Immune Cells in Colorectal Cancer: Prognostic Relevance and Role of MSI

There is growing evidence that both local and systemic inflammatory responses play an important role in the progression of a variety of solid tumors. Colorectal cancer (CRC) results from the cumulative effect of sequential genetic alterations, leading to the expression of tumor-associated antigens possibly inducing a cellular anti-tumor immune response. It is well recognized that cytotoxic lymphocytes (CTLs) constitute one of the most important effector mechanisms of anti-tumor-immunity. However, their potential prognostic influence in CRC remains controversial. In addition, other key players like natural killer cells, tumor associated macrophages and regulatory T cells play an important role in the immune attack against CRC and need further investigation. This review will mainly focus on the role of the adaptive immune system in CRC and particularly in regard to microsatellite instability.

Related papers:

title: A review of the most promising biomarkers in colorectal cancer: one step closer to targeted therapy.
abstract: Rapidly growing insights into the molecular biology of colorectal cancer (CRC) and recent developments in gene sequencing and molecular diagnostics have led to high expectations for the identification of molecular markers to be used in optimized and tailored treatment regimens. However, many of the published data on molecular biomarkers are contradictory in their findings and the current reality is that no molecular marker, other than the KRAS gene in the case of epidermal growth factor receptor (EGFR)- targeted therapy for metastatic disease, has made it into clinical practice. Many markers investigated suffer from technical shortcomings, resulting from lack of quantitative techniques to capture the impact of the molecular alteration. This understanding has recently led to the more comprehensive approaches of global gene expression profiling or genome-wide analysis to determine prognostic and predictive signatures in tumors. In this review, an update of the most recent data on promising biological prognostic and/or predictive markers, including microsatellite instability, epidermal growth factor receptor, KRAS, BRAF, CpG island methylator phenotype, cytotoxic T lymphocytes, forkhead box P3-positive T cells, receptor for hyaluronic acid-mediated motility, phosphatase and tensin homolog, and T-cell originated protein kinase, in patients with CRC is provided.

title: The chromosomal instability pathway in colon cancer.
abstract: The acquisition of genomic instability is a crucial feature in tumor development and there are at least 3 distinct pathways in colorectal cancer pathogenesis: the chromosomal instability (CIN), microsatellite instability, and CpG island methylator phenotype pathways. Most cases of colorectal cancer arise through the CIN pathway, which is characterized by widespread imbalances in chromosome number (aneuploidy) and loss of heterozygosity. It can result from defects in chromosomal segregation, telomere stability, and the DNA damage response, although the full complement of genes underlying CIN remains incompletely described. Coupled with the karyotypic abnormalities observed in CIN tumors are the accumulation of a characteristic set of mutations in specific tumor suppressor genes and oncogenes that activate pathways critical for colorectal cancer initiation and progression. Whether CIN creates the appropriate milieu for the accumulation of these mutations or vice versa remains a provocative and unanswered question. The goal of this review is to provide an updated perspective on the mechanisms that lead to CIN and the key mutations that are acquired in this pathway.

title: Microsatellite instability in colorectal cancer—the stable evidence
abstract: Microsatellite instability (MSI) is the molecular fingerprint of a deficient mismatch repair system. Approximately 15% of colorectal cancers (CRC) display MSI owing either to epigenetic silencing of MLH1 or a germline mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2. Methods to detect MSI are well established and routinely incorporated into clinical practice. A clinical and molecular profile of MSI tumors has been described, leading to the concept of an MSI phenotype in CRC. Studies have confirmed that MSI tumors have a better prognosis than microsatellite stable CRC, but MSI cancers do not necessarily have the same response to the chemotherapeutic strategies used to treat microsatellite stable tumors. Specifically, stage II MSI tumors might not benefit from 5-fluorouracil-based adjuvant chemotherapy regimens. New data suggest possible advantages of irinotecan-based regimens, but these findings require further clarification. Characterization of the molecular basis of MSI in CRC is underway and initial results show that mutations in genes encoding kinases and candidate genes with microsatellite tracts are over-represented in MSI tumors. Transcriptome expression profiles of MSI tumors and systems biology approaches are providing the opportunity to develop targeted therapeutics for MSI CRC.

title: DNA mismatch repair deficiency in sporadic colorectal cancer and Lynch syndrome
abstract: Poulogiannis G, Frayling I M & Arends M J (2010) Histopathology56, 167–179

title: The adaptive immunologic microenvironment in colorectal cancer: a novel perspective.
abstract: Colorectal cancer is one of the most common malignancies, often presenting with a poor prognosis. To date, the anatomic extent of disease has been by far the most important prognostic factor. Recently, we obtained evidence that the type, density, and location of immune cells in colorectal cancer could provide a prognostic factor superior and independent to that of criteria related to the anatomic extent of the tumor. Here, we discuss the meaning and potential implications of this novel finding.

title: ASCO 2006 update of recommendations for the use of tumor markers in gastrointestinal cancer.
abstract: To update the recommendations for the use of tumor marker tests in the prevention, screening, treatment, and surveillance of gastrointestinal cancers.



For the 2006 update, an update committee composed of members from the full Panel was formed to complete the review and analysis of data published since 1999. Computerized literature searches of Medline and the Cochrane Collaboration Library were performed. The Update Committee's literature review focused attention on available systematic reviews and meta-analyses of published tumor marker studies.



For colorectal cancer, it is recommended that carcinoembryonic antigen (CEA) be ordered preoperatively, if it would assist in staging and surgical planning. Postoperative CEA levels should be performed every 3 months for stage II and III disease for at least 3 years if the patient is a potential candidate for surgery or chemotherapy of metastatic disease. CEA is the marker of choice for monitoring the response of metastatic disease to systemic therapy. Data are insufficient to recommend the routine use of p53, ras, thymidine synthase, dihydropyrimidine dehydrogenase, thymidine phosphorylase, microsatellite instability, 18q loss of heterozygosity, or deleted in colon cancer (DCC) protein in the management of patients with colorectal cancer. For pancreatic cancer, CA 19-9 can be measured every 1 to 3 months for patients with locally advanced or metastatic disease receiving active therapy. Elevations in serial CA 19-9 determinations suggest progressive disease but confirmation with other studies should be sought. New markers and new evidence to support the use of the currently reviewed markers will be evaluated in future updates of these guidelines.

title: Effector memory T cells, early metastasis, and survival in colorectal cancer.
abstract: The role of tumor-infiltrating immune cells in the early metastatic invasion of colorectal cancer is unknown.



We studied pathological signs of early metastatic invasion (venous emboli and lymphatic and perineural invasion) in 959 specimens of resected colorectal cancer. The local immune response within the tumor was studied by flow cytometry (39 tumors), low-density-array real-time polymerase-chain-reaction assay (75 tumors), and tissue microarrays (415 tumors).



Univariate analysis showed significant differences in disease-free and overall survival according to the presence or absence of histologic signs of early metastatic invasion (P<0.001). Multivariate Cox analysis showed that an early conventional pathological tumor-node-metastasis stage (P<0.001) and the absence of early metastatic invasion (P=0.04) were independently associated with increased survival. As compared with tumors with signs of early metastatic invasion, tumors without such signs had increased infiltrates of immune cells and increased levels of messenger RNA (mRNA) for products of type 1 helper effector T cells (CD8, T-BET [T-box transcription factor 21], interferon regulatory factor 1, interferon-gamma, granulysin, and granzyme B) but not increased levels of inflammatory mediators or immunosuppressive molecules. The two types of tumors had significant differences in the levels of expression of 65 combinations of T-cell markers, and hierarchical clustering showed that markers of T-cell migration, activation, and differentiation were increased in tumors without signs of early metastatic invasion. The latter type of tumors also had increased numbers of CD8+ T cells, ranging from early memory (CD45RO+CCR7-CD28+CD27+) to effector memory (CD45RO+CCR7-CD28-CD27-) T cells. The presence of high levels of infiltrating memory CD45RO+ cells, evaluated immunohistochemically, correlated with the absence of signs of early metastatic invasion, a less advanced pathological stage, and increased survival.



Signs of an immune response within colorectal cancers are associated with the absence of pathological evidence of early metastatic invasion and with prolonged survival.

title: The essential role of the in situ immune reaction in human colorectal cancer
abstract: Colorectal cancer is the second most common cause of cancer‐related death. Novel prognostic factors should be identified and validated to refine the present tumor‐node‐metastasis system. The presence of immune cells infiltrating colorectal cancers is a common phenomenon. However, the current belief is that clinically detectable human tumors escaping immune surveillance are no longer kept in check by the immune cells of the tumor microenvironment. Despite studies showing the influence of immune cell infiltrates on the behavior of colorectal carcinomas, this parameter is not currently recognized as a reliable prognostic factor. We showed that the nature, functional orientation, density, and location of immune cells within distinct tumor regions could provide a prognostic factor superior to and independent of criteria related to the anatomic extent of the tumor. The strength of the immune reaction identified in our studies could advance our understanding of cancer evolution and have important consequences for clinical practice.

title: Cancer immunoediting and "spontaneous" tumor regression.
abstract: The combination of host protective and tumor-promoting actions of the immune system throughout tumor development is termed cancer immunoediting. This review briefly summarizes the currently vast evidence supporting the immune system's role in not only protecting against developing cancer, but also sculpting tumor immunogenicity and immune escape. We also briefly summarize the history of immunotherapy and discuss the immunoediting process in the context of spontaneous tumor regression and whether this observation can be utilized in future treatment regimens.

title: The influence of host response on colorectal cancer prognosis.
abstract: Colorectal cancer (CRC) is a leading cause of cancer-related death worldwide. Several tumor characteristics have been shown to be of prognostic significance, although stage at diagnosis continues to be the most important predictor of survival. Emerging new data suggest that the presence of a host response to CRC may also influence survival and other outcomes in CRC. This review summarizes recent evidence regarding the prognostic significance of the host response to CRC. In retrospective analyses, tumor-associated macrophages and tumor-infiltrating lymphocytes appear to be the elements most significantly associated with improved outcomes in CRC. The presence of other cells, including dendritic cells, natural killer cells, eosinophils, and mast cells, also appears to be associated with increased survival. The influence of the host response to CRC needs confirmation in prospective studies, but in the meantime should be part of risk stratification. Novel approaches to further augmenting this response merit study.

title: The immune system and cancer.
abstract: Cancer patients mount adaptive immune responses against their tumor. However, while tumor-infiltrating lymphocytes and natural-killer (NK) cells try to detect and eliminate malignant cells, they eventually fail when these malignant cells develop mechanisms to evade effective immunosurveillance. First, malignant cells produce immunosuppressive cytokines and prostaglandins that skew the immune response toward a Th2 response, resulting in a humoral response with significantly less antitumor capacities, generating a low interleukin-2 environment blocking NK cell division, T-helper cell proliferation, and T-cytotoxic cell proliferation and function. Second, immunoresistant malignant cell variants emerge through selection of major histocompatibility class I and II and antigen-processing mutants reducing antigenicity. Finally, malignant cells may actively eliminate T-cells via activation-induced cell death or by mounting a counterattack through Fas ligand expression.

title: Immunosurveillance is active in colorectal cancer as downregulation but not complete loss of MHC class I expression correlates with a poor prognosis
abstract: Many colorectal tumors lose or downregulate cell surface expression of MHC class I molecules conferring resistance to T‐cell‐mediated attack. It has been suggested that this phenomenon is due to in vivo immune‐tumor interactions. However, evidence of the impact of MHC class I loss on outcomes from colorectal cancer is scarce. In our study of more than 450 colorectal cancers in tissue microarray format, we have shown that both high levels of MHC class I expression and absent MHC class I expression are associated with similar disease‐specific survival times, possibly due to natural killer cell‐mediated clearance of MHC class I‐negative tumor cells. However, tumors with low level expression of MHC class I were found to confer a significantly poorer prognosis, retaining independent significance on multivariate analysis. The existence of these poor prognosis tumors, which may avoid both NK‐ and T‐cell‐mediated immune surveillance, has important implications for the design of immunotherapeutic strategies in colorectal cancer.

title: The role of CD8+ T cells in immune responses to colorectal cancer
abstract: Cytotoxic T lymphocytes (CTL) are essential effectors of the cell-mediated immune response. The ability of CTL to specifically recognise and lyse malignant cells expressing the relevant surface antigens under optimal in vitro conditions justifies attempts to boost their number and activity through various forms of immunotherapy. Considering the high prevalence of colorectal cancer and poor survival rates for patients with advanced-stage disease, the development of new protocols based on CTL stimulation represents a genuine and promising treatment option. Significant advances in recombinant DNA technology and molecular biology have led to the identification of a number of tumour-associated antigens (TAA). These have served as vaccine constituents and/or stimuli for obtaining CTL used for adoptive immunotherapy after in vitro stimulation and expansion. The present review describes the properties and functions of CTL as effectors of the immune response against tumours, and summarises the known TAA recognised by CTL and the current status of CTL-related immunotherapeutic interventions in colorectal cancer patients.

