Limits...
Use of formalin-fixed paraffin-embedded samples for gene expression studies in breast cancer patients.

Musella V, Callari M, Di Buduo E, Scuro M, Dugo M, Miodini P, Bianchini G, Paolini B, Gianni L, Daidone MG, Cappelletti V - PLoS ONE (2015)

Bottom Line: With the use of Affymetrix HG-U133 2.0 Plus chips on fixed samples and a quantitative polymerase chain reaction -based sample pre-assessment step, results were satisfactory in terms of biological reliability, despite the low number of present calls (M = 21%±5).Compared with the Illumina DASL WG platform, Affymetrix data showed a wider interquartile range (1.32 vs 0.57, P<2.2 E-16,) and larger fold changes.By including in the workflow the sample pre-assessment steps, 96% of the samples predicted to give good results (44/46), were in fact rated as satisfactory from the point of view of technical and biological meaningfulness.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.

ABSTRACT
To obtain gene expression profiles from samples collected in clinical trials, we conducted a pilot study to assess feasibility and estimate sample attrition rates when profiling formalin-fixed, paraffin-embedded specimens. Ten matched fresh-frozen and fixed breast cancer samples were profiled using the Illumina HT-12 and Ref-8 chips, respectively. The profiles obtained with Ref 8, were neither technically nor biologically reliable since they failed to yield the expected separation between estrogen receptor positive and negative samples. With the use of Affymetrix HG-U133 2.0 Plus chips on fixed samples and a quantitative polymerase chain reaction -based sample pre-assessment step, results were satisfactory in terms of biological reliability, despite the low number of present calls (M = 21%±5). Compared with the Illumina DASL WG platform, Affymetrix data showed a wider interquartile range (1.32 vs 0.57, P<2.2 E-16,) and larger fold changes. The Affymetrix chips were used to run a pilot study on 60 fixed breast cancers. By including in the workflow the sample pre-assessment steps, 96% of the samples predicted to give good results (44/46), were in fact rated as satisfactory from the point of view of technical and biological meaningfulness. Our gene expression profiles showed strong agreement with immunohistochemistry data, were able to reproduce breast cancer molecular subtypes, and allowed the validation of an estrogen receptor status classifier derived in frozen samples. The approach is therefore suitable to profile formalin-fixed paraffin-embedded samples collected in clinical trials, provided that quality controls are run both before (sample pre-assessment) and after hybridization on the array.

No MeSH data available.


Related in: MedlinePlus

Comparison of reliability of GEPs obtained with Affymetrix and DASL WG platforms in FFPE samples.A. Distribution plots of interquartile ranges for GEPs obtained from 12 FFPE breast cancer samples using the Affymetrix HG-U133 2.0 Plus chips (left panel) or the Illumina DASL WG platform (right panel). B. Log2 expression intensity levels for ESR1in 12 breast cancer FFPE samples obtained respectively using Affymetrix HG-U133 2.0 Plus chips (left panel, ESR1 probeset ID “205225_at”) or the Illumina DASL WG platform (right panel, ESR1 probe ID “3360095”), from the lowest to the highest. Black circles and grey squares dot represent respectively ER+ and ER- negative samples, as determined by IHC. C. Distribution plots of log2-trasnformed fold change values obtained by Class Comparison between 6 ER+ and 6 ER- FF breast cancer samples profiled with Affymetrix HG-U133 2.0 Plus chips (left panel) or with the Illumina DASL WG platform (right panel).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4386823&req=5

pone.0123194.g004: Comparison of reliability of GEPs obtained with Affymetrix and DASL WG platforms in FFPE samples.A. Distribution plots of interquartile ranges for GEPs obtained from 12 FFPE breast cancer samples using the Affymetrix HG-U133 2.0 Plus chips (left panel) or the Illumina DASL WG platform (right panel). B. Log2 expression intensity levels for ESR1in 12 breast cancer FFPE samples obtained respectively using Affymetrix HG-U133 2.0 Plus chips (left panel, ESR1 probeset ID “205225_at”) or the Illumina DASL WG platform (right panel, ESR1 probe ID “3360095”), from the lowest to the highest. Black circles and grey squares dot represent respectively ER+ and ER- negative samples, as determined by IHC. C. Distribution plots of log2-trasnformed fold change values obtained by Class Comparison between 6 ER+ and 6 ER- FF breast cancer samples profiled with Affymetrix HG-U133 2.0 Plus chips (left panel) or with the Illumina DASL WG platform (right panel).

Mentions: GEPs obtained from DASL showed a lower variability in probe signals (median IQR = 0.57 vs median IQR = 1.32, P<2.2E-18, Wilcoxon test, Fig 4A) which was also confirmed looking at the ESR1 gene alone (Fig 4B) with DASL-derived ESR1 levels (defined using the address ID “3360095”) compressed in a narrower intensity interval compared to Affymetrix ESR1 data and with a poor distinction (prediction) of ER classes (Fig 4B). Fold changes for all genes according to ER status were definitely higher in the Affymetrix data set, where a higher number of genes presented a fold change >2 or <0.5 (Fig 4C).


