Malt1-induced cleavage of regnase-1 in CD4(+) helper T cells regulates immune activation.

Regnase-1 (also known as Zc3h12a and MCPIP1) is an RNase that destabilizes a set of mRNAs, including Il6 and Il12b, through cleavage of their 3' UTRs. Although Regnase-1 inactivation leads to development of an autoimmune disease characterized by T cell activation and hyperimmunoglobulinemia in mice, the mechanism of Regnase-1-mediated immune regulation has remained unclear. We show that Regnase-1 is essential for preventing aberrant effector CD4(+) T cell generation cell autonomously. Moreover, in T cells, Regnase-1 regulates the mRNAs of a set of genes, including c-Rel, Ox40, and Il2, through cleavage of their 3' UTRs. Interestingly, T cell receptor (TCR) stimulation leads to cleavage of Regnase-1 at R111 by Malt1/paracaspase, freeing T cells from Regnase-1-mediated suppression. Furthermore, Malt1 protease activity is critical for controlling the mRNA stability of T cell effector genes. Collectively, these results indicate that dynamic control of Regnase-1 expression in T cells is critical for controlling T cell activation.

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.

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.

The Jmjd3-Irf4 axis regulates M2 macrophage polarization and host responses against helminth infection.

Polarization of macrophages to M1 or M2 cells is important for mounting responses against bacterial and helminth infections, respectively. Jumonji domain containing-3 (Jmjd3), a histone 3 Lys27 (H3K27) demethylase, has been implicated in the activation of macrophages. Here we show that Jmjd3 is essential for M2 macrophage polarization in response to helminth infection and chitin, though Jmjd3 is dispensable for M1 responses. Furthermore, Jmjd3 (also known as Kdm6b) is essential for proper bone marrow macrophage differentiation, and this function depends on demethylase activity of Jmjd3. Jmjd3 deficiency affected trimethylation of H3K27 in only a limited number of genes. Among them, we identified Irf4 as encoding a key transcription factor that controls M2 macrophage polarization. Collectively, these results show that Jmjd3-mediated H3K27 demethylation is crucial for regulating M2 macrophage development leading to anti-helminth host responses.

Semaphorins guide the entry of dendritic cells into the lymphatics by activating myosin II.

The recirculation of leukocytes is essential for proper immune responses. However, the molecular mechanisms that regulate the entry of leukocytes into the lymphatics remain unclear. Here we show that plexin-A1, a principal receptor component for class III and class VI semaphorins, was crucially involved in the entry of dendritic cells (DCs) into the lymphatics. Additionally, we show that the semaphorin Sema3A, but not Sema6C or Sema6D, was required for DC transmigration and that Sema3A produced by the lymphatics promoted actomyosin contraction at the trailing edge of migrating DCs. Our findings not only demonstrate that semaphorin signals are involved in DC trafficking but also identify a previously unknown mechanism that induces actomyosin contraction as these cells pass through narrow gaps.

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.

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.

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.

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.

TANK is a negative regulator of Toll-like receptor signaling and is critical for the prevention of autoimmune nephritis.

The intensity and duration of immune responses are controlled by many proteins that modulate Toll-like receptor (TLR) signaling. TANK has been linked to positive regulation of the transcription factors IRF3 and NF-kappaB. Here we demonstrate that TANK is not involved in interferon responses and is a negative regulator of proinflammatory cytokine production induced by TLR signaling. TLR-induced polyubiquitination of the ubiquitin ligase TRAF6 was upregulated in Tank(-/-) macrophages. Notably, Tank(-/-) mice spontaneously developed fatal glomerulonephritis owing to deposition of immune complexes. Autoantibody production in Tank(-/-) mice was abrogated by antibiotic treatment or the absence of interleukin 6 (IL-6) or the adaptor MyD88. Our results demonstrate that constitutive TLR signaling by intestinal commensal microflora is suppressed by TANK.

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.

Regulation of humoral and cellular gut immunity by lamina propria dendritic cells expressing Toll-like receptor 5.

