Recent advances in molecular profiling have enabled 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. Herein, 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.

This 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, and sufficient plasma samples for ctDNA analysis (AlphaLiquid®100 from IMBdx).

Among 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 fusions, IDH1 mutations, microsatellite instability-high, ERBB2 amplifications, PIK3CA mutations, BRCA1/2 mutations, and MET amplifications. 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).

Among 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.

Our study is the first large-scale investigation of the clinical utility of liquid biopsy, focusing on circulating tumor DNA (ctDNA), as an alternative to conventional tumor tissue analysis, among Asian patients with advanced biliary tract cancer. The results demonstrated acceptable concordance between analysis of ctDNA vs. tissue for identifying therapeutic targets and potentially actionable genetic alterations. This indicates that ctDNA analysis can provide critical insights regarding advanced biliary tract cancer treatment, particularly in cases where it is challenging to obtain or analyze tumor tissue.

Circulating tumor DNA (ctDNA) has emerged as a dynamic biomarker in cancer, as evidenced by its increasing integration into clinical practice. Carrying tumor specific characteristics, ctDNA can be used to inform treatment selection, monitor response, and identify drug resistance. In this review, we provide a comprehensive, up-to-date summary of ctDNA in monitoring treatment response with a focus on lung, colorectal, and breast cancers, and discuss current challenges and future directions.

KRAS G12C inhibitors can treat KRASG12C-mutant advanced colorectal cancers and pancreatic ductal adenocarcinomas (PDAC), but alterations in Kirsten rat sarcoma (KRAS), EGFR, BRAF, MAP2K1, and other genes bypass KRAS inhibition and reduce therapy efficacy. Our study evaluates the genetic landscape of candidate primary resistance alterations relevant to KRAS targeting in KRASG12C-mutant colorectal cancer and PDAC.

We analyzed two cohorts (national database and Mayo) of patients with advanced colorectal cancer or PDAC tested with next-generation sequencing of ctDNA via Guardant360. Cohorts were divided into three groups: KRASG12C alone (KRASG12C without a resistance gene), KRASG12C with resistance (KRASG12C and ≥1 candidate resistance gene), and KRAS not detected. Candidate resistance mutations were inferred from the reported literature.

Among the national (13,603 colorectal cancer and 5,016 PDAC cases) and Mayo (741 colorectal cancer and 422 PDAC cases) cohorts, resistance alterations were identified in a considerable number of KRASG12C cases (46.5% of national colorectal cancer, 16.4% of national PDAC, 53.8% of Mayo colorectal cancer, and 36.4% of Mayo PDAC). The presence of resistance alterations was associated with a trend toward worse overall survival in KRASG12C colorectal cancer (P = 0.05).

Putative resistance alterations are prevalent in PDAC and colorectal cancer and may limit monotherapy efficacy. Identifying these alterations has potential implications in optimal patient selection for targeted therapies and the development of combination therapy strategies to overcome primary resistance.

Glioma recurrence can be divided into in situ recurrence and non-in situ recurrence, and the mutation evolution of gliomas with different recurrence patterns is still unknown. We used sequential sequencing of circulating tumor DNA (ctDNA) to compare the somatic mutation profile and clonal evolution of gliomas with different recurrence patterns. To investigate the value of ctDNA in predicting early postoperative tumor recurrence and guiding prognosis stratification in patients with glioma.

We prospectively recruited 92 patients with near-total resection of gliomas from our center. Two hundred and thirty-four postoperative tissue and Tumor In Situ Fluid (TISF) samples from 69 eligible patients were included in ctDNA analysis.

Among the 69 patients, 37 glioblastoma (GBM) patients experienced recurrence, and the median progression-free survival (mPFS) was not significantly different between the situ recurrence group and the non-in situ recurrence group (8.6 vs. 6.1 months). The ctDNA of recurrent tissue and TISF were significantly consistent. Before and after initial treatment, TISF-ctDNA mutant allele fraction (MAF), subclonal mutation, and alterations in related pathways (lysine degradation and PI3K pathway) were negatively correlated with treatment response and PFS. Among recurrent GBM patients, EGFR mutations were the most common. Mutations related to the RTK-RAS pathway (NF1) were most common in patients with situ recurrent GBM, while mutations in the MUC family and TP53 pathway (MUC16, CHEK2) were prevalent and continuously increased in patients with non-in situ recurrent GBM.

In glioma patients undergoing primary surgery, dynamic monitoring of ctDNA and genotyping can be used for early risk stratification, efficacy monitoring, and early recurrence detection, and provide a basis for clinical research to evaluate early therapeutic intervention.

Cetuximab (Ctx), a monoclonal antibody targeting the epidermal growth factor receptor (EGFR) for colorectal cancer treatment, often faces diminished clinical efficacy due to acquired resistance driven by EGFR mutations. Here, we investigated the molecular mechanisms underlying this mutation-induced drug resistance and developed a mechanism-based strategy to restore the binding affinity of Ctx to EGFR mutants. Through molecular dynamics simulations and free energy perturbation calculations, we discovered that most resistant mutations primarily alter the electrostatic properties of the binding interface. Focusing on two key mutations, EGFR K489E and I491M, we rationally designed two Ctx variants-CtxD103R for EGFRK489E and CtxD103E_E105D for EGFRI491M-using electrostatic compensation and steric hindrance adjustment strategies. Surface plasmon resonance measurements verified that the two designed Ctx variants exhibit improved binding affinity to the corresponding EGFR mutants compared with wild-type Ctx. Additionally, western blot experiments using HEK-293 T cells showed that the designed CtxD103R effectively inhibits EGF-stimulated phosphorylation of EGFRK489E. Our findings highlight a rational design approach, empowered by interaction landscape at atomic detail, as a promising and cost-effective strategy to combat mutation-driven resistance in antibody therapies.