title: Mechanisms of lysis by cytotoxic T cells.
abstract: When a cytotoxic lymphocyte binds to an antigenic cell, there is a reorientation of the Golgi apparatus and a directed exocytosis of cytoplasmic granules toward the offending cell. In the model originally proposed by Henkart, these granules were hypothesized to contain lytic molecules that contribute to the demise of the target cell. Initially, perforin/cytolysin was believed to be the major player in the mechanism of lysis. Recent work, however, using a variety of biochemical, molecular biological, and genetic approaches, has provided convincing evidence that other granule-associated molecules contribute to the lytic pathway. Of particular note are the granzymes, whose proteolytic activity is intimately associated with the induction of DNA fragmentation and apoptosis. In this review we summarize these recent experiments and present an updated view of the granule-mediated killing mechanism. For some years the universality of the granule-killing mechanism has been challenged. Experiments reported in the last few years on the characteristics of target cell death, detailed studies with cytolytic T cell lines, and, ultimately, with lymphocytes from mice that have been genetically altered using homologous recombination, have proven the existence of a second pathway. We discuss whether the existence of this alternate, Fas antigen/Fas ligand, mechanism really deals a knock-out blow to the granule exocytosis followers. Most likely it represents an important immunoregulatory system rather than a defense against pathogenesis. The granule-mediated and Fas-dependent mechanisms of cytolysis represent, at first sight, completely different pathways. However, it is becoming increasingly apparent that once the kiss of death has been received, through either granules or Fas, the events within the target cell are remarkably similar. Considerable attention is now being focussed on a family of interleukin-1 beta-converting enzymes (ICEs) that become activated within the target cell after attack by a cytotoxic lymphocyte. These enzymes are particularly interesting as they have now been shown to be directly involved in developmentally controlled, programmed cell death.

title: Immune cells in colorectal cancer: prognostic relevance and therapeutic strategies
abstract: During the last two decades, considerable efforts have been made to improve the prevention, early diagnosis and therapy of colorectal cancer by gaining enhanced insights into disease-specific pathogenesis. Along these lines, tumor-infiltrating immune cells turned out to be critical indicators for an efficient antitumor immune response and the number and type of tumor-infiltrating immune cells determined the resulting tumor prognosis. This review aims to describe the prognostic relevance of the different subsets of tumor-infiltrating immune cells and highlights their specific function in the complex process of immune system-mediated rejection of colorectal cancer cells. Considering the clinical impact of this increased insight into tumor-associated immunology, resulting innovative approaches in the treatment of colorectal cancer are summarized and discussed immunotherapeutic critically.

title: The role of tumour‐associated macrophages in tumour progression: implications for new anticancer therapies
abstract: The role of macrophages in tumour growth and development is complex and multifaceted. Whilst there is limited evidence that tumour‐associated macrophages (TAMs) can be directly tumouricidal and stimulate the anti‐tumour activity of T cells, there is now contrasting evidence that tumour cells are able to block or evade the activity of TAMs at the tumour site. In some cases, tumour‐derived molecules even redirect TAM activities to promote tumour survival and growth. Indeed, evidence has emerged for a symbiotic relationship between tumour cells and TAMs, in which tumour cells attract TAMs and sustain their survival, with TAMs then responding to micro‐environmental factors in tumours such as hypoxia (low oxygen tension) by producing important mitogens as well as various growth factors and enzymes that stimulate tumour angiogenesis. This review presents evidence for the number and/or distribution of TAMs being linked to prognosis in different types of human malignancy. It also outlines the range of pro‐ and anti‐tumour functions performed by TAMs, and the novel therapies recently devised using TAMs to stimulate host immune responses or deliver therapeutic gene constructs to solid tumours.

title: Vascular endothelial growth factor, CD68, and epidermal growth factor receptor expression and survival in patients with Stage II and Stage III colon carcinoma
abstract: The elucidation of new therapeutic targets of prognostic significance in colon carcinoma is necessary to improve outcomes. In the current study, the authors examined the expression of epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) in primary colon carcinoma cases and VEGF in tumor‐associated macrophages (TAM)/stroma, and their correlation with survival.

title: Different occurrence of CD8+, CD45R0+, and CD68+ immune cells in regional lymph node metastases from colorectal cancer as potential prognostic predictors
abstract: To study whether there are differences in the immunohistochemical staining of CD8, CD45R0, and CD68 of immune cells in regional lymph node metastases from colorectal cancer that are of potential interest in prognostic prediction.  Analysis of archival specimens from 93 patients operated on for colorectal cancer (based on monoclonal antibodies, the ABC technique, and semiquantitative classification).  There was a significant difference in survival time between patients with respect to the number of positive immune cells. The cancer-specific 5-year survival rate was 77% for patients with high numbers of CD8+ cells, compared to 33% for those with lower numbers. The corresponding figures for patients with CD45R0+ cells were 66% vs. 33%, and for patients with CD68+ cells 60% vs. 38%. Significant differences remained among the 74 patients without adjuvant radio-/chemotherapy regarding CD8 and CD45R0 but not CD68.  The presence of CD8+, CD45R0+, and CD68+ immune cells in regional lymph node metastases may serve as predictors of patients survival in colorectal cancer Dukes' stage C.

title: Prognostic significance of CD8+ T cell and macrophage peritumoral infiltration in colorectal cancer.
abstract: The purpose of this study was to evaluate the prognostic significance of CD8+ T cell and macrophage peritumoral infiltration in patients with colorectal cancer. A total of 97 adenocarcinomas of the colon and rectum were examined. Immunohistochemical staining was performed by the standard avidin-biotin-peroxidase complex method using antibodies to CD8 and CD68. Peritumoral infiltration by CD8+ T cells or macrophages was evaluated along the invasive margin of the cancer in each specimen. The area with the most abundant infiltration was selected, and the number of immunoreactive positive cells counted at x400 magnification. Patients were divided into two groups based on the degree of infiltration by each cell type: namely those with a high level of infiltration (more than the mean number of positive cells) and those with a low level of infiltration (less than the mean number of positive cells). Patients with a low level of macrophage infiltration had a significantly deeper depth of invasion than patients with a high level of macrophage infiltration (P=0.027). The percentage of patients with a high level of macrophage infiltration was significantly higher in vascular invasion-negative cases (46.7%) than in vascular invasion-positive cases (22.7%; P=0.045), and in lymph node metastasis-negative cases (52.9%) than in lymph node metastasis-positive cases (28.3%; P=0.014). Overall survival was significantly shorter for patients with a low level of CD8+ T cell infiltration than those with a high level of CD8+ T cell infiltration (P=0.01). The survival rate for patients with a high level of both CD8+ T cell and macrophage infiltration was 100%. In conclusion, both CD8+ T cell and macrophage peritumoral infiltration indicates anti-tumoral action in patients with colorectal cancer.

title: The density of macrophages in the invasive front is inversely correlated to liver metastasis in colon cancer
abstract: BackgroundAlthough an abundance of evidence has indicated that tumor-associated macrophages (TAMs) are associated with a favorable prognosis in patients with colon cancer, it is still unknown how TAMs exert a protective effect. This study examined whether TAMs are involved in hepatic metastasis of colon cancer.Materials and methodsOne hundred and sixty cases of pathologically-confirmed specimens were obtained from colon carcinoma patients with TNM stage IIIB and IV between January 1997 and July 2004 at the Cancer Center of Sun Yat-Sen University. The density of macrophages in the invasive front (CD68TFHotspot) was scored with an immunohistochemical assay. The relationship between the CD68TFHotspot and the clinicopathologic parameters, the potential of hepatic metastasis, and the 5-year survival rate were analyzed.ResultsTAMs were associated with the incidence of hepatic metastasis and the 5-year survival rate in patients with colon cancers. Both univariate and multivariate analyses revealed that the CD68TFHotspot was independently prognostic of survival. A higher 5-year survival rate among patients with stage IIIB after radical resection occurred in patients with a higher macrophage infiltration in the invasive front (81.0%) than in those with a lower macrophage infiltration (48.6%). Most importantly, the CD68TFHotspot was associated with both the potential of hepatic metastasis and the interval between colon resection and the occurrence of hepatic metastasis.ConclusionThis study showed evidence that TAMs infiltrated in the invasive front are associated with improvement in both hepatic metastasis and overall survival in colon cancer, implying that TAMs have protective potential in colon cancers and might serve as a novel therapeutic target.

title: Intratumoral macrophage counts correlate with tumor progression in colorectal cancer
abstract: The role of intratumoral tumor‐associated macrophages (TAMs) in colorectal cancer (CRC) is not clear. We aim to examine the relationships of TAMs and the clinicopathologic features of CRC and the expression of matrix metalloproteinases (MMP)‐2 and MMP‐9.

title: Reduced beta-catenin and peroxisome proliferator-activated receptor-gamma expression levels are associated with colorectal cancer metastatic progression: correlation with tumor-associated macrophages, cyclooxygenase 2, and patient outcome.
abstract: Recent studies have reported cross talk between beta-catenin, peroxisome proliferator-activated receptor-gamma, and cyclooxygenase 2 signaling pathways. We examined whether molecular changes of these pathways could be related to colorectal cancer metastatic progression. Seventy-two sporadic colorectal cancers and the distant nonneoplastic mucosa were analyzed for beta-catenin, peroxisome proliferator-activated receptor-gamma, cyclooxygenase 2, and nuclear factor kappaB levels by immunohistochemistry and Western blot. The expression profiles were correlated with patient outcome and 5-year survival. Nuclear beta-catenin staining was detected in only 18.1% of tumors and correlated with poor survival as compared with cases showing cytosolic/membrane accumulation (59.7%, P < .05). This latter group and tumor samples showing cytosolic/nuclear peroxisome proliferator-activated receptor-gamma expression (70.8%) were significantly associated with a favorable prognosis (P < .001). Remarkably, reduction or loss of beta-catenin (22.2%) and peroxisome proliferator-activated receptor-gamma (29.2%) expression was strongly correlated with marked infiltration of tumor-associated macrophages (P < .01), presence of liver metastases, and very short survival (P = .0001). Moreover, beta-catenin and peroxisome proliferator-activated receptor-gamma levels were inversely correlated with cyclooxygenase 2 (P < .01) and nuclear factor kappaB expression (P < .05). Our results suggest that reduced expression of beta-catenin and peroxisome proliferator-activated receptor-gamma could play a key role in aggressive colorectal cancer behavior. This finding may provide a relevant prognostic tool and contribute to early identification of patients at high risk of mortality.

title: Tumor Associated Macrophages Protect Colon Cancer Cells from TRAIL-Induced Apoptosis through IL-1β- Dependent Stabilization of Snail in Tumor Cells
abstract: We recently reported that colon tumor cells stimulate macrophages to release IL-1β, which in turn inactivates GSK3β and enhances Wnt signaling in colon cancer cells, generating a self-amplifying loop that promotes the growth of tumor cells.  Here we describe that macrophages protect HCT116 and Hke-3 colon cancer cells from TRAIL-induced apoptosis. Inactivation of IL-1β by neutralizing IL-1β antibody, or silencing of IL-1β in macrophages inhibited their ability to counter TRAIL-induced apoptosis. Accordingly, IL-1β was sufficient to inhibit TRAIL-induced apoptosis. TRAIL-induced collapse of the mitochondrial membrane potential (Δψ) and activation of caspases were prevented by macrophages or by recombinant IL-1β. Pharmacological inhibition of IL-1β release from macrophages by vitamin D3, a potent chemopreventive agent for colorectal cancer, restored the ability of TRAIL to induce apoptosis of tumor cells cultured with macrophages. Macrophages and IL-1β failed to inhibit TRAIL-induced apoptosis in HCT116 cells expressing dnIκB, dnAKT or dnTCF4, confirming that they oppose TRAIL-induced cell death through induction of Wnt signaling in tumor cells. We showed that macrophages and IL-1β stabilized Snail in tumor cells in an NF-κB/Wnt dependent manner and that Snail deficient tumor cells were not protected from TRAIL-induced apoptosis by macrophages or by IL-1β, demonstrating a crucial role of Snail in the resistance of tumor cells to TRAIL.  We have identified a positive feedback loop between tumor cells and macrophages that propagates the growth and promotes the survival of colon cancer cells: tumor cells stimulate macrophages to secrete IL-1β, which in turn, promotes Wnt signaling and stabilizes Snail in tumor cells, conferring resistance to TRAIL. Vitamin D3 halts this amplifying loop by interfering with the release of IL-1β from macrophages. Accordingly, vitamin D3 sensitizes tumor cells to TRAIL-induced apoptosis, suggesting that the therapeutic efficacy of TRAIL could be augmented by this readily available chemopreventive agent.

