Purpose: Androgen receptor pathway inhibitors (ARPI) are standard of care for treatment-naïve metastatic castration-resistant prostate cancer (mCRPC), but rapid resistance is common. Early identification of resistance will improve management strategies. We investigated whether changes in circulating tumor DNA (ctDNA) fraction during ARPI treatment are linked with mCRPC clinical outcomes. Experimental Design: Plasma cell-free DNA was collected from 81 patients with mCRPC at baseline and after 4 weeks of first-line ARPI treatment during two prospective multicenter observational studies (NCT02426333; NCT02471469). ctDNA fraction was calculated from somatic mutations in targeted sequencing and genome copy-number profiles. Samples were classified into detected versus undetected ctDNA. Outcome measurements were progression-free survival (PFS) and overall survival (OS). Nondurable treatment response was defined as PFS ≤6 months. Results: ctDNA was detected in 48/81 (59%) baseline and 29/81 (36%) 4-week samples. ctDNA fraction for samples with detected ctDNA was lower at 4 weeks versus baseline (median 5.0% versus 14.5%, P = 0.017). PFS and OS were shortest for patients with persistent ctDNA at 4 weeks (univariate HR, 4.79; 95% CI, 2.62–8.77 and univariate HR, 5.49; 95% CI, 2.76–10.91, respectively), independent of clinical prognostic factors. For patients exhibiting change from detected to undetected ctDNA by 4 weeks, there was no significant PFS difference versus patients with baseline undetected ctDNA. ctDNA change had a positive predictive value of 88% and negative predictive value of 92% for identifying nondurable responses. Conclusions: Early changes in ctDNA fraction are strongly linked to duration of first-line ARPI treatment benefit and survival in mCRPC and may inform early therapy switches or treatment intensification. See related commentary by Sartor, p. 2745

Purpose: Although cell-free DNA (cfDNA) testing is expected to drive cancer precision medicine, little is known about the significance of detecting low-frequency variants in circulating cell-free tumor DNA (ctDNA) in castration-resistant prostate cancer (CRPC). We aimed to identify genomic profile including low-frequency variants in ctDNA from patients with CRPC and investigate the clinical utility of detecting variants with variant allele frequency (VAF) below 1%. Experimental Design: This prospective, multicenter cohort study enrolled patients with CRPC eligible for treatment with abiraterone or enzalutamide. We performed targeted sequencing of pretreatment cfDNA and paired leukocyte DNA with molecular barcodes, and ctDNA variants with a VAF ≥0.1% were detected using an in-house pipeline. We investigated progression-free survival (PFS) and overall survival (OS) after different ctDNA fraction cutoffs were applied. Results: One hundred patients were analyzed (median follow-up 10.7 months). We detected deleterious ATM, BRCA2, and TP53 variants even in samples with ctDNA fraction below 2%. When the ctDNA fraction cutoff value of 0.4% was applied, significant differences in PFS and OS were found between patients with and without defects in ATM or BRCA2 [HR, 2.52; 95% confidence interval (CI), 1.24–5.11; P = 0.0091] and TP53 (HR, 3.74; 95% CI, 1.60–8.71; P = 0.0014). However, these differences were no longer observed when the ctDNA fraction cutoff value of 2% was applied, and approximately 50% of the samples were classified as ctDNA unquantifiable. Conclusions: Detecting low-frequency ctDNA variants with a VAF <1% is important to identify clinically informative genomic alterations in CRPC.

The therapeutic landscape of metastatic clear cell renal cell carcinoma (ccRCC) has rapidly expanded, and there is an urgent need to develop noninvasive biomarkers that can select an optimal therapy or evaluate the response in real time. To evaluate the clinical utility of circulating tumor DNA (ctDNA) analysis in ccRCC, we established a highly sensitive assay to detect mutations in von Hippel‐Lindau gene (VHL) using a combination of digital PCR and multiplex PCR–based targeted sequencing. The unique assay could detect VHL mutations with a variant allele frequency (VAF) <1.0%. Further, we profiled the mutation status of VHL in 76 cell‐free DNA (cfDNA) and 50 tumor tissues from 56 patients with ccRCC using the assay. Thirteen VHL mutations were identified in cfDNA from 12 (21.4%) patients with a median VAF of 0.78% (range, 0.13%‐4.20%). Of the 28 patients with VHL mutations in matched tumor tissues, eight (28.6%) also had VHL mutation in cfDNA with a median VAF of 0.47% (range, 0.13%‐2.88%). In serial ctDNA analysis from one patient, we confirmed that the VAF of VHL mutation changed consistent with tumor size by radiographic imaging during systemic treatment. In conclusion, VHL mutation in cfDNA was detected only in a small number of patients even using the highly sensitive assay; nevertheless, we showed the potential of ctDNA analysis as a novel biomarker in ccRCC.

PURPOSE: Circulating tumor DNA (ctDNA) analysis is an alternative to tissue biopsy for genotyping in various cancers. We aimed to establish a plasma ctDNA sequencing assay, then evaluate its clinical utility in advanced urothelial cancer (UC). MATERIALS AND METHODS: This study included 82 patients with muscle-invasive or metastatic UC. A total of 158 blood samples and 73 tumor tissue samples were sequenced using our designed panel covering 53 UC-relevant genes and TERT promoter. We investigated the association between the proportion of ctDNA in total cell-free DNA (ctDNA fraction) and response to pembrolizumab treatment. ctDNA dynamics were explored during systemic therapy. RESULTS: In paired tissue-ctDNA samples with estimated tumor fraction, half (50.2%) of the somatic mutations were shared between both sources, while some (17.6%) mutations were found only in ctDNA. In the metastatic setting, on-treatment increase in ctDNA fraction during pembrolizumab treatment was significantly associated with a poor response rate (18.7% v 76.1%; P = .001) and progression-free survival (median 2.8 v 9.8 months; P < .001). A comparison of cancer cell fraction, the fraction of tumor cells carrying somatic mutations, between pembrolizumab initiation and on-treatment showed a strong correlation among patients where ctDNA fraction increased during treatment ( r = 0.73). By contrast, no correlation was observed in patients without ctDNA fraction increase ( r = 0.09). Most mutations newly detected at pembrolizumab resistance have already been identified in tumor tissues at earlier stages. CONCLUSION: Our newly established assay is suitable for assessing the genomic profile of ctDNA in patients with advanced UC. Our data may support future analyses of prognostic or predictive biomarkers for UC.

Genomic medicine based on comprehensive genomic profiling (CGP) has revolutionized cancer treatment. However, there are certain limitations to CGP based on tissue analysis. Liquid biopsy, particularly plasma cell-free DNA (cfDNA), has emerged as a less invasive source of information to complement tissue-based analysis. To use cfDNA analysis effectively in the clinical setting, it is important to know the characteristics and specific limitations of cfDNA analysis. Moreover, the utility of cfDNA testing differs between cancer types, which is not widely recognized. Furthermore, in addition to its use in CGP, there are broader applications for cfDNA testing, including its use in detecting minimal residual disease or even epigenomic profiling. In this review, we first describe the detailed characteristics of cfDNA and the limitations of cfDNA analysis, and then focus on the utility of cfDNA analysis in urologic malignancies.