Structure of study
This is a two-group, prospective, case-control study. First group shall comprise of newly diagnosed cancer patients, for whom treatment has not been initiated. The second group shall consist of subjects from normal population. Fasting blood sugar (FBS) shall be estimated for all subjects in both the groups. This is to rule out undiagnosed diabetes mellitus. A FBS level above 126 mg% is diagnostic of diabetes [21]. Fasting insulin level and IGF-1 level will be assayed in the same blood sample for all subjects.
Sample size
Based on an unpublished pilot study results (conducted on 63 patients) 761 patients and 191 controls are required to expect a study power of 80 %. We plan to recruit 800 patients and 200 controls. Controls would be age and sex matched to that of the patients.
Data and Sample collection
All subjects will be recruited from out patient division of M.S. Ramaiah Memorial Hospital, Bangalore, India and Cancer Institute, Chennai, India. Patients who are below age of 50, with carcinomas of breast, GIT, liver, prostate, uterus, ovaries and cervix shall be recruited. The exclusion criteria are; patients who have undergone treatment for cancer. Therapies for cancer including surgery and chemotherapy alter IGF-1 levels, growth factors and immune mediators. Patients on palliative therapy due to very advanced cancer and malnourished patients will also be excluded as these patients will have low IGF-1 levels due to cachexia [20]. Malnourishment will be assessed by Subjective Global Assessment (SGA) method [22]. Patients with endocrine related diseases, multiple endocrine neoplasia syndrome will also be excluded as IGF-1 is a secondary hormone and can be influenced by major hormone imbalances. Patients with diabetes mellitus (Type 1 and 2) will be excluded as such patients can have low IGF-1 levels due to altered nourishment. Patients with chronic liver diseases other than cancer will also be excluded as liver is a major producer of IGF-1. All patients with proven tobacco related cancers will be excluded.
Group 2 consists of subjects from normal population. Subjects without any chronic or acute diseases will be considered. Subjects under treatment for any medical diseases, subjects who are malnourished or subjects who have family history of cancer or subjects who have body mass index greater than 25 will be excluded. All the above factors affect IFG-1 levels.
After obtaining an informed consent from subjects of both groups, a detailed, relevant clinical history will be documented. A 5 ml fasting venous blood sample (by standard venous puncture) from all the recruited patients will be drawn. Free IGF-1, fasting blood sugar and fasting Insulin assays will be estimated on all samples.
Laboratory Assays
8 hour fasting plasma blood sugar will be done by glucose oxidase – peroxidase (GOD-POD) method. Fasting insulin will be assayed by (Radioimmunoassay) RIA method using commercially available insulin RIA kit supplied by Diagnostic Systems Laboratories Inc., Texas. IGF-1 will be assayed by Immunoradiometric assay (IRMA) method using a commercially available IRMA kit supplied by the same company.
Analysis
Homeostasis model assessment-insulin resistance (HOMA-IR) method will be used to calculate insulin resistance/sensitivity [23]. The same will be associated with IGF-1 levels using various statistical tools as mentioned below.
Data Management
All data elements recorded during the study period will be entered and validated in Microsoft Excel. All variables and changes will be transferred into the Satastical Package for Social Sciences (SPSS), SYSTAT™ statistical software, summarized (Counts, Minimum, Maximum, Mean, Median, Standard Deviation) within each treatment group, transferred to the statistical report and then reformatted. Suitable graphs will be generated to depict the changes in key efficacy parameters and then transferred to the statistical report.
Statistics
Descriptive Statistics
Descriptive statistics for each numerical variable will be summarized as the frequency, percentage, mean, median and standard deviations for all the subjects at each time interval in each study group.
Statistical methods
Chi-square test and Fisher exact tests will be used to analyze the frequency data, Student't' test (two-tailed) will be used to test the significance of study parameters between case and controls. Analysis of variance/Kruskal Wallis test will be used for within group analysis. Any other suitable statistical techniques will be used to find the effect of study parameters.
Control of Type -1 Error
All statistical tests will be conducted at the 0.05 alpha levels, meaning that P ≤ 0.05 will be considered "nominally significant". The Adjustment for multiple tests will be done by applying Bonferroni correction to the p-values. The p-value for the comparison of the primary efficacy endpoints between Cases and Control group will be considered to be conclusive.
Software
The Statistical software namely SPSS 11.0 and Systat 8.0 will be used for the analysis of the data and Microsoft word and Excel shall be used to generate graphs and tables.
Sub-Group Analysis
A similar analysis will be carried out on organ-specific cancers like breast, colon and rectum, depending on number of patients recruited.
Ethical Issues
The study has been approved by Ethical Review Boards of all centers involved in the study. (Approval Ref – MSRMC/ERB/2007/06-01 dated 12th June 2007).
Informed Consent Process
The investigators shall provide to the subjects, all the study related information in a language desired by the subject and at a level of complexity that is understandable to the subjects and the guardians. Prior to the subjects' participation in the study, the written informed consent form shall be signed and dated personally by the subject, the guardian, the investigator and impartial witness.
Interim Analysis
Two interim analyses shall be carried out. First analysis will be done once 30 % of patients are recruited. The second analysis will be done once 60 % of them are recruited. Recruitment will stop if the study attains positive significance at end of interim analysis.