Mutations In ctDNA Research Articles

Circulating tumour DNA in patients with hepatocellular carcinoma across tumour stages and treatments

ObjectiveCirculating tumour DNA (ctDNA) is a promising non-invasive biomarker in cancer. We aim to assess the dynamic of ctDNA in patients with hepatocellular carcinoma (HCC).DesignWe analysed 772 plasmas from 173...

Prognostic Role of Specific KRAS Mutations Detected in Aspiration and Liquid Biopsies from Patients with Pancreatic Cancer

Background/Objectives: Although the overall survival prognosis of patients in advanced stages of pancreatic ductal adenocarcinoma (PDAC) is poor, typically ranging from days to months from diagnosis, there are rare cases of patients remaining in therapy for longer periods of time. Early estimations of survival prognosis would allow rational decisions on complex therapy interventions, including radical surgery and robust systemic therapy regimens. Understandably, there is great interest in finding prognostic markers that can be used for patient stratification. We determined the role of various KRAS mutations in the prognosis of PDAC patients using biopsy samples and circulating tumor DNA. Methods: A total of 118 patients with PDAC, clinically confirmed by endoscopic ultrasound-guided fine-needle aspiration biopsy (EUS-FNB), were included in the study. DNA was extracted from cytological slides following a standard cytology evaluation to ensure adequacy (viability and quantity) and to mark the tumor cell fraction. Circulating tumor DNA (ctDNA) was extracted from plasma samples of 45 patients in stage IV of the disease. KRAS mutations in exons 12 and 13 were detected by denaturing capillary electrophoresis (DCE), revealing a minute presence of mutation-specific heteroduplexes. Kaplan–Meier survival curves were calculated for individual KRAS mutation types. Results:KRAS mutations were detected in 90% of tissue (106/118) and 44% of plasma (20/45) samples. All mutations were localized at exon 2, codon 12, with G12D (GGT > GAT) being the most frequent at 44% (47/106) and 65% (13/20), followed by other types including G12V (GGT > GTT) at 31% (33/106) and 10% (2/20), G12R (GGT > CGT) at 17% (18/106) and 10% (2/20), G12C (GGT/TGT) at 5% (5/106) and 0% (0/20) and G12S (GGT/AGT) at 1% (1/106) and 5% (1/20) in tissue and plasma samples, respectively. Two patients had two mutations simultaneously (G12V + G12S and G12D + G12S) in both types of samples (2%, 2/106 and 10%, 2/20 in tissue and plasma samples, respectively). The median survival of patients with the G12D mutation in tissues was less than half that of other patients (median survival 101 days, 95% CI: 80–600 vs. 228 days, 95% CI: 184–602), with a statistically significant overall difference in survival (p = 0.0080, log-rank test), and furthermore it was less than that of all combined patients with other mutation types (101 days, 95% CI: 80–600 vs. 210 days, 95% CI: 161–602, p = 0.0166). For plasma samples, the survival of patients with this mutation was six times shorter than that of patients without the G12D mutation (27 days, 95% CI: 8–334 vs. 161 days, 95% CI: 107–536, p = 0.0200). In contrast, patients with detected KRAS G12R in the tissue survived nearly twice as long as other patients in the aggregate (286 days, 95% CI: 70–602 vs. 162 days, 95% CI: 122–600, p = 0.0374) or patients with other KRAS mutations (286 days, 95% CI: 70–602 vs. 137 days, 95% CI: 107–600, p = 0.0257). Conclusions: Differentiation of specific KRAS mutations in EUS-FNB and ctDNA (above all, the crucial G12D and G12R) is feasible in routine management of PDAC patients and imperative for assessment of prognosis.

Concordance of ctDNA and tissue genomic profiling in advanced biliary tract cancer

