Shinji Sumiyoshi

Validation of the IASLC/ATS/ERS Lung Adenocarcinoma Classification for Prognosis and Association with EGFR and KRAS Gene Mutations: Analysis of 440 Japanese Patients

Introduction: This study aimed to validate the utility of the new histological classification proposed by the International Association for the Study of Lung Cancer (IASLC), American Thoracic Society (ATS), and European Respiratory Society (ERS) for identifying the prognostic subtypes of adenocarcinomas in Japanese patients; correlations between the classification and the presence of EGFR or KRAS mutation status were also investigated. Methods: We retrospectively reviewed 440 patients with lung adenocarcinoma, who underwent resection. The tumors were classified according to the IASLC/ATS/ERS classification. EGFR and KRAS mutations were detected using the established methods. Results: Five-year disease-free survival rates were: 100% for adenocarcinoma in situ (n = 20) and minimally invasive adenocarcinoma (n = 33), 93.8% for lepidic-predominant adenocarcinoma (n = 36), 88.8% for invasive mucinous adenocarcinoma (n = 10), 66.7% for papillary-predominant adenocarcinoma (n = 179), 69.7% for acinar-predominant adenocarcinoma (n = 61), 43.3% for solid-predominant adencoarcinoma (n = 78), and 0% for micropapillary-predominant adenocarcinoma (n = 19). Multivariate analysis revealed that the new classification was an independent predictor of disease-free survival. EGFR and KRAS mutations were detected in 90 cases (53.9%) and 21 cases (13.3%), respectively; EGFR mutations were significantly associated with adenocarcinoma in situ, minimally invasive adenocarcinoma, lepidic- and papillary-predominant adenocarcinoma, and KRAS mutations adenocarcinomas with mucinous tumor subtypes. Conclusions: We found that the IASLC/ATS/ERS classification identified prognostic histologic subtypes of lung adenocarcinomas among Japanese patients. Histologic subtyping and molecular testing for EGFR and KRAS mutations can help predict patient prognosis and select those who require adjuvant chemotherapy.

HER2 status in lung adenocarcinoma: a comparison of immunohistochemistry, fluorescence in situ hybridization (FISH), dual-ISH, and gene mutations.

OBJECTIVES While novel anti-human epidermal growth factor receptor 2 (HER2) agents have recently been developed, no definite criteria have been proposed as indications for the use of these agents in patients with lung cancer. Here, we tested HER2 alterations by using four methods and explored the concordance of these methods to improve our understanding of the accuracy of HER2 testing methods. MATERIALS AND METHODS We analyzed HER2 protein expression by immunohistochemistry (IHC) and HER2 amplification by fluorescence in situ hybridization (FISH) and dual in situ hybridization (DISH) by using a tissue microarray comprising lung adenocarcinoma specimens from 243 consecutive patients. The presence of mutations in the EGFR, KRAS, and HER2 genes were also determined. RESULTS Positive IHC (score 3+) was observed in six cases (2.5%). Amplification of HER2 was observed in five cases (2.1%) by FISH and in nine cases (3.7%) by DISH. HER2 expression by IHC and gene amplification by FISH were significantly associated (P<0.001). The overall concordance between FISH and DISH by amplification status was 96.7% (P<0.001). One hundred nine tumors (49.9%) had EGFR mutations, 25 (11.2%) had KRAS mutations, and six (2.7%) had HER2 mutations. All of these mutations were mutually exclusive. Cases having HER2 mutations were positively correlated with cases having HER2 amplification (P<0.001). Two of six cases with HER2 mutations showed amplifications by FISH and DISH tests. CONCLUSION HER2 protein overexpression, gene amplification, and gene mutations appeared to be uncommon in lung adenocarcinoma. Cases with HER2 mutations tended to show HER2 gene amplification. The results indicated that HER2 gene amplification and mutations should be tested to determine whether patients are eligible for administration of new anti-HER2 agents. In addition, DISH was better than FISH for detection of cases with HER2 amplification.

