Yoshiyuki Suehara

Response to Cabozantinib in Patients with RET Fusion-Positive Lung Adenocarcinomas

The discovery of RET fusions in lung cancers has uncovered a new therapeutic target for patients whose tumors harbor these changes. In an unselected population of non-small cell lung carcinomas (NSCLCs), RET fusions are present in 1% to 2% of cases. This incidence increases substantially, however, in never-smokers with lung adenocarcinomas that lack other known driver oncogenes. Although preclinical data provide experimental support for the use of RET inhibitors in the treatment of RET fusion-positive tumors, clinical data on response are lacking. We report preliminary data for the first three patients treated with the RET inhibitor cabozantinib on a prospective phase II trial for patients with RET fusion-positive NSCLCs (NCT01639508). Confirmed partial responses were observed in 2 patients, including one harboring a novel TRIM33-RET fusion. A third patient with a KIF5B-RET fusion has had prolonged stable disease approaching 8 months (31 weeks). All three patients remain progression-free on treatment.

Rationale for co-targeting IGF-1R and ALK in ALK fusion-positive lung cancer

Crizotinib, a selective tyrosine kinase inhibitor (TKI), shows marked activity in patients whose lung cancers harbor fusions in the gene encoding anaplastic lymphoma receptor tyrosine kinase (ALK), but its efficacy is limited by variable primary responses and acquired resistance. In work arising from the clinical observation of a patient with ALK fusion–positive lung cancer who had an exceptional response to an insulin-like growth factor 1 receptor (IGF-1R)-specific antibody, we define a therapeutic synergism between ALK and IGF-1R inhibitors. Similar to IGF-1R, ALK fusion proteins bind to the adaptor insulin receptor substrate 1 (IRS-1), and IRS-1 knockdown enhances the antitumor effects of ALK inhibitors. In models of ALK TKI resistance, the IGF-1R pathway is activated, and combined ALK and IGF-1R inhibition improves therapeutic efficacy. Consistent with this finding, the levels of IGF-1R and IRS-1 are increased in biopsy samples from patients progressing on crizotinib monotherapy. Collectively these data support a role for the IGF-1R–IRS-1 pathway in both ALK TKI–sensitive and ALK TKI–resistant states and provide a biological rationale for further clinical development of dual ALK and IGF-1R inhibitors.

Pfetin as a Prognostic Biomarker of Gastrointestinal Stromal Tumors Revealed by Proteomics

Purpose: We aimed to develop prognostic biomarkers for gastrointestinal stromal tumors (GIST) using a proteomic approach. Experimental Design: We examined the proteomic profile of GISTs using two-dimensional difference gel electrophoresis. The prognostic performance of biomarker candidates was examined using a large-scale sample set and specific antibodies. Results: We identified 43 protein spots whose intensity was statistically different between GISTs with good and poor prognosis. Mass spectrometric protein identification showed that the 43 spots corresponded to 25 distinct gene products. Eight of the 43 spots derived from pfetin, a potassium channel protein, and four of the eight pfetin spots had a high discriminative power between the two groups. Western blotting and real-time PCR showed that pfetin expression and tumor metastasis were inversely related. The prognostic performance of pfetin was also examined by immunohistochemistry on 210 GIST cases. The 5-year metastasis-free survival rate was 93.9% and 36.2% for patients with pfetin-positive and pfetin-negative tumors, respectively (P < 0.0001). Univariate and multivariate analyses revealed that pfetin expression was a powerful prognostic factor among the clinicopathologic variables examined, including risk classification and c-kit– or platelet-derived growth factor receptor A mutation status. Conclusions: These results establish pfetin as a powerful prognostic marker for GISTs and may provide novel therapeutic strategies to prevent metastasis of GIST.

Distinct clinicopathological features of NAB2-STAT6 fusion gene variants in solitary fibrous tumor with emphasis on the acquisition of highly malignant potential