title: The expression and distribution of the hypoxia-inducible factors HIF-1alpha and HIF-2alpha in normal human tissues, cancers, and tumor-associated macrophages.
abstract: The cellular response to hypoxia includes the hypoxia-inducible factor-1 (HIF-1)-induced transcription of genes involved in diverse processes such as glycolysis and angiogenesis. Induction of the HIF-regulated genes, as a consequence of the microenvironment or genetic changes, is known to have an important role in the growth of experimental tumors. Hypoxia-inducible factors 1alpha and 2alpha (HIF-1alpha and HIF-2alpha) are known to dimerize with the aryl hydrocarbon receptor nuclear translocator in mediating this response. Because regulation of the alpha chain protein level is a primary determinant of HIF activity, our aim was to investigate the distribution of HIF-1alpha and HIF-2alpha by immunohistochemistry in normal and pathological tissues using monoclonal antibodies (mAb). We raised a new mAb to detect HIF-1alpha, designated 122, and used our previously validated mAb 190b to HIF-2alpha. In the majority of solid tumors examined, including bladder, brain, breast, colon, ovarian, pancreatic, prostate, and renal carcinomas, nuclear expression of HIF-1alpha and -2alpha was observed in varying subsets of the tumor cells. HIF-2alpha was also strongly expressed by subsets of tumor-associated macrophages, sometimes in the absence of any tumor cell expression. Less frequently staining was observed in other stromal cells within the tumors and in normal tissue adjacent to tumor margins. In contrast, in normal tissue neither molecule was detectable except within subsets of bone marrow macrophages, where HIF-2alpha was strongly expressed.

title: Chemoattractant Signaling between Tumor Cells and Macrophages Regulates Cancer Cell Migration, Metastasis and Neovascularization
abstract: Tumor-associated macrophages are known to influence cancer progression by modulation of immune function, angiogenesis, and cell metastasis, however, little is known about the chemokine signaling networks that regulate this process. Utilizing CT26 colon cancer cells and RAW 264.7 macrophages as a model cellular system, we demonstrate that treatment of CT26 cells with RAW 264.7 conditioned medium induces cell migration, invasion and metastasis. Inflammatory gene microarray analysis indicated CT26-stimulated RAW 264.7 macrophages upregulate SDF-1α and VEGF, and that these cytokines contribute to CT26 migration in vitro. RAW 264.7 macrophages also showed a robust chemotactic response towards CT26-derived chemokines. In particular, microarray analysis and functional testing revealed CSF-1 as the major chemoattractant for RAW 264.7 macrophages. Interestingly, in the chick CAM model of cancer progression, RAW 264.7 macrophages localized specifically to the tumor periphery where they were found to increase CT26 tumor growth, microvascular density, vascular disruption, and lung metastasis, suggesting these cells home to actively invading areas of the tumor, but not the hypoxic core of the tumor mass. In support of these findings, hypoxic conditions down regulated CSF-1 production in several tumor cell lines and decreased RAW 264.7 macrophage migration in vitro. Together our findings suggest a model where normoxic tumor cells release CSF-1 to recruit macrophages to the tumor periphery where they secrete motility and angiogenic factors that facilitate tumor cell invasion and metastasis.

title: Activated macrophages induce metastatic behavior of colon cancer cells.
abstract: Tumor-associated macrophages were linked to the growth, angiogenesis, and metastasis of variety of cancers. However, the role of macrophages in colon cancer is elusive. In the present study, we demonstrate that activated macrophage-conditioned medium (AMCM), containing tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6, markedly induced proliferation and migration of human colon cancer cells HCT116. Furthermore, AMCM significantly increased activation of transcription factor NF-kappaB and secretion of vascular endothelial growth factor (VEGF) from colon cancer cells, which subsequently induced capillary morphogenesis of human aortic endothelial cells. In conclusion, the interruption of signaling between activated macrophages and colon cancer cells could be considered as a new therapeutic strategy.

title: Immunotherapy and immunoescape in colorectal cancer
abstract: Immunotherapy encompasses a variety of interventions and techniques with the common goal of eliciting tumor cell destructive immune responses. Colorectal carcinoma often presents as metastatic disease that impedes curative surgery. Novel strategies such as active immunization with dendritic cells (DCs), gene transfer of cytokines into tumor cells or administration of immunostimulatory monoclonal antibodies (such as anti-CD137 or anti-CTLA-4) have been assessed in preclinical studies and are at an early clinical development stage. Importantly, there is accumulating evidence that chemotherapy and immunotherapy can be combined in the treatment of some cases with colorectal cancer, with synergistic potentiation as a result of antigens cross-presented by dendritic cells and/or elimination of competitor or suppressive T lymphocyte populations (regulatory T-cells). However, genetic and epigenetic unstable carcinoma cells frequently evolve mechanisms of immunoevasion that are the result of either loss of antigen presentation, or an active expression of immunosuppressive substances. Some of these actively immunosuppressive mechanisms are inducible by cytokines that signify the arrival of an effector immune response. For example, induction of 2, 3 indoleamine dioxygenase (IDO) by IFNγ in colorectal carcinoma cells. Combinational and balanced strategies fostering antigen presentation, T-cell costimulation and interference with immune regulatory mechanisms will probably take the stage in translational research in the treatment of colorectal carcinoma.

title: CD4+CD25+FOXP3+ Regulatory T Cells Suppress Anti-Tumor Immune Responses in Patients with Colorectal Cancer
abstract: A wealth of evidence obtained using mouse models indicates that CD4+CD25+FOXP3+ regulatory T cells (Treg) maintain peripheral tolerance to self-antigens and also inhibit anti-tumor immune responses. To date there is limited information about CD4+ T cell responses in patients with colorectal cancer (CRC). We set out to measure T cell responses to a tumor-associated antigen and examine whether Treg impinge on those anti-tumor immune responses in CRC patients.  Treg were identified and characterized as CD4+CD25+FOXP3+ using flow cytometry. An increased frequency of Treg was demonstrated in both peripheral blood and mesenteric lymph nodes of patients with colorectal cancer (CRC) compared with either healthy controls or patients with inflammatory bowel disease (IBD). Depletion of Treg from peripheral blood mononuclear cells (PBMC) of CRC patients unmasked CD4+ T cell responses, as observed by IFNγ release, to the tumor associated antigen 5T4, whereas no effect was observed in a healthy age-matched control group.  These results support a rationale for manipulating Treg to enhance cancer immunotherapy.

title: Regulatory T cells in colorectal cancer patients suppress anti-tumor immune activity in a COX-2 dependent manner
abstract: ObjectiveNaturally occurring regulatory T (TR) cells suppress autoreactive T cells whereas adaptive TR cells, induced in the periphery, play an important role in chronic viral diseases and cancer. Several studies indicate that cyclooxygenase (COX) inhibitors prevent cancer development of colon adenomas and delay disease progression in patients with colorectal cancer (CRC). We have shown that adaptive TR cells express COX-2 and produce PGE2 that suppress effector T cells in a manner that is reversed by COX-inhibitors.Methods and resultsHere we demonstrate that CRC patients have elevated levels of PGE2 in peripheral blood, and CRC tissue samples and draining lymph nodes display increased numbers of FOXP3+ TR cells. Depletion of TR cells from PBMC enhanced anti-tumor T-cell responses to peptides from carcinoembryonic antigen. Furthermore, the COX inhibitor indomethacin and the PKA type I antagonist Rp-8-Br-cAMPS significantly improved the anti-tumor immune activity.ConclusionWe suggest that adaptive TR cells contribute to an immunosuppressive microenvironment in CRC and inhibit effector T cells by a COX-2–PGE2-dependent mechanism and thereby facilitate tumor growth. Therapeutic strategies targeting TR cells and the PGE2–cAMP pathway may be interesting to pursue to enhance anti-tumor immune activity in CRC patients.

title: Chemo-immunotherapy of colorectal carcinoma: preclinical rationale and clinical experience
abstract: SummaryAdvanced colorectal cancer is a common disease with an high mortality rate. For four decades, pharmacological treatment of the advanced disease was based on the use of 5-fluorouracil alone or in combination with biomodulators such as folinic acid and intereferon alpha. In the last 5 years, response to therapy has been considerably ameliorated thanks to the discovery of new drugs such as oxaliplatin and CPT-11. These agents, in combination with 5-fluorouracil, according to various schedules of treatment, have reached a significant improvement of palliation, response rate and survival. Immunotherapy is an uprising modality of treatment for human cancer including colorectal carcinoma. Its rationale is based on the knowledge that tumour cells are genetically unstable and produce molecular structures which allow their recognition and destruction by the immune-surveillance system. Therefore, humoral as well as cellular compartments of the immune system can be utilized according to a “passive” strategy (e.g. monoclonal antibody administration and adoptive immunotherapy) or an “active” approach, by using different modalities of vaccine therapy. In this context, monoclonal antibodies (mAbs) and cancer vaccines are being tested for the treatment of advanced colorectal cancer. Due to their genetic instability and extraordinary adaptative potential, tumour cells may acquire resistance to the immune effectors and mAbs exactly as they do for cytotoxic drugs. To improve the results of both immunological and chemical modality of cancer treatment, an increasing number of authors is starting to combine chemo and immunotherapy in the attempt to circumvent the limitations of both strategies.This report tries to review the possible rationale of the chemo-immunotherapy combination, illustrating preliminary results of preclinical and clinical studies.

title: Tumor Infiltrating T Lymphocytes in Colorectal Cancer: Tumor-Selective Activation and Cytotoxic Activity In Situ
abstract: To examine whether tumor-selective infiltration, activation, and cytotoxic activity of tumor infiltrating T lymphocytes (TIL) can be demonstrated in situ in colorectal cancer samples. Recent studies indicated a correlation between the presence of TIL and an improved prognosis in colorectal cancer. However, tumor-selective activation and cytotoxic activity of CD8+ TIL in situ in colorectal cancer patients have not yet been examined. Tumor samples from 49 patients and corresponding normal mucosa samples from 23 patients with colorectal cancer (UICC stages II–IV) were examined for TIL. Two-color fluorescence immunohistochemistry and multicolor flowcytometric (FACS) analysis were used for quantification of CD8+ T cells and measurement of their activation status (CD69-expression) and cytotoxic activity (CD107a-expression) in situ. Presence of tumor antigen-reactive T cells in tumor, blood, and bone marrow was evaluated by IFN-γ Elispot analysis. While absolute numbers of CD8+ T cells were similar, CD4+ T helper cells were significantly increased in tumor tissue compared with normal mucosa. There was a significantly higher proportion of activated and cytotoxically active CD8+ TIL in colorectal cancer compared with normal mucosa. Increased activation, cytotoxic activity, and functional reactivity of TIL were correlated with the presence of functional tumor antigen-reactive T cells in the blood and bone marrow. The proportion of activated TIL decreased significantly with higher tumor stage. Tumor-selective activation and cytotoxic activity of CD8+ TIL and tumor-selective migration of CD4+ T helper cells were demonstrated in colorectal cancer for the first time. Our data support the immunogenicity of colorectal cancer and suggest clinical significance of tumor-specific immune responses.

title: Active specific immunotherapy for stage II and stage III human colon cancer: a randomised trial
abstract: Colon cancer is curable by surgery, but cure rate depends on the extent of disease. We investigated whether adjuvant active specific immunotherapy (ASI) with an autologous tumour cell-BCG vaccine with surgical resection was more beneficial than resection alone in stage II and III colon cancer.



In a prospective randomised trial, 254 patients with colon cancer were randomly assigned postoperative ASI or no adjuvant treatment. ASI was three weekly vaccinations starting 4 weeks after surgery, with a booster vaccination at 6 months with 10(7) irradiated autologous tumour cells. The first vaccinations contained 10(7) BCG organisms. We followed up patients for time to recurrence, and recurrence-free and overall survival. Analysis was by intention to treat.



