Liquid biopsy for early cancer detection: technological revolutions and clinical dilemma.

INTRODUCTION: Liquid biopsy is an innovative advancement in oncology, offering a noninvasive method for early cancer detection and monitoring by analyzing circulating tumor cells, DNA, RNA, and other biomarkers in bodily fluids. This technique has the potential to revolutionize precision oncology by providing real-time analysis of tumor dynamics, enabling early detection, monitoring treatment responses, and tailoring personalized therapies based on the molecular profiles of individual patients. AREAS COVERED: In this review, the authors discuss current methodologies, technological challenges, and clinical applications of liquid biopsy. This includes advancements in detecting minimal residual disease, tracking tumor evolution, and combining liquid biopsy with other diagnostic modalities for precision oncology. Key areas explored are the sensitivity, specificity, and integration of multi-omics, AI, ML, and LLM technologies. EXPERT OPINION: Liquid biopsy holds great potential to revolutionize cancer care through early detection and personalized treatment strategies. However, its success depends on overcoming technological and clinical hurdles, such as ensuring high sensitivity and specificity, interpreting results amidst tumor heterogeneity, and making tests accessible and affordable. Continued innovation and collaboration are crucial to fully realize the potential of liquid biopsy in improving early cancer detection, treatment, and monitoring.

Recent advances in ctDNA detection using electrochemical biosensor for cancer.

In the quest for early cancer diagnosis, early identification and treatment are paramount. Recently, ctDNA detection has emerged as a viable avenue for early screening of cancer. The examination of ctDNA in fluid biopsies has gained substantial attention in tumor diagnosis and therapy. Both the scientific community and industry are actively exploring this field. However, developing cost-effective, portable, and real-time ctDNA measurement methods using conventional gene detection equipment poses a significant challenge. This challenge has led to the exploration of alternative approaches. Electrochemical biosensors, distinguished by their heightened sensitivity, remarkable specificity, affordability, and excellent portability, have emerged as a promising avenue for ctDNA detection. This review is dedicated to the specific focus on ctDNA detection, highlighting recent advancements in this evolving detection technology. We aimed to reference previous studies related to ctDNA-targeted cancer detection using electrochemical biosensors to advocate the utilization of electrochemical biosensors in healthcare diagnostics. Further research is imperative for the effective integration of ctDNA analysis into point-of-care cancer testing. Innovative approaches utilizing multiple markers need to be explored to advance this technology and make substantial contributions to societal well-being.

Analytical and clinical validation of a NGS panel in detecting targetable variants from ctDNA of metastatic NSCLC patients.

BACKGROUND: Circulating tumor DNA (ctDNA) has emerged as a promising biomarker for noninvasive cancer diagnostics, particularly in the context of metastatic non-small-cell lung cancer (NSCLC). Detecting targetable variants through ctDNA analysis offers the potential to guide treatment decisions, especially in cases where tissue samples are insufficient or unavailable. METHOD: In this study, we developed and validated a next-generation sequencing panel targeting 101 cancer-related genes (101-test) to detect somatic variants in ctDNA from a large cohort of Chinese patients with metastatic NSCLC. The performance of the 101-test was assessed by evaluating its limit of detection (LOD), accuracy, and precision in identifying molecular variants. Additionally, the concordance between ctDNA and tissue samples for detecting targetable variants was analyzed in 904 patients. RESULTS: The 101-test demonstrated a LOD of 0.38% for single-nucleotide variants (SNVs), 0.33% for insertions and deletions (InDels), and 0.33% for fusions. Sensitivity was 98.3% for SNVs, 100% for InDels, and 100% for fusions when compared to digital droplet PCR (ddPCR)/breakpoint PCR reference methods. The by-variant sensitivity for somatic variants was 97.5%, with a specificity of 99.9% between tumor-only and tumor-normal analyses. In a real-world cohort, the concordance between ctDNA and tissue samples for identifying targetable variants was 72.2% (457/633). Notably, the EGFR S768I variant, recently recommended by clinical guidelines, achieved an 80% concordance rate. Furthermore, 4.3% of patients (27/633) with targetable variants were identified exclusively through ctDNA testing. CONCLUSION: The ctDNA-based 101-test is a highly sensitive and specific tool for detecting targetable variants in metastatic NSCLC, particularly in cases with insufficient tissue samples. The findings support the use of ctDNA testing as a reliable and complementary method to traditional tissue-based molecular analysis, enhancing the precision of treatment strategies for NSCLC patients.

