BACKGROUND & AIMS Staging inadequately predicts metastatic risk in patients with colon cancer. We used a gene expression profile derived from invasive, murine colon cancer cells that were highly metastatic in an immunocompetent mouse model to identify patients with colon cancer at risk of recurrence. METHODS This phase 1, exploratory biomarker study used 55 patients with colorectal cancer from Vanderbilt Medical Center (VMC) as the training dataset and 177 patients from the Moffitt Cancer Center as the independent dataset. The metastasis-associated gene expression profile developed from the mouse model was refined with comparative functional genomics in the VMC gene expression profiles to identify a 34-gene classifier associated with high risk of metastasis and death from colon cancer. A metastasis score derived from the biologically based classifier was tested in the Moffitt dataset. RESULTS A high score was significantly associated with increased risk of metastasis and death from colon cancer across all pathologic stages and specifically in stage II and stage III patients. The metastasis score was shown to independently predict risk of cancer recurrence and death in univariate and multivariate models. For example, among stage III patients, a high score translated to increased relative risk of cancer recurrence (hazard ratio, 4.7; 95% confidence interval, 1.566–14.05). Furthermore, the metastasis score identified patients with stage III disease whose 5-year recurrence-free survival was >88% and for whom adjuvant chemotherapy did not increase survival time. CONCLUSION A gene expression profile identified from an experimental model of colon cancer metastasis predicted cancer recurrence and death, independently of conventional measures, in patients with colon cancer.

The Wilms' tumor gene WT1 is overexpressed in leukemias and various types of solid tumors, and the WT1 protein was demonstrated to be an attractive target antigen for immunotherapy against these malignancies. Here, we report the outcome of a phase I clinical study of WT1 peptide-based immunotherapy for patients with breast or lung cancer, myelodysplastic syndrome, or acute myeloid leukemia. The WT1 gene was isolated as a gene responsible for Wilms' tumor, a pediatric renal cancer, and encodes a zinc finger transcription factor, which is involved in cell proliferation and differentiation, apoptosis, and organ development (3-6). Although the WT1 gene was first categorized as a tumor suppressor gene, we have proposed that the wild-type WT1 gene functions as an oncogene rather than a tumor-suppressor gene on the basis of the following findings. The first is high expression of the wild-type WT1 gene in both leukemias and solid tumors (7-18), the second is growth inhibition of leukemic and solid tumor cells by treatment with WT1 antisense oligomers (14,19), and the third is block of differentiation, but induction of proliferation, of wild-type WT1 gene-transfected myeloid progenitor cells in response to granulocyte colony-stimulating factor (20, 21). The last two are block of thymocyte differentiation but induction of thymocyte proliferation in the transgenic mice with the lck promoter-driven WT1 gene (22), and WT1 gene expression in the majority of dimethylbenzanthracene-induced erythroblastic leukemia and a stronger tendency of the cells with high levels of WT1 to develop into leukemias (23).Expression of the wild-type WT1 gene has been found in most cases of acute myelocytic leukemia (AML), acute lymphocytic leukemia, chronic myelocytic leukemia, and myelodysplastic syndrome (MDS) at higher levels than those in normal bone marrow (BM) or peripheral blood (7-13). Furthermore, various types of solid tumors, including lung, breast, thyroid, and colorectal cancers, expressed the wild-type WT1 gene at higher levels compared to those in corresponding normal tissues (15-18). These results indicated that the wild-type WT1 gene product may be a promising target for cancer immunotherapy (24,25).We tested the potential of the WT1 gene product to serve as a target antigen for tumor-specific immunotherapy. Human WT1-specific CTLs have been found to induce lysis of endogenously WT1-expressing tumor cells in vitro, but not to cause damage to physiologically WT1-expressing normal cells (24,(26)(27)(28). We used a mouse in vivo system to demonstrate that immunization of mice with either MHC class I-restricted WT1 peptide or WT1 cDNA induced WT1-specific CTLs. We also showed that the immunized mice rejected challenges of WT1-expressing tumor cells, whereas the induced CTLs did not affect normal healthy tissues that physiologically expressed WT1 nor damaged the normal tissues (25, 29). These results indicated that the WT1 protein could be a novel tumor rejection antigen for cancer immunotherapy (24)(25)(26)(27)(28)(29)(30)(31)(32).In...

