Regulation of lymphoid-myeloid lineage bias through regnase-1/3-mediated control of Nfkbiz.

ABSTRACT: Regulation of lineage biases in hematopoietic stem and progenitor cells (HSPCs) is pivotal for balanced hematopoietic output. However, little is known about the mechanism behind lineage choice in HSPCs. Here, we show that messenger RNA (mRNA) decay factors regnase-1 (Reg1; Zc3h12a) and regnase-3 (Reg3; Zc3h12c) are essential for determining lymphoid fate and restricting myeloid differentiation in HSPCs. Loss of Reg1 and Reg3 resulted in severe impairment of lymphopoiesis and a mild increase in myelopoiesis in the bone marrow. Single-cell RNA sequencing analysis revealed that Reg1 and Reg3 regulate lineage directions in HSPCs via the control of a set of myeloid-related genes. Reg1- and Reg3-mediated control of mRNA encoding Nfkbiz, a transcriptional and epigenetic regulator, was essential for balancing lymphoid/myeloid lineage output in HSPCs in vivo. Furthermore, single-cell assay for transposase-accessible chromatin sequencing analysis revealed that Reg1 and Reg3 control the epigenetic landscape on myeloid-related gene loci in early stage HSPCs via Nfkbiz. Consistently, an antisense oligonucleotide designed to inhibit Reg1- and Reg3-mediated Nfkbiz mRNA degradation primed hematopoietic stem cells toward myeloid lineages by enhancing Nfkbiz expression. Collectively, the collaboration between posttranscriptional control and chromatin remodeling by the Reg1/Reg3-Nfkbiz axis governs HSPC lineage biases, ultimately dictating the fate of lymphoid vs myeloid differentiation.

Aberrant expression of NPPB through YAP1 and TAZ activation in mesothelioma with Hippo pathway gene alterations.

BACKGROUND: Mesothelioma is a neoplastic disease associated with asbestos exposure. It is highly malignant and has a poor prognosis; thus, early detection is desirable. Recent whole-genome analysis has revealed that mesothelioma is characterized by a high frequency of mutations in a set of genes involved in the Hippo pathway, such as NF2 and LATS2. However, a rapid, simple, and precise method for finding mesothelioma with these mutations has not yet been established. METHODS: Clustering of Hippo pathway gene alteration groups and the differential expression of each gene in mesothelioma patients were analyzed using The Cancer Genome Atlas database. Gene expression levels in various tumors and normal tissues were analyzed using public databases. Knockdown or transient expression of YAP1 or TAZ was performed to evaluate the regulation of gene expression by these genes. NT-proBNP was measured in the pleural effusions of 18 patients and was compared with NF2 expression in five cases where cell lines had been successfully established. RESULTS: NPPB mRNA expression was markedly higher in the group of mesothelioma patients with Hippo pathway gene mutations than in the group without them. NPPB expression was low in all normal tissues except heart, and was highest in mesothelioma. Mesothelioma patients in the high NPPB expression group had a significantly worse prognosis than those in the low NPPB expression group. NPPB expression was suppressed by knockdown of YAP1 or TAZ. NT-proBNP was abundant in the effusions of mesothelioma patients and was particularly high in those with impaired NF2 expression. CONCLUSIONS: NPPB, whose levels can be measured in pleural effusions of mesothelioma patients, has the potential to act as a biomarker to detect NF2-Hippo pathway gene alterations and/or predict patient prognosis. Additionally, it may provide useful molecular insights for a better understanding of mesothelioma pathogenesis and for the development of novel therapies.

Usefulness of NF2 hemizygous loss detected by fluorescence in situ hybridization in diagnosing pleural mesothelioma in tissue and cytology material: A multi-institutional study.

