Microsatellite Instability and High Content of Activated Cytotoxic Lymphocytes Identify Colon Cancer Patients with a Favorable Prognosis

Colorectal cancers with high-frequency microsatellite instability show peculiar clinicopathological features and a favorable clinical outcome. We investigated whether the improved prognosis for these cancers is related to the content of activated cytotoxic intraepithelial T lymphocytes. Microsatellite instability and the amount of activated cytotoxic lymphocytes were analyzed according to clinicopathological features, survival, and disease recurrence in 109 right-sided colon carcinomas from 245 consecutive patients with stage II/III colon cancer that underwent radical surgery. High-frequency microsatellite instability was found in 43% of stage II/III proximal colon cancers and was associated with significantly higher numbers of activated cytotoxic lymphocytes. High-frequency microsatellite instability, as well as the content of intratumoral-activated cytotoxic T lymphocytes correlated with improved overall and disease-free survival, particularly in patients with stage III tumors. Multivariate analysis revealed that patients with both features had a risk of death and relapse markedly lower than that associated with microsatellite status or intratumoral cytotoxic lymphocytes separately. The presence of local cytotoxic immune responses is probably the major determinant of the good clinical course of patients with microsatellite unstable colon cancer. Furthermore, high-frequency microsatellite instability coupled with a high content of intratumoral cytotoxic lymphocytes may identify a subset of colon cancer patients with a favorable clinical outcome, particularly in stage III disease.     

Clinical Implications of Microsatellite Instability in T1 Colorectal Cancer

Purpose

The estimation of regional lymph node metastasis (LNM) risk in T1 colorectal cancer is based on histologic examination and imaging of the primary tumor. High-frequency microsatellite instability (MSI-H) is likely to decrease the possibility of metastasis to either regional lymph nodes or distant organs in colorectal cancers. This study evaluated the clinical implications of MSI in T1 colorectal cancer with emphasis on the usefulness of MSI as a predictive factor for regional LNM.

Materials and Methods

A total of 133 patients who underwent radical resection for T1 colorectal cancer were included. Genomic DNA was extracted from normal and tumor tissues and amplified by polymerase chain reaction (PCR). Five microsatellite markers, BAT-25, BAT-26, D2S123, D5S346, and D17S250, were used. MSI and clinicopathological parameters were evaluated as potential predictors of LNM using univariate and multivariate analyses.

Results

Among 133 T1 colorectal cancer patients, MSI-H, low-frequency microsatellite instability (MSI-L), and microsatellite stable (MSS) colorectal cancers accounted for 7.5%, 6%, and 86.5%, respectively. MSI-H tumors showed a female predominance, a proximal location and more retrieved lymph nodes. Twenty-two patients (16.5%) had regional LNM. Lymphovascular invasion and depth of invasion were significantly associated with LNM. There was no LNM in 10 MSI-H patients; however, MSI status was not significantly correlated with LNM. Disease-free survival did not differ between patients with MSI-H and those with MSI-L/MSS.

Conclusion

MSI status could serve as a negative predictive factor in estimating LNM in T1 colorectal cancer, given that LNM was not detected in MSI-H patients. However, validation of our result in a different cohort is necessary.     

Prevalence and Prognostic Significance of Neuroendocrine Differentiation in Colorectal Carcinomas

The prognostic significance of neuroendocrine differentiation in colorectal carcinoma is uncertain. We analyzed 289 moderately differentiated (grades II and III) colorectal carcinomas for neuroendocrine differentiation by immunohistochemistry andin situ hybridization. The tumors were divided into three groups based on the presence of and the numbers of neuroendocrine cells, with group I having no neuroendocrine cells, group II having <1 positive cell/mm², and group III with >1 positive cell/mm².In situ hybridization with probes for chromogranin A and B detected almost twice as many neuroendocrine cells as did immunostaining with an antibody for chromogranin A. There was no prognostic difference associated with the presence or absence of neuroendocrine differentiation in this group of moderately differentiated carcinomas. These results indicate that the presence of neuroendocrine cells detected by expression of chromogranin protein or mRNA does not influence prognosis in moderately differentiated colorectal carcinomas.     

Viruses and Cytotoxic T Lymphocytes in Type 1 Diabetes

Histopathological studies on pancreas tissues from individuals with recent-onset type 1 diabetes (T1D) consistently find that CD8 T cells substantially contribute to the formation of islet lesions. CD8 T cells reactive against islet-associated antigens can also be found in blood samples from T1D patients. Mechanistic studies on the pathogenic role of this T cell subset have mostly focused on two animal models, i.e., the non-obese diabetic mouse and the virally induced rat insulin promoter–lymphocytic choriomeningitis virus model. Data were obtained in support of a role for viral infection in expanding a population of diabetogenic cytotoxic T lymphocytes. In view of the theorized association of viral infection with initiation of islet autoimmunity and progression to clinically overt disease, CD8 T cells thus represent an attractive target for immunotherapy. We will review here arguments in favor of a pivotal role for CD8 T cells in driving T1D development and speculate on etiologic agents that may provoke their aberrant activation.     

