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Background/aim: Brain metastasis, a leading cause of cancer death, is a clinical challenge. Recently, genetic characterization of brain metastatic lesions based on next generation sequencing-based advanced technologies, such as single-cell RNA sequencing, has been performed to develop novel efficient therapies. The present study aimed to investigate brain-metastasis-specific biomarkers as well as relevant prognostic factors., Patients and Methods: The genetic profiles and expression levels of immune response-associated genes and 820 cancer-associated genes were compared between primary cancer lesions and metastatic cancer lesions obtained from nine cancer patients at the Shizuoka Cancer Center. Cytokine and chemokine marker genes were analyzed via quantitative PCR. T-cell receptor (TCR) repertoire profiling was performed for the same patients. For survival analysis, survival data of 52 cancer patients with brain metastases were utilized., Results: Comparison of driver mutation profiling between primary and metastatic lesions revealed shared core mutations in both lesions and a few new mutations in metastatic lesions. A high tumor mutation burden (TMB) was detected in metastatic lesions. Volcano plot analysis revealed specific features of the metastatic tumor microenvironment, such as cancer signaling promotion and immune suppression due to decreased immune cell infiltration. Survival analysis revealed that three genes, the TREML2 gene, the BTLA gene on activated microglia and the CERS2 gene on metastatic tumor, were potent prognostic factors., Conclusion: High TMB in metastatic lesions indicates potential benefit from immune checkpoint inhibitor usage for brain metastasis and TREML2 and BTLA are factors associated with poor prognosis. Activated microglia may be novel targets for the treatment of brain metastasis., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Transcriptome-based classification, such as consensus molecular subtyping, is expected to be applied to colorectal cancer (CRC). However, the relationship between molecular profiles and classical tumor markers, which are already used in clinical practice, has not been analyzed in a large cohort and remains unclear. We classified more than 1,500 Japanese patients with CRC based on consensus molecular subtyping and investigated the clinically available blood carcinoembryonic antigen (CEA) concentrations of each subgroup. To precisely distinguish CRCs, we allocated them to five subgroups, including tumors that were difficult to classify using the consensus molecular subtypes (CMSs), and extracted a heterogeneous population with somatic mutations and expression profiles that differed from those of the CMSs 1-4. For patients allocated to the CMS4 subgroup of stage III CRCs, elevated blood CEA concentrations may identify a subgroup with highly aggressive disease and contribute to improving therapeutic decisions. Furthermore, gene expression and pathway analyses of tumor and non-tumor tissues revealed that tumor immunity was "cold" in this subgroup with high CEA concentrations. The combination of emerging molecular profiling and classical tumor markers may have greater clinical utility than either used alone.

Despite the frequent detection of KRAS driver mutations in patients with colorectal cancer (CRC), no effective treatments that target mutant KRAS proteins have been introduced into clinical practice. In this study, we identified potential effector molecules, based on differences in gene expression between CRC patients carrying wild-type KRAS (n = 390) and those carrying KRAS mutations in codon 12 (n = 240). CRC patients with wild-type KRAS harboring mutations in HRAS, NRAS, PIK3CA, PIK3CD, PIK3CG, RALGDS, BRAF, or ARAF were excluded from the analysis. At least 11 promising candidate molecules showed greater than two-fold change between the KRAS G12 mutant and wild-type and had a Benjamini-Hochberg-adjusted P value of less than 1E-08, evidence of significantly differential expression between these two groups. Among these 11 genes examined in cell lines transfected with KRAS G12 mutants, BMP4, PHLDA1, and GJB5 showed significantly higher expression level in KRAS G12A, G12D, and G12V transfected cells than in the wild-type transfected cells. We expect that this study will lead to the development of novel treatments that target signaling molecules functioning with KRAS G12-driven CRC., (© 2021. The Author(s).)

Background: Mutation analysis using next-generation sequencing highlights the features of tumors with somatic alterations. However, the mutation profile of double cancer remains unclear. Here, we analyzed tumors derived from the same patient using whole exome sequencing (WES) to investigate the coherence of somatic mutations in double cancer., Methods: First, the tumor mutational burden (TMB) was investigated using WES of 5521 tumor specimens from a Japanese pan-cancer cohort. The frequencies of mutation concordance were then compared in these cancers. Finally, we calculated the expected value of mutational concordance fitting a Poisson distribution to determine the relationship between double and metastatic cancers., Results: In all, 44, 58, and 121 paired samples were diagnosed as double cancer, multifocal lesions (derived from identical tissues), and metastasis, respectively. Our analysis revealed that common somatic mutations were almost entirely absent in double cancer, whereas primary tumors and metastatic foci harbored several identical alterations. Concordance of the mutation profile in the same patient reflects the tumor origin and development, suggesting the potential for identifying double cancer based on common somatic mutations. Furthermore, according to a Poisson distribution, double cancer could be discriminated based on paired samples from the same patient. The probability of double cancer with more than 10 mutations was ≤1 part-per-billion (ppb, 10 - 9 ). In multifocal lesions, 74% of tumor pairs accumulated ≤10 common mutations, implying a difference in tumor origin within identical tissues., Conclusions: These findings indicate that counting common somatic mutations can indicate the differences in origin between tumors derived from the same patient. Our mutation coherence analysis can thus provide beneficial information for diagnosing double cancer.

Follicular lymphoma (FL) is a germinal center-derived B-cell lymphoma that is known to proliferate in the intrafollicular region. However, lymphoma cells can be identified in the extrafollicular regions, which are related to disease dissemination. We purified the intrafollicular and extrafollicular regions of FL cells by laser microdissection and conducted microarray analysis in order to characterize the gene expression profiles of FL cells from both regions. BCL2 and genes of germinal B-cell markers clearly separated intrafollicular and extrafollicular regions of reactive follicular hyperplasia, suggesting the adequacy of the current analysis. In FL cases, cytokine-related genes were significantly enriched in extrafollicular regions compared with those in the intrafollicular regions. In intrafollicular regions of FL, cell-cycle-related genes were enriched. We found that the FL cells in the extrafollicular region more strongly expressed IL3RA and CXCL12 than those of intrafollicular regions. The cytokines might be also derived from stroma cells in the extrafollicular regions, which may initiate activation and migration of the tumor cells to this region. Our results suggest that FL cell interaction with surrounding stroma cells plays an important role in the pathophysiology of FL and that such interactions should be a good target for therapy., (© 2020 John Wiley & Sons Ltd.)

Tumor mutational burden analysis using whole-exome sequencing highlights features of tumors with various mutations or known driver alterations. Cancers with few changes in the exon regions have unclear characteristics, even though low-mutated tumors are often detected in pan-cancer analysis. In the present study, we analyzed tumors with low tumor mutational burden listed in the Japanese version of The Cancer Genome Atlas, a data set of 5020 primary solid tumors. Our analysis revealed that detection rates of known driver mutations and copy number variation were decreased in samples with tumor mutational burden below 1.0 (ultralow tumor), compared with those in samples with low tumor mutational burden (≤5 mutations/Mb). This trend was also observed in The Cancer Genome Atlas data set. In the ultralow tumor mutational burden tumors, expression analysis showed decreased TP53 inactivation and chromosomal instability. TP53 inactivation frequently correlated with PI3K/mTOR-related gene expression, implying suppression of the PI3K/mTOR pathway in ultralow tumor mutational burden tumors. In common with mutational burden, the T cell-inflamed gene expression profiling signature was a potential marker for prediction of an immune checkpoint inhibitor response, and some ultralow tumor mutational burden tumor populations highly expressed this signature. Our analysis focused on how these tumors could provide insight into tumors with low somatic alteration that are difficult to detect solely using whole-exome sequencing., (© 2020 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Although next-generation sequencing-based panel testing is well practiced in the field of cancer medicine for the identification of target molecules in solid tumors, the clinical utility and clinical issues surrounding panel testing in hematological malignancies have yet to be fully evaluated. We conducted a multicenter prospective clinical sequencing study to verify the feasibility of a panel test for hematological tumors, including acute myeloid leukemia, acute lymphoblastic leukemia, multiple myeloma, and diffuse large B-cell lymphoma. Out of 96 eligible patients, 79 patients (82%) showed potentially actionable findings, based on the clinical sequencing assays. We identified that genetic alterations with a strong clinical significance were found at a higher frequency in terms of diagnosis (n = 60; 63%) and prognosis (n = 61; 64%) than in terms of therapy (n = 8; 8%). Three patients who harbored a germline mutation in either DDX41 (n = 2) or BRCA2 (n = 1) were provided with genetic counseling. At 6 mo after sequencing, clinical actions based on the diagnostic (n = 5) or prognostic (n = 3) findings were reported, but no patients were enrolled in a clinical trial or received targeted therapies based on the sequencing results. These results suggest that panel testing for hematological malignancies would be feasible given the availability of useful diagnostic and prognostic information. This study is registered with the UMIN Clinical Trial Registry (UMIN000029879, multiple myeloma; UMIN000031343, adult acute myeloid leukemia; UMIN000033144, diffuse large B-cell lymphoma; and UMIN000034243, childhood leukemia)., (© 2020 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Leucine-rich α-2 glycoprotein 1 (LRG1) is a diagnostic marker candidate for glioblastoma. Although LRG1 has been associated with angiogenesis, it has been suggested that its biomarker role differs depending on the type of tumor. In this study, a clinicopathological examination of LRG1's role as a biomarker for glioblastoma was performed. We used tumor tissues of 155 cases with diffuse gliomas (27 astrocytomas, 14 oligodendrogliomas, 114 glioblastomas). The immunohistochemical LRG1 intensity scoring was classified into 2 groups: low expression and high expression. Mutations of IDH1, IDH2, and TERT promoter were analyzed through the Sanger method. We examined the relationship between LRG1 expression level in glioblastoma and clinical parameters, such as age, preoperative Karnofsky performance status, tumor location, extent of resection, O6-methylguanine DNA methyltransferase promoter, and prognosis. LRG1 high expression rate was 41.2% in glioblastoma, 3.7% in astrocytoma, and 21.4% in oligodendroglioma. Glioblastoma showed a significantly higher LRG1 expression than lower-grade glioma (p = 0.0003). High expression of LRG1 was an independent favorable prognostic factor (p = 0.019) in IDH-wildtype glioblastoma and correlated with gross total resection (p = 0.002) and the tumor location on nonsubventricular zone (p = 0.00007). LRG1 demonstrated multiple potential as a diagnostic, prognostic, and regional biomarker for glioblastoma., (© 2020 American Association of Neuropathologists, Inc. All rights reserved.)