Circulating tumor DNA predicts recurrence and survival in patients with resectable gastric and gastroesophageal junction cancer.

BACKGROUND: Gastric and gastroesophageal junction (GEJ) cancer represents a significant global health challenge, with high recurrence rates and poor survival outcomes. This study investigates circulating tumor DNA (ctDNA) as a biomarker for assessing recurrence risk in patients with resectable gastric and GEJ adenocarcinomas (AC). METHODS: Patients with resectable gastric and GEJ AC, undergoing perioperative chemotherapy and surgery, were prospectively enrolled. Serial plasma samples were collected at baseline, after one cycle of chemotherapy, after preoperative chemotherapy, and after surgery. ctDNA was assessed by a ddPCR test (TriMeth), which targets the gastrointestinal cancer-specific methylation patterns of the genes C9orf50, KCNQ5, and CLIP4. RESULTS: ctDNA analysis was performed on 229 plasma samples from 86 patients. At baseline, ctDNA was detected in 56% of patients, which decreased to 37% following one cycle of chemotherapy, 25% after preoperative chemotherapy and 15% after surgical resection. The presence of ctDNA after one cycle of chemotherapy was associated with reduced recurrence-free survival (RFS) (HR = 2.54, 95% confidence interval (CI) 1.33-4.85, p = 0.005) and overall survival (OS) (HR = 2.23, 95% CI 1.07-4.62, p = 0.032). Similarly, ctDNA after surgery was associated with significantly shorter RFS (HR = 6.22, 95% CI 2.39-16.2, p < 0.001) and OS (HR = 6.37, 95% CI 2.10-19.3, p = 0.001). Multivariable regression analysis confirmed ctDNA after surgery as an independent prognostic factor (p < 0.001). CONCLUSION: ctDNA analysis has the potential to identify patients at elevated risk of recurrence, thus providing personalized treatment strategies for patients with resectable gastric and GEJ cancer. Further validation in larger cohorts and ctDNA-guided interventions are needed for future clinical use.

Plasma EGFR mutation ctDNA dynamics in patients with advanced EGFR-mutated NSCLC treated with Icotinib: phase 2 multicenter trial result.

Plasma epidermal growth factor receptor mutation (EGFRm) circulating tumor DNA (ctDNA) dynamics exhibit promise in predicting outcomes in patients with EGFRm-advanced non-small cell lung cancer (NSCLC). However, there remains limited trial-level data on integrating ctDNA monitoring into clinical practice. We performed a prospective, multicenter trial to investigate the relationship between EGFRm ctDNA dynamic changes and clinical outcomes in NSCLC patients with EGFRm. Ninety-eight treatment-naive EGFRm-advanced NSCLC patients were recruited and administered icotinib until disease progression. Plasma samples were collected at baseline and four weeks after icotinib administration. ctDNA was analyzed using a droplet-digital polymerase chain reaction. At baseline, 71.4% of patients had detectable EGFRm ctDNA. Among them, 45.9% of patients' ctDNA became undetectable within four weeks of treatment. These patients demonstrated significantly longer progression-free survival (PFS) and overall survival (OS) than those with detectable ctDNA after treatment (P = 0.004 and < 0.001, respectively) and were comparable to those with undetectable ctDNA at both baseline and four weeks. ctDNA detectable at four weeks emerged as a poor independent risk factor for PFS and OS. Patients whose ctDNA became undetectable after treatment had outcomes similar to those with initially undetectable ctDNA, underscoring the predictive value of ctDNA dynamics in treatment efficacy.Registry and the Registration No. of the study/trial: ChiCTR-DDD-17013131. Date of registration: 2017-10-27.

