Timothy H. W. Chan, Calum MacAulay, Wan Lam, Stephen Lam, Kim Lonergan, Steven Jones, Marco Marra, Raymond T. Ng

1
Using the Permutation Test to Analyze Lung Cancer
SAGE Libraries
Timothy H. W. Chan, Calum MacAulay, Wan Lam,
Stephen Lam, Kim Lonergan, Steven Jones, Marco
Marra, Raymond T. Ng Department of Computer
Science, University of British Columbia The
British Columbia Cancer Research Centre
BACKGROUND
RESULTS
Permutation Test

• Previously analyzed publicly available Breast
  and Brain SAGE libraries using the permutation
  test (Ng. et al, Frontiers of Cardiovascular
  Science 2003) and had some success (60 of top
  ranked genes for breast SAGE data were verified
  to be related to the neoplastic process).
• BC Cancer Research Centre has produced various
  Lung Cancer SAGE libraries including 5 CIS
  (carcinoma in situ), 6 Invasive and 17 Normal
  libraries.
• It would be interesting to use the permutation
  test to contrast and compare the various stages
  of lung cancer and search for small
  transcriptional changes (pathway regulators,
  check points, switches).

• 1981 out 32,871 TAGS considered at 99
  confidence failed the permutation test for Normal
  vs Invasive Lung Cancer.
• 1887 TAGS out of 40,476 TAGS considered at 99
  confidence failed the permutation test for Normal
  vs CIS Lung Cancer
• 119 TAGS out of 20,077 TAGS considered failed the
  permutation test for CIS vs Invasive Lung Cancer

Verification Results
OBJECTIVES
   
   
 

• To use the permutation test on normal and
  different stages of lung cancer (CIS and
  Invasive) SAGE libraries to discover candidate
  cancer-related genes.
• To contrast and compare these two stages of lung
  cancer.
• To demonstrate the advantages and power the
  permutation test holds over the T-test.

METHODOLGY
Data Pre-Processing

• Quality of these genes is mostly dependent on
  criteria A and B. Following closely are criteria
  C and D as they are important genes in the
  neoplastic process
• Hypotheticals or genes who have no known
  function did not meet any of the criteria.
• Indicates that there exists a duplicate (more
  than one TAG match to the same gene).

99 confidence - Output
1. Gene-to-Tag Assignment

• Some tags map to more than one gene. To deal with
  this, the expression level of the tag is assigned
  to each gene the tag maps to. For instance, if
  tag A maps to genes 1, 2, and 3, all the genes
  will be assigned the tag count of tag A.

Intersections of Top Ranked Genes Between the
Inv. vs Norm. and CIS vs Norm. Results

• The null hypothesis states that there is no
  difference between the mean of the normal and the
  cancer sample. If this were the case, it would
  make no difference if we mix up the labels of
  the libraries.
• The alternative hypothesis states that it does
  make a difference and the mean of the normal and
  cancer sample are different.

2. Normalizing the Libraries

• The low intersections suggest that CIS and
  Invasive stages of cancer are different.

• To reduce comparison errors, the tag frequencies
  are normalized by scaling each library up to
  300,000.

Scoring and Ranking Genes
Power of The Permutation Test

• Higher permutation scores correspond to either
  greater differences between the two samples or
  greater differential consistencies between the
  two samples.
• For each tissue and significant genes, rank the
  genes by sorting the permutation scores in
  descending order.

CONCLUSION

• With the permutation test, the number of samples
  required for the test to be acceptable is
  relatively low compared to other statistical
  tests (ie. T-test, chi-square).

• The permutation test is great at picking out
  genes that are related to the neoplastic process.
  
• It is also much better at picking out these genes
  than the T-test.
• The permutation test between Invasive and CIS
  show that there are 119 Tags that are
  differentially expressed which suggests that the
  two stages of cancer have different genes turned
  on or off. In addition, the intersections
  between the top ranked genes between Normal vs
  Invasive And Normal vs CIS are quite low (top 200
  only 25 of the Tags intersect) which also
  suggest differences between the 2 stages.

Literature Verification

• An investigation is conducted on the top ranked
  genes for cancer-relation using the currently
  available literatures on PubMed.

Verification Criteria
FUTURE DIRECTIONS

• Continue to use the permutation test to analyze
  other SAGE libraries.
• The permutation also has the power to detect
  small transcriptional changes as long as the gene
  across all the libraries have a consistent Tag
  count. Further analysis of these low TAG count
  significant genes (with high permutation scores)
  is required as they could be vital pathway
  regulators, checkpoints or switches that may have
  led to the onset of lung cancer.
• Validate genes further by experimentation.
• Use validated genes for early cancer detection or
  derive new treatments from data.