Microfluidic Chip-Based Automatic System for Sequencing Patient-Derived Organoids at the Single-Cell Level.

Genetically sequencing patient-derived organoids (PDOs) at the single-cell level has emerged as a promising method to infer cell-level heterogeneity of original organs and improve cancer precision medicine. Unfortunately, because of the limited starting quantity and uncontrolled establishing process of PDOs, the existing single-cell sequencing technologies, either manual-operation-based or microfluid-based, are inefficient in processing PDOs originating from clinical tissue samples. To address such issues, this study presents a microfluidic chip-based automatic system for sequencing organoids at the single-cell level, named as MASSO. By performing all required procedures, including PDO establishment/culturing/digesting and single-cell isolation/lysis/whole-genome amplification, in a single microfluidic chip, the possible loss of precious PDO is avoided, and the high quality of on-chip whole-genome amplification of a single PDO cell is ensured. By automating the entire operation process, possible human error is eliminated, and the data repeatability is improved, therefore bridging the technical gap between laboratorial proof-of-concept studies and clinical practices. After characterizing the organoid single-cell whole-genome amplification chip (named as OSA-Chip) and the MASSO, the first successful attempt, to the best of our knowledge, on whole-genome sequencing lung cancer PDO at the single-cell level was performed by MASSO. The results reveal that the MASSO is capable of not only identifying common cancer-related mutations but also discovering specific mutations that affect drug responses, therefore laying the technical foundation for efficiently understanding the cell-level heterogeneities of PDOs and corresponding original organs.

Super multiple primary lung cancers harbor high-frequency BRAF and low-frequency EGFR mutations in the MAPK pathway.

The incidence of multiple primary lung cancer (MPLC) is increasing, with some of our surgical patients exhibiting numerous lesions. We defined lung cancer with five or more primary lesions as super MPLCs. Elucidating the genomic characteristics of this special MPLC subtype can help reduce disease burden and understand tumor evolution. In our cohort of synchronous super early-stage MPLCs (PUMCH-ssesMPLC), whole-exome sequencing on 130 resected malignant specimens from 18 patients provided comprehensive super-MPLC genomic landscapes. Mutations are enriched in PI3k-Akt and MAPK pathways. Their BRAF mutation frequency (31.5%) is significantly higher than MPLC with fewer lesions and early-stage single-lesion cancer, while EGFR mutations are significantly fewer (13.8%). As lesion counts increase, BRAF mutations gradually become dominant. Also, invasive lesions more tend to have classic super-MPLC mutation patterns. High-frequency BRAF mutations, especially Class II, and low-frequency EGFR mutations could be a reason for the limited effectiveness of targeted therapy in super-MPLC patients.

A Microfluidic Chip-Based Automated System for Whole-Course Monitoring the Drug Responses of Organoids.

Tumor patients-derived organoids, as a promising preclinical prediction model, have been utilized to evaluate ex vivo drug responses for formulating optimal therapeutic strategies. Detecting adenosine triphosphate (ATP) has been widely used in existing organoid-based drug response tests. However, all commercial ATP detection kits containing the cell lysis procedure can only be applied for single time point ATP detection, resulting in the neglect of dynamic ATP variations in living cells. Meanwhile, due to the limited number of viable organoids from a single patient, it is impractical to exhaustively test all potential time points in search of optimal ones. In this work, a multifunctional microfluidic chip was developed to perform all procedures of organoid-based drug response tests, including establishment, culturing, drug treatment, and ATP monitoring of organoids. An ATP sensor was developed to facilitate the first successful attempt on whole-course monitoring the growth status of fragile organoids. To realize a clinically applicable automatic system for the drug testing of lung cancer, a microfluidic chip based automated system was developed to perform entire organoid-based drug response test, bridging the gap between laboratorial manipulation and clinical practices, as it outperformed previous methods by improving data repeatability, eliminating human error/sample loss, and more importantly, providing a more accurate and comprehensive evaluation of drug effects.