OBJECTIVES: BAP1, CDKN2A, and NF2 are the most frequently altered genes in pleural mesotheliomas (PM). Discriminating PM from benign mesothelial proliferation (BMP) is sometimes challenging; it is well established that BAP1 loss, determined by immunohistochemistry (IHC), and CDKN2A homozygous deletion (HD), determined by fluorescence in situ hybridization (FISH), are useful. However, data regarding the diagnostic utility of NF2 FISH in PM is limited. Thus, we performed a multi-institutional study examining the utility of NF2 alterations determined by FISH for diagnosing PM in combination with BAP1 loss and CDKN2A HD. MATERIALS AND METHODS: Multi-institutional PM cases, including 106 surgical and 107 cell block samples as well as 37 tissue cases of benign mesothelial proliferation (BMP) and 31 cell block cases with reactive mesothelial cells (RMC), were collected and analyzed using IHC for BAP1 and FISH for CDKN2A and NF2. RESULTS: In PM, NF2 FISH revealed hemizygous loss (HL) in 54.7% of tissue cases (TC) and 49.5% of cell block cases (CBC), with about 90% of HL being monosomy. CDKN2A HD or BAP1 loss were detected in 75.5%/65.4% TC or 63.6%/60% CBC, respectively. BMP or RMC showed no BAP1 loss, CDKN2A HD, or NF2 HL. For discriminating PM from BMP, a combination of BAP1 loss, CDKN2A HD, and NF2 HL yielded enhanced sensitivity of 98.1% TC/94.4% CBC. BAP1 loss, CDKN2A HD, or NF2 HL were observed in 69%, 70%, or 58% of epithelioid PM, but in 9%, 91%, or 27% of sarcomatoid PM, respectively. Histotype, histological gradings, and CDKN2A deletion status showed significant differences in overall survival, while BAP1 loss and NF2 HL did not. CONCLUSION: NF2 HL, consisting predominantly of monosomy, can be detected by FISH in both TC and CBC of PM, and is effective for distinguishing PM from BMP, especially when combined with BAP1 loss and CDKN2A HD.

Incidence and Risk Factors of Chest Wall Metastasis at Biopsy Sites in Patients with Malignant Pleural Mesothelioma.

To investigate the incidence and risk factors of chest wall metastasis (CWM) at biopsy sites in patients with malignant pleural mesothelioma (MPM). This retrospective cohort study was conducted in 262 consecutive MPM patients who underwent multimodal treatment in which including neoadjuvant chemotherapy (NAC) and curative-intent surgery, from August 2009 to March 2021. CWM was evaluated radiologically (r-CWM) and pathologically (p-CWM). We also investigated the risk factors of p-CWM and the consistency between r-CWM and p-CWM. Of 262 patients, 25 patients were excluded from analysis due to missing data or impossibility of evaluation. Of the eligible 237 patients, pleural biopsy was performed via video-assisted thoracoscopic surgery in 197 (83.1%) and medical thoracoscopy in 40 (16.9%). Pleurodesis was performed after pleural biopsy in 74 patients (31.2%). All patients received NAC followed by curative-intent surgery. Radiological examination showed r-CWM in 43 patients (18.1%), while pathological examination showed p-CWM in 135 patients (57.0%). The incidence of p-CWM was significantly higher in the patients who received pleurodesis after pleural biopsy (77.0% vs. 47.9%, <0.001). Multivariate logistic regression analysis for p-CWM revealed that pleurodesis is an independent risk factor of p-CWM (adjusted hazard ratio, 3.46; 95% confidence interval, 1.84−6.52, <0.001). CWM at the biopsy site was pathologically proven in more than half of the patients (57.0%) who received NAC followed by curative-intent surgery, which was higher than the numbers diagnosed by radiological examinations (p-CWM: 57.0% vs. r-CWM: 18.1%). Pleurodesis after pleural biopsy is an independent risk factor of p-CWM.

