Yutaka Hatanaka

Quantitative immunohistochemical evaluation of HER2/neu expression with HercepTestTM in breast carcinoma by image analysis

HercepTestTM (DAKO A/S, Glostrup, Denmark) is an immunohistochemical assay that detects HER2/neu gene products, and evaluates the overexpression status of the HER2/neu protein in determining eligibility for the Trastuzumab (HerceptinR, Genentech, San Francisco, CA, USA) therapy. However, practically, interobserver variability of the HER2/neu interpretation of the immunostained results has caused marked disagreement with regard to the intensity of tumor staining. In this study, we quantitated HER2/neu expression by image analysis, and applied this analyzing system to help to minimize interobserver variability of the interpretation of the HercepTestTM. All the immunostained results were scored semiquantitatively on a range of 0 to 3+ in accordance with the criteria described as per the manufacturer’s instructions, and quantitatively evaluated using an image analyzing system with image processing software. Among the 92 cases, 15 were scored as 3+, six were 2+, and 32 were 1+ under intraobservers agreement. When the cases were quantitated, a high correlation was shown between the signal area extracted by image analysis and the corresponding score of staining intensity with the HercepTestTM. By converting the quantitatively extracted data into a scoring system based upon the criteria, the outcome demonstrated a strong concordance with the scoring data obtained from immunostaining. The results indicated that a quantitative scoring system performed by simple image analysis may provide to improve interobserver agreement of the interpretation of the HercepTest TM in clinical practice.

Disruption of the blood brain barrier by brain metastases of triple‐negative and basal‐type breast cancer but not HER2/neu‐positive breast cancer

Generally, the blood‐brain barrier (BBB) of brain metastasis was thought to be disrupted.

Cytometrical image analysis for immunohistochemical hormone receptor status in breast carcinomas

A cytometrical image analyzing method for nuclear protein was established using WinROOF, a commercially available, inexpensive software, to determine the status of both estrogen and progesterone receptors. Immunohistochemical evaluation of estrogen receptors (ER) and progesterone receptors (PR) was performed with the anti‐ER (clone 1D5) and the anti‐PR (clone PgR636), respectively, combined with dextran polymer reagent EnVision+, all of which are approved in vitro diagnostics in Japan. The immunostained results were captured as digital images in Windows, and then analyzed in WinROOF with macroinstructions for analyzing each captured area either immunolabeled with chromogen or counterstained with hematoxylin. This image analysis method graded the immunostained nuclei of carcinoma cells based on staining intensities, and calculated the labeling index (LI) for both ER and PR. Furthermore, the LI correlated highly with the results from a histology score (HSCORE) when 20 breast carcinomas were quantified. Regarding ER, when 20% in the LI was considered as the cut‐off point for positive, the positivity of ER in computer‐assisted analysis was 75% (15 of 20 cases), and was completely concordant with that of HSCORE‐based analysis. These results indicate that the cytometrical image analysis‐based quantification could be appropriately applied to the objective determination of the immunohistochemical status of both ER and PR.

Bright-Field Dual-Color Chromogenic In Situ Hybridization for Diagnosing Echinoderm Microtubule-Associated Protein-Like 4-Anaplastic Lymphoma Kinase-Positive Lung Adenocarcinomas

Introduction: A subset of lung cancers harbors an EML4-ALK (echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase) gene fusion, and detecting this subset may hold therapeutic implications. Many prior studies used fluorescence in situ hybridization (FISH) analysis for this detection, but FISH may have disadvantages including signal decay and dark-field examination that may obscure tissue architecture. In this study, we explored the potential of the ALK-break-apart chromogenic in situ hybridization (CISH) method to detect ALK-rearranged lung cancer. Methods: We examined 15 lung adenocarcinomas with reverse-transcriptase polymerase chain reaction-proven EML4-ALK fusion transcripts and 30 ALK-negative cases. One hundred tumor cells were evaluated by CISH and FISH for each case, and a detailed signal profile was recorded and compared. Results: CISH preserved tissue architecture and cytomorphology considerably and facilitated the signal evaluation using a routine light microscope. Positive rearrangement signals (splits or isolated 3′ signals) were identified in 13 to 78% (mean ± SD, 41% ± 19%) of tumor cells in the ALK-positive cohort and in 0 to 15% (mean ± SD, 6% ± 4%) of cells in the ALK-negative cohort. The two groups were best separated by a cutoff value of 20%, with a sensitivity of 93% and a specificity of 100%. The only false-negative tumor having only 13% CISH-positive cells displayed predominantly (76%) isolated 5′ signals unaccompanied by 3′ signals. FISH showed largely similar signal profiles, and the results were completely concordant with CISH. Conclusions: We have successfully introduced CISH for diagnosing EML4-ALK-positive lung adenocarcinoma. This method allows simultaneous visualization of genetics and tumor cytomorphology and facilitates the molecular evaluation and could be applicable in clinical practice to detect lung cancer that may be responsive to ALK inhibitors.

