Detection rate and prognostic value of circulating tumor cells and circulating tumor DNA in metastatic uveal melanoma

Circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) have been recently investigated in several cancer types, but their respective clinical significance remains to be determined. In our prospective study, we compared the detection rate and the prognostic value of these two circulating biomarkers in patients with metastatic uveal melanoma. GNAQ/GNA11 mutations were characterized in archived tumor tissue. Using a highly sensitive and mutation‐specific bidirectional pyrophosphorolysis‐activated polymerization (bi‐PAP) technique, GNAQ c.626A>T, GNAQ c.626A>C and GNA11 c.626A>T copy numbers were quantified in plasma from 12 mL of blood. CTCs were detected at the same time in 7.5 mL of blood by the CellSearch® technique. Patient characteristics and outcome were prospectively collected. CTCs (≥1) were detected in 12 of the 40 included patients (30%, range 1–20). Among the 26 patients with known detectable mutations, ctDNA was detected and quantified in 22 (84%, range 4–11,421 copies/mL). CTC count and ctDNA levels were associated with the presence of miliary hepatic metastasis (p = 0.004 and 0.03, respectively), with metastasis volume (p = 0.005 and 0.004) and with each other (p < 0.0001). CTC count and ctDNA levels were both strongly associated with progression‐free survival (p = 0.003 and 0.001) and overall survival (p = 0.0009 and <0.0001). In multivariate analyses, ctDNA appeared to be a better prognostic marker than CTC. In conclusion, ctDNA and CTC are correlated and both have poor prognostic significance. CTC detection can be performed in every patient but, in patients with detectable mutations, ctDNA was more frequently detected than CTC and has possibly more prognostic value.     

Single tube liquid biopsy for advanced non-small cell lung cancer

The need for a liquid biopsy in non-small cell lung cancer (NSCLC) patients is rapidly increasing. We studied the relation between overall survival (OS) and the presence of four cancer biomarkers from a single blood draw in advanced NSCLC patients: EpCAM^high circulating tumor cells (CTC), EpCAM^low CTC, tumor-derived extracellular vesicles (tdEV) and cell-free circulating tumor DNA (ctDNA). EpCAM^high CTC were detected with CellSearch, tdEV in the CellSearch images and EpCAM^low CTC with filtration after CellSearch. ctDNA was isolated from plasma and mutations present in the primary tumor were tracked with deep sequencing methods. In 97 patients, 21% had ≥2 EpCAM^high CTC, 15% had ≥2 EpCAM^low CTC, 27% had ≥18 tdEV and 19% had ctDNA with ≥10% mutant allele frequency. Either one of these four biomarkers could be detected in 45% of the patients and all biomarkers were present in 2%. In 11 out of 16 patients (69%) mutations were detected in the ctDNA. Two or more unfavorable biomarkers were associated with poor OS. The presence of EpCAM^high CTC and elevated levels of tdEV and ctDNA was associated with a poor OS; however, the presence of EpCAM^low CTC was not. This single tube approach enables simultaneous analysis of multiple biomarkers to explore their potential as a liquid biopsy.     

Circulating tumor DNA quantity is related to tumor volume and both predict survival in metastatic pancreatic ductal adenocarcinoma

