超低流速の液体クロマトグラフィーや質量分析の機器や技術の進歩により，数年前には考えられなかった高い感度でタンパク質の網羅的な解析が可能になってきている。本稿では国立がんセンター研究所にて行われている，このようなプロテオーム解析によるがんの早期診断・個別化医療のためのバイオマーカーの開発について概説する。 Cytometryの領域とは異なるが，定量的な解析より臨床医学や生物現象を理解しようとする点で共通性があり，本誌の読者に興味をもっていただければ幸いである。

癌組織を構成する細胞は等しく腫瘍形成能を有するのではなく，腫瘍形成能を有する癌幹細胞と腫瘍形成能を有しない細胞に区別できるという癌幹細胞仮説が造血器腫瘍で提唱され注目されている。最近，この癌幹細細胞は固形癌の代表格である消化器癌でも示された。この概念は日頃の臨床現場で腫瘍の多様性の克服が癌治療の要を成すと実感している臨床家にとって理解し易いものである。癌幹細胞は自己複製しながら腫瘍としての造腫瘍性に重要な役割を果たす。そこでは，他臓器とも共通するヒト腫瘍の検定系の課題とともに，消化器癌に特徴的なマーカーの開発，分子のメカニズム解析などが必要となっている。今後癌幹細胞仮説の導入による新たな腫瘍学に基づいた消化器癌治療法の確立が期待される。

NK cells recognize MHC class I molecules and inhibit the killing of target cells that have self MHC class I molecules through their inhibitory receptors. Therefore inhibitory NK receptor negatively regulate NK cell functions, including target cell lysis, through their binding to MHC class I molecules. The development of leukemia and malignant tumors might be induced as a consequence of the failure of immune surveillance. Leukemic cells and tumor cells can escape from immune recognition by cytotoxic T cells because of the low expression level of tumor antigen and/or HLA class I molecules. In such cases, non-tumor antigen-restricted cytotoxic cells such as NK cells and NK receptor-expressing cells may be important to overcome tolerance in the tumorbearing host. Donor allogeneic NK cells can attack patient's leukemic cell resulting in the enhancement of graft-versus-leukemia（GVL）effect and at the same time attack host antigen-presenting cells resulting in the suppression of graft-versus-host disease（GVHD）in allogeneic stem cell transplantation with Killer cell immunoglobulin-like recepto（r KIR）-ligand mismatch donor. Therefore, NK cell and KIR incompatibility may have an important role in the clinical outcome of immunological cell therapy such as allogeneic stem cell transplantation for not only hematological malignancies but also solid cancers.

Tumor cells release many kinds of immunosuppressive and pro-inflammatory factors that facilitate tumor immune escape and tumor growth. In addition, many kinds of immunosuppressive cells accumulate in the tumor microenvironment. These factors are ultimately responsible for orchestrating immunosuppressive network at the tumor site. CD1d-restricted invariant natural killer T (iNKT) cells play crucial roles in both the innate and acquired immune responses. Activation of iNKT cells leads to downstream activation of other cell types in the immune system, which is crucial to the protective anti-tumor immune responses. In this review, we discuss the contribution of iNKT-dendritic cell (DC) cross-talk governing T helper (Th) balance and the possible mechanisms by which NKT cells regulate the tumor microenvironment to enhance the anti-tumor immune responses.

Colorectal cancer (CRC) is the third most common _nd cancers in the world with a very high mortality rate. In many countries like Canada and Japan it is the 2^nd after lung cancer. Most CRC are sporadic, and others are familial or genetic. Sequence-time Fishbone of multistage colorectal cancer is associated between macro scopic developing [from adenomatous polyp, severe dysplasia, carcinoma, to metastasis] and up-regulated follow by mutation of several gene expression [APC, K-ras, p53, DCC] has been figured. The dancing of CD4- CD8 occur during the evolution of adenomatous polyp, severe dysplasia to carcinoma. Sequence APC, K-ras,p53, DCC gene mutation in the Fishbone has been transformed to a Firecrackers (up-regulated then down regulated APC gene will up-regulated the next gene K-ras, and p53. When K-ras and 53 is down-regulated DCC gene is up-regulated, when DCC gene is mutated metastasis happens). Non genetic, non gender, famil ial, geographical dependency (sporadic), also in young people, increase 2 to about 200 times in last decade ask many scientist who is the firelighter of the firecrackers gene mutation? The aim of this study was to identi fy the firelighter of the colorectal cancer development and progression and find that U-238 which emits alpha and beta ray during their decay is the agent.