CC‐chemokine ligand 17 gene therapy induces tumor regression through augmentation of tumor‐infiltrating immune cells in a murine model of preexisting CT26 colon carcinoma

Chemokines, which regulate leukocyte trafficking and infiltration of local sites, are attractive candidates for improving the efficacy of cancer immunotherapy by enhancing the accumulation of immune cells in tumor tissue. Herein, we evaluated the antitumor effects of intratumoral injection of RGD fiber‐mutant adenoviral vectors (AdRGDs) encoding the chemokines CCL17, CCL19, CCL20, CCL21, CCL22, CCL27, XCL1 or CX3CL1 in a murine model of preexisting CT26 colon carcinoma. Among these 8 chemokine‐expressing AdRGDs, injection of AdRGD‐CCL17 most effectively induced tumor regression and generated specific immunity in rechallenge experiments. Tumor elimination activity by intratumoral injection of AdRGD‐CCL17 depended on both the vector dose and the number of injections, and mainly required CD8+ CTLs in an effector phase as confirmed by analysis using BALB/c nude mice and an in vivo depletion assay. In addition, CCL17 gene transduction induced significant increases in the number of infiltrating macrophages and CD8+ T cells in CT26 tumors, and changed the tumor microenvironment to an immunologic activation state in which there was enhanced expression of lymphocyte activation markers and cell adhesion molecules. Thus, our data provide evidence that CCL17 gene transduction of local tumor sites is a promising approach for the development of a cancer immunogene therapy that can recruit activated tumor‐infiltrating immune effector cells.

Prognostic significance of epithelial-mesenchymal transition-related markers in extrahepatic cholangiocarcinoma : comprehensive immunohistochemical study using a tissue microarray

Background:Epithelial–mesenchymal transition (EMT) is characterised by the loss of cell-to-cell adhesion and gaining of mesenchymal phenotypes. Epithelial–mesenchymal transition is proposed to occur in various developmental processes and cancer progression. ‘Cadherin switch’, a process in which cells shift to express different isoforms of the cadherin transmembrane protein and usually refers to a switch from the expression of E-cadherin to N-cadherin, is one aspect of EMT and can have a profound effect on tumour invasion/metastasis. The aim of this study was to investigate the clinicopathological significance of EMT-related proteins and cadherin switch in extrahepatic cholangiocarcinoma (EHCC).Methods:We investigated the association between altered expression of 12 EMT-related proteins and clinical outcomes in patients with EHCC (n=117) using immunohistochemistry on tissue microarrays.Results:Univariate and multivariate analyses revealed that, in addition to N classification (P=0.0420), the expression of E-cadherin (P=0.0208), N-cadherin (P=0.0038) and S100A4 (P=0.0157) was each an independent and a significant prognostic factor. We also demonstrated that cadherin switch was independently associated with poor prognosis (P=0.0143) in patients with EHCC.Conclusions:These results may provide novel information for selection of patients with EHCC who require adjuvant therapy and strict surveillance.

Evaluation of HER2 gene amplification in invasive breast cancer using a dual‐color chromogenic in situ hybridization (dual CISH)

Fluorescence in situ hybridization (FISH) assay is considered the ‘gold standard’ for evaluation of HER2/neu (HER2) gene status, however, it is difficult to recognize morphologic features of tumors using fluorescence microscopy. Thus, chromogenic in situ hybridization (CISH) has been proposed as an alternative method to evaluate HER2 gene amplification. Here, we examined the dual color CISH (dual CISH) method which provides information regarding the copy number of the HER2 gene and chromosome 17 centromere from a single slide. We examined 40 cases of invasive ductal carcinomas of the breast that were resected surgically. HER2 gene status was assessed with FISH (Abbott) and dual CISH (Dako). HER2 gene amplification status was classified according to the guidelines of the American Society of Clinical Oncology and College of American Pathologists (ASCO/CAP). Comparison of the cut‐off values for HER2/chromosome 17 centromere copy number ratio obtained by dual CISH and FISH showed that there was almost perfect agreement between two methods (Kappa coefficient 0.96). The results of the two commercial products were almost consistent for evaluation of HER2 gene counts on the sections. The current study proved that dual CISH is comparable with FISH for evaluating HER2 gene status.