Background & AimsRecent advances in molecular profiling enable the identification of potential therapeutic targets for biliary tract cancer (BTC). However, in patients with BTC, molecular profiling is hindered by challenges in obtaining adequate tissue samples. Circulating tumor DNA (ctDNA) may offer an alternative to tissue-based analysis. Here we aimed to assess the concordance between ctDNA and tissue genomic profiling in a large cohort of Asian patients with advanced BTC, and to evaluate the feasibility of liquid biopsy in BTC treatment. MethodsThis study included patients with systemic treatment-naive advanced BTC, treated at CHA Bundang Medical Center between January 2019 and December 2022. We enrolled patients with available baseline tissue-based next-generation sequencing (NGS), and sufficient plasma samples for ctDNA analysis (AlphaLiquid®100 from IMBdx). ResultsAmong 102 enrolled patients, 49.0% had intrahepatic cholangiocarcinoma, 26.5% extrahepatic cholangiocarcinoma, and 24.5% gallbladder cancer. The concordance between intra-patient ctDNA and tumor tissue mutations revealed a sensitivity of 84.8%, and positive predictive value of 79.4%. ctDNA revealed targetable alterations in 34.3% of patients—including FGFR2 fusion, IDH1 mutation, microsatellite instability (MSI)-high, ERBB2 amplification, PIK3CA mutations, BRCA1/2 mutations, and MET amplification. Notably, a novel FGFR2-TNS1 fusion was identified in ctDNA, which was not targeted in the tissue NGS panel. A high maximum somatic variant allele frequency in ctDNA was associated with poor prognosis after gemcitabine/cisplatin-based chemotherapy, in terms of both overall survival (p = 6.9 × 10−6) and progression-free survival (p = 3.8 × 10−7). ConclusionsAmong patients with advanced BTC, ctDNA-based genotyping showed acceptable concordance with tissue genomic profiling. Liquid biopsy using ctDNA could be a valuable complement to tissue-based genomic analysis in BTC. Impact and implicationsOur study is the first large-scale investigation of the clinical utility of liquid biopsy, focusing on ctDNA, as an alternative to conventional tumor tissue analysis, among Asian patients with advanced BTC. The results demonstrated acceptable concordance between analysis of ctDNA versus tissue for identifying therapeutic targets and potentially actionable genetic alterations. This indicates that ctDNA analysis can provide critical insights regarding advanced BTC treatment, particularly in cases where it is challenging to obtain or analyze tumor tissue.

Circulating tumor DNA in conjunctival melanoma: landscape and surveillance value

PurposeTo evaluate the surveillance value of circulating tumor DNA (ctDNA) for detecting distant metastasis and indicating systemic therapeutic efficacy in conjunctival melanoma (CoM). DesignRetrospective, observational case series. MethodsFrom July 2021 to June 2023, 30 CoM patients in our center underwent plasma ctDNA assessment, out of which 12 individuals presented with distant metastases. We employed a 437-gene panel containing common mutations in CoM and common drug-sensitive mutations using next-generation sequencing (NGS) technology to analyze ctDNA mutations in plasma. Clinical and radiological records were used to assess tumor status. The relationship between ctDNA characteristics, tissue gene mutations, and clinical manifestations were explored. ResultsCoM-related driver mutations were detected in ctDNA of 11 patients with distant metastasis. The ctDNA were highly consistent with tissue sequencing, mutual driver mutation including BRAF, NRAS, KRAS, NF1, CTNNB1, and TP53 mutation. those with a higher VAF had shorter progression-free survival (PFS, p=0.0475) and overall survival (OS, p=0.0043). The ctDNA variant allele fraction (VAF) was not correlated with the sum of the longest diameters (SLD, p=0.8192) in distant metastasis patients. ConclusionsPositive plasma ctDNA reflected the presence of metastases. The ctDNA could be used as a complement or alternative to tissue sequencing. High VAF ctDNA might indicate rapid disease progression in distant metastasis patients.

Predicting Disease Progression in Inoperable Localized NSCLC Patients Using ctDNA Machine Learning Model.

There is an urgent clinical need to accurately predict the risk for disease progression in post-treatment NSCLC patients, yet current ctDNA mutation profiling approaches are limited by low sensitivity. We represent a non-invasive liquid biopsy assay utilizing cfDNA neomer profiling for predicting disease progression in 44 inoperable localized NSCLC patients. A total of 97 plasma samples were collected at various time points during or post-treatments (TP1: 39, TP2: 33, TP3: 25). cfDNA neomer profiling, generated based on target sequencing data, was used to fit survival support vector machine models for each time point. Leave-one-out cross-validation (LOOCV) was performed to evaluate the models' predictive performances. Our cfDNA neomer profiling assay showed excellent performance in detecting patients with a high risk for disease progression. At TP1, the high-risk patients detected by our model showed an increased risk of 3.62 times (hazard ratio [HR] = 3.62, p = 0.0026) for disease progression, compared to 3.91 times (HR = 3.91, p = 0.0022) and 4.00 times (HR = 4.00, p = 0.019) for TP2 and TP3. These neomer profiling determined HRs were higher than the ctDNA mutation-based results (HR = 2.08, p = 0.074; HR = 1.49, p = 0.61) at TP1 and TP3. At TP1, the predictive model reached 40% sensitivity at 92.9% specificity, outperforming the mutation-based method (40% sensitivity at 78.6% specificity), while the combination results reached a higher sensitivity (60%). Finally, the longitudinal analysis showed that the combination of neomer and ctDNA mutation-based results could predict disease progression with an excellent sensitivity of 88.9% at 80% specificity. In conclusion, we developed a cfDNA neomer profiling assay for predicting disease progression in inoperable NSCLC patients. This assay showed increased predicting power during and post-treatment compared to the ctDNA mutation-based method, thus illustrating a great clinical potential to guide treatment decisions in inoperable NSCLC patients. ClinicalTrials.gov: NCT04014465.

