Limits...
In situ mutation detection and visualization of intratumor heterogeneity for cancer research and diagnostics.

Grundberg I, Kiflemariam S, Mignardi M, Imgenberg-Kreuz J, Edlund K, Micke P, Sundström M, Sjöblom T, Botling J, Nilsson M - Oncotarget (2013)

Bottom Line: Activating oncogenic mutations are targets for a new generation of cancer drugs.High-throughput screening of KRAS mutation status was successfully performed on a tissue microarray.This in situ method holds great promise as a tool to investigate the role of somatic mutations during tumor progression and for prediction of response to targeted therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Rudbeck Laboratory.

ABSTRACT
Current assays for somatic mutation analysis are based on extracts from tissue sections that often contain morphologically heterogeneous neoplastic regions with variable contents of normal stromal and inflammatory cells, obscuring the results of the assays. We have developed an RNA-based in situ mutation assay that targets oncogenic mutations in a multiplex fashion that resolves the heterogeneity of the tissue sample. Activating oncogenic mutations are targets for a new generation of cancer drugs. For anti-EGFR therapy prediction, we demonstrate reliable in situ detection of KRAS mutations in codon 12 and 13 in colon and lung cancers in three different types of routinely processed tissue materials. High-throughput screening of KRAS mutation status was successfully performed on a tissue microarray. Moreover, we show how the patterns of expressed mutated and wild-type alleles can be studied in situ in tumors with complex combinations of mutated EGFR, KRAS and TP53. This in situ method holds great promise as a tool to investigate the role of somatic mutations during tumor progression and for prediction of response to targeted therapy.

Show MeSH

Related in: MedlinePlus

In situ genotyping with padlock probes and target-primed RCA(A) Schematic overview of the method. Target cDNA (black) is created by reverse transcription with an LNA-primer. Target mRNA (grey) is degraded by RNase H, except for the region that is hybridized to the LNA-part of the primer that is protected from degradation, anchoring the created cDNA to the target. Target specific padlock probes (wild-type:green, mutant:red), with similar target sites except for the single point mutated base (e.g. G/A), are hybridized to the cDNA and circularized by target-dependent ligation. The targeted transcripts act as primer for RCA and the resulting RCPs are labeled with fluorescence-labeled detection probes and visualized as bright spots in the cells or tissue. (B) In situ detection of a KRAS codon 12 (G12S) mutation in fresh frozen lung tumor tissue. Green RCPs represent wild-type KRAS transcripts (GGT), red RCPs represent mutant KRAS (AGT). The green dashed square indicates normal lymphocytes (magnified in solid green square) and the red dashed square indicates tumor cells with an activating G12S KRAS mutation (magnified in solid red square). The pie charts indicate the ratio between wild-type (green) and mutant (red) signals in respective square. Cell nuclei are shown in grey. Scale bar, 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3926836&req=5

Figure 1: In situ genotyping with padlock probes and target-primed RCA(A) Schematic overview of the method. Target cDNA (black) is created by reverse transcription with an LNA-primer. Target mRNA (grey) is degraded by RNase H, except for the region that is hybridized to the LNA-part of the primer that is protected from degradation, anchoring the created cDNA to the target. Target specific padlock probes (wild-type:green, mutant:red), with similar target sites except for the single point mutated base (e.g. G/A), are hybridized to the cDNA and circularized by target-dependent ligation. The targeted transcripts act as primer for RCA and the resulting RCPs are labeled with fluorescence-labeled detection probes and visualized as bright spots in the cells or tissue. (B) In situ detection of a KRAS codon 12 (G12S) mutation in fresh frozen lung tumor tissue. Green RCPs represent wild-type KRAS transcripts (GGT), red RCPs represent mutant KRAS (AGT). The green dashed square indicates normal lymphocytes (magnified in solid green square) and the red dashed square indicates tumor cells with an activating G12S KRAS mutation (magnified in solid red square). The pie charts indicate the ratio between wild-type (green) and mutant (red) signals in respective square. Cell nuclei are shown in grey. Scale bar, 50 μm.

Mentions: We designed padlock probes for point mutations in KRAS codons 12, 13 (G12S, G12R, G12C, G12D, G12A, G12V and G13D) and 61 (Q61H), as well as for EGFR (G719A, G719C, S768I and L858R) and TP53 (S127F and P190S). Padlock probes for the wild-type forms of the different targets were designed as well (Supplementary Table 1 and 2). The mutation-specific padlock probes were designed with identical target sequences except for the last nucleotide in the 3´-end that differ depending on genotype (Fig. 1A). Mismatches at this position are not accepted by the DNA ligase used and single nucleotide differences, like point mutations, are therefore efficiently discriminated [13]. To distinguish the RCPs from each other using detection probes labeled with different fluorescence dyes, e.g. green and red, two different sites for detection probes for wild-type and mutant padlocks were incorporated. We also included detection of the ACTB transcript in our assays, detected by an additional fluorophore, as an internal reference having a relative constant expression between cell types. A comparison of the ACTB signals across samples provided an estimation of the detection efficiency in different samples. The ACTB data was useful during the development phase of this assay, but turned out to be dispensable for mutation scoring and tissue classification. Before applying the padlock probes onto cell lines or tissues they were evaluated in vitro with synthetic templates to assure similar hybridization and ligation efficiency.


