Katsuya Nakanishi

Detection of specific genetic abnormalities by fluorescence in situ hybridization in soft tissue tumors

For the detection of chromosome translocations/chimeric genes and specific genetic abnormalities in soft tissue tumors, we conducted fluorescence in situ hybridization (FISH) analysis on 280 cases of soft tissue and other tumors using formalin‐fixed paraffin‐embedded tissue sections. The detection rate of the FISH split‐signal was 84% (129/154 cases) for the translocation‐associated soft tissue tumors, such as Ewings sarcoma/primitive neuroectodermal tumor, synovial sarcoma, alveolar rhabdomyosarcoma, myxoid liposarcoma, clear cell sarcoma and so forth. Positive split‐signals from EWSR1, SS18 and FOXO1A probes were detected in 3% (2/64) of various histological types of carcinoma, lymphoma, melanoma, meningioma and soft tissue tumors. In FISH using the INI1/CEP22 probe, the INI1 deletion signal was detected in 100% (9/9) of epithelioid sarcoma. In well‐differentiated and dedifferentiated liposarcomas, detection of MDM2 amplification signals in FISH using the MDM2/CEP12 probe were both as high as 85% (11/13) and 100% (13/13), respectively. In other adipocytic and non‐adipocytic tumors requiring differentiation from these types, detection was only 13% (5/39), and CEP12 polysomy was frequently detected. As these results demonstrate the high sensitivity and specificity of FISH, we concluded FISH to be a useful pathological diagnostic adjunct for definite and differential diagnosis of soft tissue tumors.

A novel isoform of TUCAN is overexpressed in human cancer tissues and suppresses both caspase-8- and caspase-9-mediated apoptosis.

Caspase-associated recruitment domains (CARD) are protein-protein interaction modules found extensively in proteins that play important roles in apoptosis. One of the CARD-containing proteins, TUCAN (CARD8), was reported previously as an antiapoptotic protein with a molecular weight of 48 kDa, which was up-regulated in colon cancer cells. We identified a novel isoform of TUCAN with a molecular weight of 54 kDa. The new variant of TUCAN, termed TUCAN-54, was expressed in gastric, colon, and breast cancer tissues but was barely detected in normal noncancerous tissues, whereas 48-kDa TUCAN was detected in tumor tissues and noncancerous tissues. To know the function of TUCAN-54 in the apoptosis of cancer cells, TUCAN-54 was overexpressed in tumor cells by gene transfection. Its overexpression inhibited pro-caspase-9 activation, leading to the suppression of the cell death induced by a protein kinase inhibitor, staurosporine, or a chemotherapeutic reagent, etoposide (VP-16). In contrast, specific small interfering RNA-mediated suppression of TUCAN-54 expression in tumor cells increased the VP-16-induced cell death rate, indicating that expression of TUCAN-54 might be associated with chemoresistance of tumor cells. In addition, it inhibited caspase-8 activation as well, thereby suppressing Fas-induced cell death. It was revealed that Fas-associated death domain was physically associated with TUCAN-54 but not with 48-kDa TUCAN. Thus, TUCAN-54 might be a novel tumor-specific antiapoptotic molecule expressed in a variety of human cancer tissues, which might aggravate malignant potential of cancer cells, such as chemoresistance and immunoresistance.

Localization and function in endoplasmic reticulum stress tolerance of ERdj3, a new member of Hsp40 family protein

Abstract Heat shock protein 40 (Hsp40) family proteins are known to bind to Hsp70 through their J-domain and regulate the function of Hsp70 by stimulating its adenosine triphosphatase activity. In the endoplasmic reticulum (ER), there are 5 Hsp40 family proteins known so far, 3 of which were recently identified. In this report, one of the novel Hsp40 cochaperones, ERdj3, was characterized in terms of its subcellular localization, stress response, and stress tolerance of cells. By using ERdj3-specific polyclonal antibody, endogenous ERdj3 protein was shown to reside in the ER as gene transfer–mediated exogenous ERdj3. Analysis of the expression level of endogenous ERdj3 protein revealed its moderate induction in response to various ER stressors, indicating its possible action as a stress protein in the ER. Subsequently, we analyzed whether this molecule was involved in ER stress tolerance of cells, as was the case with the ER-resident Hsp70 family protein BiP. Although overexpression of ERdj3 by gene transfection could not strengthen ER stress tolerance of neuroblastoma cells, reduction of ERdj3 expression by small interfering ribonucleic acid decreased the tolerance of cells, indicating that ERdj3 might have just a marginal role in the ER stress resistance of neuroblastoma cells. In contrast, overexpression of ERdj3 notably suppressed vero toxin–induced cell death. These data suggest that ERdj3 might have diverse roles in the ER, including that of the molecular cochaperone of BiP and an as yet unknown protective action against vero toxin.

