Estrogen receptor mutations and splice variants determined in liquid biopsies from metastatic breast cancer patients

Abstract

Mutations and splice variants in the estrogen receptor (ER) gene, ESR1, may yield endocrine resistance in metastatic breast cancer (MBC) patients. These putative endocrine resistance markers are likely to emerge during treatment, and therefore, its detection in liquid biopsies, such as circulating tumor cells (CTCs) and cell‐free DNA (cfDNA), is of great interest. This research aimed to determine whether ESR1 mutations and splice variants occur more frequently in CTCs of MBC patients progressing on endocrine treatment. In addition, the presence of ESR1 mutations was evaluated in matched cfDNA and compared to CTCs. CellSearch‐enriched CTC fractions (≥5/7.5 mL) of two MBC cohorts were evaluated, namely (a) patients starting first‐line endocrine therapy (n = 43, baseline cohort) and (b) patients progressing on any line of endocrine therapy (n = 40, progressing cohort). ESR1 hotspot mutations (D538G and Y537S/N/C) were evaluated in CTC‐enriched DNA using digital PCR and compared with matched cfDNA (n = 18 baseline cohort; n = 26 progressing cohort). Expression of ESR1 full‐length and 4 of its splice variants (∆5, ∆7, 36 kDa, and 46 kDa) was evaluated in CTC‐enriched mRNA. It was observed that in the CTCs, the ESR1 mutations were not enriched in the progressing cohort (8%), when compared with the baseline cohort (5%) (P = 0.66). In the cfDNA, however, ESR1 mutations were more prevalent in the progressing cohort (42%) than in the baseline cohort (11%) (P = 0.04). Three of the same mutations were observed in both CTCs and cfDNA, 1 mutation in CTCs only, and 11 in cfDNA only. Only the ∆5 ESR1 splice variant was CTC‐specific expressed, but was not enriched in the progressing cohort. In conclusion, sensitivity for detecting ESR1 mutations in CTC‐enriched fractions was lower than for cfDNA. ESR1 mutations detected in cfDNA, rarely present at the start of first‐line endocrine therapy, were enriched at progression, strongly suggesting a role in conferring endocrine resistance in MBC.

Liquid biopsy in breast cancer: A comprehensive review

“…Lastly, they suggested that because ESR1 mutations in cfDNA were enriched in progressed metastatic breast cancer patients compared to baseline cohort, this is indicative of ESR1 mutations' role in conferring endocrine resistance in metastatic breast cancer. 204 In another study, Mazel mutations are weak predictors. 206 Future studies and controlled trials should focus on designing patient specific panels using liquid biopsy in order to facilitate early detection of resistance.…”

“…The researchers concluded that the sensitivity for detecting ESR1 mutations in cfDNA was much higher than in CTC‐enriched fractions. Lastly, they suggested that because ESR1 mutations in cfDNA were enriched in progressed metastatic breast cancer patients compared to baseline cohort, this is indicative of ESR1 mutations' role in conferring endocrine resistance in metastatic breast cancer …”

Liquid biopsies

“…Another study was performed in 76 individual and pooled informative CTCs from 12 patients; this study has shown a 85% concordance in at least one or more prioritized somatic mutations and copy number alterations between paired CTCs and tissue metastases with potentially actionable genomic alterations identified in tissue but not CTCs, and vice versa . Very recently, using digital PCR, the presence of ESR1 mutations was evaluated in CTCs and matched cfDNA in patients with metastatic breast cancer, starting first‐line endocrine therapy and progressing on any line of endocrine therapy; according to the data presented in this study, sensitivity for detecting ESR1 mutations in CTC‐enriched fractions was lower than for cfDNA . A recent study has shown that beyond ESR1 mutations, ESR1 gene can be epigenetically silenced in CTCs through DNA methylation, and that this was strongly associated with lack of response to everolimus/exemestane regimen …”

“…60 Very recently, using digital PCR, the presence of ESR1 mutations was evaluated in CTCs and matched cfDNA in patients with metastatic breast cancer, starting first-line endocrine therapy and progressing on any line of endocrine therapy; according to the data presented in this study, sensitivity for detecting ESR1 mutations in CTC-enriched fractions was lower than for cfDNA. 61 A recent study has shown that beyond ESR1 mutations, ESR1 gene can be epigenetically silenced in CTCs through DNA methylation, and that this was strongly associated with lack of response to everolimus/exemestane regimen. 62 Besides ER, recent data have shown that AR expression in CTCs of breast cancer patients is associated with bone metastases, suggesting that therapeutic targeting of AR could offer a potential benefit for patients with metastatic breast cancer.…”

“…91 In breast cancer, the sensitivity for detecting ESR1 mutations in CTC-enriched fractions was found lower than for cfDNA. 61 In NSCLC, cfDNA and CTCs are complementary, noninvasive assays for evaluation of acquired resistance to firstline EGFR TKIs and may expand the number of patients in whom actionable genetic information can be obtained at acquired resistance.…”