Loss of CDX2 Expression and Microsatellite Instability Are Prominent Features of Large Cell Minimally Differentiated Carcinomas of the Colon

Most large bowel cancers are moderately to well-differentiated adenocarcinomas comprised chiefly or entirely of glands lined by tall columnar cells. We have identified a subset of poorly differentiated colon carcinomas with a distinctive histopathological appearance that we term large cell minimally differentiated carcinomas (LCMDCs). These tumors likely include a group of poorly differentiated carcinomas previously described by others as medullary adenocarcinomas. To better understand the pathogenesis of these uncommon neoplasms, we compared molecular features of 15 LCMDCs to those present in 25 differentiated adenocarcinomas (DACs) of the colon. Tumors were examined for alterations commonly seen in typical colorectal carcinomas, including increased p53 and beta-catenin immunoreactivity, K-ras gene mutations, microsatellite instability, and loss of heterozygosity of markers on chromosomes 5q, 17p, and 18q. In addition, tumors were evaluated by immunohistochemistry for CDX2, a homeobox protein whose expression in normal adult tissues is restricted to intestinal and colonic epithelium. Markedly reduced or absent CDX2 expression was noted in 13 of 15 (87%) LCMDCs, whereas only 1 of the 25 (4%) DACs showed reduced CDX2 expression (P < 0.001). Nine of 15 (60%) LCMDCs had the high-frequency microsatellite instability phenotype, but only 2 of 25 (8%) DACs had the high-frequency microsatellite instability phenotype (P = 0.002). Our findings provide support for the hypothesis that the molecular pathogenesis of LCMDCs is distinct from that of most DACs. CDX2 alterations and DNA mismatch repair defects have particularly prominent roles in the development of LCMDCs.     

BAT-26 and BAT-40 Instability in Colorectal Adenomas and Carcinomas and Germline Polymorphisms

Analysis of the mononucleotide repeats BAT-26 and BAT-40 has reportedly revealed significant microsatellite instability in sporadic colorectal adenomas, whereas studies with dinucleotide and tetranucleotide repeats have not. In addition, BAT-26 has been reported to be "quasimonomorphic" in the germline. We evaluated BAT-26 and BAT-40 in a series of colorectal tumors previously analyzed with a panel of tetranucleotide repeats. Instability in BAT-26 or BAT-40 was significantly associated with tetranucleotide repeat instability in sporadic adenomas and carcinomas (P < 0.0001) and was similarly much less common in adenomas than in carcinomas. Germline polymorphisms in both BAT-40 and BAT-26 were identified, and the frequency of BAT-26 polymorphisms was significantly higher in African Americans than in Caucasians (7.7% versus 0.08%, P < 0.001). BAT-26 and BAT-40 may be very useful in evaluating instability in small tumors, as sufficient DNA to be amplified by large panels of microsatellites is not always available from such lesions. Polymorphisms in these microsatellites, however, limit their utility in determinations of microsatellite instability without corresponding normal DNA.     

Loss of Heterozygosity and Microsatellite Instability in De Novo versus Ex-Adenoma Carcinomas of the Colorectum

Small adenocarcinomas of the colorectum showing no evidence of origin from an adenoma have been called de novo carcinomas, a name that implies an origin via a different molecular genetic mechanism than the usual colorectal carcinoma which develops from an adenoma. Using microsatellite analysis, 35 early (pT1) de novo and 36 pT1 ex-adenoma carcinomas were compared using 8 microsatellite loci at 6 different chromosomal loci (1p, 2p, 8p, 5q, 17p, and 18q) known or hypothesized to be important for colorectal carcinogenesis. The rate of loss of heterozygosity (LOH) at the 17p locus (near the p53 gene) was significantly higher in the de novo than in the ex-adenoma group (73 vs. 37%, P = 0.004). The rates of LOH at the other loci (including the APC and DCC genes) and the rate of MSI were not significantly different in the two groups. These results indicate that de novo carcinomas of the colorectum develop via a similar carcinogenetic pathway as conventional ex-adenoma carcinomas; however, their higher rate of LOH at 17p is evidence for a biologically more advanced lesion with more frequent p53 mutations, consistent with clinicopathological data indicating that de novo carcinomas are more aggressive than ex-adenoma carcinomas.     

Cytotoxic Effects of Activated Human Monocytes and Lymphocytes to Anti-D-treated Human Erythrocytes in Vitro

Incubation of human monocytes, derived from peripheral blood, with cell-free supernatants from mixed lymphocyte cultures resulted in morphological and functional changes in the mature macrophages. Activation of monocyte-derived macrophages by these factors resulted in a significant increase in their capacity to lyse anti-D-treated human erythrocytes. The lytic activity of both normal and activated macrophages appeared to be independent of erythrophagocytosis. T lymphocytes activated by either allogeneic cells or the mitogen phytohaemagglutinin were not cytolytic to treated erythrocytes even at high effector to target cell ratios.     