Tumor-Produced Secreted Form of Binding of Immunoglobulin Protein Elicits Antigen-Specific Tumor Immunity

Binding of immunoglobulin protein (BiP) is a major molecular chaperone localized in endoplasmic reticulum (ER). It has been demonstrated to interact with nascent Ig. However, contrary to other ER-resident heat shock proteins such as gp96, calreticulin, and ORP150, it is not clear whether tumor-derived BiP plays a role in inducing antitumor immunity. In this study, we show that the tumor-derived secreted form of BiP is capable of inducing antitumor CD8+ T cell responses. We constructed an ER-retention signal KDEL-deleted mutant of BiP cDNA and transfected it to tumor cells, which resulted in continuous secretion of tumor-derived BiP into the extracellular milieu. We show that this secreted BiP is taken up by bone marrow-derived dendritic cells, and thereafter BiP-associated Ag peptide is cross-presented in association with MHC class I molecules, resulting in elicitation of an Ag-specific CD8+ T cell response and antitumor effect. This strategy to boost antitumor immune responses shows that a tumor could be its own cellular vaccine via gene modification of the secretion of the tumor Ag–BiP complex.

Diagnostic utility of NCOA2 fluorescence in situ hybridization and Stat6 immunohistochemistry staining for soft tissue angiofibroma and morphologically similar fibrovascular tumors

Soft tissue angiofibroma (STA), a recently suggested new histologic entity, is a benign fibrovascular soft tissue tumor composed of bland spindle-shaped tumor cells with abundant collagenous to myxoid stroma and branching small vessels. The lesion has a characteristic AHRR-NCOA2 fusion gene derived from chromosomal translocation of t(5;8)(p15;q13). However, morphologically similar tumors containing abundant fibrovascular and myxoid stroma can complicate diagnosis. We designed an original DNA probe for detecting NCOA2 split signals on fluorescence in situ hybridization (FISH) and estimated its utility with 20 fibrovascular tumors: 4 each of STAs, solitary fibrous tumors (SFTs), and cellular angiofibromas and 3 each of low-grade myxofibrosarcomas, myxoid liposarcomas, and low-grade fibromyxoid sarcomas. We also performed FISH for 13q14 deletion and immunohistochemistry (IHC) staining for estrogen receptor, progesterone receptor, retinoblastoma protein, and MUC-4 expression. Furthermore, IHC for Stat6 was conducted in the 20 cases analyzed by FISH and in an additional 26 SFTs. We found moderate to strong nuclear Stat6 expression in all SFTs but no expression in the other tumors. Both estrogen receptor and progesterone receptor expressions were observed in STAs, SFTs, and cellular angiofibromas. Expression of retinoblastoma protein was found in less than 10% of cells in all tumor types except myxoid liposarcoma. The low-grade fibromyxoid sarcomas were strongly positive for MUC-4. All STAs showed NCOA2 split signals on FISH. All tumors, regardless of histologic type, had 13q14 deletion. The NCOA2 FISH technique is a practical method for confirming STA diagnosis. The combination of NCOA2 FISH and Stat6 IHC proved effective for the differential diagnosis of STA, even when using small biopsy specimens.

A novel nuclear DnaJ protein, DNAJC8, can suppress the formation of spinocerebellar ataxia 3 polyglutamine aggregation in a J-domain independent manner.

Polyglutamine (polyQ) diseases comprise neurodegenerative disorders caused by expression of expanded polyQ-containing proteins. The cytotoxicity of the expanded polyQ-containing proteins is closely associated with aggregate formation. In this study, we report that a novel J-protein, DNAJ (HSP40) Homolog, Subfamily C, Member 8 (DNAJC8), suppresses the aggregation of polyQ-containing protein in a cellular model of spinocerebellar ataxia type 3 (SCA3), which is also known as Machado-Joseph disease. Overexpression of DNAJC8 in SH-SY5Y neuroblastoma cells significantly reduced the polyQ aggregation and apoptosis, and DNAJC8 was co-localized with the polyQ aggregation in the cell nucleus. Deletion mutants of DNAJC8 revealed that the C-terminal domain of DNAJC8 was essential for the suppression of polyQ aggregation, whereas the J-domain was dispensable. Furthermore, 22-mer oligopeptide derived from C-termilal domain could suppress the polyQ aggregation. These results indicate that DNAJC8 can suppress the polyQ aggregation via a distinct mechanism independent of HSP70-based chaperone machinery and have a unique protective role against the aggregation of expanded polyQ-containing proteins such as pathogenic ataxin-3 proteins.

Ovarian high-grade endometrioid stromal sarcoma with YWHAE and NUTM2B rearrangements