The Wilms' tumor gene WT1 is overexpressed in most types of leukemias and various kinds of solid tumors, including lung and breast cancer, and participates in leukemogenesis and tumorigenesis. WT1 protein has been reported to be a promising tumor antigen in mouse and human. In the present study, a single amino-acid substitution, M-->Y, was introduced into the first anchor motif at position 2 of the natural immunogenic HLA-A*2402-restricted 9-mer WT1 peptide (CMTWNQMNL; a.a. 235-243). This substitution increased the binding affinity of the 9-mer WT1 peptide to HLA-A*2402 molecules from 1.82 x 10(-5) to 6.40 x 10(-7) M. As expected from the increased binding affinity, the modified 9-mer WT1 peptide (CYTWNQMNL) elicited WT1-specific cytotoxic T lymphocytes (CTL) more effectively than the natural 9-mer WT1 peptide from peripheral blood mononuclear cells (PBMC) of HLA-A*2402-positive healthy volunteers. CTL induced by the modified 9-mer WT1 peptide killed the natural 9-mer WT1 peptide-pulsed CIR-A*2402 cells, primary leukemia cells with endogenous WT1 expression and lung cancer cell lines in a WT1-specific HLA-A*2402-restricted manner. These results showed that this modified 9-mer WT1 peptide was more immunogenic for the induction of WT1-specific CTL than the natural 9-mer WT1 peptide, and that CTL induced by the modified 9-mer WT1 peptide could effectively recognize and kill tumor cells with endogenous WT1 expression. Therefore, cancer immunotherapy using this modified 9-mer WT1 peptide should provide efficacious treatment for HLA-A*2402-positive patients with leukemias and solid tumors.

IntroductionWilms tumor gene, WT1, is responsible for the tumorigenesis of a childhood renal neoplasm, Wilms tumor, which is thought to arise as a result of the inactivation of both alleles of the WT1 gene. 1,2 The WT1 gene has been considered a tumor-suppressor gene on the basis of findings such as intragenic deletions or mutations in Wilms tumor, germline mutations in patients with leukemia predisposition syndromes, and WT1-mediated growth suppression of Wilms tumor cells. [3][4][5][6][7] This gene encodes a zinc finger transcription factor involved in tissue development, in cell proliferation and differentiation, and in apoptosis. 8 The WT1 gene product represses the transcription of growth factor (platelet-derived growth factor ␣ chain, 9 colony-stimulating factor-1, 10 and insulinlike growth factor-II [IGF-II] 11 ) and growth factor receptor genes (IGF-IR 12 and EGFR 13 ), and the other genes (RAR-␣, 14 c-myb, 15 c-myc, 16 bcl-2, 16 ornithine decarboxylase, 17 and N-myc 18 ), whereas it activates the transcription of some genes (retinoblastoma suppressor-associated protein 46,20 and bcl-2 21 ). Unlike tumor-suppressor genes such as Rb and p53 that are ubiquitously expressed, WT1 gene expression is restricted to a limited set of tissues, including gonads, uterus, kidney, mesothelium, and hematopoietic progenitors. [22][23][24] WT1 knock-out mice have been shown to have defects in the urogenital system and to die at embryonic day 13.5, probably because of heart failure. 25 The WT1 gene was originally defined as a tumor-suppressor gene, as mentioned earlier. However, we recently proposed that the wild-type WT1 gene performs an oncogenic rather than a tumorsuppressor function in leukemogenesis and tumorigenesis in various types of solid tumors on the basis of the following findings: (1) high expression of the wild-type WT1 gene in leukemias [26][27][28][29][30][31] and various types of solid tumors, including ovarian tumors, Leydig cell tumors, mesothelioma, gastric cancer, colon cancer, lung cancer, and breast cancer 23,[32][33][34][35][36][37][38][39][40][41][42][43] ; (2) growth inhibition of leukemic 44,45 and solid tumor cells 41 by treatment with WT1 antisense oligomers; (3) promotion of cell growth, but blocking of cell differentiation, in the myeloid progenitor cell line 32D 46 and in normal bone marrow myeloid cells 47 as a result of constitutive WT1 gene expression caused by transfection with the wild-type WT1 gene; and (4) WT1 expression detected in most 7,12-dimethylbenzanthracene-induced erythroblastic leukemias and a tendency for cells with high levels of WT1 expression to develop into leukemias. 48 Stimulation in vitro of HLA-A2.1-positive or -A24.2-positive peripheral blood mononuclear cells with 9-mer WT1 peptides containing major histocompatibility complex (MHC) class 1 binding anchor motifs elicited WT1-specific cytotoxic T lymphocytes (CTLs). [49][50][51] These CTLs specifically killed WT1-expressing tumor cells in an HLA class 1-restricted manner and inhibited colony (1) cancer-testis ant...