An elevated CEA, confirmed by retesting, needs further evaluation for metastatic disease, but does not justify the institution of adjuvant therapy or systemic therapy for presumed metastatic disease. It is because of CEA has only moderate 70% sensitivity and specificity. Decreasing CD4, CD8 and ratio of CD4/CD8 increase the specificity of this CEA function. This study figured a fishbone and then transformed to firecrackers of APC, K-ras, p53, DCC mutation, microsatellite instability which has commonly occured during the evolution of adenomatous polyp, severe dysplasia to carcinoma. The position of the dancing of CD4 and CD8-PE is put on this firecrackers corridor. Using the dancing of CD4 and CD8-PE is along with T-reg (T4reg and T8reg) in the time sequence firecrackers with upregulated-downregulated gen sequences. Up and down of CD4 and CD8 react in between each gene mutation stage., increase the sensitivity and specificity of CEA (CD66e) for CRC prognosis. Quasi experimental got ten patients pre- and post-operative CRC. Analyzing were done with Fisher exact test for categorical determination. Decreasing CD8+ and CD4/CD8 has increased the sensitivity and specificity of CEA to 100%.

Introduction:

Cancer immunotherapy has been used in clinical trials and shows a powerful life force. But if it is benefit for the treatment of gastric cancer is still not well studied. Present study discussed the antitumor efficiency of individualized immunotherapy induced by dendritic cells transfected with total antigen of autologouse gastric cancer cells and reported the initial results of our pilot clinical trail.

Methods:

Autologous DCs were isolated from PBMC and proliferated in supplement with GM-CSF and IL-4. Resulting immature DCs were loaded with autologous tumor antigen (tumor lysate or total RNA) and matured in supplement with TNF-α (tu-DC). Autologous T lymphocytes were isolated from PBMC and cocultured with tu-DC to prepare antigen-specific CTLs. Autologous primary cancer cells were obtained from resected specimens of the patients with gastric cancer and cultured as target cells. Phenotypic characters of DCs were detected and evaluated by FCM. The efficiency of cytotoxicity and cytokine release was measured and estimated by using assays of Cytotoxicity and ELISA.

Humanized immune system was reconstructed on 4-week-old nude mice by intraperitoneal (i.p.) injection of human PBMC with cell number of 3×10⁷ each. Prophylactic assay and therapeutic assay were performed by subcutaneously challenge of primary cancer cells after or before autologous DC vaccination, respectively, and the tumor burden were observed.

Nine patients with gastric cancer were subjected to a pilot clinical trail. Two patients with stage Ib-IIIa underwent a radical D2 resection, 5 patients with stage IIIb-IV underwent palliative resection, and 2 patients with stage IV underwent an open-and-close operation. Autologous tumor lysate were prepared from fresh specimens of the patients. DC vaccines and tumor specific CTLs were prepared by using the methods above mentioned. To suppress Treg and to promote calreticulin (CRT) exposure, CTX 1000 mg and ADM 60 mg were intravenously infused a day before immunotherapy. Tumor-specific CTLs were therapied by intravenous infusing with one day interval, and DC vaccines were inoculated to the axillary lymph nodes with two therapies interval. Thymosin alpha 1 (Tα1) was injected once a day subcutaneously during the therapy. The levels of Treg were measured before and after chemotherapy by FCM, and the Incidence of recurrence, Karnofsky performance scale, tumor burden, and side effects were observed and evaluated.