Early diagnostic strategies for colorectal cancer.

At present, cancer is still an important factor threatening human health. Colorectal cancer (CRC) is one of the top three most common cancers worldwide and one of the deadliest malignancies in humans. The latest data showed that CRC incidence and mortality rank third and second, respectively, among global malignancies. Early and accurate diagnosis is crucial to reduce the morbidity, mortality and improve survival of patients with CRC, but the current early diagnostic methods have limitations. The effectiveness and compliance of diagnostic methods have a certain impact on whether people choose screening. In this editorial, we explore strategies for the early diagnosis of CRC, including stool-based, blood-based, direct visualization, and imaging examinations.

Genomic-Based Early Detection Screening: A Literature Review of Prospective Trials and Emerging Strategies for Gastrointestinal Cancers.

Numerous genomic-based early detection screening tests are being developed. These tests have the potential to revolutionize current single-organ screening paradigms, especially in gastrointestinal cancers. In this review, we underscore the performance of these genomic-based early detection tests based on prospective clinical trials. Moreover, we discuss a professional advancement for gastroenterologists in the diagnostic assessment of individuals who are cancer signal positive.

Potential value of detection of minimal residual disease in colorectal cancer following radical resection.

Although there has been significant advancement in the identification and management of colorectal cancer (CRC) in recent years, there is still room for improvement in the current standard treatment regimen. One area of concern is the lack of reliable tumor markers to predict treatment efficacy and guide tailored care. Due to its dynamic, effective, and non-invasive benefits over tissue biopsy, the detection of minimal or molecular residual lesions (MRD) based on circulating tumor DNA (ctDNA) is beneficial to the clinical development of drugs for patients with CRC after radical treatment, as well as for continuous monitoring of tumor recurrence and malignancy molecular gene evolution. The detection of ctDNA can currently be used to guide individual postoperative auxiliary treatment decisions (upgrade or downgrade treatment) in CRC, stratify the risk of clinical recurrence more precisely, and predict the risk of recurrence in advance of imaging examination, according to a large number of observational or prospective clinical studies. With increasing clarity comes the possibility of selecting a regimen of treatment based on postoperative ctDNA, which also improves the accuracy of clinical recurrence risk assessment for CRC. Therefore, it is anticipated that the identification of ctDNA would alter the current framework for dealing with CRC and lead to individualized, stratified precision therapy; however, additional confirmation will require subsequent high-quality, prospective, large-scale randomized controlled studies. This article will provide an overview of the definition and clinical significance of MRD, the primary indications and technological challenges for MRD detection, along with the advancement in clinical research about ctDNA detection following radical resection of the CRC.

Circulating tumor DNA-based stratification strategy for chemotherapy plus PD-1 inhibitor in advanced non-small-cell lung cancer.

Stratification strategies for chemotherapy plus PD-1 inhibitors in advanced non-small-cell lung cancer (NSCLC) are critically demanded. We performed high-throughput panel-based deep next-generation sequencing and low-pass whole genome sequencing on prospectively collected circulating tumor DNA (ctDNA) specimens from 460 patients in the phase 3 CHOICE-01 study at different time points. We identified predictive markers for chemotherapy plus PD-1 inhibitor, including ctDNA status and genomic features such as blood-based tumor mutational burden, intratumor heterogeneity, and chromosomal instability. Furthermore, we established an integrated ctDNA-based stratification strategy, blood-based genomic immune subtypes (bGIS) scheme, to distinguish patients who benefit from the addition of PD-1 inhibitor to first-line chemotherapy. Moreover, we demonstrated potential applications for the dynamic monitoring of ctDNA. Overall, we proposed a potential therapeutic algorithm based on the ctDNA-based stratification strategy, shedding light on the individualized management of immune-chemotherapies for patients with advanced NSCLC.

