Plasma Circulating Tumor DNA and Clonal Hematopoiesis in Metastatic Renal Cell Carcinoma

BackgroundThere is a lack of molecularly-informed biomarkers for patients with metastatic renal cell carcinoma (RCC). Plasma cell-free DNA (cfDNA) sequencing is a minimally-invasive alternative to tissue for profiling the genome in other cancers but relevance in metastatic RCC remains unclear.Materials and MethodsWhole blood was collected from 55 patients with metastatic RCC. Plasma cfDNA and leukocyte DNA were subjected to targeted sequencing across 981 cancer genes. Matched tumor tissue from 14 patients was analyzed.ResultsThirty-three percent of patients had evidence for RCC-derived circulating tumor DNA (ctDNA), significantly lower than patients with metastatic prostate or bladder cancer analyzed using the same approach. Among ctDNA-positive patients, ctDNA fraction averaged only 3.9% and showed no strong association with clinical variables. In these patients, the most commonly mutated genes were VHL, BAP1, and PBRM1, and matched tissue concordance was 77%. Evidence of somatic expansions unrelated to RCC, such as clonal hematopoiesis of indeterminate potential, were detected in 43% of patients. Pathogenic germline mutations in DNA repair genes were detected in 11% of patients. CtDNA-positive patients had shorter overall survival and progression-free survival on first-line therapy. Patients with evidence of clonal hematopoiesis of indeterminate potential had an intermediate prognosis compared with ctDNA-positive and -negative patients.ConclusionsCfDNA sequencing enables straightforward characterization of the somatic RCC genome in a minority of patients with metastatic RCC. Owing to low ctDNA abundance, and the presence of non-RCC derived somatic clones in circulation, cfDNA sequencing may not be a simple pan-patient alternative to tissue biopsy in metastatic RCC.     

Circulating Tumor DNA Analysis for Patients with Oncogene-Addicted NSCLC With Isolated Central Nervous System Progression

IntroductionIn patients with oncogene-addicted NSCLC and isolated central nervous system progression (iCNS), tissue biopsy is challenging, and the clinical utility of plasma liquid biopsy (i.e., circulating tumor DNA [ctDNA]) is unknown.MethodsPatients with advanced NSCLC with known baseline genomic alteration (GA) (EGFR, ALK, BRAF, KRAS, HER2, ROS1, MET, PIK3CA, STK11, TP53) on tissue were divided into three groups on the basis of their disease progression pattern: iCNS, extra-CNS only (noCNS), or both (cCNS). All patients with available plasma ctDNA were included and were analyzed by next-generation sequencing InVisionFirst-Lung. ctDNA was considered positive if at least one GA was detected. Cell-free tumor DNA was analyzed in cerebrospinal fluid when available.ResultsOut of 517 patients screened, 247 were included: 54 had iCNS, 99 had noCNS, and 94 had cCNS progressive disease (64, 128, and 110 ctDNA samples, respectively). CtDNA was positive in 52% iCNS versus 84% in noCNS and 92% in cCNS (p < 0.00001), with lower detection of driver (37% versus 77% and 73%, respectively) and resistance alterations (6% versus 45% and 44%). Patients with iCNS and positive ctDNA were more at risk of extra-CNS progression (32% versus 7%, p = 0.026). In 12 patients with iCNS, ctDNA was positive in six (50%) plasma and in 10 (83%) paired cerebrospinal fluid (p = 0.193).ConclusionsAlthough tagged amplicon-based next-generation sequencing has high detection rates of GA in plasma ctDNA in patients with NSCLC with extra-CNS disease, detection rate of GAs (52%) is lower in the subset of patients with iCNS disease. Complementary tests such as cerebrospinal fluid cell-free DNA may be useful. Further evidence would be beneficial to understand the genomic landscape in patients with NSCLC and iCNS.     

Parallel Evaluation of Circulating Tumor DNA and Circulating Tumor Cells in Metastatic Colorectal Cancer

Background

Tissue biopsy is the gold standard for tumor genotyping, but it is an invasive procedure providing a single snapshot into tumor heterogeneity. Liquid biopsy approaches, encompassing the analysis of circulating tumor DNA (ctDNA) or circulating tumor cells (CTCs), have been proposed as an alternative, with the potential of providing a comprehensive portrait of the tumor molecular landscape. In metastatic colorectal cancer (mCRC), both CTCs and ctDNA analysis have been investigated, but comparative analyses are limited.