In situ mutation detection and visualization of intratumor heterogeneity for cancer research and diagnostics.

Grundberg I, Kiflemariam S, Mignardi M, Imgenberg-Kreuz J, Edlund K, Micke P, Sundström M, Sjöblom T, Botling J, Nilsson M - Oncotarget (2013)

In situ genotyping with padlock probes and target-primed RCA(A) Schematic overview of the method. Target cDNA (black) is created by reverse transcription with an LNA-primer. Target mRNA (grey) is degraded by RNase H, except for the region that is hybridized to the LNA-part of the primer that is protected from degradation, anchoring the created cDNA to the target. Target specific padlock probes (wild-type:green, mutant:red), with similar target sites except for the single point mutated base (e.g. G/A), are hybridized to the cDNA and circularized by target-dependent ligation. The targeted transcripts act as primer for RCA and the resulting RCPs are labeled with fluorescence-labeled detection probes and visualized as bright spots in the cells or tissue. (B) In situ detection of a KRAS codon 12 (G12S) mutation in fresh frozen lung tumor tissue. Green RCPs represent wild-type KRAS transcripts (GGT), red RCPs represent mutant KRAS (AGT). The green dashed square indicates normal lymphocytes (magnified in solid green square) and the red dashed square indicates tumor cells with an activating G12S KRAS mutation (magnified in solid red square). The pie charts indicate the ratio between wild-type (green) and mutant (red) signals in respective square. Cell nuclei are shown in grey. Scale bar, 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3926836&req=5

Figure 1: In situ genotyping with padlock probes and target-primed RCA(A) Schematic overview of the method. Target cDNA (black) is created by reverse transcription with an LNA-primer. Target mRNA (grey) is degraded by RNase H, except for the region that is hybridized to the LNA-part of the primer that is protected from degradation, anchoring the created cDNA to the target. Target specific padlock probes (wild-type:green, mutant:red), with similar target sites except for the single point mutated base (e.g. G/A), are hybridized to the cDNA and circularized by target-dependent ligation. The targeted transcripts act as primer for RCA and the resulting RCPs are labeled with fluorescence-labeled detection probes and visualized as bright spots in the cells or tissue. (B) In situ detection of a KRAS codon 12 (G12S) mutation in fresh frozen lung tumor tissue. Green RCPs represent wild-type KRAS transcripts (GGT), red RCPs represent mutant KRAS (AGT). The green dashed square indicates normal lymphocytes (magnified in solid green square) and the red dashed square indicates tumor cells with an activating G12S KRAS mutation (magnified in solid red square). The pie charts indicate the ratio between wild-type (green) and mutant (red) signals in respective square. Cell nuclei are shown in grey. Scale bar, 50 μm.
Mentions: We designed padlock probes for point mutations in KRAS codons 12, 13 (G12S, G12R, G12C, G12D, G12A, G12V and G13D) and 61 (Q61H), as well as for EGFR (G719A, G719C, S768I and L858R) and TP53 (S127F and P190S). Padlock probes for the wild-type forms of the different targets were designed as well (Supplementary Table 1 and 2). The mutation-specific padlock probes were designed with identical target sequences except for the last nucleotide in the 3´-end that differ depending on genotype (Fig. 1A). Mismatches at this position are not accepted by the DNA ligase used and single nucleotide differences, like point mutations, are therefore efficiently discriminated [13]. To distinguish the RCPs from each other using detection probes labeled with different fluorescence dyes, e.g. green and red, two different sites for detection probes for wild-type and mutant padlocks were incorporated. We also included detection of the ACTB transcript in our assays, detected by an additional fluorophore, as an internal reference having a relative constant expression between cell types. A comparison of the ACTB signals across samples provided an estimation of the detection efficiency in different samples. The ACTB data was useful during the development phase of this assay, but turned out to be dispensable for mutation scoring and tissue classification. Before applying the padlock probes onto cell lines or tissues they were evaluated in vitro with synthetic templates to assure similar hybridization and ligation efficiency.

Bottom Line: Activating oncogenic mutations are targets for a new generation of cancer drugs.High-throughput screening of KRAS mutation status was successfully performed on a tissue microarray.This in situ method holds great promise as a tool to investigate the role of somatic mutations during tumor progression and for prediction of response to targeted therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Rudbeck Laboratory.

ABSTRACT
Current assays for somatic mutation analysis are based on extracts from tissue sections that often contain morphologically heterogeneous neoplastic regions with variable contents of normal stromal and inflammatory cells, obscuring the results of the assays. We have developed an RNA-based in situ mutation assay that targets oncogenic mutations in a multiplex fashion that resolves the heterogeneity of the tissue sample. Activating oncogenic mutations are targets for a new generation of cancer drugs. For anti-EGFR therapy prediction, we demonstrate reliable in situ detection of KRAS mutations in codon 12 and 13 in colon and lung cancers in three different types of routinely processed tissue materials. High-throughput screening of KRAS mutation status was successfully performed on a tissue microarray. Moreover, we show how the patterns of expressed mutated and wild-type alleles can be studied in situ in tumors with complex combinations of mutated EGFR, KRAS and TP53. This in situ method holds great promise as a tool to investigate the role of somatic mutations during tumor progression and for prediction of response to targeted therapy.

Show MeSH
Related in: MedlinePlus