Tumor location and immunity play important roles in the progression of colorectal cancer (CRC). This study aimed to investigate the differences in the immunosurveillance pattern between right- and left-sided CRC and analyze their association with clinicopathologic features, including mismatch repair (MMR) status. We included surgically resected stage II/III CRC cases and evaluated the immunohistochemical findings of HLA class I, HLA class II, programmed cell death-ligand 1 (PD-L1), PD-1, CTLA-4, CD3, CD4, CD8, TIA-1, T-bet, GATA3, RORγT, Foxp3, and CD163. A total of 117 patients were included in the analyses; of these, 30 and 87 had right- and left-sided cancer, respectively. Tumor immunity varied according to the tumor location in the overall cohort. Analysis of the tumors excluding those with DNA mismatch repair (MMR) deficiency also revealed that tumor immunity differed according to the tumor location. In right-sided colon cancer (CC), high expression of Foxp3 (P = .0055) and TIA-1 (P = .0396) were associated with significantly better disease-free survival (DFS). High CD8 (P = .0808) and CD3 (P = .0863) expression tended to have better DFS. Furthermore, in left-sided CRC, only high PD-L1 expression in the stroma (P = .0426) was associated with better DFS. In multivariate analysis, high Foxp3 expression in right-sided CC was an independent prognostic factor for DFS (hazard ratio, 7.6445; 95% confidence interval, 1.2091-150.35; P = .0284). In conclusion, the immunosurveillance pattern differs between right- and left-sided CRC, even after adjusting for MMR deficiency., (© 2020 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Background: Studies to identify predictive biomarkers of adjuvant chemotherapy with S-1 after gastrectomy in Stage II/III gastric cancer patients have been done; however, more clarity and understanding are needed. Our aim in the present study was to identify biomarkers predicting benefit due to S-1 adjuvant chemotherapy using comprehensive gene expression analysis., Methods: We retrospectively analyzed 102 patients receiving adjuvant chemotherapy with S-1 and 46 patients not receiving S-1 adjuvant chemotherapy after gastrectomy for gastric cancer treatment between January 2014 and December 2016. Hierarchical clustering analysis was performed based on the gene expression data obtained using cDNA microarray. Differentially expressed genes (DEGs) were identified using thresholds of absolute fold changes of > 4.0 and a false discovery rate P value of < 0.01. Gene Ontology (GO) analysis and GO network visualization were performed using the ClueGO app in Cytoscape., Results: Hierarchical clustering analysis in patients treated with S-1 adjuvant chemotherapy revealed two clusters with favorable and unfavorable survival outcomes. We identified 147 upregulated DEGs and 192 downregulated DEGs in the favorable outcome group. GO analysis to identify significantly upregulated genes showed enrichment in immune-related genes and GO terms. Upregulation of these immune-related genes was not associated with survival in patients not receiving S-1 adjuvant chemotherapy., Conclusions: The upregulation and enrichment of immune-related genes and GO terms may be predictive biomarkers in patients who would benefit from adjuvant S-1 chemotherapy to treat Stage II/III gastric cancer.

Adult T-cell leukemia/lymphoma (ATLL) in Japan presents at a median age of 70 years and only 5% of patients are <50 years of age. We conducted RNA and targeted DNA sequencing of 8 ATLLs from Japanese patients <50 years of age and identified 3 (37.5%) with both CTLA4-CD28 and inducible costimulator (ICOS)-CD28 fusions. Mutations of PLCG1, PRKCB, and STAT3, which were frequent in other ATLL-sequencing studies, were not identified. Differential expression analysis identified the negative checkpoint molecule LAG3 as the most downregulated gene among cases with the fusions. Immunohistochemistry demonstrated expression of CD80 and CD86, the ligands for CTLA4 and CD28, on ATLL cells and tumor-associated macrophages, respectively. Expression of CTLA4-CD28 in Ba/F3 cells conferred cytokine-independent growth when cocultured with Raji cells that express CD80 and CD86. Growth was associated with recruitment of the p85 subunit of phosphatidylinositol 3-kinase to CTLA4-CD28 and phosphorylation of AKT and extracellular signal-regulated kinase. A CTLA4-blocking antibody reduced cytokine-independent growth in a dose-dependent manner. Together, these results suggest that young Japanese ATLL cases have a unique biology dependent on cell-nonautonomous interactions that drive CD28 signaling. Assessment for CD28 fusions and treatment with CTLA4 blockade should be considered in younger patients with relapsed/refractory ATLL., (© 2020 by The American Society of Hematology.)

This study aimed to establish the Japanese Cancer Genome Atlas (JCGA) using data from fresh frozen tumor tissues obtained from 5143 Japanese cancer patients, including those with colorectal cancer (31.6%), lung cancer (16.5%), gastric cancer (10.8%) and other cancers (41.1%). The results are part of a single-center study called "High-tech Omics-based Patient Evaluation" or "Project HOPE" conducted at the Shizuoka Cancer Center, Japan. All DNA samples and most RNA samples were analyzed using whole-exome sequencing, cancer gene panel sequencing, fusion gene panel sequencing and microarray gene expression profiling, and the results were annotated using an analysis pipeline termed "Shizuoka Multi-omics Analysis Protocol" developed in-house. Somatic driver alterations were identified in 72.2% of samples in 362 genes (average, 2.3 driver events per sample). Actionable information on drugs that is applicable in the current clinical setting was associated with 11.3% of samples. When including those drugs that are used for investigative purposes, actionable information was assigned to 55.0% of samples. Germline analysis revealed pathogenic mutations in hereditary cancer genes in 9.2% of samples, among which 12.2% were confirmed as pathogenic mutations by confirmatory test. Pathogenic mutations associated with non-cancerous hereditary diseases were detected in 0.4% of samples. Tumor mutation burden (TMB) analysis revealed 5.4% of samples as having the hypermutator phenotype (TMB ≥ 20). Clonal hematopoiesis was observed in 8.4% of samples. Thus, the JCGA dataset and the analytical procedures constitute a fundamental resource for genomic medicine for Japanese cancer patients., (© 2019 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Viral infection induces potent cellular immunity and activated intracellular signaling, which may dictate the driver events involved in immune escape and clonal selection of virus-associated cancers, including Epstein-Barr virus (EBV)-positive lymphomas. Here, we thoroughly interrogated PD-L1/PD-L2-involving somatic aberrations in 384 samples from various lymphoma subtypes using high-throughput sequencing, particularly focusing on virus-associated lymphomas. A high frequency of PD-L1/PD-L2-involving genetic aberrations was observed in EBV-positive lymphomas [33 (22%) of 148 cases], including extranodal NK/T-cell lymphoma (ENKTL, 23%), aggressive NK-cell leukemia (57%), systemic EBV-positive T-cell lymphoproliferative disorder (17%) as well as EBV-positive diffuse large B-cell lymphoma (DLBCL, 19%) and peripheral T-cell lymphoma-not otherwise specified (15%). Predominantly causing a truncation of the 3'-untranslated region, these alterations represented the most prevalent somatic lesions in ENKTL. By contrast, the frequency was much lower in EBV-negative lymphomas regardless of histology type [12 (5%) of 236 cases]. Besides PD-L1/PD-L2 alterations, EBV-positive DLBCL exhibited a genetic profile distinct from EBV-negative one, characterized by frequent TET2 and DNMT3A mutations and the paucity of CD79B, MYD88, CDKN2A, and FAS alterations. Our findings illustrate unique genetic features of EBV-associated lymphomas, also suggesting a potential role of detecting PD-L1/PD-L2-involving lesions for these lymphomas to be effectively targeted by immune checkpoint blockade.