MAPK pathway inhibitors (MAPKi) are increasingly used in the treatment of advanced colorectal cancer, but often produce short-lived responses in patients. Although acquired resistance by de novo mutations in tumors have been found to reduce response in some patients, additional mechanisms underlying the limited response durability of MAPK targeting therapy remain unknown. Here, we denote new contributory tumor biology and provide insight on the impact of tumor plasticity on therapy response. Analysis of MAPKi treated patients revealed activation of stemness programs and increased ASCL2 expression, which are associated with poor outcomes. Greater ASCL2 with MAPKi treatment was also seen in patient-derived CRC models, independent of driver mutations. We find ASCL2 denotes a distinct cell population, arising from phenotypic plasticity, with a proliferative, stem-like phenotype, and decreased sensitivity to MAPKi therapy, which were named adaptive plasticity tumor (APT) cells. MAPK pathway suppression induces the APT phenotype in cells, resulting in APT cell enrichment in tumors and limiting therapy response in preclinical and clinical data. APT cell depletion improved MAPKi treatment efficacy and extended MAPKi response durability in mice. These findings uncover a cellular program that mitigates the impact of MAPKi therapies and highlights the importance of addressing tumor plasticity to improve clinical outcomes.

The epidermal growth factor receptor (EGFR) is a validated therapeutic target for RAS/RAF wild-type colorectal cancer (CRC). However, monoclonal antibody-based anti-EGFR therapies such as cetuximab have limited effectiveness. Herein, it is identified that EGFR internalization is associated with poor treatment response and prognosis in patients with CRC, based on a retrospective analysis of patients treated with cetuximab. It is further demonstrated that the endocytosis inhibitor prochlorperazine (PCZ) can move EGFR, which is hidden inside the cell, to the cell surface to improve therapeutic antibody binding. Thus, a thermosensitive hydrogel co-loaded with cetuximab and PCZ (Gel@Cmab/PCZ) is constructed for sustained inhibition of endocytosis and effective cetuximab delivery. Peritumoral injection of Gel@Cmab/PCZ shows strong antitumor efficacy in subcutaneous and orthotopic CRC tumor models and completely abrogated liver metastasis when combined with chemotherapy. In a humanized patient-derived xenograft model, a single injection of Gel@Cmab/PCZ with one-third of the conventional cetuximab dose achieved 91% tumor growth inhibition, which promoted NK cell infiltration into tumor tissues and their antibody-dependent cell-mediated cytotoxicity effect. This study represents a novel strategy to boost the monoclonal antibody-mediated anti-tumor response in CRC.

Panitumumab has been extensively applied in antitumor therapy, and the regulation of its adverse drug reactions (ADRs) has become extremely important. This study utilized the U.S. Food and Drug Administration Adverse Event Reporting System (FAERS) database to extract real-world panitumumab ADR signals and provide relevant information for drug safety.

ROR, PRR, BCPNN, and MGPS were used to identify real-world ADR signals associated with panitumumab.

Analysis of 9,033 patients identified 263 ADR signals across 20 MedDRA System Organ Classifications. New signals including peripheral sensory neuropathy, gene mutation, decreased neutrophil count, polyneuropathy, ileus, neutropenia, and febrile neutropenia. Age and sex subgroup analyses revealed specific risks, such as polyneuropathy and gene mutation in those under 65 years of age, decreased neutrophil count and peripheral sensory neuropathy in those over 65 years of age, and febrile neutropenia in men. Ileus was highlighted as a novel ADR in gastrointestinal disorders, with no significant age or sex differences.

This study identified new signals of ADR associated with panitumumab, providing valuable information for the clinical use of panitumumab.

MET amplification (amp) is a driver of acquired resistance to epidermal growth factor receptor (EGFR) antibodies in patients with RAS wild-type (WT) metastatic colorectal cancer (mCRC). Savolitinib is an oral small molecule tyrosine kinase inhibitor that has demonstrated anti-tumor activity in MET-driven advanced solid tumors. We report the results of a phase 2 study of savolitinib in patients with mCRC with MET amp detected by circulating cell free (cf)DNA.

Patients with chemotherapy refractory mCRC and MET amp detected by cfDNA were treated with savolitinib until unacceptable toxicity or disease progression. The primary endpoint was objective response rate. Secondary endpoints were clinical activity and safety.

Five patients were enrolled and treated. Best overall response was stable disease (SD) in two patients, progressive disease (PD) in two patients, and one patient unevaluable for response. The majority of treatment-emergent adverse events (TEAEs) were grade 1 or 2. The most common TEAEs included fatigue (n = 3) and nausea (n = 3). There were no grade 4 or 5 TEAEs.

Savolitinib was well tolerated; however, in this small group of biomarker-selected patients, we observed no evidence of anti-tumor activity.

Clinicaltrials.gov Identifier: NCT03592641. Registered on July 17, 2018.