The 5.3 year median follow-up (range 8 months to 8 years 11 months) showed 44% (95% CI 7-66) risk reduction for recurrence in the recurrence-free period in all patients receiving ASI (p=0.023). Overall, there were 40 recurrences in the control group and 25 in the ASI group. Analysis by stage showed no significant benefit of ASI in stage III disease. The major impact of ASI was seen in patients with stage II disease, with a significantly longer recurrence-free period (p=0.011) and 61% (18-81) risk reduction for recurrences. Recurrence-free survival was significantly longer with ASI (42% risk reduction for recurrence or death [0-68], p=0.032) and there was a trend towards improved overall survival.



ASI gave significant clinical benefit in surgically resected patients with stage II colon cancer. ASI has minimal adverse reactions and should be considered in the management of stage II colon cancer.

title: Lymphocytic infiltration and survival in rectal cancer.
abstract: Lymphocytic infiltration was assessed semiquantitatively in 447 specimens of rectal cancer. Corrected five year survivals for pronounced, moderate, and little lymphocytic infiltration were 92%, 65% and 36%, respectively. Grading was shown to be reproducible in an intraobserver study (observed agreement 81%, expected agreement 34%, kappa coefficient 0.72). In the multivariate survival analysis of grade and stage related variables lymphocytic infiltration was the only grade related variable to be accepted within the prognostic model. This model also included the number of lymph node metastases and the extent of tumour spread. It is suggested that the conservation of the normal interactive traffic between epithelium and mucosa associated lymphoid tissue (MALT) signals a low grade growth. This is supported by the association between lymphocytic infiltration and the expression of secretory component by malignant epithelium.

title: Prognostic value of tumour‐infiltrating lymphocytes (TILs) in colorectal cancer
abstract: Clinical follow‐up data of 276 colorectal adenocarcinoma patients treated in Kuopio University Hospital between 1976 and 1986 and followed up for a mean of 14 years were analysed. The clinical findings were correlated with tumour‐infiltrating lymphocytes (TILs) and with histological and quantitative factors including nuclear parameters and volume‐corrected mitotic index. In univariate survival analysis, TNM classification, Dukes' stage, histological grade, and TILs were significant predictors of survival. TNM classification, Dukes' stage, and TILs also predicted recurrence‐free survival. In multivariate analysis, TILs were an independent prognostic factor of survival in all cases, as well as in patients with T1‐4N0‐3M0 and T1‐4N1‐3M1. TILs also independently predicted recurrence‐free survival. TILs can provide important prognostic information in colorectal cancer to be used in evaluating for adjuvant therapy in different tumour stages.

title: Focus on TILs: prognostic significance of tumor infiltrating lymphocytes in human colorectal cancer.
abstract: Human colorectal cancer tissues are infiltrated by various immune/inflammatory cells, usually along the invasive margin. These responses tend be regarded as "non-specific". However, it is now clear that these cellular responses, particularly lymphocytic reactions, are independent prognostic factors for a better survival. Immunohistochemical subset analyses have generally disclosed that the number of T-lymphocytes is important. The effects of these T cells tend to be more manifest when the observation periods are longer. These data suggest that some degrees of anti-tumor immunity exist in human colorectal carcinomas. However, human tumors are generally composed of various histologic subtypes, which sometimes complicates these analyses and simple explanations may be misleading. Considered with precaution, these morphologic analyses on immune cell subtypes are important tools to understand the immune responses in human cancers.

title: Lymphocyte subsets in peripheral blood as prognostic factors in colorectal cancer.
abstract: To evaluate changes in different parameters of the immune status of colorectal cancer patients before their surgical treatment and to look for a possible impact these parameters could exert on overall survival.



Forty patients with histologically confirmed colorectal cancer in stage II (n=22), III and IV (n=18) were eligible for inclusion in the present study. Heparinized venous blood (5 ml) of patients was examined 5 days before surgery. Their preoperative white blood cell (WBC) count was >/=3.0*10(9)/l, hemoglobin >/=10 g/l, and platelets >/=180*10(9)/l. The indices of cellular immunity determined for all of the patients were the following: total leukocyte number, absolute number and percents of total lymphocytes, monocytes and neutrophils. The percentage and absolute number of lymphocytes subsets CD3(+), T-helpers (CD4(+)), T-cytotoxic cells (CD8(+)), immunoregulation index (CD4(+)/CD8(+)), B-lymphocytes (CD20(+)), natural killer (NK) cells (CD16(+)) were measured by immunofluorescence methods.



Cox regression analysis showed no dependence of the survival on estimated cellular immunity parameters of colorectal cancer patients in stage II. However, the number of circulating lymphocytes and in particular of T cells and NK cells was an independent prognostic variable for overall survival of stage III and IV patients. Analyzing the dependence of survival on immunological indices of colorectal cancer patients, a significant dependence of survival was determined on the absolute preoperative number of total lymphocytes and lymphocyte subsets (total lymphocytes levels >/=1.2*10(9)/l, >/=CD3(+) >/=0.8*10(9)/l, CD4(+) >/=0.3* 10(9)/l, CD8(+) >/=0.3*10(9)/l, and CD16(+)>/=0.25*10(9)/l). Cox regression analysis showed that higher absolute number of lymphocyte subpopulations may be associated with longer survival of colorectal cancer patients in stage III and IV.



This study suggests total number of lymphocytes >/=1.2*10(9)/l, CD3(+) >/=0.8*10(9)/l, CD4(+) >/=0.3* 10(9)/l, CD8(+) >/=0.3*10(9)/l, and CD16(+) >/=0.25*10(9)/l before surgery have a beneficial effect on overall survival of colorectal cancer patients in advanced stages (III and IV) of the disease.

title: The prognostic significance of peripheral T-lymphocyte subsets and natural killer cells in patients with colorectal cancer.
abstract: To evaluate the prognostic significance of preoperative T-lymphocyte subsets ad natural killer (NK) cells and their correlan with other prognostic factors in patients with colorectal cancer who underwent surgical treatment.



The numbers of T-lymphocytes and NK cells in the peripheral blood of patients with colorectal carcinoma were counted by flow cytometry, and the correlations between indicators of cellular immunity and clinicopathological characteristics and the prognosis of patients after surgical treatment were analyzed by univariate and multivariate analyses.



In the multivariate regression analysis, all potential prognostic factors essentially reflected TNM stage, and tumor resection, NK lymphocyte counts, and peripheral CD3+ and CD4+/CD8+ counts were significant (p < 0.05) independent prognostic indicators of overall survival; patients with higher CD3+, CD4+/CD8+, and NK levels had longer overall survival rates than those with lower CD3+, CD4+/CD8+, and NK levels.



Measurement of cellular immunity in the peripheral blood of patients with colorectal cancer allowed us to identify associations between immune status and clinical outcomes.

title: In situ cytotoxic and memory T cells predict outcome in patients with early-stage colorectal cancer.
abstract: Many patients who present with early-stage colorectal cancer (International Union Against Cancer TNM stages I and II) are nevertheless at high risk of relapse. We hypothesized that intratumoral immune reaction could influence their prognosis.



The intratumoral immune reaction was investigated in 29 tumors by large-scale real-time polymerase chain reaction. Cytotoxic (CD8) and memory (CD45RO) T cells were quantified by immunohistochemical analyses of tissue microarrays from the center (CT) and the invasive margin (IM) of the 602 tumors from two independent cohorts. The results were correlated with tumor recurrence and patient survival.



Patients with a strong infiltration of CD45RO(+) cells in the tumor exhibited an increased expression of T-helper 1 and cytotoxicity-related genes. Densities of CD45RO(+) and CD8(+) cells in tumor regions (CT/IM) classified the patients into four distinct prognostic groups based on the presence of high density of each marker in each tumor region. The four groups were associated with dramatic differences in disease-free, disease-specific, and overall survival (all P < .0001). Five years after diagnosis, only 4.8% (95% CI, 0.6% to 8.8%) of patients with high densities of CD8(+) plus CD45RO(+) cells had tumor recurrence, and 86.2% (CI, 79.4% to 93.6%) survived. In contrast, the tumor recurred in 75% (95% CI, 17% to 92.5%) of patients with low densities of these cells, and only 27.5% (95% CI, 10.5% to 72%) survived (all P < .0001). Multivariate analyses showed that the immune criteria had independent effects on the rates of complete remission and survival.



The combined analysis of CD8(+) plus CD45RO(+) cells in specific tumor regions could provide a useful criterion for the prediction of tumor recurrence and survival in patients with early-stage colorectal cancer.

title: The T-box transcription factor eomesodermin controls CD8 T cell activity and lymph node metastasis in human colorectal cancer
abstract: An efficient cytolytic T cell function is essential for immune mediated rejection of colorectal cancer. However, the molecular mechanisms driving T cell mediated cancer rejection are still poorly understood. Here, we assessed the relevance of the T-box transcription factor eomesodermin in colorectal cancer.  By analysing tissue probes from 88 different colorectal tumours, a significant (p<0.02) inverse correlation between eomesodermin expression in colorectal cancers and the presence of lymph node metastases could be shown, whereas no such correlation was noted for the master transcription factor of regulatory T cells, FoxP3 and CD8alpha expression. To evaluate whether this effect might be due to effects of eomesodermin on tumour infiltrating CD8 T cells, we subsequently analysed the regulated expression and function of this transcription factor in human T cells. Whereas overexpression of this factor induced perforin but not granzyme expression, siRNA mediated suppression of eomesodermin expression led to significantly reduced IFN-&ggr; production, perforin levels and cytolytic activity of CD8 T cells. Furthermore, TGF-&bgr; and IL4 could be identified as important inducer of eomesodermin expression.  These data define for the first time a regulatory role of eomesodermin for CD8 T cell activity in humans. Our findings are consistent with a model in which eomesodermin expression in tumour infiltrating T cells regulates cytolytic functions of CD8 T cells via perforin expression. These data provide novel insights into control mechanisms governing the functional activity of human CD8 T lymphocytes via T-box transcription factors in cancer.

title: CD3+ cells at the invasive margin of deeply invading (pT3-T4) colorectal cancer and risk of post-surgical metastasis: a longitudinal study.
abstract: The density of tumour-infiltrating lymphocytes (TIL) has been proposed as an independent predictor of outcome in patients with colorectal cancer. However, the relative roles of TIL density, nodal status, and microsatellite instability (MSI) in predicting tumour progression to metachronous metastasis remain to be elucidated. The aim of this study was to assess the relationship between the density of CD3+ TIL and the postsurgical occurrence of distant-organ metastases in a large series of patients with deeply invading and MSI-typed colorectal cancer.



Per cent areas of immunoreactivity due to CD3+ TIL at the invasive margin of the tumour (CD3+ TIL(IM)) were measured by computer-assisted image analysis in 286 tissue specimens from pT3 or pT4 MSI-tested colorectal cancer. Tissue samples were taken from consecutive patients who underwent resection at the IRCCS Istituto Clinico Humanitas, Rozzano, Milan, Italy, from January, 1997, to November, 2004, for colorectal cancer with no evidence of metastasis at diagnosis. Occurrence of metachronous metastasis, disease-specific survival (DSS), and disease-free survival (DFS), were assessed retrospectively in relation to per cent immunoreactivity.



CD3+ TIL(IM) density was higher in MSI colorectal cancer than in mismatch repair-system-proficient tumours (6.53%vs 2.19%; p<0.0001). At Cox analysis, higher CD3+ TIL(IM) densities, colonic site, and absence of nodal involvement were significantly associated with a lower risk of metachronous metastasis, but only the interaction between CD3+ TIL(IM) density and N-stage was significant on multivariate analysis (p=0.002). On separate analysis of node-negative colorectal cancer, increasing percentage of CD3+ immunoreactive area progressively reduced the risk of metachronous metastasis (<1%, reference; 1-5%, HR 0.28, 95% CI 0.10-0.81, p=0.02; >5%, 0.06, 0.01-0.48, p=0.008). Conversely, no significant association was seen between CD3+ immunoreactive area and risk of metachronous metastasis in node-positive colorectal cancer. Accordingly, CD3+ TIL(IM) density was associated with a better DSS (p=0.01) and DFS (p=0.006) only in patients with node-negative colorectal cancer. In primary tumours that had progressed to metachronous metastasis, stage III tumours had higher CD3+ TIL(IM) densities than stage II tumours (p=0.0004).



Metachronous metastases are unlikely to arise from node-negative colorectal cancers with a high-density CD3+ TIL(IM), whereas high densities of CD3+ TIL(IM) are not associated with the absence of postsurgical metastasis in patients with node-positive colorectal cancer. Our data suggest that densities of CD3+ TIL(IM) cannot be used as an independent predictor of clinical outcome in patients with stage III colorectal cancer and, at least for now, the tumour-node-metastasis classification should remain the preferred prognostic system. Our findings are consistent with a relationship between nodal involvement and tumour immunoevasion.