Pulmonary adenocarcinomas with micropapillary component significantly correlate with recurrence, but can be well controlled with EGFR tyrosine kinase inhibitors in the early stages

Pulmonary adenocarcinoma with a micropapillary component (PA-MPC) is known to exhibit biologically aggressive behavior. The aim of this study was to evaluate the clinicopathological characteristics of early-stage PA-MPC and to investigate the correlation between PA-MPC and epidermal growth factor receptor (EGFR) or KRAS mutation status. We reviewed 440 PA patients who underwent resection. We defined PA-MPC as adenocarcinoma with MPC occupying at least 5% of the entire tumor. EGFR and KRAS mutations were detected using established methods. Of the 440 cases, 256 cases were classified as stage IA, of which 53 cases (20.7%) had MPC. The 5-year disease-free survival rates in the MPC-negative and MPC-positive groups of patients with stage IA tumors were 92.1% and 77.6%, respectively. The difference in these rates was statistically significant (p = 0.003), whereas the difference in overall survival between the groups was not statistically significant (p = 0.973). The mean percentage of MPC was 20.4% in the recurrent group and 18.3% in the non-recurrent group, with no significant correlation (p = 0.996). Of the 10 recurrent cases, 6 cases exhibited EGFR mutations; the 5 cases treated with a tyrosine kinase inhibitor (TKI) achieved long survival (median, 64.6 months). No KRAS mutations were detected in any of the 10 cases. PA-MPCs were strongly associated with recurrence, but were not influenced by the MPC percentage even in early-stage lesions. Moreover, PA-MPCs with recurrence were associated with relatively better survival. These findings indicate that PA-MPCs were biologically aggressive but could be controlled with EGFR-TKIs.

CD8+tumor-infiltrating lymphocytes at primary sites as a possible prognostic factor of cutaneous angiosarcoma

Tumor‐infiltrating lymphocytes (TILs) have been reported as a prognostic factor in various cancers and are a promising target for immunotherapy. To investigate whether TILs have any impact on the prognosis of angiosarcoma patients, 55 non‐treated patients (40 patients at stage 1 with cutaneous localized tumors, 4 patients at stage 2 with lymph node metastases and 11 patients at stage 3 with distant metastases) with angiosarcoma were evaluated retrospectively by immunohistochemistry stained CD4, CD8, FOXP3 and Ki67. The Kaplan–Meier method was used to estimate overall survival with patients at stage 1. Survival differences were analyzed by the log‐rank test. Patients with higher numbers of CD8+ TILs in their primary tumors survived significantly longer compared with patients with lower values. Moreover, the number of CD8 in TILs was positively correlated with a distant metastasis‐free period. The total number of primary TILs (CD4 plus CD8) and CD8+ primary TILs of stage 3 patients with distant metastases was positively correlated with their overall survival. To evaluate whether CD8+ effector T cells are activated or differentiated, flow cytometric analysis of peripheral blood mononuclear cells (PBMC) was performed. The percentages of CD8+ T cells producing IFN‐γ in PBMC were significantly higher in patients with angiosarcoma (n = 10) compared not only with that of healthy controls (n = 20) but also patients with advanced melanoma (n = 11). These results suggest that anti‐tumor immunity is clinically relevant in angiosarcoma.

The Expression Profile of Phosphatidylinositol in High Spatial Resolution Imaging Mass Spectrometry as a Potential Biomarker for Prostate Cancer

High-resolution matrix-assisted laser desorption/ionization imaging mass spectrometry (HR-MALDI-IMS) is an emerging application for the comprehensive and detailed analysis of the spatial distribution of ionized molecules in situ on tissue slides. HR-MALDI-IMS in negative mode in a mass range of m/z 500–1000 was performed on optimal cutting temperature (OCT) compound-embedded human prostate tissue samples obtained from patients with prostate cancer at the time of radical prostatectomy. HR-MALDI-IMS analysis of the 14 samples in the discovery set identified 26 molecules as highly expressed in the prostate. Tandem mass spectrometry (MS/MS) showed that these molecules included 14 phosphatidylinositols (PIs), 3 phosphatidylethanolamines (PEs) and 3 phosphatidic acids (PAs). Among the PIs, the expression of PI(18:0/18:1), PI(18:0/20:3) and PI(18:0/20:2) were significantly higher in cancer tissue than in benign epithelium. A biomarker algorithm for prostate cancer was formulated by analyzing the expression profiles of PIs in cancer tissue and benign epithelium of the discovery set using orthogonal partial least squares discriminant analysis (OPLS-DA). The sensitivity and specificity of this algorithm for prostate cancer diagnosis in the 24 validation set samples were 87.5 and 91.7%, respectively. In conclusion, HR-MALDI-IMS identified several PIs as being more highly expressed in prostate cancer than benign prostate epithelium. These differences in PI expression profiles may serve as a novel diagnostic tool for prostate cancer.