Clinical Utility of Circulating Tumor DNA for Detecting Lung Cancer Mutations by Targeted Next-Generation Sequencing With Insufficient Tumor Samples.

Circulating tumor deoxyribonucleic acid (ctDNA) is increasingly applied in clinical practice. This study aimed to explore clinical utility of a minimal invasive and sensitive way of ctDNA for next-generation sequencing in non-small cell lung cancer (NSCLC) with inadequate tumor samples. Targeted DNA sequencing was performed on tissue biopsies and matched plasma samples from 60 patients with NSCLC. A total of 13 driving genes were detected in 60 matched tissue DNA (tDNA) and ctDNA samples. Overall concordance rate was 75.47%, with 77.55% sensitivity and 50% specificity. Epidermal growth factor receptor (EGFR) mutations were the most common in both tDNA and ctDNA samples. Among other mutated genes were tumor protein p53 (TP53), erb-b2 receptor tyrosine kinase 2 (ERBB2), anaplastic lymphoma kinase (ALK), cyclin-dependent kinase inhibitor 2A (CDKN2A), ros proto-oncogene 1, and receptor tyrosine kinase (ROS1). Mutations in b-raf proto-oncogene, serine/threonine kinase (BRAF), cluster of differentiation 274 (CD274), neurotrophin receptor tyrosine kinase 1 (NTRK1), and rearranged during transfection (RET) occurred only in plasma. The majority of mutations in both samples were single-nucleotide variants. Deletions were found in EGFR, BRAF, and TP53 in ctDNA, whereas in tDNA, deletions were only found in EGFR. In ALK, single nucleic acid-site amplification occurred simultaneously in tissue and plasma, but insertions and copy number variations were detected only in plasma. Identifying ctDNA mutations by targeted sequencing in plasma is feasible, showing the clinical value of ctDNA-targeted sequencing in NSCLC patients when tumor tissue sampling is insufficient or even impossible.

Abstract A009: Mutant KRAS in Circulating Tumor DNA Assessed by Digit Droplet PCR as a Biomarker in Pancreatic Cancer in Patients Treated with Neoadjuvant Chemotherapy

Abstract Introduction: The detectability and prognostic significance of circulating tumor DNA (ctDNA) in patients with pancreatic ductal adenocarcinoma (PDAC) after neoadjuvant chemotherapy (NAC) is unclear. The objective of this study was to investigate the rate of detection and prognostic value of KRAS mutant ctDNA in PDAC patients after treatment with NAC as assessed by digital droplet polymerase chain reaction (ddPCR). Methods: Patients with newly diagnosed, localized, PDAC were enrolled in a prospective cohort study. Peripheral blood samples were collected at diagnosis, after NAC, and after resection. DNA was extracted from patient plasma samples using the QIAamp Circulating Nucleic Acid Kit #55114 (Qiagen N.V., Venlo Netherlands). Each sample was probed (catalog number 10049550) for KRAS codon 12 exon 2 glycine substitutions for aspartic acid (G12D), valine (G12V), and arginine (G12R). Kaplan-Meier, log-rank, and Cox regression survival analyses were performed. Results: 84 patients were included in the analysis. Patients were most commonly male (53.6%) and had tumors that were radiographically resectable (57.1%). After diagnosis, 79.8% of patients received NAC and 57.1% of patients received curative-intent surgical resection. Samples were collected for analysis from 59 (70.2%), 56 (83.6%), and 45 (93.8%) patients at diagnosis, after NAC, and after resection, respectively. Mutant KRAS ctDNA was detected in 49.3% of patients at diagnosis, 69.6% of patients after NAC, and 54.1% of patients after resection, respectively. The median OS of the cohort was 25.1 months (12.0-not reached [NR]). Detection (hazard ratio [HR] 36.8, 95% confidence interval [CI] 2.9-461.4), copy number (HR 4.0 95% CI 1.6-10.2), and percentage mutation (HR 2.9 95% CI 1.2-6.8) of KRAS G12V ctDNA after resection were all independently associated with shorter OS. Mutant KRAS ctDNA detection was not associated with and OS at other study timepoints. There were 15 (17.9%) patients that cleared mutational ctDNA over the course of treatment. Clearance of ctDNA during NAC was associated with improved overall survival (OS) (18.4 mo. vs NR). Conclusions: In patients with PDAC treated with NAC, KRAS mutations are detectable in ctDNA by ddPCR in the majority of patients over the course of treatment. Detection, copy number, and percentage mutation of KRAS G12V after NAC and resection were each independently associated with shorter OS. Clearance of ctDNA is associated with improved OS. The results demonstrate that KRAS mutant ctDNA predicts prognosis after NAC and resection. Citation Format: Dominic Vitello, Dhavan Shah, Amy Wells, Larissa Masnyk, Madison Cox, Lauren Janczewski, John Abad, Kevin Dawravoo, Arlene D'Souza, Grace Suh, Robert Bayer, Massimo Cristofanilli, David Bentrem, Yingzhe Liu, Hui Zhang, Lucas Santana-Santos, Lawrence Jennings, Qiang Zhang, Akhil Chawla. Mutant KRAS in Circulating Tumor DNA Assessed by Digit Droplet PCR as a Biomarker in Pancreatic Cancer in Patients Treated with Neoadjuvant Chemotherapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A009.