MIUR (Ministero dell'Istruzione, dell'Università e della Ricerca), Target Project Oncologia 2006, and Alleanza Contro il Cancro.

title: T cell‐mediated immunoregulation in the gastrointestinal tract
abstract: In the intestinal tract, only a single layer of epithelial cells separates innate and adaptive immune effector cells from a vast amount of antigens. Here, the immune system faces a considerable challenge in tolerating commensal flora and dietary antigens while preventing the dissemination of potential pathogens. Failure to tightly control immune reactions may result in detrimental inflammation. In this respect, ‘conventional’ regulatory CD4+ T cells, including naturally occurring and adaptive CD4+ CD25+ Foxp3+ T cells, Th3 and Tr1 cells, have recently been the focus of considerable attention. However, regulatory mechanisms in the intestinal mucosa are highly complex, including adaptations of nonhaematopoietic cells and innate immune cells as well as the presence of unconventional T cells with regulatory properties such as resident TCRγδ or TCRαβ CD8+ intraepithelial lymphocytes. This review aims to summarize the currently available knowledge on conventional and unconventional regulatory T cell subsets (Tregs), with special emphasis on clinical data and the potential role or malfunctioning of Tregs in four major human gastrointestinal diseases, i.e. inflammatory bowel diseases, coeliac disease, food allergy and colorectal cancer. We conclude that the clinical data confirms some but not all of the findings derived from experimental animal models.

title: Combination of microsatellite instability and lymphocytic infiltrate as a prognostic indicator for adjuvant therapy in colon cancer.
abstract: Microsatellite instability (MSI) and lymphocytic infiltrate (LI) in colon cancer are associated with less aggressive biological characteristics. Patients with stage II disease who are negative for MSI and LI have been found to have a less favorable prognosis. These patients may be candidates for more aggressive adjuvant therapy.



To evaluate the outcomes of patients with colon cancer treated with and without adjuvant chemotherapy on the basis of stage, MSI, and LI.



Prospective evaluation of MSI and LI status with retrospective analysis of chemotherapy regimen.



Community hospital system.



A total of 167 patients with colon cancer.



Definitive resection of colorectal cancer with or without chemotherapy.  with and without chemotherapy according to combined MSI and LI status.



Data on MSI and LI status and chemotherapeutic regimens were available for 140 patients. The 5-year DFS was 50% for patients with stage II disease who underwent chemotherapy vs 76% for those who did not (P = .02). In the group negative for MSI and LI, 5-year DFS was 29% for those undergoing chemotherapy and 91% for those who did not (P = .001).



Forgoing adjuvant chemotherapy should be considered in patients with stage II colon cancer who are negative for MSI and LI. The MSI and LI status shows promise as a combined prognostic marker and may prove particularly useful in selecting patients with stage II disease for adjunctive therapy.

title: Frameshift peptide‐derived T‐cell epitopes: A source of novel tumor‐specific antigens
abstract: Microsatellite instability (MSI) caused by defective DNA mismatch repair (MMR) is a hallmark of hereditary nonpolyposis colorectal cancers (HNPCC) but also occurs in about 15% of sporadic tumors. If instability affects microsatellites in coding regions, translational frameshifts lead to truncated proteins often marked by unique frameshift peptide sequences at their C‐terminus. Since MSI tumors show enhanced lymphocytic infiltration and our previous analysis identified numerous coding mono‐ and dinucleotide repeat‐bearing candidate genes as targets of genetic instability, we examined the role of frameshift peptides in triggering cellular immune responses. Using peptide pulsed autologous CD40‐activated B cells, we have generated cytotoxic T lymphocytes (CTL) that specifically recognize HLA‐A2.1‐restricted peptides derived from frameshift sequences. Among 16 frameshift peptides predicted from mutations in 8 different genes, 3 peptides conferred specific lysis of target cells exogenously loaded with cognate peptide. One peptide derived from a (−1) frameshift mutation in the TGFβIIR gene gave rise to a CTL bulk culture capable of lysing the MSI colorectal cancer cell line HCT116 carrying this frameshift mutation. Given the huge number of human coding microsatellites and assuming only a fraction being mutated and encoding immunologically relevant peptides in MSI tumors, frameshift protein sequences represent a novel subclass of tumor‐specific antigens. It is tempting to speculate that a frameshift peptide‐directed vaccination approach not only could offer new treatment modalities for existing MSI tumors but also might benefit asymptomatic  individuals in HNPCC families by a prophylactic vaccination strategy.

title: Tumour‐infiltrating lymphocytes in colorectal cancer with microsatellite instability are activated and cytotoxic
abstract: Patients with colorectal cancer that display high‐level microsatellite instability (MSI‐H) appear to have a better prognosis. This may be explained by the pronounced T cell infiltrate seen in MSI‐H tumours that is related to a specific antigen‐driven immune response. The nature of tumour‐infiltrating lymphocytes in colorectal cancers was investigated using quantitative real‐time polymerase chain reaction (PCR) and immunohistochemistry.

title: Frameshift-mutation-derived peptides as tumor-specific antigens in inherited and spontaneous colorectal cancer
abstract: The functional role and specificity of tumor infiltrating lymphocytes (TIL) is generally not well characterized. Prominent lymphocyte infiltration is the hallmark of the most common form of hereditary colon cancer, hereditary nonpolyposis colon cancer (HNPCC) and the corresponding spontaneous colon cancers with the microsatellite instability (MSI) phenotype. These cancers are caused by inherited or acquired defects in the DNA mismatch–repair machinery. The molecular mechanism behind the MSI phenotype provides a clue to understanding the lymphocyte reaction by allowing reliable prediction of potential T cell epitopes created by frameshift mutations in candidate genes carrying nucleotide repeat sequences, such as TGFβRII and BAX. These tumors therefore represent an interesting human system for studying TIL and characterizing tumor-specific T cells. We here describe T cell reactivity against several T helper cell epitopes, representing a common frameshift mutation in TGFβRII, in TIL and peripheral blood lymphocytes from patients with MSI+ tumors. The peptide SLVRLSSCVPVALMSAMTTSSSQ was recognized by T cells from two of three patients with spontaneous MSI+ colon cancers and from all three patients with HNPCC. Because such mutations are present in 90% of cancers within this patient group, these newly characterized epitopes provide attractive targets for cancer vaccines, including a prophylactic vaccine for individuals carrying a genetic disposition for developing HNPCC.

title: The immunogenicity of colorectal cancers with high-degree microsatellite instability
abstract: BackgroundHigh-degree microsatellite instability (MSI-H) is a feature of approximately 15% of sporadic colorectal cancers. Patients with MSI-H cancers have been reported to have a better prognosis than those with non-MSI-H cancers. The MSI-H subset is also characterised by a dense infiltrate of intra-epithelial lymphocytes and the hypothesis that the latter represents an efficacious immune response contributing to improved outcome is very attractive.MethodsData for this review were identified by searches of MEDLINE, PubMed, and cross references from relevant articles using the search terms 'microsatellite instability', 'colorectal cancer' and 'immunology', 'immune response' or 'immunogenicity'. total of 38 articles were identified by the search criteria and a further 95 articles by cross-referencing. The relevance of the articles to be interviewed was established by hand searching. Out of a total of 133 articles identified, 47 articles were rejected due to lack of relevance. A total of 86 articles were included in the review, pertaining to microsatellite instability in colorectal cancer, and immune mechanisms in colorectal cancer.ConclusionIt is suggested that this distinct group of colorectal cancers may have inherent immunogenic properties and that further elucidation of these may be invaluable to the development of successful immunotherapy.

title: Tumor-specific immunological recognition of frameshift-mutated peptides in colon cancer with microsatellite instability.
abstract: Colorectal cancers with microsatellite instability (MSI+ CRCs) caused by dysfunction of DNA mismatch repair have unique clinicopathological characteristics including good prognosis with T-cell infiltration in tumor. To identify tumor antigens that induce immune response against MSI+ CRC, SEREX (serological analysis of recombinant cDNA expression cloning) was applied. By screening a lambda phage cDNA library constructed from three MSI+ CRC cell lines with serum from a patient with MSI+ CRC with abundant T-cell infiltrates in tumor, 64 antigens were isolated. Immunogenicity of each antigen was evaluated by screening sera from patients with various cancers and from healthy individuals, and specific IgG antibodies (Abs) for 49 antigens were detected only in MSI+ CRC patients. A frameshift mutation in the repetitive G sequences (microsatellite) in the coding region of CDX2, one of the identified antigens, was found in the tumor tissue of the patient who had anti-CDX2 serum Ab. The Ab recognized both the COOH-terminal tumor-specific peptides created by the frameshift mutation and the NH(2)-terminal normal peptides of CDX2 when Western blot analysis was performed using various bacterial recombinant CDX2 proteins including the normal and altered peptides, which indicated that immune response could be raised against tumor-specific peptides generated through MSI. The anti-CDX2 Ab was detected only in the patient with the CDX2 frameshift mutation in tumor and disappeared 7 years after the curative resection, suggesting that this immune response may also be useful as a tumor marker. No altered subcellular localization and transcription ability was demonstrated in the mutated CDX2, although decreased expression was suggested in immunohistochemical analysis. Therefore, tumor-specific peptides generated by MSI may be involved in antitumor immune responses and may be useful for the development of diagnostic and therapeutic methods for patients with MSI+ CRC.

title: Immune response against frameshift-induced neopeptides in HNPCC patients and healthy HNPCC mutation carriers.
abstract: Colorectal cancers (CRC) associated with the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome display high-level microsatellite instability (MSI-H) as a consequence of mismatch repair deficiency. HNPCC-associated CRC frequently show features of a pronounced immune response, most likely resulting from the MSI-induced generation of novel tumor-specific carboxy-terminal frameshift peptides (FSPs). However, the role of FSP-specific immune surveillance mechanisms in HNPCC are unknown at present.



The efficacy of tumor-infiltrating T cells isolated from MSI-H CRCs (n = 3) was examined by in vitro killing assays. FSP-specific T-cell responses were measured by enzyme-linked immunospot in the peripheral blood from patients with MSI-H CRC (n = 32), healthy HNPCC mutation carriers (n = 16), patients with microsatellite stable (MSS) CRC (n = 17), and healthy donors (n = 22).



Tumor-infiltrating T cells isolated from MSI-H CRCs specifically recognized MSI-induced FSPs and showed cytotoxic activity against MSI-H but not MSS CRC cells. FSP-specific T-cell responses were detected in the majority of peripheral blood samples from patients with MSI-H but not MSS CRC. Interestingly, FSP-specific T-cell reactivity was already detectable in the peripheral blood of healthy HNPCC family members with germline mutations but without history of tumor development.



