Low NCOR2 levels in multiple myeloma patients drive multidrug resistance via MYC upregulation.

MYC upregulation is associated with multidrug refractory disease in patients with multiple myeloma (MM). We, isolated patient-derived MM cells with high MYC expression and discovered that NCOR2 was down-regulated in these cells. NCOR2 is a transcriptional coregulatory protein and its role in MM remains unknown. To define the role of NCOR2 in MM, we created NCOR2 knockout human myeloma cell lines and demonstrated that NCOR2 knockout led to high MYC expression. Furthermore, NCOR2 knockout conferred resistance to pomalidomide, BET and HDAC inhibitors, independent of Cereblon (CRBN), indicating high MYC expression as a cause of multidrug resistance. Moreover, NCOR2 interacted with the nucleosome remodeling and deacetylase (NuRD) complex and repressed the expression of CD180 by directly binding to its promoter and inducing MYC expression. Next, we generated lenalidomide-resistant and pomalidomide-resistant human myeloma cell lines. Whole-exome sequencing revealed that these cell lines acquired the same exonic mutations of NCOR2. These cell lines showed NCOR2 downregulation and MYC upregulation independent of CRBN and demonstrated resistance to BET and HDAC inhibitors. Our findings reveal a novel CRBN independent molecular mechanism associated with drug resistance. Low NCOR2 expression can serve as a potential biomarker for drug resistance and needs further validation in larger prospective studies.

KCTD12 is negatively regulated by Kit in gastrointestinal stromal tumors.

Our group has previously demonstrated that pfetin, encoded by the KCTD12 gene, is a strong prognostic biomarker for gastrointestinal stromal tumors (GISTs). However, the underlying mechanisms that control pfetin expression remain unknown. To elucidate the regulatory mechanisms of KCTD12 in GIST, in addition to a possible association between KCTD12 alterations and protein expression, we examined 76 patients with GISTs for KCTD12 mutations by PCR-direct sequence, and compared these results with clinicopathologic data. The function of pfetin in GIST progression was also revealed using GIST T1 cells. In this series, pfetin expression was not observed in 15 cases, and loss of pfetin expression was associated with higher mitotic rate (>5/50HPFs: p = 0.029). There was also a trend between presence of necrosis and loss of pfetin expression but this was not statistically significant (p = 0.09). KCTD12 mutations were frequently observed in 22 out of 76 GISTs (28.9%); however, they did not affect protein expression and were not associated with patients' prognosis. KCTD12 in vitro knockdown resulted in the accelerated growth of GIST T1 cells, confirming that pfetin functions as a tumor suppressor. KIT knockdown significantly inhibited cellular growth and upregulated the expression of pfetin at both the mRNA and protein level. These findings suggest that GISTs with loss of pfetin expression has proliferative advantage and that higher pfetin expression in GISTs may be indicative of lower expression levels of KIT. This relationship confirms that pfetin is a useful prognostic marker in GISTs.

PPP2R1A regulated by PAX3/FOXO1 fusion contributes to the acquisition of aggressive behavior in PAX3/FOXO1-positive alveolar rhabdomyosarcoma.

To better characterize the oncogenic role of the PAX3-FOXO1 fusion protein in the acquisition of aggressive behavior in ARMS, we employed a proteomic approach using a PAX3-FOXO1 knockdown system in ARMS cell lines. This approach revealed a protein list consisting of 107 consistently upregulated and 114 consistently downregulated proteins that were expected to be regulated by PAX3-FOXO1 fusion protein. Furthermore, we identified 16 upregulated and 17 downregulated critical proteins based on a data-mining analysis. We also evaluated the function of PPP2R1A in ARMS cells. The PPP2R1A expression was upregulated at both the mRNA and protein levels by PAX3-FOXO1 silencing. The silencing of PPP2R1A significantly increased the cell growth of all four ARMS cells, suggesting that PPP2R1A still has a tumor suppressive function in ARMS cells; however, the native expression of PPP2R1A was low in the presence of PAX3-FOXO1. In addition, the activation of PP2A-part of which was encoded by PPP2R1A-by FTY720 treatment in ARMS cell lines inhibited cell growth. On the human phospho-kinase array analysis of 46 specific Ser/Thr or Tyr phosphorylation sites on 39 selected proteins, eNOS, AKT1/2/3, RSK1/2/3 and STAT3 phosphorylation were decreased by FTY-720 treatment. These findings suggest that PPP2R1A is a negatively regulated by PAX3-FOXO1 in ARMS. The activation of PP2A-probably in combination with kinase inhibitors-may represent a therapeutic target in ARMS. We believe that the protein expression profile associated with PAX3-FOXO1 would be valuable for discovering new therapeutic targets in ARMS.