[Application and Research Progress of Lung Cancer Organoid in Precision Medicine 
for Lung Cancer].

The continuous advancement of molecular detection technology has greatly propelled the development of precision medicine for lung cancer. However, tumor heterogeneity is closely associated with tumor metastasis, recurrence, and drug resistance. Additionally, different lung cancer patients with the same genetic mutation may exhibit varying treatment responses to different therapeutic strategies. Therefore, the development of modern precision medicine urgently requires the precise formulation of personalized treatment strategies through personalized tumor models. Lung cancer organoid (LCO) can highly simulate the biological characteristics of tumor in vivo, facilitating the application of innovative drugs such as antibody-drug conjugate in precision medicine for lung cancer. With the development of co-culture model of LCO with tumor microenvironment and tissue engineering technology such as microfluidic chip, LCO can better preserve the biological characteristics and functions of tumor tissue, further improving high-throughput and automated drug sensitivity experiment. In this review, we combine the latest research progress to summarize the application progress and challenges of LCO in precision medicine for lung cancer.
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The EEG-Based Fusion Entropy-Featured Identification of Isometric Contraction Forces under the Same Action.

This study explores the important role of assessing force levels in accurately controlling upper limb movements in human-computer interfaces. It uses a new method that combines entropy to improve the recognition of force levels. This research aims to differentiate between different levels of isometric contraction forces using electroencephalogram (EEG) signal analysis. It integrates eight different entropy measures: power spectrum entropy (PSE), singular spectrum entropy (SSE), logarithmic energy entropy (LEE), approximation entropy (AE), sample entropy (SE), fuzzy entropy (FE), alignment entropy (PE), and envelope entropy (EE). The findings emphasize two important advances: first, including a wide range of entropy features significantly improves classification efficiency; second, the fusion entropy method shows exceptional accuracy in classifying isometric contraction forces. It achieves an accuracy rate of 91.73% in distinguishing between 15% and 60% maximum voluntary contraction (MVC) forces, along with 69.59% accuracy in identifying variations across 15%, 30%, 45%, and 60% MVC. These results illuminate the efficacy of employing fusion entropy in EEG signal analysis for isometric contraction detection, heralding new opportunities for advancing motor control and facilitating fine motor movements through sophisticated human-computer interface technologies.

Clonal expansion of shared T cell receptors reveals the existence of immune commonality among different lesions of synchronous multiple primary lung cancer.

The increase in the detection rate of synchronous multiple primary lung cancer (MPLC) has posed remarkable clinical challenges due to the limited understanding of its pathogenesis and molecular features. Here, comprehensive comparisons of genomic and immunologic features between MPLC and solitary lung cancer nodule (SN), as well as different lesions of the same patient, were performed. Compared with SN, MPLC displayed a lower rate of EGFR mutation but higher rates of BRAF, MAP2K1, and MTOR mutation, which function exactly in the upstream and downstream of the same signaling pathway. Considerable heterogeneity in T cell receptor (TCR) repertoire exists among not only different patients but also among different lesions of the same patient. Invasive lesions of MPLC exhibited significantly higher TCR diversity and lower TCR expansion than those of SN. Intriguingly, different lesions of the same patient always shared a certain proportion of TCR clonotypes. Significant clonal expansion could be observed in shared TCR clonotypes, particularly in those existing in all lesions of the same patient. In conclusion, this study provided evidences of the distinctive mutational landscape, activation of oncogenic signaling pathways, and TCR repertoire in MPLC as compared with SN. The significant clonal expansion of shared TCR clonotypes demonstrated the existence of immune commonality among different lesions of the same patient and shed new light on the individually tailored precision therapy for MPLC.

A dual-functional microfluidic chip for guiding personalized lung cancer medicine: combining EGFR mutation detection and organoid-based drug response test.