Liquid biopsy using circulating tumor DNA (ctDNA) has emerged as a minimally invasive, timely approach to provide molecular diagnosis and monitor tumor evolution in patients with cancer. Since the molecular landscape of metastatic colorectal cancer (mCRC) is substantially heterogeneous and dynamic over space and time, ctDNA holds significant advantages as a biomarker for this disease. Numerous studies have demonstrated that ctDNA broadly recapitulates the molecular profile of the primary tumor and metastases, and have mainly focused on the genotyping of RAS and BRAF, that is propaedeutic for anti-EGFR treatment selection. However, ctDNA soon broadened its scope towards the assessment of early tumor response, as well as the identification of drug resistance biomarkers to drive potential molecular actionability. In this review article, we provide an overview of the current state-of-the-art of this methodology and its applications, focusing on ongoing clinical trials that employ ctDNA to prospectively guide treatment in patients with mCRC.

Background/Objectives: Biliary tract cancer (BTC) is a rare but aggressive malignancy that requires surgical treatment. However, postoperative recurrence rates are high, and reliable predictors of recurrence are limited. This study aimed to investigate the effectiveness of cell-free DNA (cfDNA) and circulating tumor cells (CTCs) in predicting early recurrence after curative surgery and complete adjuvant therapy in patients with BTC. Methods: Twenty-four patients who underwent R0 and R1 resections and completed adjuvant therapy for BTC between September 2019 and March 2022 were followed up until March 2024. Patients were categorized into early recurrence (ER) and non-ER groups, using one year as the cutoff for recurrence. Results: The combination score derived from ultrashort fragments of cfDNA, vimentin-positive CTCs, and carbohydrate antigen (CA) 19-9 levels showed a statistically significant difference between the ER and non-ER groups (p-value < 0.001). The receiver operating characteristic curve from the combination score and CA 19-9 levels yielded areas under the curve of 0.891 and 0.750, respectively. Conclusions: Although further research is required, these findings suggest that cfDNA and CTCs may increase the accuracy of predicting postoperative recurrence in patients with BTC.

This study aimed to compare tumor tissue DNA (ttDNA) and circulating tumor DNA (ctDNA) to explore the clinical applicability of ctDNA and to better understand clonal evolution in patients with metastatic colorectal cancer undergoing palliative first-line systemic therapy.

We performed targeted sequencing analysis of 88 cancer-associated genes using germline DNA, ctDNA at baseline (baseline-ctDNA), and ctDNA at progressive disease (PD-ctDNA). The results were compared with ttDNA data.

Among 208 consecutively enrolled patients, we selected 84 (41 males; median age, 59 years; range, 35 to 90 years) with all four sample types available. A total of 202 driver mutations were found in 34 genes. ttDNA exhibited the highest mutation frequency (n=232), followed by baseline-ctDNA (n=155) and PD-ctDNA (n=117). Sequencing ctDNA alongside ttDNA revealed additional mutations in 40 patients (47.6%). PD-ctDNA detected 13 novel mutations in 10 patients (11.9%) compared to ttDNA and baseline-ctDNA. Notably, seven mutations in five patients (6.0%) were missense or nonsense mutations in APC, TP53, SMAD4, and CDH1 genes. In baseline-ctDNA, higher maximal variant allele frequency (VAF) values (p=0.010) and higher VAF values of APC (p=0.012), TP53 (p=0.012), and KRAS (p=0.005) mutations were significantly associated with worse overall survival.

While ttDNA remains more sensitive than ctDNA, our ctDNA platform demonstrated validity and potential value when ttDNA was unavailable. Post-treatment analysis of PD-ctDNA unveiled new pathogenic mutations, signifying cancer's clonal evolution. Additionally, baseline-ctDNA's VAF values were prognostic after treatment.

To investigate the technical feasibility of RT-PCR and direct sequencing to quantify HPV DNA in the saliva of patients with Human-Papilloma-Virus related oropharyngeal cancer (HPV-OPC), the level of which is known to predict prognosis after treatment.

Nine patients with locally advanced HPV-OPC treated with definitive radiotherapy with chemotherapy or cetuximab, or radiotherapy alone between April 2016 and September 2017, were enrolled, two of whom also received induction chemotherapy. Saliva was collected before (baseline), during (mid-RT) and after (post-RT) radiotherapy. HPV-16 DNAs (E6 and E7) in saliva were quantified by RT-PCR and sequencing, the latter using a custom cancer panel. Correlations between HPV DNA levels and clinical outcomes were assessed.

Compared to the baseline, the relative cycle threshold (Ct) value of E6 and E7 reduced at the point of mid-RT in the majority of the patients (100% and 75% for E6 and E7, respectively). Similarly, the relative Ct value from the baseline to post-RT reduced in 86% and 100% of the patients for E6 and E7, respectively. During the follow-up period, three patients (33%) experienced disease progression. The relative baseline Ct values of these three patients were in the top 4 of all the patients. The sequences of HPV DNA were detected in five (83%) of six samples of the baseline saliva that underwent DNA sequencing, along with several gene mutations, such as TP53,CDKN2A and PIK3CA.

This study demonstrates that, in addition to detection and quantification of HPV DNA by RT-PCR, detection by sequencing of HPV-DNA using a customized cancer panel is technically possible.