Regnase-1 Prevents Pulmonary Arterial Hypertension Through mRNA Degradation of Interleukin-6 and Platelet-Derived Growth Factor in Alveolar Macrophages.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a type of pulmonary hypertension (PH) characterized by obliterative pulmonary vascular remodeling, resulting in right-sided heart failure. Although the pathogenesis of PAH is not fully understood, inflammatory responses and cytokines have been shown to be associated with PAH, in particular, with connective tissue disease-PAH. In this sense, Regnase-1, an RNase that regulates mRNAs encoding genes related to immune reactions, was investigated in relation to the pathogenesis of PH. METHODS: We first examined the expression levels of ZC3H12A (encoding Regnase-1) in peripheral blood mononuclear cells from patients with PH classified under various types of PH, searching for an association between the ZC3H12A expression and clinical features. We then generated mice lacking Regnase-1 in myeloid cells, including alveolar macrophages, and examined right ventricular systolic pressures and histological changes in the lung. We further performed a comprehensive analysis of the transcriptome of alveolar macrophages and pulmonary arteries to identify genes regulated by Regnase-1 in alveolar macrophages. RESULTS: ZC3H12A expression in peripheral blood mononuclear cells was inversely correlated with the prognosis and severity of disease in patients with PH, in particular, in connective tissue disease-PAH. The critical role of Regnase-1 in controlling PAH was also reinforced by the analysis of mice lacking Regnase-1 in alveolar macrophages. These mice spontaneously developed severe PAH, characterized by the elevated right ventricular systolic pressures and irreversible pulmonary vascular remodeling, which recapitulated the pathology of patients with PAH. Transcriptomic analysis of alveolar macrophages and pulmonary arteries of these PAH mice revealed that Il6, Il1b, and Pdgfa/b are potential targets of Regnase-1 in alveolar macrophages in the regulation of PAH. The inhibition of IL-6 (interleukin-6) by an anti-IL-6 receptor antibody or platelet-derived growth factor by imatinib but not IL-1ß (interleukin-1ß) by anakinra, ameliorated the pathogenesis of PAH. CONCLUSIONS: Regnase-1 maintains lung innate immune homeostasis through the control of IL-6 and platelet-derived growth factor in alveolar macrophages, thereby suppressing the development of PAH in mice. Furthermore, the decreased expression of Regnase-1 in various types of PH implies its involvement in PH pathogenesis and may serve as a disease biomarker, and a therapeutic target for PH as well.

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.

Enhancement of Regnase-1 expression with stem loop-targeting antisense oligonucleotides alleviates inflammatory diseases.

Regnase-1 is an ribonuclease that plays essential roles in restricting inflammation through degrading messenger RNAs (mRNAs) involved in immune reactions via the recognition of stem-loop (SL) structures in the 3' untranslated regions (3'UTRs). Dysregulated expression of Regnase-1 is associated with the pathogenesis of inflammatory and autoimmune diseases in mice and humans. Here, we developed a therapeutic strategy to suppress inflammatory responses by blocking Regnase-1 self-regulation, which was mediated by the simultaneous use of two antisense phosphorodiamidate morpholino oligonucleotides (MOs) to alter the binding of Regnase-1 toward the SL structures in its 3'UTR. Regnase-1-targeting MOs not only enhanced Regnase-1 expression by stabilizing mRNAs but also effectively reduced the expression of multiple proinflammatory transcripts that were controlled by Regnase-1 in macrophages. Intratracheal administration of Regnase-1-targeting MOs ameliorated acute respiratory distress syndrome and chronic fibrosis through suppression of inflammatory cascades. In addition, intracranial treatment with Regnase-1-targeting MOs attenuated the development of experimental autoimmune encephalomyelitis by promoting the expansion of homeostatic microglia and regulatory T cell populations. Regnase-1 expression was inversely correlated with disease severity in patients with multiple sclerosis, and MOs targeting human Regnase-1 SL structures were effective in mitigating cytokine production in human immune cells. Collectively, MO-mediated disruption of the Regnase-1 self-regulation pathway is a potential therapeutic strategy to enhance Regnase-1 abundance, which, in turn, provides therapeutic benefits for treating inflammatory diseases by suppressing inflammation.

Short 57 kb CDKN2A FISH probe effectively detects short homozygous deletion of the 9p21 locus in malignant pleural mesothelioma.

Homozygous deletion (homo-d) of the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene is frequently found in malignant pleural mesothelioma (MPM). Fluorescence in situ hybridization (FISH) is commonly used to detect chromosomal deletion, and sometimes reveals more frequent heterozygous deletion (hetero-d) compared with homo-d. In clinical practice, such CDKN2A FISH results belong to the 'borderline' homo-d rate, which makes it difficult to definitively diagnose MPM. Microdeletion, [<200 kilobase (kb)], can induce a 'pseudo' hetero-d signal in FISH assays with long probes owing to redundant probe reactivity. Thus, the present study hypothesized that shorter FISH probes can effectively detect the small deletion status of the CDKN2A gene and increase homo-d rate in MPM, which has high hetero-d and low homo-d status. The present study aimed to evaluate the effectiveness of a shorter CDKN2A FISH probe in diagnosing MPM. CDKN2A FISH with either a 222 kb long probe (L-probe) or a 57 kb short probe (S-probe) was performed in four MPM cases with high hetero-d and low homo-d patterns. Furthermore, immunohistochemistry for methylthioadenosine phosphorylase (MTAP) and quantitative (q)PCR analyses were performed to confirm the microdeletion of the 9p21 locus. The results demonstrated that all four MPM cases retained MTAP protein expression. CDKN2A FISH with L-probe revealed high hetero-d (cases 1-4; 73.3, 37.1, 59.2 and 64.8%, respectively) and low homo-d (cases 1-4; 12.1, 12.4, 25.4 and 22.2%, respectively). CDKN2A FISH with S-probe revealed high homo-d (cases 1-4; 96.8, 90.0, 87.5 and 82.6%, respectively), with low hetero-d (cases 1-4; 0.0, 1.2, 1.2 and 4.3%, respectively). qPCR analysis demonstrated no allele deletions of the MTAP gene and two-allele deletions of the CDKN2A gene in 3/4 cases. Taken together, these results suggest that the S-probe detects the short homo-d of the 9p21 locus more effectively than the L-probe in MPM. This can assist in solving diagnostic difficulties in cases involving high hetero-d with low homo-d.

