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.     

Ultra-deep next-generation sequencing of plasma cell-free DNA in patients with advanced lung cancers: results from the Actionable Genome Consortium

BackgroundNoninvasive genotyping using plasma cell-free DNA (cfDNA) has the potential to obviate the need for some invasive biopsies in cancer patients while also elucidating disease heterogeneity. We sought to develop an ultra-deep plasma next-generation sequencing (NGS) assay for patients with non-small-cell lung cancers (NSCLC) that could detect targetable oncogenic drivers and resistance mutations in patients where tissue biopsy failed to identify an actionable alteration.Patients and methodsPlasma was prospectively collected from patients with advanced, progressive NSCLC. We carried out ultra-deep NGS using cfDNA extracted from plasma and matched white blood cells using a hybrid capture panel covering 37 lung cancer-related genes sequenced to 50 000× raw target coverage filtering somatic mutations attributable to clonal hematopoiesis. Clinical sensitivity and specificity for plasma detection of known oncogenic drivers were calculated and compared with tissue genotyping results. Orthogonal ddPCR validation was carried out in a subset of cases.ResultsIn 127 assessable patients, plasma NGS detected driver mutations with variant allele fractions ranging from 0.14% to 52%. Plasma ddPCR for EGFR or KRAS mutations revealed findings nearly identical to those of plasma NGS in 21 of 22 patients, with high concordance of variant allele fraction (r = 0.98). Blinded to tissue genotype, plasma NGS sensitivity for de novo plasma detection of known oncogenic drivers was 75% (68/91). Specificity of plasma NGS in those who were driver-negative by tissue NGS was 100% (19/19). In 17 patients with tumor tissue deemed insufficient for genotyping, plasma NGS identified four KRAS mutations. In 23 EGFR mutant cases with acquired resistance to targeted therapy, plasma NGS detected potential resistance mechanisms, including EGFR T790M and C797S mutations and ERBB2 amplification.ConclusionsUltra-deep plasma NGS with clonal hematopoiesis filtering resulted in de novo detection of targetable oncogenic drivers and resistance mechanisms in patients with NSCLC, including when tissue biopsy was inadequate for genotyping.     

Prospective blinded study of somatic mutation detection in cell-free DNA utilizing a targeted 54-gene next generation sequencing panel in metastatic solid tumor patients

Sequencing of the mutant allele fraction of circulating cell-free DNA (cfDNA) derived from tumors is increasingly utilized to detect actionable genomic alterations in cancer.We conducted a prospective blinded study of a comprehensive cfDNA sequencing panel with 54 cancer genes. To evaluate the concordance between cfDNA and tumor DNA (tDNA), sequencing results were compared between cfDNA from plasma and genomic tumor DNA (tDNA). Utilizing next generation digital sequencing technology (DST), we profiled approximately 78,000 bases encoding 512 complete exons in the targeted genes in cfDNA from plasma. Seventy-five patients were prospectively enrolled between February 2013 and March 2014, including 61 metastatic cancer patients and 14 clinical stage II CRC patients with matched plasma and tissue samples. Using the 54-gene panel, we detected at least one somatic mutation in 44 of 61 tDNA (72.1%) and 29 of 44 (65.9%) cfDNA. The overall concordance rate of cfDNA to tDNA was 85.9%, when all detected mutations were considered. We collected serial cfDNAs during cetuximab-based treatment in 2 metastatic KRAS wild-type CRC patients, one with acquired resistance and one with primary resistance. We demonstrate newly emerged KRAS mutation in cfDNA 1.5 months before radiologic progression. Another patient had a newly emerged PIK3CA H1047R mutation on cfDNA analysis at progression during cetuximab/irinotecan chemotherapy with gradual increase in allele frequency from 0.8 to 2.1%. This blinded, prospective study of a cfDNA sequencing showed high concordance to tDNA suggesting that the DST approach may be used as a non-invasive biopsy-free alternative to conventional sequencing using tumor biopsy.     