Glioblastomas are highly aggressive brain tumors with a particularly poor prognosis. Glucose transporter-1 (GLUT1/SLC2A1), a uniporter that is expressed by various carcinomas and may be involved in malignant neoplasm glycometabolism, may also be related to prognosis in glioblastomas. GLUT1 is essential to central nervous system glycometabolism. To clarify the exact role of GLUT1 in glioblastoma, we assessed the expression and localization of GLUT1 in patient samples by immunohistochemistry and in situ RNA hybridization. This revealed that GLUT1 was mainly expressed on perivascular and pseudopalisaded tumor cell membranes. All samples expressed GLUT1 to some degree, with 30.8% showing stronger staining. On the basis of these data, samples were divided into high and low expression groups, although SLC2A1 mRNA expression was also higher in the high GLUT1 expression group. Kaplan-Meier survival curves revealed that high GLUT1 expression associated with lower overall survival (log-rank test, p = 0.001) and worse patient prognoses (p = 0.001). Finally, MIB-1 staining was stronger in high GLUT1 expression samples (p = 0.0004), suggesting a link with proliferation. We therefore hypothesize that GLUT1 expression in glioblastomas may enhance glycolysis, affecting patient prognosis. Examination of GLUT1 in patients with glioblastomas may provide a new prognostic tool to improve outcome., (© 2019 American Association of Neuropathologists, Inc.)

Exosomes are small vesicles found in extracellular environments including blood, urine, and cell culture medium. Their contents are cell‑type specific, and molecules embedded in exosomes can be useful fluid‑based clinical biomarkers. To identify proteins with metastatic marker potential, we conducted a comparative exosomal proteome analysis using human pancreatic cancer cell lines derived from metastasis, ascites, and primary tumors. Metastatic potential of cell lines was assessed by migratory and invasive activities. A pancreatic cancer cell line from metastasis (SU.86.86) revealed 23‑fold and 20‑fold increases in cell migratory and invasive activities, respectively, compared to the MIA PaCa‑2 cell line derived from primary tumor cells. Liquid chromatography‑mass spectrometry‑based proteome analysis and subsequent validation by immunoblot analysis revealed that epidermal growth factor receptor pathway substrate 8 (Eps8) was highly abundant in exosomes from metastasis‑derived SU.86.86 cells. Comparison of 12 pancreatic cancer cell lines derived from different stages of malignancy revealed a strong relationship between exosomal Eps8 protein levels and cell motile activities (migration: r=0.85, P=4.2x10‑4; invasion: r=0.60, P=3.2x10‑2). Conversely, relationships between intracellular Eps8 protein levels and cell motile activities were moderate (migration: r=0.65, P=2.0x10‑2; invasion: r=0.51, P=9.2x10‑2). It was therefore concluded that exosomal Eps8 protein levels were correlated with the migratory cell potential of human pancreatic cancer cells, indicating that exosomal Eps8 has the potential to be a metastatic biomarker for human pancreatic cancer.

GNA13 is a G protein involved in modulating tumor proliferative capacity, infiltration, metastasis, and migration. Genomic alteration of GNA13 was frequently observed in follicular lymphoma (FL). In this study, we examined 167 cases of FL by immunostaining of GNA13 using tissue microarray to evaluate the clinical significance. There were 26 GNA13-positive cases (15.6%) and 141 GNA13-negative cases (84.4%). GNA13-positive cases had a higher incidence of early progression of disease for which disease progression was recognized within 2 years compared with GNA13-negative cases (P=0.03). There were no significant differences in other clinicopathologic factors including histological grade, BCL2-IGH translocation, immunohistochemical phenotype, and Follicular Lymphoma International Prognostic Index. In addition, overall survival and progression-free survival were poorer in GNA13-positive cases than in GNA13-negative cases (P=0.009 and 0.005, respectively). In multivariate analysis, GNA13 positivity was found to be a poor prognostic factor for overall survival and progression-free survival. Thus, GNA13 protein expression was an independent prognostic factor and may affect disease progression in FL.

Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) is cytologically and phenotypically heterogeneous. Retinoic acid-related orphan receptor-γt (RORγt) is a transcription factor that regulates the differentiation of naïve CD4 + helper T cells to Th17 cells. In the present study, we immunohistochemically confirmed the expression of RORγt in PTCL-NOS. Pathological and clinical investigations were performed for 170 cases of PTCL-NOS. RORγt-positive cases accounted for 17.6% (30/170) of the total cases, and they showed a significantly higher frequency of CD8 positivity (P = .033), lower counts of white blood cells (P = .030) and neutrophils (P = .039) in the peripheral blood, higher levels of hypergammaglobulinemia (P = .031), a higher frequency of a complete response (P = .009), and a tendency for a lower International Prognostic Index (P = .061) and better overall survival (P = .0806). These results suggest that RORγt-positive PTCL-NOS could be a subpopulation of PTCL-NOS. Further research associated with this genomic abnormality at the transcriptional level is needed to confirm the results of this study., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Transforming acidic coiled-coil containing protein 3 (TACC3) is expressed during the mitotic phase of nuclear division and regulates microtubules. Recently, high TACC3 expression in tumor cells of various cancers including soft tissue sarcoma has been reported. However, its role in osteosarcoma remains unknown. Because we have few prognostic markers for survival in osteosarcoma, we wanted to investigate the potential role of TACC3 in human osteosarcoma and determine if it is associated with survival., Questions/purposes: (1) Is there a relationship between TACC3 expression and clinicopathologic characteristics such as sex, age (< 20 or ≥ 20 years), histologic type (osteoblastic or others), tumor location (femur or others), American Joint Committee on Cancer staging system (AJCC stage IIA or IIB), tumor necrosis percentage after chemotherapy (< 90% or ≥ 90%), p53 expression (low or high), and Ki-67 expression (low or high)? (2) Is TACC3 expression associated with event-free and overall survival in patients with osteosarcoma?, Methods: Forty-six conventional patients with osteosarcoma were treated at our institution from 1989 to 2013. Patients were excluded because of unresectable primary site (two patients) and no chemotherapy (two patients). Patients with metastasis at the initial visit (five patients), without pretreatment biopsy samples (two patients), or clinical charts (two patients) were also excluded. The left 33 patients who received neoadjuvant and adjuvant chemotherapy, which consisted of cisplatin/doxorubicin/methotrexate or cisplatin/doxorubicin/methotrexate/ifosfamide, and completed surgical resection with histologic wide tumor margins. Primary tumor samples before chemotherapy were used in this study. We investigated TACC3 expression using immunohistochemical staining and statistically analyzed the TACC3 expression, clinicopathologic characteristics, and event-free and overall survival in patients with osteosarcoma., Results: High TACC3 expression was observed in 19 of 33 osteosarcoma specimens (58%), and this was associated with larger tumor size (ie, AJCC stage IIB in this study; p = 0.002), higher p53 expression (p = 0.007), and higher Ki-67 expression (p = 0.002). The estimated metastasis-free survival at 5 years was 21% (95% confidence interval [CI], 7%-41%) in patients with high TACC3 expression and 79% (95% CI, 47%-93%) in patients with low TACC3 expression (p < 0.001), and the estimated overall survival at 5 years was 34% (95% CI, 13%-56%) in patients with high TACC3 expression and 86% (95% CI, 54%-96%) in patients with low TACC3 expression (p < 0.001). Furthermore, high TACC3 expression was an independent poor prognostic factor for metastasis-free survival with a hazard ratio of 3.89 (95% CI, 1.07-19.78; p = 0.039) as well as overall survival with 4.41 (95% CI, 1.01-32.97; p = 0.049)., Conclusions: High TACC3 expression was associated with aggressive clinicopathologic features and unfavorable prognosis in these patients with osteosarcoma. Our preliminary results suggest that further analysis about mutation or an inactive form of TACC3 would be useful to understand the mechanism of abnormal TACC3 expression in patients with osteosarcoma. If these findings are substantiated in larger studies, TACC3 might be useful for predicting survival and a potential therapeutic target for osteosarcoma., Level of Evidence: Level III, therapeutic study.