Tumor suppressive efficacy through augmentation of tumor-infiltrating immune cells by intratumoral injection of chemokine-expressing adenoviral vector

Our goal in the present study was to evaluate antitumor effects and frequency of tumor-infiltrating immune cells upon intratumoral injection of RGD fiber-mutant adenoviral vector (AdRGD) encoding the chemokines CCL17, CCL19, CCL20, CCL21, CCL22, CCL27, XCL1, and CX3CL1. Among eight kinds of chemokine-expressing AdRGDs, AdRGD-CCL19 injection most efficiently induced infiltration of T cells into established B16BL6 tumor parenchyma, whereas most of these T cells were perforin-negative in immunohistochemical analysis. Additionally, the growth of AdRGD-CCL19-injected tumors decreased only slightly as well as that of other tumors treated with each chemokine-expressing AdRGD, which indicated that accumulation of naive T cells in tumor tissue does not effectively damage the tumor cells. Tumor-bearing mice, in which B16BL6-specific T cells were elicited by dendritic cell-based immunization, demonstrated that intratumoral injection of AdRGD-CCL17, -CCL22, or -CCL27 could considerably suppress tumor growth and attract activated T cells. On the other hand, AdRGD-CCL19-injection in the immunized mice showed slight increase of tumor-infiltrating T cells compared to treatment using control vector. Collectively, although AdRGD-mediated chemokine gene transduction into established tumors would be very useful for augmentation of tumor-infiltrating immune cells, a combinational treatment that can systemically induce tumor-specific effector T cells is necessary for satisfactory antitumor efficacy.

Immunohistochemical analysis of cancer stem cell markers in pancreatic adenocarcinoma patients after neoadjuvant chemoradiotherapy

BackgroundCancer stem cells (CSCs) have been reported to play an important role in chemoradiation resistance. Although the association of CSC markers with clinicopathological outcomes after neoadjuvant chemoradiotherapy (NACRT) has been reported in various types of cancers, there have been no such reports for pancreatic cancer. Here we examined the sequential changes in CSC marker expressions after NACRT in patients with pancreatic adenocarcinoma (PA) and the impact of these changes on the prognosis.MethodsWe used immunohistochemistry to evaluate the expressions of the CSC markers epithelial cell adhesion molecule (EpCAM), CD24, CD44, CD133, CXCR4 and Aldehyde dehydrogenase 1 (ALDH1) in resected specimens obtained from 28 PA patients, and we compared these expressions with the patients’ clinicopathological parameters and survival data.ResultsThe expression frequencies of CD44 and ALDH1 were significantly higher in the NACRT group (n = 17) compared to the non-NACRT group (n = 11), but the CD133 expression was significantly lower in the NACRT group. In the NACRT group, the expression of CD133 was inversely correlated with that of ALDH1, and CD133+/ALDH1- expression was associated with an unfavorable patient outcome.ConclusionThis is the first report showing that NACRT may influence the expression frequencies of CD44, CD133 and ALDH1 in PA patients. Moreover, CD133 and ALDH1 expressions may be useful predictors of prognosis in PA patients who have received NACRT.

Cotransduction of CCL27 gene can improve the efficacy and safety of IL-12 gene therapy for cancer

Interleukin-12 (IL-12) is a potent antitumoral cytokine, but high doses are toxic. Herein, we demonstrate that combinational transduction of IL-12 and CC-chemokine ligand-27 (CCL27) genes into pre-existing murine OV-HM ovarian carcinoma and Meth-A fibrosarcoma, by using RGD fiber-mutant adenoviral vectors, could induce tumor regression and relieve systemic side effects more effectively than either treatment alone. The antitumor activity of the IL-12 and CCL27 combination treatment was T-cell-dependent, and development of long-term specific immunity was confirmed in rechallenge experiments. Immunohistochemical analysis of tumors transduced with CCL27 gene alone or cotransduced with IL-12 and CCL27 genes showed significant increases in numbers of infiltrating CD3+ T cells, which included both CD4+ and CD8+ cells. Additionally, cotransduction with IL-12 and CCL27 genes could more efficiently activate tumor-infiltrating immune cells than transduction with CCL27 alone, as determined by the frequency of perforin-positive cells and expression levels of IFN-γ. Furthermore, mice treated with the IL-12 and CCL27 combination compared with those treated with IL-12 alone showed milder pathological changes, for example, lymphocyte infiltration and extramedullary hematopoiesis, in lung, liver and spleen. Our data provide evidence that combinational in vivo transduction with IL-12 and CCL27 genes is a promising approach for the development of cancer immunogene therapy that can simultaneously recruit and activate tumor-infiltrating immune cells.