The intestinal cell types responsible for defense against pathogenic organisms remain incompletely characterized. Here we identify a subset of CD11c(hi)CD11b(hi) lamina propria dendritic cells (LPDCs) that expressed Toll-like receptor 5 (TLR5) in the small intestine. When stimulated by the TLR5 ligand flagellin, TLR5(+) LPDCs induced the differentiation of naive B cells into immunoglobulin A-producing plasma cells by a mechanism independent of gut-associated lymphoid tissue. In addition, by a mechanism dependent on TLR5 stimulation, these LPDCs promoted the differentiation of antigen-specific interleukin 17-producing T helper cells and type 1 T helper cells. Unlike spleen DCs, the LPDCs specifically produced retinoic acid, which, in a dose-dependent way, supported the generation and retention of immunoglobulin A-producing cells in the lamina propria and positively regulated the differentiation interleukin 17-producing T helper cells. Our findings demonstrate unique properties of LPDCs and the importance of TLR5 for adaptive immunity in the intestine.

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.

Endosomal sorting by Semaphorin 4A in retinal pigment epithelium supports photoreceptor survival.

Photoreceptor cell death is the hallmark of a group of human inherited retinal degeneration. Although the causative genetic mutations are often known, the mechanisms leading to photoreceptor degeneration remain poorly defined. Here, we show that Semaphorin 4A (Sema4A), a member of axonal guidance molecule semaphorin, plays a role in Rab11/FIP2-mediated endosomal sorting in retinal pigment epithelial cells to support photoreceptor function. In response to oxidative stress, Sema4A switches the endosomal sorting of the lysosomal precursor protein prosaposin from the lysosome to the exosomal release, which prevents light-induced photoreceptor apoptosis. In the absence of oxidative stress, Sema4A sorts retinoid-binding proteins with retinoids between the cell surface and endoplasmic reticulum, by which 11-cis-retinal, a chromophore for phototransduction, is regenerated and transported back to photoreceptors. Owing to defects in these processes, Sema4A-deficient mice exhibit marked photoreceptor degeneration. Our findings therefore indicate that Sema4A regulates two distinct endosomal-sorting pathways that are critical for photoreceptor survival and phototransduction during the transition between daylight and darkness.

Regnase-1 and Roquin Nonredundantly Regulate Th1 Differentiation Causing Cardiac Inflammation and Fibrosis.

Regnase-1 and Roquin are RNA binding proteins that are essential for degradation of inflammatory mRNAs and maintenance of immune homeostasis. Although deficiency of either of the proteins leads to enhanced T cell activation, their functional relationship in T cells has yet to be clarified because of lethality upon mutation of both Regnase-1 and Roquin. By using a Regnase-1 conditional allele, we show that mutations of both Regnase-1 and Roquin in T cells leads to massive lymphocyte activation. In contrast, mutation of either Regnase-1 or Roquin affected T cell activation to a lesser extent than the double mutation, indicating that Regnase-1 and Roquin function nonredundantly in T cells. Interestingly, Regnase-1 and Roquin double-mutant mice suffered from severe inflammation and early formation of fibrosis, especially in the heart, along with the increased expression of Ifng, but not Il4 or Il17a Consistently, mutation of both Regnase-1 and Roquin leads to a huge increase in the Th1, but not the Th2 or Th17, population in spleens compared with T cells with a single Regnase-1 or Roquin deficiency. Regnase-1 and Roquin are capable of repressing the expression of a group of mRNAs encoding factors involved in Th1 differentiation, such as Furin and Il12rb1, via their 3' untranslated regions. Moreover, Regnase-1 is capable of repressing Roquin mRNA. This cross-regulation may contribute to the synergistic control of T cell activation/polarization. Collectively, our results demonstrate that Regnase-1 and Roquin maintain T cell immune homeostasis and regulate Th1 polarization synergistically.

High-density array-CGH with targeted NGS unmask multiple noncontiguous minute deletions on chromosome 3p21 in mesothelioma.

