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Currently, approximately 30%-55% of the patients with non-small cell lung cancer (NSCLC) develop recurrence due to minimal residual disease (MRD) after receiving surgical resection of the tumor. This study aims to develop an ultrasensitive and affordable fragmentomic assay for MRD detection in patients with NSCLC. A total of 87 patients with NSCLC, who received curative surgical resections (23 patients relapsed during follow-up), enrolled in this study. A total of 163 plasma samples, collected at 7 days and 6 months postsurgical, were used for both whole-genome sequencing (WGS) and targeted sequencing. WGS-based cell-free DNA (cfDNA) fragment profile was used to fit regularized Cox regression models, and leave-one-out cross-validation was further used to evaluate models' performance. The models showed excellent performances in detecting patients with a high risk of recurrence. At 7 days postsurgical, the high-risk patients detected by our model showed an increased risk of 4.6 times, while the risk increased to 8.3 times at 6 months postsurgical. These fragmentomics determined higher risk compared with the targeted sequencing-based circulating mutations both at 7 days and 6 months postsurgical. The overall sensitivity for detecting patients with recurrence reached 78.3% while using both fragmentomics and mutation results from 7 days and 6 months postsurgical, which increased from the 43.5% sensitivity by using only the circulating mutations. The fragmentomics showed great sensitivity in predicting patient recurrence compared with the traditional circulating mutation, especially after the surgery for early-stage NSCLC, therefore exhibiting great potential to guide adjuvant therapeutics., Significance: The circulating tumor DNA mutation-based approach shows limited performance in MRD detection, especially for landmark MRD detection at an early-stage cancer after surgery. Here, we describe a cfDNA fragmentomics-based method in MRD detection of resectable NSCLC using WGS, and the cfDNA fragmentomics showed a great sensitivity in predicting prognosis., (© 2023 The Authors; Published by the American Association for Cancer Research.)

Background: For locally advanced rectal cancer (LARC) patients who receive neoadjuvant chemoradiotherapy (nCRT), there are no reliable indicators to accurately predict pathological complete response (pCR) before surgery. For patients with clinical complete response (cCR), a "Watch and Wait" (W&W) approach can be adopted to improve quality of life. However, W&W approach may increase the recurrence risk in patients who are judged to be cCR but have minimal residual disease (MRD). Magnetic resonance imaging (MRI) is a major tool to evaluate response to nCRT; however, its ability to predict pCR needs to be improved. In this prospective cohort study, we explored the value of circulating tumor DNA (ctDNA) in combination with MRI in the prediction of pCR before surgery and investigated the utility of ctDNA in risk stratification and prognostic prediction for patients undergoing nCRT and total mesorectal excision (TME)., Methods and Findings: We recruited 119 Chinese LARC patients (cT3-4/N0-2/M0; median age of 57; 85 males) who were treated with nCRT plus TME at Fudan University Shanghai Cancer Center (China) from February 7, 2016 to October 31, 2017. Plasma samples at baseline, during nCRT, and after surgery were collected. A total of 531 plasma samples were collected and subjected to deep targeted panel sequencing of 422 cancer-related genes. The association among ctDNA status, treatment response, and prognosis was analyzed. The performance of ctDNA alone, MRI alone, and combining ctDNA with MRI was evaluated for their ability to predict pCR/non-pCR. Ranging from complete tumor regression (pathological tumor regression grade 0; pTRG0) to poor regression (pTRG3), the ctDNA clearance rate during nCRT showed a significant decreasing trend (95.7%, 77.8%, 71.1%, and 66.7% in pTRG 0, 1, 2, and 3 groups, respectively, P = 0.008), while the detection rate of acquired mutations in ctDNA showed an increasing trend (3.8%, 8.3%, 19.2%, and 23.1% in pTRG 0, 1, 2, and 3 groups, respectively, P = 0.02). Univariable logistic regression showed that ctDNA clearance was associated with a low probability of non-pCR (odds ratio = 0.11, 95% confidence interval [95% CI] = 0.01 to 0.6, P = 0.04). A risk score predictive model, which incorporated both ctDNA (i.e., features of baseline ctDNA, ctDNA clearance, and acquired mutation status) and MRI tumor regression grade (mrTRG), was developed and demonstrated improved performance in predicting pCR/non-pCR (area under the curve [AUC] = 0.886, 95% CI = 0.810 to 0.962) compared with models derived from only ctDNA (AUC = 0.818, 95% CI = 0.725 to 0.912) or only mrTRG (AUC = 0.729, 95% CI = 0.641 to 0.816). The detection of potential colorectal cancer (CRC) driver genes in ctDNA after nCRT indicated a significantly worse recurrence-free survival (RFS) (hazard ratio [HR] = 9.29, 95% CI = 3.74 to 23.10, P < 0.001). Patients with detectable driver mutations and positive high-risk feature (HR&#95;feature) after surgery had the highest recurrence risk (HR = 90.29, 95% CI = 17.01 to 479.26, P < 0.001). Limitations include relatively small sample size, lack of independent external validation, no serial ctDNA testing after surgery, and a relatively short follow-up period., Conclusions: The model combining ctDNA and MRI improved the predictive performance compared with the models derived from individual information, and combining ctDNA with HR&#95;feature can stratify patients with a high risk of recurrence. Therefore, ctDNA can supplement MRI to better predict nCRT response, and it could potentially help patient selection for nonoperative management and guide the treatment strategy for those with different recurrence risks., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: HB, XF, and XW are the employees of Geneseeq Technology Inc. YX, HB and YS are the employees of Nanjing Geneseeq Technology Inc.