Myeloid sarcoma (MS) is a rare condition and is an extramedullary tumour of immature myeloid cells. It is now known that the programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) pathway suppresses the host antitumor responses and that these products are expressed on both tumour cells and tumour-infiltrating cells in various malignancies. However, little is known about the significance of PD-1/PD-L1 expression on tumour cells and tumour microenvironmental cells in MS. To investigate the clinicopathological significance of PD-1/PD-L1 expression in MS, we analyzed 98 patients by immunohistochemistry. Of these, 10.2% of cases had neoplastic tumour cells positive for PD-L1 (nPD-L1 + ). However, the rate of nPD-L1 + was <5% (range: 0.27 to 2.97%). On the other hand, PD-L1 expression on 1 or more of stromal cells in the tumour microenvironment (miPD-L1 + ) was observed in 37.8% of cases. Because all nPD-L1 + cases expressed PD-1 on less than 5% of tumour cells, we compared the miPD-L1 + and miPD-L1 - groups. There was a correlation between miPD-L1 + status and the number of PD-1-expressing tumour -infiltrating lymphocytes (PD-1 + TILs; P = .0229). miPD-L1 + was found to be associated with poorer overall survival and progression-free survival (P = .00392, P = .00261, respectively). Multivariate analysis also confirmed miPD-L1 + to be an independent poor prognostic factor. In conclusion, our study indicated that the immunotherapy blocking the PD-1/PD-L1 pathway may improve the clinical outcome of MS., (Copyright © 2018 John Wiley & Sons, Ltd.)

Aims: Adult T cell leukaemia/lymphoma (ATLL) is an aggressive malignancy with a poor prognosis. Human leucocyte antigen (HLA) and β2 microglobulin (β2M) serve as key molecules in tumour immunity, and their expression is reduced frequently in tumour cells. Programmed cell death (PD)-1/PD-ligand1 (PD-L1) interactions play a role in escape of tumour cells from T cell immunity. Therefore, this study aimed to determine the clinicopathological relevance of HLA and β2M expressions in ATLL cells and PD-L1 expression in lymphoma or stromal cells and predict the overall survival of patients with ATLL., Methods and Results: We analysed a total of 123 biopsy samples from patients newly diagnosed with ATLL by using immunohistochemical analysis. Of the patients enrolled, 91 (74%) were positive for HLA (in cell membrane, 60 patients), 89 (72%) were positive for β2M (in cell membrane, 54 patients) and 48 (39%) were positive for both HLA and β2M in the cell membrane (HLA m+ β2M m+ ). No significant clinical differences other than prognosis were found between the HLA m+ β2M m+ group and the other groups. Immunophenotypical evaluation revealed significantly higher rates of CD30-positive lymphoma cells (P = 0.003) and PD-L1-positive stromal cells in microenvironments (miPD-L1 high ) (P = 0.011) of the HLA m+ β2M m+ group than in the other groups. The HLA m+ β2M m+ group had a significantly better prognosis that the other groups (P = 0.0096), and patients showing HLA m+ β2M m+ with miPD-L1 high had the most favourable prognosis among all groups., Conclusions: The membranous expression of HLA and β2M is likely to reflect the immune response and would be useful to predict prognosis before starting ATLL therapy., (© 2018 John Wiley & Sons Ltd.)

Project High-tech Omics-based Patient Evaluation (HOPE), including comprehensive whole-exome sequencing (WES) and gene expression profiling (GEP) using freshly resected tumor specimens, has been in progress since its implementation in 2014. Among a total of 1,685 cancer patients, 13 melanoma patients were registered in the HOPE Project and were characterized using multi-omics analyses. Among the 13 melanoma patients, 4 were deceased, and 9 were alive. The mean overall survival (OS) and relapse‑free survival (RFS) times of the melanoma patients were 16.9 and 14.7 months, respectively. Previously, we developed an immune response‑associated gene list, which consisted of 164 genes in Project HOPE, for evaluating the immunological status. In the present study, the association of immune response‑associated gene expression with immunological parameters, such as programmed death-ligand 1 (PD-L1) and CD8 expression levels, single nucleotide variant (SNV) number, and Vogelstein driver gene mutation number, was investigated. With respect to PD-L1 expression, both immuno-suppression and immuno-stimulation-related genes were upregulated in PD-L1-positive melanomas. In contrast, regarding Vogelstein driver mutations, several T-cell activation-related genes were significantly downregulated in the high driver gene mutation group. In addition, many T-cell activation-related genes were upregulated in the CD8-positive melanomas. The correlation of immune response-associated gene expression with the survival time of the melanoma patients was investigated. Eight specific genes were commonly identified as genes that were significantly correlated for both the overall OS and RFS time, which could be possible prognostic factors for melanoma patients. These results revealed that an immune response-associated gene panel could be an informative tool for evaluating the immunological status prior to clinical immunotherapy in the upcoming era of genomic cancer medicine.

Peripheral T cell lymphoma, not otherwise specified (PTCL-NOS), is a heterogeneous disease with respect to clinicopathological features. Cell adhesion molecule 1 (CADM1) has been reported to be ectopically expressed in adult T cell leukaemia/lymphoma (ATLL). However, the frequency of CADM1 expression remains unknown in peripheral T cell lymphomas. In the current study, CADM1 expression was analysed in 88 PTCL-NOS patients. CADM1 was expressed in 14 of 88 (15.9%) PTCL-NOS cases, and its expression was associated with C-C chemokine receptor type 4 (CCR4) expression and nuclear atypia. CADM1-positive PTCL-NOS cases (10/74) had a significantly poorer prognosis than CADM1-negative cases (64/74) (P = 0.001). Multivariate analysis confirmed that CADM1 expression was an independent prognostic factor in PTCL-NOS. These findings suggest that CADM1 expression is a novel prognostic factor for PTCL-NOS.

We identified a case of lymphadenopathy of metastatic papillary thyroid carcinoma (PTC) with CD20 expression, which was also expressed by the primary tumor. CD20 expression was identified using immunohistochemistry (IHC) in metastatic PTC biopsy samples from a 58-year-old woman. CD20 expression was initially determined using a CD20-recognizing L26 clone. To validate this phenomenon, we performed IHC with another antibody that recognizes the N-terminus of CD20 and fluorescent double staining using anti-TTF-1 and anti-CD20 antibodies. Taken together, we concluded metastatic PTC expressed CD20. We also examined 21 additional PTC cases and found four more cases that were CD20 positive. Therefore, five of the 22 (23%) cases were positive for CD20. In the positive cases, four cases were classical papillary thyroid carcinoma and one case was a follicular variant of papillary thyroid carcinoma. CD20 is an important target for molecularly targeted therapy for a subset of B-cell lymphomas. Complement-dependent and antibody-dependent cellular cytotoxicities are important effector mechanisms of anti-CD20 therapy. Here, for the first time, we report PTC with expression of CD20. Our findings provide a rationale for treating CD20-positive PTC patients with anti-CD20 therapy., (© 2017 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

Primary malignant pericardial sarcomatoid mesothelioma (PMPSM) is an extremely rare tumor with poor prognosis. We present an autopsy case in an 80-year-old man admitted for heart failure after one month of treatment at an outpatient clinic. He died three months after symptom onset. A complete autopsy revealed localization of the tumor to the pericardium without other lesions. Histologically, mainly spindle-shaped atypical cells with hyperchromatic nuclei and nucleoli were observed. Immunohistochemical markers for mesothelioma were positive for calretinin, cytokeratin AE1/AE3, and cytokeratin CAM5.2. Thus, we diagnosed primary sarcomatoid malignant mesothelioma of the pericardium. To our knowledge, only four PMPSM cases have been reported in the English literature in the past 30 years. Although PMPSM is rare, clinicians and pathologists should recognize it as a possible diagnosis of pericardial tumors. It is necessary to accumulate clinical and pathological diagnostic findings to establish early detection methods for this extremely rare disease., (© 2017 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

GATA binding protein-3 (GATA3) is a transcription factor that regulates cell differentiation and maintenance in some types of normal cells. This study aimed to investigate the association between GATA3 expression and primary lung adenocarcinoma and to clarify the clinical significance of GATA3 expression in lung adenocarcinoma. Immunohistochemical GATA3 expression was evaluated using completely resected lung adenocarcinoma samples from 95 cases. GATA3 immunohistochemical staining was performed and scored. Associations between clinicopathological factors and GATA3 expression were analyzed by using the χ 2 test and Fisher exact test. The Kaplan-Meier method was used to analyze overall survival (OS) and disease-free survival (DFS). Forty-nine cases expressed high levels of GATA3, which were associated with lymphatic invasion (P=.003). In univariate and multivariate analyses, vascular invasion (P<.001) and high GATA3 expression (P=.023) were identified as independent risk factors for OS. Higher pathological stages (P=.012), vascular invasion (P=.010), and high GATA3 expression (P=.009) were identified as independent risk factors for DFS. The high GATA3 expression group exhibited statistically worse OS (P=.031) and DFS (P=.011) than the low-expression group based on the Kaplan-Meier curves. In resected lung adenocarcinoma, high GATA3 expression is associated with poorer prognosis for both OS and DFS. Therefore, the immunohistochemical evaluation of GATA3 represents a potentially useful prognostic tool for postoperative patients., (Copyright © 2017 Elsevier Inc. All rights reserved.)