Improved EGFR mutation detection using combined exosomal RNA and circulating tumor DNA in NSCLC patient plasma

BackgroundA major limitation of circulating tumor DNA (ctDNA) for somatic mutation detection has been the low level of ctDNA found in a subset of cancer patients. We investigated whether using a combined isolation of exosomal RNA (exoRNA) and cell-free DNA (cfDNA) could improve blood-based liquid biopsy for EGFR mutation detection in non-small-cell lung cancer (NSCLC) patients.Patients and methodsMatched pretreatment tumor and plasma were collected from 84 patients enrolled in TIGER-X (NCT01526928), a phase 1/2 study of rociletinib in mutant EGFR NSCLC patients. The combined isolated exoRNA and cfDNA (exoNA) was analyzed blinded for mutations using a targeted next-generation sequencing panel (EXO1000) and compared with existing data from the same samples using analysis of ctDNA by BEAMing.ResultsFor exoNA, the sensitivity was 98% for detection of activating EGFR mutations and 90% for EGFR T790M. The corresponding sensitivities for ctDNA by BEAMing were 82% for activating mutations and 84% for T790M. In a subgroup of patients with intrathoracic metastatic disease (M0/M1a; n = 21), the sensitivity increased from 26% to 74% for activating mutations (P = 0.003) and from 19% to 31% for T790M (P = 0.5) when using exoNA for detection.ConclusionsCombining exoRNA and ctDNA increased the sensitivity for EGFR mutation detection in plasma, with the largest improvement seen in the subgroup of M0/M1a disease patients known to have low levels of ctDNA and poses challenges for mutation detection on ctDNA alone.Clinical TrialsNCT01526928     

Analysis of ctDNA to predict prognosis and monitor treatment responses in metastatic pancreatic cancer patients

Cell‐free circulating tumor DNA (ctDNA) in plasma has been used as a potential noninvasive biomarker for various tumors. Our study was performed to evaluate the clinical implications of ctDNA detection in patients with metastatic pancreatic cancer. First, we attempted to prospectively screen a panel of 60 genes in cell‐free DNA (cfDNA) from ten metastatic pancreatic cancer patients via exome sequencing. Second, droplet digital PCR (ddPCR) was used to identify potential mutations in a cohort of 188 patients with metastatic pancreatic cancer. Finally, to preliminary evaluate the potential role of ctDNA in monitoring tumor responses following chemotherapy, we detected the presence of ctDNA in serial plasma samples from 13 metastatic pancreatic cancer patients (Clinical trial: NCT02017015). The analysis revealed five somatic mutations at BRCA2, EGFR, KDR and ERBB2 gene loci. The frequencies of ctDNA mutation at BRCA2, KDR, EGFR, ERBB2 exon17 and ERBB2 exon27 were 11.7%, 13.8%, 13.3%, 13.3% and 6.4% respectively. Univariate and multivariate analyses identified the ERBB2 exon17 mutation (p = 0.035, HR = 1.61) as an independent factor associated with overall survival among metastatic pancreatic cancer patients. Furthermore, the rate of coincident detection of ctDNA and response to treatment as assessed by CT imaging was 76.9% (10 of 13 cases), and the presence of ctDNA provided the earliest measure of treatment in 6 of 10 patients (60%). ctDNA sequencing may have clinical value for determining metastatic pancreatic cancer treatment and monitoring the tumor response.     

Comprehensive Analysis of the Discordance of EGFR Mutation Status between Tumor Tissues and Matched Circulating Tumor DNA in Advanced Non–Small Cell Lung Cancer

