In an all-comer cohort, tumour burden estimates and outperform any perturbation to infer prognosis.

Circulating tumor DNA (ctDNA) has emerged as a potential new biomarker with diagnostic, predictive, and prognostic applications for various solid tumor types. Before beginning large prospective clinical trials to prove the added value of utilizing ctDNA in clinical practice, it is essential to investigate the effects of various preanalytical conditions on the quality of cell‐free DNA (cfDNA) in general and of ctDNA in particular in order to optimize and standardize these conditions. Whole blood samples were collected from patients with metastatic cancer bearing a known somatic variant. The following preanalytical conditions were investigated: (a) different time intervals to plasma isolation (1, 24, and 96 h) and (b) different preservatives in blood collection tubes (EDTA, CellSave, and BCT). The quality of cfDNA/ctDNA was assessed by DNA quantification, digital polymerase chain reaction (dPCR) for somatic variant detection and a β‐actin fragmentation assay for DNA contamination from lysed leukocytes. In 11 (69%) of our 16 patients, we were able to detect the known somatic variant in ctDNA. We observed a time‐dependent increase in cfDNA concentrations in EDTA tubes, which was positively correlated with an increase in wild‐type copy numbers and large DNA fragments (> 420 bp). Using different preservatives did not affect somatic variant detection ability, but did stabilize cfDNA concentrations over time. Variant allele frequency was affected by fluctuations in cfDNA concentration only in EDTA tubes at 96 h. Both CellSave and BCT tubes ensured optimal ctDNA quality in plasma processed within 96 h after blood collection for downstream somatic variant detection by dPCR.

Cell-free DNA Metastatic colorectal cancer Somatic mutations Next-generation sequencing A B S T R A C TAssessing circulating tumor DNA (ctDNA) is a promising method to evaluate somatic mutations from solid tumors in a minimally-invasive way. In a group of twelve metastatic colorectal cancer (mCRC) patients undergoing liver metastasectomy, from each patient DNA from cell-free DNA (cfDNA), the primary tumor, metastatic liver tissue, normal tumor-adjacent colon or liver tissue, and whole blood were obtained. Investigated was the feasibility of a targeted NGS approach to identify somatic mutations in ctDNA. This targeted NGS approach was also compared with NGS preceded by mutant allele enrichment using synchronous coefficient of drag alteration technology embodied in the OnTarget assay, and for selected mutations with digital PCR (dPCR). All tissue and cfDNA samples underwent IonPGM sequencing for a CRC-specific 21-gene panel, which was analyzed using a standard and a modified calling pipeline. In addition, cfDNA, whole blood and normal tissue DNA were analyzed with the OnTarget assay and with dPCR for specific mutations in cfDNA as detected in the corresponding primary and/or metastatic tumor tissue. NGS with modified calling was superior to standard calling and detected ctDNA in the cfDNA of 10 patients harboring mutations in APC, ATM, CREBBP, FBXW7, KRAS, KMT2D, PIK3CA and TP53. Using this approach, variant allele frequencies in plasma ranged predominantly from 1 to 10%, resulting in limited concordance between ctDNA and the primary tumor (39%) and the metastases (55%). Concordance between ctDNA and tissue markedly improved when ctDNA was evaluated for KRAS, PIK3CA and TP53 mutations by the OnTarget assay (80%) and digital PCR (93%). Additionally, using these techniques mutations were observed in tumor-adjacent tissue with normal morphology in the majority of patients, which were not observed in whole blood. In conclusion, in these mCRC patients with

Mutations in the gene coding for the estrogen receptor (ER), ESR1, have been associated with acquired endocrine resistance in patients with ER-positive metastatic breast cancer (MBC). Functional studies revealed that these ESR1 mutations lead to constitutive activity of the ER, meaning that the receptor is active in absence of its ligand estrogen, conferring resistance against several endocrine agents. While recent clinical studies reported that the occurrence of ESR1 mutations is rare in primary breast cancer tumors, these mutations are more frequently observed in metastatic tissue and circulating cell-free DNA of MBC patients pretreated with endocrine therapy. Given the assumed impact that the presence of ESR1 mutations has on outcome to endocrine therapy, assessing ESR1 mutations in MBC patients is likely to be of significant interest to further individualize treatment for MBC patients. Here, ESR1 mutation detection methods and the most relevant pre-clinical and clinical studies on ESR1 mutations regarding endocrine resistance are reviewed, with particular interest in the ultimate goal of guiding treatment decision-making based on ESR1 mutations.

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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.

BACKGROUND: Preclinical and clinical studies have reported that human epidermal growth factor receptor 2 (HER2) overexpression yields resistance to endocrine therapies. Here the prevalence and prognostic impact of HER2-positive circulating tumor cells (CTCs) were investigated retrospectively in metastatic breast cancer (MBC) patients with a HER2-negative primary tumor receiving endocrine therapy. Additionally, the prevalence and prognostic significance of HER2-positive CTCs were explored in a chemotherapy cohort, as well as the prognostic impact of the estrogen receptor (ER) CTC status in both cohorts. METHODS: Included were MBC patients with a HER2-negative primary tumor, with ≥1 detectable CTC, starting a new line of treatment. CTCs were enumerated using the CellSearch system, characterized for HER2 with the CellSearch anti-HER2 phenotyping reagent, and characterized for ER mRNA expression. Primary end point was progression-free rate after 6 months (PFR6months) of endocrine treatment in HER2-positive versus HER2-negative CTC patients. RESULTS: HER2-positive CTCs were present in 29% of all patients. In the endocrine cohort (n = 72), the PFR6months was 53% for HER2-positive versus 68% for HER2-negative CTC patients (P = .23). In the chemotherapy cohort (n = 82), no prognostic value of HER2-positive CTCs on PFR6months was observed either. Discordances in ER status between the primary tumor and CTCs occurred in 25% of all patients but had no prognostic value in exploratory survival analyses. CONCLUSION: Discordances regarding HER2 status and ER status between CTCs and the primary tumor occurred frequently but had no prognostic impact in our MBC patient cohorts.

Recent reports have emphasized the clinical relevance of detecting AR-V7 in circulating tumor cells (CTCs). Our aim was to set up a validated multicenter pipeline to measure AR-V7 by quantitative RT-PCR (RT-qPCR) in RNA isolated from CellSearch-enriched CTCs to provide an AR-V7-positive or AR-V7-negative score in a clinically acceptable time range. CellSearch-enirched CTCs from patients with metastatic castration-resistant prostate cancer were characterized by RT-qPCR. After optimization, it was prospectively tested whether it was possible to report the AR-V7 status within 11 days (PRELUDE study). In the range of the RNA equivalent of 0.2 to 12 VCaP cells, the CV for AR-V7 was 9% (n = 37). The limit of detection was 0.3, and the limit of quantitation was 3 cells in the final RT-qPCR. No differences were observed between AR-V7 data generated by five technicians or in two different laboratories. For the 45 patients in PRELUDE, 13 patients were ineligible, 22 patients were AR-V7 negative, and 10 were AR-V7 positive. The median time to inform the physician of the test result was 7 days (range, 2 to 11 days). This assay can establish the AR-V7 status in CTCs from patients with metastatic castration-resistant prostate cancer. Furthermore, it was possible to provide an AR-V7 outcome within 11 days, indicating that it may be used to choose between an anti-androgen receptor or taxane-based cabazitaxel treatment.