Circulating tumor DNA correlates with tumor burden and predicts outcome in pancreatic cancer irrespective of tumor stage

IntroductionCirculating tumor DNA (ctDNA) represents a promising tool for diagnosis, prognosis and treatment monitoring of several malignancies. Its association with tumor burden in pancreatic ductal cancer (PDAC), especially in localized disease, is not fully explored yet. We aimed to investigate the association of pretherapeutic ctDNA levels in localized and metastatic PDAC with tumor volume and clinical outcomes.Material and methodsLiquid biopsy for ctDNA detection was prospectively obtained from patients with localized or disseminated PDAC prior to either resection or systemic treatment. Detection rates and levels of ctDNA (digital droplet PCR) were correlated to tumor volume, relapse rate and survival.Results60 patients with localized and 47 patients with metastatic PDAC were included. ctDNA was detected in 10% of localized and 57.4% of metastasized PDAC samples. In localized disease, ctDNA detection significantly correlated with the numbers of involved locoregional lymph nodes (p = 0.030). Primary tumor volume did not correlate with ctDNA levels in neither localized (p = 0.573) nor metastasized disease (p = 0.878). In disseminated disease, ctDNA levels correlated with total tumor volume (p = 0.026) and especially with liver metastases volume (p = 0.004), but not with other metastases. Detection of pretherapeutic ctDNA was associated with shorter DFS in localized (3.3 vs. 18.1 months, p = 0.000), whereas ctDNA levels were associated with worse survival in metastatic PDAC (5.7 vs. 7.8 months, p = 0.036).ConclusionctDNA positivity indicates major nodal involvement or even presence of undetected distant metastases associated with early recurrence in localized PDAC. Moreover, it predicts worse clinical outcome in both localized and metastatic disease.     

The Clinical Impact of Comprehensive Genomic Testing of Circulating Cell-Free DNA in Advanced Lung Cancer

Introduction

Next-generation sequencing (NGS) of cell-free circulating tumor DNA (cfDNA) enables noninvasive genomic analysis of NSCLC patients. Although plasma-detected genomic alterations (GAs) have been shown to predict targeted therapy response, evidence of durability of response is lacking or limited to small cohorts as is the impact of cfDNA NGS results on clinical decisions.

Capture-Based Targeted Ultradeep Sequencing in Paired Tissue and Plasma Samples Demonstrates Differential Subclonal ctDNA-Releasing Capability in Advanced Lung Cancer

IntroductionCirculating tumor DNA (ctDNA), which represents an unbiased way to assess tumor genetic profile noninvasively, facilitates studying intratumor heterogeneity. Although intratumor heterogeneity has been elucidated substantially in a few cancer types, including NSCLC, how it influences the ability of tumor cells harboring different genetic abnormalities in releasing their DNA remains elusive. We designed a capture-based panel targeting NSCLC to detect and quantify genetic alterations from plasma samples by using deep sequencing. By applying the panel to paired biopsy and plasma samples, we imputed and compared the ctDNA-releasing efficiency in subclones harboring distinct genetic variants.MethodsWe collected 40 pairs of matched biopsy and plasma samples from patients with advanced lung cancer and applied capture-based sequencing using our LungPlasma panel, which consists of critical exons and introns of 168 genes. We derived a normalized relative allelic fraction score (NRAFS) to reflect ctDNA-releasing efficiency.ResultsBy using mutations detected in biopsy samples as a reference, we achieved 87.2% by-variant sensitivity, including for single-nucleotide variants, insertions or deletions, and gene fusions. Furthermore, the by-variant sensitivity for the seven most critical and actionable genes was 96.2%. The average NRAFS for subclones carrying mutations from seven actionable genes was 0.877; in contrast, the average NRAFS for other mutations was 0.658. Mutations from four genes involved in cell cycle pathways had a particularly low NRAFS (0.480) compared with the other two groups (p = 0.07).ConclusionsWe have demonstrated that subclones carrying driver mutations are more prone to release DNA. We have also demonstrated the quantitative ability of capture-based sequencing, paving its way for routine utilization in clinical settings.     