Many efforts have been paid to advance the effectiveness of personalized medicine for lung cancer patients. Sequencing-based molecular diagnosis of EGFR mutations has been widely used to guide the selection of anti-lung-cancer drugs. Organoid-based assays have also been developed to ex vivo test individual responses to anti-lung-cancer drugs. After addressing several technical difficulties, a new combined strategy, in which anti-cancer medicines are first selected based on molecular diagnosis and then ex vivo tested on organoids, has been realized in a single dual-functional microfluidic chip. A DNA-based nanoruler has been developed to detect the existence of EGFR mutations and shrink the detection period from weeks to hours, compared with sequencing. The employment of the DNA-based nanoruler creates a possibility to purposively test anti-cancer drugs, either EGFR-TKIs or chemotherapy drugs, not both, on limited amounts of organoids. Moreover, a DNA-based nanosensor has been developed to recognize intracellular ATP variation without harming cell viability, realizing in situ monitoring of the whole course growth status of organoids for on-chip drug response test. The dual-functional microfluidic chip was validated by both cell lines and clinical samples from lung cancer patients. Furthermore, based on the dual-functional microfluidic chip, a fully automated system has been developed to span the divide between experimental procedures and therapeutic approaches. This study constitutes a novel way of combining EGFR mutation detection and organoid-based drug response test on an individual patient for guiding personalized lung cancer medicine.

[Relationship between Bacteria in the Lower Respiratory Tract/Lung Cancer 
and the Development of Lung Cancer as well as Its Clinical Application].

Due to the advancement of 16S rRNA sequencing technology, the lower respiratory tract microbiota, which was considered non-existent, has been revealed. The correlation between these microorganisms and diseases such as tumor has been a hot topic in recent years. As the bacteria in the surrounding can infiltrate the tumors, researchers have also begun to pay attention to the biological behavior of tumor bacteria and their interaction with tumors. In this review, we present the characteristic of the lower respiratory tract bacteria and summarize recent research findings on the relationship between these microbiota and lung cancer. On top of that, we also summarize the basic feature of bacteria in tumors and focus on the characteristic of the bacteria in lung cancer. The relationship between bacteria in lung cancer and tumor development is also been discussed. Finally, we review the potential clinical applications of bacterial communities in the lower respiratory tract and lung cancer, and summarize key points of sample collection, sequencing, and contamination control, hoping to provide new ideas for the screening and treatment of tumors.
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Adjuvant crizotinib treatment selected by patient-derived organoids in a patient with stage IIIA adenocarcinoma with novel LRRTM4-ALK fusion: a case report.

Background: Over 90 different anaplastic lymphoma kinase (ALK) fusions have been reported, and patients with different ALK fusion partners exhibit different responses to targeted therapy. Patient-derived organoid (PDO), a kind of 3-dimensional culture, is a promising model for drug-sensitivity testing for personalized treatment decision-making. It further has the potential to provide treatment strategy for patients with novel mutations, rare mutations, and concomitant mutations, serving as a supplement to evidence-based medicine. Case Description: We report a case in which a man with stage IIIA adenocarcinoma had pleural effusion 1 month after surgery. A novel leucine-rich repeat transmembrane neuronal protein 4 (LRRTM4)-ALK fusion was unveiled by next-generation sequencing (NGS), and PDOs were used in drug-sensitivity testing to select a proper adjuvant therapy for this patient. We chose crizotinib based on result of the test and drugs' availability in China and helped the patient achieve a more than 3-year-long disease-free survival (DFS). Higher variant allele frequencies (VAFs) of the driver mutation were also found in PDOs and their waste culture medium, indicating that the PDO model could filter out cells with driver genes or stemness and help us to identify the critical cancer cell colony in treatment decision-making. Conclusions: For the first time, we report the case of a LRRTM4-ALK fusion. The patient achieved a more than 3-year long-term DFS under crizotinib treatment, which was selected by an emerging PDO drug-sensitivity test model. We also discovered the enrichment of a low-abundance driver mutation in PDO and its waste culture medium, providing a new direction for future research.

