Kenichi Kohashi

Inflammatory myofibroblastic tumor versus IgG4-related sclerosing disease and inflammatory pseudotumor: a comparative clinicopathologic study.

Inflammatory pseudotumor (IPT) is a heterogeneous group of lesions occurring in various organs, which is histologically characterized by fibroblastic and myofibroblastic proliferation with inflammatory infiltrate. Inflammatory myofibroblastic tumor (IMT) is a neoplastic counterpart of IPT, which shows aberrant expression of ALK and its gene translocation. In contrast, the concept “immunoglobulin (Ig)G4-related IPT” in the lung, liver, and pancreas has recently been proposed as a member of IgG4-related sclerosing disease. In this study, we compared the histopathologic features with an emphasis on IgG4 expression between 22 cases of IMT and 16 cases of IgG4-related sclerosing disease, including chronic sclerosing sialadenitis (n=8), mass-forming autoimmune pancreatitis (n=3), sclerosing cholangitis (n=1), retroperitoneal fibrosis (n=2), and chronic sclerosing dacryoadenitis (n=2). Bland-looking spindle cell proliferation with fibrosis and inflammatory infiltrate of lymphocytes and plasma cells was the common morphologic feature in both lesions. Obstructive phlebitis was observed in all of the IgG4-related sclerosing lesions, but in only 1/22 (4.5%) of IMT. The immunohistochemical expression of ALK was observed in 15/22 (68.2%) of IMT and 0/16 (0%) of IgG4-related sclerosing disease. The number of IgG4-positive plasma cells and the ratio of IgG4+/IgG+ plasma cells were each significantly lower in IMT than in IgG4-related sclerosing disease [mean 6.4/HPF vs. 178.3/HPF (P<0.0001), 3.0% vs. 67.5% (P<0.0001), respectively]. The results suggest that IgG4 does not play an important role in the pathogenesis of IMT. In addition, the evaluation of IgG4+ plasma cells and the ratio of IgG4+/IgG+ plasma cells and the presence of obstructive phlebitis may be useful for the differential diagnosis between IMT and IgG4-related sclerosing disease.

Infrequent SMARCB1/INI1 gene alteration in epithelioid sarcoma: a useful tool in distinguishing epithelioid sarcoma from malignant rhabdoid tumor ☆

Loss of SMARCB1/INI1 protein expression is considered useful for confirming a histologic diagnosis of malignant rhabdoid tumor. However, loss of SMARCB1/INI1 protein expression has recently been reported in other tumors as well, including a few cases of epithelioid sarcoma. In addition, the histopathologic differences between proximal-type epithelioid sarcoma and malignant rhabdoid tumor have not been conclusively defined. We analyzed SMARCB1/INI1 protein expression in 54 epithelioid sarcoma (proximal-type, 25; distal-type, 29) and examined alterations of the SMARCB1/INI1 gene in the cases lacking protein expression. We found that 19 (76.0%) proximal-type epithelioid sarcoma and 27 (93.1%) distal-type epithelioid sarcoma showed loss of SMARCB1/INI1 protein expression. Analysis of 39 cases with loss of protein expression revealed 4 cases (10.3%) with SMARCB1/INI1 gene alterations at the DNA level (homozygous deletion, 2; 1- or 2-bp deletion, 2) that could have induced the loss of gene products, and all 4 of these were proximal-type epithelioid sarcoma. Epithelioid sarcoma was thus associated with a high frequency of loss of SMARCB1/INI1 protein expression similar to that in malignant rhabdoid tumor. However, the frequency of SMARCB1/INI1 gene alteration at the DNA level in proximal-type epithelioid sarcoma was significantly lower than that in malignant rhabdoid tumor. In addition, the prognosis of patients with malignant rhabdoid tumor is significantly worse than that of patients with proximal-type epithelioid sarcoma (P = .001). Therefore, proximal-type epithelioid sarcoma and malignant rhabdoid tumor are suggested to be distinctive tumors with respect to the mechanism of the loss of SMARCB1/INI1 protein expression. Analysis of alterations in the SMARCB1/INI1 gene may thus be a useful diagnostic tool to distinguish proximal-type epithelioid sarcoma from malignant rhabdoid tumor.