MUM1/IRF4 expression is detected in 18% to 41% of Burkitt lymphoma (BL). However, only a few studies of MUM1-positive (MUM1) BL have been reported, and its characteristics still remain controversial. To highlight the features of MUM1 BL, we compared the clinicopathologic characteristics of 37 cases of MUM1 and 51 cases of MUM1-negative (MUM1) BL in Japan. Compared with MUM1 BL, patients with MUM1 BL showed significantly younger onset (P=0.0062) and a higher ratio of females (P=0.013). We have also revealed the difference in the involved sites. The MUM1 group showed lower incidences of involvement of stomach (P=0.012) and tonsil (P=0.069). There was a more tendency in MUM1 group to involve colon (P=0.072), breast (P=0.073), and kidney (P=0.073). Regarding the prognosis, a trend toward a lower overall survival for MUM1 group was noted (P=0.089). Notably, comparing MUM1 and MUM1 BL cases of adults (age16 y old and above), the former showed significantly worse prognosis (P=0.041). Among the BL patients treated with the intensive chemotherapy, a standard therapy for BL, MUM1 cases showed worse prognosis (P=0.056). In conclusion, MUM1 BL showed worse prognosis, particularly in adult cases, compared with MUM1 BL. In addition, the difference of the onset age, sex ratio, and involved sites between the 2 groups was noted. Our results demonstrate that MUM1 expression might predict worse prognosis of BL, and MUM1 BL should be distinguished from MUM1 BL.

Recent genetic analysis has identified frequent mutations in ten-eleven translocation 2 (TET2), DNA methyltransferase 3A (DNMT3A), isocitrate dehydrogenase 2 (IDH2) and ras homolog family member A (RHOA) in nodal T-cell lymphomas, including angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma, not otherwise specified. We examined the distribution of mutations in these subtypes of mature T-/natural killer cell neoplasms to determine their clonal architecture. Targeted sequencing was performed for 71 genes in tumor-derived DNA of 87 cases. The mutations were then analyzed in a programmed death-1 (PD1)-positive population enriched with tumor cells and CD20-positive B cells purified by laser microdissection from 19 cases. TET2 and DNMT3A mutations were identified in both the PD1+ cells and the CD20+ cells in 15/16 and 4/7 cases, respectively. All the RHOA and IDH2 mutations were confined to the PD1+ cells, indicating that some, including RHOA and IDH2 mutations, being specific events in tumor cells. Notably, we found that all NOTCH1 mutations were detected only in the CD20+ cells. In conclusion, we identified both B- as well as T-cell-specific mutations, and mutations common to both T and B cells. These findings indicate the expansion of a clone after multistep and multilineal acquisition of gene mutations., Competing Interests: The authors declare no conflict of interest.

Marginal zone lymphoma (MZL) is a low-grade B-cell lymphoma derived from marginal zone B cells. Because of a lack of specific immunohistochemical markers, MZL is mainly diagnosed based on the cytological appearance and growth pattern of the tumor. Marginal zone B cells were recently shown to selectively express immunoglobulin superfamily receptor translocation-associated 1 (IRTA1), but the antibody used in that study is not commercially available. We therefore investigated the IRTA1 expression in nonneoplastic lymphoid tissues and 261 malignant lymphomas, examining the ability of a commercially available antibody to accurately diagnose MZL. Among 37 MZLs, 23 of 25 extranodal MZLs of mucosa-associated lymphoid tissue (MALT lymphomas), 3 of 6 splenic MZLs and 3 of 6 nodal MZLs were positive for IRTA1. Among the 98 diffuse large B-cell lymphomas, 33 were positive for IRTA1, including 1 of 38 follicular lymphomas, and all precursor B-lymphoblastic (2/2) and T-lymphoblastic (7/7) leukemia/lymphomas. Other mature B-cell and T-cell lymphomas, and Hodgkin lymphoma were negative for IRTA1. In MALT lymphoma, positive cells were detected mainly in intraepithelial and subepithelial marginal zone B cells. In 1 case of grade 3 follicular lymphoma, IRTA1 was also expressed in the area of large cell transformation. When tumors were classified as germinal center B cell-like (GCB) or non-GCB using the algorithm of Hans, positive expression of IRTA1 was correlated significantly with non-GCB diffuse large B-cell lymphomas (P < .05). These results demonstrated the ability of the commercially available IRTA1 antibody to distinguish MALT lymphoma from other low-grade B-cell lymphomas., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Purpose: The immune checkpoint of the programmed death 1/programmed death ligand 1 (PD-1/PD-L1) pathway is believed to play an important role in evasion of host antitumor immune surveillance in various malignancies; however, little is known about its role in thymic carcinoma. This study investigated PD-1/PD-L1 expression and its association with clinicopathologic features, the expression of immune-related proteins in tumor-infiltrating lymphocytes (TIL), and patient prognosis., Experimental Design: PD-L1 and PD-1 expression was evaluated by IHC in 25 thymic carcinoma tissue specimens. Copy number alterations of the PD-L1 gene in 11 cases were assessed in formalin-fixed, paraffin-embedded material using qRT-PCR., Results: Compared with normal subjects, 3 thymic carcinoma patients showed an increase in PD-L1 copy number, whereas 8 did not. PD-L1 was significantly overexpressed in cases with copy number gain as compared with normal cases. High PD-L1 expression was associated with higher disease-free and overall survival rates as compared to cases with low expression. Prognostic analysis revealed low PD-L1 expression and high number of PD-1(+) TILs as significant predictors of poor survival, together with Masaoka-Koga stage IVa/IVb disease and incomplete resection. In the quantitative analysis of TILs, PD-L1 expression correlated proportionally with the number of infiltrating CTLs., Conclusions: Here, for the first time, we report that PD-L1 and PD-1 expression might be useful prognostic predictors in thymic carcinoma. Further studies are expected to substantiate the prognostic value of PD-L1 and PD-1 expression, and the potential efficacy of targeting the PD-1/PD-L1 pathway in thymic carcinoma via immunotherapy. Clin Cancer Res; 22(18); 4727-34. ©2016 AACR., (©2016 American Association for Cancer Research.)

Programmed cell death ligand 1 (PD-L1) is expressed on both tumor and tumor-infiltrating nonmalignant cells in lymphoid malignancies. The programmed cell death 1 (PD-1)/PD-L1 pathway suppresses host antitumor responses, although little is known about the significance of PD-1/PD-L1 expression in the tumor microenvironment. To investigate the clinicopathological impact of PD-L1 expression in adult T-cell leukemia/lymphoma (ATLL), we performed PD-L1 immunostaining in 135 ATLL biopsy samples. We observed 2 main groups: 1 had clear PD-L1 expression in lymphoma cells (nPD-L1(+), 7.4% of patients), and the other showed minimal expression in lymphoma cells (nPD-L1(-), 92.6%). Within the nPD-L1(-) group, 2 subsets emerged: the first displayed abundant PD-L1 expression in nonmalignant stromal cells of the tumor microenvironment (miPD-L1(+), 58.5%) and the second group did not express PD-L1 in any cell (PD-L1(-), 34.1%). nPD-L1(+) ATLL (median survival time [MST] 7.5 months, 95% CI [0.4-22.3]) had inferior overall survival (OS) compared with nPD-L1(-) ATLL (MST 14.5 months, 95% CI [10.1-20.0]) (P = .0085). Among nPD-L1(-) ATLL, miPD-L1(+) ATLL (MST 18.6 months, 95% CI [11.0-38.5]) showed superior OS compared with PD-L1(-) ATLL (MST 10.2 months, 95% CI [8.0-14.7]) (P = .0029). The expression of nPD-L1 and miPD-L1 maintained prognostic value for OS in multivariate analysis (P = .0322 and P = .0014, respectively). This is the first report describing the clinicopathological features and outcomes of PD-L1 expression in ATLL. More detailed studies will disclose clinical and biological significance of PD-L1 expression in ATLL., (© 2016 by The American Society of Hematology.)