Background: The correlation between blood-based tumor mutation burden (bTMB) and tissue-based tumor mutation burden(tTMB) has not been broadly tested in a multicancer cohort. Here, we assess the correlation between bTMB with tTMB in phase I trial patients treated with immunotherapy. As an exploratory analysis, we evaluated circulating tumor DNA (ctDNA) dynamics in responders., Methods: Patients treated with immunotherapy at the Princess Margaret phase I trials unit were enrolled. Pretreatment plasma ctDNA and matched normal blood controls were collected. Available archival tissue formalin-fixed paraffin-embedded (FFPE) samples were analyzed. A 425-gene panel was used to sequence both ctDNA and FFPE samples. Samples with TMB within the highest tertile were considered as high TMB., Results: Thirty-eight patients were accrued from 25 different trials, 86.8% of which involved an anti-PD-1/PD-L1 agent. Thirty patients (78.9%) had detectable mutations in ctDNA, of which the median (range) bTMB was 5 (1-53) mutations per megabase (mut/Mb). Of the 22 patients with available FFPE samples, mutations were detected in 21 (95.4%); the median (range) tTMB was 6 (2-124) mut/Mb. Among the 16 patients with detectable mutations in both FFPE and ctDNA, a statistically significant correlation between bTMB and tTMB was observed ( ρ  = 0.71; P  = .002). High TMB was not associated with better survival. All 3 responders had a decrease in the variant allele frequency of mutations detected in ctDNA at a second timepoint relative to baseline, indicating a potential early marker of response., Conclusions: In this small series, bTMB correlated with tTMB. An on-treatment decrease in VAF of mutations detected in ctDNA at baseline was observed in responders. Larger studies to verify our findings are warranted., (© The Author(s) 2021. Published by Oxford University Press.)

Background: Previous studies found that cell-free DNA (cfDNA) generated from tumors was shorter than that from healthy cells, and selecting short cfDNA could enrich for tumor cfDNA and improve its usage in early cancer diagnosis and treatment monitoring; however, the underlying mechanism of shortened tumor cfDNA was still unknown, which potentially limits its further clinical application., Results: Using targeted sequencing of cfDNA in a large cohort of solid tumor patient, sequencing reads harboring tumor-specific somatic mutations were isolated to examine the exact size distribution of tumor cfDNA. For the majority of studied cases, 166 bp remained as the peak size of tumor cfDNA, with tumor cfDNA showing an increased proportion of short fragments (100-150 bp). Less than 1% of cfDNA samples were found to be peaked at 134/144 bp and independent of tumor cfDNA purity. Using whole-genome sequencing of cfDNA, we discovered a positive correlation between cfDNA shortening and the magnitude of chromatin inaccessibility, as measured by transcription, DNase I hypersensitivity, and histone modifications. Tumor cfDNA shortening occurred simultaneously at both 5' and 3' ends of the DNA wrapped around nucleosomes., Conclusions: Tumor cfDNA shortening exhibited two distinctive modes. Tumor cfDNA purity and chromatin inaccessibility were contributing factors but insufficient to trigger a global transition from 166 bp dominant to 134/144 bp dominant phenotype.