Circulating tumor DNA predicts venous thromboembolism in patients with cancers

BackgroundDespite rapid advances in liquid biopsy for circulating tumor DNA (ctDNA), its prognostic value for venous thromboembolism (VTE) in patients with cancer is underexplored, particularly in underserved and minoritized populations. Objectives▪. MethodsWe analyzed data from 1038 cancer patients who underwent ctDNA measurement for oncologic care at a large safety-net hospital system in the United States. We investigated the association between ctDNA and VTE after adjusting for cancer type, stage, treatment, and time from initial diagnosis using Fine–Gray models. We further assessed the discrimination of the genetic, clinical-only, and combined models using the area under the time-dependent receiver operating characteristic curve (AUC). ResultsThe presence of pathogenic ctDNA was independently associated with VTE after adjusting for clinical variables. Independent of tumor type, the number of pathogenic ctDNA mutations was predictive of future VTE risk (adjusted subdistribution hazard ratios of 2.75, 1.94, and 1.38 for ≥3, 2, and 1 pathogenic mutation, respectively, compared with none; P < .0001). The association was primarily driven by mutations in KRAS, PTEN, CDKN2A, NF1, and EGFR genes. Compared with the clinical-only model (AUC, 0.71; 95% CI, 0.64-0.76), the combined clinical and ctDNA model had a numerically higher time-dependent AUC (AUC, 0.74; 95% CI, 0.67-0.80). ConclusionctDNA testing may serve as an adjunctive tool to clinical risk assessment models in cancer patients to improve personalized VTE risk assessment and management.

Loss of Heterozygosity and Mutations in the RAS-ERK Pathway Genes in Tumor Cells of Various Loci in Multiple Myeloma.

Multiple myeloma (MM) is a disease characterized by spatiotemporal heterogeneity of tumor clones. Different genetic aberrations can be observed simultaneously in tumor cells from different loci, and as the disease progresses, new subclones may appear. The role of liquid biopsy, which is based on the analysis of tumor DNA circulating in the blood plasma, continues to be explored in MM. Here, we present an analysis of the STR profiles and mutation status of the KRAS, NRAS, and BRAF genes, evaluated in plasma free circulating tumor DNA (ctDNA), CD138+ bone marrow cells, and plasmacytomas. The prospective single-center study included 97 patients, with a median age of 55 years. Of these, 94 had newly diagnosed symptomatic MM, and three had primary plasma cell leukemia. It should be noted that if mutations were detected only in ctDNA, "non-classical" codons were more often affected. A variety of adverse laboratory and clinical factors have been associated with the detection of rare KRAS or NRAS gene mutations in bone marrow or ctDNA, suggesting that these mutations may be factors of an unfavorable prognosis for MM. Liquid biopsy studies provide undeniable fundamental information about tumor heterogeneity and clonal evolution in MM. Moreover, we focus on using liquid biopsy to identify new high-risk factors for MM.

From haystack to high precision: advanced sequencing methods to unraveling circulating tumor DNA mutations.

Identifying mutations in cancer-associated genes to guide patient treatments is essential for precision medicine. Circulating tumor DNA (ctDNA) offers valuable insights for early cancer detection, treatment assessment, and surveillance. However, a key issue in ctDNA analysis from the bloodstream is the choice of a technique with adequate sensitivity to identify low frequent molecular changes. Next-generation sequencing (NGS) technology, evolving from parallel to long-read capabilities, enhances ctDNA mutation analysis. In the present review, we describe different NGS approaches for identifying ctDNA mutation, discussing challenges to standardized methodologies, cost, specificity, clinical context, and bioinformatics expertise for optimal NGS application.