Circulating tumor DNA (ctDNA) is assumed to reflect tumor burden and has been suggested as a tool for prognostication and follow-up in patients with metastatic pancreatic ductal adenocarcinoma (mPDAC). However, the prognostic value of ctDNA and its relation with tumor burden has yet to be substantiated, especially in mPDAC. In this retrospective analysis of prospectively collected samples, cell-free DNA from plasma samples of 58 treatment-naive mPDAC patients was isolated and sequenced using a custom-made pancreatobiliary NGS panel. Pathogenic mutations were detected in 26/58 (44.8%) samples. Cross-check with droplet digital PCR showed good agreement in Bland–Altman analysis (p = 0.217, nonsignificance indicating good agreement). In patients with liver metastases, ctDNA was more frequently detected (24/37, p < 0.001). Tumor volume (3D reconstructions from imaging) and ctDNA variant allele frequency (VAF) were correlated (Spearman's ρ = 0.544, p < 0.001). Median overall survival (OS) was 3.2 (95% confidence interval [CI] 1.6–4.9) versus 8.4 (95% CI 1.6–15.1) months in patients with detectable versus undetectable ctDNA (p = 0.005). Both ctDNA VAF and tumor volume independently predicted OS after adjustment for carbohydrate antigen 19.9 and treatment regimen (hazard ratio [HR] 1.05, 95% CI 1.01–1.09, p = 0.005; HR 1.00, 95% CI 1.01–1.05, p = 0.003). In conclusion, our study showed that ctDNA detection rates are higher in patients with larger tumor volume and liver metastases. Nevertheless, measurements may diverge and, thus, can provide complementary information. Both ctDNA VAF and tumor volume were strong predictors of OS.     

Postoperative recurrence detection using individualized circulating tumor DNA in upper tract urothelial carcinoma

Biomarkers that could detect the postoperative recurrence of upper tract urothelial carcinoma (UTUC) have not been established. In this prospective study, we aim to evaluate the utility of individualized circulating tumor DNA (ctDNA) monitoring using digital PCR (dPCR) as a tumor recurrence biomarker for UTUC in the perioperative period. Twenty-three patients who underwent radical nephroureterectomy (RNU) were included. In each patient, whole exome sequencing by next-generation sequencing and TERT promoter sequencing of tumor DNA were carried out. Case-specific gene mutations were selected from sequencing analysis to examine ctDNA by dPCR analysis. We also prospectively collected plasma and urine ctDNA from each patient. The longitudinal variant allele frequencies of ctDNA during the perioperative period were plotted. Case-specific gene mutations were detected in 22 cases (96%) from ctDNA in the preoperative samples. Frequently detected genes were TERT (39%), FGFR3 (26%), TP53 (22%), and HRAS (13%). In all cases, we obtained plasma and urine samples for 241 time points and undertook individualized ctDNA monitoring for 2 years after RNU. Ten patients with intravesical recurrence had case-specific ctDNA detected in urine at the time of recurrence. The mean lead time of urinary ctDNA in intravesical recurrence was 60 days (range, 0–202 days). Two patients with distal metastasis had case-specific ctDNA in plasma at the time of metastasis. In UTUC, tumor-specific gene mutations can be monitored postoperatively as ctDNA in plasma and urine. Individualized ctDNA might be a minimally invasive biomarker for the early detection of postoperative recurrence.     

Mutational profiling of circulating tumor DNA and clinical characteristics in lymphoma: Based on next generation sequencing

Liquid biopsy has been experimented with to identify the mutation of lymphoma based on next-generation sequencing (NGS). We applied NGS analysis to circulating tumor DNA (ctDNA) in 20 lymphoma patients. Then, we compared treatment outcomes, and clinical characteristics among these patients, then investigated mutational profiling. Two independent cohorts of 241 patients with mature B cell lymphoma in Mature B-cell malignancies data set (MBN) data set and 50 diffuse large B-cell lymphoma (DLBCL) patients in DLBCL data set, were used to examine the association between gene mutations and prognosis. We found ctDNA positive group had significantly more relapsed/PD (7/12, 58.3%) and less CR/PR patients (1/12, 8.3%) compared to negative group (0, 0%) (5/8, 62.5%) (p < 0.001). Somatic alterations were identified in 12 of 20 patients and the total 11 mutations were: Ataxia telangiectasia mutated (ATM), TP53, BCL2, BTG2, CD28, EP300, IDH2, IRF8, JAK3, NOTCH1, and NRAS. ATM (S2168L) was found in SLL and TLBL for the first time. BTG2 (c.292_293del), CD28 (P119T), IRF8 (E74D) and NOTCH1 (c.4348 G > A) were newly detected in DLBCL, angioimmunoblastic T-cell lymphoma, primary central nervous system lymphoma, and BCL for the first time respectively. We also disclosed an unreported mutation EP300 (c.1058_1059insC) in DLBCL. Our cases implied ctDNA detection consistent with the FISH of tissue samples to some extent, speculating new molecular subtypes of DLBCL, finding some potential drug-resistant mutations, and suggesting disease recurrence. Moreover, in MBN and DLBCL datasets, patients with TP53 mutation had a significantly shorter OS (all p < 0.05) in both circulating free DNA and tumor tissue. The mutations (no SNP) of NOTCH1 (all p < 0.05) significantly contributed to worse OS in the two cohorts.     

