Hanifa Bouzourene

Exhaustion of tumor-specific CD8+ T cells in metastases from melanoma patients

In chronic viral infections, CD8⁺ T cells become functionally deficient and display multiple molecular alterations. In contrast, only little is known of self- and tumor-specific CD8⁺ T cells from mice and humans. Here we determined molecular profiles of tumor-specific CD8⁺ T cells from melanoma patients. In peripheral blood from patients vaccinated with CpG and the melanoma antigen Melan-A/MART-1 peptide, we found functional effector T cell populations, with only small but nevertheless significant differences in T cells specific for persistent herpesviruses (EBV and CMV). In contrast, Melan-A/MART-1-specific T cells isolated from metastases from patients with melanoma expressed a large variety of genes associated with T cell exhaustion. The identified exhaustion profile revealed extended molecular alterations. Our data demonstrate a remarkable coexistence of effector cells in circulation and exhausted cells in the tumor environment. Functional T cell impairment is mediated by inhibitory receptors and further molecular pathways, which represent potential targets for cancer therapy.

Methylation silencing and mutations of the p14ARF and p16INK4a genes in colon cancer.

The INK4a-ARF locus encodes two tumor suppressor proteins involved in cell-cycle regulation, p16INK4a and p14ARF, whose functions are inactivated in many human cancers. The aim of this study was to evaluate p14ARF and p16INK4a gene inactivation and its association with some clinocopathological parameters in colon cancer. The mutational and methylation status of the p14ARF and p16INK4a genes was analyzed in 60 primary colon carcinomas and 8 colon cancer cell lines. We have identified the first two reported mutations affecting exon 1β of p14ARF in the HCT116 cell line and in one of the primary colon carcinomas. Both mutations occur within the N-terminal region of p14ARF, documented as important for nucleolar localization and interaction with Mdm2. Tumor-specific methylation of the p14ARF and p16INK4a genes was found in 33% and 32% of primary colon carcinomas, respectively. Methylation of the p14ARF was inversely correlated with p53 overexpression (p = 0.02). p14ARF and p16INK4a gene methylation was significantly more frequent in right-sided than in left-sided tumors (p = 0.02). Methylation of the p14ARF gene occurred more frequently in well-differentiated adenocarcinomas (p = 0.005), whereas the p16INK4a gene was more often methylated in poorly differentiated adenocarcinomas (p = 0.002). The present results underline the role of p14ARF and p16INK4a gene inactivation in the development of colon carcinoma. They suggest that the methylation profile of specific genes, in particular p14ARF and p16INK4a, might be related to biologically distinct subsets of colon carcinomas and possibly to different tumorigenic pathways.

Intratumor genetic heterogeneity in advanced human colorectal adenocarcinoma.

Colorectal carcinogenesis is widely accepted as one of the best‐characterized examples of stepwise progression. The existing colorectal carcinogenesis model assumes genetic homogeneity of individual tumors for the main known genetic alterations: K‐ras and p53 genes point mutations and loss of heterozygosity (LOH) of chromosome 5q and 18q. The object of the present study was to demonstrate the existence of an intratumor genetic heterogeneity in advanced sporadic colorectal carcinoma for these genetic alterations. Using improved tissue microdissection and DNA extraction, for each tumor, amplifiable DNA was obtained from 15 to 20 areas, of which 1 to 2 concerned lymph node metastases (LNM). This study revealed that 10 of 15 (67%) analyzed tumors were heterogeneous for at least 1 genetic alteration, with between 2 and 6 genotypically different clones detected per tumor. No correlation was observed between the genotype of these subclones and histological differentiation or invasive propensity. Intratumor heterogeneity was more frequently observed for LOH than for point mutations, 67% and 58% for LOH at APC and DCC locus, and 20% for mutation of either the K‐ras or p53 gene. In 5 of the 9 (56%) heterogeneous cases with available LNM, the genotype observed in the LNM was different from that of the main clone in the primary tumor, and moreover, 2 of the LNM displayed a genotype undetected in the primary tumor. In conclusion, intratumor genetic heterogeneity was demonstrated in advanced sporadic colorectal carcinoma and was represented as topographically distinct genotypic subclones. Taking into account such a significant genetic heterogeneity of colorectal tumors, the use of genetic markers for prognosis management should be reconsidered.

