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With Sequencing Approach, Researchers Characterize Diversity of Breast Cancer Subclones

NEW YORK (GenomeWeb) – Researchers led by the Wellcome Trust Sanger Institute's Peter Campbell have characterized the diversity of subclones within more than four dozen primary breast cancers, as they reported today in Nature Medicine.

A single tumor is often an amalgamation of various clones possibly harboring different mutations that are competing with one another. Tailoring therapeutics to patients' specific, personal mutations — such as ER or HER mutations — requires, the researchers noted, knowing just what mutations are present and how frequent they are.

Campbell and his colleagues turned to both whole-genome and targeted sequencing of multiple samples from 50 patients' breast tumors to characterize their subclonal structure. They found that subclonal diversity varied from patient to patient and that about a quarter of tumors had mutations in subclones that were potentially targetable.

"Understanding subclonality is fundamental to improving cancer care but will require the prospective integration of genomics studies into clinical trials," the researchers wrote in their paper.

All in all, the researchers sequenced 290 samples from their 50-tumor cohort to high coverage, focusing on 360 known cancer genes, including 40 genes recurrently mutated in breast cancer. This set of tumors included 27 estrogen receptor-positive, but HER2-negative tumors, three ER+ HER2+ tumors, and 20 triple-negative tumors.

The researchers sliced a subset of a dozen tumors in half and collected six needle biopsy samples from each half to examine the distribution of subclones. Eight of these 12 tumors exhibited significant spatial heterogeneity of point mutations and a further two tumors displayed heterogeneity of copy number changes, the researchers reported.

By overlaying the spatial information atop mutational data, the researchers noted that local, geographically constrained expansion was the prevalent pattern of heterogeneity, as 10 of the 12 tumors had at least one mutation that was only found in neighboring regions.

For all 12, the researchers uncovered at least one clonal somatic driver mutation or copy number variant that all the samples shared, and in four cancers, they noted subclonal driver mutations, including TP53 missense mutations, MYC amplifications, and nonsense BRCA2 mutations.

Nearly 60 percent of these cases had subclonal mutations that the researchers said could be mistaken for clonal mutations if only a single biopsy were analyzed.

For all 50 tumors, Campbell and his colleagues examined intratumoral heterogeneity, finding that nearly half of the tumors didn't exhibit any difference in point mutations across tumor sub-regions. Three cancers had, though, what the researchers called profound heterogeneity, especially of private mutations. The rest, though, had intermediate levels of heterogeneity.

They noted, though, that the majority of driver mutations were present in all the lesions they sequenced, suggesting they arose before the cancer's most recent common ancestor. But just when these mutations cropped up varied from tumor to tumor.

In 13 of the tumors, subclonal mutations affected genes that are potential drug targets.

For 18 cancers, the researchers also sequenced residual disease that was found in the patients after they underwent neoadjuvant chemotherapy. In six of these, the mutations the researchers uncovered were subclonal in both the pre- and post-treatment samples, but for five, the residual disease samples harbored a subclone that was not evident in the pre-treatment samples. These treatment-resistant subclones had potential driver mutations that included CDK6, MYC, and FGFR2 amplifications.

The branch lengths of the pre- and post-treatment subclones on phylogenetic trees were of similar lengths, suggesting they are of similar ages. Thus, clones only found in residual disease were likely subclones that were present prior to treatment.

For two other cases, the researchers examined the whole genomes of both primary tumor biopsies and ones from metastases. For these, they found that the metastases arose from subclones of the primary tumor.

"This finding has clinical relevance: if metastatic disease arose from a very early branch of the phylogenetic tree, before all subclonal diversification within the primary tumor, treating actionable mutations that were subclonal in the primary tumor would not help prevent disease relapse," the researchers noted.

Such findings, Campbell and his colleagues said, should inform the development and testing of cancer drugs. "Drug development is increasingly 'rational,' based on an improved understanding of each tumor's individual biology; drug testing should follow this lead, incorporating the biology of cancer evolution into trial design and evaluation," they added.