IntroductionThis study aimed to address the underlying reasons for and clinical significance of the discordant EGFR mutation (EGFRm) status between tumor tissue (TT) and circulating tumor DNA (ctDNA).MethodsThree groups of EGFR tyrosine kinase inhibitor (EGFR TKI)-treated patients whose EGFRm status was determined by the amplification refractory mutation system (ARMS) were included (group A, TT-positive/ctDNA-positive EGFRm status; group B, TT-negative/ctDNA-positive EGFRm status; and group C, TT-positive/ctDNA-negative EGFRm status). Patients with discordant EGFRm status were reevaluated by droplet digital polymerase chain reaction (ddPCR) and next-generation sequencing. Meanwhile, surgical tumor specimens were microdissected for EGFRm detection by ddPCR.ResultsOf the 2463 patients with matched TT and ctDNA specimens, 1017 patients carried EGFRm in TT and/or ctDNA by the ARMS. Of these 1017 patients, 472 received EGFR TKIs, including 264, 28, and 180 in groups A, B, and C, respectively. The median progression-free survivals of those receiving EGFR TKIs across the three groups were similar (p = 0.062). Through ddPCR and next-generation sequencing of biopsy specimens (n = 22) and microdissected surgical specimens (n = 5), 27 patients in group B were identified as harboring EGFRm. After reevaluation by ddPCR, 64 patients in group C tested positive for EGFRm in their ctDNA. ctDNA as a screen for EGFRm then tissues as supplement (ctDNA→TT pattern) had similar detection efficiency and saved about 30% of TT compared with TT for initial EGFRm detection followed by ctDNA (TT→ctDNA pattern).ConclusionsIntratumor heterogeneity and the relatively low sensitivity of the ARMS contributed to discordant EGFRm status between TT specimens and ctDNA. The ctDNA→TT pattern might be a rational clinical procedure for EGFRm determination.     

Clinical Utility of Liquid Biopsy (Cell-free DNA) Based EGFR Mutation Detection Post treatment Initiation as a Disease Monitoring Tool in Patients With Advanced EGFR-mutant NSCLC

IntroductionPlasma cfDNA-based mutation analysis has shown disease-monitoring potential in various cancers. We assessed the potential of cfDNA-based EGFR mutation testing as a monitoring tool in patients with NSCLC.Patients and MethodsPatients with NSCLC harboring EGFR mutations receiving first-line treatment as per institutional protocol were enrolled. EGFR mutation status was determined using plasma samples at baseline and post treatment initiation. Patients in whom EGFR mutation was detected or persisted after treatment initiation were considered circulating tumor DNA (ctDNA)-positive. Progression-free survival (PFS) and overall survival (OS) for ctDNA-positive and negative patients post treatment initiation were the primary endpoints; concordance for baseline EGFR status between tissue and plasma and proportion of patients who were ctDNA-positive post treatment initiation were the secondary endpoints.ResultsWe enrolled 158 patients; 76 received gefitinib, and 82 received gefitinib plus chemotherapy. Median follow-up duration was 42 months. About 25% of patients were ctDNA-positive post treatment initiation. Median PFS for ctDNA-negative patients post treatment initiation was 14 (95% confidence interval [CI], 12.0-17.0) months, while that for ctDNA-positive patients was 8 (95% CI, 6.0-10.0) months. Median OS for ctDNA-negative patients post treatment initiation was 27 (95% CI, 24.0-32.0) months, while that for ctDNA-positive patients was 15 (95% CI, 11.0-19.0) months. Concordance at baseline between tissue and plasma samples was 75.4%.ConclusionPlasma-based EGFR mutation detection post treatment initiation can be used as a predictive marker for outcome in patients with EGFR-mutant NSCLC receiving first-line treatment.     

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.     

High concordance of mutation patterns in 10 common mutated genes between tumor tissue and cell-free DNA in metastatic colorectal cancer

The concordance of mutation patterns between cell-free DNA (cfDNA) and tumor DNA varies in colorectal cancers (CRCs). Next-generation sequencing (NGS) by targeted sequencing can detect novel genes. We aimed to use NGS to test the concordance between cfDNA and tumor DNA in metastatic CRCs. A total of 95 paired tumor and peripheral blood samples from metastatic CRC patients were included. The tumor DNA and cfDNA were analyzed with a 10-gene NGS panel (Illumina HiSeq2500 system). The median number of mutations in tumor samples was 3 (range 0-7). The most commonly mutated gene was TP53 (63.2%), followed by APC (49.5%), KRAS (35.8%) and FAT4 (15.8%). The concordance of mutation patterns in these 10 genes was as high as 91% between cfDNA and tumor samples in these metastatic CRC patients. A sensitivity of 88.2% and specificity of 100% was found when using KRAS mutation status of cfDNA to predict KRAS mutation in tumor tissue. For tumor DNA with TP53, KRAS, or APC mutations, right-sided CRCs were more likely to develop peritoneal metastases, while for tumor DNA with TP53 mutations, left-sided tumors were more likely to have lung metastases. For cfDNA with TP53 or KRAS mutations, right-sided CRCs were more likely to have peritoneal metastases. Due to the high concordance of mutation patterns between cfDNA and tumor samples, monitoring the mutation pattern of cfDNA may be applicable in the treatment of metastatic CRC.     

