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Quantitative measurement of cancer tissue biomarkers in the lab and in the clinic.

Carvajal-Hausdorf DE, Schalper KA, Neumeister VM, Rimm DL - Lab. Invest. (2014)

Bottom Line: Detection of biomolecules in tissues provides contextual information and the possibility to assess the interaction of different cell types and markers.Routine qualitative assessment of immune- and oligonucleotide-based methods in research and the clinic has been associated with assay variability because of lack of stringent validation and subjective interpretation of results.As a result, the vast majority of in situ assays in clinical usage are nonquantitative and, although useful, often of questionable scientific validity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Yale University School of Medicine, New Haven, CT, USA.

ABSTRACT
Detection of biomolecules in tissues provides contextual information and the possibility to assess the interaction of different cell types and markers. Routine qualitative assessment of immune- and oligonucleotide-based methods in research and the clinic has been associated with assay variability because of lack of stringent validation and subjective interpretation of results. As a result, the vast majority of in situ assays in clinical usage are nonquantitative and, although useful, often of questionable scientific validity. Here, we revisit the reporters and methods used for single- and multiplexed in situ visualization of protein and RNA. Then we examine methods for the use of quantitative platforms for in situ measurement of protein and mRNA levels. Finally, we discuss the challenges of the transition of these methods to the clinic and their potential role as tools for development of companion diagnostic tests.

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Measurement of PTEN mRNA in FFPE tissues using in situ hybridization with the paired probes assay (RNAscope) coupled to quantitative fluorescence(A) Representative fluorescence microphotographs showing in situ detection of PTEN mRNA (upper left, red fluorescence channel), Ubiquitin C mRNA (UbC, red channel, middle panel) and DapB mRNA (red channel, right panel) in archival breast cancer specimens. The lower panels show the cytokeratin stain in each tissue sample (green fluorescence channel). UbC was used as positive control for the presence of measurable mRNA and bacterial DapB was used as negative control and noise indicator for each sample and in each run. (B) Chart showing the levels of PTEN mRNA (blue columns), UbC mRNA (red columns) and DapB (green columns) in archival FFPE breast cancer samples. Serial sections from a tissue microarray including samples from 238 breast carcinomas (YTMA128) were stained simultaneously for each mRNA target and with cytokeratin protein. The levels of each marker were measured in the tumor compartment using the AQUA technology and are expressed as arbitrary units of fluorescence (Y axis). Only spots including available scores for all 3 mRNA markers are included in the chart.
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Figure 2: Measurement of PTEN mRNA in FFPE tissues using in situ hybridization with the paired probes assay (RNAscope) coupled to quantitative fluorescence(A) Representative fluorescence microphotographs showing in situ detection of PTEN mRNA (upper left, red fluorescence channel), Ubiquitin C mRNA (UbC, red channel, middle panel) and DapB mRNA (red channel, right panel) in archival breast cancer specimens. The lower panels show the cytokeratin stain in each tissue sample (green fluorescence channel). UbC was used as positive control for the presence of measurable mRNA and bacterial DapB was used as negative control and noise indicator for each sample and in each run. (B) Chart showing the levels of PTEN mRNA (blue columns), UbC mRNA (red columns) and DapB (green columns) in archival FFPE breast cancer samples. Serial sections from a tissue microarray including samples from 238 breast carcinomas (YTMA128) were stained simultaneously for each mRNA target and with cytokeratin protein. The levels of each marker were measured in the tumor compartment using the AQUA technology and are expressed as arbitrary units of fluorescence (Y axis). Only spots including available scores for all 3 mRNA markers are included in the chart.

Mentions: Perhaps the most prominently used method is the paired-probes method for mRNA ISH (a.k.a. as Z-probes or branched probes) is based on the contiguous hybridization of various pairs of 14–20-long RNA oligonucleotides spanning typically a ~1kb area. Each probe is designed with a target specific sequence, a spacer and a tail sequence that is recognized by the signal amplification HRP or AP-based system only when serially aligned with its partner probe (and not with potentially non-specific single-bounded probes) (56). The major advantages of this method are the high level signal amplification, the noise reduction achieved by the paired Z probe method and the facilitation of the parallel use of positive and negative control probes (e.g Ubiquitin C or GAPDH as positive controls; and bacterial DapB as negative control) to determine sample integrity and experimental quality. Figure 2 shows an example of PTEN, UbC and DapB stained in serial TMA sections and quantified using the AQUA® method by multiplexing with pancytokeratin stain to define the tumor compartment. The paired-probe system is available in 2 commercial assays (RNAscope® and QuantiGene RNAview®) providing a vast array of target probes and possibilities for customized probe design. The 2 commercial platforms share the paired-probe design, but may differ in their signal detection method.