Clinical applications of circulating tumor DNA in hematological malignancies: From past to the future.

Liquid biopsy, particularly circulating tumor DNA (ctDNA), has drawn a lot of attention as a non- or minimal-invasive detection approach for clinical applications in patients with cancer. Many hematological malignancies are well suited for serial and repeated ctDNA surveillance due to relatively high ctDNA concentrations and high loads of tumor-specific genetic and epigenetic abnormalities. Progress of detecting technology in recent years has improved sensitivity and specificity significantly, thus broadening and strengthening the potential utilities of ctDNA including early diagnosis, prognosis estimation, treatment response evaluation, minimal residual disease monitoring, targeted therapy selection, and immunotherapy surveillance. This manuscript reviews the detection methodologies, clinical application and future challenges of ctDNA in hematological malignancies, especially for lymphomas, myeloma and leukemias.

Liquid Biopsy in Progressing Prostate Cancer Patients Starting Docetaxel with or Without Enzalutamide: A Biomarker Study of the PRESIDE Phase 3b Trial.

BACKGROUND AND OBJECTIVE: The PRESIDE (NCT02288247) randomized trial demonstrated prolonged progression-free survival (PFS) with continuing enzalutamide beyond progression in metastatic castration-resistant prostate cancer (mCRPC) patients starting docetaxel. This study aims to test the associations of PFS and circulating tumor DNA (ctDNA) prior to and after one cycle (cycle 2 day 1 [C2D1]) of docetaxel and with a liquid biopsy resistance biomarker (LBRB; plasma androgen receptor [AR] gain and/or circulating tumor cells [CTCs] expressing AR splice variant 7 [CTC-AR-V7]) prior to continuation of enzalutamide/placebo. METHODS: Patients consenting to the biomarker substudy and donating blood before starting docetaxel with enzalutamide/placebo (N = 157) were included. Sequential plasma DNA samples were characterized with a prostate-cancer bespoke next-generation-sequencing capture panel (PCF_SELECT), and CTCs were assessed for AR-V7 (Epic Sciences, San Diego, CA, USA). Cox models, Kaplan-Meier, and restricted mean survival time (RMST) at 18 mo were calculated. KEY FINDINGS AND LIMITATIONS: There was a significant association of worse PFS with pre-docetaxel ctDNA detection (N = 86 (55%), 8.1 vs 10.8 mo hazard ratio [HR] = 1.78, p = 0.004) or persistence/rise of ctDNA at C2D1 (N = 35/134, 5.5 vs 10.9 mo, HR = 1.95, 95% confidence interval [CI] = 1.15-3.30, p = 0.019). LBRB-positive patients (N = 62) had no benefit from continuing enzalutamide with docetaxel (HR = 0.78, 95% CI = 0.41-1.48, p = 0.44; RMST: 7.9 vs 7.1 mo, p = 0.50). Conversely, resistance biomarker-negative patients (N = 87) had significantly prolonged PFS (HR = 0.49, 95% CI = 0.29-0.82, p = 0.006; RMST: 11.5 vs 8.9 mo, p = 0.005). Eight patients were unevaluable. An exploratory analysis identified increased copy-number gains (CDK6/CDK4) at progression on docetaxel. Limitations included relatively low detection of CTC-AR-V7. Validation of impact on overall survival is required. CONCLUSIONS AND CLINICAL IMPLICATIONS: Liquid biopsy gives an early indication of docetaxel futility, could guide patient selection for continuing enzalutamide, and identifies cell cycle gene alterations as a potential cause of docetaxel resistance in mCRPC. PATIENT SUMMARY: In the PRESIDE biomarker study, we found that detecting circulating tumor DNA in plasma after starting treatment with docetaxel (chemotherapy) for metastatic prostate cancer resistant to androgen deprivation therapy can predict early how long patients will take to respond to treatment. Patients negative for a liquid biopsy resistance biomarker (based on the status of androgen receptor (AR) gene and AR splice variant 7 in circulating tumor cells) benefit from continuing enzalutamide in combination with docetaxel, while patients positive for the resistance biomarker did not. Additionally, we identified alterations in the cell cycle genes CDK6 and CDK4 as a potential genetic cause of resistance to docetaxel, which may support testing of specific drugs targeting these alterations.