[Recent Advances and Controversies in Minute Pulmonary Meningothelial-like Nodules].

Minute pulmonary meningothelial-like nodules (MPMNs) are benign small lesions in the lungs, with similar pathological characteristics to the meningeal epithelium. MPMNs have similar imaging manifestations to malignant tumors, which can lead to misdiagnosis in clinical practice. There is no consensus on the pathogenesis of MPMNs, with some suggest that MPMNs derive from reactive proliferation, while others suggest that MPMNs share a common origin and molecular mechanism with meningiomas in the central nervous system. Understanding the characteristics of MPMNs and studying their pathogenesis will help improve the understanding and diagnosis of MPMNs. In this article, we reviewed the clinical, pathological, imaging characteristics, differential diagnosis and pathogenesis of MPMNs. We also analyze the existing research advances regarding the pathogenesis and propose prospects for further research.
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Rapid determination of the presence of EGFR mutations with DNA-based nanocalipers.

Selecting 1st-line treatment for lung cancer is currently a binary choice, either chemotherapy or targeted medicine, depending on whether EGFR mutations exist. Next-generation sequencing is fully capable of accurately identifying EGFR mutations and guiding the usage of tyrosine kinase inhibitors, but it is highly expensive. Moreover, as the sequencing is not helpful for patients with wild-type EGFR, the long wait for sequencing may delay the chemotherapy and correspondingly increase the risks of cancer progression. To address this issue, a new method for rapidly determining the presence of EGFR mutations is developed in this study. A series of DNA origami-engineered nanocalipers are designed and constructed to determine the EGFR spatial distribution of either mutated EGFR or wild-type EGFR lung cancer cells. The experimental results on cancer cell lines and 9 clinical tissue samples show that compared with wild-type EGFR cells, mutated EGFR cells have narrower EGFR spacing. Hence, the DNA nanocalipers are demonstrated to be capable of determining the presence of EGFR mutations and shrinking the detection period from weeks to hours, compared with sequencing. For determining EGFR mutation status in 9 clinical samples, DNA nanocalipers show 100% consistency with next-generation sequencing.

Real-time monitoring ATP variation in human cancer organoids for a long term by DNA-based nanosensor.

Cancer organoids have become promising tools for predicting drug responses on many different types of cancer. Detecting the adenosine triphosphate (ATP) has currently been considered as a decisive test to profile the growth status and drug responses of organoids. ATP profiling using commercial ATP detection kits, which involve cell lysis, can be performed at a single time spot, causing a clinical dilemma of selecting the optimal time spot to adopt diverse cancer types and patients. This study provides a feasible solution to this dilemma by developing a DNA-based ATP nanosensor to realize real-time ATP monitoring in organoids for a long term. The employment of DNA materials ensures high biocompatibility and low cytotoxicity, which are crucial for fragile organoids; The usage of tetrahedral DNA framework ensures cell permeability and intracellular ATP detection; The introduction of ATP-mediated molecular replacement ensures the high sensitivity and selectivity of ATP recognition. These features result in the first successful attempt on real-time monitoring ATP in organoids for up to 26 days and gaining growth status curves for the whole duration of a drug sensitivity test on human lung cancer organoids.

Dual targeting PET tracer [68Ga]Ga-FAPI-RGD in patients with lung neoplasms: a pilot exploratory study.