SMARCB1/INI1 protein expression in round cell soft tissue sarcomas associated with chromosomal translocations involving EWS: a special reference to SMARCB1/INI1 negative variant extraskeletal myxoid chondrosarcoma.

Several previous studies have demonstrated the lack of SMARCB1/INI1 protein expression in only the malignant rhabdoid tumor (MRT). Several sarcoma groups are associated with a tumor-specific translocation involving EWS. Moreover, the EWS and SMARCB1/INI1 genes are located on the same 22q chromosome. We analyzed the status of SMARCB1/INI1 protein expression in 93 cases of sarcomas associated with chromosomal translocation involving EWS, comprising 52 Ewings sarcoma/primitive neuroectodermal tumors, 24 extraskeletal myxoid chondrosarcomas (EMCS), 14 clear cell sarcomas of soft tissue, 2 desmoplastic small round cell tumors, and 1 myxoid/round cell liposarcoma. In addition, we analyzed the detailed SMARCB1/INI1 gene alteration in cases, which lacked its protein expression. Consequently, 4 EMCS showed no SMARCB1/INI1 expression, and 2 of these 4 cases revealed homozygous deletion and frameshift mutation of the SMARCB1/INI1 gene, respectively. These cases showed histologic findings compatible with EMCS, according to the most recent WHO classification, but no major fusion gene transcripts were detected. Moreover, 3 out of 4 SMARCB1/INI1 negative variant EMCS disclosed rhabdoid features. Therefore, the lack of SMARCB1/INI1 protein expression may be associated with rhabdoid features. The immunohistochemical result of the SMARCB1/INI expression is not an absolute diagnostic criteria for MRT and careful histologic evaluation is required to make a precise diagnosis of MRT.

Prognostic significance of AKT/mTOR and MAPK pathways and antitumor effect of mTOR inhibitor in NF1-related and sporadic malignant peripheral nerve sheath tumors

Purpose: Malignant peripheral nerve sheath tumor (MPNST) is a rare soft tissue sarcoma with poor prognosis. MPNSTs occur frequently in patients with neurofibromatosis type 1 (NF1), in which NF1 gene deficiency leads to Ras hyperactivation. Ras activation causes the subsequent activation of the AKT/mTOR and Raf/MEK/ERK pathways and regulates cellular functions. However, the activation profiles of the AKT/mTOR and MAPK pathways in MPNSTs are poorly understood. The purposes of this study are to examine the correlation between the activation of these pathways and clinicopathologic or prognostic factors and to identify candidate target molecules in MPNST. Moreover, we assessed the antitumor effects of the inhibitor of candidate target. Experimental Design: Immunohistochemistry was conducted to evaluate the activation profiles of AKT/mTOR and MAPK pathways using 135 tumor specimens. Immunohistochemical expressions were confirmed by Western blotting. Then, an in vitro study was conducted to examine the antitumor effect of the mTOR inhibitor on MPNST cell lines. Results: Phosphorylated-AKT (p-AKT), p-mTOR, p-S6RP, p-p70S6K, p-4E-BP1, p-MEK1/2, and p-ERK1/2 expressions were positive in 58.2%, 47.3%, 53.8%, 57.1%, 62.6%, 93.4%, and 81.3% of primary MPNSTs, respectively. Positivity for each factor showed no difference between NF1-related and sporadic MPNSTs. Univariate prognostic analysis revealed that p-AKT, p-mTOR, and p-S6RP expressions were associated with poor prognosis. Furthermore, activation of each p-mTOR and p-S6RP was an independent poor prognostic factor by multivariate analysis. mTOR inhibition by Everolimus showed antitumor activity on MPNST cell lines in vitro. Conclusion: mTOR inhibition is a potential treatment option for both NF1-related and sporadic MPNSTs. Clin Cancer Res; 19(2); 450–61. ©2012 AACR.