Detection of Minimal Residual Disease Using ctDNA in Lung Cancer: Current Evidence and Future Directions

Advances in DNA sequencing methods have significantly expanded the potential clinical applications of analyzing circulating tumor DNA (ctDNA). This genetic information can identify the presence of targetable mutations and has been explored for cancer screening purposes. ctDNA can be obtained without the risks inherent to biopsy, allowing for serial assessments over time. Several studies have additionally suggested that ctDNA can be used to detect the presence of minimal residual disease (MRD) after surgical resection in several cancer types, including lung cancer. The ability to detect MRD would allow clinicians to tailor adjuvant therapies, which carry risks of significant toxicities and may benefit only select groups of patients. Here, we review the current state of ctDNA profiling methods and evaluate the evidence supporting the use of ctDNA analysis to assess for MRD. We discuss how MRD detection could help identify patients at increased risk of disease recurrence and thus guide treatment decisions for resectable lung cancer. Finally, we propose future steps to validate such approaches and expand the utility of these rapidly progressing technologies.     

Phase II Study Evaluating the Mechanisms of Resistance on Tumor Tissue and Liquid Biopsy in Patients With EGFR-mutated Non-pretreated Advanced Lung Cancer Receiving Osimertinib Until and Beyond Radiologic Progression: The MELROSE Trial

BackgroundOsimertinib, a third-generation tyrosine kinase inhibitor, is a new therapeutic option in epidermal growth factor receptor (EGFR)-mutated non-pretreated advanced non–small-cell lung cancer (NSCLC). The tumor escape mechanisms after first-line treatment with osimertinib are partially known; most of the data being obtained by analysis of circulating tumor DNA (ctDNA) from the FLAURA phase III trial.Study DesignThe MELROSE study, a French multicentric, open label, phase II trial (ClinicalTrials.gov NCT03865511) plans to enroll 150 patients with treatment-naive advanced EGFR-mutated (L858R or exon 19 deletion) NSCLC, age ≥ 18 years, with an Eastern Cooperative Oncology Group performance status 0 or 1. All patients will receive osimertinib at the dose of 80 mg/d. Tumor assessment according to Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 criteria will be performed every 3 months, with brain and thoracoabdominal computed tomographic scan. The continuation of osimertinib is at the discretion of the referring physician, particularly if clinical benefit is observed. The primary objective is the genetic tumor profile, both on tissue biopsy and ctDNA analyses, at the time of disease progression. Other endpoints include kinetic studies of ctDNA, biological progression-free survival (bPFS) (time from first study dose to first biological event on ctDNA), median PFS according to RECIST criteria 1.1 (called radiological [r] PFS), and median clinical (c) PFS (time from the first study dose to off-osimertinib). This study started in April 2019, and 18 centers in France are participants.     

Liquid Biopsy for Advanced NSCLC: A Consensus Statement From the International Association for the Study of Lung Cancer

Although precision medicine has had a mixed impact on the clinical management of patients with advanced-stage cancer overall, for NSCLC, and more specifically for lung adenocarcinoma, the advances have been dramatic, largely owing to the genomic complexity and growing number of druggable oncogene drivers. Furthermore, although tumor tissue is historically the “accepted standard” biospecimen for these molecular analyses, there are considerable innate limitations. Thus, liquid biopsy represents a practical alternative source for investigating tumor-derived somatic alterations. Although data are most robust in NSCLC, patients with other cancer types may also benefit from this minimally invasive approach to facilitate selection of targeted therapies. The liquid biopsy approach includes a variety of methodologies for circulating analytes. From a clinical point of view, plasma circulating tumor DNA is the most extensively studied and widely adopted alternative to tissue tumor genotyping in solid tumors, including NSCLC, first entering clinical practice for detection of EGFR mutations in NSCLC. Since the publication of the first International Association for the Study of Lung Cancer (IASLC) liquid biopsy statement in 2018, several additional advances have been made in this field, leading to changes in the therapeutic decision-making algorithm for advanced NSCLC and prompting this 2021 update. In view of the novel and impressive technological advances made in the past few years, the growing clinical application of plasma-based, next-generation sequencing, and the recent Food and Drug and Administration approval in the United States of two different assays for circulating tumor DNA analysis, IASLC revisited the role of liquid biopsy in therapeutic decision-making in a recent workshop in October 2020 and the question of “plasma first” versus “tissue first” approach toward molecular testing for advanced NSCLC. Moreover, evidence-based recommendations from IASLC provide an international perspective on when to order which test and how to interpret the results. Here, we present updates and additional considerations to the previous statement article as a consensus from a multidisciplinary and international team of experts selected by IASLC.     

