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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

Detection of two EGFR point mutations, G719C and S768I, using mutation-specific padlock probes giving rise to differently colored RCPs; green RCPs represent wild-type EGFR, red RCPs G719C mutants and blue RCPs S768I mutantsCell nuclei are shown in grey. The left part of the tissue represents benign respiratory epithelium of a small bronchus that expresses wild-type EGFR whereas the right part shows a representative tumor area with cancer cells displaying RCPs from the EGFR wild-type, G719C and S768I padlock probes. The pie charts indicate the ratio between wild-type (green), G719C mutant (red) and S768I (blue) signals in two different areas of the tissue (dotted line denotes border). Scale bar, 50 μm.
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Figure 6: Detection of two EGFR point mutations, G719C and S768I, using mutation-specific padlock probes giving rise to differently colored RCPs; green RCPs represent wild-type EGFR, red RCPs G719C mutants and blue RCPs S768I mutantsCell nuclei are shown in grey. The left part of the tissue represents benign respiratory epithelium of a small bronchus that expresses wild-type EGFR whereas the right part shows a representative tumor area with cancer cells displaying RCPs from the EGFR wild-type, G719C and S768I padlock probes. The pie charts indicate the ratio between wild-type (green), G719C mutant (red) and S768I (blue) signals in two different areas of the tissue (dotted line denotes border). Scale bar, 50 μm.

Mentions: To further study intratumor heterogeneity, we designed probes for tumors that were known to harbor multiple point mutations. As a proof-of-concept that intratumor heterogeneity can be studied, we established individualized in situ mutation assays for screening of FFPE cases carrying unique combinations of mutations in EGFR, KRAS, and TP53 (Table 1). One lung cancer case was positive for the activating EGFR mutation G719C, as well as the EGFR S768I mutation that is associated with resistance to anti-EGFR therapy (Fig. 6 and Supplementary Fig. 11A). The expression of the G719C transcript was high compared to the S768I transcript throughout the tumor section. This balance between the expressed mutated alleles might be expected as this case represents a patient that had not received anti-EGFR therapy so no selection pressure for increased expression of the resistance mutation was present.


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)

Detection of two EGFR point mutations, G719C and S768I, using mutation-specific padlock probes giving rise to differently colored RCPs; green RCPs represent wild-type EGFR, red RCPs G719C mutants and blue RCPs S768I mutantsCell nuclei are shown in grey. The left part of the tissue represents benign respiratory epithelium of a small bronchus that expresses wild-type EGFR whereas the right part shows a representative tumor area with cancer cells displaying RCPs from the EGFR wild-type, G719C and S768I padlock probes. The pie charts indicate the ratio between wild-type (green), G719C mutant (red) and S768I (blue) signals in two different areas of the tissue (dotted line denotes border). Scale bar, 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Detection of two EGFR point mutations, G719C and S768I, using mutation-specific padlock probes giving rise to differently colored RCPs; green RCPs represent wild-type EGFR, red RCPs G719C mutants and blue RCPs S768I mutantsCell nuclei are shown in grey. The left part of the tissue represents benign respiratory epithelium of a small bronchus that expresses wild-type EGFR whereas the right part shows a representative tumor area with cancer cells displaying RCPs from the EGFR wild-type, G719C and S768I padlock probes. The pie charts indicate the ratio between wild-type (green), G719C mutant (red) and S768I (blue) signals in two different areas of the tissue (dotted line denotes border). Scale bar, 50 μm.
Mentions: To further study intratumor heterogeneity, we designed probes for tumors that were known to harbor multiple point mutations. As a proof-of-concept that intratumor heterogeneity can be studied, we established individualized in situ mutation assays for screening of FFPE cases carrying unique combinations of mutations in EGFR, KRAS, and TP53 (Table 1). One lung cancer case was positive for the activating EGFR mutation G719C, as well as the EGFR S768I mutation that is associated with resistance to anti-EGFR therapy (Fig. 6 and Supplementary Fig. 11A). The expression of the G719C transcript was high compared to the S768I transcript throughout the tumor section. This balance between the expressed mutated alleles might be expected as this case represents a patient that had not received anti-EGFR therapy so no selection pressure for increased expression of the resistance mutation was present.

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