Reduced expression of SMARCB1/INI1 protein in synovial sarcoma

Synovial sarcoma is classified as a tumor of uncertain differentiation, and some synovial sarcomas have rhabdoid cells. In previous studies, all malignant rhabdoid tumors and renal medullary carcinomas, some extraskeletal myxoid chondrosarcomas, almost all epithelioid sarcomas and half of epithelioid malignant peripheral nerve sheath tumors showed a loss of SMARCB1/INI1 protein expression in tumor cells and all of these tumors are also known to have rhabdoid cells. We analyzed the immunohistochemical and mRNA expression of SMARCB1/INI1 in 95 synovial sarcomas (73 monophasic fibrous type, 18 biphasic type and 4 poorly differentiated type) and 30 spindle cell sarcomas (3 adult fibrosarcomas, 7 fibrosarcomas arising in dermatofibrosarcoma protuberans, 10 leiomyosarcomas and 10 malignant peripheral nerve sheath tumors) resembling monophasic fibrous synovial sarcoma. The results have shown that 66 of the 95 synovial sarcoma cases (69%) had reduced SMARCB1/INI1 protein expression, whereas the remaining 29 cases (31%) and all 30 spindle cell sarcomas showed preserved this protein expression. No case with a complete loss of SMARCB1/INI1 protein expression was recognized. The median values of SMARCB1/INI1 mRNA expression in non-tumor skeletal muscle and synovial sarcoma with reduced protein expression were 12.86 and 134.01, respectively, and a statistically significant difference was detected between these two groups (P=0.0000004). However, there was no statistically significant difference of prognosis between the synovial sarcoma group with reduced and that with preserved SMARCB1/INI1 protein expression (P=0.46). Therefore, it was suggested that there is a post-transcriptional SMARCB1/INI1 regulatory mechanism in the tumor cells of synovial sarcoma.

The Akt/mammalian target of rapamycin pathway is activated and associated with adverse prognosis in soft tissue leiomyosarcomas.

The Akt/mammalian target of rapamycin (mTOR) pathway mediates cell survival and proliferation and contributes to tumor progression. Soft tissue leiomyosarcoma continues to show poor prognosis, and little is known about its mechanisms of tumor progression. Here the authors investigated the significance of activation of the Akt/mTOR pathway in soft tissue leiomyosarcomas.

Prognostic significance of p14ARF, p15INK4b, and p16INK4a inactivation in malignant peripheral nerve sheath tumors

Purpose:p14ARF, p15INK4b, and p16INK4a are tumor suppressor genes that are located closely at 9p21 and are often coinactivated by genetic or epigenetic alterations. Malignant peripheral nerve sheath tumor (MPNST) is a rare sarcoma with poor prognosis. However, the prognostic implications of inactivation of p14ARF, p15INK4b, and p16INK4a in MPNSTs have not been adequately investigated. Here we carried out a genetic, epigenetic, and expression analysis of p14ARF, p15INK4b, and p16INK4a, and clarified the prognostic significance of their inactivation in MPNSTs. Experimental Design: p14ARF, p15INK4b, and p16INK4a protein expressions were assessed by immunohistochemistry in 129 formalin-fixed samples of MPNST including 85 primary tumors. Thirty-nine samples, for which frozen material was available, were also investigated by Western blotting and quantitative reverse transcription PCR (RT-PCR) to detect p14ARF, p15INK4b, and p16INK4a protein and mRNA expression, and by multiplex real-time PCR, PCR single strand conformation polymorphism and methylation-specific PCR to detect p14ARF, p15INK4b, and p16INK4a gene alterations. Results: Immunohistochemically decreased expressions of p14ARF, p15INK4b, and p16INK4a were observed in 48%, 54%, and 49% of primary MPNSTs, respectively, and were significantly correlated with their concordant mRNA levels. As for gene alterations, homozygous deletion of CDKN2A was detected in one third of the cases. Inactivation of p14ARF and p16INK4a was associated with poor prognosis by both univariate and multivariate analyses. Furthermore, cases with inactivation of all p14ARF, p15INK4b, and p16INK4a genes showed the worst prognosis in a combined prognostic assessment. Conclusion: A comprehensive analysis of p14ARF, p15INK4b, and p16INK4a inactivation status provides useful prognostic information in MPNSTs. Clin Cancer Res; 17(11); 3771–82. ©2011 AACR.