Monitoring of cancer patients via next‐generation sequencing of patient‐derived circulating tumor cells and tumor DNA

Liquid biopsy of circulating tumor cells (CTC) and circulating tumor DNA (ctDNA) is gaining attention as a method for real‐time monitoring in cancer patients. Conventional methods based upon epithelial cell adhesion molecule (EpCAM) expression have a risk of missing the most aggressive CTC subpopulations due to epithelial‐mesenchymal transition and may, thus, underestimate the total number of actual CTC present in the bloodstream. Techniques utilizing a label‐free inertial microfluidics approach (LFIMA) enable efficient capture of CTC without the need for EpCAM expression. In this study, we optimized a method for analyzing genetic alterations using next‐generation sequencing (NGS) of extracted ctDNA and CTC enriched using an LFIMA as a first‐phase examination of 30 patients with head and neck cancer, esophageal cancer, gastric cancer and colorectal cancer (CRC). Seven patients with advanced CRC were enrolled in the second‐phase examination to monitor the emergence of alterations occurring during treatment with epidermal growth factor receptor (EGFR)‐specific antibodies. Using LFIMA, we effectively captured CTC (median number of CTC, 14.5 cells/mL) from several types of cancer and detected missense mutations via NGS of CTC and ctDNA. We also detected time‐dependent genetic alterations that appeared during anti–EGFR therapy in CTC and ctDNA from CRC patients. The results of NGS analyses indicated that alterations in the genomic profile revealed by the liquid biopsy could be expanded by using a combination of assays with CTC and ctDNA. The study was registered with the University Hospital Medical Information Network Clinical Trials Registry (ID: UMIN000014095).     

Application of plasma circulating KRAS mutations as a predictive biomarker for targeted treatment of pancreatic cancer

Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations in circulating tumor deoxyribonucleic acid (ctDNA) have been reported as representative noninvasive prognostic markers for pancreatic ductal adenocarcinoma (PDAC). Here, we aimed to evaluate single KRAS mutations as prognostic and predictive biomarkers, with an emphasis on potential therapeutic approaches to PDAC. A total of 128 patients were analyzed for multiple or single KRAS mutations (G12A, G12C, G12D, G12R, G12S, G12V, and G13D) in their tumors and plasma using droplet digital polymerase chain reaction (ddPCR). Overall, KRAS mutations were detected by multiplex ddPCR in 119 (93%) of tumor DNA and 68 (53.1%) of ctDNA, with a concordance rate of 80% between plasma ctDNA and tumor DNA in the metastatic stage, which was higher than the 44% in the resectable stage. Moreover, the prognostic prediction of both overall survival (OS) and progression-free survival (PFS) was more relevant using plasma ctDNA than tumor DNA. Further, we evaluated the selective tumor-suppressive efficacy of the KRAS G12C inhibitor sotorasib in a patient-derived organoid (PDO) from a KRAS G12C-mutated patient using a patient-derived xenograft (PDX) model. Sotorasib showed selective inhibition in vitro and in vivo with altered tumor microenvironment, including fibroblasts and macrophages. Collectively, screening for KRAS single mutations in plasma ctDNA and the use of preclinical models of PDO and PDX with genetic mutations would impact precision medicine in the context of PDAC.     