Dynamic bTMB combined with residual ctDNA improves survival prediction in locally advanced NSCLC patients with chemoradiotherapy and consolidation immunotherapy.

Background: Liquid biopsy-based biomarkers, including circulating tumor DNA (ctDNA) and blood tumor mutational burden (bTMB), are recognized as promising predictors of prognoses and responses to immune checkpoint inhibitors (ICIs), despite insufficient sensitivity of single biomarker detection. This research aims to determine whether the combinatorial utility of longitudinal ctDNA with bTMB analysis could improve the prognostic and predictive effects. Methods: This prospective two-center cohort trial, consisting of discovery and validation datasets, enrolled unresectable locally advanced non-small-cell lung cancer (LA-NSCLC) patients and assigned them to chemoradiotherapy (CRT) or CRT + consolidation ICI cohorts from 2018 to 2022. Blood specimens were collected pretreatment, 4 weeks post-CRT, and at progression to assess bTMB and ctDNA using 486-gene next-generation sequencing. Dynamic ∆bTMB was calculated as post-CRT bTMB minus baseline bTMB levels. Decision curve analyses were performed to calculate Concordance index (C-index). Results: One hundred twenty-eight patients were enrolled. In the discovery dataset (n = 73), patients treated with CRT and consolidation ICI had significantly longer overall survival (OS; median not reached [NR] vs 20.2 months; P < 0.001) and progression-free survival (PFS; median 25.2 vs 11.4 months; P = 0.011) than those without ICI. Longitudinal analysis demonstrated a significant decrease in ctDNA abundance post-CRT (P < 0.001) but a relative increase with disease progression. Post-CRT detectable residual ctDNA correlated with significantly shorter OS (median 18.3 months vs NR; P = 0.001) and PFS (median 7.3 vs 25.2 months; P < 0.001). For patients with residual ctDNA, consolidation ICI brought significantly greater OS (median NR vs 14.8 months; P = 0.005) and PFS (median 13.8 vs 6.2 months; P = 0.028) benefit, but no significant difference for patients with ctDNA clearance. Dynamic ∆bTMB was predictive of prognosis. Patients with residual ctDNA and increased ∆bTMB (∆bTMB > 0) had significantly worse OS (median 9.0 vs 23.0 months vs NR; P < 0.001) and PFS (median 3.4 vs 7.3 vs 25.2 months; P < 0.001). The combinatorial model integrating post-CRT ctDNA with ∆bTMB had optimal predictive effects on OS (C-index = 0.723) and PFS (C-index = 0.693), outperforming individual features. In the independent validation set, we confirmed residual ctDNA predicted poorer PFS (median 50.8 vs 14.3 months; P = 0.026) but identified more consolidation ICI benefit (median NR vs 8.3 months; P = 0.039). The combined model exhibited a stable predictive advantage (C-index = 0.742 for PFS). Conclusions: The multiparameter assay integrating qualitative residual ctDNA testing with quantitative ∆bTMB dynamics improves patient prognostic risk stratification and efficacy predictions, allowing for personalized consolidation therapy for LA-NSCLC.

Advances in blood DNA methylation-based assay for colorectal cancer early detection: a systematic updated review.