Absence of human herpesvirus-8 and Epstein-Barr virus in inflammatory myofibroblastic tumor with anaplastic large cell lymphoma kinase fusion gene.

Inflammatory myofibroblastic tumor (IMT) is clinically and histologically characterized by inflammation. Some populations of IMT have anaplastic large cell lymphoma kinase (ALK) gene rearrangements. Infection with Epstein–Barr virus (EBV) and human herpesvirus‐8 (HHV‐8) in tumor cells of IMT has been reported; these reports, however, have been limited to ALK‐negative IMT. The purpose of the present paper was to evaluate 21 cases of IMT for the presence of EBV and HHV‐8. Immunohistochemically, 15 cases were ALK positive and six were negative. Of eight cases analyzed using reverse transcription–polymerase chain reaction, tropomyosin 3 (TPM3)‐ALK, TPM4‐ALK and clathrin heavy chain‐ALK fusion genes were detected in one, two and two cases, respectively. All 21 IMT, irrespective of ALK expression, were negative for EBV by in situ hybridization for EBV‐encoded RNA and immunohistochemical stain for latent membrane antigen‐1. HHV‐8 was also negative in all IMT by PCR for HHV‐8 DNA sequence (KS330/233) and immunohistochemical stain for latent nuclear antigen. These results suggest that IMT may be a heterogeneous group in terms of pathogenesis, and EBV and HHV‐8 do not play a major role in the pathogenesis of ALK‐positive tumor.

ALK, ROS1 and NTRK3 gene rearrangements in inflammatory myofibroblastic tumours.

The aim of this study was to elucidate the pathological features of inflammatory myofibroblastic tumour (IMT) with gene rearrangement other than ALK.

Highly aggressive behavior of malignant rhabdoid tumor: a special reference to SMARCB1/INI1 gene alterations using molecular genetic analysis including quantitative real-time PCR

PurposeSMARCB1/INI1, which negatively regulates cell cycle progression from G0/G1 into the S-phase via the p16INK4a-RB-E2F pathway, has been reported to be inactivated homozygously by deletion and/or mutations in malignant rhabdoid tumor (MRT). In the current study, we investigated the alteration of the SMARCB1/INI1 gene using simple methods, and its gene product at the protein level. Moreover, we investigated the status of hyperphosphorylation in RB protein, known as a key cell cycle molecule.MethodsThree cell lines and 11 formalin-fixed, paraffin-embedded specimens of MRT were investigated. SMARCB1/INI1 gene alteration was analyzed with simple methods as a quantitative real-time PCR and direct sequencing method. Furthermore, SMARCB1/INI1 and RB protein were immunohistochemically evaluated.ResultsIn 12 of 14 cases, we detected genetic alterations comprised of nine (including three cell lines) homozygous deletions and three mutations, which can induce abnormal expression of gene products. At the protein level, SMARCB1/INI1 immunohistochemical expressions were not detected in any cases. Twelve out of 14 cases showed high-level (+5) expression of tRB (both hyperphosphorylated and underphosphorylated RB), combined with low-level (+1) expression of uRB (underphosphorylated RB), indicating a high rate of hyperphosphorylation.ConclusionsWe could analyze the SMARCB1/INI1 gene alteration with simple methods, and SMARCB1/INI1 gene alteration was found in 12 of 14 cases. Especially, quantitative real-time PCR was a convenient and accurate method. In addition, a high rate of hyperphosphorylation of RB gene was recognized. These results suggest that the clinically aggressive character of MRT is caused by the inactivation of the SMARCB1/INI1 gene.