OX-40 Protein Expression in the Sentinel Lymph Nodes of Patients With Cancer

• OX-40 expression [ Time Frame: During immunostaining ] [ Designated as safety issue: No ]
  The first ten cases will be reviewed to assess the spectrum of OX-40 expression and to devise a semiquantitative scoring system. Each slide will be scanned in its entirety and then scored according to representative views with the greatest expression of immunostaining
  
  
• Disease-free survival [ Time Frame: During data analysis ] [ Designated as safety issue: No ]
  Kaplan-Maier method will be used to estimate the disease-free survival, and a log-rank test will be used to compare the disease-free survival between high vs. low OX-40 groups
  
  

Genetic: protein expression analysis
The first ten cases will be reviewed to assess the spectrum of OX-40 expression and to devise a semiquantitative scoring system. Each slide will be scanned in its entirety and then scored according to representative views with the greatest expression of immunostaining. Two independent viewers, who will be blinded to clinical data and outcome at the time of scoring, will review all slides. Scores from these independent readings will be compared, and differences were resolved upon mutual review of given cases. We will estimate the distribution of OX-40 IHC score by tumor type and tumor stage. OX-40 IHC score will be correlated with the IHC scores of other markers using Spearman's correlation coefficient. Kruskal-Wallis test will be used to test whether OX-40 IHC score is comparable between tumor type and tumor stage
Other: immunohistochemistry staining method
5-micron sections will be cut, mounted on capillary gap slides and dried in a 60°C oven. The slides will then be then deparaffinized in xylene baths and gradually hydrated in graded alcohol. For epitope retrieval, the slides are placed in baths of 0.5 M Tris buffer at pH 10 and subjected to microwave irradiation for at least 19 minutes. The slides will be cooled and washed with deionized water and 0.05% TWEEN. They are then placed in a blocking solution of 0.3% bovine serum albumin, drained, and placed in wells of their primary antibody in a humidity chamber at room temperature overnight. Primary mouse antihuman antibodies will include anti-OX-40, anti-CD4, and anti-HLA class II. Anti-rat CD45 will be used as a negative control for each section. The slides will then be then washed and placed in their appropriate biotinylated anti-mouse secondary antibody for 24 minutes. Endogenous peroxidase in the samples is blocked using a solution of 3% hydrogen peroxide.
Other: laboratory biomarker analysis
Lymphocytes will be collected from fresh tissue and peripheral blood (PBLs) for use in marker analysis and in vitro assays of immune function. Tissues will be dissociated to obtain lymphocytes
Procedure: biopsy
De-identified tissue samples will be obtained from the OHSU Pathology Department. Tissues will consist of paraffin blocks collected either prospectively for previous cases, or fresh tissue collected prospectively for patients enrolled in the study. For either case, tissue will be collected only after tissue necessary for the adequate rendering of the pathologic diagnosis has been collected by the pathology Department. Thus, the collection of tissue will not compromise patient diagnosis/care.

OBJECTIVES:

• Determine the qualitative and quantitative expression of OX-40 and related markers in sentinel lymph nodes previously removed from patients with cancer.
• Determine the qualitative and quantitative expression of OX-40 and related markers in sentinel lymph nodes freshly removed from patients with cancer.
• Correlate the expression of OX-40 and related markers in sentinel lymph node tissue with expression in nonsentinel tissue from primary tumors and nonsentinel lymph nodes, tumor stage, and patient outcomes.

OUTLINE: Sections of primary tumors, nonsentinel lymph nodes, and sentinel lymph nodes will be analyzed for expression of OX-40 by immunohistochemistry. Sections of negative (no tumor metastases) lymph nodes will be used as internal controls. Fresh tissue and blood will be tested for marker analysis and in vitro assays of immune function.

PROJECTED ACCRUAL: A total of 100 specimens from sentinel lymph nodes previously removed from cancer patients and 100 specimens from sentinel lymph nodes freshly removed from cancer patients will be accrued for this study.