Circulating tumor cell telomerase activity as a prognostic marker for overall survival in SWOG 0421: A phase III metastatic castration resistant prostate cancer trial

Circulating tumor cells (CTC) are promising biomarkers in metastatic castration resistant prostate cancer (mCRPC), and telomerase activity (TA) is a recognized cancer marker. Therefore, we hypothesized that CTC TA may be prognostic of overall survival (OS) in mCRPC. To test this, we used a novel Parylene‐C slot microfilter to measure live CTC TA in S0421, a phase III SWOG‐led therapeutic trial. Blood samples underwent CTC capture and TA measurement by microfilter, as well as parallel enumeration by CellSearch (Janssen/J&J). Cox regression was used to assess baseline (pre‐treatment) TA versus OS, and recursive partitioning was used to explore potential prognostic subgroups and to generate Kaplan‐Meier (KM) OS curves. Samples were obtained from 263 patients and generated 215 TA measures. In patients with baseline CTC count ≥5 (47% of patients), higher CTC TA was associated with hazard ratio 1.14 (p = 0.001) for OS after adjusting for other clinical covariates including CTC counts and serum PSA at study entry. Recursive partitioning identified new candidate risk groups with KM OS curve separation based on CTC counts and TA. Notably, in men with an intermediate range baseline CTC count (6–54 CTCs/7.5 ml), low versus high CTC TA was associated with median survival of 19 versus 12 months, respectively (p = 0.009). Baseline telomerase activity from CTCs live‐captured on a new slot microfilter is the first CTC‐derived candidate biomarker prognostic of OS in a large patient subgroup in a prospective clinical trial. CTC telomerase activity thus merits further study and validation as a step towards molecular CTC‐based precision cancer management.     

Detection of circulating tumor DNA without a tumor-informed search using next-generation sequencing is a prognostic biomarker in pancreatic ductal adenocarcinoma

The confounding effects of next-generation sequencing (NGS) noise on detection of low frequency circulating tumor DNA (ctDNA) without a priori knowledge of solid tumor mutations has limited the applications of circulating cell-free DNA (ccfDNA) in clinical oncology. Here, we use a 118 gene panel and leverage ccfDNA technical replicates to eliminate NGS-associated errors while also enhancing detection of ctDNA from pancreatic ductal adenocarcinomas (PDACs). Pre-operative ccfDNA and tumor DNA were acquired from 14 patients with PDAC (78.6% stage II-III). Post-operative ccfDNA was also collected from 11 of the patients within 100 days of surgery. ctDNA detection was restricted to variants corresponding to pathogenic mutations in PDAC present in both replicates. PDAC-associated pathogenic mutations were detected in pre-operative ccfDNA in four genes (KRAS, TP53, SMAD4, ALK) from five patients. Of the nine ctDNA variants detected (variant allele frequency: 0.08%-1.59%), five had a corresponding mutation in tumor DNA. Pre-operative detection of ctDNA was associated with shorter survival (312 vs. 826 days; χ2=5.4, P = 0.021). Guiding ctDNA detection in pre-operative ccfDNA based on mutations present in tumor DNA yielded a similar survival analysis. Detection of ctDNA in the post-operative ccfDNA with or without tumor-informed guidance was not associated with outcomes. Therefore, the detection of PDAC-derived ctDNA during a broad and untargeted survey of ccfDNA with NGS may be a valuable, non-invasive, prognostic biomarker to integrate into the clinical assessment and management of patients prior to surgery.     

Characterization of metastatic breast cancer patients with nondetectable circulating tumor cells

Circulating tumor cells (CTC) are an independent prognostic factor in metastatic breast cancer patients (MBC). However, CTC are undetectable in one third of patients. The aim of this study was to assess the prognostic factors in MBC patients without detectable CTC. This retrospective study included 292 MBC patients evaluated between January 2004 and December 2007. CTC were enumerated before patients started a new line of treatment using the CellSearch?. Overall survival (OS) was calculated from the date of CTC measurement and estimated by the Kaplan‐Meier product limit method. CTC were not detected in 35.96% patients, whereas 40.75% patients had CTC ≥ 5. Undetectable CTC status was positively correlated with presence of brain metastasis (OR: 6.17, 95%CI = 2.14–17.79; p = 0.001), and inversely correlated with bone metastasis (OR: 0.47; 95%CI = 0.27–0.80; p = 0.01). In multivariate analysis, hormone receptors, number of metastatic sites and lines of therapy were independent prognostic factors for OS in patients without detectable CTC. Patients without detectable CTC before starting of a new line of therapy comprise a heterogeneous group with substantially different prognosis. We showed that some important metastatic disease characteristics are predictive of undetectable CTC status in MBC.     