Hybrid capture-based genomic profiling of circulating tumor DNA from patients with estrogen receptor-positive metastatic breast cancer

BackgroundGenomic changes that occur in breast cancer during the course of disease have been informed by sequencing of primary and metastatic tumor tissue. For patients with relapsed and metastatic disease, evolution of the breast cancer genome highlights the importance of using a recent sample for genomic profiling to guide clinical decision-making. Obtaining a metastatic tissue biopsy can be challenging, and analysis of circulating tumor DNA (ctDNA) from blood may provide a minimally invasive alternative.Patients and methodsHybrid capture-based genomic profiling was carried out on ctDNA from 254 female patients with estrogen receptor-positive breast cancer. Peripheral blood samples were submitted by clinicians in the course of routine clinical care between May 2016 and March 2017. Sequencing of 62 genes was carried out to a median unique coverage depth of 7503×. Genomic alterations (GAs) in ctDNA were evaluated and compared with matched tissue samples and genomic datasets of tissue from breast cancer.ResultsAt least 1 GA was reported in 78% of samples. Frequently altered genes were TP53 (38%), ESR1 (31%) and PIK3CA (31%). Temporally matched ctDNA and tissue samples were available for 14 patients; 89% of mutations detected in tissue were also detected in ctDNA. Diverse ESR1 GAs including mutation, rearrangement and amplification, were observed. Multiple concurrent ESR1 GAs were observed in 40% of ESR1-altered cases, suggesting polyclonal origin; ESR1 compound mutations were also observed in two cases. ESR1-altered cases harbored co-occurring GAs in PIK3CA (35%), FGFR1 (16%), ERBB2 (8%), BRCA1/2 (5%), and AKT1 (4%).ConclusionsGAs relevant to relapsed/metastatic breast cancer management were identified, including diverse ESR1 GAs. Genomic profiling of ctDNA demonstrated sensitive detection of mutations found in tissue. Detection of amplifications was associated with ctDNA fraction. Genomic profiling of ctDNA may provide a complementary and possibly alternative approach to tissue-based genomic testing for patients with estrogen receptor-positive metastatic breast cancer.     

Monitoring levels of circulating cell‐free DNA in patients with metastatic colorectal cancer as a potential biomarker of responses to regorafenib treatment

Circulating cell‐free DNA (cfDNA) contains circulating tumor DNA (ctDNA), which can be obtained from serial liquid biopsies to enable tumor genome analysis throughout the course of treatment. We investigated cfDNA and mutant ctDNA as potential biomarkers to predict the best outcomes of regorafenib‐treated metastatic colorectal cancer (mCRC) patients. We analyzed longitudinally collected plasma cfDNA of 43 mCRC patients prospectively enrolled in the phase II TEXCAN trial by IntPlex qPCR. Qualitative (KRAS, NRAS, BRAF^V600E mutations) and quantitative (total cfDNA concentration, mutant ctDNA concentration, mutant ctDNA fraction) parameters were correlated with overall survival (OS) and progression‐free survival (PFS). When examined as classes or continuous variables, the concentrations of total cfDNA, mutant ctDNA, and, partly, mutant ctDNA fraction prior to regorafenib treatment correlated with OS. Patients with baseline cfDNA > 26 ng·mL⁻¹ had shorter OS than those with cfDNA value below this threshold (4.0 vs 6.9 months; log‐rank P = 0.0366). Patients with baseline mutant ctDNA > 2 ng·mL⁻¹ had shorter OS than those with mutant ctDNA below this threshold (log‐rank P = 0.0154). We show that pretreatment cfDNA and mutant ctDNA levels may identify mCRC patients that may benefit from regorafenib treatment.     