The impact of NGFI-A binding protein 2 (NAB2)-signal transducer and activator of transcription 6 (STAT6) fusion on the biological behavior and the mechanism of acquisition of malignant phenotype in solitary fibrous tumor (SFT) is not well understood. We examined variations of the NAB2-STAT6 fusion gene in 40 cases of SFT using formalin-fixed, paraffin-embedded tissues and secondary genetic alterations of tumor protein p53 (TP53),, platelet-derived growth factor receptor, β polypeptide (PDGFRB), and telomerase reverse transcriptase (TERT) promoters. These gene variations were compared with the clinicopathological features. The 2-year and 5-year disease-free survival rates (DFSRs) were 91% and 83%, respectively. All 40 samples demonstrated nuclear staining for STAT6, including CD34-negative cases. Moreover, p53-positive staining was associated with a lower DFSR and was significantly associated with higher Ki-67 label index, higher mitotic rate (mitosis, >4/high-power field), and the presence of nuclear atypia/pleomorphism. NAB2-STAT6 fusions were detected in all of the cases; the NAB2 exon 4-STAT6 exon 2, the most common genotype, appeared in 18 cases, which was associated with thoracic tumor location and the less aggressive phenotype. In contrast, tumors with NAB2 exon 6-STAT6 exon 16/18 demonstrated an aggressive phenotype. Mutations in TP53 and PDGFRB were detected in 2 and 3 cases respectively, and these occurred in a mutually exclusive fashion. TERT promoter hot spot mutations were observed in 5 cases, which were associated with shorter DFSR. Two dedifferentiated SFT cases harbored both TP53 and TERT promoter mutations. TP53 mutations, which result in its overexpression, in combination with TERT promoter mutations seem to play an important role in the dedifferentiation process.

Clinical proteomics identified ATP-dependent RNA helicase DDX39 as a novel biomarker to predict poor prognosis of patients with gastrointestinal stromal tumor.

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal malignancy of the gastrointestinal tract, comprising a wide spectrum from a curable disorder to highly malignant disease. GIST is characterized by tyrosine kinase mutations, and molecular targeting therapies against these abnormal enzymes require prognostic biomarkers. To identify candidate prognostic biomarkers, we examined proteomic features corresponding to metastasis after surgery. Using two-dimensional difference gel electrophoresis with a large format gel, we compared the primary tumor tissues of GIST patients free of metastasis for two years after surgery (eight cases) with those of patients who developed metastasis within one year after surgery (nine cases). We found the intensities of 38 protein spots to differ significantly between the two groups. Mass spectrometric protein identification revealed that these corresponded to 25 unique genes. Immunohistochemical validation demonstrated ATP-dependent RNA helicase DDX39 to be significantly associated with metastasis and poor clinical outcomes in a group of 72 GIST patients. In conclusion, we have established a novel prognostic utility of ATP-dependent RNA helicase DDX39 in GIST.ATP-dependent RNA helicase DDX39, a novel biomarker for GIST likely to be associated with metastatic disease, can identify patients likely to benefit from new therapeutic strategies such as tyrosine kinase inhibitors.

Global Protein-expression Analysis of Bone and Soft Tissue Sarcomas

AbstractAnalysis of global protein expression, an approach known as expression proteomics, can offer important clues for understanding tumor biology that cannot be obtained by other approaches (e.g., genome or transcriptome analysis). Using two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry, we performed global protein expression studies of bone and soft tissue sarcomas to develop novel diagnostic and therapeutic biomarkers and allow molecular classification of the tumors. Among 1500 protein variants identified in the two-dimensional gel, 67 proteins correctly distinguished the eight subtypes of 99 histologically classified soft tissue sarcomas. Hierarchical clustering demonstrated leiomyosarcoma and MFH shared a similar protein expression profile, and clear cell sarcoma, synovial sarcoma, and MPNST could be grouped according to their protein expression patterns. Pleomorphic leiomyosarcoma and MFH showed similar tropomyosin isoform expression patterns. Patients with gastrointestinal stromal tumors expressing pfetin protein had better survival than those whose tumors lacked it. We identified 10 protein spots associated with the chemosensitivity of osteosarcoma to preoperative chemotherapy. These 10 spots could be new diagnostic and prognostic markers for osteosarcoma and new therapeutic targets for the disease. Proteomic analysis using 2D-DIGE provides novel information on the biology of bone and soft tissue sarcomas that could be used to diagnosis and treat these tumors. Level of Evidence: Level II, diagnostic study. See the Guidelines for Authors for a complete description of levels of evidence.

Peroxiredoxin 2 as a chemotherapy responsiveness biomarker candidate in osteosarcoma revealed by proteomics

Purpose: We aimed to identify novel chemotherapy responsiveness biomarkers for osteosarcoma (OS) by investigating the global protein expression profile of 12 biopsy samples from OS patients. Experimental design: Six patients were classified as good responders and six as poor responders, according to the Huvos grading system. The protein expression profiles obtained by 2‐D DIGE consisted of 2250 protein spots. Results: Among them, we identified 55 protein spots whose intensity was significantly different (Bonferroni adjusted p‐value<0.01) between the two patient groups. Mass spectrometric protein identification demonstrated that the 55 spots corresponded to 38 distinct gene products including peroxiredoxin 2 (PRDX 2). Use of a specific antibody against PRDX 2 confirmed the differential expression of PRDX 2 between good and poor responders, while PRDX 2 levels as measured by Western blotting correlated highly with their corresponding 2‐D DIGE values. The predictive value of PRDX 2 expression was further confirmed by examining an additional four OS cases using Western blotting. Conclusions and clinical relevance: These results establish PRDX 2 as a candidate for chemotherapy responsiveness marker in OS. Measuring PRDX 2 in biopsy samples before treatment may contribute to more effective management of OS.