Analysis of colorectal cancer‐related mutations by liquid biopsy: Utility of circulating cell‐free DNA and circulating tumor cells

We recruited 56 colorectal cancer patients and compared the mutational spectrum of tumor tissue DNA, circulating cell‐free DNA (ccfDNA) and circulating tumor cell (CTC) DNA (ctcDNA) to evaluate the potential of liquid biopsy to detect heterogeneity of cancer. Tumor tissue DNA, ccfDNA, and ctcDNA were extracted from each patient and analyzed using next‐generation sequencing (NGS) and digital PCR. To maximize yields of CTC, three antibodies were used in the capture process. From 34 untreated patients, 53 mutations were detected in tumor tissue DNA using NGS. Forty‐seven mutations were detected in ccfDNA, including 20 not detected in tissues. Sixteen mutations were detected in ctcDNA, including five not detected in tissues. In 12 patients (35.3%), mutations not found in tumor tissues were detected by liquid biopsy: nine (26.5%) in ccfDNA only and three (8.8%) in ctcDNA only. Combination analysis of the two liquid biopsy samples increased the sensitivity to detect heterogeneity. From 22 stage IV patients with RAS mutations in their primary tumors, RAS mutations were detected in 14 (63.6%) ccfDNA and in eight (36.4%) ctcDNA using digital PCR. Mutations not detected in primary tumors can be identified in ccfDNA and in ctcDNA, indicating the potential of liquid biopsy in complementing gene analysis. Combination analysis improves sensitivity. Sensitivity to detect cancer‐specific mutations is higher in ccfDNA compared with ctcDNA.     

Clinical significance of the mutational landscape and fragmentation of circulating tumor DNA in renal cell carcinoma

Reliable biomarkers for renal cell carcinoma (RCC) have yet to be determined. Circulating tumor DNA (ctDNA) is an emerging resource to detect and monitor molecular characteristics of various tumors. The present study aims to clarify the clinical utility of ctDNA for RCC. Fifty‐three patients histologically diagnosed with clear cell RCC were enrolled. Targeted sequencing was carried out using plasma cell‐free DNA (cfDNA) and tumor DNA. We applied droplet digital PCR (ddPCR) to validate detected mutations. cfDNA fragment size was also evaluated using a microfluidics‐based platform and sequencing. Proportion of cfDNA fragments was defined as the ratio of small (50‐166 bp) to large (167‐250 bp) cfDNA fragments. Association of mutant allele frequency of ctDNA with clinical course was analyzed. Prognostic potential was evaluated using log‐rank test. A total of 38 mutations across 16 (30%) patients were identified from cfDNA, including mutations in TP53 (n = 6) and VHL (n = 5), and median mutant allele frequency of ctDNA was 10%. We designed specific ddPCR probes for 11 mutations and detected the same mutations in both cfDNA and tumor DNA. Positive ctDNA was significantly associated with a higher proportion of cfDNA fragments (P = .033), indicating RCC patients with ctDNA had shorter fragment sizes of cfDNA. Interestingly, the changes of mutant allele frequency in ctDNA concurrently correlated with clinical course. Positive ctDNA and fragmentation of cfDNA were significantly associated with poor cancer‐specific survival (P < .001, P = .011). In conclusion, our study shows the clinical utility of ctDNA status and cfDNA fragment size as biomarkers for prognosis and disease monitoring in RCC.     

Circulating tumor cells in metastatic breast cancer

BACKGROUND.

The aim of the current study was to assess the prognostic value of baseline circulating tumor cells (CTCs) in a large cohort of patients with newly diagnosed metastatic breast cancer (MBC).