Aim: A systematic review was conducted to summarize the methylated circulating tumor DNA (ctDNA) markers reported over the last decade for early detection of colorectal cancer (CRC) and to identify the main technical challenges that are impeding their clinical implementation. Background: CRC is a major cause of cancer deaths worldwide, but early detection is key for successful treatment. Non-invasive methods such as methylated ctDNA testing show promise for improving detection and monitoring of CRC. Methods: A comprehensive search was performed using Web of Science, PubMed, and Scopus up to December 30, 2023, limited to articles published in the last 10 years (after 2012), while including advanced adenoma/stage 0 or stage I/II samples in biomarker validation. Results: After identifying 694 articles, removing duplicates and screening titles, abstracts, and full texts, a total of 62 articles were found to meet the inclusion criteria. Among the single biomarkers, MYO1-G, SEPT9, SDC2, and JAM3 revealed the highest sensitivity for polyps and stage I/II CRC. For multi-biomarkers with suitable sensitivity, combinations of SFRP1, SFRP2, SDC2, PRIMA1, or ALX4, BMP3, NPTX2, RARB, SDC2, SEPT9, VIM or ZFHX4, ZNF334, ELOVL2, UNC5C, LOC146880, SFMBT2, GFRA1 were identified for polyps and stage I/II CRC. Conclusion: Enhancing sensitivity and specificity of molecular screening methods is crucial for improving CRC detection. Identifying a select few valuable biomarkers is key to reducing costs, despite challenges posed by low ctDNA levels in plasma, particularly in early-stage cancers.

Circulating Tumor DNA in Patients with Desmoid Fibromatosis during Active Surveillance.

BACKGROUND: Sporadic desmoid fibromatosis (DF) is a rare locally aggressive tumor characterized by mutation in exon 3 of CTNNB1 (T41A, S45F, and S45P). Standard of care is active surveillance (AS), but 30% require treatment. DF clinical course is unpredictable and identification of prognostic markers is needed to tailor strategy. In this prospective study, we investigated the consistency between mutation detected in tumor biopsies with that detected in plasma by digital droplet PCR (ddPCR) and the association between circulating tumor DNA (ctDNA) abundancy with clinical outcome. PATIENTS AND METHODS: A total of 56 patients and 10 healthy donors were included. CTNNB1 mutation status of DF biopsies was determined by Sanger and in case of WT CTNNB1 with NGS. In matched plasma samples at enrollment and during AS at specific timepoints, we evaluated cfDNA quantity and ctDNA. RESULTS: ctDNA levels were measured in 46 patients with CTNNB1 mutation. Detection rate for T41A, S45F and S45P was 68%, 42% and 100%, respectively. S45P variant has been detected in all patients with S45P mutation. Longitudinal assessment of ctDNA during AS in nine patients (four with regression and five with progression as first event according to RECIST) showed a concordance between the event and ctDNA level change in six out of nine patients tested (4/5 with progression and 2/4 with regression). CONCLUSIONS: Results of ctDNA analysis support its potential clinical implementation as diagnostic tool in specific clinical scenarios where biopsy can be challenging. A prospective clinical trial needs to be performed to evaluate the potential role of ctDNA as predictive biomarker.

Optimizing ctDNA: An Updated Review of a Promising Clinical Tool for the Management of Uveal Melanoma.

Uveal melanoma (UM) is the most common primary malignant intraocular tumor in adults. Distant metastasis is common, affecting around 50% of patients. Prognostic accuracy relies on molecular characterization of tumor tissue. In these patients, however, conventional biopsy can be challenging due to the difficulty of obtaining sufficient tissue for the analysis due to the small tumor size and/or post-brachytherapy shrinkage. An alternative approach is liquid biopsy, a non-invasive technique that allows for real-time monitoring of tumor dynamics. Liquid biopsy plays an increasingly prominent role in precision medicine, providing valuable information on the molecular profile of the tumor and treatment response. Liquid biopsy can facilitate early detection and can be used to monitor progression and recurrence. ctDNA-based tests are particularly promising due to their ease of integration into clinical practice. In this review, we discuss the application of ctDNA in liquid biopsies for UM. More specifically, we explore the emerging technologies in this field and the advantages and disadvantages of using different bodily fluids for liquid biopsy. Finally, we discuss the current barriers to routine clinical use of this technique.