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.

Clinicopathological features of radiological early malignant pleural mesothelioma with no apparent tumor or pleural thickening.

BACKGROUND: We occasionally encounter malignant pleural mesothelioma (MPM) of no apparent tumor or pleural thickening that is radiological early MPM. This study aimed to examine the clinicopathological outcomes of radiological early MPM. METHODS: Patients with MPM treated with neoadjuvant chemotherapy and planned surgery at the time of diagnosis between July 2004 and December 2019 were retrospectively examined. Pretreatment maximal pleural thickness of all patients was measured on chest computed tomography. We extracted and investigated the patients who exhibited a lack of pleural thickening or visible tumor, which was defined as radiological early MPM. Survival was analyzed by the Kaplan-Meier method. RESULTS: Of 296treated patients, 16 (5.4%) exhibited radiological early MPM. Fourteen (87.5%) of these patients underwent pleurectomy/decortication and 2 (12.5%) underwent extrapleural pneumonectomy. Pathological stage T1 disease was diagnosed in 14 (87.5%) patients; 2 (12.5%) exhibited pulmonary parenchymal invasion (pathological stage T2). Lymphatic invasion was detected in only 1 patient. Lymph node metastases and vascular invasion were not detected. Median follow-up was 42 months. Median progression-free survival and median overall survival were 40.7 and 56.1 months, respectively. The 3-year progression-free survival and overall survival rates were 84.8% and 83.6%, respectively. CONCLUSIONS: Radiological early MPM occurs in approximately 1 of every 20 patients treated with neoadjuvant chemotherapy and surgery planned at the time of diagnosis in an experienced center. Radiological early MPM was associated with early pathological stage and long-term survival.

Aicardi-Goutières syndrome-like encephalitis in mutant mice with constitutively active MDA5.

MDA5 is a cytoplasmic sensor of viral RNA, triggering type I interferon (IFN-I) production. Constitutively active MDA5 has been linked to autoimmune diseases such as systemic lupus erythematosus, Singleton-Merten syndrome (SMS) and Aicardi-Goutières syndrome (AGS), a genetically determined inflammatory encephalopathy. However, AGS research is challenging due to the lack of animal models. We previously reported lupus-like nephritis and SMS-like bone abnormalities in adult mice with constitutively active MDA5 (Ifih1G821S/+), and herein demonstrate that these mice also exhibit high lethality and spontaneous encephalitis with high IFN-I production during the early postnatal period. Increases in the number of microglia were observed in MDA5/MAVS signaling- and IFN-I-dependent manners. Furthermore, microglia showed an activated state with an increased phagocytic capability and reduced expression of neurotrophic factors. Although multiple auto-antibodies including lupus-related ones were detected in the sera of the mice as well as AGS patients, Ifih1G821S/+Rag2-/- mice also exhibited up-regulation of IFN-I, astrogliosis and microgliosis, indicating that auto-antibodies or lymphocytes are not required for the development of the encephalitis. The IFN-I signature without lymphocytic infiltration observed in Ifih1G821S/+ mice is a typical feature of AGS. Collectively, our results suggest that the Ifih1G821S/+ mice are a model recapitulating AGS and that microglia are a potential target for AGS therapy.

Profibrotic function of pulmonary group 2 innate lymphoid cells is controlled by regnase-1.

