Detection of breast cancer stem cell gene mutations in circulating free DNA during the evolution of metastases

Abstract

Purpose: Limited knowledge exists on the detection of breast cancer stem cell (BCSC)-related mutations in circulating free DNA (cfDNA) from patients with advanced cancers. Identification of new cancer biomarkers may allow for earlier detection of disease progression and treatment strategy modifications.

Methods: We conducted a prospective study to determine the feasibility and prognostic utility of droplet digital polymerase chain reaction (ddPCR)-based BCSC gene mutation analysis of cfDNA in patients with breast cancer.

Results: Detection of quantitative BCSC gene mutation in cfDNA by ddPCR mirrors disease progression and thus may represent a valuable and cost-effective measure of tumor burden. We have previously shown that hematological and neurological expressed 1-like (HN1L), ribosomal protein L39 (RPL39), and myeloid leukemia factor 2 (MLF2) are novel targets for BCSC self-renewal, and targeting these genetic alterations could be useful for personalized genomic-based therapy.

Conclusion: BCSC mutation detection in cfDNA may have important implications for diagnosis, prognosis, and serial monitoring.

Keywords: Breast carcinoma; Droplet digital polymerase chain reaction; Metastasis; Mutation; Stem cell.

Conflict of interest statement

Conflict of interests: The authors declare no potential conflicts of interest with the material reported in this manuscript and have no financial relationship with the organization that funded this study. Individual conflict of interest unrelated to the data presented here are listed in the conflict of interest form.

Figures

Fig. 1. Flow diagram of mutation analysis of tumor DNA and cfDNA

RNA-Seq was performed to detect the presence of BCSC-specific gene mutations in 8 lung metastatic breast cancer samples. ddPCR analysis of the four BCSC gene mutations was performed on 28 FFPE lung metastatic samples, 8 matched primary breast and lung metastatic pairs, and 24 matched primary breast and other metastases. Plasma cfDNA was analyzed from 210 individuals (86 normal volunteers, and 124 cancer patients with active stage I, II, III, and IV breast cancer). BCSC, breast cancer stem cell; cfDNA, circulating free DNA; ddPCR, droplet digital polymerase chain reaction; FFPE, formalin-fixed, paraffin-embedded.

Fig. 2. Mutational spectrum in non-matched lung metastases (n=28)

(a) Mutational frequency in non-matched lung metastatic tumor samples. The frequency of each mutation was calculated as the ratio of mutation-positive samples to total samples. (b) Fractional abundances of BCSC gene alterations in 28 lung metastatic tissue samples. For each sample, the fractional abundance (percentage of mutant DNA molecules) was calculated by dividing the number of mutant DNA molecules (copies/μl) by the number of total DNA molecules (mutant plus WT, copies/μl). (c) Metastasis-free probability by Kaplan-Meier analysis of TTM according to BCSC gene mutation status of FFPE lung metastatic samples. BCSC, breast cancer stem cell; FFPE, formalin-fixed paraffin-embedded; TTM, time-to-metastasis; WT, wild-type.

Fig. 3. Mutational analysis in matched primary breast/lung metastatic pairs and primary breast/other metastatic pairs

ddPCR analysis of BCSC gene alterations was performed on 8 matched pairs of primary breast cancer and lung metastases, and with 24 pairs of breast and other metastatic sites. (a) Shared mutations, mutations only found in primary breast and lung metastatic samples are shown. Shared mutation indicates mutations expressed in both primary breast cancer and matched lung metastases. Enrichment indicates a higher fractional abundance of mutant gene copies in lung metastases. (b) Analysis of all other metastatic sites was done for 24 matched pairs, and enrichment with higher fractional abundance of mutant gene copies was observed in metastases. BCSC, breast cancer stem cell; ddPCR, droplet digital polymerase chain reaction.

Fig. 4. BCSC and gene mutation frequency in plasma cfDNA from normal controls (n=86) and patients with stage I~IV breast cancer (n=124)

(a) Normalized mutation frequencies of the 4 BCSC in plasma cfDNA samples from 124 patients with stage I~IV breast cancer. Mutation frequencies in patient cfDNA samples were normalized based on the mean fractional abundances of the individual BCSC gene mutations in normal control cfDNA samples. (b) Normalized mutation frequencies of the 4 BCSC in plasma cfDNA samples from 86 normal control cfDNA samples and 124 patients with stage I~IV breast cancer, as well as 28 FFPE samples from lung metastases. Patients with breast cancer were divided into early (stage I or stage II) and late (stage III or stage IV) groups. At least 1 of the 4 BCSC gene mutations defined as stem cell mutations was calculated. *** represented statistically different among 4 groups (normal, stage I or II, stage III or IV). (c) Sensitivity and specificity of BCSC gene mutation detection in plasma cfDNA and FFPE lung metastatic samples from breast cancer patients compared with plasma cfDNA samples from normal controls. (d) ROC curve analysis of the BCSC gene mutations in cfDNA samples from breast cancer patients according to stage and tumor DNA of lung metastatic samples. AUC, area under the curve; BCSC, breast cancer stem cell; cfDNA, circulating free DNA; ddPCR, droplet digital polymerase chain reaction; FFPE, formalin-fixed paraffin-embedded; ROC, receiver operating characteristic.

Fig. 5. Kaplan-Meier analysis according to cfDNA BCSC gene mutation status in 40 patients with stage IV breast cancer

BCSC, breast cancer stem cell; cfDNA, circulating free DNA; PFS, progression-free survival.