12 Concordance Analysis of Tissue and Circulating Tumor DNA (ctDNA) in Renal Cell Carcinoma (RCC): Insights from a Multimodal Real-World Database

Abstract Background Next-generation sequencing (NGS) of circulating tumor DNA (ctDNA) has emerged as a powerful complement to tissue NGS, offering a noninvasive and serially conductible test. Its application holds promise in enhancing the assessment of spatial and temporal molecular tumor heterogeneity, thus providing valuable insights into cancer progression and treatment response. In this study, we explore the mutational landscape of renal cell carcinoma (RCC) patients through comprehensive profiling of mutations in ctDNA and matched tissue samples, aiming to elucidate the concordance and clinical significance of molecular alterations detected in both circulating and tissue-derived DNA. Methods From the Tempus multimodal database, we retrospectively analyzed de-identified NGS data from patients with RCC that had dual tissue (Tempus xT, 648 genes) and ctDNA testing (Tempus xF, 105 genes). Patients with matched samples (collected +/- 90 days of one another) were included. We evaluated socio-demographic and clinical characteristics and select pathogenic somatic short variants (PSSV) and copy number variants [(amplifications and deletions, two copy number losses (CNL)]. Concordance analyses were restricted to the 105 genes tested on the ctDNA panel and further restricted to short variants, with the exception of amplifications and CNL detected by both xF and xT. Analysis was further stratified by metastatic status (n=260, metastatic, n=120 non-metastatic) prior to collection of both xT and xF. Results Among all patients (n=393), the median age was 61 years, and 71% were male. The patient cohort comprised a diverse population based on race, with 75% white, 12% African American, 4.8% Asian and 8% others. The median time from tissue to blood collection was 21 days (IQR, 7, 39). 67% (n=265) and 68% (n=266) had metastatic disease at the time of tissue and blood collection, respectively. The most common tissue sites were kidney (49%, n=189), bone (11%, n=43), lung (9%, n=34), lymph node (8%, n=29), liver (6%, n=23), and brain/CNS (4%, n=17). Genes harboring the most common PSSV in tissue included VHL (59% n=232), PBRM1 (31%, n=123), SETD2 (23%, n=91), TP53 (14%, n=54), BAP1 (12%, n=46) and TERT (11%, n=45). Genes with common PSSV in ctDNA included TP53 (23%, n=91), VHL (18%, n=69), BAP1 (6%, n=23), PBRM1 (5%, n=21), PTEN (4%, n=15), KRAS (4%, n=14) and NF2 (4%, n=14). The combination of tissue and ctDNA testing increased the detection of mutations (Table 1). There was higher concordance between somatic alterations in select genes among patients with metastases vs. non-metastases, including BAP1 (55.9% vs. 9.1%), TP53 (36.8% vs. 9.1%), VHL (32.3% vs. 12.5%), ARID1A (25% vs. 16.7%), and ATM (25% vs. 0%). Table 1: Detection of genetic mutations in tissue and ctDNA testing Conclusions The analysis conducted in this study highlights the complementary nature of ctDNA profiling alongside tissue-based NGS in RCC, demonstrating an increased detection of mutations. Particularly, we observed a higher concordance between ctDNA and tissue profiling in individuals with metastatic disease, suggesting the potential utility of ctDNA analysis in advanced stages of RCC. Further research is warranted to elucidate how longitudinal ctDNA analysis can delineate biomarkers of response and resistance at both the mutation and ctDNA fraction levels. Understanding these dynamics could offer valuable insights into disease progression and guide personalized treatment strategies for RCC patients. DOD CDMRP Funding: no

Kinetic Profiling of RAS Mutations With Circulating Tumor DNA in the Canadian Cancer Trials Group CO.26 Trial Suggests the Loss of RAS Mutations in Neo-RAS-Wildtype Metastatic Colorectal Cancer Is Transient.

In metastatic colorectal cancer (mCRC), RAS mutations drive resistance to anti-epidermal growth factor receptor antibodies. It is unclear whether RAS mutations ever become clonally undetectable. CO.26 was a phase II clinical trial that assessed durvalumab + tremelimumab in heavily pretreated mCRC. RAS mutation status was tracked over time using circulating tumor DNA (ctDNA) sequencing at baseline, week 8, and on progression. Among the 95 patients with KRAS/NRAS mutations in their archival tumor tissue, 6.3% (6/95) had undetectable RAS mutations in ctDNA collected at baseline or week 8 of the CO.26 study. Of these, 67% (4/6) of disappearances were transient, with the same mutation reappearing with progressive disease. In three cases, the simultaneous persistence of other preexisting CRC-associated truncal mutations could not be demonstrated, suggestive of low tumor shedding of ctDNA, leaving the incidence of true clonal reversion to RAS-wildtype (WT) possibly as low as 3.2% (3/95). Fewer patients in the neo-RAS-WT group (33%) had greater than four lesions at trial baseline compared with patients with persistent RAS mutations (75%), P = .046. The likelihood of synchronous metastases at cancer diagnosis (33% v 63%; P = .15) or liver metastases at trial baseline (50% v 68.5%; P = .17) was not significantly different between patients with disappearing versus persistent RAS mutations. Overall survival from stage IV diagnosis (hazard ratio, 0.77 [95% CI, 0.35 to 1.72]; P = .52) was not significantly different between those with disappearing versus persistent RAS mutations. The disappearance of RAS mutations was not associated with primary tumor sidedness (P = .41), archival BRAF/MEK/ERK-mutant status (P = .16/1.00/.09), nor baseline ctDNA HER2 amplifications (P = 1.00). We identified a 3.2%-6.3% prevalence of the neo-RAS-WT phenomenon in the CO.26 trial. However, 67% of apparent cases were transient with subsequent re-emergence.