Background: One of the major challenges in managing invasive breast cancer (BC) is the lack of reliable biomarkers to track response. Circulating tumor DNA (ctDNA) from liquid biopsy, as a candidate biomarker, provides a valuable assessment of BC patients. In this retrospective study, we evaluated the utility of ctDNA to reflect the efficacy of treatment and to monitor resistance mechanisms., Methods: Targeted next-generation sequencing (NGS) of 416 cancer-relevant genes was performed on 41 plasma biopsy samples of 19 HER2+ and 12 HER2- BC patients. Longitudinal ctDNA samples were analyzed in three BC patients over the treatment course for detecting acquired mutations., Results: In HER2+ BC patients, ERBB2 somatic copy numbers in ctDNA samples were significantly higher in patients progressed on HER2-targeted therapy than those who were still responding to the treatment. Recurrent acquired mutations were detected in genes including ERBB2, TP53, EGFR, NF1, and SETD2, which may contribute to trastuzumab resistance. In longitudinal analyses, the observed mutation allele frequencies were tracked closely in concordance with treatment responses. A novel ERBB2 p.(Leu869Arg) mutation was acquired in one patient upon resistant to trastuzumab therapy, which was further validated as an oncogenic mutation in vitro and contributed to resistance. In HER2- BC patients with chemotherapy resistance, genetic alterations on TP53, PIK3CA, and DNA damage repair genes were frequently observed., Conclusions: In summary, ctDNA monitoring, particularly longitudinal analyses, provides valuable insights into the assessment of targeted therapy efficacy and gene alterations underlying trastuzumab resistance and chemotherapy resistance in HER2+ and HER2- BC patients, respectively., (© 2019 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Crizotinib has been used to counter MET gene amplification in a number of different human malignancies. Transient response to crizotinib in MET-amplified gastric cancer has been reported, but the mechanisms of resistance are not well studied. Here, we reported a stage IV gastric cancer patient with high levels of MET amplification. The implementation of crizotinib treatment led to significant symptomatic improvement in the first 2 months, but was followed by rapid disease progression. Periodic mutation profiling of patient's circulating tumor DNA (ctDNA) by next generation sequencing (NGS) revealed a number of genetic alterations including re-occurrence of MET amplification, multiple secondary MET mutations, a dramatic increase of FGFR2 gene relative copy number as well as mutations in other downstream and bypassing elements, which may collectively related to the patient's cancer progression. Our results illustrate the complex and heterogeneous molecular mechanisms for crizotinib resistance in this patient, and demonstrate the great potential of ctDNA profiling for treatment decision-making and prognosis in clinical practice.

Cancer is a disease of complex genetic alterations, and comprehensive genetic diagnosis is beneficial to match each patient to appropriate therapy. However, acquisition of representative tumor samples is invasive and sometimes impossible. Circulating tumor DNA (ctDNA) is a promising tool to use as a non-invasive biomarker for cancer mutation profiling. Here we implemented targeted next generation sequencing (NGS) with a customized gene panel of 382 cancer-relevant genes on 605 ctDNA samples in multiple cancer types. Overall, tumor-specific mutations were identified in 87% of ctDNA samples, with mutation spectra highly concordant with their matched tumor tissues. 71% of patients had at least one clinically-actionable mutation, 76% of which have suggested drugs approved or in clinical trials. In particular, our study reveals a unique mutation spectrum in Chinese lung cancer patients which could be used to guide treatment decisions and monitor drug-resistant mutations. Taken together, our study demonstrated the feasibility of clinically-useful targeted NGS-based ctDNA mutation profiling to guide treatment decisions in cancer.