These data suggest that FSPs presented by DNA mismatch repair-deficient CRC cells are effectively recognized by the patient's immune system and may explain the characteristic clinicopathologic features of HNPCC-associated but also sporadic MSI-H CRCs. These observations are of high relevance for the development of FSP-based vaccination approaches, particularly for the preventive application in HNPCC mutation carriers.

title: Tumor-infiltrating lymphocytes in colorectal cancers with microsatellite instability are correlated with the number and spectrum of frameshift mutations
abstract: Colorectal cancers with microsatellite instability are characterized by an important density of tumor-infiltrating lymphocytes and a good prognosis. Microsatellite instability results from the inactivation of the DNA mismatch repair system and induces secondary somatic frameshift mutations within target genes harboring repeat sequences in their coding frame. By disrupting the open reading frame, frameshift mutations can result in the appearance of potentially immunogenic neopeptides. To determine the frameshift mutations inducing a T-cell response during the development of a tumor with microsatellite instability, we studied in 61 colorectal cancer patients with microsatellite instability, using a fluorescent multiplex PCR comparative analysis, the relative frequency of frameshift mutations within 19 target genes and analyzed the correlation of these frameshift mutations with the density of CD3+ tumor-infiltrating lymphocytes. The four most frequently mutated genes were ACVR2 (92%), TAF1B (84%), ASTE1/HT001 (80%) and TGFBR2 (77%). The vast majority (95%) of the tumors exhibited at least three frameshift mutations, and the number of frameshift mutations was associated with tumor progression (TNM stage, wall invasion and tumor diameter). Tumor-infiltrating lymphocyte density was associated with the overall number of frameshift mutations and with the presence of frameshift mutations within two target genes, namely ASTE1/HT001 and PTEN. These results strongly argue for the clinical relevance of immunotherapy of colorectal cancers with microsatellite instability.

title: High prevalence of activated intraepithelial cytotoxic T lymphocytes and increased neoplastic cell apoptosis in colorectal carcinomas with microsatellite instability.
abstract: Microsatellite instability (MSI) characterizes colorectal carcinomas (CRCs) in hereditary nonpolyposis colorectal cancer (HNPCC) syndrome and a proportion of sporadic CRCs. These MSI+ CRCs share several clinicopathological features, including a reputation for better survival rates than MSI- cases and a pronounced stromal inflammatory reaction of still undefined nature. In the present study, the presence, spatial distribution, and activation status of infiltrating cytotoxic effectors were investigated comparatively in 18 MSI+ and 37 MSI- CRCs by immunohistochemistry. The frequency of apoptosis was also evaluated by morphology and in situ end-labeling. MSI+ cases carried significantly higher numbers of cytotoxic lymphocytes infiltrating within neoplastic epithelial structures, as shown by immunostaining for CD3 (15.1 +/- 6.2 versus 4.6 +/- 4.1, P < 0.001), CD8 (13 +/- 6.4 versus 3.7 +/- 3.8, P < 0.001), and TIA-1 (11.2 +/- 6.5 versus 1.9 +/- 1.7, P < 0.001). These cytotoxic effectors were globally more activated in MSI+ than in MSI- tumors, as revealed by the expression of granzyme B (5.3 +/- 4.5 versus 0.6 +/- 1.3, P < 0.001). In MSI+ CRCs, the number of intraepithelial activated cytotoxic lymphocytes was significantly correlated with the proximal location of the tumor, a poorly differentiated phenotype, and the presence of peritumor lymphoid nodules. Multivariate analysis revealed that MSI was the major determinant of the presence of activated cytotoxic intraepithelial lymphocytes. Moreover, MSI+ CRCs also showed a significantly higher percentage of tumor cells undergoing apoptotic cell death (4.1 +/- 2.1 versus 2.6 +/- 1.1, P < 0.0001, by the TUNEL method), often located in close proximity of activated cytotoxic lymphocytes. These results are consistent with the presence of anti-tumor cytotoxic immune responses in most of MSI+ CRCs, a phenomenon that may at least in part contribute to the survival advantage ascribed to these patients.

title: Microsatellite instability and high content of activated cytotoxic lymphocytes identify colon cancer patients with a favorable prognosis.
abstract: 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.

title: Tumour infiltrating lymphocytes and apoptosis are independent features in colorectal cancer stratified according to microsatellite instability status.
abstract: The presence of high level DNA microsatellite instability (MSI-H) in colorectal cancer is associated with an improved prognosis, as is the presence of tumour infiltrating lymphocytes (TILs). It is not clear if TILs contribute directly to the survival advantage associated with MSI-H cancers through activation of an antitumour immune response.



To correlate TIL and apoptosis rates in colorectal cancer stratified by MSI status.



The distribution of TILs was characterised and quantified in a selected series of 102 sporadic colorectal cancers classified according to levels of MSI as 32 MSI-H, 30 MSI-low (MSI-L), and 40 microsatellite stable (MSS). Archival blocks were immunostained using the T cell markers CD3 and CD8, and the B cell marker CD20. Apoptosis of malignant epithelial cells was quantified by immunohistochemistry with the M30 CytoDEATH antibody.



Positive staining with anti-CD3 and negative staining with anti-CD20 identified virtually all TILs as T cells. The majority of CD3+ TILs (>75%) also stained with anti-CD8. TILs were most abundant in MSI-H colorectal cancers in which 23/32 (72%) scored as TIL positive. Only 5/40 (12.5%) MSS tumours and 9/30 (30%) MSI-L cancers were TIL positive (p<0.0001). MSI-H cancers showed a twofold higher rate of apoptosis (mean (SD) 3.52 (0.34)%) than the MSS cancers (1.53 (0.23)%) while the MSI-L subgroup had an intermediate level (2.52 (0.35)%) (p<0.0001). Overall, there was a small (r=0.347) but significant linear correlation between CD3+ and M30+ random apoptosis counts (p<0.001). However, TILs and apoptosis showed little colocalisation.



While TILs might be expected to explain the increased apoptotic rate and improved prognosis of MSI-H cancers, it is likely that TILs and apoptosis are independent characteristics of MSI-H cancers.

title: Microsatellite instability in colorectal cancer is associated with local lymphocyte infiltration and low frequency of distant metastases
abstract: Colorectal carcinomas (CRCs) with high microsatellite instability (MSI-H) share clinicopathological features distinctly different from their microsatellite stable (MSS) counterparts. Unlike MSS cancers, MSI-H CRCs occur predominantly in the right-sided colon and are often characterised by a strong lymphocyte infiltration. A poor differentiation pattern is found in most MSI-H CRCs, even though patients with MSI-H carcinomas seem to have a significantly longer survival after surgical resection. To clarify which factors contribute to the obvious paradoxon of a more favourable prognosis of MSI tumours, several clinical and histopathological features as well as the microsatellite status were evaluated in 120 colorectal cancer cases fulfilling clinical criteria (Bethesda) indicative for familial colorectal cancer. Microsatellite instablity status and lymphocyte infiltration were related to tumour stage and patients' follow-up. Statistical analysis confirmed well-known relations, such as enhanced lymphocyte infiltration accompanied by Crohn's like reaction (CLR) in MSI-H cancers (CLR+ in 27 out of 47 MSI-H vs 14 out of 71 MSS CRCs, P<0.001). However, after stratification for depth of local invasion and penetration of the primary tumour, T3 tumours displaying MSI had a significantly lower rate of distant metastases (M1 in four out of 35 MSI-H vs 20 out of 41 MSS CRCs, P<0.001). A similar tendency was observed for CLR-positive CRCs (M1 in six out of 29 CLR+ vs 17 out of 45 CLR− CRCs, P=0.13). In a logistic regression model, the MSI-H phenotype and the presence of CLR were independent predictors of a low UICC stage (P=0.006 and 0.04, respectively). These data, together with the recent definition of highly immunogenic neo-antigens expressed in MSI-H tumour cells, suggest that MSI-H CRCs elicit a protective host response that may prevent metastasis formation.

title: The "Fas counterattack" is not an active mode of tumor immune evasion in colorectal cancer with high-level microsatellite instability.
abstract: Microsatellite instability (MSI) is an alternative pathway of colorectal carcinogenesis. It is found in 10% to 15% of sporadic colorectal neoplasms and is characterized by failure of the DNA mismatch-repair system. High-level MSI (MSI-H) is associated with tumor-infiltrating lymphocytes (TILs) and a favorable prognosis. Expression of Fas ligand (FasL/CD95L) by cancer cells may mediate tumor immune privilege by inducing apoptosis of antitumor immune cells. The aim of this study was to investigate the relationship between FasL expression and MSI status in primary colon tumors. Using immunohistochemistry, we detected FasL expression in 91 colorectal carcinoma specimens, previously classified according to the level of MSI as MSI-H (n = 26), MSI-low (MSI-L) (n = 29), and microsatellite stable (n = 36). Tumor-infiltrating lymphocyte density was quantified by immunohistochemical staining for CD3. MSI-H tumors were significantly associated with reduced frequency (P = .04) and intensity (P = .066) of FasL expression relative to non-MSI-H (ie, microsatellite stable and MSI-L) tumors. Higher FasL staining intensity correlated with reduced TIL density (P = .059). Together, these findings suggest that the abundance of TILs found in MSI-H tumors may be due to the failure of these tumor cells to up-regulate FasL and may explain, in part, the improved prognosis associated with these tumors.

title: Tumour budding in colorectal carcinoma
abstract: The term tumour budding denotes that at the invasion front of colorectal adenocarcinomas tumour cells, singly or in small aggregates, become detached from the neoplastic glands. This morphological feature is increasingly being recognized as a strong and robust adverse prognostic factor. Biologically, tumour budding is closely related to the epithelial–mesenchymal transition. In this review the morphological features of tumour budding are discussed, as observed by the surgical pathologist reporting colorectal carcinoma resection specimens. The morphological features are put into context with the rapidly expanding knowledge of the epithelial–mesenchymal transition in general, and the molecular pathology of colorectal carcinoma in particular. Finally, a systematic analysis of the relevant published clinicopathological studies emphasizes the potential of tumour budding as a prognostic factor for routine surgical pathology.

title: Tumor Budding is a Strong and Reproducible Prognostic Marker in T3N0 Colorectal Cancer
abstract: Tumor budding along the advancing front of colorectal adenocarcinoma is an early event in the metastatic process. A reproducible, prognostic budding scoring system based on outcomes in early stage colorectal cancer has not been established.  One hundred twenty-eight T3N0M0 colorectal carcinoma patients with known outcome were identified. Tumor budding was defined as isolated tumor cells or clusters of <5 cells at the invasive tumor front. Tumor bud counts were generated in 5 regions at 200× by 2 pathologists (conventional bud count method). The median bud count per case was used to divide cases into low (median=0) and high budding (median ≥1) groups. Forty cases were reevaluated to assess reproducibility using the conventional and a novel rapid bud count method.  Fifty-seven (45%) carcinomas had high and 71 (55%) had low budding scores. High budding was associated with an infiltrative growth pattern (P<0.0001) and lymphovascular invasion (P=0.005). Five-year cancer-specific survival was significantly poorer in high compared with low budding groups: 63% versus 91%, respectively, P<0.0001. Multivariate analysis demonstrated tumor budding to be independently prognostic (hazard ratio=4.76, P<0.001). Interobserver agreement was at least equivalent comparing the conventional to the rapid bud count methods: 87.5% agreement (κ=0.75) versus 92.5% agreement (κ=0.85), respectively.  Tumor budding is a strong, reproducible, and independent prognostic marker of outcome that is easily assessed on hematoxylin and eosin slides. This may be useful for identifying the subset of T3N0M0 patients at high risk of recurrence who may benefit from adjuvant therapy.

title: CD8+ lymphocytes/ tumour-budding index: an independent prognostic factor representing a ‘pro-/anti-tumour' approach to tumour host interaction in colorectal cancer
abstract: The tumour-host interaction at the invasive front of colorectal cancer, including the epithelial–mesenchymal transition and its hallmark ‘tumour budding’, is an important area of investigation in terms of prognosis. The aim of this study was to determine the prognostic impact of a ‘pro-/anti-tumour’ approach defined by an established ‘pro-tumour’ (tumour budding) and host-related ‘anti-tumour’ factor of the adaptive immunological microenvironment (CD8+ lymphocytes).Methods:Double immunostaining for CK22/CD8 on whole tissue sections (n=279; Cohort 1) and immunohistochemistry for CD8+ using tissue microarrays (n=191; Cohort 2) was carried out. Tumour buds, CD8+ and CD8+ T-lymphocytes : tumour buds indices were evaluated per high-power field.In Cohort 1, a low-CD8+/ buds index was associated with lymph node metastasis (P<0.001), vascular invasion (P=0.009), worse survival in univariate (P<0.001) and multivariable (P<0.001) analysis, and furthermore in lymph node-negative patients (P=0.002). In Cohort 2, the CD8+/ buds index was associated with T stage (P<0.001), N stage (P=0.041), vascular invasion (P=0.005) and survival in patients with TNM stage II (P=0.019), stage III (P=0.004), and adjuvantly untreated (P=0.009) and treated patients (The CD8+ lymphocyte : tumour-budding index is an independent prognostic factor in colorectal cancer and a promising approach for a future prognostic score for patients with this disease.

title: Differential significance of tumour infiltrating lymphocytes in sporadic mismatch repair deficient versus proficient colorectal cancers: a potential role for dysregulation of the transforming growth factor-beta pathway.
abstract: The prognostic importance of tumour infiltrating lymphocytes (TILs) in colorectal cancer (CRC) remains controversial. The present study was undertaken to evaluate the role of TILs as prognostic indicators and to investigate the role of transforming growth factor-beta (TGF-beta) in TIL infiltration. Immunohistochemical staining for components in the TGF-beta pathway was performed on a tissue microarray of 1420 unselected CRCs with complete clinico-pathological data. Statistical analyses were carried out on samples stratified by mismatch repair (MMR) proficiency status and TIL counts. TIL infiltration was found to correlate with multiple clinico-pathological features but was a prognostic marker only in MMR proficient CRCs. In all CRCs, findings indicative of insensitivity to TGF-beta and increased TGF-beta secretion were independent predictors of high TIL counts, suggesting that perturbations in the TGF-beta signalling pathway play an important role in the recruitment and retention of TILs within CRC epithelium.