IRE1α-XBP1 inhibitors exerted anti-tumor activities in Ewing's sarcoma.

Ewing's sarcoma (ES) is the second-most frequent pediatric bone tumor. Chromosomal translocation t(11;22)(q24:q12) results in the formation of EWS/FLI1 gene fusion, which is detected in approximately 90% of tumors of the Ewing family. Several transcriptome studies have provided lists of genes associated with EWS/FLI1 expression. However, the protein expression profiles associated with EWS/FLI1 have yet to be elucidated. In this study, to identify the regulated proteins associated with EWS/FLI1 and therapeutic targets in ES, we conducted proteomic studies using EWS/FLI1 knockdown in four Ewing's sarcoma cell lines and human mesenchymal stem cells (hMSCs) expressing EWS/FLI1. Isobaric tags for relative and absolute quantitation (i-TRAQ) analyses identified more than 2,000 proteins regulated by the EWS/FLI1 fusion. In addition, the network analyses identified several critical pathways, including XBP1, which was ranked the highest. XBP1 is a protein well known to play an important role in the unfolded protein response (UPR) to endoplasmic reticulum (ER) stress through the IRE1α-XBP1 pathway. We confirmed the high mRNA expression of XBP1 (spliced XBP1 and unspliced XBPl) in surgical samples and cell lines in ES. The silencing of XBP1 significantly suppressed the cell viabilities in ES cell lines. In the inhibitor assays using IRE1α-XBP1 inhibitors, including toyocamycin, we confirmed that these agents significantly suppressed the cell viabilities, leading to apoptosis in ES cells both in vitro and in vivo. Our findings suggested that IRE1α-XBP1 inhibitors might be useful for developing novel therapeutic strategies in ES.

Proteomic signatures corresponding to the SS18/SSX fusion gene in synovial sarcoma.

Synovial sarcoma (SS) is a malignant soft tissue lesion and most commonly arises in young adults. Chromosomal translocation t(X;18)(p11;q11) results in the formation of SS18/SSX by gene fusion of the SS18 gene on chromosome 18 to either SSX1, SSX2, or SSX4 gene located on chromosome X, which is detected in more than 95% of SSs. Although multiple lines of evidence suggest that the SS18/SSX fusion is the oncogene in this tumor, the protein expression profiles associated with SS18/SSX have yet to be elucidated. In this study, we conducted proteomic studies using SS18/SSX knockdown in three SS cell lines to identify the regulated proteins associated with SS18/SSX in SS. Isobaric tags for relative and absolute quantitation (i-TRAQ) analyses identified approximate 1700-2,000 proteins regulated by the SS18/SSX fusion in each SS cell line. We also analyzed the three profiles to identify proteins that were similarly altered in all 3 cell lines and found 17 consistently upregulated and 18 consistently downregulated proteins, including TAGLN and ACTN4. In addition, network analyses identified several critical pathways including RUNX2 and SMARCA4. RUNX2 and SMARCA4 had the highest ranking in these identified pathways. In addition, we found that expression of TAGLN inhibited cell viability in SS cell lines. Our data suggest that the differentiation and cell growth of SS may be enhanced by the identified proteins induced by SS18/SSX. We believe that the findings obtained in the present functional analyses will help to improve our understanding of the relationship between SS18/SSX and malignant behavior in SS.

Cabozantinib and dastinib exert anti-tumor activity in alveolar soft part sarcoma.

