nuclear atypia and mitotic count into a grade of I-III using the published system. Nuclear grade was assessed by one pathologist for three institutions, the remaining were scored independently. The presence or absence of necrosis and predominant growth pattern were also evaluated. Two additional scoring systems were evaluated, one combining nuclear grade and necrosis and the other mitotic count and necrosis. Median overall survival was the primary endpoint. A total of 776 cases were identified including 301 (39%) nuclear grade I tumors, 354 (45%) grade II tumors and 121 (16%) grade III tumors. The overall survival was 16 months, and correlated independently with age (P=0.006), sex (0.015), necrosis (0.030), mitotic count (0.001), nuclear atypia (0.009), nuclear grade (<0.0001), and mitosis and necrosis score (<0.0001). The addition of necrosis to nuclear grade further stratified overall survival, allowing classification of epithelioid malignant pleural mesothelioma into four distinct prognostic groups: nuclear grade I tumors without necrosis (29 months), nuclear grade I tumors with necrosis and grade II tumors without necrosis (16 months), nuclear grade II tumors with necrosis (10 months) and nuclear grade III tumors (8 months). The mitosis-necrosis score stratified patients by survival, but not as well as the combination of necrosis and nuclear grade. This study confirms that nuclear grade predicts survival in epithelioid malignant pleural mesothelioma, identifies necrosis as factor that further stratifies overall survival, and validates the grading system across multiple institutions and among both biopsy and resection specimens. An alternative scoring system, the mitosis-necrosis score is also proposed.

SMARCB1/INI1/BAF47- deficient pleural malignant mesothelioma with rhabdoid features.

Malignant mesothelioma (MM) with rhabdoid features is an MM variant. Fifteen cases have been reported previously, all of which were combined with other types of MM. Herein, we report an autopsy case of pleural MM with monomorphic rhabdoid features. The patient was a 62-year-old male without a history of asbestos exposure. An autopsy revealed a soft, granular tumor that replaced the entire left pleura and had invaded to the diaphragm and lower lobe of the lung. The tumor cells, which had eosinophilic plump cytoplasm and eccentric nuclei, were loosely cohesive. Immunohistochemistry showed that the cells were diffusely positive for calretinin, D2-40, vimentin, CAM5.2, and AE1/AE3; and negative for WT-1, TTF-1, CK7, CEA, desmin, CD34, BCL-2, S100 protein, and p40. Neither homozygous deletion of p16 nor BAP-1 protein loss was observed. Loss of INI1/BAF47 protein, an indicator of malignant rhabdoid tumor, was observed. Therefore, MM with rhabdoid features was confirmed.

Midkine is a potential novel marker for malignant mesothelioma with different prognostic and diagnostic values from mesothelin.

BACKGROUND: We evaluated possible diagnostic and prognostic values of serum midkine in malignant pleural mesothelioma in comparison with those of serum mesothelin, a well-established diagnostic biomarker. METHODS: Serum mesothelin and midkine levels were determined with an enzyme-linked immunosorbent assay. We examined specimens from 95 Turkish cases with malignant pleural mesothelioma, 56 metastatic cancers to pleura, 27 other types of benign pleural diseases and 20 benign asbestos pleurisy. The cut-off values were 1.5 nmol/L for mesothelin and 421 pg/mL for midkine. RESULTS: Sensitivity and specificity of mesothelin were 51.6 and 71.4%, 51.6 and 85.2%, and 51.6 and 85% for differentiating mesothelioma from metastatic cancers to pleura, other benign pleural diseases and benign asbestos pleurisy, respectively. Sensitivity and specificity of midkine were 61.1 and 41.1%, 61.1 and 48.1%, and 61.1 and 75% to distinguish mesothelioma from metastatic cancers to pleura, other benign pleural diseases and benign asbestos pleurisy, respectively. Combination of both biomarkers did not improve the differential diagnostic efficacy. Mesothelin levels were elevated in the epitheloid type and in the advanced cases, but were not related to the prognosis. In contrast, elevated baseline levels of midkine were independently associated with a poor prognosis of mesothelioma patients after adjusting for the stage, the histological subtypes and treatment schedules (HR = 1.84; 95% CI: 1.09-3.09) (p = 0.022). CONCLUSIONS: Serum mesothelin showed moderate sensitivity and high specificity to differentiate malignant pleural mesothelioma from metastatic malignancy to pleura and from benign pleural diseases. In contrast, midkine was a useful marker for predicting prognosis of mesothelioma patients.