Treatment of Non–Small-Cell Lung Cancer Based on Circulating Cell-Free DNA and Impact of Variation Allele Frequency

BackgroundNext-generation sequencing of circulating cell-free DNA (cfDNA) can identify sensitizing and resistance mutations in non–small-cell lung cancer (NSCLC). cfDNA is helpful when tissue is insufficient for genomic testing or repeat biopsy is not feasible or poses unacceptable risk. Here we report the experience of cfDNA testing at the time of diagnosis and how this intervention can help avoid further invasive interventions, how it can be used to determine initiation of therapy, and how variation allele frequency of the somatic alteration affects response to subsequent treatment.Patients and MethodsThis is a single-institution retrospective study of patients with advanced NSCLC who had cfDNA from plasma tested using the Guardant360 panel, which identifies somatic genomic alterations by massive parallel sequencing of target genes. An institutional Clinical Laboratory Improvement Amendments tissue panel using fluorescence in situ hybridization (for MET, RET, ROS1, and ALK) and next-generation sequencing for selected genes was used for tissue analysis. Actionable mutations are those with US Food and Drug Administration–approved targeted therapies (EGFR, ALK, ROS, BRAF, NTRK fusions) or therapies soon to be approved (RET fusions and MET amplifications, or MET exon 14 skipping mutation).ResultsA total of 163 blood samples from 143 patients were evaluated, 82 at diagnosis and 81 at disease progression. A total of 94 cases had tissue and cfDNA testing performed within 12 weeks of each other. Seventy-six (81%) of 94 cases were concordant, of which 22 cases were concordantly positive and 54 concordantly negative. Eighteen (19%) of 94 cases were discordant, of which 11 had negative blood and positive tissue results, and 7 had positive blood and negative tissue results. cfDNA testing had a sensitivity of 67% (95% confidence interval [CI], 51%, 83%), specificity of 89% (95% CI, 81%, 97%), negative predictive value of 83% (95% CI, 74%, 92%), and positive predictive value of 76% (95% CI, 60%, 91%). Nineteen (21%) of 82 cfDNA samples analyzed at diagnosis had actionable mutations identified (4 EGFR exon 19 deletion, 2 EGFR exon 21 L858R, 2 EGFR L861Q, 1 L861R, 4 EML4-ALK fusion, 2 CD74-ROS1 fusion, 2 MET exon 14 skipping mutation, 2 KIF5B-RET fusion). Of the 82 patients with cfDNA testing performed at the time of diagnosis, 8 patients (10%) initiated targeted therapy on the basis of cfDNA results only, with 6 patients experiencing partial response, 1 patient complete response, and 1 patient stable disease. The response rate for patients who initiated targeted therapies on the basis of cfDNA only at diagnosis was 88%. Variant allele frequency had no impact on response.ConclusionsInitiation of targeted therapy for advanced NSCLC was feasible based only on identification of actionable mutations by cfDNA testing in 9% of the cases for which tissue diagnosis could not be obtained. Actionable targets were identified by cfDNA in 20% of the samples sent at diagnosis. A substantial number of patients benefited from cfDNA testing at initial diagnosis because it identified actionable mutations that led to appropriate targeted treatments.     

Monitoring of somatic mutations in circulating cell-free DNA by digital PCR and next-generation sequencing during afatinib treatment in patients with lung adenocarcinoma positive for EGFR activating mutations