BACKGROUND: Alveolar soft part sarcoma (ASPS) is an extremely rare metastatic soft tissue tumor with a poor prognosis for which no effective systemic therapies have yet been established. Therefore, the development of novel effective treatment approaches is required. Tyrosine kinases (TKs) are being increasingly used as therapeutic targets in a variety of cancers. The purpose of this study was to identify novel therapeutic target TKs and to clarify the efficacy of TK inhibitors (TKIs) in the treatment of ASPS. EXPERIMENTAL DESIGN: To identify novel therapeutic target TKs in ASPS, we evaluated the antitumor effects and kinase activity of three TKIs (pazopanib, dasatinib, and cabozantinib) against ASPS cells using an in vitro assay. Based on these results, we then investigated the phosphorylation activities of the identified targets using western blotting, in addition to examining antitumor activity through in vivo assays of several TKIs to determine both the efficacy of these substances and accurate targets. RESULTS: In cell proliferation and invasion assays using pazopanib, cabozantinib, and dasatinib, all three TKIs inhibited the cell growth in ASPS cells. Statistical analyses of the cell proliferation and invasion assays revealed that dasatinib had a significant inhibitory effect in cell proliferation assays, and cabozantinib exhibited marked inhibitory effects on cellular functions in both assays. Through western blotting, we also confirmed that cabozantinib inhibited c-MET phosphorylation and dasatinib inhibited SRC phosphorylation in dose-dependent fashion. Mice that received cabozantinib and dasatinib had significantly smaller tumor volumes than control animals, demonstrating the in vivo antitumor activity of, these substances. CONCLUSIONS: Our findings suggest that cabozantinib and dasatinib may be more effective than pazopanib against ASPS cells. These in vitro and in vivo data suggest that c-MET may be a potential therapeutic target in ASPS, and cabozantinib may be a particularly useful therapeutic option for patients with ASPS, including those with pazopanib-resistant ASPS.

Reduced argininosuccinate synthetase expression in refractory sarcomas: Impacts on therapeutic potential and drug resistance.

BACKGROUND: Treating drug-resistant sarcomas remains a major challenge. The present study aimed to identify a novel therapy for drug-resistant sarcomas based on metabolic errors involving argininosuccinate synthetase1 (ASS1). RESULTS: ASS1 expression was reduced in Dox-resistant sarcoma cells. Immunohistochemistry and real-time PCR showed an inverse correlation between ASS1 and P-gp expressions. The inhibition of cellular proliferation with G1-arrest was shown to lead to autophagy with arginine deprivation. In addition, the combination of an autophagy inhibitor plus arginine deprivation was more effective than arginine deprivation alone. In cells with suppressed ASS1 expression, P-gp expression was upregulated as compared to that in negative controls. DISCUSSION: These results indicate that the reduced ASS1 expression in Dox-resistant sarcomas may contribute to drug resistance in association with the expression of P-gp. ASS1 deficiency is a potential target for novel drug therapies. The combination of arginine-deprivation therapy and an autophagy inhibitor may have anti-tumor effects in refractory sarcomas. METHODS: We assessed the expressions of ASS1 and P-glycoprotein (P-gp) in clinical specimens and cell lines of osteosarcoma (KHOS), doxorubicin (Dox)-resistant osteosarcoma (KHOSR2), epithelioid sarcomas (ES-X and VAESBJ) and alveolar soft part sarcoma (ASPS-KY). Each cell line was cultured in arginine-containing and arginine-free media. Cell growth was assessed using an XTT assay and flow cytometry. We analyzed the induction of autophagy in arginine-free medium. Moreover, we assessed the expression of P-gp after suppressing ASS1 in Dox-sensitive cells (MCF-7 and KHOS) and after transfecting ASS1 into Dox-resistant cells (ES-X, VAESBJ, ASPS-KY and KHOSR2).

Clinicopathological effects of protein phosphatase 2, regulatory subunit A, alpha mutations in gastrointestinal stromal tumors.