Rationale: Early discovery, accurate diagnosis, and staging of lung cancer is essential for patients to receive appropriate treatment. PET/CT has become increasingly recognized as a valuable imaging modality for these patients, but there remains room for improvement in PET tracers. We aimed to evaluate the feasibility of using [68Ga]Ga-FAPI-RGD, a dual-targeting heterodimeric PET tracer that recognizes both fibroblast activation protein (FAP) and integrin αvß3 for detecting lung neoplasms, by comparing it with [18F]FDG and single-targeting tracers [68Ga]Ga-RGD and [68Ga]Ga-FAPI. Methods: This was a pilot exploratory study of patients with suspected lung malignancies. All 51 participants underwent [68Ga]Ga-FAPI-RGD PET/CT, of which: 9 participants received dynamic scans, 44 participants also underwent [18F]FDG PET/CT scan within two weeks, 9 participants underwent [68Ga]Ga-FAPI PET/CT scan and 10 participants underwent [68Ga]Ga-RGD PET/CT scan. The final diagnosis was made based on histopathological analyses and clinical follow-up reports. Results: Among those who underwent dynamic scans, the uptake of pulmonary lesions increased over time. The optimal timepoint for a PET/CT scan was identified to be 2 h post-injection. [68Ga]Ga-FAPI-RGD had a higher detection rate of primary lesions than [18F]FDG (91.4% vs. 77.1%, p < 0.05), higher tumor uptake (SUVmax, 6.9 ± 5.3 vs. 5.3 ± 5.4, p < 0.001) and higher tumor-to-background ratio (10.0 ± 8.4 vs. 9.0 ± 9.1, p < 0.05), demonstrated better accuracy in mediastinal lymph node evaluation (99.7% vs. 90.9%, p < 0.001), and identified more metastases (254 vs. 220). There was also a significant difference between the uptake of [68Ga]Ga-FAPI-RGD and [68Ga]Ga-RGD of primary lesions (SUVmax, 5.8 ± 4.4 vs. 2.3 ± 1.3, p < 0.001). Conclusion: In our small scale cohort study, [68Ga]Ga-FAPI-RGD PET/CT gave a higher primary tumor detection rate, higher tracer uptake, and improved detection of metastases compared with [18F]FDG PET/CT, and [68Ga]Ga-FAPI-RGD also had advantages over [68Ga]Ga-RGD and was non-inferior to [68Ga]Ga-FAPI. We thus provide proof-of-concept for using [68Ga]Ga-FAPI-RGD PET/CT for diagnosing lung cancer. With the stated advantages, the dual-targeting FAPI-RGD should also be explored for therapeutic use in future studies.

Footprints: Stamping hallmarks of lung cancer with patient-derived models, from molecular mechanisms to clinical translation.

The conventional two-dimensional (2D) tumor cell lines in Petri dishes have played an important role in revealing the molecular biological mechanism of lung cancer. However, they cannot adequately recapitulate the complex biological systems and clinical outcomes of lung cancer. The three-dimensional (3D) cell culture enables the possible 3D cell interactions and the complex 3D systems with co-culture of different cells mimicking the tumor microenvironments (TME). In this regard, patient-derived models, mainly patient-derived tumor xenograft (PDX) and patient-derived organoids discussed hereby, are with higher biological fidelity of lung cancer, and regarded as more faithful preclinical models. The significant Hallmarks of Cancer is believed to be the most comprehensive coverage of current research on tumor biological characteristics. Therefore, this review aims to present and discuss the application of different patient-derived lung cancer models from molecular mechanisms to clinical translation with regards to the dimensions of different hallmarks, and to look to the prospects of these patient-derived lung cancer models.

Efficacy and Safety of Epidermal Growth Factor Receptor (EGFR)-Tyrosine Kinase Inhibitor Combination Therapy as First-Line Treatment for Patients with Advanced EGFR-Mutated, Non-Small Cell Lung Cancer: A Systematic Review and Bayesian Network Meta-Analysis.