Adoptive Transfer of Herpesvirusspecific Cytotoxic T Lymphocytes in Transplant Recipients

Profound suppression of T-cell immunity observed after transplantation may permit the emergence of life-threatening viral complications. Prevention or treatment of viral infections in immunocompromised patients through the infusion of small numbers of antigen-specific T-cell lines or clones is the most successful example of adoptive immunotherapy in humans. In fact, adoptive transfer of T cells of donor origin has been demonstrated to be able to restore protective immunity towards these infectious agents and to control established disease. This article reviews human trials of adoptive immunotherapy for herpesvirus-related complications, with the aim of defining principles and problems encountered so far to aid the design of future clinical studies.     

宫颈癌细胞增殖及凋亡与bcl-2表达的研究

本研究旨在探求宫颈癌变中细胞增殖和细胞凋亡的变化规律,以及凋亡出现频率与ｂｃｌ－２癌蛋白的关系。结果发现宫颈癌组织的增殖指数（ＰＩ）和凋亡指数（ＡＩ）明显高于正常宫颈组织和宫颈不典型增生组织（Ｐ＜０．０１）,而ＡＩ／ＰＩ显著低于两者（Ｐ＜０．０１）。ｂｃｌ－２癌蛋白常见于正常宫颈上皮和不典型增生上皮的基底细胞层,宫颈癌组织中其表达随临床期别升高呈下降趋势,ｂｃｌ－２阳性组的ＡＩ与阴性组无显著差异。证实宫颈癌变是细胞无限增殖,凋亡异常被抑制的结果。ｂｃｌ－２是宫颈癌变的早期特征,但不是调节细胞凋亡的唯一因素     

Cell Mediated Lympholysis in Man: An Attempt to Type with Cytotoxic Lymphocytes

From approximately 3,000 CML combinations, originally established in order to evaluate the qualitative and quantitative influence of the serologically defined HLA-A, B, and C antigens on cellular, complement independent cytolysis, 12 combinations were selected yielding reproducible positive cytolysis on allogenic target cells, although no HLA-antigenic sharing could be demonstrated between stimulator and target lymphocytes. These 12 CytoToxic Lymphocytes (CTL's) have been tested in parallell as "CML typing combinations" against lymphocytes from a random population sample of 100 unrelated Danes. Based on a pairwise analysis 11 of these CTL's could be classified into two groups of significantly correlated CTL's. These two groups do not define monospecific traits of allelic genetic origin as judged by a mutually positive correlation and a poor fit to Hardy-Weinberg equilibrium. The traits defined by these groups may be either partially identical or governed by closely linked loci. The same groups were identified and the same conclusions reached after exclusion of those individuals in the population sample where HLA-A, B, C, or D antigens may be targets for destruction. Thus, this study gives direct evidence that known HLA antigens are not sole target determinants in CML or that cytotoxic lymphocytes recognize HLA molecules in a different way than lymphocytotoxic antibodies. The studies underline the immunogenetic complexity of CML although this reaction is most probably governed by genes in the HLA region. It is suggested that cytotoxic lymphocytes may recognize "backbone structures" of the HLA molecules.     

The Role of Calcium in Stimulation of Activated T Lymphocytes with Interleukin 2

In a study of the role of Ca++ in the stimulation of activated T lymphocytes with interleukin 2 (IL-2) it was found that IL-2-induced proliferation can occur independently of extracellular calcium. Further, there was no correlation between triggering of DNA synthesis and an increase in free cytoplasmic calcium. However, IL-2 induced an increased uptake of 45Ca++ from the extracellular medium. Since there is no increase in free cytoplasmic calcium, it must be assumed that this is caused by an increase in membrane-associated calcium. Further, the calcium channel-blocking agent, verapamil, and TMB-8, a putative inhibitor of mobilization of calcium from intracellular pools, both exerted a dose-dependent inhibition of IL-2-induced DNA synthesis in activated T lymphocytes. We conclude that calcium is not a second messenger in activated T lymphocytes stimulated by IL-2, but our results indicate that calcium may play a role at membrane level.     

Clones of Human Cytotoxic T Lymphocytes Derived from an Allosensitized Individual: HLA Specificity and Cell Surface Markers

By planned immunization of a volunteer, two stable (greater than or equal to 6 months), specific, alloreactive cytolytic T-cell clones have been established from his peripheral blood lymphocytes. One clone reacts with all serologically defined HLA-Cw3 cells from our panel, whereas the other defines a split within the serological HLA-B40 specificity. The two cytotoxic clones are SmIg-negative, E-rosette positive, EA and EAC rosette-negative, HLA-A, -B and -C- positive, and also HLA-DR- or 'Ia like'-positive. In addition, they present very similar patterns of iodinated cell surface molecules as analysed by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), contrasting with that of an EBV cell line derived from the same donor.