Recently, several studies have reported that dysfunctions in protein phosphatase 2A (PP2A) caused by alterations in protein phosphatase 2 regulatory subunit A, alpha (PPP2R1A) are responsible for tumorigenesis and tumor progression in several types of cancers. The impact of PPP2R1A mutations remains unknown in gastrointestinal stromal tumors (GISTs), although mutations in KIT and PDGFRA, which result in constitutive activation of the receptor tyrosine kinase pathway, are important in GIST tumorigenesis. In this study, we performed mutation analysis of PPP2R1A to examine the frequency of PPP2R1A mutations and their clinicopathological correlation in 94 GIST cases. In addition, we performed an in vitro analysis to investigate the effects of PPP2R1A mutations on cell proliferation and kinase phosphorylation in GIST cells. Seventeen GIST cases (18%) harbored mutations in PPP2R1A. All but one of these 17 cases harbored a KIT, PDGFRA, HRAS, NRAS, or KRAS mutation as the oncogenic driver mutation, and the remaining case was immunohistochemically negative for succinate dehydrogenase B (SDHB). Multivariate analysis showed that larger tumor size, higher mitotic rate, and PPP2R1A mutation are independent prognostic factors for overall survival; however, PPP2R1A mutation was not an independent prognostic factor for disease-free survival. The transduction of GIST cells with mutant PPP2R1A induced an accelerated growth rate via increased phosphorylation of Akt1/2, ERK1/2, and WNK1, a kinase associated with angiogenesis. In addition, the transduction of GIST cells with mutant PPP2R1A caused increased c-kit phosphorylation, suggesting that c-kit is also a target of PP2A, reinforcing the tumorigenic capabilities of c-kit. Furthermore, the transducing GIST cells with wild-type PP2A dephosphorylated mutant c-kit. This study provides a new insight into the biology of GISTs and their phosphatase activity, and activated PP2A could be a therapeutic target in GISTs.

Expression of F-actin-capping protein subunit beta, CAPZB, is associated with cell growth and motility in epithelioid sarcoma.

BACKGROUND: A previous proteomics study demonstrated the overexpression of F-actin capping protein subunit beta (CAPZB) in tissue specimens of epithelioid sarcoma (EpiS). The aim of the present study was to elucidate the function of CAPZB in EpiS. METHODS: Cellular functional assays were performed in two EpiS cell lines using CAPZB siRNAs. In addition, comparative protein expression analyses using Isobaric Tags for Relative and Absolute Quantitation (i-TRAQ) method were performed to identify the specific proteins whose expression was dysregulated by CAPZB, and analysed the data with the Ingenuity Pathways Analysis (IPA) system using the obtained protein profiles to clarify the functional pathway networks associated with the oncogenic function of CAPZB in EpiS. Additionally, we performed functional assays of the INI1 protein using INI1-overexpressing EpiS cells. RESULTS: All 15 EpiS cases showed an immunohistochemical expression of CAPZB, and two EpiS cell lines exhibited a strong CAPZB expression. Silencing of CAPZB inhibited the growth, invasion and migration of the EpiS cells. Analysis of protein profiles using the IPA system suggested that SWI/SNF chromatin-remodeling complexes including INI1 may function as a possible upstream regulator of CAPZB. Furthermore, silencing of CAPZB resulted in a decreased expression of INI1 proteins in the INI1-positive EpiS cells, whereas the induction of INI1 in the INI1-deficient EpiS cells resulted in an increased CAPZB mRNA expression. CONCLUSIONS: CAPZB is involved in tumor progression in cases of EpiS, irrespective of the INI1 expression, and may be a potential therapeutic target. The paradoxical relationship between the tumor suppressor INI1 and the oncoprotein CAPZB in the pathogenesis of EpiS remains to be clarified.

Protein Expression Profiling of Giant Cell Tumors of Bone Treated with Denosumab.