title: Tumor infiltrating lymphocytes: an intriguing player in the survival of colorectal cancer patients
abstract: BackgroundThere is growing evidence that both local and systemic inflammatory responses play an important role in the progression of a variety of solid tumors. Colorectal cancer results from the cumulative effect of sequential genetic alterations, leading to the expression of tumor associated antigens possibly inducing a cellular anti-tumor immune response. It is well recognized that cytotoxic lymphocytes constitute one of the most important effector mechanisms of anti-tumor-immunity. However, their potential prognostic influence in colorectal cancer remains controversial. Aim of the study was to examine infiltration of CD3+ and CD8+ lymphocytes in colorectal cancer and their prognostic potential.Two-hundred-fifteen colorectal cancer cases, previously analyzed for microsatellite instability (MSI), were selected for immunohistochemical detection of CD3+, CD8+ infiltration and the expression of granzyme B. Prognostic relevance was assessed by survival analysis.ResultsStrong correlations were found between the infiltration of lymphocytes and several clinicopathological variables. Survival analysis revealed that intra-epithelial infiltration of CD3+ and CD8+ T lymphocytes and stromal infiltration of CD3+ lymphocytes had a major impact on the patients' overall survival in the univariate analysis, however independent of their association with MSI-status. In addition, it was also demonstrated that there was an important disease specific survival advantage for patients with microsatellite stable (MSS) tumors containing intraepithelial CD8+ tumor infiltrating lymphocytes. When samples were analyzed for colon cancer and rectal cancer separately, the results of the overall population were confirmed in colon cancer only. When entered into a multiple Cox regression analysis adjusting for other possible important confounding factors, the strong impact of lymphocyte infiltration on overall survival was not maintained. Only early stage and young age (borderline significant for overall population only) were associated with a better overall survival (early disease with disease-free survival also).ConclusionsIn conclusion our results suggest a role for infiltrating CD3+ and CD8+ T lymphocytes in colorectal cancer whereby tumor infiltration could reflect a general principle of antitumor immunity, irrespective of the MSI-status.

title: Prognostic role of CD8+ tumor-infiltrating lymphocytes in stage III colorectal cancer with and without microsatellite instability.
abstract: Previous studies have identified high numbers of intraepithelial T lymphocytes to be associated with good prognosis in various types of cancer. Few studies addressing this issue have been published for colorectal cancer. In a simulated prospective approach ("phase II prognostic factor study"), all nonmetachronous International Union Against Cancer (UICC) stage III colorectal cancers that were accessioned in the years 1994 to 1999 were included in the study (152 cases). Follow-up information as to vital status and occurrence of metachronous metastases could be obtained for all patients in the years 2001 and 2002. CD8+ intratumoral lymphocytes were quantified after immunostaining and referred to tumor cell area (CD8+ densities). Microsatellite status was determined by using the Bethesda panel of microsatellite markers. CD8+ densities ranged from 0 per square millimeter to 1436 per square millimeter of tumor area in a nonnormal distribution that was skewed toward low values. Univariate survival analyses revealed the 66th percentile as a stringent cutoff (CD8+high versus CD8+low), with CD8+high cases taking a significantly better clinical course. This prognostic impact appeared even more pronounced in the subset of patients with colon carcinomas who were receiving 5-fluouracil/leucovorin as adjuvant treatment (79 patients). Seventeen patients had carcinomas with high microsatellite instability (MSI-H). MSI-H-CD8+high cases (n = 11) showed an excellent prognosis, with tumor-free survival for 9 of the 11 patients. The prognostic effect of CD8+high was retained in Cox regression analyses when including UICC substages (IIIA to IIIC). Our results identify CD8+ tumor-infiltrating lymphocytes as a promising candidate for further evaluation in the ongoing search for prognostic and predictive factors of colorectal cancer, particularly if combined with microsatellite status.

title: Targeted immunotherapy for colorectal cancer: monoclonal antibodies and immunotoxins
abstract: Colorectal cancer (CRC) is a major health concern worldwide. It is the third most frequently diagnosed cancer and the second leading cause of cancer death. There currently are a number of treatment options for CRC, however many of them have failed to demonstrate desired therapeutic benefit. Therefore, significant efforts are being directed towards the development of new biological therapies with improved efficacy. Immunotherapy is an emerging treatment modality for a variety of cancers. Several promising treatments have already been approved by the US FDA and are being tested in clinical trials. Antibodies have been proved to be useful in cancer therapy due to their ability to recognize tumor-associated antigens expressed at higher density on malignant cells in comparison with those that are normal. Antibodies can be used as a single therapy or in combination with other therapies. A large variety of monoclonal antibodies have been developed. However, only a very few are able to kill a sufficient number of malignant cells and cause tumor regression. Hence, it is often necessary to arm the antibody with a cytotoxic agent to enhance the efficacy of the anti-tumor activity. This review provides a brief overview of some of the current agents being employed in targeted immunotherapy for CRC.

title: Efficiency of adjuvant active specific immunization with Newcastle disease virus modified tumor cells in colorectal cancer patients following resection of liver metastases: results of a prospective randomized trial
abstract: PurposeMetastatic disease is a major cause of mortality in colorectal cancer patients. Even after complete resection of isolated liver metastases, recurrence develops in the majority of patients. Therefore, development of strategies to prevent recurrent liver metastases is of major clinical importance. The present prospectively randomised phase III trial investigates the efficiency of active specific immunotherapy (ASI) after liver resection for hepatic metastases of colorectal cancer.MethodsPatients with histologically confirmed liver metastases from colorectal cancer were randomised to the vaccination or control group. After complete resection of liver metastases, patients randomised to the vaccination group received six doses of Newcastle disease virus (NDV) infected autologous tumour cell vaccine (ATV-NDV). The primary end-point was overall survival, secondary end-points were disease-free survival and metastases-free survival.ResultsFifty-one patients were enrolled in the study with 50 patients available for analysis. The follow-up period was 116.1 ± 23.8 month in the vaccination arm and 112.4 ± 18.5 month in the control group. In the total patient group, no differences in the primary and secondary end-points were detected. Most interestingly, subgroup analysis revealed a significant advantage for vaccinated colon cancer patients with respect to overall survival [hazard ratio: 3.3; 95%, confidence interval (CI): 1.0–10.4; P = 0.042] and metastases-free survival (hazard ratio: 2.7; 95%, CI: 1.0–7.4; P = 0.047) in the intention-to-treat analysis.ConclusionActive specific immunotherapy in unselected colorectal cancer patients was not effective for prevention of recurrent metastatic disease. However, in colon cancer patients, ASI with ATV-NDV appears to be beneficial prolonging overall and metastases-free survival.

title: Active Specific Immunotherapy with Autologous Tumor cell vaccines for Stage II Colon Cancer: Logistics, Efficacy, Safety and Immunological Pharmacodynamics
abstract: In the area of cancer treatment, immunotherapy with vaccines has suffered in the last 5 years, due to many clinical trial failures. One must keep in mind however, that many of the clinical trials conducted in the past decade were performed without the benefit of sound regulatory guidance or validated and compliant manufacturing processes. This has clearly been the case for patient specific, tumor cell vaccine therapy. The safety concerns that emanated within the regulatory agencies from the Somatic Cell Therapy concepts, translated to active specific immunotherapy with vaccines. Fortunately, in the past 5 years advances in understanding the immune system, improved design of clinical trials, improvement and compliance of manufacturing processes provided opportunities to significantly improve efficacy and safety. Clearly, the vaccine research establishment has learned the importance of not just selecting antigens but the requirement of tumor associated immunogens that can stimulate a functional immune response. Also, it has become clear that immunotherapy works best in situations of minimal residual disease. Finally, more realistic endpoints in clinical trials have been recognized and accepted by oversight review committees. This commentary describes the “trials and tribulations” of developing a patient specific, autologous tumor cell vaccine for therapy of Stage II colon cancer.

title: Immunotherapy targeting fibroblast activation protein inhibits tumor growth and increases survival in a murine colon cancer model
abstract: Murine studies have shown that immunological targeting of fibroblast activation protein (FAP) can elicit protective immunity in the absence of significant pathology. Fibroblast activation protein is a product overexpressed by tumor‐associated fibroblasts (TAF) and is the predominant component of the stoma in most types of cancer. Tumor‐associated fibroblasts differ from normal adult tissue fibroblasts, and instead resemble transient fetal and wound healing‐associated fibroblasts. Tumor‐associated fibroblasts are critical regulators of tumorigenesis, but differ from tumor cells by being more genetically stable. Therefore, in comparison to tumor cells, TAF may represent more viable therapeutic targets for cancer immunotherapy. To specifically target TAF, we constructed a DNA vaccine directed against FAP. This vaccine significantly suppressed primary tumor and pulmonary metastases primarily through CD8+ T‐cell‐mediated killing in tumor‐bearing mice. Most importantly, tumor‐bearing mice vaccinated against FAP exhibited a 1.5‐fold increase in lifespan and no significant pathology. These results suggest that FAP, a product preferentially expressed by TAF, could function as an effective tumor rejection antigen.

title: Neoadjuvant immunotherapy enhances radiosensitivity through natural killer cell activation.
abstract: We investigated whether natural killer (NK) cells in the tumor microenvironment have a radiosensitization effect. The radiosensitization effect of combined CpG and Herceptin((R)) (Genentech, Inc., South San Francisco, CA) (CpG/Herceptin), given before or after radiation, was evaluated by using a murine colon cancer cell line overexpressing human HER2/neu, CT26HER2/neu. In vitro radiosensitization effects were investigated by coculture of CT26HER2/neu with splenocytes, CpG, and Herceptin before applying radiation. Tumor cells, cocultured with CpG-pretreated splenocytes and Herceptin, were more vulnerable to radiation damage. In BALB/c mice injected with CT26HER2/neu, CpG/Herceptin administered before radiotherapy was associated with a better retardation of tumor growth than when administered after radiotherapy. The radiosensitization effect was significantly abrogated by NK-cell depletion, indicating that NK cells play an essential role in it. Further, surviving mice treated with CpG or CpG/Herceptin and reverse transcriptase were resistant to renewed tumor challenge, suggesting the presence of an induced immune response to the tumor. Neoadjuvant immunotherapy with CpG/Herceptin may improve response to radiotherapy of HER2/neu-expressing tumors.

title: Combination of CTL‐associated Antigen‐4 Blockade and Depletion of CD25+ Regulatory T Cells Enhance Tumour Immunity of Dendritic Cell‐based Vaccine in a Mouse Model of Colon Cancer
abstract: Immune regulation has been shown to be involved in the progressive growth of some murine tumours. Interruption of immune regulatory pathways via CTL‐associated antigen‐4 (CTLA‐4) blockade or removal of CD4+ CD25+ regulatory T (Treg) cells appears to be a promising strategy for cancer immunotherapy. In this study, we tested the hypothesis that the combination of CTLA‐4 blockade and depletion of Treg cells would improve the potency of dendritic cell (DC)‐based vaccine in a clinically relevant mouse model, which is transgenic for both carcinoembryonic antigen (CEA) and HLA‐A2 for the treatment of colon carcinoma in a therapeutic setting. We found that administration of anti‐CD25 antibody prior to vaccination or systemic administration of anti‐CTLA‐4 antibody with the vaccine improved tumour‐free survival against CEA‐expressing tumours compared with mice immunized with DC‐based vaccine alone. However, the efficacy of the vaccine proved to be most effective when anti‐CTLA‐4 antibody was combined with Treg inhibition. This vaccination strategy dramatically improved the tumour‐free survival and allowed the development of long‐lasting immune responses. The combined vaccination strategy resulted in increased secretion of IFN‐γ and enhanced HLA‐A2‐restricted CEA‐specific CTL responses. Furthermore, coadministration of anti‐CD25 and anti‐CTLA‐4 antibodies along with the vaccine was effective against more advanced tumours. These results provide evidence that simultaneous blockade of T‐cell regulatory pathways is a promising approach for the induction of therapeutic antitumour immunity against CEA+ colon carcinoma.