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Purpose: Some studies suggest that the Cluster of Differentiation 8 (CD8+ T cells) is down-regulated and this reduction may be involved in immune evasion in cancer patients. The present study was designed to investigate CD8+ T lympho cytes and its relationship to Carcinoembryonic Antigen (CEA) in colorectal cancer patients. CEA is correlated with volume tumor with response therapy anti tumor and associated with remnant tumor after resection (sensitivity and specificity 70-80%). High CEA level in preoperative figured probability of post operative recurrent. CEA is not useful for screening general population or early colon Ca but adjunct toward the diagnostic of patient with Colon Carcinoma (Association between CEA level and the stage of malignancy). CEA should not be used to rule out the presence of a malignancy. The best clinical use is in assessing the prognosis of patients with colorectal carcinoma. Plus the drop of CD8, the sensitivity and specificity of recurrent become forward 100%. Experimental Design: CD8+ T cells was evaluated by multicolor flow cytometry. CD8-PE was count with the background of CD3-FITC and CD45-PerCP. CEA in the sera was quantitated by ELISA. Ca Colorecti were included from Top Referral Hospital of Indonesia: Cipto Mangunkusumo Hospital. CEA> 5 ng/ml as D+ and Ratio CD4/CD8 range 1.5-2.2 and increasing CD8 as TResult: Sensitivity become 80% and specificity 100% Conclusions: CD8-PE has an important role to increase sensitivity and specificity of CEA in colorectal carcinoma prognosis.

Although many genomic/epigenomic alterations involved in pathogenesis of gastrointestinal tumors have been reported, their precise molecular mechanisms remain unclear. Using those alterations as landmarks, genes, which are involved in the cancer development/progression and useful for diagnostic markers as well as therapeutic targets in personalized medicine, can be identified.

We have focused on genomic copy-number aberrations, especially the remarkanle aberrations, i.e., local high-level amplifications and homozygous deletions, and tried to identify novel tumor-related genes as targets for those alterations in various types of neoplasms including gastrointestinal cancers. We applied conventional and in-house bacterial artificial chromosome (BAC)-array-based comparative genomic hybridization (CGH). As amplification targets, many genes were identified, and some of them, such as cIAP1 and ZASC1 identified from esophageal cancer and CCND3 from colon cancer, seem to be important as prognosticators as well as therapeutic targets. From homozygously deleted regions detected in esophageal and gastric cancer, several genes were also identified as tumor suppressors frequently silenced through homozygous loss (genetic) or promoter hypermethylation (epigenetic). In addition, BAC array-based methylated CpG island amplification (BAMCA), a newly developed method to screen abnormally methylated sequences using BAC-array, accelerated the identification of tumor suppressor genes whose expression was altered by DNA methylation.

Genome-wide surveys of genetic and epigenetic abnormalities interpreting them in the context of broader knowledge of cancers facilitates the identification of crucial genes and pathways involved in the gastrointestinal carcinogenesis, resulting in the development of new diagnostic methods and molecular targeting therapies of gastrointestinal cancers.

Cancer stem-like cell subpopulations, referred to as “side-population” SP cells have been identified in several tumors based on their ability to efflux the fluorescent dye Hoechst 33342. While SP cells have been identified in the normal human endometrium, they have not been characterized in endometrial cancer. In this study, we isolated and characterized the SP cells in an endometrial cancer cell line and in rat endometrial cells expressing oncogenic [¹²Val] human K-Ras protein. These SP cells showed 1) reductions in the expression levels of differentiation markers 2) long-term repopulating properties 3) self-renewal capacity 4) enhancement of migration and podia formation 5) enhancement of tumorigenicity 6) bipotent developmental potential (tumor cells and stroma-like cells). In nude mice, SP cells formed large, invasive tumors, which were composed of both tumor and extracellular matrix enriched stromal-like cells. The expression levels of vimentin,αsmooth muscle actin and collagen III were enhanced in SP-tumors compared with the levels in non-SP-tumors. Analysis of microdissected samples and fluorescence in situ hybridization showed that the stromal -like cells with enriched ECM contained human DNA, confirming that the stromal-like cells were derived from the inoculated cells. In a matrigel assay, SP cells differentiated into _-SMA -expressing cells. These findings demonstrate that SP cells have cancer stem like cell features including the potential to differentiate into the mesenchymal cell lineage.