Giant cell tumors of bone (GCTB) are locally aggressive osteolytic bone tumors. Recently, some clinical trials have shown that denosumab is a novel and effective therapeutic option for aggressive and recurrent GCTB. This study was performed to investigate the molecular mechanism underlying the therapeutic effect of denosumab. Comparative proteomic analyses were performed using GCTB samples which were taken before and after denosumab treatment. Each expression profile was analyzed using the software program to further understand the affected biological network. One of identified proteins was further evaluated by gelatin zymography and an immunohistochemical analysis. We identified 13 consistently upregulated proteins and 19 consistently downregulated proteins in the pre- and post-denosumab samples. Using these profiles, the software program identified molecular interactions between the differentially expressed proteins that were indirectly involved in the RANK/RANKL pathway and in several non-canonical subpathways including the Matrix metalloproteinase pathway. The data analysis also suggested that the identified proteins play a critical functional role in the osteolytic process of GCTB. Among the most downregulated proteins, the activity of MMP-9 was significantly decreased in the denosumab-treated samples, although the residual stromal cells were found to express MMP-9 by an immunohistochemical analysis. The expression level of MMP-9 in the primary GCTB samples was not correlated with any clinicopathological factors, including patient outcomes. Although the replacement of tumors by fibro-osseous tissue or the diminishment of osteoclast-like giant cells have been shown as therapeutic effects of denosumab, the residual tumor after denosumab treatment, which is composed of only stromal cells, might be capable of causing bone destruction; thus the therapeutic application of denosumab would be still necessary for these lesions. We believe that the protein expression patterns and the results of the network analysis will provide a better understanding of the effects of denosumab administration in patients with GCTB.

TERT promoter mutations are rare in bone and soft tissue sarcomas of Japanese patients.

Recurrent hot-spot mutations in the telomerase reverse transcriptase (TERT) promoter have been reported in various types of tumor. In several tumor types, TERT promoter mutations are associated with poor clinical outcomes. TERT promoter mutations are reported to be rare in soft tissue tumors, with the exception of myxoid liposarcoma (MLS). Our previous study reported that TERT promoter mutations occurred in a subset of solitary fibrous tumors (SFTs) and were associated with adverse clinical outcomes in Japanese individuals. The site-specific frequency (e.g. central nervous or soft tissue origin) of TERT promoter mutations in our SFT cases appeared to be different from previously reported values in a European population. These findings prompted the present study to elucidate the potential role of ethnic background in the different frequencies of TERT promoter mutations in bone and soft tissue sarcomas. In the present study, TERT promoter mutations were examined in 180 cases of bone and soft tissue sarcomas. TERT promoter region mutations were identified in 10 cases [5 SFTs, 3 MLSs, 1 undifferentiated pleomorphic sarcoma (UPS) and 1 malignant granular cell tumor]. All mutations were C228T. The frequencies of TERT promoter mutation in MLS and UPS were 23.1 (3/13) and 5% (1/20), respectively. Only 1/5 patients with TERT-mutated tumors experienced local recurrence or distant metastasis. The present study revealed the first case of a malignant granular cell tumor with a TERT promoter mutation and revealed that the frequency of TERT promoter mutations in MLSs of Japanese patients is lower compared with that reported in German patients, providing evidence of a possible ethnic difference in the frequency of TERT promoter mutations.

Sarcomatous transformation of EGFR and TP53 mutation-positive metastatic adenocarcinoma of the lungs, masquerading as a primary pleomorphic sarcoma of the proximal femur.

We investigated a case of metastatic adenocarcinoma of the lungs at the left proximal femur, masquerading as a primary pleomorphic sarcoma. A 72-year-old woman presented with pain in her left thigh in conjunction with a mass that had been gradually growing over a few months. She was being treated with gefitinib for lung adenocarcinoma positive for the epidermal growth factor receptor (EGFR) mutation L858R, and had multiple bone metastases. The lung adenocarcinoma and metastases had stabilized with the treatment. The metastatic lesions in the bone had also received radiation; however, a tumor in the proximal femur kept growing despite treatment. A biopsy specimen from the proximal femur revealed the proliferation of spindle-shaped cells without an epithelial glandular component. The patient underwent en bloc resection of the proximal femur that was replaced by prosthesis. Histologically, the resected tumor was entirely composed of pleomorphic cells and tumor giant cells exhibiting no apparent glandular structures. Tumor cells were diffusely positive for p53 and focally positive for epithelial markers and EGFR, but were negative for thyroid transcription factor-1, suggesting an initial diagnosis of primary pleomorphic sarcoma. Genetic examination revealed mutations in EGFR and p53 that were of the same type as the lung tumor, leading to the final diagnosis of the femoral mass as a sarcomatous transformation of metastatic lung adenocarcinoma. However, secondary genetic alterations that might explain the acquired resistance to gefitinib could not be found in the proximal femoral tumor. The patient remains alive and the remaining lesions are well controlled.