Liquid biopsies can accurately identify molecular alterations in patients with colorectal cancer with high concordance with tissue analysis and shorter turnaround times. Circulating tumor (ct) DNA analysis can be used for diagnosing and monitoring tumor evolution in patients with metastatic colorectal cancer who are treated with EGFR inhibitors. In this article, we reported three clinical cases to illustrate the relevance of RAS mutations identified in ctDNA samples of patients with wild-type metastatic colorectal cancer who received an EGFR inhibitor plus chemotherapy as first-line treatment. The identification of RAS mutations in these patients is one of the most frequently identified mechanisms of acquired resistance. However, detecting a KRAS mutation via liquid biopsy can be caused by inter-tumor heterogeneity or it can be a false positive due to clonal hematopoiesis. More research is needed to determine whether ctDNA monitoring may help guide therapy options in metastatic colorectal cancer patients. We performed a literature review to assess the technologies that are used for analysis of RAS mutations on ctDNA, the degree of agreement between tissue and plasma and the importance of tissue/plasma discordant cases.

Timely accurate and cost-efficient detection of colorectal cancer (CRC) is of great clinical importance. This study aims to establish prediction models for detecting CRC using plasma cell-free DNA (cfDNA) fragmentomic features. Whole-genome sequencing (WGS) was performed on cfDNA from 620 participants, including healthy individuals, patients with benign colorectal diseases and CRC patients. Using WGS data, three machine learning methods were compared to build prediction models for the stratification of CRC patients. The optimal model to discriminate CRC patients of all stages from healthy individuals achieved a sensitivity of 92.31% and a specificity of 91.14%, while the model to separate early-stage CRC patients (stage 0-II) from healthy individuals achieved a sensitivity of 88.8% and a specificity of 96.2%. Additionally, the cfDNA fragmentation profiles reflected disease-specific genomic alterations in CRC. Overall, this study suggests that cfDNA fragmentation profiles may potentially become a noninvasive approach for the detection and stratification of CRC.

Targeted therapies have improved outcomes for patients with metastatic colorectal cancer, but their impact is limited by rapid emergence of resistance. We hypothesized that an understanding of the underlying genetic mechanisms and intrinsic tumor features that mediate resistance to therapy will guide new therapeutic strategies and ultimately allow the prevention of resistance.

We assembled a series of 52 patients with paired pretreatment and progression samples who received therapy targeting EGFR (n = 17), BRAF V600E (n = 17), KRAS G12C (n = 15), or amplified HER2 (n = 3) to identify molecular and clinical factors associated with time on treatment (TOT).

All patients stopped treatment for progression and TOT did not vary by oncogenic driver (P = 0.5). Baseline disease burden (≥3 vs. <3 sites, P = 0.02), the presence of hepatic metastases (P = 0.02), and gene amplification on baseline tissue (P = 0.03) were each associated with shorter TOT. We found evidence of chromosomal instability (CIN) at progression in patients with baseline MAPK pathway amplifications and those with acquired gene amplifications. At resistance, copy-number changes (P = 0.008) and high number (≥5) of acquired alterations (P = 0.04) were associated with shorter TOT. Patients with hepatic metastases demonstrated both higher number of emergent alterations at resistance and enrichment of mutations involving receptor tyrosine kinases.

Our genomic analysis suggests that high baseline CIN or effective induction of enhanced mutagenesis on targeted therapy underlies rapid progression. Longer response appears to result from a progressive acquisition of genomic or chromosomal instability in the underlying cancer or from the chance event of a new resistance alteration.

Adagrasib, an irreversible, selective KRASG12C inhibitor, may be an effective treatment in KRASG12C-mutated colorectal cancer, particularly when combined with an anti-EGFR antibody. In this analysis of the KRYSTAL-1 trial, patients with previously treated KRASG12C-mutated unresectable or metastatic colorectal cancer received adagrasib (600 mg twice daily) plus cetuximab. The primary endpoint was objective response rate (ORR) by blinded independent central review. Ninety-four patients received adagrasib plus cetuximab. With a median follow-up of 11.9 months, ORR was 34.0%, disease control rate was 85.1%, and median duration of response was 5.8 months (95% confidence interval [CI], 4.2-7.6). Median progression-free survival was 6.9 months (95% CI, 5.7-7.4) and median overall survival was 15.9 months (95% CI, 11.8-18.8). Treatment-related adverse events (TRAEs) occurred in all patients; grade 3-4 in 27.7% and no grade 5. No TRAEs led to adagrasib discontinuation. Exploratory analyses suggest circulating tumor DNA may identify features of response and acquired resistance.

Adagrasib plus cetuximab demonstrates promising clinical activity and tolerable safety in heavily pretreated patients with unresectable or metastatic KRASG12C-mutated colorectal cancer. These data support a potential new standard of care and highlight the significance of testing and identification of KRASG12C mutations in patients with colorectal cancer. This article is featured in Selected Articles from This Issue, p. 897.

Colon cancer (CC) is one of the most frequent cancers worldwide being responsible for over 500 thousand deaths in 2022. Its financial and human burden is expected to increase in the next decades accompanying the growing and aging of the global population. Much of this burden could be alleviated considering that the lethality of CC is mostly due to its late diagnosis and failure in the individualized management of patients. Coordinated government actions and implementation of better diagnostic tools capable of detecting CC earlier and of tracking tumoral evolution are mandatory to achieve a reduction in CC's social impact. CtDNA-based liquid biopsy (LB) has great potential to contribute to patients' screening adhesion, CC earlier detection, and to longitudinal tumor follow-up. In this review, we will discuss the latest epidemiological data on CC disease, diagnostic, subtypes, genetics, and treatment management focusing on the advantages and limitations of ctDNA-based LB, including important bottlenecks and solutions necessary for its clinical translation. The latest ctDNA-directed CC clinical trials will also be examined.