RB1 circulating-tumor DNA in the blood of Retinoblastoma patients increases in untreated patients.

Circulating tumor DNA (ctDNA) in plasma has been identified in many cancers, including retinoblastoma at diagnosis. We have previously shown that with treatment (enucleation or ophthalmic artery chemosurgery (OAC)) all ctDNA disappears; and if there is persistent plasma ctDNA after treatment metastases develop. The purpose of this study was to determine how the ctDNA RB1 variant allele frequency (VAF) changes in patients with retinoblastoma who have delayed treatment. Circulating tumor DNA RB1 was detected and VAF was measured at diagnosis and again prior to any intervention at some time later ranging from 2 to 28 days. Four patients with five ctDNA RB1 mutations were detected at diagnosis and VAF was increased on re-evaluation of the same RB1 mutations in ctDNA. In this small cohort, every patient (4) and every RB1 mutation (5) plasma level VAF% increased when measured at two time periods before treatment was instituted suggesting that growing tumors demonstrate increasing plasma ctDNA.

Different efficacy of tyrosine kinase inhibitors by KIT and PGFRA mutations identified in circulating tumor DNA for the treatment of refractory gastrointestinal stromal tumors

BackgroundWhile advanced gastrointestinal stromal tumors (GISTs) are primarily treated with tyrosine kinase inhibitors (TKIs), acquired resistance from specific mutations in KIT or PDGFRA frequently occurs. We aimed to assess the utility of circulating tumor DNA (ctDNA) as a modality of therapeutic decision-making in advanced GIST.MethodsWe conducted a pooled analysis of SCRUM-Japan studies for advanced GIST patients. We compared patient characteristics analyzed with tissue and blood samples, assessed gene alteration profiles, and evaluated prognostic implications from ctDNA status.ResultsIn 133 patients, tissue and blood samples were analyzed for 89 and 44 patients, respectively. ctDNA was detected in 72.7% of cases; no prior treatment or progressive disease was significantly associated with ctDNA-positivity. ctDNA-positive patients had significantly shorter progression-free survival compared with ctDNA-negative patients (hazard ratio = 3.92; P = 0.007). ctDNA genotyping revealed a complex landscape of gene alterations, characterized by multi-exonic mutations in KIT, compared with tissue-based analysis. Patients who received TKIs matched to the identified KIT mutation in ctDNA demonstrated significantly longer PFS than those with unmatched treatment (median, 8.23 vs. 2.43 months; P < 0.001).ConclusionsctDNA-based analysis facilitates assessment of disease status and genomic profiles, thus potentially assisting in identifying optimal therapeutic strategies for advanced GIST patients.

Early Evaluation of Risk Stratification and Clinical Outcomes for Patients with Advanced Breast Cancer through Combined Monitoring of Baseline Circulating Tumor Cells and DNA.

Early evaluation of tumor heterogeneity related to metastasis and outcomes is a major challenge in the management of advanced breast cancer (BCa) in the clinic. In this study, we introduced the value of baseline circulating tumor cells (CTC) and ctDNA for early differentiation of clinical stages, tumor heterogeneity, and prognosis in clinic. A total of 292 patients with BCa were enrolled in this study, including 254 Stage IV and 38 Stage III patients, and examined the baseline levels of CTCs, CTC-clusters, and plasma ctDNA before initiating therapies. Outcomes including progression-free survival (PFS) and overall survival were evaluated using proportional hazards regression analysis. The baseline CTCs, including HER2+ CTCs, in Stage IV patients were approximately 9.5 times higher than those detected in Stage III patients. Baseline CTC counts with a cutoff of 5 were significantly associated with the prognosis. Within each stage, patients with <5 CTCs had significantly longer PFS. Stage III patients with no CTCs exhibited the longest survival compared with patients with ≥1 CTC. CTC-clusters were only found in Stage IV patients, among whom 15 Stage IV patients with ≥5 CTC-clusters had the worst PFS compared with the 239 Stage IV patients with <5 CTC-clusters. Similar outcomes were observed in 28 out of 254 Stage IV patients who had at least one CTC-cluster detected, as these patients had shorter PFS compared with CTC-cluster negative group. The major differences in ctDNA mutations between patients with Stage III and Stage IV BCa were in PIK3CA and ESR1, which were associated with specific organ metastasis and worse outcomes. Assessing the baseline levels of CTCs, CTC-clusters, and mutational ctDNA profile could reliably aid in differentiation of clinical stage and early prediction of metastasis and outcomes in advanced BCa.