Use of formalin-fixed paraffin-embedded samples for gene expression studies in breast cancer patients.

Musella V, Callari M, Di Buduo E, Scuro M, Dugo M, Miodini P, Bianchini G, Paolini B, Gianni L, Daidone MG, Cappelletti V - PLoS ONE (2015)

Comparison of reliability of GEPs obtained with Affymetrix and DASL WG platforms in FFPE samples.A. Distribution plots of interquartile ranges for GEPs obtained from 12 FFPE breast cancer samples using the Affymetrix HG-U133 2.0 Plus chips (left panel) or the Illumina DASL WG platform (right panel). B. Log2 expression intensity levels for ESR1in 12 breast cancer FFPE samples obtained respectively using Affymetrix HG-U133 2.0 Plus chips (left panel, ESR1 probeset ID “205225_at”) or the Illumina DASL WG platform (right panel, ESR1 probe ID “3360095”), from the lowest to the highest. Black circles and grey squares dot represent respectively ER+ and ER- negative samples, as determined by IHC. C. Distribution plots of log2-trasnformed fold change values obtained by Class Comparison between 6 ER+ and 6 ER- FF breast cancer samples profiled with Affymetrix HG-U133 2.0 Plus chips (left panel) or with the Illumina DASL WG platform (right panel).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4386823&req=5

pone.0123194.g004: Comparison of reliability of GEPs obtained with Affymetrix and DASL WG platforms in FFPE samples.A. Distribution plots of interquartile ranges for GEPs obtained from 12 FFPE breast cancer samples using the Affymetrix HG-U133 2.0 Plus chips (left panel) or the Illumina DASL WG platform (right panel). B. Log2 expression intensity levels for ESR1in 12 breast cancer FFPE samples obtained respectively using Affymetrix HG-U133 2.0 Plus chips (left panel, ESR1 probeset ID “205225_at”) or the Illumina DASL WG platform (right panel, ESR1 probe ID “3360095”), from the lowest to the highest. Black circles and grey squares dot represent respectively ER+ and ER- negative samples, as determined by IHC. C. Distribution plots of log2-trasnformed fold change values obtained by Class Comparison between 6 ER+ and 6 ER- FF breast cancer samples profiled with Affymetrix HG-U133 2.0 Plus chips (left panel) or with the Illumina DASL WG platform (right panel).
Mentions: GEPs obtained from DASL showed a lower variability in probe signals (median IQR = 0.57 vs median IQR = 1.32, P<2.2E-18, Wilcoxon test, Fig 4A) which was also confirmed looking at the ESR1 gene alone (Fig 4B) with DASL-derived ESR1 levels (defined using the address ID “3360095”) compressed in a narrower intensity interval compared to Affymetrix ESR1 data and with a poor distinction (prediction) of ER classes (Fig 4B). Fold changes for all genes according to ER status were definitely higher in the Affymetrix data set, where a higher number of genes presented a fold change >2 or <0.5 (Fig 4C).

Bottom Line: With the use of Affymetrix HG-U133 2.0 Plus chips on fixed samples and a quantitative polymerase chain reaction -based sample pre-assessment step, results were satisfactory in terms of biological reliability, despite the low number of present calls (M = 21%±5).Compared with the Illumina DASL WG platform, Affymetrix data showed a wider interquartile range (1.32 vs 0.57, P<2.2 E-16,) and larger fold changes.By including in the workflow the sample pre-assessment steps, 96% of the samples predicted to give good results (44/46), were in fact rated as satisfactory from the point of view of technical and biological meaningfulness.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.

ABSTRACT
To obtain gene expression profiles from samples collected in clinical trials, we conducted a pilot study to assess feasibility and estimate sample attrition rates when profiling formalin-fixed, paraffin-embedded specimens. Ten matched fresh-frozen and fixed breast cancer samples were profiled using the Illumina HT-12 and Ref-8 chips, respectively. The profiles obtained with Ref 8, were neither technically nor biologically reliable since they failed to yield the expected separation between estrogen receptor positive and negative samples. With the use of Affymetrix HG-U133 2.0 Plus chips on fixed samples and a quantitative polymerase chain reaction -based sample pre-assessment step, results were satisfactory in terms of biological reliability, despite the low number of present calls (M = 21%±5). Compared with the Illumina DASL WG platform, Affymetrix data showed a wider interquartile range (1.32 vs 0.57, P<2.2 E-16,) and larger fold changes. The Affymetrix chips were used to run a pilot study on 60 fixed breast cancers. By including in the workflow the sample pre-assessment steps, 96% of the samples predicted to give good results (44/46), were in fact rated as satisfactory from the point of view of technical and biological meaningfulness. Our gene expression profiles showed strong agreement with immunohistochemistry data, were able to reproduce breast cancer molecular subtypes, and allowed the validation of an estrogen receptor status classifier derived in frozen samples. The approach is therefore suitable to profile formalin-fixed paraffin-embedded samples collected in clinical trials, provided that quality controls are run both before (sample pre-assessment) and after hybridization on the array.

No MeSH data available.


Related in: MedlinePlus