In the past decade, immune checkpoint inhibitors (ICI) have been approved for treatment of genitourinary malignancies and have revolutionized the treatment landscape of these tumors. However, despite the remarkable success of these therapies in some GU malignancies, many patients' tumors do not respond to these therapies, and others may experience significant side effects, such as immune-related adverse events (iRAEs). Accordingly, biomarkers and improved prognostic tools are critically needed to help predict which patients will respond to ICI, predict and mitigate risk of developing immune-related adverse events, and inform personalized choice of therapy for each patient. Ongoing clinical and preclinical studies continue to provide an increasingly robust understanding of the mechanisms of the response to immunotherapy, which continue to inform biomarker development and validation. Herein, we provide a comprehensive review of biomarkers of the response to immunotherapy in GU tumors and their role in selection of therapy and disease monitoring.

Colorectal cancer (CRC) is one of the most common causes of tumor-related deaths globally. Despite recent improvements in the comprehensive therapy of malignancy, metastatic CRC continues to have a poor prognosis. Human epidermal growth factor receptor 2 (HER2) is an established oncogenic driver, which is successfully targeted for breast and gastric cancers. Approximately 5% of CRC patients carry somatic HER2 mutations or gene amplification. In 2019, the U.S. Food and Drug Administration have approved trastuzumab and pertuzumab in combination with chemotherapy for the treatment of HER2-positive metastatic CRC. This approval marked a significant milestone in the treatment of CRC, as HER2-positive patients now have access to targeted therapies that can improve their outcomes. Yet, assessment for HER2 overexpression/ amplification in CRC has not been standardized. The resistance mechanisms to anti-HER2 therapy have been not clearly investigated in CRC. Although many unknowns remain, an improved understanding of these anti-HER2 agents will be essential for advanced CRC. In this review, we provide an overview of the role of HER2 in CRC as an oncogenic driver, a prognostic and predictive biomarker, and a clinically actionable target, as well as the current progress and challenges in the field.

Lung adenocarcinoma is the most common source of brain metastasis (BM), resulting in significant morbidity and mortality. We aimed to identify patients with high BM risk who possibly benefit from brain-penetrant drugs, prophylactic cranial irradiation, or close brain magnetic resonance imaging surveillance.

Metastatic lung adenocarcinoma patients with extracranial tumor samples profiled by a next-generation sequencing panel targeting 425 tumor-related genes were retrospectively enrolled between February 2008 and July 2021. We compared BM and non-BM patients' genomic and clinical features and studied their associations with BM risk. Two external cohorts were used for result validation and molecular mechanisms investigation, respectively.

We included 174 eligible patients, including 90 having developed BM by the end of follow-up. Age≤60, EGFR activating mutations, and high-level apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) mutational signatures were associated with elevated BM risk. Similar findings in BM-free survival were obtained by fitting Fine-Gray subdistribution hazard models addressing competing risks. Increased BM risk related to APOBEC mutational signatures was validated in an external cohort (N = 440). RNA sequencing data analyses performed in another external cohort (N = 230) revealed that expressions of metastasis-related pathways such as transforming growth factor (TGF)β and epithelial-mesenchymal transition (EMT) were upregulated in the patients with high-level APOBEC mutational signatures.

APOBEC mutational signatures related to upregulated TGFβ and EMT, could serve as an independent risk factor for BM and BM-free survival in metastatic lung adenocarcinoma patients. Further investigations are warranted to tailor personalized treatments to improve the susceptible patient's outcomes.

Metastatic colorectal cancer requires a multidisciplinary and individualized approach. Herein, we reported the case of a young woman diagnosed with metastatic rectal cancer who received an individualized multimodal treatment strategy that resulted in a remarkable survival. There were several particular aspects of this case, such as the early onset of the disease, the successful use of conversion therapy, the application of liquid biopsy to guide treatment, and the specific nature of the bone metastasis. To offer more insights for navigating such challenges in patients with metastatic colorectal cancer, we have conducted a literature review to find more data related to the particularities of this case. The incidence of early onset colorectal cancer is on the rise. Data suggests that it differs from older-onset colorectal cancer in terms of its pathological, epidemiological, anatomical, metabolic, and biological characteristics. Conversion therapy and surgical intervention provide an opportunity for cure and improve outcomes in metastatic colorectal cancer. It is important to approach each case individually, as every patient with limited liver disease should be considered as a candidate for secondary resection. Moreover, liquid biopsy has an important role in the individualized management of metastatic colorectal cancer patients, as it offers additional information for treatment decisions.

