Table 8Evidence Table. Analytic validity, MammaPrint®

Study, yearMeasureConclusions
Ach, 2007, 57Context: MammaPrint assay was used to evaluate the intra- and inter-laboratory reproducibility of the assay involving three laboratories. Variation in RNA amplification and labeling, hybridization and wash, and slide scanning was measured on 4 tumors, dye-flip design, 24 slides (8 per site).The authors showed very low influence on sample-to-reference ratios based on averaged triplicate measurements in the two-color experiments;
Methods: To assess reproducibility in this study, ANOVA P values and Pearson's correlation were used.RNA labeling was the largest contributor to inter-laboratory variation;
Results:
  • Replicate hybridizations Pearson's correlation at the same site:
Overall, despite this variation, measurement of 70-gene signature in three different laboratories was found to be highly robust;
 For 1 tumor in 1 sub-array = 0.983
 For 2 tumor in 2 sub-arrays = 0.988
 For all the other technical replicates > 0.993
  • Scanning reproducibility across sites:
 Cy3: Pearson correlation >0.995, slope = 0.97
 Cy5: less reproducible (data not shown)
  • 70-gene signature reproducibility:
 No differences between hybridization sites
 No differences between hybridization days (regardless of site)
 Statistically significant difference (P value <0.05) between labeling sites for two tumors
Buyse, 200659Context: The MammaPrint assay was used. Out of the 403 eligible samples the analysis was carried on 326 samples.
Methods: Standard MammaPrint assay protocols were used, using fresh-frozen specimens
Results:
  • 326 patients out of 403 successfully analyzed, for the following reasons:
 Poor RNA yield: 77/403 patients (19.1%)
 Successful assays: 326/403 patients (80.9%)
Glas, 200658Context: MammaPrint assay development through re-analysis of patients from the van't Veer21 and van de Vijver25 cohorts for which an RNA aliquot or the frozen specimen were available. A different reference RNA was used, as well as a different quantification method.The authors demonstrate for the first time that microarray technology can be used as a reliable diagnostic tool;
Methods:
  • 162 total samples from fresh-frozen specimens:
The MammaPrint assay performed similarly to the original 70-genes signature
 84 patients from the van de Vijver25 cohort
 All 78 patients form the van't Veer cohort21
 A combination of the two population above: 145 (84+61) LN-negative patients
 Re-analysis on 49 patients
  • Dye-swap hybridization over reference RNA from 105 sample from the van de Vijver series (balanced in terms of Good and Poor)
  • Relative gene expression quantified as the error-weighted average over triplicate spots of the log10 ratio
  • Pearson's correlation to assess correlation with original data and reproducibility
  • ANOVA analysis to model variability in repeated experiments using the 70 genes of the signature
  • Reproducibility in time was assessed by repeated measurements of RNA aliquots:
 1 patient with cosine correlation to Good profile = 0.61, for 12 months
 1 patient with cosine correlation to Good profile = -0.44, for 12 months
 1 border-line with cosine correlation to Good profile = 0.43, for 4 months
Results:
  • Comparison to original 70-gene signature data, Pearson's Correlations and in repeated measurements:
 78 van't Veer21 patients, r = 0.92, p value < 0.0001
 145 (84+61) van de Vijver25 LN-negative patients: r = 0.88 p value < .0001
 49 patients analyzed twice, r = 0.995
  • Reproducibility results from ANOVA analysis:
 No variation within individuals (p value = 0.96)
 Significant variation between individuals and genes
  • Reproducibility in time analysis results:
 For both patients assessed over a period of 12 months measurements SD was 0.028 of the cosine correlation
 For the 1 border-line sample assessed over a period of 4 months measurements SD was 0.027 of the cosine correlation
 This latter sample was miss-classified 6 times (15%)
van't Veer21Context: In this study the 70-gene signature was developed and no analytic validity data about the MammaPrint assay are reported.
van de Vijver25Context: In this study the 70-gene signature was developed and no analytic validity data about the MammaPrint assay are reported.

RNA= ribonucleic acid; ANOVA= analysis of variance; Cy3= the green fluorescent dyes commonly used in two colors design microarray hybridization; Cy5= the red fluorescent dyes commonly used in two colors design microarray hybridization; LN= lymph node; SD= standard deviation;

From: Appendix I: Evidence Tables

Cover of Impact of Gene Expression Profiling Tests on Breast Cancer Outcomes
Impact of Gene Expression Profiling Tests on Breast Cancer Outcomes.
Evidence Reports/Technology Assessments, No. 160.
Marchionni L, Wilson RF, Marinopoulos SS, et al.

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