Metformin produces growth inhibitory effects in combination with nutlin-3a on malignant mesothelioma through a cross-talk between mTOR and p53 pathways.

BACKGROUND: Mesothelioma is resistant to conventional treatments and is often defective in p53 pathways. We then examined anti-tumor effects of metformin, an agent for type 2 diabetes, and combinatory effects of metformin and nutlin-3a, an inhibitor for ubiquitin-mediated p53 degradation, on human mesothelioma. METHODS: We examined the effects with a colorimetric assay and cell cycle analyses, and investigated molecular events in cells treated with metformin and/or nutlin-3a with Western blot analyses. An involvement of p53 was tested with siRNA for p53. RESULTS: Metformin suppressed cell growth of 9 kinds of mesothelioma including immortalized cells of mesothelium origin irrespective of the p53 functional status, whereas susceptibility to nutlin-3a was partly dependent on the p53 genotype. We investigated combinatory effects of metformin and nutlin-3a on, nutlin-3a sensitive MSTO-211H and NCI-H28 cells and insensitive EHMES-10 cells, all of which had the wild-type p53 gene. Knockdown of p53 expression with the siRNA demonstrated that susceptibility of MSTO-211H and NCI-H28 cells to nutlin-3a was p53-dependent, whereas that of EHMES-10 cells was not. Nevertheless, all the cells treated with both agents produced additive or synergistic growth inhibitory effects. Cell cycle analyses also showed that the combination increased sub-G1 fractions greater than metformin or nutlin-3a alone in MSTO-211H and EHMES-10 cells. Western blot analyses showed that metformin inhibited downstream pathways of the mammalian target of rapamycin (mTOR) but did not activate the p53 pathways, whereas nutlin-3a phosphorylated p53 and suppressed mTOR pathways. Cleaved caspase-3 and conversion of LC3A/B were also detected but it was dependent on cells and treatments. The combination of both agents in MSTO-211H cells rather suppressed the p53 pathways that were activated by nutrin-3a treatments, whereas the combination rather augmented the p53 actions in NCI-H28 and EHMES-10 cells. CONCLUSION: These data collectively indicated a possible interactions between mTOR and p53 pathways, and the combinatory effects were attributable to differential mechanisms induced by a cross-talk between the pathways.

Cytotoxicity of replication-competent adenoviruses powered by an exogenous regulatory region is not linearly correlated with the viral infectivity/gene expression or with the E1A-activating ability but is associated with the p53 genotypes.

BACKGROUND: Replication-competent adenoviruses (Ad) produced cytotoxic effects on infected tumors and have been examined for the clinical applicability. A biomarkers to predict the cytotoxicity is valuable in a clinical setting. METHODS: We constructed type 5 Ad (Ad5) of which the expression of E1A gene was activated by a 5' regulatory sequences of survivin, midkine or cyclooxygenase-2, which were highly expressed in human tumors. We also produced the same replication-competent Ad of which the fiber-knob region was replaced by that of Ad35 (AdF35). The cytotoxicity was examined by a colorimetric assay with human tumor cell lines, 4 kinds of pancreatic, 9 esophageal carcinoma and 5 mesothelioma. Ad infectivity and Ad-mediated gene expression were examined with replication-incompetent Ad5 and AdF35 which expressed the green fluorescence protein gene. Expression of cellular receptors for Ad5 and AdF35 was also examined with flow cytometry. A transcriptional activity of the regulatory sequences was investigated with a luciferase assay in the tumor cells. We then investigated a possible correlation between Ad-mediated cytotoxicity and the infectivity/gene expression, the transcriptional activity or the p53 genotype. RESULTS: We found that the cytotoxicity was greater with AdF35 than with Ad5 vectors, but was not correlated with the Ad infectivity/gene expression irrespective of the fiber-knob region or the E1A-activating transcriptional activity. In contrast, replication-competent Ad produced greater cytotoxicity in p53 mutated than in wild-type esophageal carcinoma cells, suggesting a possible association between the cytotoxicity and the p53 genotype. CONCLUSIONS: Sensitivity to Ad-mediated cytotoxic activity was linked with the p53 genotype but was not lineally correlated with the infectivity/gene expression or the E1A expression.