Stromal plasma cells expressing immunoglobulin G4 subclass in non-small cell lung cancer.

Inflammatory cell infiltration in tumor stroma may represent the interaction between the tumor and the immune system. The significance of immunoglobulin (Ig) G4+ plasmacytic infiltration, however, is poorly understood. Here, we analyzed the number of stromal IgG4+ plasma cells and the IgG4/IgG ratio of plasma cells in 294 primary non-small cell lung cancers (NSCLCs) using tissue microarray (TMA) and conventional surgical specimens. In TMA, 35 (12%) cases of NSCLC revealed more than 20 IgG4+ plasma cells per high-power field. In surgical specimens, most (97%) of those IgG4+ plasma cell-enriched cases showed obliterative phlebitis or arteritis, one of the key morphologic features of IgG4-related disease, within or at the periphery of the tumor. Clinically, none of the patients showed symptoms associated with IgG4-related systemic diseases. In patients with stage I squamous cell carcinoma, IgG4-enriched stroma was significantly associated with a favorable prognosis (P = .04). In conclusion, considerable IgG4+ plasma cell infiltration can be seen in a minority of cases of NSCLC and might contribute to prognostic modulation of NSCLC.

Application of complement component 4d immunohistochemistry to ABO‐compatible and ABO‐incompatible liver transplantation

Antibody‐mediated rejection (AMR) is difficult to diagnose after ABO‐compatible or ABO‐identical (ABO‐C) liver transplantation. To determine whether complement component 4d (C4d) immunostaining would be useful for diagnosing AMR, we compared the results of C4d immunohistochemistry for allograft biopsy samples with assays for anti‐donor antibodies performed at the time of biopsy. One hundred fourteen patients with ABO‐C grafts and 29 patients with ABO‐incompatible (ABO‐I) grafts were included. Linear C4d endothelial staining (identifiable with a 4× objective lens) or staining seen in 50% or more of the portal tracts was considered positive. Five of the 114 patients (4%) with ABO‐C grafts and 15 of the 29 patients (52%) with ABO‐I grafts showed C4d positivity. In the ABO‐C cases, C4d positivity in late biopsy samples (≥30 days after transplantation) was associated with stage 2 or higher fibrosis (METAVIR score; P = 0.01) and with the presence of donor‐specific anti–human leukocyte antigen DR antibodies (HLA‐DR DSAs) with a mean fluorescence intensity > 5000 according to the Luminex single‐antigen bead assay (P = 0.04). Conversely, the presence of HLA‐DR DSAs was associated with the presence of stage 2 or higher fibrosis, acute cellular rejection, and C4d positivity. During the 2‐year follow‐up, neither C4d positivity nor HLA‐DR DSAs were related to graft loss. Among ABO‐I patients, C4d positivity was not associated with allograft dysfunction or fibrosis. Only 3 of the 15 C4d‐positive patients (20%) showed periportal hemorrhagic edema, which could be a histological sign of AMR in ABO‐I grafts, and they were the only cases associated with elevations in anti‐donor A/B antibody titers. In conclusion, C4d endothelial positivity among ABO‐C patients is an uncommon event that could be associated with chronic graft damage with or without clinical AMR. C4d positivity is common among ABO‐I patients and may not be associated with allograft dysfunction if alloantibody titers are not elevated. Liver Transpl 20:200‐209, 2014.