To the Editor: Here, we present an extremely rare case of primary ovarian high-grade endometrioid stromal sarcoma (OHGESS) with YWHAE and NUTM2B rearrangements in an elderly woman. A 65-year-old woman with a small mass in the right submandibular gland had undergone resection of the mass in a hospital about 2 years earlier. The pathological diagnosis of the mass was diffuse large B-cell lymphoma (DLBCL). A month after the surgery, postoperative assessment by positron emission tomographycomputed tomography and magnetic resonance imaging (MRI) revealed a multinodular right ovarian mass compressing the anterior wall of the atrophic uterine corpus (Fig. S1a). Axial T2-weighted imaging also showed a multinodular mass with a homogeneous intermediateintensity signal on the right side and a heterogeneous irregular-intensity signal on the left side corresponding to degeneration (Fig. S1b). MRI additionally revealed multiple enlarged neck, mediastinal, and para-aortic lymph nodes and a nodule in the left gluteus medius. Based on a clinical diagnosis of primary right ovarian tumor and systematic involvement of DLBCL in the lymph nodes, she underwent hysterectomy and bilateral salpingo-oophorectomy in the Department of Gynecology, Sapporo Medical University Hospital. A month after the operation, she was treated with four cycles of rituximab, cyclophosphamide, doxorubicin, and prednisone (R-CHOP) therapy against DLBCL in the residual para-aortic lymph nodes. However, radiological evaluation by CT revealed no therapeutic effect in the lymph nodes. Based on the pathological diagnosis of OHGESS, we anticipated lymph node metastasis of the tumor, so she also underwent additional para-aortic and pelvic lymph node dissection with omentectomy. About 3 weeks after the second operation, an adjuvant chemotherapy regimen consisting of carboplatin and paclitaxel (TC) was started. After 9 courses of TC chemotherapy, multiple liver metastases emerged and the left gluteal nodule increased in size. She subsequently received second-line chemotherapy consisting of gemcitabine and docetaxel (GD) about 4 weeks after TC chemotherapy. After 3 courses of GD chemotherapy, the gluteal nodule and pelvic lymph nodes increased in size; she received further chemotherapy consisting of pegylated liposomal doxorubicin. About 2 years after the first operation, the patient died of the disease with multiple metastases of the tumor. No autopsy was performed. The right ovary was markedly enlarged and measured 12 7 5 cm in size. On cross-section, the tumor was milky-whitish, solid, and degenerated, with necrotic areas (Fig. S2a). There was no tumor in the uterus. Histologically, the tumor consisted of tight nests of mediumto large-sized round tumor cells separated by delicate vasculatures (Fig. 1a). The tumor cells had round to irregularly shaped nuclei with coarse chromatin, conspicuous nucleoli, and abundant eosinophilic cytoplasm (Fig. 1b). Mitotic figures were frequently observed (50/10 high-powered fields). The tumor cells diffusely metastasized to para-aortic and pelvic lymph nodes, and not to the greater omentum. On immunohistochemistry, the tumor cells were strongly positive for vimentin, c-kit (Fig. S2b), CD34 (Fig. S2c), BRG1, and cyclin D1 (approximately 70% in tumor nuclei) (Fig. 1c) and were negative for pan-cytokeratin AE1/AE3, CK7, CK20, EMA, desmin, a-SMA, muscle-specific actin HHF-35, S-100 protein, ER, PgR, DOG1, LCA, CD3, CD20, CD10, CD79a, CD43, CD56, chromogranin A, and synaptophysin. We performed FISH to detect a specific chimeric gene of YWHAE-NUTM2 by a custom dual-color, split-signal YWHAE probe set for the YWHAE locus on chromosome 17p13 (Chromosome Science Lab, Inc., Sapporo, Japan). We also used a custom break-apart probe for the NUTM2A locus on chromosome 10q23 and the NUTM2B locus on chromosome 10q22.3 (Agilent Technologies, Santa Clara, CA, USA). The former probe design consisted of a 501 kbp probe targeting the 50 region of the NUTM2A gene and a 397 kbp probe targeting the 30 region. The probes comprised 42250 (50 probe) and 41831 (30 probe) unique 100-200-mer oligonucleotides complementary to the targeted region and were labeled with FITC (50 probe) and Cy3 (30 probe) fluorophores. The latter probe design consisted of a 392 kbp probe targeting the 50 region of the NUTM2B gene and a 558 kbp probe targeting the 30 region. The probes comprised 39628 (50 probe) and 42108 (30 probe) unique 100-200-mer oligonucleotides complementary to the targeted region and were labeled with FITC (30 probe) and Cy3 (50 probe) fluorophores. These probes were synthesized using oligo-based SureFISH technology (Agilent). We counted 50 tumor nuclei for each probe set and observed numerous polyploid nuclei in 70% and 64% of nuclei by YWHAE and NUTM2B FISH, respectively. We detected two fused signals (yellow) and one split-signal for YWHAE rearrangement (isolated red and green) in 60% of tumor nuclei (Fig. 1d). In addition, we observed NUTM2B

Successful treatment of childhood hypocellular acute myeloid leukemia.

Hypocellular acute myeloid leukemia (AML) is extremely rare in childhood. We report on a 7-year-old girl with hypocellular AML who was treated successfully with granulocyte-colony stimulating factor (G-CSF) and combined chemotherapy. High-dose G-CSF induced complete remission and she subsequently received reduced intensity conditioning and unrelated cord blood transplantation; however, this resulted in early rejection. After a complete hematological recovery, she received 3 courses of combination chemotherapy oriented toward AML. She has remained in complete remission for over 1 year after the completion of the therapy. G-CSF effectively induced remission, and combination chemotherapy has been proven to be feasible for patients with childhood hypocellular AML.