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Acquisition of biologically relevant gene expression data by Affymetrix microarray analysis of archival formalin-fixed paraffin-embedded tumours.

Linton KM, Hey Y, Saunders E, Jeziorska M, Denton J, Wilson CL, Swindell R, Dibben S, Miller CJ, Pepper SD, Radford JA, Freemont AJ - Br. J. Cancer (2008)

Bottom Line: Arrays from two paired frozen and FFPET samples were compared: although FFPET sensitivity was low ( approximately 50%), high specificity (95%) and positive predictive value (92%) suggest that transcript detection is reliable.RT-PCR and immunohistochemistry on independent cases validated prognostic significance for several genes including RECQL4, FRRS1, CFH and MET - whose combined expression carried greater prognostic value than tumour grade - and cmet and TRKB proteins.Reliable clinically relevant data can be obtained from archival FFPET, but protocol amendments are needed to improve the sensitivity and broad application of this approach.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK, Department of Medical Oncology, Christie Hospital NHS Foundation Trust, Wilmslow Road, Withington, Manchester, M20 4BX, UK. kim.linton@christie.nhs.uk

ABSTRACT
Robust protocols for microarray gene expression profiling of archival formalin-fixed paraffin-embedded tissue (FFPET) are needed to facilitate research when availability of fresh-frozen tissue is limited. Recent reports attest to the feasibility of this approach, but the clinical value of these data is poorly understood. We employed state-of-the-art RNA extraction and Affymetrix microarray technology to examine 34 archival FFPET primary extremity soft tissue sarcomas. Nineteen arrays met stringent QC criteria and were used to model prognostic signatures for metastatic recurrence. Arrays from two paired frozen and FFPET samples were compared: although FFPET sensitivity was low ( approximately 50%), high specificity (95%) and positive predictive value (92%) suggest that transcript detection is reliable. Good agreement between arrays and real time (RT)-PCR was confirmed, especially for abundant transcripts, and RT-PCR validated the regulation pattern for 19 of 24 candidate genes (overall R(2)=0.4662). RT-PCR and immunohistochemistry on independent cases validated prognostic significance for several genes including RECQL4, FRRS1, CFH and MET - whose combined expression carried greater prognostic value than tumour grade - and cmet and TRKB proteins. These molecules warrant further evaluation in larger series. Reliable clinically relevant data can be obtained from archival FFPET, but protocol amendments are needed to improve the sensitivity and broad application of this approach.

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Illustration of cmet immunostaining and computerised image analysis detection of immunostained features in a case of synovial sarcoma. (A) Immunopositive areas are dark grey (SG chromogen) and background is light grey, (B) computer detected features are marked with blue overlay, which confirms excellent detection of immunopositive areas shown in A.
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fig4: Illustration of cmet immunostaining and computerised image analysis detection of immunostained features in a case of synovial sarcoma. (A) Immunopositive areas are dark grey (SG chromogen) and background is light grey, (B) computer detected features are marked with blue overlay, which confirms excellent detection of immunopositive areas shown in A.

Mentions: One digital image per immunostained tissue core was acquired using a Leica RMDB Research Microscope coupled with a DeltaPix camera, and analysed using Quantimet 550 software and an in-house macro written in QUIPS. This enabled automatic computer detection and measurement of immunopositive features (Figure 4). Images were converted to grey scale and each pixel was allocated a value corresponding to its ‘grey shade'. The range of values representing positive staining was previously determined for each antibody by examining staining in positive and negative controls. The sum of all grey values (‘sum grey') was used to compute quantitative protein expression. For qualitative purposes, samples were considered to have high protein expression if sum grey exceeded the median expression value.


Acquisition of biologically relevant gene expression data by Affymetrix microarray analysis of archival formalin-fixed paraffin-embedded tumours.

Linton KM, Hey Y, Saunders E, Jeziorska M, Denton J, Wilson CL, Swindell R, Dibben S, Miller CJ, Pepper SD, Radford JA, Freemont AJ - Br. J. Cancer (2008)

Illustration of cmet immunostaining and computerised image analysis detection of immunostained features in a case of synovial sarcoma. (A) Immunopositive areas are dark grey (SG chromogen) and background is light grey, (B) computer detected features are marked with blue overlay, which confirms excellent detection of immunopositive areas shown in A.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2361698&req=5

fig4: Illustration of cmet immunostaining and computerised image analysis detection of immunostained features in a case of synovial sarcoma. (A) Immunopositive areas are dark grey (SG chromogen) and background is light grey, (B) computer detected features are marked with blue overlay, which confirms excellent detection of immunopositive areas shown in A.
Mentions: One digital image per immunostained tissue core was acquired using a Leica RMDB Research Microscope coupled with a DeltaPix camera, and analysed using Quantimet 550 software and an in-house macro written in QUIPS. This enabled automatic computer detection and measurement of immunopositive features (Figure 4). Images were converted to grey scale and each pixel was allocated a value corresponding to its ‘grey shade'. The range of values representing positive staining was previously determined for each antibody by examining staining in positive and negative controls. The sum of all grey values (‘sum grey') was used to compute quantitative protein expression. For qualitative purposes, samples were considered to have high protein expression if sum grey exceeded the median expression value.

Bottom Line: Arrays from two paired frozen and FFPET samples were compared: although FFPET sensitivity was low ( approximately 50%), high specificity (95%) and positive predictive value (92%) suggest that transcript detection is reliable.RT-PCR and immunohistochemistry on independent cases validated prognostic significance for several genes including RECQL4, FRRS1, CFH and MET - whose combined expression carried greater prognostic value than tumour grade - and cmet and TRKB proteins.Reliable clinically relevant data can be obtained from archival FFPET, but protocol amendments are needed to improve the sensitivity and broad application of this approach.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK, Department of Medical Oncology, Christie Hospital NHS Foundation Trust, Wilmslow Road, Withington, Manchester, M20 4BX, UK. kim.linton@christie.nhs.uk

ABSTRACT
Robust protocols for microarray gene expression profiling of archival formalin-fixed paraffin-embedded tissue (FFPET) are needed to facilitate research when availability of fresh-frozen tissue is limited. Recent reports attest to the feasibility of this approach, but the clinical value of these data is poorly understood. We employed state-of-the-art RNA extraction and Affymetrix microarray technology to examine 34 archival FFPET primary extremity soft tissue sarcomas. Nineteen arrays met stringent QC criteria and were used to model prognostic signatures for metastatic recurrence. Arrays from two paired frozen and FFPET samples were compared: although FFPET sensitivity was low ( approximately 50%), high specificity (95%) and positive predictive value (92%) suggest that transcript detection is reliable. Good agreement between arrays and real time (RT)-PCR was confirmed, especially for abundant transcripts, and RT-PCR validated the regulation pattern for 19 of 24 candidate genes (overall R(2)=0.4662). RT-PCR and immunohistochemistry on independent cases validated prognostic significance for several genes including RECQL4, FRRS1, CFH and MET - whose combined expression carried greater prognostic value than tumour grade - and cmet and TRKB proteins. These molecules warrant further evaluation in larger series. Reliable clinically relevant data can be obtained from archival FFPET, but protocol amendments are needed to improve the sensitivity and broad application of this approach.

Show MeSH
Related in: MedlinePlus