Regnase-1 is an RNase critical for post-transcriptional control of pulmonary immune homeostasis in mice by degrading immune-related mRNAs. However, little is known about the cell types Regnase-1 controls in the lung, and its relevance to human pulmonary diseases.Regnase-1-dependent changes in lung immune cell types were examined by a competitive bone marrow transfer mouse model, and group 2 innate lymphoid cells (ILC2s) were identified. Then the associations between Regnase-1 in ILC2s and human diseases were investigated by transcriptome analysis and a bleomycin-induced pulmonary fibrosis mouse model. The clinical significance of Regnase-1 in ILC2s was further assessed using patient-derived cells.Regnase-1-deficiency resulted in the spontaneous proliferation and activation of ILC2s in the lung. Intriguingly, genes associated with pulmonary fibrosis were highly upregulated in Regnase-1-deficient ILC2s compared with wild-type, and supplementation of Regnase-1-deficient ILC2s augmented bleomycin-induced pulmonary fibrosis in mice. Regnase-1 suppresses mRNAs encoding transcription factors Gata3 and Egr1, which are potent to regulate fibrosis-associated genes. Clinically, Regnase-1 protein levels in ILC2 negatively correlated with the ILC2 population in bronchoalveolar lavage fluid. Furthermore, idiopathic pulmonary fibrosis (IPF) patients with ILC2s >1500 cells·mL-1 peripheral blood exhibited poorer prognosis than patients with lower numbers, implying the contribution of Regnase-1 in ILC2s for the progression of IPF.Collectively, Regnase-1 was identified as a critical post-transcriptional regulator of the profibrotic function of ILC2s both in mouse and human, suggesting that Regnase-1 may be a novel therapeutic target for IPF.

Development of mesothelioma in situ and its progression to invasive disease observed in a patient with uncontrolled pleural effusions for 15 years.

Mesothelioma in situ (MIS) has recently been investigated as a distinct phase of mesothelioma carcinogenesis. The diagnostic criteria proposed for MIS include a loss of BRCA1-associated protein 1 (BAP1) expression detected by immunohistochemistry (IHC). However, the length of time that MIS typically remains an in situ lesion before progression to invasive disease is still unclear. Herein, we report a case of a Japanese woman in her early seventies who had suffered from recurrent pleural effusions for 15 years, during which MIS developed and progressed to invasive mesothelioma. Retrospective diagnosis of partial MIS, fully developed MIS, and invasive disease was made using BAP1 IHC on three biopsy specimens and via clinical observations with radiological images. MIS and invasive lesions revealed BAP1 loss. The interval from partial or full MIS to invasive disease was 14 and 7 years, respectively. These results support a diagnostic strategy combining histomorphology with genomic-based assays including BAP1 IHC in biopsy tissues from a patient with recurrent pleural effusions.

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.

Clinical feature of diagnostic challenging cases for pleural biopsy in patient with malignant pleural mesothelioma.

OBJECTIVE: Pleural biopsy through video-assisted thoracic surgery (VATS pleural biopsy) is the most reliable diagnostic procedure for malignant pleural mesothelioma (MPM). However, definitive diagnosis of MPM is occasionally difficult to establish. This study aims to investigate clinicopathological features of MPM patients who failed diagnosis by the first VATS pleural biopsy. METHODS: Four hundred consecutive patients with suspected MPM who received VATS pleural biopsy between March 2004 and July 2017 were enrolled in this retrospective study. Patients, whose histological diagnoses were not definitive in the first VATS pleural biopsy, were followed up as atypical mesothelial proliferation (AMP) or non-specific pleuritis (NSP). Re-examination was performed in cases strongly suspected of having MPM. RESULTS: Of the 400 patients, 267 (66.8%) were pathologically diagnosed with MPM, 25 with metastatic carcinoma and 6 with benign pleural disease by the first VATS pleural biopsy. Of the remaining 102 patients diagnosed with AMP or NSP, 10 patients (9.8%) were subsequently diagnosed with MPM. Analysis of the clinical course revealed that only insufficient tissue for diagnosis was obtained via VATS pleural biopsy in all cases and that it was caused by very early stage without visible tumour in 4 patients, intrathoracic inflammation in 4 and desmoplastic MPM in 2. CONCLUSIONS: In our review, 9.8% of patients diagnosed with AMP or NSP in first VATS pleural biopsy were subsequently diagnosed with MPM due to insufficient tissue for diagnosis. Definitive diagnosis via VATS pleural biopsy is sometimes challenging in following situation; very early stage, intrathoracic inflammation and desmoplastic MPM.