Extended co-expression of inhibitory receptors by human CD8 T-cells depending on differentiation, antigen-specificity and anatomical localization.

Inhibitory receptors mediate CD8 T-cell hyporesponsiveness against cancer and infectious diseases. PD-1 and CTLA-4 have been extensively studied, and blocking antibodies have already shown clinical benefit for cancer patients. Only little is known on extended co-expression of inhibitory receptors and their ligands. Here we analyzed the expression of eight inhibitory receptors by tumor-antigen specific CD8 T-cells. We found that the majority of effector T-cells simultaneously expressed four or more of the inhibitory receptors BTLA, TIM-3, LAG-3, KRLG-1, 2B4, CD160, PD-1 and CTLA-4. There were major differences depending on antigen-specificity, differentiation and anatomical localization of T-cells. On the other hand, naive T-cells were only single or double positive for BTLA and TIM-3. Extended co-expression is likely relevant for effector T-cells, as we found expression of multiple ligands in metastatic lesions of melanoma patients. Together, our data suggest that naive T-cells are primarily regulated by BTLA and TIM-3, whereas effector cells interact via larger numbers of inhibitory receptors. Blocking multiple inhibitory receptors simultaneously or sequentially may improve T-cell based therapies, but further studies are necessary to clarify the role of each receptor-ligand pair.

Interleukin-17 Is a Critical Mediator of Vaccine-Induced Reduction of Helicobacter Infection in the Mouse Model

BACKGROUND & AIMS Despite the proven ability of immunization to reduce Helicobacter infection in mouse models, the precise mechanism of protection has remained elusive. This study explores the possibility that interleukin (IL)-17 plays a role in the reduction of Helicobacter infection following vaccination of wild-type animals or in spontaneous reduction of bacterial infection in IL-10-deficient mice. METHODS In mice, reducing Helicobacter infection, the levels and source of IL-17 were determined and the role of IL-17 in reduction of Helicobacter infection was probed by neutralizing antibodies. RESULTS Gastric IL-17 levels were strongly increased in mice mucosally immunized with urease plus cholera toxin and challenged with Helicobacter felis as compared with controls (654 +/- 455 and 34 +/- 84 relative units for IL-17 messenger RNA expression [P < .01] and 6.9 +/- 8.4 and 0.02 +/- 0.04 pg for IL-17 protein concentration [P < .01], respectively). Flow cytometry analysis showed that a peak of CD4(+)IL-17(+) T cells infiltrating the gastric mucosa occurred in immunized mice in contrast to control mice (4.7% +/- 0.3% and 1.4% +/- 0.3% [P < .01], respectively). Gastric mucosa-infiltrating CD4(+)IL-17(+) T cells were also observed in IL-10-deficient mice that spontaneously reduced H felis infection (4.3% +/- 2.3% and 2% +/- 0.6% [P < .01], for infected and noninfected IL-10-deficient mice, respectively). In wild-type immunized mice, intraperitoneal injection of anti-IL-17 antibodies significantly inhibited inflammation and the reduction of Helicobacter infection in comparison with control antibodies (1 of 12 mice vs 9 of 12 mice reduced Helicobacter infection [P < .01], respectively). CONCLUSIONS IL-17 plays a critical role in the immunization-induced reduction of Helicobacter infection from the gastric mucosa.

Dukes B colorectal cancer: distinct genetic categories and clinical outcome based on proximal or distal tumor location.