Quantitative measurement of cancer tissue biomarkers in the lab and in the clinic.

Carvajal-Hausdorf DE, Schalper KA, Neumeister VM, Rimm DL - Lab. Invest. (2014)

Measurement of PTEN mRNA in FFPE tissues using in situ hybridization with the paired probes assay (RNAscope) coupled to quantitative fluorescence(A) Representative fluorescence microphotographs showing in situ detection of PTEN mRNA (upper left, red fluorescence channel), Ubiquitin C mRNA (UbC, red channel, middle panel) and DapB mRNA (red channel, right panel) in archival breast cancer specimens. The lower panels show the cytokeratin stain in each tissue sample (green fluorescence channel). UbC was used as positive control for the presence of measurable mRNA and bacterial DapB was used as negative control and noise indicator for each sample and in each run. (B) Chart showing the levels of PTEN mRNA (blue columns), UbC mRNA (red columns) and DapB (green columns) in archival FFPE breast cancer samples. Serial sections from a tissue microarray including samples from 238 breast carcinomas (YTMA128) were stained simultaneously for each mRNA target and with cytokeratin protein. The levels of each marker were measured in the tumor compartment using the AQUA technology and are expressed as arbitrary units of fluorescence (Y axis). Only spots including available scores for all 3 mRNA markers are included in the chart.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4383674&req=5

Figure 2: Measurement of PTEN mRNA in FFPE tissues using in situ hybridization with the paired probes assay (RNAscope) coupled to quantitative fluorescence(A) Representative fluorescence microphotographs showing in situ detection of PTEN mRNA (upper left, red fluorescence channel), Ubiquitin C mRNA (UbC, red channel, middle panel) and DapB mRNA (red channel, right panel) in archival breast cancer specimens. The lower panels show the cytokeratin stain in each tissue sample (green fluorescence channel). UbC was used as positive control for the presence of measurable mRNA and bacterial DapB was used as negative control and noise indicator for each sample and in each run. (B) Chart showing the levels of PTEN mRNA (blue columns), UbC mRNA (red columns) and DapB (green columns) in archival FFPE breast cancer samples. Serial sections from a tissue microarray including samples from 238 breast carcinomas (YTMA128) were stained simultaneously for each mRNA target and with cytokeratin protein. The levels of each marker were measured in the tumor compartment using the AQUA technology and are expressed as arbitrary units of fluorescence (Y axis). Only spots including available scores for all 3 mRNA markers are included in the chart.
Mentions: Perhaps the most prominently used method is the paired-probes method for mRNA ISH (a.k.a. as Z-probes or branched probes) is based on the contiguous hybridization of various pairs of 14–20-long RNA oligonucleotides spanning typically a ~1kb area. Each probe is designed with a target specific sequence, a spacer and a tail sequence that is recognized by the signal amplification HRP or AP-based system only when serially aligned with its partner probe (and not with potentially non-specific single-bounded probes) (56). The major advantages of this method are the high level signal amplification, the noise reduction achieved by the paired Z probe method and the facilitation of the parallel use of positive and negative control probes (e.g Ubiquitin C or GAPDH as positive controls; and bacterial DapB as negative control) to determine sample integrity and experimental quality. Figure 2 shows an example of PTEN, UbC and DapB stained in serial TMA sections and quantified using the AQUA® method by multiplexing with pancytokeratin stain to define the tumor compartment. The paired-probe system is available in 2 commercial assays (RNAscope® and QuantiGene RNAview®) providing a vast array of target probes and possibilities for customized probe design. The 2 commercial platforms share the paired-probe design, but may differ in their signal detection method.

Bottom Line: Detection of biomolecules in tissues provides contextual information and the possibility to assess the interaction of different cell types and markers.Routine qualitative assessment of immune- and oligonucleotide-based methods in research and the clinic has been associated with assay variability because of lack of stringent validation and subjective interpretation of results.As a result, the vast majority of in situ assays in clinical usage are nonquantitative and, although useful, often of questionable scientific validity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Yale University School of Medicine, New Haven, CT, USA.

ABSTRACT
Detection of biomolecules in tissues provides contextual information and the possibility to assess the interaction of different cell types and markers. Routine qualitative assessment of immune- and oligonucleotide-based methods in research and the clinic has been associated with assay variability because of lack of stringent validation and subjective interpretation of results. As a result, the vast majority of in situ assays in clinical usage are nonquantitative and, although useful, often of questionable scientific validity. Here, we revisit the reporters and methods used for single- and multiplexed in situ visualization of protein and RNA. Then we examine methods for the use of quantitative platforms for in situ measurement of protein and mRNA levels. Finally, we discuss the challenges of the transition of these methods to the clinic and their potential role as tools for development of companion diagnostic tests.

Show MeSH
Related in: MedlinePlus