(1) Background: Several randomized controlled trials (RCTs) have been conducted in combination with Efficacy and Safety of Epidermal Growth Factor Receptor(EGFR)-Tyrosine Kinase Inhibitor (TKI) for the first-line treatment of patients with advanced non-small cell lung cancer; however, head-to-head comparisons of combination therapies are still lacking. Therefore, this study aims to compare the efficacy and safety of various combination treatments. (2) Methods: We conducted a systematic review and Bayesian network meta-analysis by searching MEDLINE, EMBASE, and COCHRANE for relevant RCTs. (3) Results: TKI combined with antiangiogenic therapy, chemotherapy, or radiation achieved a significant benefit compared with TKI alone for progression free survival (PFS). A combination with radiation yielded better benefits in PFS than any other treatment. In terms of overall survival (OS), only the combination with pemetrexed and carboplatin (HR = 0.63, 95% credible interval 0.43-0.86)/radiation (0.44, 0.23-0.83) was superior to TKI alone. All of the combination therapies may increase the incidence of ≥Grade 3 AEs, as the pooled RRs are over 1; different toxicity spectrums were revealed for individual treatments. (4) Conclusions: The TKI combination of radiation/pemetrexed and carboplatin could provide the best antitumor effects among the first generation TKI-based treatments. Considering safety, ramucirumab and bevacizumab may be the ideal additions to TKIs (systematic review registration: PROSPERO CRD42022350474).

Comprehensive analysis of T cell receptor repertoire in patients with KRAS mutant non-small cell lung cancer.

Background: Kirsten rat sarcoma viral oncogene homolog (KRAS) is one of the most frequently mutated oncogenes in non-small cell lung cancer (NSCLC). The administration of immunotherapy has demonstrated significant efficacy in prolonging the overall survival of patients with KRAS mutation in recent years. However, the efficacy of immunotherapy in KRAS mutant NSCLC is variable. Analysis of T cell receptor (TCR) repertoire may contribute to a better understanding of the mechanisms behind such differential outcomes. Methods: A total of 47 patients with KRAS mutant NSCLC were enrolled in this study. Deep sequencing of the TCR ß chain complementarity-determining regions in tumor tissue and paired peripheral blood specimens was conducted. Comprehensive analysis of TCR repertoire metrics was performed with different KRAS mutation subtypes and concomitant mutations. Moreover, the associations between TCR repertoire metrics and tumor mutation burden (TMB), as well as programmed death-ligand 1 were explored, respectively. Results: TCR repertoire metrics, including Shannon index, Clonality, and Morisita index (MOI), showed no significant differences among different KRAS mutation subtypes. The similar results were observed between patients with tumor protein p53 (TP53) mutation and those with wild-type TP53. In contrast, although no significant differences were found in Shannon index and Clonality, patients with KRAS/serine/threonine kinase 11 (STK11) comutation showed a significantly higher MOI compared to their STK11 wild-type counterparts (P=0.012). In addition, TCR repertoire metrics were neither associated with TMB nor programmed death-ligand 1 expression in KRAS mutant NSCLC. Conclusions: This retrospective study comprehensively described the TCR repertoire in KRAS mutant NSCLC. A higher MOI represented more overlap of the TCR repertoire between tumor tissue and paired peripheral blood, indicating distinctive immunological features in NSCLC with KRAS/STK11 comutation.

[Research Progress of Angiogenesis Inhibitors Plus EGFR-TKI in EGFR-mutated 
Advanced Non-small Cell Lung Cancer].

Lung cancer is one of the leading causes of cancer-related morbidity and mortality. Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) have become the standard treatment for EGFR-mutated advanced non-small cell lung cancer (NSCLC). Unfortunately, drug resistance is inevitable in most cases. EGFR-TKI combined with angiogenesis inhibitors is a treatment scheme being explored to delay the therapeutic resistance, which is called "A+T treatment". Several clinical trials have demonstrated that the A+T treatment can improve the progression free survival (PFS) of the NSCLC patients. However, compared to EGFR-TKI monotherapy, the benefits of the A+T treatment based on different EGFR-TKIs, as well as its safety and exploration prospects are still unclear. Therefore, we reviewed the literature related to all three generations EGFR-TKIs combined with angiogenesis inhibitors, and summarized the mechanism, benefit, safety, optimal target population of A+T treatment.
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Machine Learning-Assisted Dual-Marker Detection in Serum Small Extracellular Vesicles for the Diagnosis and Prognosis Prediction of Non-Small Cell Lung Cancer.