Background: Gastric cancer is the fifth most common cancer type. Most patients are diagnosed at advanced stages with poor prognosis. A non-invasive assay for the detection of early-stage gastric cancer is highly desirable for reducing associated mortality. Methods: We collected a prospective study cohort of 110 stage I–II gastric cancer patients and 139 non-cancer individuals. We performed whole-genome sequencing with plasma samples and profiled four types of cell-free DNA (cfDNA) characteristics, fragment size pattern, copy number variation, nucleosome coverage pattern, and single nucleotide substitution. With these differential profiles, we developed an ensemble model to detect gastric cancer signals. Further, we validated the assay in an in-house first validation cohort of 73 gastric cancer patients and 94 non-cancer individuals and an independent second validation cohort of 47 gastric cancer patients and 49 non-cancer individuals. Additionally, we evaluated the assay in a hypothetical 100,000 screening population by Monte Carlo simulation. Results: Our cfDNA-based assay could distinguish early-stage gastric cancer from non-cancer at an AUROC of 0.962 (95% CI: 0.942–0.982) in the study cohort, 0.972 (95% CI: 0.953–0.992) in the first validation cohort and 0.937 (95% CI: 0.890–0.983) in the second validation cohort. The model reached a specificity of 92.1% (128/139) and a sensitivity of 88.2% (97/110) in the study cohort. In the first validation cohort, 91.5% (86/94) of non-cancer individuals and 91.8% (67/73) of gastric cancer patients were correctly identified. In the second validation cohort, 89.8% (44/49) of non-cancer individuals and 87.2% (41/47) of gastric cancer patients were accurately classified. Conclusions: We introduced a liquid biopsy assay using multiple dimensions of cfDNA characteristics that could accurately identify early-stage gastric cancer from non-cancerous conditions. As a cost-effective non-invasive approach, it may provide population-wide benefits for the early detection of gastric cancer. Trial registration: This study was registered on ClinicalTrials.gov under the identifier NCT05269056 on March 7, 2022. [ABSTRACT FROM AUTHOR]

Hormonal therapy is an important treatment option for estrogen receptor (ER)-positive patients with advanced ovarian cancer. Although ER overexpression has been previously used as an indicator for hormonal therapy, the clinical outcomes of advanced ovarian cancer patients receiving hormonal therapy remain unsatisfactory. Additional biomarkers for screening patients are needed to improve its efficacy. In this study, we reported a metastatic ovarian cancer case with estrogen receptor 1 (ESR1) gene amplification and protein overexpression, which showed sustained partial response to hormonal therapy, including letrozole and tamoxifen, and displayed an overall survival of 47 months. The response to the therapy was evaluated by imageological examinations, cancer antigen-125 (CA-125) tests, and circulating tumor DNA (ctDNA) sequencing using capture-based hybrid next-generation sequencing. Our clinical data suggested that ESR1 amplification might be a potential predictor of response to hormonal therapy in ovarian cancer. The combination of tumor detection techniques including imaging, CA-125 and ctDNA would enable confirmation of tumor response with high confidence. [ABSTRACT FROM AUTHOR]

Introduction: Pembrolizumab is widely used in advanced non-small-cell lung cancer (NSCLC) patients with positive programmed death-ligand 1 (PD-L1). However, efficacy evaluation along treatment by serial monitoring of circulating tumor DNA (ctDNA) using next-generation sequencing remained to be well studied. Methods: Nine PD-L1 positive advanced NSCLC patients were prospectively enrolled and received pembrolizumab monotherapy. Pretreatment tissue and/or plasma samples were collected as baseline reference. Serial plasma samples were collected after 3 and 6 weeks of treatment as well as at disease progression. All samples underwent targeted next-generation sequencing. Results: The median progression-free survival (mPFS) and median overall survival (mOS) were 4.43 and 25.53 months, respectively. In total, 3 patients achieved partial response (PR) or stable disease (SD) for more than 6 months and were thus classified into the durable clinical benefit (DCB) group, whereas the rest 6 were grouped as nondurable benefit (NDB) patients. Molecular profiling of baseline samples revealed that TP53 and APC were the 2 most frequently mutated genes in all patients, whereas POT1 and SETD2 mutations were enriched in DCB and NDB groups, respectively. Higher tumor mutational burden (TMB) was observed in DCB patients than NDB group. During serial ctDNA monitoring, 2 DCB patients showed a dramatic ctDNA reduction while 75% of NDB patients' ctDNA concentration increased at week 6. Several acquired mutations might contribute to the pembrolizumab resistance, including CDKN2A frameshift and MITF nonsense mutations. Conclusions: Genomic profiling of peripheral blood samples can be applied to dynamically monitor disease progression. The reduction in ctDNA concentration during treatment implied DCBs. [ABSTRACT FROM AUTHOR]