Skeletal Metastasis of Unknown Primary Origin at the Initial Visit: A Retrospective Analysis of 286 Cases.

BACKGROUND: Skeletal metastasis is a common metastatic event for several carcinomas, and the treatment for skeletal metastasis of unknown primary (SMUP) are a critical issue in cancer therapy. Making a diagnosis of the primary site is the most crucial step in the treatment of SMUP; however, the procedures are sometimes difficult and time-consuming, and the primary site often remains unknown. Therefore, to establish optimal diagnostic strategies and elucidate the overall survival rates of SMUP, we conducted this retrospective study. METHODS: We retrospectively analyzed the clinical data for 286 SMUP cases from a total of 2,641 patients with skeletal metastases who were treated between 2002 and 2014 at our initiations. RESULTS: The primary sites were identified in 254/286 patients (88.8%), while 32 (11.2%) primary sites were not detected by our diagnostic strategies. Lung cancer was identified in 72 (25.2%) cases, and was the most frequently observed primary lesion. The median survival time of the SMUP patients was 20.0 months, while the median survival times of solitary bone metastasis cases and multi-bone metastasis cases were 39.0 months and 16.0 months, respectively. The median survival times of prostate cancer cases was over 120 months, that of patients with primary lung cancers was 9.0 months and the median survival time of cases who were finally diagnosed with an unknown primary was 11.0 months. CONCLUSIONS: We believe that our study would contribute to establishing an optimal strategy for diagnosing the primary site in SMUP patients, and our data provide definite indications for the survival times for different SMUP situations.

TERT promoter mutations are a rare event in gastrointestinal stromal tumors.

Recently, the impact of telomere dysregulation on malignant progression has been reported in many cancers. A few studies have examined TERT promoter mutations in gastrointestinal stromal tumors (GISTs). Irregular telomerase activation can be maintained by TERT hot spot alterations and alternative lengthening of telomeres (ALT) characterized by inactivation of either the alpha-thalassemia/mental retardation syndrome X-linked (ATRX) or death domain-associated protein (DAXX). To elucidate the clinicopathological impact of telomere dysregulation in GISTs, we examined 92 cases of GISTs for TERT promoter hot spot mutations along with immunohistochemical analysis of ATRX and DAXX expression, and compared these findings with the clinicopathological features. Univariate clinicopathological analysis revealed that tumor site, smaller tumor size, presence of necrosis, higher mitotic rate (>5/50 high-power fields) and risk classification were prognostic factors for either disease-free survival or overall survival. Two of 92 informative cases (2.2 %) were found to have heterozygous TERT promoter mutations (C228T), and these mutations occurred in a low-risk and a high-risk tumor, respectively. On immunohistochemical analysis for ATRX and DAXX, 16 (17.4 %) and 3 (3.3 %) of 92 cases showed loss of expression of ATRX and DAXX, respectively. Loss of expression of ATRX and DAXX were mutually exclusive except for one case. TERT promoter mutations were also mutually exclusive of the ALT phenotype. Telomere dysregulation was not associated with patient survival; however, telomere dysregulation was frequently observed in tumors of extra-gastric origin, which have an adverse outcome compared to those of gastric origin.

Proteomics study of open biopsy samples identifies peroxiredoxin 2 as a predictive biomarker of response to induction chemotherapy in osteosarcoma.

