Studying Tissue Samples From Patients With Stage II Colon Cancer Treated on Clinical Trial CLB-9581

PRIMARY OBJECTIVES:

I. To evaluate the impact of loss of p21 and mutations in p53 on the relationship between
higher intake of a Western dietary pattern (manifested by higher red meat and total fat
intake and lower n-3 polyunsaturated fatty acids, fruit and vegetable intake) and colon
cancer recurrence/mortality. (B1)

II. To evaluate the impact of mutated v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog
(K-ras), mutated p53, phorphorylated (phospho)-v-akt murine thymoma viral oncogene homolog 1
(Akt) overexpression, and p27 loss on the relationship between each of obesity, physical
activity and dietary glycemic intake and colon cancer recurrence/mortality. (B1)

III. To evaluate the impact of tumoral cyclooxygenase-2 (COX-2) and vascular endothelial
growth factor (VEGF) overexpression, high tumor microvessel density and intact p21 on the
relationship between aspirin use and colon cancer recurrence. (B1)

IV. To define the association of type and extent of genomic instability with clinical outcome
in stage III colon cancers treated with resection and adjuvant chemotherapy (CALGB 89803).
(B2)

V. To assess the ability of a prognostic gene expression signature to stratify stage II
colorectal cancer patients into those who will experience relapse within five years post
surgery (high risk) and those who will experience five-year disease free survival (low risk),
without additional treatment. (B3)

VI. To determine whether there is a significant relationship between the risk of recurrence
and the continuous 12-gene recurrence score (RS) as measured by the growth hormone
insufficiency (GHI) assay of gene expression, using the pre-specified genes and Recurrence
Score algorithm. (B4) VII. To determine whether there is a significant relationship between
the risk of recurrence and the continuous 15-gene RS2 as measured by the GHI assay of gene
expression, using the pre-specified genes and second-generation Recurrence Score algorithm.
(B4)

VIII. To assess the methylation status of mutL homolog 1 (MLH1), o-6-methylguanine-DNA
methyltransferase (MGMT), and Werner syndrome (WRN) in tumors obtained from patients enrolled
in both treatment arms of CALGB 89803. (B5)

IX. Determine the expression of DNA repair pathway proteins assessed by methylation-specific
polymerase chain reaction (MSP) assays as well as the expression of DNA repair proteins that
are known to interact with these epigenetically silenced DNA repair proteins using validated
immunostaining assays. (B5)

X. Recognition of cytosine phosphate guanine (CpG) island methylator phenotype (CIMP) genes.
(B5)

XI. To understand the relationship between tumor gene methylation status and epigenetic
silencing of DNA repair pathway genes. (B5)

XII. To examine newly identified prognostic biomarkers (long interspersed nucleotide element
1 [LINE-1] methylation, phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit
alpha [PIK3CA] mutation, v-raf murine sarcoma viral oncogene homolog B1 [BRAF] mutation,
fatty acid synthase [FASN] expression and vitamin D receptor [VDR] expression) in stage III
colon cancers (CALGB 89803) with detailed lifestyle, treatment and follow-up data and
correlate them with survival. (B6)

SECONDARY OBJECTIVES:

I. To define the association of type and extent of genomic instability with microsatellite
instability and p53 mutations, which have previously been characterized in these tumors by
Drs. Warren and Bertagnolli. (B2)

II. Associations with other markers will be determined at some future time once they have
been characterized by collaborators. (B2)

III. To determine whether the 12-gene RS provides significant information beyond clinical and
pathologic measures, including T stage, mismatch repair system (MMR) status, number of lymph
nodes examined, tumor grade, and lymphovascular invasion. (B4)

IV. To determine whether the 15-gene RS2 provides significant information beyond the clinical
and pathologic measures, including T stage, MMR status, number of lymph nodes examined, tumor
grade, and lymphovascular invasion. (B4)

V. To compare the risk of recurrence between the high and low recurrence risk groups based on
pre-specified percentile cut-points for the 12-gene RS. (B4)

VI. To compare the risk of recurrence between the high and low recurrence risk groups based
on pre-specified percentile cut-points for the 15-gene RS2. (B4)

VII. To determine, for each of a panel of selected new genes (up to 768 genes), whether there
is a significant relationship between gene expression and recurrence-free interval (RFI).
(B4)

VIII. To define the association of CIMP and methylated genes with other genetic alterations
and tumor-specific characteristics. (B5)

IX. To study the influence of diet and other lifestyle factors on cancer recurrence and
treatment-related toxicity in patients participating in this trial. (B6)

OUTLINE:

Previously collected tissue samples are analyzed for K-ras mutations; COX-2, phospho-AKT, and
VEGF overexpression; microvessel density; association of genomic instability with
microsatellite instability and p53 mutations; and methylation status of MLH1, MGMT, and WRN
and to identify prognostic biomarkers by LINE-1 hypomethylation, PIK3CA mutation, BRAF
mutation, FASN expression, and VDR expression via immunohistochemistry, polymerase chain
reaction (PCR), RT-PCR, and microarray.

Inclusion Criteria:

- Registration to CALGB 9581 or 89803

- Samples present within the CALGB Pathology Coordinating Office (PCO) or at the
institutions providing treatment that are sufficient to meet study aims

- Institutional Review Board (IRB) review and approval at the institution where the
laboratory work will be performed is required

- CALGB does not require that a separate consent form be signed for this study:

- The subject population to be studied in this protocol includes patients selected
from either of the following CALGB treatment protocols: CALGB 9581 or 89803; all
such patients have signed (or will sign) a written informed consent document
meeting all federal, state, and institutional guidelines as part of entry into
those trials

- All samples to be studied are obtained and stored as part of the patient's
respective treatment trial; the material and data obtained from the patient's
protocol record will be used to obtain appropriate clinical information; in no
instance will the patient be contacted directly

- There should be no physical, psychological, social, or legal risks associated
with this study; no invasive procedures are recommended or requested

- All appropriate and necessary procedures will be utilized to maintain
confidentiality; all patients who have had samples submitted for analysis will
have their CALGB study number used to identify specimens

- This study does not require direct patient contact and no specific risk or
benefits to individuals involved in the trial are anticipated; it is likely,
however, that the information gained will substantially help similar patients in
the future