Adult T cell leukaemia/lymphoma (ATLL) is an intractable T cell neoplasm caused by human T cell leukaemia virus type 1. Next-generation sequencing-based comprehensive mutation studies have revealed recurrent somatic CCR4 mutations in ATLL, although clinicopathological findings associated with CCR4 mutations remain to be delineated. In the current study, 184 cases of peripheral T cell lymphoma, including 113 cases of ATLL, were subjected to CCR4 mutation analysis. This sequence analysis identified mutations in 27% (30/113) of cases of ATLL and 9% (4/44) of cases of peripheral T cell lymphoma not otherwise specified. Identified mutations included nonsense (NS) and frameshift (FS) mutations. No significant differences in clinicopathological findings were observed between ATLL cases stratified by presence of CCR4 mutation. All ATLL cases with CCR4 mutations exhibited cell-surface CCR4 positivity. Semi-quantitative CCR4 protein analysis of immunohistochemical sections revealed higher CCR4 expression in cases with NS mutations of CCR4 than in cases with wild-type (WT) CCR4. Furthermore, among ATLL cases, FS mutation was significantly associated with a poor prognosis, compared with NS mutation and WT CCR4. These results suggest that CCR4 mutation is an important determinant of the clinical course in ATLL cases, and that NS and FS mutations of CCR4 behave differently with respect to ATLL pathophysiology., (Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

In order to clarify the role of endothelin B receptors (ETBRs) in gliomas, we analyzed cell cultures and surgical specimens of gliomas using RT-PCR and immunohistochemistry. RT-PCR measured the absolute expression of ETBR mRNA in twelve samples, which included gliomas that were classified using the World Health Organization (WHO) classification system Grade I-IV, as well as two glioblastoma cell lines (CCF-STTG1 and U87-MG). Using immunohistochemistry, 77 glioma specimens were evaluated for their expression of ETBR and infiltrating T lymphocytes, including an analysis of cytotoxic T cells (CTLs) and regulatory T lymphocytes (Tregs). The number of ETBR-positive vessels in the glioblastomas (Grade IV) was significantly higher than in other grades of gliomas (comparisons to Grade IV, Grade I: p = 0.0323, Grade II: p = 0.0009, Grade III: p = 0.0273). The ETBR expression rate (defined as the number of ETBR-positive blood vessels divided by the total number of blood vessels) in the glioblastomas was higher than the ETBR expression rate in the low-grade gliomas (compared to Grade IV, Grade I: p = 0.0132, Grade II: p = 0.0018, Grade III: p = 0.0745). In addition, the cases which had an ETBR expression rate of 50 % or higher exhibited fewer infiltrating CTLs and more infiltrating Tregs compared to the cases with an ETBR expression rate <50 % (CTLs: p = 0.0342; Tregs: p = 0.0175). Isocitrate dehydrogenase 1 (IDH-1) mutations were identified in 21 cases, but there was no correlation between ETBR expression and IDH-1 mutations for any WHO grade. These results suggest that ETBR expression during neo-angiogenesis may interfere with the homing of CTLs around the tumor and be involved in the immune escape mechanism of gliomas.

We analyzed serum ProGRP levels in patients with Ewing sarcoma, and found that 5 out of 9 patients had elevated levels; the values range equally with those of patients with limited disease of small-cell lung carcinoma. Serum ProGRP levels in patients with bone and soft tissue malignancies other than Ewing sarcoma are not elevated. Immunohistochemical studies demonstrated that ProGRP-like immunoreactivities were detected in Ewing sarcoma tissues obtained from 2 patients with elevated serum ProGRP levels, suggesting that ProGRP is a product of tumor cells of Ewing sarcoma. These results indicate that serum ProGRP could serve as a specific tumor marker for Ewing sarcoma. Since ProGRP is a major hormonal product of tumor cells of small-cell lung carcinoma, a typical neuroendocrine carcinoma, it is reasonable to postulate that the present study provides an evidence for Ewing sarcoma to possess neuroendocrine differentiation.

Aims: Epstein-Barr virus (EBV)-positive diffuse large B cell lymphoma (DLBCL) of the elderly is characterised by frequent extranodal involvement, a morphological spectrum from polymorphous to monomorphous and a poor prognosis. The frequency is higher in Japan and Korea but lower in the West, while the status in Taiwan has not been reported yet., Methods: We conducted a retrospective study of DLBCL in a single institute in Taiwan by immunohistochemistry and in situ hybridisation for EBV., Results: Of the 424 consecutive DLBCL cases, 332 cases were studied for EBV. 15 (4.5%) were EBV-positive and 13 (3.9%) fulfil WHO criteria of EBV-positive DLBCL of the elderly with a median age of 75. Of these 15 cases, extranodal presentation occurred in 11 (73%) patients with predominance in the gastrointestinal tract and 6 (40%) were of germinal centre B cell phenotype. There was no difference between EBV-positive and -negative patients in terms of age, gender, nodal versus extranodal presentation, and immunophenotypical profile. EBV-positive patients showed a trend for a shorter median survival time (5.0 vs 39.3 months; p=0.058). Of all DLBCL patients, multivariable analysis revealed a significantly worse overall survival for patients older than 50 (p=0.001) and for those with bcl-6-negative tumours (p=0.003) but not with other clinicopathological factors including EBV status., Conclusions: EBV-positive DLBCL of the elderly is relatively rare in Taiwan, with an incidence intermediate between Japan/Korea and the West. Further studies are warranted to clarify the association of EBV and the clinicopathological features and the prognostic significance in patients with DLBCL.

The Project HOPE (High-tech Omics-based Patient Evaluation) for cancer medicine aims to evaluate biological characteristics of each cancer tissue as well as diathesis of each patient in around 1,000 consecutive cases per year, who receive operations at the Shizuoka Cancer Center. Cancer tissues are investigated by whole-exome sequencing for 18,835 genes, focusing on 12,776 in-house cancer hotspots from 483 cancer-associated genes. To confirm cancer-specific genetic changes, we analyzed blood cells to collate with data of cancer tissues, and we reevaluate cancer tissues by comprehensive cancer panel for 409 genes. In order to investigate diathesis of the patients, we evaluate 43,015 hotspots associated with non-cancerous diseases. In terms of gene expression profiling, we analyze cancer-specific alterations for 29,833 genes using tumor and adjacent normal tissues. If and when necessary, we investigate tumor and normal tissues by proteomics and metabolomics. The model experiments using glioblastoma cell lines demonstrated that the method is appropriate for clinical application. The Project HOPE makes it possible to implement individualized medicine and to practice preventive and presymptomatic medicine for cancer patients. Furthermore, the project can create important seeds for research and development in cancer medicine.

Acute megakaryoblastic leukaemia (AMGL) is an uncommon disease with poor prognosis. Histopathologically, AMGL cases show variable degree of fibrosis and the presence of uniform blasts or mature dysplastic megakaryocytes. Here we examined 18 cases of AMGL, including idiopathic (n = 9) and secondary (n = 9) cases. Fourteen cases were males and four were females, ranging in age from 14 to 87 years (median, 58). All cases had anaemia, but leukocyte and platelet counts varied. Blast cells were detected in the peripheral blood of 14 cases. Fourteen of 16 cases showed chromosomal abnormalities. The median survival was 6 months (range, 1-48 months). Survival rates did not correlate with the severity of fibrosis, proportion of blast cells and cause of AMGL. Nine of the 11 cases examined immunohistochemically were positive for platelet-derived growth factor (PDGF)(-BB), especially megakaryoblasts and a few fibroblasts. The PDGF-positive cases showed various degrees of fibrosis, while the negative cases showed no evidence of fibrosis. Our results confirmed the poor prognosis of patients with AMGL, irrespective of the degrees of fibrosis, and demonstrated that PDGF could play an important role in the pathogenesis of marrow fibrosis., (© 2013 The Authors. Pathology International © 2013 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd.)