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.

Does circulating tumor DNA apply as a reliable biomarker for the diagnosis and prognosis of head and neck squamous cell carcinoma?

Oral cavity cancer is the most common type of head and neck cancer. There is no definitive standard diagnosis, prognosis, or treatment response biomarker panel based on simple, specific, non-invasive, and reliable methods for head and neck squamous cell carcinoma (HNSCC) patients. On the other hand, the frequent post-treatment biopsies make it challenging to discriminate residual disease or recurrent tumors following postoperative reparative and post-radiation changes. Saliva, blood plasma, and serum samples were commonly used to monitor HNSCC through liquid biopsies. Based on the evidence, the most prominent molecular-based fluid biomarker, such as circulating tumor DNA (ctDNA), has potential applications for early cancer diagnosis, screening, patient management, and surveillance. ctDNA showed genomic and epigenomic changes and the status of human papillomavirus (HPV) with the real-time monitoring of tumor status through cancer therapy. Due to the intra and inter-tumor heterogeneity of tumor cells like cancer stem cells (CSCs) and tumor microenvironment (TME) in HNSCC, the tiny tissue biopsy cannot reflect all genomic and transcriptomic abnormality. Most liquid biopsies are applied to detect circulating molecular biomarkers consisting of cell-free DNA (cfDNA), ctDNA, microRNA, mRNA, and exosome for monitoring tumor progression. Based on the results of previous studies, liquid biopsy can be applied for comprehensive multi-omic discovery by assessing the predictive value of ctDNA in both early and advanced cancers. Liquid biopsy can be used to evaluate molecular signature profiles in HNSCC patients, with great potential to help in early diagnosis, prognosis, surveillance, and treatment monitoring of tumors. These happen by designing longitudinal extensive cohort studies and the utility of organoid technology that promotes the context of personalized and precision cancer medicine.

The Unholy Grail of cancer screening: or is it just about the Benjamins?

The biotechnology company Grail developed a non-invasive blood test (Galleri test) which is claimed to detect 50 types of cancer at early and potentially curable stages. The initially promising results from prospective studies, and the anticipated financial success of Grail led the sequencing giant Illumina to purchase Grail for $8 billion (2021). Following this event, Grail collaborated with the UK National Health System to further clarify the test's capability, in a 3-year prospective trial, along with the standard of care. At the end of the first year, UK-NHS announced that they will stop the trial due to unsatisfactory clinical performance and until they analyze the data for the first year (which already enrolled 140,000 participants). Legal and financial issues between the interested parties are currently in flux. The Grail case is reminiscent of the multi-billion Theranos scandal, which sent a few people to jail and led to company dissolution. We previously expressed concerns about the sensitivity and specificity of the Galleri test. In this Perspective, we revisit the hyped technology, and we provide new suggestions on the use of this test.

Survival benefit of adjuvant chemotherapy based on molecular residual disease detection in resected colorectal liver metastases: subgroup analysis from CIRCULATE-Japan GALAXY.