Emergence of acquired resistance limits the efficacy of the anti-EGFR therapies cetuximab and panitumumab in metastatic colorectal cancer. In the last decade, preclinical and clinical cohort studies have uncovered genomic alterations that confer a selective advantage to tumor cells under EGFR blockade, mainly downstream re-activation of RAS-MEK signaling and mutations in the extracellular domain of EGFR (EGFR-ECD). Liquid biopsies (genotyping of ctDNA) have been established as an excellent tool to easily monitor the dynamics of genomic alterations resistance in the blood of patients and to select patients for rechallenge with anti-EGFR therapies. Accordingly, several clinical trials have shown clinical benefit of rechallenge with anti-EGFR therapy in genomically-selected patients using ctDNA. However, alternative mechanisms underpinning resistance beyond genomics -mainly related to the tumor microenvironment-have been unveiled, specifically relevant in patients receiving chemotherapy-based multi-drug treatment in first line. This review explores the complexity of the multifaceted mechanisms that mediate secondary resistance to anti-EGFR therapies and potential therapeutic strategies to circumvent acquired resistance.

Mutation detection for therapy monitoring in cell-free DNA (cfDNA) is used clinically for some malignancies. Gallbladder carcinoma (GBC) presents a diagnostic challenge and has limited late-stage treatment options. To our knowledge, this novel study examines, for the first time, genomic alterations in cfDNA from GBC to assess diagnostic accuracy and therapeutic options. The concordance of somatic genomic changes in cfDNA and DNA from paired tumor tissue was analyzed. Paired serum and tissue samples from 40 histologically proven GBC, 20 cholecystitis, and 4 normal (noninflamed gallbladder) controls were included. Targeted next-generation sequencing with a 22-gene panel (Colon and Lung Cancer Research Panel v2, Thermo Scientific) in cfDNA and tumor tissue with high depth and uniform coverage on ION Personal Genome Machine (ION, PGM) was performed. A spectrum of 223 mutations in cfDNA and 225 mutations in formalin-fixed paraffin-embedded tissue DNA were identified in 22 genes. Mutations ranged from 1 to 17 per case. In cfDNA frequent alterations were in TP53 (85.0%), EGFR (52.5%), MET (35%) CTNNB1, SMAD4, BRAF (32.5%), PTEN (30%), FGFR3 and PIK3CA (27.5%), NOTCH1 (25.0%), and FBXW7 and ERBB4 (22.5%). At least one clinically actionable mutation was identified in all cfDNA samples. Paired samples shared 149 of 225 genetic abnormalities (66.2%). Individual gene mutation concordance ranged from 44.44% to 82.0% and was highest for EGFR (82.0%), BRAF and NOTCH1 (80.0%), TP53 (73.08%), MET (72.22%), and ERBB4 (71.42%) with a significant level of correlation (Spearman r = 0.91, P ≤ .0001). The sensitivity and specificity of the TP53 gene at the gene level was the highest (94.44% and 100.0%, respectively). Overall survival was higher for ERBB4 and ERBB2 mutant tumors. The adenocarcinoma subtype revealed specific genetic changes in ERBB4, SMAD4, ERBB2, PTEN, KRAS, and NRAS. NGS-based cfDNA mutation profiling can be used to diagnose GBC before surgery to guide treatment decisions. Targeted therapy identified in GBC included SMAD4, ERBB2, ERBB4, EGFR, KRAS, BRAF, PIK3CA, MET, and NRAS.

Progression occurs in approximately two-thirds of patients with locally advanced non-small cell lung cancer (LA-NSCLC) receiving chemoradiation and consolidation immunotherapy. Molecular indicators for outcome prediction are under development. A novel metric, the ratio of mean to max variant allele frequency (mmVAF), was derived from 431 pre-treatment tissue biopsies from The Cancer Genome Atlas and evaluated in serial circulating tumor DNA (ctDNA) from 70 LA-NSCLC patients receiving definitive radiotherapy/chemoradiotherapy (RT/CRT) with/without immunotherapy. High mmVAFs in pre-treatment tissue biopsies, indicating clonal predominant tumors (P < 0.01), were associated with inferior overall survival [OS, hazard ratio (HR): 1.48, 95 % confidence interval (CI): 1.11-1.98]. Similar associations of mmVAF with clonality (P < 0.01) and OS (HR: 2.24, 95 % CI: 0.71-7.08) were observed in pre-treatment ctDNA. At 1-month post-RT, ctDNA mmVAF-high patients receiving consolidation immunotherapy exhibited improved progression-free survival (PFS) compared to those who did not (HR: 0.14, 95 % CI: 0.03-0.67). From the baseline to week 4 of RT and/or 1-month post-RT, survival benefits from consolidation immunotherapy were exclusively observed in ctDNA mmVAF-increased patients (PFS, HR: 0.39, 95 % CI: 0.14-1.15), especially in terms of distant metastasis (HR: 0.11, 95 % CI: 0.01-0.95). In summary, our longitudinal data demonstrated the applicability of ctDNA-defined clonality for prognostic stratification and immunotherapy benefit prediction in LA-NSCLC.

Colorectal cancer (CRC) is one of the most heterogeneous and deadly diseases, with a global incidence of 1.5 million cases per year. Genomics has revolutionized the clinical management of CRC by enabling comprehensive molecular profiling of cancer. However, a deeper understanding of the molecular factors is needed to identify new prognostic and predictive markers that can assist in designing more effective therapeutic regimens for the improved management of CRC. Recent breakthroughs in single-cell analysis have identified new cell subtypes that play a critical role in tumor progression and could serve as potential therapeutic targets. Spatial analysis of the transcriptome and proteome holds the key to unlocking pathogenic cellular interactions, while liquid biopsy profiling of molecular variables from serum holds great potential for monitoring therapy resistance. Furthermore, gene expression signatures from various pathways have emerged as promising prognostic indicators in colorectal cancer and have the potential to enhance the development of equitable medicine. The advancement of these technologies for identifying new markers, particularly in the domain of predictive and personalized medicine, has the potential to improve the management of patients with CRC. Further investigations utilizing similar methods could uncover molecular subtypes specific to emerging therapies, potentially strengthening the development of personalized medicine for CRC patients.

