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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.

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In situ mutation detection of codon 12 and 13 KRAS mutations on (A-D) fresh frozen colon and lung tissues and (E, F) FFPE colon tissues using padlock probes and RCAThe tissues display KRAS mutant (red) and wild-type (green) RCPs. Cell nuclei are shown in grey. KRAS G12D mutation analysis in fresh frozen (A) mutant and (B) wild-type colon tumor tissue, in (C) mutant and (D) wild-type lung tumor tissue, and on FFPE colon tissues with reported (E) G12C or (F) G13D KRAS mutations. The pie charts indicate the ratio between wild-type (green) and mutant (red) signals in respective tissue. The images were acquired with 10x or 20x objective. Scale bar, 50 μm. See Supplementary Fig. 2-4 for the complete set of analyzed samples.
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Figure 2: In situ mutation detection of codon 12 and 13 KRAS mutations on (A-D) fresh frozen colon and lung tissues and (E, F) FFPE colon tissues using padlock probes and RCAThe tissues display KRAS mutant (red) and wild-type (green) RCPs. Cell nuclei are shown in grey. KRAS G12D mutation analysis in fresh frozen (A) mutant and (B) wild-type colon tumor tissue, in (C) mutant and (D) wild-type lung tumor tissue, and on FFPE colon tissues with reported (E) G12C or (F) G13D KRAS mutations. The pie charts indicate the ratio between wild-type (green) and mutant (red) signals in respective tissue. The images were acquired with 10x or 20x objective. Scale bar, 50 μm. See Supplementary Fig. 2-4 for the complete set of analyzed samples.

Mentions: The selectivity of the padlock probes was first tested in situ on KRAS wild-type- and mutant cell lines (Supplementary Fig. 1). After confirmation of the quality of the probes, the in situ genotyping method was applied to ten fresh frozen human colon and lung cancer tissues with known KRAS status (Fig. 2A-D and Supplementary Fig. 2 and 3). In this validation phase, each mutation specific probe-pair was tested individually. The samples represented all codon 12 and 13 mutations except for the rarest one, G12R (Table 1), but the performance of the G12R mutation assay was verified on one of the tested cell lines (Supplementary Fig. 1F). Scoring for KRAS status was done by microscopic inspection in a fashion similar to regular fluorescent in situ hybridization (FISH). Scoring criteria are further discussed in Supplementary Note 2.


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 mutation detection of codon 12 and 13 KRAS mutations on (A-D) fresh frozen colon and lung tissues and (E, F) FFPE colon tissues using padlock probes and RCAThe tissues display KRAS mutant (red) and wild-type (green) RCPs. Cell nuclei are shown in grey. KRAS G12D mutation analysis in fresh frozen (A) mutant and (B) wild-type colon tumor tissue, in (C) mutant and (D) wild-type lung tumor tissue, and on FFPE colon tissues with reported (E) G12C or (F) G13D KRAS mutations. The pie charts indicate the ratio between wild-type (green) and mutant (red) signals in respective tissue. The images were acquired with 10x or 20x objective. Scale bar, 50 μm. See Supplementary Fig. 2-4 for the complete set of analyzed samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: In situ mutation detection of codon 12 and 13 KRAS mutations on (A-D) fresh frozen colon and lung tissues and (E, F) FFPE colon tissues using padlock probes and RCAThe tissues display KRAS mutant (red) and wild-type (green) RCPs. Cell nuclei are shown in grey. KRAS G12D mutation analysis in fresh frozen (A) mutant and (B) wild-type colon tumor tissue, in (C) mutant and (D) wild-type lung tumor tissue, and on FFPE colon tissues with reported (E) G12C or (F) G13D KRAS mutations. The pie charts indicate the ratio between wild-type (green) and mutant (red) signals in respective tissue. The images were acquired with 10x or 20x objective. Scale bar, 50 μm. See Supplementary Fig. 2-4 for the complete set of analyzed samples.
Mentions: The selectivity of the padlock probes was first tested in situ on KRAS wild-type- and mutant cell lines (Supplementary Fig. 1). After confirmation of the quality of the probes, the in situ genotyping method was applied to ten fresh frozen human colon and lung cancer tissues with known KRAS status (Fig. 2A-D and Supplementary Fig. 2 and 3). In this validation phase, each mutation specific probe-pair was tested individually. The samples represented all codon 12 and 13 mutations except for the rarest one, G12R (Table 1), but the performance of the G12R mutation assay was verified on one of the tested cell lines (Supplementary Fig. 1F). Scoring for KRAS status was done by microscopic inspection in a fashion similar to regular fluorescent in situ hybridization (FISH). Scoring criteria are further discussed in Supplementary Note 2.

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