BackgroundAnalysis of circulating cell-free DNA (cfDNA) is under intensive investigation for its potential to identify tumor somatic mutations. We have now explored the usefulness of such liquid biopsy testing with both the digital polymerase chain reaction (dPCR) and next-generation sequencing (NGS) during treatment of patients with the epidermal growth factor receptor (EGFR) inhibitor afatinib.Patients and methodsEligible patients had advanced lung adenocarcinoma with EGFR activating mutations and were treated with afatinib. Plasma samples were collected before and during (4 and 24 weeks) afatinib treatment as well as at disease progression. Tumor and plasma DNA were analyzed by dPCR and NGS.ResultsThirty-five patients were enrolled. The objective response rate and median progression-free survival (PFS) were 77.1% and 13.8 months, respectively. Tumor and plasma DNA were available for 32 patients. dPCR and NGS detected EGFR activating mutations in 81.3% and 71.9% of baseline cfDNA samples, respectively. In 19 patients treated with afatinib for ≥24 weeks, the number of EGFR mutant alleles detected in cfDNA by dPCR declined rapidly and markedly after treatment onset, becoming undetectable or detectable at only a low copy number (<10 copies per milliliter) at 4 weeks. Median PFS was slightly longer for patients with undetectable EGFR mutant alleles in cfDNA at 4 weeks than for those in whom such alleles were detectable (14.3 versus 10.0 months). A total of 45 somatic mutations was identified in baseline tumor DNA, and 30 (66.7%) of these mutations were identified in cfDNA by NGS. Allele frequency for somatic mutations in cfDNA determined by NGS changed concordantly during afatinib treatment with the number of EGFR mutant alleles determined by dPCR.ConclusionsMonitoring of cfDNA by dPCR is informative for prediction of afatinib efficacy, whereas that by NGS is reliable and has the potential to identify mechanisms of treatment resistance.     

Genomewide copy number alteration screening of circulating plasma DNA: potential for the detection of incipient tumors

BackgroundEarly cancer diagnosis might improve survival rates. As circulating tumor DNA (ctDNA) carries cancer-specific modifications, it has great potential as a noninvasive biomarker for detection of incipient tumors.Patients and methodsWe collected cell-free DNA (cfDNA) samples of 1002 elderly without a prior malignancy, carried out whole-genome massive parallel sequencing and scrutinized the mapped sequences for the presence of (sub)chromosomal copy number alterations (CNAs) predictive for a malignancy. When imbalances were detected, 6-monthly clinical follow-up was carried out.ResultsIn 3% of participants chromosomal imbalances were detected. Follow-up analyses, including whole-body MRI screening, confirmed the presence of five hematologic malignancies: one Hodgkin lymphoma (HL), stage II; three non-HL (type chronic lymphocytic leukemia, Rai I–Binet A; type SLL, stage III; type mucosa-associated lymphoid tissue, stage I) and one myelodysplastic syndrome with excess blasts, stage II. The CNAs detected in cfDNA were tumor-specific. Furthermore, one case was identified with monoclonal B-cell lymphocytosis, a potential precursor of B-cell malignancy. In 24 additional individuals, CNAs were identified but no cancer diagnosis was made. For 9 of them, the aberrant cfDNA profile originated from peripheral blood cells. For 15 others the origin of aberrations in cfDNA remains undetermined.Conclusion(s)Genomewide profiling of cfDNA in apparently healthy individuals enables the detection of incipient hematologic malignancies as well as clonal mosaicism with unknown clinical significance. CNA screening of cellular DNA of peripheral blood in elderly has established that clonal mosaicism for these chromosomal anomalies predicts a 5- to 10-fold enhanced risk of a subsequent cancer. We demonstrate that cfDNA screening detects CNAs, which are not only derived from peripheral blood, but even more from other tissues. Since the clinical relevance of clonal mosaics in other tissues remains unknown, long-term follow-up is warranted. Taken together, this study demonstrates that genomewide cfDNA analysis has potential as an unbiased screening approach for hematological malignancies and premalignant conditions.     

Monitoring gastric cancer progression with circulating tumour DNA

Background:

Circulating tumour DNA (ctDNA) is an emerging candidate biomarker for malignancies and may be useful for monitoring the disease status of gastric cancer.

Methods:

We performed targeted deep sequencing of plasma cell-free DNA (cfDNA) by massively parallel sequencing in patients with tumours harbouring TP53 mutations. The quantitative values of TP53-ctDNA during the clinical course were compared with the tumour status.

Results:

Three out of ten patients with TP53 mutations in primary tumours showed detectable TP53 mutation levels in preoperative cfDNA. Although the cfDNA concentrations were not always reflective of the disease course, the ctDNA fraction correlated with the disease status.

Conclusions:

ctDNA may serve as a useful biomarker to monitor gastric cancer progression and residual disease.     