Small extracellular vesicles (sEVs) carry molecular information from their source cells and are desired biomarkers for cancer diagnosis. We establish a machine learning-assisted dual-marker detection method to analyze the expression of epidermal growth factor receptor (EGFR) and C-X-C chemokine receptor 4 (CXCR4) in serum sEVs for the diagnosis and prognosis prediction of non-small cell lung cancer (NSCLC). We find that the serum sEV EGFR and CXCR4 are significantly higher in advanced stage NSCLC (A/NSCLC) patients compared to early stage NSCLC (E/NSCLC) patients and the healthy donors (HDs). A receiver operating characteristic curve (ROC) analysis demonstrates that the combination of EGFR and CXCR4 in serum sEVs as an efficient diagnostic index and malignant degree indicator for NSCLC. Machine learning further shows a diagnostic accuracy of 97.4% for the training cohort and 91.7% for the validation cohort based on the combinational marker. Moreover, this machine leaning-assisted serum sEV analysis successfully predicts the possibility of tumor relapse in three NSCLC patients by comparing their serum sEVs before and three days after surgery. This study provides an intelligent serum sEV-based assay for the diagnosis and prognosis prediction of NSCLC, and will benefit the precision management of NSCLC.

DNA methylation patterns at and beyond the histological margin of early-stage invasive lung adenocarcinoma radiologically manifested as pure ground-glass opacity.

BACKGROUND: Early-stage lung cancers radiologically manifested as ground-glass opacities (GGOs) have been increasingly identified, among which pure GGO (pGGO) has a good prognosis after local resection. However, the optimal surgical margin is still under debate. Precancerous lesions exist in tumor-adjacent tissues beyond the histological margin. However, potential precancerous epigenetic variation patterns beyond the histological margin of pGGO are yet to be discovered and described. RESULTS: A genome-wide high-resolution DNA methylation analysis was performed on samples collected from 15 pGGO at tumor core (TC), tumor edge (TE), para-tumor tissues at the 5 mm, 10 mm, 15 mm, 20 mm beyond the tumor, and peripheral normal (PN) tissue. TC and TE were tested with the same genetic alterations, which were also observed in histologically normal tissue at 5 mm in two patients with lower mutation allele frequency. According to the difference of methylation profiles between PN samples, 2284 methylation haplotype blocks (MHBs), 1657 differentially methylated CpG sites (DMCs), and 713 differentially methylated regions (DMRs) were identified using reduced representation bisulfite sequencing (RRBS). Two different patterns of methylation markers were observed: Steep (S) markers sharply changed at 5 mm beyond the histological margin, and Gradual (G) markers changed gradually from TC to PN. S markers composed 86.2% of the tumor-related methylation markers, and G markers composed the other 13.8%. S-marker-associated genes enriched in GO terms that were related to the hallmarks of cancer, and G-markers-associated genes enriched in pathways of stem cell pluripotency and transcriptional misregulation in cancer. Significant difference in DNA methylation score was observed between peripheral normal tissue and tumor-adjacent tissues 5 mm further from the histological margin (p < 0.001 in MHB markers). DNA methylation score at and beyond 10 mm from histological margin is not significantly different from peripheral normal tissues (p > 0.05 in all markers). CONCLUSIONS: According to the methylation pattern observed in our study, it was implied that methylation alterations were not significantly different between tissues at or beyond P10 and distal normal tissues. This finding explained for the excellent prognosis from radical resections with surgical margins of more than 15 mm. The inclusion of epigenetic characteristics into surgical margin analysis may yield a more sensitive and accurate assessment of remnant cancerous and precancerous cells in the surgical margins.