A system for detecting high impact-low frequency mutations in primary tumors and metastases

Abstract

Tumor complexity and intratumor heterogeneity contribute to subclonal diversity. Despite advances in next-generation sequencing (NGS) and bioinformatics, detecting rare mutations in primary tumors and metastases contributing to subclonal diversity is a challenge for precision genomics. Here, in order to identify rare mutations, we adapted a recently described epithelial reprograming assay for short-term propagation of epithelial cells from primary and metastatic tumors. Using this approach, we expanded minor clones and obtained epithelial cell-specific DNA/RNA for quantitative NGS analysis. Comparative Ampliseq Comprehensive Cancer Panel sequence analyses were performed on DNA from unprocessed breast tumor and tumor cells propagated from the same tumor. We identified previously uncharacterized mutations present only in the cultured tumor cells, a subset of which has been reported in brain metastatic but not primary breast tumors. In addition, whole-genome sequencing identified mutations enriched in liver metastases of various cancers, including Notch pathway mutations/chromosomal inversions in 5/5 liver metastases, irrespective of cancer types. Mutations/rearrangements in FHIT, involved in purine metabolism, were detected in 4/5 liver metastases, and the same four liver metastases shared mutations in 32 genes, including mutations of different HLA-DR family members affecting OX40 signaling pathway, which could impact the immune response to metastatic cells. Pathway analyses of all mutated genes in liver metastases showed aberrant tumor necrosis factor and transforming growth factor signaling in metastatic cells. Epigenetic regulators including KMT2C/MLL3 and ARID1B, which are mutated in >50% of hepatocellular carcinomas, were also mutated in liver metastases. Thus, irrespective of cancer types, organ-specific metastases may share common genomic aberrations. Since recent studies show independent evolution of primary tumors and metastases and in most cases mutation burden is higher in metastases than primary tumors, the method described here may allow early detection of subclonal somatic alterations associated with metastatic progression and potentially identify therapeutically actionable, metastasis-specific genomic aberrations.

The lysine‐specific methyltransferase KMT 2C/ MLL 3 regulates DNA repair components in cancer

“…Thus, in tumor evolution, promoter methylation of KMT2C may provide a selective advantage to emerging KMT2C ‐mutated cells by reducing wild‐type protein levels. In support of this model, KMT2C mutations were recently identified as late events in subclones of lung adenocarcinomas during tumor evolution and in metastatic breast cancer subclones .…”

“…Student's t-test was used. ** designates P-value < 0.01 and ****P-value < 0.0001. subclones of lung adenocarcinomas during tumor evolution [66] and in metastatic breast cancer subclones [67]. Functionally, KMT2C and the related KMT2D protein direct H3K4 monomethylation, which poises enhancers for activation and transcription factor binding, thus regulating the transcription of neighbor genes [68,69].…”

Dual TGFβ/BMP Pathway Inhibition Enables Expansion and Characterization of Multiple Epithelial Cell Types of the Normal and Cancerous Breast

“…We had previously demonstrated that short-term culturing followed by sequencing of cultured tumor cells could detect functionally important mutations that are otherwise undetectable upon direct sequencing of tumors (36). Culturing for short duration as such did not introduce mutations.…”

“…Metastasis is the main cause of cancer-related death and several studies have shown distinct differences between primary tumors and metastasis (34,35). We had previously shown that epithelial reprogramming assay could be used to grow and perform genomics of liver metastasis of various cancers (36). However, because mouse embryonic feeder layer cells were needed to grow cancer cells, sorting of cancer cells by flow cytometry was required before sequencing, and bioinformatics analyses of genomic data required additional steps to ensure that sequence reads are not from residual contaminating mouse DNA.…”

Moving Breast Cancer Therapy up a Notch

“…Notch2/3 inhibition (Tarextumab) decreased CSC numbers in the UM-PE13 breast cancer cells ( 144 ). Furthermore, mutations in Notch2 could facilitate development of liver metastasis ( 145 ). However, other studies showed that, Notch2 mutations do not unequivocally associate with better prognosis and therapy efficacy ( 144 , 146 ).…”