PURPOSE: The aim of this study was to determine whether tumor location proximal or distal to the splenic flexure is associated with distinct molecular patterns and can predict clinical outcome in a homogeneous group of patients with Dukes B (T3–T4, N0, M0) colorectal cancer. It has been hypothesized that proximal and distal colorectal cancer may arise through different pathogenetic mechanisms. Althoughp53 and Ki-ras gene mutations occur frequently in distal tumors, another form of genomic instability associated with defective DNA mismatch repair has been predominantly identified in the proximal colon. To date, however, the clinical usefulness of these molecular characteristics remains unproven. METHODS: A total of 126 patients with a lymph node-negative sporadic colon or rectum adenocarcinoma were prospectively assessed with the endpoint of death by cancer. No patient received either radiotherapy or chemotherapy. p53 protein was studied by immunohistochemistry using DO-7 monoclonal antibody, andp53 and Ki-ras gene mutations were detected by single strand conformation polymorphism assay. RESULTS: During a mean follow-up of 67 months, the overall five-year survival was 70 percent. Nuclearp53 staining was found in 57 tumors (47 percent), and was more frequent in distal than in proximal tumors (55vs. 21 percent; chi-squared test,P<0.001). For the whole group, p53 protein expression correlated with poor survival in univariate and multivariate analysis (log-rank test,P=0.01; hazard ratio=2.16; 95 percent confidence interval=1.12−4.11,P=0.02). Distal colon tumors and rectal tumors exhibited similar molecular patterns and showed no difference in clinical outcome. In comparison with distal colorectal cancer, proximal tumors were found to be statistically significantly different on the following factors: mucinous content (P=0.008), degree of histologic differentiation (P=0.012), p53 protein expression, and gene mutation (P=0.001 and 0.01 respectively). Finally, patients with proximal tumors had a marginally better survival than those with distal colon or rectal cancers (log-rank test,P=0.045). CONCLUSION: In this series of Dukes B colorectal cancers, p53 protein expression was an independent factor for survival, which also correlated with tumor location. Eighty-six percent ofp53-positive tumors were located in the distal colon and rectum. Distal colon and rectum tumors had similar molecular and clinical characteristics. In contrast, proximal neoplasms seem to represent a distinct entity, with specific histopathologic characteristics, molecular patterns, and clinical outcome. Location of the neoplasm in reference to the splenic flexure should be considered before group stratification in future trials of adjuvant chemotherapy in patients with Dukes B tumors.

Immunization of BALB/c Mice With Helicobacter Urease B Induces a T helper 2 Response Absent in Helicobacter Infection

BACKGROUND & AIMS Infection with Helicobacter induces a T helper type 1 response in mice and humans. Mice can be cured or protected from infection with Helicobacter by mucosal immunization with recombinant H. pylori urease B subunit (rUreB). This study characterizes the immune response of infected mice immunized with rUreB. METHODS BALB/c mice were infected with H. felis. Two weeks later, they were orally immunized four times with rUreB and cholera toxin (CT) at weekly intervals. Controls were only infected or sham-immunized with CT. Animals were killed at various times after immunization. Splenic CD4(+) cells were obtained and cultured in vitro with rUreB to evaluate antigen-specific proliferation and induction of interferon gamma and interleukin 4 secretion. RESULTS All rUreB-immunized mice (n = 8) were cured from infection 3 weeks after the fourth immunization. Immunization induced a proliferative response of splenic CD4(+) cells, a progressive decrease in interferon gamma secretion, and a concomitant increase in interleukin 4 secretion after each immunization. A simultaneous increase in rUreB specific serum immunoglobulin G1 levels was observed in infected/immunized mice. CONCLUSIONS In BALB/c mice, therapeutic mucosal immunization with rUreB induces progressively a Th2 CD4(+) T cell response resulting in the elimination of the pathogen.