We attempted to identify biomarkers that would predict responsiveness of osteosarcoma (OS) to induction chemotherapy. Tumor tissues obtained by open biopsy before induction chemotherapy were investigated. On the basis of histological observations at the time of surgery and the Huvos grading system, 7 patients were classified as good responders and the other 6 as poor responders. Protein expression profiling was performed by two-dimensional difference gel electrophoresis. Among 3494 protein spots observed, the intensity of 33 spots was found to differ significantly between the two patient groups. The proteins for these 33 protein spots were identified by mass spectrometry. The higher expression of peroxiredoxin 2 (PRDX2) in poor responders was confirmed by Western blotting. Gene silencing assay demonstrated that reduced expression of PRDX2 was associated with increased sensitivity of OS cells to chemotherapeutic drugs such as methotrexate, doxorubicin and cisplatin. Moreover, siRNA-induced silencing of PRDX2 resulted in a decrease of cell proliferation, invasion and migration. These findings indicated that PRDX2 would be a candidate biomarker of response to induction chemotherapy. Measurement of PRDX2 in open biopsy samples before treatment may contribute to risk stratification therapy for OS. BIOLOGICAL SIGNIFICANCE: The response of osteosarcoma patients to induction chemotherapy is critical because the prognosis of responders is quite favorable, whereas that of non-responders is poor. Although there are many therapeutic options for osteosarcoma, no parameter for predicting the response to induction chemotherapy has been available. We conducted a proteomics study aimed at developing a biomarker that would predict the response of osteosarcoma to induction chemotherapy. Using open biopsy samples obtained before chemotherapy, we conducted 2D-DIGE with our originally devised large-format electrophoresis apparatus and identified peroxiredoxin 2 (PRDX2) as a novel predictive biomarker. The diagnostic performance of PRDX2 was confirmed by ROC analysis, and its functional properties were investigated in a series of in vitro functional assays. Our findings indicate the possible application of PRDX2 as a predictive biomarker in patients with osteosarcoma.

The prognostic value of pfetin: a validation study in gastrointestinal stromal tumors using a commercially available antibody.

OBJECTIVE: Adjuvant treatment with imatinib mesylate is an effective treatment for gastrointestinal stromal tumor. However, 50% of patients with gastrointestinal stromal tumor can be cured by surgery alone; hence, risk stratification for therapy with imatinib mesylate is the next challenge. Previously, using a proteomic approach, we discovered a potential prognostic biomarker for gastrointestinal stromal tumor, pfetin, and immunohistochemically validated its clinical utility using our original monoclonal antibody. In the present study, we examine the usefulness of a commercially available polyclonal antibody against pfetin. METHODS: Western blotting and immunohistochemistry were performed using surgical specimens of primary tissues from gastrointestinal stromal tumor patients using a polyclonal antibody against pfetin and our original monoclonal antibody. Formalin-fixed and paraffin-embedded primary tissue sections from 112 gastrointestinal stromal tumor patients were subjected to immunohistochemistry. The immunohistochemistry results were integrated with the clinico-pathological observations. RESULTS: Western blotting revealed that both antibodies recognized multiple post-translationally modified pfetin isoforms. The immunohistochemical study with the commercial antibody demonstrated that the disease-free survival rate was 88 and 56% for pfetin-positive and pfetin-negative patients, respectively. Univariate and multivariate analyses showed that pfetin expression as measured by the commercial antibody was a significant and independent prognostic factor among the clinico-pathological parameters examined. Of the 112 gastrointestinal stromal tumor cases examined, 13 yielded discordant results between the commercial antibody and our original antibody, and there were no significantly different clinical or pathological factors to account for this discrepancy. CONCLUSIONS: Our observations suggest that the pfetin expression level assessed by the commercial antibody could be a prognostic biomarker in gastrointestinal stromal tumors.

Proteomic approach toward personalized sarcoma treatment: lessons from prognostic biomarker discovery in gastrointestinal stromal tumor.

Sarcomas range from curable tumors to those causing death via metastasis and recurrence. Thus, there is an urgent need for biomarker identification in order to assess the degree of malignancy, predict prognosis, and evaluate possible therapies. Various proteomic approaches and different clinical materials have been used to this end, and candidate biomarkers have been reported for the different types of sarcomas. However, the sample size used in these biomarker studies was generally insufficient, and thus far, no biomarker has been proved useful in clinics. Given that sarcomas are rare, biomarker validation in this setting is more challenging than in other malignancies. In gastrointestinal stromal tumor, adjuvant therapy has proven to be effective. However, only 40% patients experience metastasis after curative surgery alone, and the rest of the patients may not need adjuvant therapy. Using a proteomic approach, we identified pfetin (potassium channel tetramerization domain containing 12, KCTD 12) as a novel prognostic biomarker for sarcoma, and immunohistochemically confirmed its clinical usefulness by a multiinstitutional validation study. Here, we describe our experience and discuss the critical points in the discovery of this biomarker.