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Heterogeneity of PIK3CA mutational status at the single cell level in circulating tumor cells from metastatic breast cancer patients.

ABSTRACT: Circulating Tumor Cells (CTCs) represent a "liquid biopsy of the tumor" which might allow real-time monitoring of cancer biology and therapies in individual patients. CTCs are extremely rare in the blood stream and their analysis is technically challenging. The CellSearch(®) system provides the enumeration of CTCs with prognostic significance in patients with metastatic breast cancer (mBC), but it does not allow their molecular characterization, which might be useful to identify therapeutically relevant targets for individualized treatment. Combining the CellSearch(®) and DEPArray™ technologies allows the recovery of single CTCs as a pure sample for molecular analysis. The purpose of the study was to investigate the heterogeneity of PIK3CA mutational status within single CTCs isolated from individual mBC patients. CTCs were enriched and enumerated by CellSearch(®) in blood samples collected from 39 mBC patients. In 20 out of 39 patients enriched samples with ≥5 CTCs were sorted using DEParray™ to isolate single CTCs or pools of CTCs to be submitted to Whole Genome Amplification (WGA) before sequencing analysis. In 18 out of 20 patients, it was possible to perform PIK3CA sequencing on exons 9 and 20. Twelve subjects were wild type (wt) for the PIK3CA gene. PIK3CA status could also be assessed in pools of CTCs in seven of these patients, with consistent wt status found. Six patients (33%) had a PIK3CA mutation identified. In 2 of the six patients, molecular heterogeneity was detected when mutational analysis was performed on more than one single CTC, including one patient with loss of heterozygosity on both single and pooled CTCs, and one patient with three different PIK3CA variants on single CTCs but PIK3CA wt status on pooled CTC samples. In six out of the 18 cases PIK3CA status was also evaluable on a primary tumor sample. In one of the six cases a discordance in PIK3CA status between the primary (wild-type) and the matched CTC (exon 20 mutation) was observed. This study demonstrates the feasibility of a non-invasive approach based on the liquid biopsy in mBC patients. Moreover, our data suggest the importance of characterizing CTCs at the single cell level in order to investigate the molecular heterogeneity within cells from the same patient.

SUBMITTER: Pestrin M

PROVIDER: S-EPMC5528771 | biostudies-other | 2015 Apr

REPOSITORIES: biostudies-other

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Project description:Breast cancer is the most frequent cancer in women and consists of heterogeneous types of tumours that are classified into different histological and molecular subtypes1-3. Pik3ca and p53 are the two most frequently mutated genes and are associated with different types of human breast cancers4. The cellular origin and the mechanisms leading to Pik3ca-induced tumour heterogeneity remain unknown. Here, we used a genetic approach in mice to define the cellular origin of Pik3ca-derived tumours and its impact on tumour heterogeneity. Surprisingly, oncogenic Pik3ca-H1047R expression at physiological levels5 in basal cells (BCs) using K5CREERT2 induced the formation of luminal ER+PR+ tumours, while its expression in luminal cells (LCs) using K8CREERT2 gave rise to luminal ER+PR+ tumours or basal-like ER-PR- tumours. Concomitant deletion of p53 and expression of Pik3ca-H1047R accelerated tumour development and induced more aggressive mammary tumours. Interestingly, expression of Pik3ca-H1047R in unipotent BCs gave rise to luminal-like cells, while its expression in unipotent LCs gave rise to basal-like cells before progressing into invasive tumours. Transcriptional profiling of cells that have undergone cell fate transition upon Pik3ca-H1047R expression in unipotent progenitors demonstrate a profound oncogene-induced reprogramming of these newly formed cells and identified gene signatures, characteristic of the different cell fate switches that occur upon Pik3ca-H1047R expression in BC and LCs, which correlated with the cell of origin, tumour type and different clinical outcomes. Altogether our study identifies the cellular origin of Pik3ca-induced tumours and reveals that oncogenic Pik3ca-H1047R activates a multipotent genetic program in normally lineage-restricted populations at the early stage of tumour initiation, setting the stage for future intratumoural heterogeneity. These results have important implications for our understanding of the mechanisms controlling tumour heterogeneity and the development of new strategies to block PIK3CA breast cancer initiation. Luminal and basal cells, or tumour cells, from mice in which expression of PIK3CA-H1047R and YFP (and in some conditions loss of p53) was targeted in basal cells using K5CREERT2 or in luminal cells using K8CREERT2 were FACS isolated and RNA was extracted before being hybridized Affymetrix microarrays.

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Heterogeneity of PIK3CA mutational status at the single cell level in circulating tumor cells from metastatic breast cancer patients.

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