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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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Related in: MedlinePlus

In situ detection of KRAS mutations on prospective clinical samples with unknown mutation statusThe tissues display KRAS mutant (red) and wild-type (green) RCPs and cell nuclei are shown in grey. KRAS detection in lung FFPE tissues with (A) a G12C mutation and in (B) a KRAS wild-type sample. Mutation detection in prospective colon touch tumor imprints in (C) G12D and (D) G12R mutated samples and in (E, F) imprint samples with wild-type KRAS. The pie charts indicate the ratio between wild-type (green) and mutant (red) signals in respective tissue. Scale bar, 50 μm. See Supplementary Fig. 7 and 8 for the complete set of analyzed samples.
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Figure 3: In situ detection of KRAS mutations on prospective clinical samples with unknown mutation statusThe tissues display KRAS mutant (red) and wild-type (green) RCPs and cell nuclei are shown in grey. KRAS detection in lung FFPE tissues with (A) a G12C mutation and in (B) a KRAS wild-type sample. Mutation detection in prospective colon touch tumor imprints in (C) G12D and (D) G12R mutated samples and in (E, F) imprint samples with wild-type KRAS. The pie charts indicate the ratio between wild-type (green) and mutant (red) signals in respective tissue. Scale bar, 50 μm. See Supplementary Fig. 7 and 8 for the complete set of analyzed samples.

Mentions: To investigate whether multiple mutations could be tested in the same reaction, we combined all KRAS probes and compared the in situ mutation detection result to that of individual mutation-specific probes (Supplementary Fig. 6). Detection efficiency or selectivity was not affected noticeably when multiple probes competed for the two-codon target site. The combined analysis can thus provide a rapid answer to whether the tumor harbors an activating KRAS mutation or not. Multiplex mutation detection was thereafter demonstrated on eight prospective lung FFPE tissues (Table 2 and Supplementary Fig. 7) with unknown KRAS mutation status. In situ mutation analysis suggested that three of the eight cases were mutated (Fig. 3A and Supplementary Fig. 7A-C). The results were compared to pyrosequencing of DNA extracts from the same tissues, and the suggested genotypes were confirmed to be correct in every case (Supplementary Fig. 12).


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 detection of KRAS mutations on prospective clinical samples with unknown mutation statusThe tissues display KRAS mutant (red) and wild-type (green) RCPs and cell nuclei are shown in grey. KRAS detection in lung FFPE tissues with (A) a G12C mutation and in (B) a KRAS wild-type sample. Mutation detection in prospective colon touch tumor imprints in (C) G12D and (D) G12R mutated samples and in (E, F) imprint samples with wild-type KRAS. The pie charts indicate the ratio between wild-type (green) and mutant (red) signals in respective tissue. Scale bar, 50 μm. See Supplementary Fig. 7 and 8 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 3: In situ detection of KRAS mutations on prospective clinical samples with unknown mutation statusThe tissues display KRAS mutant (red) and wild-type (green) RCPs and cell nuclei are shown in grey. KRAS detection in lung FFPE tissues with (A) a G12C mutation and in (B) a KRAS wild-type sample. Mutation detection in prospective colon touch tumor imprints in (C) G12D and (D) G12R mutated samples and in (E, F) imprint samples with wild-type KRAS. The pie charts indicate the ratio between wild-type (green) and mutant (red) signals in respective tissue. Scale bar, 50 μm. See Supplementary Fig. 7 and 8 for the complete set of analyzed samples.
Mentions: To investigate whether multiple mutations could be tested in the same reaction, we combined all KRAS probes and compared the in situ mutation detection result to that of individual mutation-specific probes (Supplementary Fig. 6). Detection efficiency or selectivity was not affected noticeably when multiple probes competed for the two-codon target site. The combined analysis can thus provide a rapid answer to whether the tumor harbors an activating KRAS mutation or not. Multiplex mutation detection was thereafter demonstrated on eight prospective lung FFPE tissues (Table 2 and Supplementary Fig. 7) with unknown KRAS mutation status. In situ mutation analysis suggested that three of the eight cases were mutated (Fig. 3A and Supplementary Fig. 7A-C). The results were compared to pyrosequencing of DNA extracts from the same tissues, and the suggested genotypes were confirmed to be correct in every case (Supplementary Fig. 12).

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