Rahul K. Hejmadi

The effects of CCR5 inhibition on regulatory T-cell recruitment to colorectal cancer

Background:Regulatory T cells (Treg) are enriched in human colorectal cancer (CRC) where they suppress anti-tumour immunity. The chemokine receptor CCR5 has been implicated in the recruitment of Treg from blood into CRC and tumour growth is delayed in CCR5−/− mice, associated with reduced tumour Treg infiltration.Methods:Tissue and blood samples were obtained from patients undergoing resection of CRC. Tumour-infiltrating lymphocytes were phenotyped for chemokine receptors using flow cytometry. The presence of tissue chemokines was assessed. Standard chemotaxis and suppression assays were performed and the effects of CCR5 blockade were tested in murine tumour models.Results:Functional CCR5 was highly expressed by human CRC infiltrating Treg and CCR5high Treg were more suppressive than their CCR5low Treg counterparts. Human CRC-Treg were more proliferative and activated than other T cells suggesting that local proliferation could provide an alternative explanation for the observed tumour Treg enrichment. Pharmacological inhibition of CCR5 failed to reduce tumour Treg infiltration in murine tumour models although it did result in delayed tumour growth.Conclusions:CCR5 inhibition does not mediate anti-tumour effects as a consequence of inhibiting Treg recruitment. Other mechanisms must be found to explain this effect. This has important implications for anti-CCR5 therapy in CRC.

Wnt signalling in adenomas of familial adenomatous polyposis patients

Background:Epigenetic silencing of Wnt antagonists and expression changes in genes associated with Wnt response pathways occur in early sporadic colorectal tumourigenesis, indicating that tumour cells are more sensitive to Wnt growth factors and respond differently. In this study, we have investigated whether similar changes occur in key markers of the Wnt response pathways in the genetic form of the disease, familial adenomatous polyposis (FAP).Methods:We investigated epigenetic and expression changes using pyrosequencing and real-time RT-PCR in samples from seven patients without neoplasia, and matched normal and tumour tissues from 22 sporadic adenoma and 14 FAP patients.Results:We found that 17 out of 24 (71%) FAP adenomas were hypermethylated at sFRP1, compared with 20 out of 22 (91%) of sporadic cases. This was reflected at the level of sFRP1 transcription, where 73% of FAP and 100% of sporadic cases were down-regulated. Increased expression levels of c-myc and FZD3 were less common in FAP (35 and 46% respectively) than sporadic tumours (78 and 67% respectively).Conclusion:Overall, the changes in expression and methylation were comparable, although the degree of change was generally lower in the FAP adenomas. Molecular heterogeneity between multiple adenomas from individual FAP patients may reflect different developmental fates for these premalignant tumours.

Evaluation of serum lysyl oxidase as a blood test for colorectal cancer

AIMS Lysyl oxidase (LOX) expression is elevated in colorectal cancer (CRC) tissue and associated with disease progression. A blood test may form a more acceptable diagnostic test for CRC although LOX has not previously been measured in the serum. We therefore sought to determine the clinical usefulness of a serum LOX test for CRC in a symptomatic population. METHODS Adult patients referred to a hospital colorectal clinic with bowel symptoms completed a questionnaire and provided a blood sample for serum LOX measurement. Associations between presenting symptoms, serum LOX concentrations and outcomes of investigations were tested by univariate and multivariate analyses to determine if serum LOX was clinically useful in the prediction of CRC. LOX expression in CRC and adjacent colon biopsies was evaluated by ELISA and immunohistochemistry. RESULTS Thirty-one cases of colorectal cancer and 16 high-risk polyps were identified from a total of 962 participants. There was no association between serum LOX concentration and the presence of CRC, high-risk polyps or cancers at any site. LOX expression was significantly increased in CRC tissue compared to adjacent colon. CONCLUSION Despite overexpression of LOX in CRC tissue, elevated serum levels could not be demonstrated. Serum LOX measurement is therefore not a clinically useful test for CRC.