Concordance of blood- and tumor-based detection of RAS mutations to guide anti-EGFR therapy in metastatic colorectal cancer

BackgroundCirculating tumor DNA (ctDNA) is a potential source for tumor genome analysis. We explored the concordance between the mutational status of RAS in tumor tissue and ctDNA in metastatic colorectal cancer (mCRC) patients to establish eligibility for anti-epidermal growth factor receptor (EGFR) therapy.Patients and methodsA prospective-retrospective cohort study was carried out. Tumor tissue from 146 mCRC patients was tested for RAS status with standard of care (SoC) PCR techniques, and Digital PCR (BEAMing) was used both in plasma and tumor tissue.ResultsctDNA BEAMing RAS testing showed 89.7% agreement with SoC (Kappa index 0.80; 95% CI 0.71 − 0.90) and BEAMing in tissue showed 90.9% agreement with SoC (Kappa index 0.83; 95% CI 0.74 − 0.92). Fifteen cases (10.3%) showed discordant tissue-plasma results. ctDNA analysis identified nine cases of low frequency RAS mutations that were not detected in tissue, possibly due to technical sensitivity or heterogeneity. In six cases,RAS mutations were not detected in plasma, potentially explained by low tumor burden or ctDNA shedding. Prediction of treatment benefit in patients receiving anti-EGFR plus irinotecan in second- or third-line was equivalent if tested with SoC PCR and ctDNA. Forty-eight percent of the patients showed mutant allele fractions in plasma below 1%.ConclusionsPlasma RAS determination showed high overall agreement and captured a mCRC population responsive to anti-EGFR therapy with the same predictive level as SoC tissue testing. The feasibility and practicality of ctDNA analysis may translate into an alternative tool for anti-EGFR treatment selection.     

Clonal dynamics of circulating tumor DNA during immune checkpoint blockade therapy for melanoma

Assessment of treatment efficacy of immune checkpoint inhibitors in melanoma patients is difficult as the response to these therapies varies among patients or lesions. The clonal evolution of cancer during immune checkpoint blockade therapy could cause treatment resistance. We investigated the potential of liquid biopsy in monitoring the mutational profiles of metastatic melanoma during immunotherapy. Plasma samples collected from 21 Japanese metastatic melanoma patients before immune checkpoint blockade therapy were subjected to whole-exome sequencing (WES). Furthermore, 14 Japanese patients with melanoma were enrolled for longitudinal analysis of circulating tumor DNA (ctDNA). Plasma samples were collected prospectively before and during therapy and sequenced. WES of the pretreatment plasma from Japanese melanoma patients showed detectable ctDNA levels with wide ranges of variant allele frequencies within a sample, suggesting clonal and subclonal mutations in ctDNA. In targeted sequencing using longitudinal samples, ctDNA levels correlated with increased tumor size, while ctDNA content immediately decreased after a surge in a patient exhibiting pseudo-progression, suggesting the potential of ctDNA analysis in discriminating between pseudo- and true progression. Mutant ctDNA levels showed different patterns within the clinical course of specific patients, suggesting that these mutations were derived from different tumor clones with distinct therapeutic responses. During further investigation, WES of plasma samples from 1 patient showed marked differences in the mutational profiles of ctDNA, including expansive tumor evolution during an acute exacerbation. Immunotherapy may induce characteristic clonal evolutions of tumors; longitudinal analysis of ctDNA has the potential of determining these tumor evolution patterns and therapeutic responses.     

Mutated circulating tumor DNA as a liquid biopsy in lung cancer detection and treatment

Over the past decade, substantial developments have been made in the detection of circulating tumor DNA (ctDNA)—cell‐free DNA (cfDNA) fragments released into the circulation from tumor cells and displaying the genetic alterations of those cells. As such, ctDNA detected in liquid biopsies serves as a powerful tool for cancer patient stratification, therapy guidance, detection of resistance, and relapse monitoring. In this Review, we describe lung cancer diagnosis and monitoring strategies using ctDNA detection technologies and compile recent evidence regarding lung cancer‐related mutation detection in liquid biopsy. We focus not only on epidermal growth factor receptor (EGFR) alterations, but also on significant co‐mutations that shed more light on novel ctDNA‐based liquid biopsy applications. Finally, we discuss future perspectives of early‐cancer detection and clonal hematopoiesis filtering strategies, with possible inclusion of microbiome‐driven liquid biopsy.     