Liquid biopsy refers to a set of pathological samples retrieved from non-solid sources, such as blood, cerebrospinal fluid, urine, and saliva through non-invasive or minimally invasive approaches. In the recent decades, an increasing number of studies have focused on clinical applications and improving technological investigation of liquid biopsy biosources for diagnostic goals particularly in cancer. Materials extracted from these sources and used for medical evaluations include cells like circulating tumor cells (CTCs), tumor-educated platelets (TEPs), cell-free nucleic acids released by cells, such as circulating tumor DNA (ctDNA), cell-free DNA (cfDNA), cell-free RNA (cfRNA), and exosomes. Playing significant roles in the pathogenesis of human malignancies, analysis of these sources can provide easier access to genetic and transcriptomic information of the cancer tissue even better than the conventional tissue biopsy. Notably, they can represent the inter- and intra-tumoral heterogeneity and accordingly, liquid biopsies demonstrate strengths for improving diagnosis in early detection and screening, monitoring and follow-up after therapies, and personalization of therapeutical strategies in various types of human malignancies. In this review, we aim to discuss the roles, functions, and analysis approaches of liquid biopsy sources and their clinical implications in human malignancies with a focus on colorectal cancer.

Activating RAS gene mutations occur in approximately 55% of patients with metastatic colorectal cancer (mCRC) and are associated with poorer clinical outcomes due to epidermal growth factor receptor (EGFR) blockade resistance. Combined EGFR and mitogen-activated protein kinase (MEK) inhibition may extend response to EGFR inhibition and overcome acquired resistance. This phase Ib/II dose escalation trial evaluated the safety and activity of dual inhibition with binimetinib (MEK1/2 inhibitor) and panitumumab (EGFR inhibitor [EGFRi]) in patients with RAS mutant or BRAF wild type (WT)/RAS WT mCRC.

Phase Ib dose escalation started with binimetinib 45 mg twice daily plus panitumumab 6 mg/kg administered every 2 weeks. In the phase II study, patients with measurable mCRC were enrolled into 4 groups based on previous anti-EGFR monoclonal antibody therapy and RAS mutational status.

No patients in the phase Ib portion (n = 10) had a response; 70% of patients had stable disease. In the phase II portion (n = 43), overall response rate (ORR, confirmed) was 2.3% with one partial response in the RAS WT group, DCR was 30.2%, and median progression-free survival was 1.8 months (95%CI, 1.6-3.3). All patients experienced ≥1 adverse event, with the most common being diarrhea (71.7%), vomiting (52.8%), nausea (50.9%), fatigue (49.1%), dermatitis acneiform (43.4%), and rash (41.5%). Most patients required treatment interruption or dose reduction due to difficulties tolerating treatment.

The combination of binimetinib and panitumumab had substantial toxicity and limited clinical activity for patients with mutant or WT RAS mCRC, independent of EGFRi treatment history (Trial registration: NCT01927341).

In the evolving molecular treatment landscape of metastatic colorectal cancer (mCRC), the identification of druggable alterations is pivotal to achieve the best therapeutic opportunity for each patient. Because the number of actionable targets is expanding, there is the need to timely detect their presence or emergence to guide the choice of different available treatment options. Liquid biopsy, through the analysis of circulating tumor DNA (ctDNA), has proven safe and effective as a complementary method to address cancer evolution while overcoming the limitations of tissue biopsy. Even though data are accumulating regarding the potential for ctDNA-guided treatments applied to targeted agents, still major gaps in knowledge exist as for their application to different areas of the continuum of care. In this review, we recapitulate how ctDNA information could be exploited to drive different targeted treatment strategies in mCRC patients, by refining molecular selection before treatment by addressing tumor heterogeneity beyond tumor tissue biopsy; longitudinally monitoring early-tumor response and resistance mechanisms to targeted agents, potentially leading to tailored, molecular-driven, therapeutic options; guiding the molecular triage towards rechallenge strategies with anti-EGFR agents, suggesting the best time for retreatment; and providing opportunities for an "enhanced rechallenge" through additional treatments or combos aimed at overcoming acquired resistance. Besides, we discuss future perspectives concerning the potential role of ctDNA to fine-tune investigational strategies such as immuno-oncology.

Colorectal carcinoma (CRC) is the third most common cancer in terms of diagnosis and the second in terms of mortality. Recent studies have shown that various proteins, extracellular vesicles (i.e., exosomes), specific genetic variants, gene transcripts, cell-free DNA (cfDNA), circulating tumor DNA (ctDNA), microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and altered epigenetic patterns, can be used to detect, and assess the prognosis of CRC. Over the last decade, a plethora of conventional methodologies (e.g., polymerase chain reaction [PCR], direct sequencing, enzyme-linked immunosorbent assay [ELISA], microarray, in situ hybridization) as well as advanced analytical methodologies (e.g., microfluidics, electrochemical biosensors, surface-enhanced Raman spectroscopy [SERS]) have been developed for analyzing genetic and epigenetic biomarkers using both optical and non-optical tools. Despite these methodologies, no gold standard detection method has yet been implemented that can analyze CRC with high specificity and sensitivity in an inexpensive, simple, and time-efficient manner. Moreover, until now, no study has critically reviewed the advantages and limitations of these methodologies. Here, an overview of the most used genetic and epigenetic biomarkers for CRC and their detection methods are discussed. Furthermore, a summary of the major biological, technical, and clinical challenges and advantages/limitations of existing techniques is also presented.