METHODS.

This retrospective study included 185 patients with newly diagnosed MBC evaluated between 2001 and 2007. CTCs were isolated and enumerated before patients started first‐line treatment using the CellSearch system. Overall survival (OS) was calculated from the date of CTC measurement, estimated by the Kaplan‐Meier product limit method, and compared between groups with the log‐rank test. Cox proportional hazards models were fitted to determine the association between CTC levels and OS after controlling for other prognostic factors.

RESULTS.

The median age of the patients at the time of MBC diagnosis was 49 years. Fifty‐six (30.3%) patients presented with de novo metastatic disease, and 129 (69.7%) presented with newly recurrent breast cancer. A total of 114 patients (61.6%) had CTC <5, and 71 (38.4%) had CTC ≥5. The median OS was 28.3 months and 15 months (P < .0001) for patients with CTC <5 and CTC ≥5, respectively. Superior survival among patients with CTC <5 was observed regardless of hormone receptor and HER‐2/neu status, site of first metastases, or whether the patient had recurrent or de novo metastatic disease. In the multivariate model, patients with CTC ≥5 had a hazards ratio of death of 3.64 (95% confidence interval, 2.11‐6.30) compared with patients with CTC <5.

CONCLUSIONS.

The results of this large retrospective study confirms that CTCs are a strong independent predictor of survival among women with either de novo or newly recurrent MBC. CTCs should be considered as a new stratification method for women with newly diagnosed MBC. Cancer 2008. © 2008 American Cancer Society.     

Post-radiation circulating tumor DNA as a prognostic factor in locally advanced esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is a highly malignant and deadly tumor. Radiation therapy is one of the primary treatments for locally advanced ESCC. However, the biomarkers for prognosis of definitive radiation remain undefined. Peripheral blood circulating tumor (ct)DNA provides information of tumor genetic alterations and has been confirmed as a potential non-invasive biomarker for several types of cancer. The present study investigated the clinical implications of ctDNA detection in patients with ESCC and receiving definitive radiation therapy. Patients with locally advanced ESCC were retrospectively recruited. Plasma samples were collected before, during and following radiation therapy. Next-generation sequencing was performed to identify somatic mutations in 180 genes. A total of 69 baseline and post-radiation plasma samples were collected from 25 patients. A total of 59 non-silent single nucleotide variants were present in 33 genes. All pre-radiation and 58.3% (14/24) of post-radiation samples had at least one mutation. Patients with lymph node metastases (LNM) exhibited a higher number of pre-radiation mutations compared with those without LNM. The variables, progression-free survival (PFS) and overall survival (OS) of the patients with one baseline mutation were not significantly different compared with that in patients with more than one baseline mutation. Patients with initial ctDNA-positive post-radiation samples exhibited significantly reduced PFS (P=0.047) and OS (P=0.005) compared with that in patients with ctDNA-negative samples. The post-radiation plasma ctDNA status was an independent prognostic factor from univariate and multivariate analyses. Dynamic monitoring of ctDNA during follow-up was examined. The results indicated that ctDNA was a predictive and prognostic marker in patients with ESCC and receiving definitive radiation therapy, which may guide subsequent treatment.     

Ultradeep targeted sequencing of circulating tumor DNA in plasma of early and advanced breast cancer