Refined Stratification Based on Baseline Concomitant Mutations and Longitudinal Circulating Tumor DNA Monitoring in Advanced EGFR-Mutant Lung Adenocarcinoma Under Gefitinib Treatment

IntroductionThe optimal treatment for EGFR-mutant lung adenocarcinoma (LUAD) remains challenging because of intratumor heterogeneity. We aimed to explore a refined stratification model based on the integrated analysis of circulating tumor DNA (ctDNA) tracking.MethodsctDNA was prospectively collected at baseline and at every 8 weeks in patients with advanced treatment-naive EGFR-mutant LUAD under gefitinib treatment enrolled in a phase 2 trial and analyzed using next-generation sequencing of a 168-gene panel.ResultsThree subgroups categorized by baseline comutations—EGFR-sensitizing mutations (59, 32.8%), EGFR-sensitizing mutations with tumor suppressor mutations (97, 53.9%), and EGFR-sensitizing mutations with other driver mutations (24, 13.3%)—exhibited distinct progression-free survival (13.2 [11.3–15.2] versus 9.3 [7.6–10.5] versus 4.0 [2.4–9.3] months) and overall survival (32.0 [29.2–41.5] versus 21.7 [19.3–27.0] versus 15.5 [10.5–33.7] months, respectively), providing evidence for initial stratification. A total of 63.7% of the patients achieved week 8 ctDNA clearance, with significant difference noted among the three subgroups (74.5% versus 64.0% versus 29.4%, respectively, p = 0.004, Fisher’s exact test). Patients without week 8 ctDNA clearance had worse progression-free survival (clearance versus nonclearance 11.2 [9.9–13.2] versus 7.4 [5.6–9.6] months, p = 0.016, Cox regression], especially in the second subgroup [5.8 (5.6–11.5) months], suggesting the necessity of adaptive stratification during treatment. During follow-up, 56.0% and 20.8% of the patients eventually harbored p.T790M and non-p.T790M mutations, respectively, with a significant difference in non-p.T790M mutations among the three subgroups (7.5% versus 15.7% versus 80.0%, respectively, p < 0.001, Fisher’s exact test), giving clues to postline treatment.ConclusionsThe patients with baseline comutations and ctDNA nonclearance at first visit might require combined therapy because of the limited survival benefit of EGFR tyrosine kinase inhibitor monotherapy. We proposed a refined stratification mode for the whole-course management of EGFR-mutant LUAD.     

Analysis on Early Detection of Lung Cancer by PET/CT Scan

Background: This systemic analysis was conducted to to evaluate the application value of positron emissiontomography/computed tomography (PET/CT) in early diagnosis of lung cancer. Methods: Clinical studiesevaluating the application value of PET/CT for patients underwent PET/CT imaging. The histological diagnosisserved as the standard of truth. Results: Four clinical studies which including 1330 patients with pulmonary spaceoccupyinglesions were considered eligible for inclusion. Systemic analysis suggested that, in all 1330 patients,pooled sensitivity was 98.7% (1313.2/1330) and specificity was 58.2%(276.85/476). Conclusion: This systemicanalysis suggests that integrated PET/CT imaging provides high sensitivity, and reasonably high specificity, andcould be applied for early diagnosis of lung cancer.     