Background: Alternative splicing of pre-mRNA transcripts not only plays a role in normal molecular processes but is also associated with cancer development. While normal transcripts are ubiquitously expressed in normal tissues, splice variants created through abnormal alternative splicing events are often expressed in cancer cells. Although the Rho GDP dissociation inhibitor β (ARHGDIB) gene has been found to be ubiquitously expressed in normal tissues and involved in cancer development, the presence of splice variants of ARHGDIB has not yet been investigated., Results: Validation analysis for the presence of and exon structures of splice variants of ARHGDIB, performed using reverse-transcriptase polymerase chain reaction and DNA sequencing, successfully identified novel splice variants of ARHGDIB, that is, 6a, 6b, and 6c, in colon, pancreas, stomach, and breast cancer cell lines. Quantitative real-time polymerase chain reaction analysis showed that these variants were also highly expressed in normal placental tissue but not in other types of normal tissue., Conclusions: Expression of ARHGDIB variants 6a, 6b, and 6c appears to be restricted to cancer cells and normal placental tissue, suggesting that these variants possess cancer-specific functions and, as such, are potential cancer-related biomarkers.

Background: Classical Hodgkin's lymphoma is characterized by Hodgkin and Reed Sternberg cells, which are of B-cell origin in many cases. We recently highlighted the adverse prognostic significance of cytotoxic molecule expression in patients with classical Hodgkin's lymphoma. However, the clinical characteristics of cytotoxic molecule-positive classical Hodgkin's lymphoma remain controversial., Design and Methods: We investigated the clinicopathological profiles of 32 patients with cytotoxic molecule-positive Hodgkin's lymphoma, comprising 23 with nodular sclerosis and 9 with mixed cellularity, and compared these profiles with those of 55 patients with cytotoxic molecule-positive nodal peripheral T-cell lymphoma, not otherwise specified and 439 patients with cytotoxic molecule-negative Hodgkin's lymphoma., Results: The patients with cytotoxic molecule-positive Hodgkin's lymphoma consisted of 20 men and 12 women with a median age of 50 years (range, 19 to 81). All these patients had lymphadenopathy at presentation, and 14 showed mediastinal involvement. Physical findings included hepatomegaly and splenomegaly in six patients each. Four patients had a bulky mass, and nine showed stage IV disease. The tumor cells of patients with cytotoxic molecule-positive Hodgkin's lymphoma had a prototypic immunophenotype of CD15(+) CD30(+) CD45RO(-) fascin(+), with positivity for Epstein-Barr virus in 39% of cases. All patients were negative for Pax5. In comparison with patients with cytotoxic molecule-positive nodal peripheral T-cell lymphomas, not otherwise specified, patients with cytotoxic-positive Hodgkin's lymphoma had relatively mild clinical symptoms, similar to those of patients with cytotoxic molecule-negative Hodgkin's lymphoma. Regarding prognosis, the survival of patients with cytotoxic molecule-positive Hodgkin's lymphoma was worse than that of patients with cytotoxic molecule-negative Hodgkin's lymphoma (P = 0.0003) but better than that of patients with cytotoxic molecule-positive peripheral T-cell lymphomas, not otherwise specified (P = 0.002)., Conclusions: Cytotoxic molecule-positive Hodgkin's lymphoma is characterized by an unfavorable prognosis, even if its clinicopathological features are within the boundaries of classical Hodgkin's lymphoma. More effective chemotherapy for cytotoxic molecule-positive Hodgkin's lymphoma is clearly required.

Splicing variation enhances proteome diversity and modulates cancer-associated proteins. Thus, the identification of alternative splice forms is significant for discovery of new cancer-related biomarkers. However, relatively few screening approaches of alternative splicing via proteomics have been reported. In the present study, we describe a combined analysis with proteome and transcriptome to simultaneously identify cancer-related splicing variants and splicing variant-derived protein fragments that are differentially expressed in a highly metastatic gastric cancer cell line MKN45P versus its parental cell line MKN45. We found three potential alternative-spliced genes using MS-based shotgun method and two different microarray platforms. Among them, aldolase C, fructose-bisphosphate (ALDOC) was predicted to have novel alternative splice forms. We successfully identified and validated novel splice forms of ALDOC gene by RT-PCR and DNA sequencing analyses, the expression level of which were higher in MKN45P than in MKN45. Furthermore, the protein fragment derived from the validated splicing variant was identified using custom-built data set including sequences of ALDOC variants in MS/MS analysis. Our combined analysis will be a promising technique for screening of cancer-related splicing variants and their protein isoforms., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Differentiating intrahepatic cholangiocarcinoma (CCA) from other hepatic malignancies is crucial in deciding on treatment modalities and predicting clinical outcomes in patients. Periostin is a secreted protein from stromal cells and regulates the development of cancer cells through interaction with the extracellular matrix. Given that proliferation of fibrous stromal cells is a pathological feature of CCA, we examined the potential use of periostin as a serodiagnostic marker for this disease. Our study enrolled a total of 79 patients including liver cirrhosis (n=26), hepatocellular carcinoma (HCC, n=24), CCA (n=8), other hepatic malignancies (n=13) and histologically normal livers (normal control, n=8). Periostin expression was evaluated using immunohistochemistry and serum periostin level was determined via enzyme-linked immunoassay. The diagnostic performance of serum periostin levels for distinguishing CCA patients from others was also assessed. Strong expression of periostin was noted only in the fibrous stroma of CCA tissue. Serum periostin levels (median) were significantly higher in patients with CCA (513 ng/ml) compared to those patients with normal liver, liver cirrhosis, HCC and other malignancies (120, 146, 155, 213 ng/ml, respectively, all P<0.05). The area under receiver operating characteristics curve of serum periostin level was 0.94 [95% confidence interval (CI), 0.85-1.00, P<0.001]. With optimal cut-off value of 302 ng/ml, diagnostic performances for CCA were as follows: sensitivity, 0.88 (95% CI, 0.47-0.99); specificity, 0.92 (0.83-0.96); accuracy, 0.91 (0.83-0.96); positive predictive value, 0.54 (0.25-0.81); negative predictive value, 0.98 (0.92-0.99); positive-likelihood ratio, 10.4 (4.8-13.4); and negative-likelihood ratio, 0.13 (0.03-0.49). We demonstrated increased expression of periostin in the stroma of CCA tissue. Serum periostin levels were significantly elevated in patients with CCA and enable distinction between CCA and other hepatic malignancies.

Peripheral T- and NK-cell lymphomas (PT/NKCLs) are relatively rare, and few studies have validated the International Prognostic Index (IPI) for PT/NKCLs in prospective clinical trials. Histopathological specimens from 136 patients, enrolled in six prospective multicenter trials of doxorubicin-containing regimens, with PT/NKCLs were reviewed by six hematopathologists following the WHO classification. This combined analysis demonstrated that the IPI was not predictive of prognosis for patients with PT/NKCLs as previously shown by GELA. In a univariate analysis, low total serum protein (TP) and albumin levels, gastrointestinal tract involvement, and histologic subtype (extranodal NK/T-cell lymphoma, nasal type, and peripheral T-cell lymphoma, unspecified) were significantly associated with reduced survival. In a multivariate analysis, TP (p = 0.004) and histologic subtype (p = 0.024) remained significant. We discuss the need to establish the importance and meaning of TP and to develop new strategies for patients with PT/NKCLs allowing for TP, especially with worse histologic subtypes.

Peripheral T cell lymphoma not otherwise specified (PTCL-N) and ALK-negative anaplastic large cell lymphoma (ALCL) are heterogeneous categories with poor diagnostic reproducibility. To clarify the biologic features of these categories, we investigated the expression of two chemokine receptors, type 1 (Th1/Tc1)-associated CXCR3 and type 2 (Th2/Tc2)-associated CCR4 in 110 PTCL-N and 35 ALK-negative ALCL cases, as well as the expression of cytotoxic molecules (CM). CXCR3 and CCR4 were expressed in 69 (63%) and 37 (34%) of PTCL-N, and in 12 (34%) and 6 (17%) of ALK-negative ALCL, respectively. In PTCL-N, type 1 pattern (CXCR3(+)CCR4(-)) was dominant (52%), whereas in ALK-negative ALCL, 54% were negative for both (P < 0.0001). CM was expressed in 38% of PTCL-N and 51% of ALK-negative ALCL. CM-positive PTCL-N consisted mostly of type 1 disease, which shows type 1 phenotype. In contrast, type 2 pattern (CXCR3(-)CCR4(+)) was recognized in the CM-negative group only. Among type 1 disease, CM-positive cases had a higher female ratio and more aggressive clinical features than CM-negative cases and a poorer prognosis (P = 0.006). Multivariate analysis confirmed that the type 1 phenotype with CM expression was an independent prognostic factor. In both PTCL-N and ALK-negative ALCL, CM-positive type 1 disease had an extremely poor prognosis.

