The presence of 18q loss of heterozygosity (LOH) predicts decreased disease-free and overall survival in stage II colon cancer: A study of CALGB Protocol 9581 M.M. Bertagnolli 1,4, D. Niedzwiecki 2, M. Hall 2, S.D. Jewell 3, R.J. Mayer 4, R.M. Goldberg 5, T.A. Colacchio 6, R. S. Warren 7, M. Redston 1 1 Brigham and Women’s Hospital, Boston, MA; 2 CALGB Statistical Center, Duke University Durham, NC; 3 CALGB Pathology Coordinating Office, Ohio State University, Columbus, OH; 4 Dana Farber Cancer Institute, Boston, MA, 5 University of North Carolina-Chapel Hill, Chapel Hill, NC; 6 Dartmouth-Hitchcock Medical Center, Dartmouth, NH; 7 University of California-San Francisco, San Francisco, CA  Overall treatment trial result: no difference in 5-year DFS or OS between the two study arms Colacchio, et al, manuscript in prep Scoring Algorithm For any marker:  If the ratio was ≥0.66 and ≤1.35, then the marker indicated that heterozygosity was present (18q intact)  If the ratio is outside this range, then the marker indicated LOH (LOH present) Case calls:  If LOH was present at any of the 5 markers, then the patient’s tumor was called LOH+ (LOH present)  The case was called s non-informative for 18q if either the non-tumor sample was not heterozygous for the marker or if DNA amplification failure occurred CALGB 9581 Tissue Bank Tumor and normal tissue obtained from CALGB Pathology Coordinating Office Pathology review, dissection to achieve optimal tumor and normal samples, then DNA extraction by standard methods Tumors without either MSI-H or loss of expression of MLH1 or MSH2 genotyped to detect 18qLOH using theD18S55 marker Multiplex PCR using fluorescent dye labeled primers; analysis using 310 Genetic Analyzer with GeneScan/Genotyper software Results  Chromosomal location 18q is a common site of loss of heterozygosity in colon cancer  18q contains several genes implicated in tumor progression including:  SMAD4 – encodes a nuclear transcription factor mediating TGF-  signaling  SMAD22 – encodes a gene associated with endodermal differentiation  DCC – a site encoding a netrin-1 receptor; loss of expression of DCC protein by immunohistochemistry was associated with poor prognosis in stage II and III colon cancers (Shibata, et al)  A retrospective analysis of tissues from randomized phase III cooperative group trials showed that 18qLOH was associated with significantly worse survival in high risk stage II and III colon cancer patients treated with 5- fluorouracil-based chemotherapy (Watanabe et al).  No studies to date have prospectively validated the utility of 18qLOH as a prognostic marker for early stage colon cancer. Background  Saltz LB, Niedzwiecki D, Hollis D, Goldberg RM, Hantel A, Thomas JP, Fields AL, Mayer RJ. Irinotecan fluorouracil plus leucovorin is not superior to fluorouracil plus leucovorin alone as adjuvant treatment for stage II colon cancer: results of CALGB J Clin Oncol 2007; 25:3456.  Shibata D, Reale MA, Lavin P, Silverman M. Fearon ER, Steele G, Jessup JM, Loda M,Summerhayes IC. The DCC protein and prognosis in colorectal cancer. N Engl J Med 1996; 335:1727.  Watanabe T, Wu TT, Catalano PJ, Ueki T, Satriano R, Haller DG, Benson AB 3 rd, Hamilton SR. Molecular patterns of survival after adjuvant chemotherapy for colon cancer. N Engl J Med 2001; 344:1196. References Study Cohort Treatment Trial Result Primary Objective: To evaluate a panel of prognostic markers in order to determine the relationship between pre- treatment tumor marker status, overall clinical outcome, and response to MoAb 17-1A Hypothesis:  Patients whose tumors demonstrate 18qLOH will have poorer study outcomes (DFS and OS) than those whose tumors do not demonstrate 18qLOH Other markers tested:  Microsatellite instability by genotyping and MLH1, MSH2 immunohistochemistry, Crohn’s-like peritumoral inflammatory cell infiltrate  The original study design called for analysis of tumor DCC expression, which was ultimately not completed due to lack of DCC antibody availability Correlative Science Protocol Conclusions D18S55 Marker N= 537 stage II colon cancers: DFS: OS: Analysis of tumor microsatellite instability status by either genotyping using Bethesda markers to detect high levels of microsatellite instability (termed MSI-H) or immunohistochemistry to detect loss of expression of MLH1 or MSH2 (termed mismatch repair protein deficient, or MMR-D) D18S55 Marker N= 537 stage II colon cancers: DFS: OS:  Preliminary results using D18S55 as a single marker support a role for 18qLOH as a prognostic marker in stage II colon cancer  The high rate of non-informative cases in this series likely results from use of a single 18q marker  Interrogation of 4 additional 18q markers, including D18S58, D18S61, D18S64, and D18S69, is underway. Tumor Analysis Method