An image cytometric technique is a concise method to detect adenoviruses and host cell proteins and to monitor the infection and cellular responses induced.

BACKGROUND: Genetically modified adenoviruses (Ad) with preferential replications in tumor cells have been examined for a possible clinical applicability as an anti-cancer agent. A simple method to detect viral and cellular proteins is valuable to monitor the viral infections and to predict the Ad-mediated cytotoxicity. METHODS: We used type 5 Ad in which the expression of E1A gene was activated by 5'-regulatory sequences of genes that were augmented in the expression in human tumors. The Ad were further modified to have the fiber-knob region replaced with that derived from type 35 Ad. We infected human mesothelioma cells with the fiber-replaced Ad, and sequentially examined cytotoxic processes together with an expression level of the viral E1A, hexon, and cellular cleaved caspase-3 with image cytometric and Western blot analyses. RESULTS: The replication-competent Ad produced cytotoxicity on mesothelioma cells. The infected cells expressed E1A and hexon 24 h after the infection and then showed cleavage of caspase-3, all of which were detected with image cytometry and Western blot analysis. Image cytometry furthermore demonstrated that increased Ad doses did not enhance an expression level of E1A and hexon in an individual cell and that caspase-3-cleaved cells were found more frequently in hexon-positive cells than in E1A-positive cells. Image cytometry thus detected these molecular changes in a sensitive manner and at a single cell level. We also showed that an image cytometric technique detected expression changes of other host cell proteins, cyclin-E and phosphorylated histone H3 at a single cell level. CONCLUSIONS: Image cytometry is a concise procedure to detect expression changes of Ad and host cell proteins at a single cell level, and is useful to analyze molecular events after the infection.

A Case of Solitary Well-Differentiated Papillary Mesothelioma with Invasive Foci in the Pleura.

Well-differentiated papillary mesothelioma (WDPM) is a rare, distinct tumor consisting of mesothelial cells with a papillary architecture, bland cytological features, and a tendency toward superficial spread without invasion. Rare cases with superficial invasion are termed WDPM with invasive foci. We report a case of solitary WDPM with invasive foci in the pleura. A 61-year-old woman presented with a lung adenocarcinoma. A small papillary lesion measuring 29 × 10 × 8 mm was incidentally found in the parietal pleura during a lobectomy for the lung adenocarcinoma. The fibrovascular core of the small papillary lesion was surrounded by a single layer of cuboidal cells with mild to moderate atypia and large nucleoli. Atypical mesothelial cells focally invaded the submesothelial layer. The cells of the papillary lesion were positive for cytokeratins and mesothelial markers. The Ki67 index was <1 %. The lesion did not show p16 loss on fluorescence in situ hybridization. We could not detect atypical mesothelial cells in the specimen from an extrapleural pneumonectomy. WDPM with invasive foci is prone to multifocality; however, our case represents a solitary case in the pleura.

Usefulness of p16/CDKN2A fluorescence in situ hybridization and BAP1 immunohistochemistry for the diagnosis of biphasic mesothelioma.

Malignant mesothelioma is a highly aggressive neoplasm, and the histologic subtype is one of the most reliable prognostic factors. Some biphasic mesotheliomas are difficult to distinguish from epithelioid mesotheliomas with atypical fibrous stroma. The aim of this study was to analyze p16/CDKN2A deletions in mesotheliomas by fluorescence in situ hybridization (FISH) and BAP1 immunohistochemistry to evaluate their potential role in the diagnosis of biphasic mesothelioma. We collected 38 cases of pleural mesotheliomas. The results of this study clearly distinguished 29 cases of biphasic mesothelioma from 9 cases of epithelioid mesothelioma. The proportion of biphasic mesotheliomas with homozygous deletions of p16/CDKN2A in total was 96.6% (28/29). Homozygous deletion of p16/CDKN2A was observed in 18 (94.7%) of 19 biphasic mesotheliomas with 100% concordance of the p16/CDKN2A deletion status between the epithelioid and sarcomatoid components in each case. Homozygous deletion of the p16/CDKN2A was observed in 7 (77.8%) of 9 epithelioid mesotheliomas but not in fibrous stroma. BAP1 loss was observed in 5 (38.5%) of 13 biphasic mesotheliomas and in both epithelioid and sarcomatoid components. BAP1 loss was observed in 5 (62.5%) of 8 epithelioid mesotheliomas but not in fibrous stroma. Homozygous deletion of p16/CDKN2A is common in biphasic mesotheliomas, and the analysis of only one component of mesothelioma is sufficient to show that the tumor is malignant. However, compared with histology alone, FISH analysis of the p16/CDKN2A status and BAP1 immunohistochemistry in the spindled mesothelium provide a more objective means to differentiate between biphasic mesothelioma and epithelioid mesothelioma with atypical stromal cells.