Naturally Acquired MAGE-A10- and SSX-2-Specific CD8+ T Cell Responses in Patients with Hepatocellular Carcinoma

Immunotherapy is being proposed to treat patients with hepatocellular carcinoma (HCC). However, more detailed knowledge on tumor Ag expression and specific immune cells is required for the preparation of highly targeted vaccines. HCC express a variety of tumor-specific Ags, raising the question whether CTL specific for such Ags exist in HCC patients. Indeed, a recent study revealed CTLs specific for two cancer-testis (CT) Ags (MAGE-A1 and MAGE-A3) in tumor infiltrating lymphocytes of HCC patients. Here we assessed the presence of T cells specific for additional CT Ags: MAGE-A10, SSX-2, NY-ESO-1, and LAGE-1, which are naturally immunogenic as demonstrated in HLA-A2+ melanoma patients. In two of six HLA-A2+ HCC patients, we found that MAGE-A10- and/or SSX-2-specific CD8+ T cells naturally responded to the disease, because they were enriched in tumor lesions but not in nontumoral liver. Isolated T cells specifically and strongly killed tumor cells in vitro, providing evidence that these CTL were selected in vivo for high avidity Ag recognition. Therefore, besides melanoma, HCC is the second solid human tumor with clear evidence for in vivo tumor recognition by T cells, providing the rational for specific immunotherapy, based on immunization with CT Ags such as MAGE-A10 and SSX-2.

Inflammatory Gene Profiles in Gastric Mucosa during Helicobacter pylori Infection in Humans

Helicobacter pylori infection is associated with an inflammatory response in the gastric mucosa, ultimately leading to cellular hyperproliferation and malignant transformation. Hitherto, only expression of a single gene, or a limited number of genes, has been investigated in infected patients. cDNA arrays were therefore used to establish the global pattern of gene expression in gastric tissue of healthy subjects and of H. pylori-infected patients. Two main gene expression profiles were identified based on cluster analysis. The data obtained suggest a strong involvement of selected Toll-like receptors, adhesion molecules, chemokines, and ILs in the mucosal response. This pattern is clearly different from that observed using gastric epithelial cell lines infected in vitro with H. pylori. The presence of a “Helicobacter-infection signature,” i.e., a set of genes that are up-regulated in biopsies from H. pylori-infected patients, could be derived from this analysis. The genotype of the bacteria (presence of genes encoding cytotoxin-associated Ag, vacuolating cytotoxin, and blood group Ag-binding adhesin) was analyzed by PCR and shown to be associated with differential expression of a subset of genes, but not the general gene expression pattern. The expression data of the array hybridization was confirmed by quantitative real-time PCR assays. Future studies may help identify gene expression patterns predictive of complications of the infection.

Aberrant crypt foci in patients with neoplastic and nonneoplastic colonic disease

Aberrant crypt foci (ACF) are putative preneoplastic lesions that might represent the earliest morphological lesion visible in colonic carcinogenesis. However, findings concerning the growth and morphological features of these lesions in human studies suggest that ACF are highly heterogeneous in nature. In this study, we evaluated the morphological features of a large number of ACF in colon mucosa of 26 patients with colorectal carcinoma (CRC), four patients with adenoma as well as seven patients with nonneoplastic colonic diseases. By dissecting microscope, 508 ACF were identified, and of these, 378 were sampled for histological examination. The median ACF density (number of ACF/cm2) was significantly higher in the left colon than in the right colon (0.047 v 0.014 ACF/cm2). Unexpectedly, in our series, the overall ACF density was higher in the nonneoplastic colonic diseases than in CRC (0.13 v 0.032 ACF/cm2, P=.0087), cases of nonneoplastic diseases, however, being limited to 7 patients. ACF were significantly larger in colons with CRC or adenoma than in colons with nonneoplastic disease (P < .03). On histological examination, we observed 133 ACF with normal epithelium, 189 ACF with hyperplasia, 27 ACF with atypical hyperplasia, and 29 ACF with dysplasia. We noted a progressive increase of median ACF size from normal mucosa to hyperplasia, atypical hyperplasia, and dysplasia. Dysplastic ACF were more frequently observed in patients with CRC or adenoma and showed predominantly elongated crypt orifices (P < .0001). We conclude that ACF are histologically heterogeneous, encompass a spectrum of lesions of which only a subset are associated with dysplasia and then represent an early step in colorectal carcinogenesis. ACF with dysplasia are characterized by larger size, elongated crypt orifices, and an association with CRC.