We used a custom-made comparative genomic hybridization array (aCGH; average probe interval 254 bp) to screen 33 malignant mesothelioma (MM) biopsies for somatic copy number loss throughout the 3p21 region (10.7 Mb) that harbors 251 genes, including BRCA1 (breast cancer 1)-associated protein 1 (BAP1), the most commonly mutated gene in MM. We identified frequent minute biallelic deletions (<3 kb) in 46 of 251 genes: four were cancer-associated genes: SETD2 (SET domain-containing protein 2) (7 of 33), BAP1 (8 of 33), PBRM1 (polybromo 1) (3 of 33), and SMARCC1 (switch/sucrose nonfermentable- SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily c, member 1) (2 of 33). These four genes were further investigated by targeted next-generation sequencing (tNGS), which revealed sequence-level mutations causing biallelic inactivation. Combined high-density aCGH and tNGS revealed biallelic gene inactivation in SETD2 (9 of 33, 27%), BAP1 (16 of 33, 48%), PBRM1 (5 of 33, 15%), and SMARCC1 (2 of 33, 6%). The incidence of genetic alterations detected is much higher than reported in the literature because minute deletions are not detected by NGS or commercial aCGH. Many of these minute deletions were not contiguous, but rather alternated with segments showing oscillating copy number changes along the 3p21 region. In summary, we found that in MM: (i) multiple minute simultaneous biallelic deletions are frequent in chromosome 3p21, where they occur as distinct events involving multiple genes; (ii) in addition to BAP1, mutations of SETD2, PBRM1, and SMARCC1 are frequent in MM; and (iii) our results suggest that high-density aCGH combined with tNGS provides a more precise estimate of the frequency and types of genes inactivated in human cancer than approaches based exclusively on NGS strategy.

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.

BAP1 immunohistochemistry and p16 FISH results in combination provide higher confidence in malignant pleural mesothelioma diagnosis: ROC analysis of the two tests.

Differentiation of malignant pleural mesothelioma (MPM) from benign mesothelial proliferation remains problematic. Loss of nuclear staining of BRCA1-associated protein 1 (BAP1; detected using immunohistochemistry (IHC)) and homozygous deletion (HD) of p16 (detected using fluorescence in situ hybridization (FISH)) are useful for differentiation of MPM from reactive mesothelial hyperplasia (RMH), but the correlation between BAP1 expression loss and p16 HD has not been fully described. We performed BAP1 IHC and p16-specific FISH for 40 MPM and 20 RMH cases, and measured proportions of cells showing BAP1 expression and p16 HD for each case. The diagnostic accuracy for MPM and the cut-off values of the two methods were assessed using receiver operating characteristic (ROC) analysis. BAP1 expression loss, p16 HD and coexistence of both were present in 27 (67.5 %), 27 (67.5 %) and 17 (42.5 %) MPM cases, respectively. Three MPM cases (7.5 %) and all 20 RMH cases had neither BAP1 loss nor p16 HD. There was no correlation between the results of the two methods. Their combination showed higher sensitivity (92.5 %, 37/40) and estimated probability than BAP1 IHC and p16-specific FISH used alone. BAP1 IHC and p16-specific FISH have independent diagnostic value, and have increased reliability when used in combination, for MPM diagnosis.

Trimodality strategy for treating malignant pleural mesothelioma: results of a feasibility study of induction pemetrexed plus cisplatin followed by extrapleural pneumonectomy and postoperative hemithoracic radiation (Japan Mesothelioma Interest Group 0601 Trial).

PURPOSE: We conducted a prospective multi-institutional study to determine the feasibility of trimodality therapy (TMT) comprising induction chemotherapy followed by extrapleural pneumonectomy (EPP) and radiation therapy in Japanese patients with malignant pleural mesothelioma (MPM). METHODS: Major eligibility criteria were histologically confirmed diagnosis of MPM, including clinical subtypes T0-3, N0-2, M0 disease; no prior treatment for the disease; age 20-75 years; Eastern Cooperative Oncology Group performance status 0 or 1; predicted postoperative forced expiratory volume >1000 ml in 1 s; written informed consent. Treatment methods comprised induction chemotherapy using pemetrexed (500 mg/m(2)) plus cisplatin (60 mg/m(2)) for three cycles, followed by EPP and postoperative hemithoracic radiation therapy (54 Gy). Primary endpoints were macroscopic complete resection (MCR) rate for EPP and treatment-related mortality for TMT. RESULTS: Forty-two eligible patients were enrolled: median age 64.5 (range 43-74) years; M:F = 39:3, clinical stage I:II:III = 14:13:15; histological type epithelioid were sarcomatoid; biphasic; others = 28:1:9:4. Of 42 patients, 30 completed EPP with MCR and 17 completed TMT. The trial met the primary endpoints, with an MCR rate of 71 % (30/42) and treatment-related mortality of 9.5 % (4/42). Overall median survival time and 2-year survival rate for 42 registered patients were 19.9 months and 42.9 %, respectively. Two-year relapse-free survival rate of 30 patients who completed EPP with MCR was 37.0 %. CONCLUSION: This phase II study met the predefined primary endpoints, but its risk/benefit ratio was not satisfactory.