We present a study to evaluate the feasibility and clinical utility of amplicon‐based Oncomine Pan‐Cancer cell‐free assay to detect circulating tumor DNA (ctDNA) in patients with early or advanced breast cancer. In this study, 109 early and metastatic breast cancer patients were recruited before the initiation of treatment. ctDNA mutation profiles were assessed through unique molecular tagging (UMT) and ultradeep next generation sequencing (NGS). For patients with mutations, DNA from corresponding white blood cells (WBC) was sequenced to exclude variants of clonal‐hematopoietic (CH) origin. UMT targeted sequencing from plasma of 109 patients achieved a median total coverage of 55 498X and a median molecular coverage of 4187X. Among 53 ctDNA positive samples, 38% were mutation positive by WBC sequencing, indicating potentially false‐positive results contributed by CH origin. Prevalence of CH‐related mutations was associated with age (P = 7.51 × 10⁻⁴). After exclusion of CH mutations, ctDNA detection rates were 37% for local or locally advanced breast cancer (stage I‐III) and 81% for metastatic or recurrent breast cancer. The ctDNA detection rate correlated with disease stage (P = 2.60 × 10⁻⁴), nodal spread (P = 6.49 × 10⁻³) and the status of distant metastases (P = 5.00 × 10⁻⁴). ctDNA variants were detected mostly in TP53, PIK3CA and AKT1 genes, with variants showing therapeutic relevance. This pilot study endorses the use of targeted NGS for non‐invasive molecular profiling of breast cancer. Paired sequencing of plasma ctDNA and WBC should be implemented to improve accurate interpretation of liquid biopsy.     

Presence of EpCAM‐positive circulating tumor cells as biomarker for systemic disease strongly correlates to survival in patients with hepatocellular carcinoma

Current imaging technologies do not sufficiently detect micrometastasis and therefore do not allow adequate stratification of patients with hepatocellular carcinoma (HCC) for curative or systemic therapy. In HCC, presence of stem cell‐like, epithelial cell adhesion molecule (EpCAM)‐positive cells correlates with tumor aggressiveness and formation of metastasis. Therefore, we investigated the prognostic relevance of EpCAM‐positive circulating tumor cells (CTCs) in patients with HCC. Blood from 78 patients (19 patients in the control cohort and 59 patients with HCC) was tested for CTCs with the CellSearch? system. Correlation analysis to overall survival (OS), the Barcelona Clinic Liver Cancer (BCLC) staging system, macroscopic and microscopic vascular invasion and alpha‐fetoprotein (AFP) levels were performed. We detected ≥1 CTC in 18/59 HCC patients and in 1/19 patients with cirrhosis or benign hepatic tumor (p = 0.026). OS was significantly shorter (460 vs. 746 days) in the CTC‐positive cohort (p = 0.017). Comparing BCLC stages, significant differences in CTC detection rates were also observed: BCLC stages A 1/9, B 6/31 and C 11/19 (p = 0.006). Ten of 18 patients with macroscopic and 10/16 patients with microscopic vascular invasion exhibited positive findings in CTC testing (p = 0.004 and p = 0.006). Furthermore, CTC results correlated to AFP (cutoff > 400 ng/mL) levels (p = 0.050). Our study demonstrates frequent presence of EpCAM‐positive CTC in patients with intermediate or advanced HCC and its prognostic value for OS with possible implications for future treatment stratification.     

Serial monitoring of genomic alterations in circulating tumor cells of ER‐positive/HER2‐negative advanced breast cancer: feasibility of precision oncology biomarker detection

Nearly all estrogen receptor (ER)‐positive (POS) metastatic breast cancers become refractory to endocrine (ET) and other therapies, leading to lethal disease presumably due to evolving genomic alterations. Timely monitoring of the molecular events associated with response/progression by serial tissue biopsies is logistically difficult. Use of liquid biopsies, including circulating tumor cells (CTC) and circulating tumor DNA (ctDNA), might provide highly informative, yet easily obtainable, evidence for better precision oncology care. Although ctDNA profiling has been well investigated, the CTC precision oncology genomic landscape and the advantages it may offer over ctDNA in ER‐POS breast cancer remain largely unexplored. Whole‐blood (WB) specimens were collected at serial time points from patients with advanced ER‐POS/HER2‐negative (NEG) advanced breast cancer in a phase I trial of AZD9496, an oral selective ER degrader (SERD) ET. Individual CTC were isolated from WB using tandem CellSearch^®/DEPArray™ technologies and genomically profiled by targeted single‐cell DNA next‐generation sequencing (scNGS). High‐quality CTC (n = 123) from 12 patients profiled by scNGS showed 100% concordance with ctDNA detection of driver estrogen receptor α (ESR1) mutations. We developed a novel CTC‐based framework for precision medicine actionability reporting (MI‐CTCseq) that incorporates novel features, such as clonal predominance and zygosity of targetable alterations, both unambiguously identifiable in CTC compared to ctDNA. Thus, we nominated opportunities for targeted therapies in 73% of patients, directed at alterations in phosphatidylinositol‐4,5‐bisphosphate 3‐kinase catalytic subunit alpha (PIK3CA), fibroblast growth factor receptor 2 (FGFR2), and KIT proto‐oncogene, receptor tyrosine kinase (KIT). Intrapatient, inter‐CTC genomic heterogeneity was observed, at times between time points, in subclonal alterations. Our analysis suggests that serial monitoring of the CTC genome is feasible and should enable real‐time tracking of tumor evolution during progression, permitting more combination precision medicine interventions.     