Colorectal cancer (CRC) represents 10% of all cancer types, making it the third leading cause of cancer-related deaths globally. Metastasis is the primary factor causing mortality in CRC patients. Approximately 22% of CRC-related deaths have metastasis present at diagnosis, with approximately 70% of these cases recurring. Recently, with the application of novel targeted drugs, targeted therapy has become the first-line option for individualized and comprehensive treatment of CRC. The management of these patients remains a significant medical challenge. The most prevalent targeted therapies for CRC in clinical practice focus on anti-vascular endothelial growth factor and its receptor, epidermal growth factor receptor (EGFR), and multi-target kinase inhibitors. In the wake of advancements in precision diagnosis and widespread adoption of second-generation sequencing (NGS) technology, rare targets such as BRAF V600E mutation, KRAS mutation, HER2 overexpression/amplification, and MSI-H/dMMR in metastatic colorectal cancer (mCRC) are increasingly being discovered. Simultaneously, new therapeutic drugs targeting these mutations are being actively investigated. This article reviews the progress in clinical research for developing targeted therapeutics for CRC, in light of advances in precision medicine and discovery of new molecular target drugs.

Non-invasive biomarkers for immunotherapy response remain a compelling unmet medical need. POLARIS-03 is a multicenter phase II trial to evaluate the safety and efficacy of toripalimab (anti-programmed cell death 1) in refractory metastatic urothelial carcinoma (mUC). We assessed the predictive utility of longitudinal circulating tumor DNA (ctDNA) analysis from a single-institution biomarker cohort. Twenty-seven mUC patients receiving toripalimab (3 mg/kg Q2W) at Ren Ji Hospital were enrolled. Serial plasma specimens were obtained at baseline and then every two cycles during treatment. The 600-gene panel (PredicineATLAS™) liquid biopsy assay was applied to probe somatic variants and cancer cell fraction (CCF). Low-pass whole genome sequencing was used to determine the copy number abnormality (CNA) score. Across the entire cohort, we observed different degrees of concordance between somatic aberrations detected by ctDNA and those inferred by matched tumor samples. Although the baseline CCF or CNA had limited predictive value, early ctDNA response at week 8 was associated with toripalimab efficacy and prolonged patient survival. Integrating CCF and CNA decrease achieved a superior accuracy of 90.5% in classifying responders and non-responders and predicted long-term benefit from toripalimab. Dynamic changes in the CCF and CNA in blood exquisitely reflected radiographic assessment of malignant lesions, including those with FGFR3-TACC3 gene fusion or microsatellite instability. This study demonstrates the feasibility and effectiveness of integrated longitudinal ctDNA profiling as a potential biomarker in mUC patients undergoing immunotherapy and supports further clinical evaluation of minimally invasive liquid biopsy assays for treatment stratification and therapy monitoring. © 2023 The Pathological Society of Great Britain and Ireland.

Serial analysis of circulating tumor DNA (ctDNA) levels is a promising tool for both relapse prediction in the curative setting, as well as predicting clinical benefit from systemic treatment in metastasic colorectal cancer (mCRC). Most data in this context are derived from treatment naive patients.

To predict progressive disease (PD) as early as possible through monitoring of changes in ctDNA levels during systemic treatment in pretreated patients with mCRC.

A prospective, single-center, observational study.

Patients treated beyond first-line were prospectively included between February 2020 and September 2021. Blood for ctDNA detection was taken before every treatment cycle from start of treatment until first restaging by CT-scan. ctDNA was detected by mutation- (mut-ctDNA) and methylation-specific ddPCR. Receiver Operating Characteristic (ROC)-analysis was used to describe sensitivity and specificity for prediction of PD at restaging for all time points.

A total of 42 patients were included who all carried a mutation in tumor tissue. Detection rate of mut-ctDNA was 88.1% and 74.4% for meth-ctDNA. Absolute ctDNA levels before treatment were prognostic in terms of overall survival. Levels of ctDNA were significantly higher in patients with PD at restaging. Median time from start of treatment to restaging was 93 days (95% CI 88.8-96). After a median of 19 days of treatment (95% CI 16.1-20.2), a decline of either mutation- or methylation-specific ctDNA levels of ⩽58% predicted PD at restaging with a sensitivity/specificity of 92.9/85.7% and 85.7/100%, respectively. Median time to restaging was 66 days (95% CI 56.8-75.2). There was no significant increase of sensitivity/specificity at later time points of ctDNA measurements.

Monitoring early changes of ctDNA levels either by mut- or meth-ctDNA allows for early prediction of PD in pretreated patients with mCRC. This has the potential to complement RECIST-based treatment assessment with the aim to switch potentially insufficient treatments as early as possible, which is of particular interest in higher treatment lines.