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

Reciprocal correlation and PCA plots for GEPs obtained with DASL WG from 12 FFPE samples.A. Heat map reporting the reciprocal correlations (Pearson correlation coefficient) between GEPs derived from 12 FFPE breast cancer samples using the Illumina DASL WG platform. For each sample ER status and sample ID (to allow comparability with panel B) were also reported. B. PCA plots of GEPS of 12 FFPE breast cancer samples profiled using the Illumina DASL WG platform. Black circles represent ER+ samples and grey squares represent ER- samples. Only samples with concordant ER status by RLA and IHC were included. Sample ID is reported.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123194.g003: Reciprocal correlation and PCA plots for GEPs obtained with DASL WG from 12 FFPE samples.A. Heat map reporting the reciprocal correlations (Pearson correlation coefficient) between GEPs derived from 12 FFPE breast cancer samples using the Illumina DASL WG platform. For each sample ER status and sample ID (to allow comparability with panel B) were also reported. B. PCA plots of GEPS of 12 FFPE breast cancer samples profiled using the Illumina DASL WG platform. Black circles represent ER+ samples and grey squares represent ER- samples. Only samples with concordant ER status by RLA and IHC were included. Sample ID is reported.

Mentions: RNA aliquots derived from the same samples were in parallel processed with the DASL platform. This time, detection rates were high and fairly similar in all samples (median 65%, range 60–70%). Reciprocal correlations and PCA (Fig 3) suggested some separations in GEPS, which did not however reflect ER status. Furthermore, there were two samples whose GEPs were poorly correlated with the remaining samples, but contrary to what expected, they did not coincide with those samples rated as poor quality when processed with Affymetrix chips.


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)

Reciprocal correlation and PCA plots for GEPs obtained with DASL WG from 12 FFPE samples.A. Heat map reporting the reciprocal correlations (Pearson correlation coefficient) between GEPs derived from 12 FFPE breast cancer samples using the Illumina DASL WG platform. For each sample ER status and sample ID (to allow comparability with panel B) were also reported. B. PCA plots of GEPS of 12 FFPE breast cancer samples profiled using the Illumina DASL WG platform. Black circles represent ER+ samples and grey squares represent ER- samples. Only samples with concordant ER status by RLA and IHC were included. Sample ID is reported.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123194.g003: Reciprocal correlation and PCA plots for GEPs obtained with DASL WG from 12 FFPE samples.A. Heat map reporting the reciprocal correlations (Pearson correlation coefficient) between GEPs derived from 12 FFPE breast cancer samples using the Illumina DASL WG platform. For each sample ER status and sample ID (to allow comparability with panel B) were also reported. B. PCA plots of GEPS of 12 FFPE breast cancer samples profiled using the Illumina DASL WG platform. Black circles represent ER+ samples and grey squares represent ER- samples. Only samples with concordant ER status by RLA and IHC were included. Sample ID is reported.
Mentions: RNA aliquots derived from the same samples were in parallel processed with the DASL platform. This time, detection rates were high and fairly similar in all samples (median 65%, range 60–70%). Reciprocal correlations and PCA (Fig 3) suggested some separations in GEPS, which did not however reflect ER status. Furthermore, there were two samples whose GEPs were poorly correlated with the remaining samples, but contrary to what expected, they did not coincide with those samples rated as poor quality when processed with Affymetrix chips.

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