Diffuse intestinal angiomatosis as a possible paraneoplastic manifestation of small cell lung cancer: a case of small bowel angiomatosis

Vascular malformations are rare, incompletely understood and heterogeneous in presentation and clinical course. They are known to be associated with a number of benign syndromes, commonly presenting in childhood. Angiomatosis is a form of vascular malformation, hardly documented in the English literature, and has only rarely been described in the small bowel. We present a case of a middle-aged female who developed small bowel obstruction secondary to diffuse small bowel angiomatosis and subsequently developed aggressive multifocal small cell lung cancer 2 months later. Her condition rapidly deteriorated with multiple metastases and she passed away 4 months later secondary to brain metastases and diffuse disease. Small cell lung cancer is well known for its association with paraneoplastic syndromes and has been reported to cause a rise in vascular endothelial growth factor. We postulate that in this case angiomatosis presented as a paraneoplastic syndrome associated with small cell lung cancer.

Endometriosis of the appendix: the experience of general surgeons in a large teaching hospital

Endometriosis presents frequently to the general surgeon both electively and as an emergency. One reason for this is that the symptoms from endometriosis may mimic appendicitis. Endometriosis is not closely associated with appendicitis but numerous case reports of appendiceal endometriosis exist in the literature. We reviewed all cases of appendiceal endometriosis from a large UK teaching hospital over a ten-year period to determine how this rare entity may present. Seven cases were identified and the case notes retrieved. Based on histology findings, two patients were found to have appendiceal endometriosis in association with acute appendicitis. In three cases, endometrial deposits were found in their appendix with evidence of previous endometriosis-associated inflammation. The finding of appendiceal endometriosis was incidental in a further two cases. Evidence for an association between endometriosis and appendicitis and the evidence for appendicectomy at the time of laparoscopic-diagnosis of endometriosis is discussed.

Abstract A72: VAP-1 expression is significantly reduced in colorectal cancer compared to normal colon tissue despite elevated sVAP-1 levels.

Introduction: Lymphocyte infiltration in human colorectal cancer is associated with favorable outcome, both in terms of overall survival and recurrence-free survival (1). Lymphocytes are recruited to tissues after binding to receptors on endothelium. These include classical adhesion molecules belong to the immunoglobulin superfamily but also other molecules including the ectoenzyme vascular adhesion protein-1 (VAP-1) which is implicated in lymphocyte trafficking to the gut and liver. The role of VAP-1 in lymphocyte recruitment to colorectal cancer is unknown and tissue VAP-1 expression in colorectal cancer has not been documented. The aim of this study was to determine if increased VAP-1 expression in CRC supports lymphocyte recruitment to the tumor. The infiltration of specific lymphocyte subsets in colorectal cancer has a profound effect on prognosis and VAP may provide a means of cancer-specific lymphocyte recruitment. Methods: Normal colon and colorectal cancer tissue was obtained from patients undergoing resection for colorectal cancer having given informed consent. The study was approved by the Local Research and Ethics Committee. Paraffin-embedded sections were stained for VAP-1 and CD31 and visualised using chromogenic methods or by dual-color immunofluorescence. VAP-1 gene expression in whole tumor and colon tissue was analyzed by real-time PCR. Tissue lysates were used to estimate VAP-1 protein expression by Western blotting. CD31 positive cells were isolated from fresh tissue by a preliminary immunomagnetic depletion of contaminating EpCAM+ epithelial cells followed by a positive immunomagnetic selection of CD31+ cells. Cultured CD31+ cells were then stained for VAP-1 and analyzed by flow cytometry. Results: VAP-1 gene expression is reduced in colorectal cancer tissue compared to matched colon. VAP-1 protein was detected in the colonic mucosa to a far greater extent than in matched colorectal cancer for 8 matched samples. Densitometric analysis of protein bands showed that this difference was highly statistically significant. Ten matched colon and colorectal cancer sections demonstrated a striking paucity of VAP-1 staining in cancer tissue. Positive VAP-1 staining was demonstrated in colonic tissue, specifically the muscularis muscosae and surrounding lamina proprial and submucosal vessels. Dual-colour immunofluorescence demonstrated co-localization of VAP-1 with alpha-SMA rather than CD31. Cultured endothelial cells from colon and tumor expressed CD31 but no VAP-1 by flow cytometry. Conclusion: sVAP levels have been shown to be elevated in the serum of colorectal cancer compared to healthy controls. We have demonstrated by gene expression analysis, protein detection and immunohistochemistry that little VAP-1 is expressed in colorectal cancer tissue or tumor endothelial cells. This suggests that downregulation of VAP-1 may be a mechanism of tumor escape from immune surveillance by reducing the recruitment of effector lymphocytes to the tumor. Previously reported elevated sVAP levels could be explained by increased turnover and cleaving of tissue VAP-1 in CRC by matrix-metalloproteinases -2 and -9 which are elevated in colorectal cancer. Citation Format: Stephen T. Ward, Christopher J. Weston, Emma L. Shepherd, Elizabeth A. Hepburn, Ka-kit Li, Rahul K. Hejmadi, Tariq Ismail, David H. Adams. VAP-1 expression is significantly reduced in colorectal cancer compared to normal colon tissue despite elevated sVAP-1 levels. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A72.