Colorectal cancer is the third most common cancer worldwide, and its incidence continues to grow. Approximately one-third of patients with colorectal cancer develop liver metastases during the natural course of disease. Complete surgical resection is associated with very low mortality in colorectal liver metastasis patients, but only a small fraction of colorectal liver metastasis patients fulfill the selection criteria for surgical treatment. We herein describe a high-risk stage-IV rectal carcinoma patient who was initially unresectable according to the National Comprehensive Cancer Network guidelines with a clinical risk score of 4 but received conversion surgery combined with systemic chemotherapy and achieved a favorable long-term clinical outcome (pathologic complete response) of approximately 28 months. Furthermore, serial circulating tumor DNA monitoring using next-generation sequencing provided a comprehensive view of the patient's clinical and pathologic status for better clinical decision support over the course of the disease. The absence of circulating tumor DNA/cells after conversion surgery was correlated with pathologic complete response. This case study not only demonstrated that a curative oncosurgical approach could be considered for high-risk colorectal liver metastasis patients under specific circumstances but also highlighted the role of circulating tumor DNA monitoring to gain further insight into the evolution of a patient's response over time. [ABSTRACT FROM AUTHOR]

Ibrutinib‐based combination therapy with high‐dose methotrexate (HD‐MTX) has recently shown clinical activity against relapse/refractory (R/R) primary central nervous system lymphoma (PCNSL). Herein, we report our real‐world experience of treating 11 newly diagnosed PCNSL patients with the ibrutinib/MTX combination. HD‐MTX was given at 3.5 g/m2 every 2‐week for eight doses. Ibrutinib was held upon HD‐MTX infusion until clearance and was administered daily post‐induction until disease progression, intolerable toxicity, or death. Nine out of 11 patients completed the induction phase and received ibrutinib as maintenance therapy. An objective response rate (ORR) of 82% (9/11) was observed including complete response (64%) and partial response (18%). The median progression‐free survival (PFS) was 7.4 months while the median overall survival (OS) was not reached. The ibrutinib/MTX combination was well tolerated in these treatment‐naïve PCNSL patients with an acceptable safety profile. Moreover, the longitudinal analysis of cerebrospinal fluid (CSF) circulating tumor DNA (ctDNA) revealed that CSF ctDNA detection was closely associated with tumor response, and sustained tumor responses correlated with the clearance of ctDNA from the CSF. In sum, our data not only demonstrated the clinical benefit of the ibrutinib and HD‐MTX combination regimen in treating newly diagnosed PCNSL patients in a real‐world setting, but also highlighted the significance of liquid biopsy including CSF ctDNA in tracing tumor burden and assessing treatment response. [ABSTRACT FROM AUTHOR]

EML4-ALK fusions are targetable oncogenic drivers in a subset of advanced non-small cell lung cancer (NSCLC) patients that can benefit from selected ALK inhibitors. Precise detection of ALK fusions may yield critical information for selection of appropriate therapy and hence improve patient survival. Analysis of circulating tumor DNA (ctDNA) in liquid biopsies using next generation sequencing (NGS) prior to or during treatment hold great promise for disease monitoring and treatment guidance of various cancers including NSCLC. Herein, we report a case of a 21-year-old advanced lung adenocarcinoma patient with a low abundance (0.03%) of EML4-ALK rearrangement identified in plasma ctDNA upon progression on two lines of chemotherapy that demonstrated long-term complete response to alectinib (>13 months) including metastatic brain tumors. Patient's clinical and pathologic characteristics, computerized tomography (CT) scans and brain magnetic resonance imaging (MRI) were reviewed retrospectively. Taken together, our report not only reinforces the translational utility of NGS-based genomic sequencing of liquid biopsy in guiding clinical practice, but also highlights the superior efficacy of alectinib than chemotherapy in ALK + NSCLC with brain metastases, albeit at a low variant allele abundance. [ABSTRACT FROM AUTHOR]