Targeted next-generation sequencing of circulating-tumor DNA for tracking minimal residual disease in localized colon cancer

BackgroundA high percentage of patients diagnosed with localized colon cancer (CC) will relapse after curative treatment. Although pathological staging currently guides our treatment decisions, there are no biomarkers determining minimal residual disease (MRD) and patients are at risk of being undertreated or even overtreated with chemotherapy in this setting. Circulating-tumor DNA (ctDNA) can to be a useful tool to better detect risk of relapse.Patients and methodsOne hundred and fifty patients diagnosed with localized CC were prospectively enrolled in our study. Tumor tissue from those patients was sequenced by a custom-targeted next-generation sequencing (NGS) panel to characterize somatic mutations. A minimum variant allele frequency (VAF) of 5% was applied for variant filtering. Orthogonal droplet digital PCR (ddPCR) validation was carried out. We selected known variants with higher VAF to track ctDNA in the plasma samples by ddPCR.ResultsNGS found known pathological mutations in 132 (88%) primary tumors. ddPCR showed high concordance with NGS (r-=-0.77) for VAF in primary tumors. Detection of ctDNA after surgery and in serial plasma samples during follow-up were associated with poorer disease-free survival (DFS) [hazard ratio (HR), 17.56; log-rank P-=-0.0014 and HR, 11.33; log-rank P-=-0.0001, respectively]. Tracking at least two variants in plasma increased the ability to identify MRD to 87.5%. ctDNA was the only significantly independent predictor of DFS in multivariable analysis. In patients treated with adjuvant chemotherapy, presence of ctDNA after therapy was associated with early relapse (HR 10.02; log-rank P-<-0.0001). Detection of ctDNA at follow-up preceded radiological recurrence with a median lead time of 11.5-months.ConclusionsPlasma postoperative ctDNA detected MRD and identified patients at high risk of relapse in localized CC. Mutation tracking with more than one variant in serial plasma samples improved our accuracy in predicting MRD.     

Tracking aCAD-ALK gene rearrangement in urine and blood of a colorectal cancer patient treated with an ALK inhibitor

BackgroundMonitoring response and resistance to kinase inhibitors is essential to precision cancer medicine, and is usually investigated by molecular profiling of a tissue biopsy obtained at progression. However, tumor heterogeneity and tissue sampling bias limit the effectiveness of this strategy. In addition, tissue biopsies are not always feasible and are associated with risks due to the invasiveness of the procedure. To overcome these limitations, blood-based liquid biopsy analysis has proven effective to non-invasively follow tumor clonal evolution.Patients and methodsWe exploited urine cell-free, trans-renal DNA (tr-DNA) and matched plasma circulating tumor DNA (ctDNA) to monitor a metastatic colorectal cancer patient carrying aCAD-ALK translocation during treatment with an ALK inhibitor.ResultsUsing a custom next generation sequencing panel we identified the genomicCAD-ALK rearrangement and aTP53 mutation in plasma ctDNA. Sensitive assays were developed to detect both alterations in urine tr-DNA. The dynamics of theCAD-ALK rearrangement in plasma and urine were concordant and paralleled the patient’s clinical course. Detection of theCAD-ALK gene fusion in urine tr-DNA anticipated radiological confirmation of disease progression. Analysis of plasma ctDNA identifiedALK kinase mutations that emerged during treatment with the ALK inhibitor entrectinib.ConclusionWe find that urine-based genetic testing allows tracing of tumor-specific oncogenic rearrangements. This strategy could be effectively applied to non-invasively monitor tumor evolution during therapy. The same approach could be exploited to monitor minimal residual disease after surgery with curative intent in patients whose tumors carry gene fusions. The latter could be implemented without the need of patient hospitalization since urine tr-DNA can be self-collected, is stable over time and can be shipped at specified time-points to central labs for testing.