Myxoinflammatory fibroblastic sarcoma

Acral myxoinfl ammatory fi broblastic sarcoma (MIFS) was fi rst described in 1998 as a new entity in three independent reports by pathologists, Meis-Kindblom and Kindblom, Michal, and Montgomery et al. It occurs primarily in adults, with a peak incidence in the fourth and fi fth decades of life, and presents as a painless fi rm mass of the distal extremities, predominantly the hands and feet. Recently, proximally located MIFS has also been reported, and some authors have suggested dropping the adjective “acral” from the name of the tumor because of its possibly misleading nature. Histologically, MIFS is a poorly circumscribed and typically multinodular tumor. The most striking feature is infl ammatory infi ltration associated with a prominent myxoid matrix in variable proportions and the presence of bizarre virocyte or Reed-Sternberg-like cells and multivacuolated cells simulating lipoblasts. Surgical excision with a wide margin is considered the treatment of choice, as the tumor shows a high rate of local recurrence. Meis-Kindblom and Kindblom reported a 67% local recurrence rate within a median follow-up period of 5 years. However, the tumor is frequently diffi cult to diagnose correctly at the time of initial presentation because of its apparently benign nature, being a slowgrowing, small and painless mass in the distal extremity. As a result, there is a tendency for the tumor to be treated inadequately by referring physicians. Surgeons need to be mindful of MIFS, its nature, and the appropriate treatment necessary for this unique tumor. Case reports

Secernin-1 as a novel prognostic biomarker candidate of synovial sarcoma revealed by proteomics

We aimed to develop prognostic biomarkers for synovial sarcoma employing a proteomic approach. We examined the proteomic profile of synovial sarcoma using two-dimensional difference gel electrophoresis (2D-DIGE). We identified 20 protein spots whose intensity was statistically different (p<0.01) between a group of eight patients who were alive and continuously disease-free for over five years and a group of five patients who died of the disease within two years post diagnosis. Mass spectrometric protein identification demonstrated that these 20 spots corresponded to 17 distinct gene products. Three of the 20 spots corresponded to secernin-1 and had higher intensity in the good prognosis group. The prognostic performance of secernin-1 was further examined immunohistochemically in 45 synovial sarcoma cases. The 5-year survival rate was 77.6% and 21.8% for patients with secernin-1 positive and negative primary tumors respectively (p=0.0015). The metastasis-free survival was significantly higher in the patient group with high secernin-1 expression compared to that with low expression (p=0.0012). Uni- and multivariate analyses revealed that secernin-1 expression was a powerful prognostic factor compared to other clinico-pathological parameters examined. These results indicate that secernin-1 may be used as a biomarker to predict the overall and metastasis-free survival in synovial sarcoma patients.

Pfetin as a Prognostic Biomarker in Gastrointestinal Stromal Tumor: Novel Monoclonal Antibody and External Validation Study in Multiple Clinical Facilities

OBJECTIVE The clinical course of gastrointestinal stromal tumor (GIST) spans a wide spectrum from a curable disorder to a highly malignant disease that leads to metastasis and death. To develop prognostic modalities for GIST patients, we developed a mouse monoclonal antibody against pfetin, the prognostic value of which has been previously reported. METHODS The reactivity of the monoclonal antibody against pfetin was examined by western blotting and immunohistochemistry. RESULTS Western blotting demonstrated that the monoclonal antibody was specific to pfetin. The immunohistochemical study demonstrated that the 5-year disease-free survival rate was 93.2% and 94.5% for GIST patients with pfetin-positive tumors and 70.0% and 80.7% for those with pfetin-negative tumors in the 159 cases from the National Cancer Center Hospital (P < 0.0001) and in the 100 cases from Niigata University Medical and Dental Hospital (P < 0.0001), respectively. Uni- and multivariate analyses revealed that pfetin expression was a powerful prognostic factor among the clinico-pathological parameters examined. CONCLUSIONS These results establish pfetin as a practical prognostic marker for GIST patients after surgery. Pfetin may also present a novel therapeutic target to prevent recurrence of GIST.