Papillary thyroid carcinoma (PTC) is the most common malignancy of the thyroid gland, with a relatively high cure rate. Distant metastasis (DM) of PTC is uncommon, but when it occurs, it significantly decreases the survival of PTC patients. The molecular mechanisms of DM in PTC have not been systematically studied. We performed whole exome sequencing and GeneseeqPrime (425 genes) panel sequencing of the primary tumor, plasma and matched white blood cell samples from 20 PTC with DM and 46 PTC without DM. We identified somatic mutations, gene fusions and copy number alterations and analyzed their relationships with DM of PTC. BRAF‐V600E was identified in 73% of PTC, followed by RET fusions (14%) in a mutually exclusive manner (P < 0.0001). We found that gene fusions (RET, ALK or NTRK1) (P < 0.01) and chromosome 22q loss (P < 0.01) were independently associated with DM in both univariate and multivariate analyses. A nomogram model consisting of chromosome 22q loss, gene fusions and three clinical variables was built for predicting DM in PTC (C‐index = 0.89). The plasma circulating tumor DNA (ctDNA) detection rate in PTC was only 38.9%; however, it was significantly associated with the metastatic status (P = 0.04), tumor size (P = 0.001) and invasiveness (P = 0.01). In conclusion, gene fusions and chromosome 22q loss were independently associated with DM in PTC and could serve as molecular biomarkers for predicting DM. The ctDNA detection rate was low in non–DM PTC but significantly higher in PTC with DM. [ABSTRACT FROM AUTHOR]

Tumour‐induced osteomalacia (TIO) is a very rare paraneoplastic syndrome with bone pain, fractures and muscle weakness, which is mostly caused by phosphaturic mesenchymal tumours (PMTs). Cell‐free DNA (cfDNA) has been regarded as a non‐invasive liquid biopsy for many malignant tumours. However, it has not been studied in benign tumours, which prompted us to adopt the targeted next‐generation sequencing approach to compare cfDNAs of 4 TIO patients, four patients with bone metastasis (BM) and 10 healthy controls. The mutational landscapes of cfDNA in TIO and BM groups were similar in the spectrum of allele frequencies and mutation types. Markedly, deleterious missense mutations in FGFR1 and loss‐of‐function mutations in MED12 were found in 3/4 TIO patients but none of BM patients. The gene ontology analysis strongly supported that these mutated genes found in TIOs would play a potential role in PMTs' process. The genetic signatures and corresponding change in expression of FGFR1 and FGF23 were further validated in PMT tissues from a test cohort of another three TIO patients. In summary, we reported the first study of the mutational landscape and genetic signatures of cfDNA in TIO/PMTs. [ABSTRACT FROM AUTHOR]

Epidermal growth factor receptor (EGFR)-positive non-small cell lung cancer (non-SCLC) benefits from first-line treatment with first generation tyrosine kinase inhibitors (TKIs). However, drug resistance is inevitable through different mechanisms and is dominated by the acquisition of the T790M mutation within EGFR, which occurs in ~50% of cases. The present study reports the case of a patient originally diagnosed with stage IA lung adenocarcinoma, with a recurrent tumor lesion in each side of the lungs following the surgical removal of the primary tumor. Erlotinib treatment of the recurrent tumors eliminated the tumor on the right side of the lung and resulted in the histological transformation of the tumor on the left side to SCLC following 6 years of treatment. Genetic profiling of the SCLC lesions using targeted next-generation sequencing identified different genetic alterations from the primary tumor, characterized by the newly acquired copy number loss of tumor protein p53 and transcriptional coreceptor 1, and the copy number gain of SRY-box 2. Continuation of treatment with chemotherapy and erlotinib demonstrated moderate disease control for ~1 year prior to the outbreak of a new lung lesion. Liquid biopsy profiling of circulating tumor DNA revealed the acquisition of KRAS proto-oncogene, GTPase (KRAS) p.G12C mutation, indicating the occurrence of another resistance mechanism to erlotinib. During erlotinib treatment, the lung adenocarcinoma progressed through two atypical mechanisms, notably from the transformation to SCLC and the acquisition of the KRAS mutation to surpass EGFR inhibition. However, the combinational and interchanging usage of chemotherapy and TKI resulted in persistent and effective disease control. [ABSTRACT FROM AUTHOR]