Abstract A73: The selective recruitment and retainment of regulatory T cells in human colorectal cancer.

Background: Colorectal cancer (CRC) is the third most common malignancy in the United States and United Kingdom and deadly in one third of patients. Tumor lymphocytic infiltration is associated with improved survival and analysis of some lymphocyte subsets has revealed that their prognostic ability rivals the TNM tumor staging system. Regulatory T cells (Tregs) are known to be enriched in CRC and it is postulated that they promote immunological tumor escape by suppression of effector T cell responses. Little is known however about the signals controlling entry of Tregs into CRC. Methods: Matched CRC, distal colonic tissue, draining lymph node and blood were obtained from patients undergoing resection of CRC having given informed consent. The study was approved by the Local Research and Ethics Committee. Tumor-infiltrating lymphocytes were isolated from fresh tissue and phenotyped for chemokine receptors and various other markers using multicolor flow cytometry. The presence of tissue chemokines was analyzed using real-time PCR and Western blotting. CD3+ cells were isolated from tumor tissue and placed in a transwell system to measure chemotaxis in response to various chemokines. Migrated and non-migrated T cells were phenotyped to establish differential migration of T cell subsets. CD4+CD127lowCD25+ T cells were isolated by fluorescent-activated cell sorting from tumor tissue and incubated with fluorescent-labeled responder cells in standard suppression assays. Peripheral blood lymphocytes were co-cultured with tumor supernatant and effects on lymphocyte phenotype and proliferation were analyzed by flow cytometry. Results: The proportion of T cells with a suppressive phenotype (Treg, CD4+CD127lowCD25+) was significantly increased in CRC compared to matched distal colon. More than 95% of this cell population expressed the transcription factor, foxp3. The chemokine receptor CCR5 was found to be markedly upregulated on Treg compared to other effector T cells and in CRC compared to distal colon. CCR4 and to a lesser extent, CCR6, were also upregulated on Treg compared to other T cells. The ligands for CCR5 (CCL3, CCL4 and CCL5) were overexpressed in CRC tissue compared to matched colon. CCL4 was found to localize to the tumor endothelium. The ligands for other chemokine receptors were not overexpressed in the tumor tissue compared to the colon and were therefore not investigated further. Tumor-resident Treg migrated in response to CCR5 ligands and blockade of CCR5 inhibited this migration. Co-culture experiments demonstrated that CCR5 was upregulated on peripheral blood lymphocytes, and especially Treg, in mixed lymphocyte reactions. This effect that could be augmented by the addition of tumor supernatant. Conclusion: Conditions exist to actively recruit CCR5+ Treg into colorectal cancer tissue in humans. CCR5 upregulation is also promoted by the tumor microenvironment that may provide a tumor-retention signal. CCR5 inhibition may prove to be a novel immunotherapy for colorectal cancer by blocking the selective recruitment and/or egress of suppressive Treg, thereby promoting an anti-tumor immune response. Citation Format: Stephen T. Ward, Elizabeth A. Hepburn, Ka-kit Li, Stuart M. Curbishley, Rahul K. Hejmadi, Tariq Ismail, Roy Bicknell, Antal Rot, David H. Adams. The selective recruitment and retainment of regulatory T cells in human colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A73.