We evaluated the usefulness of prognostic markers in patients with diffuse large B-cell lymphoma (DLBCL) treated with cyclophosphamide, vincristine, doxorubicin, and prednisolone (CHOP) +/- rituximab (R-CHOP) in Japan. We studied 730 patients with DLBCL; 451 received CHOP and 279 R-CHOP. We analyzed biopsy samples immunohistochemically for markers of germinal center B cells (CD10, Bcl-6), postgerminal center B cells (Multiple myeloma-1), and apoptosis (Bcl-2). The median follow-up period for surviving patients was 56.4 months for the CHOP group and 25.2 months for the R-CHOP group. DLBCL were categorized as germinal center B (GCB) subtype (352/730; 48.2%) or non-GCB subtype (378/730; 51.8%). In the CHOP group, the high expression of CD10 (P = 0.022) or Bcl-6 (P = 0.021), or GCB subtype (P = 0.05) was associated with better overall survival, whereas the high expression of Bcl-2 (P = 0.001) or MUM1 (P = 0.011), or non-GCB subtype (P = 0.05) was associated with worse overall survival. In the R-CHOP group, however, these biomarkers except Bcl-6 were not significant prognostic factors. The patients with non-GCB subtype showed improved survival in the R-CHOP group (P = 0.756). The International Prognostic Index was a useful clinical marker of survival in the CHOP group (P < 0.001) and also in the R-CHOP group (P < 0.001). Results of improved survival with rituximab addition indicate that the relevance of previously recognized prognostic factors should be re-evaluated.

Burkitt's lymphoma and atypical Burkitt/Burkitt-like lymphoma (BL/BLL) are considered highly aggressive B-cell lymphomas with a rapid proliferative rate and high rate of apoptosis. The aim of the present study was to confirm whether apoptotic and cell proliferative factors affect BL/BLL clinical outcomes. We retrospectively analyzed the relationship between the clinical and immunophenotypic features of 43 BL/BLL patients by immunohistochemical staining for bcl-2 and double staining for Ki-67 plus caspase-3. In double staining experiments, all patients were divided into high and low groups for the expression of caspase-3, Ki-67, and both Ki-67 and caspase-3, by using the medians of their percentages as limits. The 43 BL/BLL patients were divided into high caspase-3 (n = 19) and low caspase-3 (n = 24) groups. There was a significant difference in the overall survival between the high (77%) and low caspase-3 (33%) groups; the survival rate of patients in the low caspase-3 group who received aggressive short-term chemotherapy (58%) was significantly better than that of patients who received cyclophosphamide, doxorubicin, vincristine and prednisolone (CHOP) therapy (17%). All patients positive for bcl-2 were in the low caspase-3 group (high caspase-3 group, 0%; low caspase-3 group, 42%). The overall survival tended to be better in the high caspase-3 and bcl-2-negative group (76%) than in the low caspase-3 and bcl-2-negative (50%) group. In addition, the low caspase-3 and bcl-2-positive group tended to show the worst prognosis (16%). We suggest that caspase-3 may function as an indicator of the prognosis of BL/BLL. Furthermore, intensive short-term chemotherapeutic regimens may improve the prognosis of the patients in the low caspase-3 group.

Diffuse large B-cell lymphoma (DLBCL) accounts for 30% of non-Hodgkin's lymphomas and is known to comprise heterogeneous groups. We previously reported that CD5+ DLBCL is a clinically distinct subgroup of these tumors that is associated with poor prognosis. In our current study, we have used gene expression profiling technology in an attempt to identify new markers and to further characterize the biological features of CD5+ DLBCL. Candidate genes, which showed the greatest difference in expression between 22 CD5+ and 26 CD5- DLBCL cases, were selected from our screening and subjected to clustering analysis. This resulted in identification of a specific mRNA profile (a CD5 signature) for CD5+ DLBCL. The CD5 signature included downregulated extracellular matrix genes such as POSTN, SPARC, COL1A1, COL3A1, CTSK, MMP9 and LAMB3, and comprised upregulated genes including TRPM4. We tested this CD5 signature for its potential use as a relevant marker for CD5+ DLBCL and found that it did indeed recognize this subgroup. The tumors identified by the CD5 signature contained most of the CD5+ DLBCL cases and some CD5- DLBCL cases. Moreover, the subgroup of cases with this CD5 signature showed a poorer prognosis. The subsequent application of the CD5 signature to the analysis of an independent series of DLBCL microarray data resulted in identification of a subgroup of DLBCL cases with a similar clinical outcome, further suggesting that the CD5 signature can be used as a clinically relevant marker of this disease.

Esophageal small cell carcinoma (SmCC) is a rarer, more highly aggressive, and more rapidly growing neoplasm than esophageal squamous cell carcinoma (SqCC). SmCC and SqCC also differ in terms of chemotherapy of choice, response to therapy, and prognosis. Accordingly, it is important to differentiate the 2 carcinomas. We studied the histology and immunohistochemical profiles of 6 cases of esophageal SmCC to elucidate the correct diagnosis of this tumor. We performed immunohistochemical analysis antibodies against cytokeratins (CKAE1/AE3, CKCAM5.2, CK34betaE12, CK7, CK8, CK10/13, and CK19), epithelial membrane antigen (EMA), neural cell adhesion molecule (NCAM; CD56), neuron-specific enolase (NSE), chromogranin-A, S-100 protein, carcinoembryonic antigen (CEA), E-cadherin, thyroid transcription factor-1 (TTF-1), and p53. In 3 of the 6 SmCCs, heterogeneous components of in situ or invasive SqCC were observed. SqCC was found in the mucosa adjacent to the main SmCC, and the boundary between SmCC and SqCC was distinct, with no transitional features. Staining for NCAM, NSE, and chromogranin-A was positive in SmCCs, but negative in SqCCs. Both SmCCs and SqCCs were positive for CKAE1/AE3, CKCAM5.2, CK8, and EMA, but only SqCCs were positive for CK34betaE12 and CK19. Moreover, SmCCs containing SqCC components were positive for CEA and E-cadherin, whereas SmCCs without SqCC were negative. Our study suggests that NCAM and NSE are useful markers in diagnosing esophageal SmCC, and CK34betaE12 and CK19 are useful for differentiating SqCC components from SmCC.

Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase located in the cell nucleus which catalyses the polymerization of deoxynucleotides at the 3'hydroxyl ends of oligo- or polydeoxynucleotide initiators without a template. TdT is known as a useful marker for the diagnosis of acute lymphoblastic leukaemia/lymphoma, but its detection usually requires fresh tissue specimens or cell suspensions, using either an enzyme analysis or immuno-fluorescence or -peroxidase staining. Until the recent development of the use of microwave-treated paraffin sections for immunoperoxidase staining, detection of TdT in paraffin sections required rather complicated processes. This new simple technique was applied to paraffin sections from the tumour tissue specimens of 16 patients with lymphoblastic lymphoma and of seven patients with non-endemic Burkitt's lymphoma, which is sometimes difficult to differentiate from lymphoblastic lymphoma because of their similar clinicopathological characteristics. In addition, as a control, ten cases each were examined of adult T-cell leukaemia/lymphoma (ATLL) and angioimmunoblastic lymphoma (AILD), which are both peripheral T-cell lymphomas. The tumour cells from 15 of the 16 (94 per cent) patients with lymphoblastic lymphoma were found to be TdT-positive. The specificity of the anti-TdT antibody used was confirmed by immunoblot and the specific 60 kD band was detected only in a specimen of lymphoblastic lymphoma. These results show that the immunostaining of TdT on paraffin-embedded sections is a useful method for differentiating lymphoblastic lymphoma from other lymphomas. This method is applicable to a routine diagnostic service.

A case of heterogeneous acute lymphoblastic leukemia of the Burkitt type (L3-ALL) is reported. A 58-year-old woman was admitted to our hospital with complaints of palpitation and shortness of breath in December 1988. On admission, the peripheral blood showed severe pancytopenia. The bone marrow was hypercellular with abnormal lymphoblasts; the cytoplasm was moderately abundant and intensely basophilic with prominent vacuoles. A diagnosis of L3-ALL was made. Cytogenetic analysis of bone marrow cells revealed a karyotype of 46,XX. The leukemic blasts were HLA-DR+, CD10+, CD19+ and CD20+, but no surface or cytoplasmic immunoglobulins were detected. This is the first report in Japan of a case of L3-ALL without chromosome abnormality and lacking surface and cytoplasmic immunoglobulins.