Quality assessment metrics for whole genome gene expression profiling of paraffin embedded samples.
BACKGROUND
Formalin fixed, paraffin embedded tissues are most commonly used for routine pathology analysis and for long term tissue preservation in the clinical setting. Many institutions have large archives of Formalin fixed, paraffin embedded tissues that provide a unique opportunity for understanding genomic signatures of disease. However, genome-wide expression profiling of Formalin fixed, paraffin embedded samples have been challenging due to RNA degradation. Because of the significant heterogeneity in tissue quality, normalization and analysis of these data presents particular challenges. The distribution of intensity values from archival tissues are inherently noisy and skewed due to differential sample degradation raising two primary concerns; whether a highly skewed array will unduly influence initial normalization of the data and whether outlier arrays can be reliably identified.


RESULTS
Two simple extensions of common regression diagnostic measures are introduced that measure the stress an array undergoes during normalization and how much a given array deviates from the remaining arrays post-normalization. These metrics are applied to a study involving 1618 formalin-fixed, paraffin-embedded HER2-positive breast cancer samples from the N9831 adjuvant trial processed with Illumina's cDNA-mediated Annealing Selection extension and Ligation assay.


CONCLUSIONS
Proper assessment of array quality within a research study is crucial for controlling unwanted variability in the data. The metrics proposed in this paper have direct biological interpretations and can be used to identify arrays that should either be removed from analysis all together or down-weighted to reduce their influence in downstream analyses.
Biomedical Statistics and Informatics, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA. mahoney@mayo.edu
Douglas W
DW
Mahoney
Terry M
TM
Therneau
S Keith
SK
Anderson
Jin
J
Jen
Jean-Pierre A
JP
Kocher
Monica M
MM
Reinholz
Edith A
EA
Perez
Jeanette E
JE
Eckel-Passow
D061067:Antibodies, Monoclonal, Humanized / Q000627:therapeutic use; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism; D017326:Clinical Trials, Phase III as Topic; D003520:Cyclophosphamide / Q000627:therapeutic use; D018076:DNA, Complementary / Q000378:metabolism; D004317:Doxorubicin / Q000627:therapeutic use; D005260:Female; D020869:Gene Expression Profiling; D015894:Genome, Human; D006801:Humans; D015233:Models, Statistical; D020411:Oligonucleotide Array Sequence Analysis; D017239:Paclitaxel / Q000627:therapeutic use; D016612:Paraffin Embedding / Q000379:methods*; D011786:Quality Control; D012313:RNA / Q000032:analysis*; D016032:Randomized Controlled Trials as Topic; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics*; D000068878:Trastuzumab
D061067:Antibodies, Monoclonal, Humanized; D003520:Cyclophosphamide; D018076:DNA, Complementary; D004317:Doxorubicin; D017239:Paclitaxel; D012313:RNA; D018719:Erb-b2 Receptor Tyrosine Kinases; D000068878:Trastuzumab