Cell-Free Tumor DNA (ctDNA) Utility in Detection of Original Sensitizing and Resistant EGFR Mutations in Non-Small Cell Lung Cancer (NSCLC)

Background: Recent studies have demonstrated the utility of cell-free tumor DNA (ctDNA) from plasma as an alternative source of genomic material for detection of sensitizing and resistance mutations in NSCLC. We hypothesized that the plasma level of ctDNA is an effective biomarker to provide a non-invasive and thus a less risky method to determine new resistance mutations and to monitor response to treatment and tumor progression in lung cancer patients. Methods: This prospective cohort study was approved and conducted at the Peter Brojde Lung Cancer Centre, Montreal. Blood was collected in STRECK tubes at four time points. DNA was extracted from plasma, and ctDNA was analyzed for the presence of mutations in the EGFR gene using the COBAS^® EGFR v2 qPCR (Roche) test. Results: Overall, 75 pts were enrolled in the study. In total, 23 pts were TKI-naïve, and 52 were already receiving first-line TKI treatment. ctDNA detected the original mutations (OM) in 35/75 (48%) patients. Significantly higher detection rates were observed in TKI-naïve patients compared to the TKI-treated group, 70% versus 37%, respectively (p = 0.012). The detection of the original mutation at the study baseline was a negative predictor of progression-free survival (PFS) and overall survival (OS). The resistance mutation (T790M) was detected in 32/74 (43%) patients. In 27/32 (84%), the T790M was detected during treatment with TKI: in 25/27 patients, T790M was detected at the time of radiologic progression, in one patient, T790M was detected before radiologic progression, and in one patient, T790M was detected four weeks after starting systemic chemotherapy post progression on TKI. At the time of progression, the detection of T790M significantly correlates with the re-appearance of OM (p = 0.001). Conclusion: Plasma ctDNA is a noninvasive patient-friendly test that can be used to monitor response to treatment, early progression, and detection of acquired resistant mutations. Monitoring of clearance and re-emergence of driver mutations during TKI treatment effectively identifies progression of the disease. As larger NGS panels are available for ctDNA testing, these findings may also have implications for other biomarkers. The results from ongoing and prospective studies will further determine the utility of plasma testing to diagnose, monitor for disease progression, and guide treatment decisions in NSCLC.     

Longitudinal Evaluation of Circulating Tumor DNA Using Sensitive Amplicon-Based Next-Generation Sequencing to Identify Resistance Mechanisms to Immune Checkpoint Inhibitors for Advanced Urothelial Carcinoma

Serial evaluation of circulating tumor DNA may allow noninvasive assessment of drivers of resistance to immune checkpoint inhibitors (ICIs) in advanced urothelial cancer (aUC). We used a novel, amplicon-based next-generation sequencing assay to identify genomic alterations (GAs) pre- and post-therapy in 39 patients with aUC receiving ICI and 6 receiving platinum-based chemotherapy (PBC). One or more GA was seen in 95% and 100% of pre- and post-ICI samples, respectively, commonly in TP53 (54% and 54%), TERT (49% and 59%), and BRCA1/BRCA2 (33% and 33%). Clearance of ≥1 GA was seen in 7 of 9 patients responding to ICI, commonly in TP53 (n = 4), PIK3CA (n = 2), and BRCA1/BRCA2 (n = 2). A new GA was seen in 17 of 20 patients progressing on ICI, frequently in BRCA1/BRCA2 (n = 6), PIK3CA (n = 3), and TP53 (n = 3), which seldom emerged in patients receiving PBC. These findings highlight the potential for longitudinal circulating tumor DNA evaluation in tracking response and resistance to therapy.     

Real-World Clinical Outcomes after Genomic Profiling of Circulating Tumor DNA in Patients with Previously Treated Advanced Non-Small Cell Lung Cancer

Comprehensive genomic profiling for advanced non-small cell lung cancer (NSCLC) can identify patients for molecularly targeted therapies that improve clinical outcomes. We analyzed data from 3084 patients (median age 65 years, 72.9% with adenocarcinoma) with advanced NSCLC registered in a real-world healthcare claims database (GuardantINFORM^TM, Guardant Health) who underwent next-generation sequencing (NGS)-based circulating tumor DNA (ctDNA) testing (Guardant360^®, Guardant Health) after first-line therapy (28.0% with agents targeted against genomic alterations). ctDNA was detected in 2771 samples (89.9%), of which 41.9% harbored actionable alterations, most commonly EGFR (epidermal growth factor receptor) mutations (29.7%). Actionable alterations were detected in 26.7% of patients (534/2001) previously treated with non-targeted agents. Emerging potentially targetable mutations were found in 40.1% (309/770) of patients previously treated with targeted therapies. Among patients with qualifying alterations detected by ctDNA testing, the time to treatment discontinuation (median 8.8 vs. 4.2 months; hazard ratio 1.97, p < 0.001) and overall survival (median 36.1 vs. 16.6 months; hazard ratio 2.08, p < 0.001) were longer for those who received matched second-line therapy versus unmatched second-line therapy. In real-world practice, results of a blood-based NGS assay prior to second-line treatment inform therapeutic decisions that can improve clinical outcomes for patients with advanced NSCLC.     