A phase II trial evaluating the efficacy and safety of perioperative pirfenidone for prevention of acute exacerbation of idiopathic pulmonary fibrosis in lung cancer patients undergoing pulmonary resection: West Japan Oncology Group 6711 L (PEOPLE Study).

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) often accompanies lung cancer, and life-threatening acute exacerbation (AE) of IPF (AE-IPF) is reported to occur in 20 % of IPF patients who undergo lung cancer surgery. Pirfenidone is an anti-fibrotic agent known to reduce disease progression in IPF patients. A phase II study was conducted to evaluate whether perioperative pirfenidone treatment could reduce the incidence of postoperative AE-IPF patients with lung cancer. METHODS: Pirfenidone was orally administered to IPF patients who were candidates for lung cancer surgery; pirfenidone was dosed at 600 mg/day for the first 2 weeks, followed by 1200 mg/day. Surgery was performed after at least 2 weeks of 1200-mg/day administration. The primary endpoint was non-AE-IPF rate during postoperative days 0-30, compared to the null value of 80 %, and the secondary endpoint was safety. Radiologic and pathologic diagnoses of IPF and AE-IPF were confirmed by an independent review committee. RESULTS: From June 2012 to January 2014, 43 cases were enrolled, and 39 were eligible (full analysis set [FAS]). Both pirfenidone treatment and surgery were performed in 36 patients (per protocol set [PPS]). AE-IPF did not occur in 37/39 patients (94.9 % [95 % confidential interval: 82.7-99.4 %, p = 0.01]) in the FAS, and in 38/39 patients (97.2 % [95 % confidential interval: 85.5-99.9 %, p = 0.004] in the PPS. A grade 5 adverse event (death) occurred in 1 patient, after AE-IPF; no other grade 3-5 adverse events were observed. CONCLUSIONS: Perioperative pirfenidone treatment is safe, and is promising for reducing AE-IPF after lung cancer surgery in IPF patients. TRIAL REGISTRATION: This clinical trial was registered with the University Hospital Medical Information Network (UMIN) on April 16th, 2012 (REGISTRATION NUMBER: UMIN000007774 ).

Combination of a third generation bisphosphonate and replication-competent adenoviruses augments the cytotoxicity on mesothelioma.

BACKGROUND: Approximately 80 % of mesothelioma specimens have the wild-type p53 gene, whereas they contain homozygous deletions in the INK4A/ARF locus that encodes p14 (ARF) and the 16 (INK4A) genes. Consequently, the majority of mesothelioma is defective of the p53 pathways. We examined whether zoledronic acid (ZOL), a third generation bisphosphonate, and adenoviruses with a deletion of the E1B-55kD gene (Ad-delE1B55), which augments p53 levels in the infected tumors, could produce combinatory anti-tumor effects on human mesothelioma cells bearing the wild-type p53 gene. METHODS: Cytotoxicity of ZOL and Ad-delE1B55 was assessed with a WST assay. Cell cycle changes were tested with flow cytometry. Expression levels of relevant molecules were examined with western blot analysis to investigate a possible mechanism of cytotoxicity. Furthermore, the expressions of Ad receptors on target cells and infectivity were estimated with flow cytometry. Viral replication was assayed with the tissue culture infection dose method. RESULTS: A combinatory use of ZOL and Ad-delE1B55 suppressed cell growth and increased sub-G1 or S-phase populations compared with a single agent, depending on cells tested. The combinatory treatment up-regulated p53 levels and subsequently enhanced the cleavage of caspase-3, 8, 9 and poly (ADP-ribose) polymerase, but expression of molecules involved in autophagy pathways were inconsistent. ZOL-treated cells also increased Ad infectivity with a dose-dependent manner and augmented Ad replication although the expression levels of integrin molecules, one of the Ad receptors, were down-regulated. CONCLUSIONS: These findings indicated that ZOL and Ad-delE1B55 achieved combinatory anti-tumor effects through augmented apoptotic pathways or increased viral replication.