Cell free DNA in patients with pancreatic adenocarcinoma: clinicopathologic correlations

Detection of circulating tumor DNA (ctDNA) from plasma cell free DNA (cfDNA) has shown promise for diagnosis, therapeutic targeting, and prognosis. This study explores ctDNA detection by next generation sequencing (NGS) and associated clinicopathologic factors in patients with pancreatic adenocarcinoma (PDAC). Patients undergoing surgical exploration or resection of pancreatic lesions were enrolled with informed consent. Plasma samples (4–6 ml) were collected prior to surgery and cfDNA was recovered from 95 plasma samples. Adequate cfDNA for NGS (20 ng) was obtained from 81 patients. NGS was performed using the Oncomine Lung cfDNA assay on the Ion Torrent S5 sequencing platform. Twenty-five patients (30.9%) had detectable mutations in KRAS and/or TP53 with allele frequencies ranging from 0.05 to 8.5%, while mutations in other genes were detected less frequently and always along with KRAS or TP53. Detectable ctDNA mutations were more frequent in patients with poorly differentiated tumors, and patients without detectable ctDNA mutations showed longer survival (medians of 10.5 months vs. 18 months, p = 0.019). The detection of circulating tumor DNA in pancreatic adenocarcinomas is correlated with worse survival outcomes.

Clinical significance of circulating biomarkers of immune-checkpoint molecules with atezolizumab plus bevacizumab therapy in unresectable hepatocellular carcinoma.

The aims of this study were to identify clinically significant biomarkers of a response to atezolizumab plus bevacizumab (ATZ + BV) therapy and to develop target strategies against unresectable hepatocellular carcinoma (u-HCC). We first investigated the potential of circulating tumor DNA (ctDNA) to serve as a biomarker for predicting the therapeutic outcome in 24 u-HCC patients treated with ATZ + BV therapy. Next, we analyzed levels of immune-related cytokines in blood samples from 134 u-HCC patients who received ATZ + BV. For this, serum immune-related molecules or cancer-immune cycle-related molecules that have been reported in HCC patient sera, namely CD274, LAG-3, CCL2, 4, 5, CXCL1, 9, 10, 12, 13, CX3CL1, CCR5, IFNγ and IL-6, 8 were measured using enzyme-linked immunosorbent assay. More than 1% of variant read frequency (VRF) mutations were found in TP53, APC, PIK3CA and VHL, although with no correlation with treatment response. Among the 15 cytokines evaluated, CXCL9 and LAG-3 levels were significantly different between patients with objective response (OR), stable disease (SD), and progressive disease (PD) following ATZ + BV treatment. Receiver-operating characteristic curve analyses of CXCL9 (cut-off value: 419.1pg/ml) and LAG-3 (cut-off value: 3736.3pg/ml) indicated areas of 0.779 and 0.697, respectively, for differentiating PD from non-PD and OR from non-OR. In multivariate analysis of progression-free survival (PFS) and overall survival (OS), high serum CXCL9 (hazard ratio (HR) and 95% confidence interval (CI): 0.412 (0.251-0.677) (p = 0.0005) for PFS and 0.252 (0.125-0.508) (p = 0.0001) for OS), and low serum LAG-3 (HR and 95% CI 0.419 (0.249-0.705) (p = 0.0011) for PFS and 0.294 (0.140-0.617) (p = 0.0012) for OS) were independent positive predictive factors. Although, as far as we examined, no ctDNA mutations in blood were found to be related to ATZ + BV treatment efficacy, serum CXCL9 and LAG-3 levels, which are related to the cancer-immune cycle, were associated with treatment efficacy and could be predictive markers of the efficacy of ATZ + BV treatment in HCC patients.

Engineering of a DNA/γPNA Hybrid Nanoreporter for ctDNA Mutation Detection via γPNA Urinalysis.

Detection of circulating tumor DNA (ctDNA) mutations, which are molecular biomarkers present in bodily fluids of cancer patients, can be applied for tumor diagnosis and prognosis monitoring. However, current profiling of ctDNA mutations relies primarily on polymerase chain reaction (PCR) and DNA sequencing and these techniques require preanalytical processing of blood samples, which are time-consuming, expensive, and tedious procedures that increase the risk of sample contamination. To overcome these limitations, here the engineering of a DNA/γPNA (gamma peptide nucleic acid)hybrid nanoreporter is disclosed for ctDNA biosensing via in situ profiling and recording of tumor-specific DNA mutations. The low tolerance of γPNA to single mismatch in base pairing with DNA allows highly selective recognition and recording of ctDNA mutations in peripheral blood. Owing to their remarkable biostability, the detached γPNA strands triggered by mutant ctDNA will be enriched in kidneys and cleared into urine for urinalysis. It is demonstrated that the nanoreporter has high specificity for ctDNA mutation in peripheral blood, and urinalysis of cleared γPNA can provide valuable information for tumor progression and prognosis evaluation. This work demonstrates the potential of the nanoreporter for urinary monitoring of tumor and patient prognosis through in situ biosensing of ctDNA mutations.