Involvement of Rev1 in alkylating agent-induced loss of heterozygosity in Oryzias latipes.

Translesion synthesis (TLS) polymerases mediate DNA damage bypass during replication. The TLS polymerase Rev1 has two important functions in the TLS pathway, including dCMP transferase activity and acting as a scaffolding protein for other TLS polymerases at the C-terminus. Because of the former activity, Rev1 bypasses apurinic/apyrimidinic sites by incorporating dCMP, whereas the latter activity mediates assembly of multipolymerase complexes at the DNA lesions. We generated rev1 mutants lacking each of these two activities in Oryzias latipes (medaka) fish and analyzed cytotoxicity and mutagenicity in response to the alkylating agent diethylnitrosamine (DENA). Mutant lacking the C-terminus was highly sensitive to DENA cytotoxicity, whereas mutant with reduced dCMP transferase activity was slightly sensitive to DENA cytotoxicity, but exhibited a higher tumorigenic rate than wild-type fish. There was no significant difference in the frequency of DENA-induced mutations between mutant with reduced dCMP transferase activity and wild-type cultured cell. However, loss of heterozygosity (LOH) occurred frequently in cells with reduced dCMP transferase activity. LOH is a common genetic event in many cancer types and plays an important role on carcinogenesis. To our knowledge, this is the first report to identify the involvement of the catalytic activity of Rev1 in suppression of LOH.

The mTOR Signaling Pathway Is Associated With the Prognosis of Malignant Pleural Mesothelioma After Multimodality Therapy.

BACKGROUND/AIM: We performed multimodality therapy comprising preoperative chemotherapy, extrapleural pneumonectomy (EPP), and radiation therapy for patients with malignant pleural mesothelioma (MPM). Although multimodality therapy resulted in good prognosis, further improvement is required. Therefore, herein, we analysed the prognostic factors using surgical specimens and searched for suitable molecular targets to improve the prognosis after multidisciplinary treatment. PATIENTS AND METHODS: Forty-six patients with MPM underwent multimodality therapy. Paraffin-embedded surgical samples were used for immunohistochemistry to evaluate the expression of phosphorylated (p-) AKT, extracellular signal-regulated kinase (ERK), mammalian target of rapamycin (mTOR), mitogen-activated protein kinase (MAPK), eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), and S6 ribosomal protein (S6RP). RESULTS: On univariate and multivariate analyses, significant differences were observed according to the histological type, pathological stage, and p-mTOR expression rate. CONCLUSION: The prognosis of MPM is affected by p-mTOR expression, suggesting that molecular-targeted treatment might be used during multimodal therapy for MPM.

Molecular pathways and diagnosis in malignant mesothelioma: A review of the 14th International Conference of the International Mesothelioma Interest Group.

The pathologist plays a central role in the diagnosis and management of malignant mesothelioma, including definitive tissue-based diagnosis in conjunction with clinical and radiographic data; diverse ancillary studies of diagnostic, prognostic, and predictive importance; and research efforts to better define the pathobiology of mesothelioma and develop novel clinical applications. The pivotal role of pathology in care of mesothelioma patients was on display at the recent meeting of the International Mesothelioma Interest Group (iMig) in Ottawa, Canada. This review summarizes the key findings of the "Molecular Pathways and Diagnosis in Malignant Mesothelioma" plenary session, including a large multi-institutional validation of a composite nuclear grading system for pleural mesothelioma, including incorporation of tumor necrosis as an additional independent prognostic factor; the correlation between nuclear grading in small biopsies and paired resection specimens in pleural mesothelioma; a multi-institutional study of important clinical and pathologic prognostic factors in peritoneal mesothelioma; the diagnostic role of HEG1 immunohistochemistry as a highly sensitive and specific marker of mesothelial lineage; the prevalence and diagnostic significance of MET protein overexpression in mesothelioma, as well as the correlation between MET protein overexpression and MET gene amplification; and the prognostic role of EZH2 protein overexpression in mesothelioma, together with data indicating an important pathogenic role for EZH2 in mesothelioma tumorigenesis. Special consideration is given to the convergence of diagnostic, prognostic, and predictive tools and their role in guiding highly personalized patient-centered management, and to the translation of novel research findings to practical techniques for routine pathologic practice.