Circulating cKIT and PDGFRA DNA indicates disease activity in Gastrointestinal Stromal Tumor (GIST)

This prospective trial aimed to investigate whether tumor-specific cKIT and PDGFRA mutations can be detected and quantified in circulating tumor (ct)DNA in patients with active GIST, and whether detection indicates disease activity. We included 25 patients with active disease and cKIT or PDGFRA mutations detected in tissue. Mutant ctDNA was detected in the peripheral blood plasma using allele-specific ligation (L-)PCR and droplet digital (d)PCR. CtDNA harboring tumor-specific cKIT or PDGFRA mutations was detected at least once in 16 out of 25 patients using L-PCR (64%) and in 20 out of 25 patients with dPCR (80%). Using dPCR, the absolute numbers of ctDNA fragments (DNA copies/ml) and the mutant allele frequency (MAF; in percent of wild-type control) strongly correlated with tumor size expressed as RECIST1.1 sum of diameter (SOD) in mm (ρ = 0.3719 and 0.408, respectively, p < 0.0001) and response status (ρ = 0.3939 and 0.392, respectively, p < 0.0001 and p < 0.001). Specificity of dPCR for detection of progression was 79.2% with a sensitivity of 55.2% and dPCR discriminated CR from active disease with a specificity of 96% and s sensitivity of 44.7%. With L-PCR, correlations of MAF with tumor size and response status were less prominent. Serial ctDNA measurement reflected individual disease courses over time. Targeted panel sequencing of four patients detected additional driver mutations in all cases and secondary resistance mutations in two cases. Thus, ctDNA indicates disease activity in patients with GIST and should be incorporated as companion biomarker in future prospective trials.     

Targeted Next-Generation Sequencing of Circulating Tumor DNA Mutations among Metastatic Breast Cancer Patients

Liquid biopsy through the detection of circulating tumor DNA (ctDNA) has potential advantages in cancer monitoring and prediction. However, most previous studies in this area were performed with a few hotspot genes, single time point detection, or insufficient sequencing depth. In this study, we performed targeted next-generation sequencing (NGS) with a customized panel in metastatic breast cancer (MBC) patients. Fifty-four plasma samples were taken before chemotherapy and after the third course of treatment for detection and analysis. Paired lymphocytes were also included to eliminate clonal hematopoiesis (CH)-related alternatives. A total of 1182 nonsynonymous mutations in 419 genes were identified. More ctDNA mutations were detected in patients with tumors > 3 cm (p = 0.035) and HER2(−) patients (p = 0.029). For a single gene, the distribution of ctDNA mutations was also correlated with clinical characteristics. Multivariate regression analysis revealed that HER2 status was significantly associated with mutation burden (OR 0.02, 95% CI 0–0.62, p = 0.025). The profiles of ctDNA mutations exhibited marked discrepancies between two time points, and baseline ctDNA was more sensitive and specific than that after chemotherapy. Finally, elevated ctDNA mutation level was positively correlated with poor survival (p < 0.001). Mutations in ctDNA could serve as a potential biomarker for the evaluation, prediction, and clinical management guidance of MBC patients with chemotherapy.