title: Tumor-Infiltrating Lymphocytes and Perforation in Colon Cancer Predict Positive Response to 5-Fluorouracil Chemotherapy
abstract: The major pathologic markers of prognosis in colorectal cancer include vascular invasion by tumor cells, invasion of adjacent lymph nodes, and perforation of the serosal wall. Recent work suggests that a high density of tumor-infiltrating lymphocytes (TIL) is associated with good outcome independently of these established prognostic markers. The aim of the present study was to investigate the prognostic significance of TILs and other routinely reported pathologic features in colon cancer, particularly in relation to the use of adjuvant chemotherapy.  Pathologic markers, disease-specific survival, and the use of adjuvant chemotherapy were recorded in a retrospective, population-based series of 1,156 stage III colon cancer patients with a median follow-up time of 52 months.  In patients treated by surgery alone (n = 851), markers with significant prognostic value included poor histologic grade, T4 stage, N2 nodal status, vascular invasion, and perforation, but not the presence of TILs. In patients treated with 5-fluorouracil–based chemotherapy (n = 305), TILs were associated with significantly improved survival [hazard ratio (HR), 0.52; 95% confidence interval, 0.30-0.91; P = 0.02] and perforation with a trend for improved survival (HR, 0.67; 95% confidence interval, 0.27-1.05; P = 0.16). Patients with TILs or perforation seemed to gain more survival benefit from chemotherapy (HR, 0.22 and 0.21, respectively) than patients without these features (HR, 0.84 and 0.82, respectively).  The apparent survival advantage from 5-fluorouracil associated with TILs and perforation requires confirmation in prospective studies. Because the presence of TILs reflects an adaptive immune response and perforation is associated with inflammatory response, these results suggest that there may be interactions between the immune system and chemotherapy leading to improved survival of colon cancer patients.

title: In situ analysis of FOXP3+ regulatory T cells in human colorectal cancer
abstract: The immune system spontaneously responds to tumor-associated antigens in peripheral blood of colorectal cancer (CRC) patients. Regulatory T cells (Treg) are suspected of influencing the interaction between the tumor and immune system and thus the course of malignant diseases. However, the function of Tregs in the development of T cell responses and on the clinical course of CRC is not clear. We analyzed Treg infiltration (FOXP3 staining) in situ in 40 CRC patients and investigated whether there is a correlation to disease stage, systemic T cell response, and survival. Treg infiltration was significantly higher in CRC than in healthy colon. Stromal Treg infiltration was significantly higher than epithelial infiltration in CRC. Furthermore, Treg infiltration in the tumor was significantly higher in limited disease than in metastatic CRC. The average Treg infiltration rate in the tumor was non-significantly higher in patients without systemic TAA-specific T cell response. Survival did not differ between patients with high Treg infiltration and those with low Treg infiltration. In conclusion, a direct link between Treg infiltration in the tumor and the development of a systemic T cell response in CRC cannot be proven. However, local Treg infiltration was significantly higher in limited disease, in which a systemic TAA-directed T cell responses is less frequently observed.

title: Increased frequency of regulatory T cells in peripheral blood and tumour infiltrating lymphocytes in colorectal cancer patients.
abstract: Recent results have shown a correlation between survival and frequency of tumour infiltrating T lymphocytes in colorectal cancer patients. However, it remains unclear whether the frequency of regulatory T cells is higher in colorectal cancer as compared to normal colon. To address this question we analysed the frequency and function of regulatory T cells in the peripheral blood and tumour infiltrating lymphocytes of colorectal cancer patients. The proportion of regulatory T cells in the peripheral blood of colorectal cancer patients (mean 8%) was significantly higher than that in normal controls (mean 2.2%). There were significantly more regulatory T cells in tumour infiltrating lymphocytes (mean 19.2%) compared to lymphocytes from an autologous non-malignant portion of the colon (mean 9%). Regulatory T cells from colorectal cancer patients were FOXP3 positive and suppressed the proliferation of autologous CD4+ CD25- cells. A higher density of tumour infiltrating regulatory T cells was found in patients with advanced as compared to early disease. These results support the hypothesis that increased numbers of regulatory T cells in the blood and tumours of colorectal cancer patients may influence the immune response against cancer and suggest that strategies to overcome regulatory T cell activity may be beneficial in the treatment of human colorectal cancer.

title: Tumor-infiltrating FOXP3+ T regulatory cells show strong prognostic significance in colorectal cancer.
abstract: To determine the prognostic significance of FOXP3(+) lymphocyte (Treg) density in colorectal cancer compared with conventional histopathologic features and with CD8(+) and CD45RO(+) lymphocyte densities.



Tissue microarrays and immunohistochemistry were used to assess the densities of CD8(+), CD45RO(+), and FOXP3(+) lymphocytes in tumor tissue and normal colonic mucosa from 967 stage II and stage III colorectal cancers. These were evaluated for associations with histopathologic features and patient survival.



FOXP3(+) Treg density was higher in tumor tissue compared with normal colonic mucosa, whereas CD8(+) and CD45RO(+) cell densities were lower. FOXP3(+) Tregs were not associated with any histopathologic features, with the exception of tumor stage. Multivariate analysis showed that stage, vascular invasion, and FOXP3(+) Treg density in normal and tumor tissue were independent prognostic indicators, but not CD8(+) and CD45RO(+). High FOXP3(+) Treg density in normal mucosa was associated with worse prognosis (hazard ratio [HR] = 1.51; 95% CI, 1.07 to 2.13; P = .019). In contrast, a high density of FOXP3(+) Tregs in tumor tissue was associated with improved survival (HR = 0.54; 95% CI, 0.38 to 0.77; P = .001).



FOXP3(+) Treg density in normal and tumor tissue had stronger prognostic significance in colorectal cancer compared with CD8(+) and CD45RO(+) lymphocytes. The finding of improved survival associated with a high density of tumor-infiltrating FOXP3(+) Tregs in colorectal cancer contrasts with several other solid cancer types. The inclusion of FOXP3(+) Treg density may help to improve the prognostication of early-stage colorectal cancer.

title: Intratumoral CD8+ T/FOXP3+ cell ratio is a predictive marker for survival in patients with colorectal cancer
abstract: The human immune system consists of a balance between immune surveillance against non-self antigens and tolerance of self-antigens. CD8+ T cells and CD4+ regulatory T cells (Tregs) are the main players for immune surveillance and tolerance, respectively. We examined immunohistochemically the immunological balance at the tumor site using 94 surgically resected colorectal cancer tissues. Forkhead box P3 (FOXP3)+ cells were considered to be Tregs in the present study. The number of intratumoral FOXP3+ cells (itFOXP3+ cells) was positively correlated with lymph node metastases (P = 0.030). itCD8+ T/itFOXP3+ cell ratio negatively correlated with pathological stages (P = 0.048). Next, relationship between the number of itCD8+ T cells or itFOXP3+ cells and survival prognosis in 94 patients who underwent a curative resection was analyzed. Only itCD8+ T/itFOXP3+ cell ratio positively correlated with disease-free survival (0.023) and overall survival (P = 0.010). Multivariate analysis indicated that itCD8+ T/itFOXP3+ cell ratio is an independent prognostic factor (P = 0.035) of overall survival. The number of itFOXP3+ cells positively correlated with transforming growth factor-beta TGF-β production at the tumor site (P = 0.020). In conclusion, itCD8+ T/itFOXP3+ cell ratio is a predictive marker for both disease-free survival time and overall survival time in patients with colorectal cancer. Importantly, itCD8+ T/itFOXP3+ cell ratio may be an independent prognostic factor. And, tumor-producing TGF-β may contribute to the increased number of itFOXP3+ cells.

title: Clinical implications of microsatellite instability in sporadic colon cancers
abstract: To review data demonstrating the prognostic and predictive impact of microsatellite instability (MSI) in human colon carcinomas. Recent findings MSI is a molecular marker of defective DNA mismatch repair that is detected in approximately 15% of sporadic colon cancers. Most, but not all retrospective studies, have shown that colon cancers with MSI have better stage-adjusted survival rates compared with non-MSI tumors. Furthermore, analyses of colon cancers from participants in randomized adjuvant therapy trials have suggested that MSI tumors do not benefit from treatment with 5-fluorouracil. Recent studies, including a pooled analysis, validate prior data demonstrating the prognostic and predictive impact of MSI status in colon cancer.  MSI is a molecular marker that can provide valuable prognostic and predictive information in colon cancer patients. In the appropriate clinical setting, MSI data can be used in clinical decision-making. Specifically, the favorable outcome of stage II colon cancers with MSI indicates that such patients should not receive adjuvant chemotherapy. Although data for stage III colon cancers with MSI suggest a lack of benefit from 5-fluorouracil alone, the benefit of the current standard treatment, 5-fluorouracil, leucovorin, and oxaliplatin, in this subgroup remains unknown and awaits further study.

title: Risk factors: CD8+:FOXP3+ cell ratio is a novel survival marker for colorectal cancer
abstract: the tumor infiltrating CD8+:FoXP3+ cell ratio may be a predictive marker of diseasefree survival in patients with colorectal cancer, according to a recent study. a team at Kyushu university, Japan analyzed the relationships of intratumoral CD8+ t cells and FoXP3+ cells with traditional pathological measurements for surgically resected specimens from 95 patients with primary colorectal cancer. the researchers did not detect any correlation between the number of intratumoral CD8+ t cells with any pathological parameter; however, lymph-node metastasis was significantly associated with the number of intratumoral FoXP3+ cells. Furthermore, suzuki noted that “the ratio of the number of [intratumoral] CD8+ t cells to the number of [intratumoral] FoXP3+ cells (itCD8+:itFoXP3+ cell ratio) was negatively correlated with pathological stages.” although the number of CD8+ t cells and FoXP3+ cells was not associated with Risk factoRs

title: Foxp3 and microsatellite stability phenotype in colorectal cancer
abstract: Colorectal tumourigenesis is in the majority of cases regarded as a multistep process resulting in chromosomal instability.1 High-frequency microsatellite instability (MSI-H) can be detected in ∼15–20% of all sporadic colorectal cancers (CRCs) and represents the hallmark of the hereditary non-polyposis colorectal cancer (HNPCC) syndrome. CRCs with MSI-H are caused by inactivation of DNA mismatch repair (MMR) genes leading to hundreds of insertion or deletion mutations in short segments of repeated nucleotide sequences, but no chromosomal instability. It has been speculated that abnormal peptides are generated as a consequence of these frameshift mutations, and one would expect a stimulation of the antitumour response by these tumour-associated antigens (TAAs).2 3 In fact, CRCs with MSI-H are associated with a markedly increased number of tumour-infiltrating lymphocytes (TILs) as compared with microsatellite-stable (MSS) tumours, and the favourable prognosis of patients with MSI-H cancer has been attributed to the high content of cytotoxic T 

Over the past few years, CD4+CD25+ regulatory T cells (Treg cells) expressing the transcription factor forkhead box P3 (Foxp3) have been demonstrated to suppress T cell-mediated immunity to TAAs in various human tumours—for example, ovarian cancer.5 In mouse models, depletion of Treg cells using treatment with CD25-, GITR- or CTLA4-specific antibodies has resulted in improved antitumour immunity.6 In human CRC tissue samples, increased numbers of Treg cells as compared with normal mucosa have also been described, with a Treg cell infiltration rate slightly higher in patients without a systemic TAA-specific immune reaction.7 Despite successful induction of an immunological response, current protocols of immunotherapy and vaccination in CRC are not satisfactory.8 The poor response rate in patients with CRC may in part be due to the fact that immunosuppressive mechanisms conferred by Treg cells outweigh the beneficial effects of …

title: High density of FOXP3-positive T cells infiltrating colorectal cancers with microsatellite instability
abstract: High-level microsatellite instability (MSI-H) in colorectal cancer accounts for about 12% of colorectal cancers and is typically associated with a dense infiltration with cytotoxic CD8-positive lymphocytes. The role of regulatory T cells that may interfere with the host's antitumoural immune response in MSI-H colorectal cancers has not been analysed yet. Using an antibody directed against the regulatory T-cell marker transcription factor forkhead box P3 (FOXP3), regulatory T cells were examined in 70 colorectal cancers with known MSI status (MSI-H, n=37; microsatellite stable, n=33). In MSI-H colorectal cancers, we found a significantly higher intraepithelial infiltration with FOXP3-positive cells (median: 8.5 cells per 0.25 mm2 vs 3.1 cells per 0.25 mm2 in microsatellite stable, P<0.001), and a significantly elevated ratio of intraepithelial to stromal infiltration (0.05 vs 0.01 in microsatellite stable, P<0.001). CD8-positive cell counts were related positively to the number of FOXP3-positive cells (Spearman's ρ=0.56 and 0.55, respectively). Our results show that the elevated number of CD8-positive lymphocytes found in MSI-H colorectal cancers is paralleled by an enhanced infiltration with CD8-negative FOXP3-positive cells. These data suggest that FOXP3-positive cells may play a role in the regulation of the immune response directed against MSI-H colorectal cancers at the primary tumour site.