Telomere shortening and karyotypic alterations in hepatocytes in long‐term transplanted human liver allografts

The long‐term fate of aged liver allografts in young recipients who received grafts from older donors is unknown. We evaluated graft aging by analyzing hepatocytic telomere length and karyotypic changes. Seventeen pediatric individuals who underwent living‐donor liver transplantation for congenital biliary diseases were selected. At a median of 10.4 years post‐transplant, ten had tolerated grafts with weaned off immunosuppressants, and seven had idiopathic post‐transplantation hepatitis. Fluorescence in situ hybridization was used to evaluate the telomere signal intensity (TI) and karyotypic changes. First, we measured predictive age‐dependent TI decline with regression analysis of donor livers. The mean TI at the earliest (within a year) and latest biopsies was significantly lower than the predicted TI of the studied allografts. With univariate analysis, a higher abnormal karyotype ratio in the donor liver was correlated with development of idiopathic post‐transplantation hepatitis. With multivariate analysis that included clinical parameters, a greater TI decline at the earliest biopsy was correlated with the development of idiopathic post‐transplantation hepatitis. In conclusion, graft aging as measured by TI decline and donor abnormal karyotype ratio was associated with idiopathic post‐transplantation hepatitis of long‐term transplanted liver allografts.

Non-terminal respiratory unit type lung adenocarcinoma has three distinct subtypes and is associated with poor prognosis

OBJECTIVES The characteristics of non-terminal respiratory unit (TRU) type lung adenocarcinoma are still unclear. The aim of the present study was to characterize non-TRU type lung adenocarcinoma. MATERIALS AND METHODS We analyzed the expression of mucins MUC5B and MUC5AC, as well as thyroid transcription factor-1 (TTF-1), using a tissue microarray comprising lung adenocarcinoma specimens from 244 consecutive patients. The presence of mutations in EGFR and KRAS were also determined. RESULTS TTF-1, MUC5B, and MUC5AC were detected in 219 (89.8%), 75 (30.7%), and 33 cases (13.5%), respectively. Cluster analysis of protein expression profiles and EGFR and KRAS mutations yielded five groups of tumors as follows: TRU1-type [TTF-1(+), MUC5B(-), MUC5AC(-), EGFR mutations(-)]; TRU2-type [TTF-1(+), MUC5B(-), MUC5AC(-), EGFR mutations(+)]; Combined-type [TTF-1(+), MUC5B(+), and/or MUC5AC(+)]; Bronchiolar-type [TTF-1(-), MUC5B(+) and/or MUC5AC(+)]; and Null-type [TTF-1(-), MUC5B(-), MUC5AC(-), EGFR mutations(-), KRAS mutations(-)]. TRU-type tumors, which include TRU1- and TRU2-type tumors, were significantly associated with TRU morphology, whereas Bronchiolar-type tumors were associated with non-TRU morphology. Combined-type cases exhibited intermediate morphologies between TRU-type and Bronchiolar-type cases. TRU-type was associated with significantly better prognosis, followed by Combined-type, Bronchiolar-type, and Null-type (disease-free survival [DFS] P = 0.017; overall survival [OS], P = 0.002). Multivariate analyses indicated that non-TRU type tumors, which include Bronchiolar-, Combined-, Null-type tumors, were significantly correlated with poorer prognoses for DFS (hazard ratio = 1.785; 95% CI, 1.041-3.063; P = 0.035) and OS (hazard ratio = 1.928; 95% CI, 1.084-3.421; P = 0.025). CONCLUSION This study revealed three distinct subtypes of non-TRU type adenocarcinomas. Additionally, non-TRU type tumors were associated with worse prognoses than TRU type tumors. The results presented here may be useful for select patients should appropriate therapies become available.

Intraductal tubulopapillary neoplasm with expansile invasive carcinoma of the pancreas diagnosed by endoscopic ultrasonography‐guided fine needle aspiration: A case report

Intraductal tubulopapillary neoplasm (ITPN) of the pancreas, a novel entity included in the World Health Organization 2010 classification, accounts for <1% of all pancreatic exocrine neoplasms and the number of reported cases is limited in the English literature. Herein we describe the cytologic features of ITPN with invasive carcinoma showing expansile growth on endoscopic ultrasonography‐guided fine needle aspiration (EUS‐FNA) cytology.