BACKGROUND: The prognostic role of circulating tumor DNA (ctDNA)-based molecular residual disease (MRD) detection and its utility for postsurgical risk stratification has been reported in colorectal cancer. In this study, we explored the use of ctDNA-based MRD detection in patients with colorectal liver metastases (CLM), for whom the survival benefit of adjuvant chemotherapy (ACT) after surgical resection remains unclear. METHODS: Patients with CLM without extrahepatic disease from the GALAXY study (UMIN000039205) were included. The disease-free survival (DFS) benefit of ACT was evaluated in MRD-positive and -negative groups after adjusting for age, gender, number, and size of liver metastases, RAS status, and previous history of oxaliplatin for primary cancer. ctDNA was detected using a personalized, tumor-informed 16-plex polymerase chain reaction-next-generation sequencing (mPCR-NGS) assay. ctDNA-based MRD status was evaluated 2-10 weeks after curative surgery, before the start of ACT. RESULTS: Among 6061 patients registered in GALAXY, 190 surgically resected CLM patients without any preoperative chemotherapy were included with a median follow-up of 24 months (1-48 months). ctDNA positivity in the MRD window was 32.1% (61/190). ACT was administered to 25.1% (48/190) of patients. In the MRD-positive group, 24-month DFS was higher for patients treated with ACT [33.3% versus not reached, adjusted hazard ratio (HR): 0.07, P < 0.0001]; whereas no benefit of ACT was seen in the MRD-negative group (24-month DFS: 72.3% versus 62.2%, adjusted HR: 0.68, P = 0.371). Multivariate analysis showed that the size of liver metastases (HR: 3.94, P = 0.031) was prognostic of DFS in the MRD-positive group. In the MRD-negative group, however, none of the clinicopathological factors were prognostic of DFS. CONCLUSIONS: Our data suggest that ACT may offer notable clinical benefits in MRD-positive patients with CLM. MRD status-based risk stratification could be potentially incorporated in future clinical trials for CLM.

The prognostic significance of circulating tumor DNA in patients with positive lymph node disease after robotic-assisted radical cystectomy: A contemporary analysis.

BACKGROUND AND OBJECTIVE: Neoadjuvant therapy followed by radical cystectomy with lymphadenectomy remains the gold standard of treatment in patients with muscle-invasive bladder cancer. Pathologically positive lymph node (pN+) disease is known to convey a poor prognosis. Tumor-informed circulating tumor DNA (ctDNA) has emerged as a possible novel prognostic biomarker in the field. We seek to assess recurrence-free survival (RFS) for patients undergoing robotic-assisted radical cystectomy (RARC) with extended pelvic lymphadenectomy (ePLND) and to assess whether ctDNA status can be a prognostic marker for RFS outcomes in patients with pN+ disease. METHODS: Patients who underwent RARC + ePLND during 2015 to 2023 were included. A sub-group analysis (n = 109) of patients who had prospectively collected serial-longitudinal tumor-informed ctDNA analyses during 2021-2023 was performed. Survival analysis and Cox-regression models were conducted. RESULTS: Included were 458 patients with a median age of 69 (IQR 63-76), and a median follow-up time of 20 months (IQR 10-37). RFS for pN0 (n = 353) and pN+ (n = 105) at 12, 24 and 36 months were 87% vs. 54%, 80% vs. 39%, and 74% vs. 35%, respectively (log-rank, P < 0.0001). On Cox multivariate analysis ≥pT3 disease (Hazzard ratio [HR] = 3.36 [2.18-5.18], P < 0.001), pN+ disease (HR = 2.39 [1.55-3.7], P < 0.001), and recipients of neoadjuvant treatment (HR = 1.61 [1.11-2.34], P = 0.013) were predictive of disease relapse. Patients with pN+ disease and undetectable precystectomy or postcystectomy ctDNA status had similar RFS to patients with pN0 with undetectable ctDNA. On Cox-regression multivariate sub-group analysis, detectable precystectomy ctDNA status (HR = 3.89 [1.32-11.4], P = 0.014), detectable ctDNA status in the minimal residual disease window ([MRD], HR = 2.89 [1.12-7.47], P = 0.028), and having ≥pT3 with pN+ disease (HR = 4.2 [1.43-12.3], P = 0.009) were predictive of disease relapse. CONCLUSIONS: Patients with pN+ .after RARC had worse oncological outcomes than patients with pN0 disease. Undetectable ctDNA status was informative of RFS regardless of nodal status at both the precystectomy and the MRD window. Patients with undetectable ctDNA status and pN+ disease may benefit from treatment de-escalation.