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

Ida Grundberg,Sara Kiflemariam,Johan Botling,Mats Nilsson,Juliana Imgenberg-Kreuz,Patrick Micke,Tobias Sjöblom,Karolina Edlund,Marco Mignardi,Magnus Sundström

doi:10.18632/oncotarget.1527

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.

Highlights

Therapeutic targeting of oncogenic mutations in signal transduction pathways has opened a new era in oncology and created a need for efficient mutational analyses in routine pathology
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)
We here report the establishment of a multiplex in situ assay that targets point mutations on tumor tissue sections and on cytological preparations. cDNA copies of transcripts, synthesized by reverse transcription of mRNA in situ, are targeted with mutant- or wild-type specific padlock probes and amplified to a detectable level with rolling-circle amplification (RCA)

Summary

Introduction

Therapeutic targeting of oncogenic mutations in signal transduction pathways has opened a new era in oncology and created a need for efficient mutational analyses in routine pathology. The complexity of cancer tissues is not taken into account in clinical mutation diagnostics which is performed on crude tissue extracts. All different cell types present in a tumor sample – normal parenchymal cells, stromal cells, inflammatory cells, pre-neoplastic and fully developed malignant cells – contribute their wild-type and mutated alleles to the analysis. The data support the concept of clonal evolution where different mutations can be enriched in different sub-clones due to an array of selection mechanisms at work in different compartments of the tumor bulk – hypoxia, inflammation, necrosis, inflammation and organ specific environmental factors [1]. To characterize intratumor heterogeneity in a routine diagnostic setting, tumor cells can be enriched by manual microdissection, but more detailed analysis of specific tumor regions requires laborious laser-assisted microdissection. Methods that offer in situ mutation detection directly on tissue sections are highly warranted

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