Highly consistency of PIK3CA mutation spectrum between circulating tumor DNA and paired tissue in lung cancer patients

BackgroundPhosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutations are associated with drug resistance and prognosis in lung cancer; however, the consistency and clinical value of PIK3CA mutations between tissue and liquid samples are unknown. MethodsCirculating tumor DNA (ctDNA) and matched tumor tissue samples from 405 advanced lung cancer patients were collected at Jilin Cancer Hospital between 2018 and 2022, and the PIK3CA mutation status was sequenced using next-generation sequencing based on a 520 gene panel. The viability of different mutant lung cancer cells was detected using MTT assay. ResultsPIK3CA mutations were detected in 46 (5.68 %) of 810 lung cancer samples, with 21 (5.19 %) of 405 plasma samples and 25 (6.17 %) of 405 matched tissues. p.Glu542Lys, p.Glu545Lys, and p.His1047Arg were the most common mutation types of PIK3CA in both the ctDNA and tissue samples. The concordance of PIK3CA mutations was 97.53 % between ctDNA and matched tissues (kappa: 0.770, P = 0.000), with sensitivity/true positive rate of 72.0 %, specificity/true negative rate of 99.2 %, and negative predictive value and positive predictive value of 0.982 and 0.857, respectively (AUC = 0.856, P = 0.000). Furthermore, the concordance of PIK3CA mutations was 98.26 % in lung adenocarcinoma and 96.43 % in lung squamous cell carcinoma. TP53 and EGFR were the most common concomitant mutations in ctDNA and tissues. Patients with PIK3CA mutations showed a high tumor mutational burden (TMB) (P < 0.001) and a significant correlation between bTMB and tTMB (r = 0.5986, P = 0.0041). For the tPIK3CAmut/ctDNA PIK3CAmut cohort, PI3K pathways alteration was associated with male sex (P = 0.022), old age (P = 0.007), and smoking (P = 0.001); tPIK3CAmut/ctDNA PIK3CAwt patients harbored clinicopathological factors of adenocarcinoma stage IV, with low PS score (≤1) and TMB. ConclusionThis study showed that ctDNA is highly concordant and sensitive for identifying PIK3CA mutations, suggesting that PIK3CA mutation detection in liquid samples may be an alternative clinical practice for tissues.

Evaluation of the diagnostic and prognostic clinical values of circulating tumor DNA and cell-free DNA in pancreatic malignancies: a comprehensive meta-analysis.

The diagnostic and prognostic clinical value of circulating tumor DNA (ctDNA) and cell-free DNA (cfDNA) in pancreatic malignancies are unclear. Herein, we aimed to perform a meta-analysis to evaluate ctDNA and cfDNA as potential diagnostic and prognostic biomarkers. PRISMA reporting guidelines were followed closely for conducting the current meta-analysis. The PubMed/Medline, Scopus, and Web of Science (WoS) databases were scanned in detail to identify eligible papers for the study. A quality assessment was performed in accordance with the REMARK criteria. The risk ratios (RRs) of the diagnostic accuracy of ctDNA compared to that of carbohydrate antigen 19.9 (CA 19.9) in all disease stages and the hazard ratios (HRs) of the prognostic role of ctDNA in overall survival (OS) were calculated with 95% confidence intervals (CIs). A total of 18 papers were evaluated to assess the diagnostic accuracy and prognostic value of biomarkers related to pancreatic malignancies. The pooled analysis indicated that CA19.9 provides greater diagnostic accuracy across all disease stages than ctDNA or cfDNA (RR = 0.64, 95% CI: 0.50-0.82, p < 0.001). Additionally, in a secondary analysis focusing on prognosis, patients who were ctDNA-positive were found to have significantly worse OS (HR = 2.00, 95% CI: 1.51-2.66, p < 0.001). The findings of this meta-analysis demonstrated that CA19-9 still has greater diagnostic accuracy across all disease stages than KRAS mutations in ctDNA or cfDNA. Nonetheless, the presence of detectable levels of ctDNA was associated with worse patient outcomes regarding OS. There is a growing need for further research on this topic. https://doi.org/10.37766/inplasy2023.12.0092, identifier INPLASY2023120092.