Hybrid Capture–Based Genomic Profiling of Circulating Tumor DNA from Patients with Advanced Non–Small Cell Lung Cancer

Introduction

Genomic profiling informs selection of matched targeted therapies as part of routine clinical care in NSCLC. Tissue biopsy is the criterion standard; however, genomic profiling of blood-derived circulating tumor DNA (ctDNA) has emerged as a minimally invasive alternative.

Molecular Analysis of Plasma From Patients With ROS1-Positive NSCLC

Introduction

Circulating tumor DNA analysis is an emerging genotyping strategy that can identify tumor-specific genetic alterations in plasma including mutations and rearrangements. Detection of ROS1 fusions in plasma requires genotyping approaches that cover multiple breakpoints and target a variety of fusion partners. Compared to other molecular subsets of NSCLC, experience with detecting ROS1 genetic alterations in plasma is limited.

Expert Consensus Recommendations on Biomarker Testing in Metastatic and Nonmetastatic NSCLC in Asia

IntroductionMost published guidelines for genomic biomarker testing in NSCLC reflect the disease epidemiology and treatments readily available in Europe and North America. Nevertheless, 60% of annual global NSCLC cases occur in Asia, where patient characteristics, tumor molecular profiles, and treatments vary greatly from the Western world. For example, mutations in the EGFR occur at a higher prevalence in Asia than in other world regions. Although medical associations such as the International Association for the Study of Lung Cancer, European Society for Medical Oncology, and American Society of Clinical Oncology have described principles for tumor genomic biomarker testing in NSCLC, there is a need for recommendations specific for Asia.MethodsThis report provides consensus recommendations for NSCLC biomarker testing from Asian lung cancer experts for clinicians working in Asia to improve patient care. Biomarker testing approaches for actionable genetic alterations in EGFR, ALK, ROS1, and others are discussed.ResultsThese recommendations are divided into nonmetastatic and metastatic forms of adenocarcinoma and squamous cell carcinoma. Owing to the higher prevalence of EGFR mutations in Asia, the experts emphasized the need for EGFR testing to include not just common mutations (exon 19 deletions and L858R substitutions) but also other uncommon EGFR mutations. In addition to the assessment of biomarkers in the tumor tissue, the role of assessing tumor biomarkers by liquid biopsy is discussed.ConclusionThis consensus provides practical recommendations for biomarker testing in nonmetastatic and metastatic Asian NSCLC patients.     

Significance of Preoperative Parameters for Predicting Tumor Volume in Nonpalpable Prostate Cancer

Preoperative parameters were assessed for their usefulness inpredicting tumor volume in 26 cases of nonpalpable prostatecancer. The number of positive biopsy cores was found to bethe parameter which predicted most accurately tumor volume insurgical specimens. All 14 patients with multiple positive coreshad an index tumor of 0.5 cm³ or more in radical prostatectomyspecimens. Only six tumors with a single positive core weresubstantial. Three of the patients showed a serum level of prostatespecific antigen (PSA) of 4.0 ng/ml or more. The pathologicalstage and prostatectomy Gleason sum could not be predicted accuratelyby this variable (P < 0.05). To predict index tumor volumesof 1.0 cm³ or greater, stepwise logistic regression analysiswas conducted, and on the basis of the results it was possibleto single out multiple positive biopsies as the only significantvariable. The number of positive biopsies appeared reliablefor predicting tumor volume of surgical specimens in cases ofnonpalpable disease. The presence of significant tumors despite"normal" levels of PSA warrants further investigation of variablesthat can predict such prostate cancers. An adequate model forpredicting significant tumors in oriental male populations shouldbe established using samples of larger size.