Approximately 85% colorectal cancers (CRCs) are thought to evolve through the adenoma‐to‐carcinoma sequence associated with specific molecular alterations, including the 5‐hydroxymethylcytosine (5hmC) signature in circulating cell‐free DNA (cfDNA). To explore colorectal disease progression and evaluate the use of cfDNA as a potential diagnostic factor for CRC screening, here, we performed genome‐wide 5hmC profiling in plasma cfDNA and tissue genomic DNA (gDNA) acquired from 101 samples (63 plasma and 38 tissues), collected from 21 early‐stage CRC patients, 21 AD patients, and 21 healthy controls (HC). The gDNA and cfDNA 5hmC signatures identified in gene bodies and promoter regions in CRC and AD groups were compared with those in HC group. All the differential 5hmC‐modified regions (DhMRs) were gathered into four clusters: Disease‐enriched, AD‐enriched, Disease‐lost, and AD‐lost, with no overlap. AD‐related clusters, AD‐enriched and AD‐lost, displayed the unique 5hmC signals in AD patients. Disease‐enriched and Disease‐lost clusters indicated the general 5hmC changes when colorectal lesions occurred. Cancer patients with a confirmable adenoma history segmentally gathered in AD‐enriched clusters. KEGG functional enrichment and GO analyses determined distinct differential 5hmC‐modified profiles in cfDNA of HC individuals, AD, and CRC patients. All patients had comprehensive 5hmC signatures where Disease‐enriched and Disease‐lost DhMR clusters demonstrated similar epigenetic modifications, while AD‐enriched and AD‐lost DhMR clusters indicated complicated subpopulations in adenoma. Analysis of CRC patients with adenoma history showed exclusive 5hmC‐gain characteristics, consistent with the ‘parallel’ evolution hypothesis in adenoma, either developed through the adenoma‐to‐carcinoma sequence or not. These findings deepen our understanding of colorectal disease and suggest that the 5hmC modifications of different pathological subtypes (cancer patients with or without adenoma history) could be used to screen early‐stage CRC and assess adenoma malignancy with large‐scale follow‐up studies in the future.

Esophageal cancer is a highly incidence and deadly disease with a poor prognosis, especially in developing countries. Owing to the lack of specific symptoms and early diagnostic biomarkers, most patients are diagnosed with advanced disease, leading to a 5-year survival rate of less than 15%. Early (n = 50) and middle-advanced (n = 50) esophageal squamous cell carcinoma (ESCC) patients, as well as 71 healthy individuals, underwent 5-hydroxymethylcytosine (5hmC) sequencing on their plasma cell-free DNA (cfDNA). A Northern Chinese cohort of cfDNA 5hmC dataset of 150 ESCC patients and 183 healthy individuals were downloaded for validation. A diagnostic model was developed using cfDNA 5hmC signatures and then improved by low-pass whole genome sequencing (WGS) features of cfDNA. Conserved cfDNA 5hmC modification motifs were observed in the two independent ESCC cohorts. The diagnostic model with 5hmC features achieved an AUC of 0.810 and 0.862 in the Southern and Northern cohorts, respectively, with sensitivities of 69.3–74.3% and specificities of 82.4–90.7%. The performance was well maintained in Stage I to Stage IV, with accuracy of 70–100%, but low in Stage 0, 33.3%. Low-pass WGS of cfDNA improved the AUC to 0.934 with a sensitivity of 82.4%, a specificity of 88.2%, and an accuracy of 84.3%, particularly significantly in Stage 0, with an accuracy up to 80%. 5hmC and WGS could efficiently differentiate very early ESCC from healthy individuals. These findings imply a non-invasive and convenient method for ESCC detection when clinical treatments are available and may eventually prolong survival.

Objective: ROS1 fusions have been identified in 1– 2% of non-small-cell lung cancer (NSCLC) patients; they are validated as a driver of carcinogenesis and could be subjected to inhibition by crizotinib. However, previous studies suggested a variable progression-free survival (PFS) ranging from 9.1 to 20.0 months for crizotinib treatment in ROS1-rearranged NSCLC. Here, we reported a 45-year-old female diagnosed with stage IVB lung adenocarcinoma with multiple lymph nodes and bone metastasis carrying a novel MPRIP-ROS1 fusion, which was identified by RNA-based NGS (next-generation sequencing) and was sensitive to crizotinib treatment. Materials and Methods: A targeted NGS panel was used to analyze genomic DNA and total RNA isolated from formalin-fixed paraffin-embedded (FFPE) tissue block of the patient. An RNA fusion panel based on amplicon sequencing was designed for detection fusion variation. Fusion results were validated using reverse transcriptase polymerase chain reaction and Sanger sequencing. Results: We reported a novel MPRIP-ROS1 fusion identified in this advanced lung adenocarcinoma case. The rearrangement was generated by exons 1– 21 of MPRIP at chr17: p11.2 joined to exons 35– 43 of ROS1 at chr6: q22.1, which retained an intact kinase domain of ROS1. The primary tumor and metastatic lymph nodes were eliminated on computed tomographic (CT) scan imaging after 2 months’ crizotinib treatment, and the multiple bone metastatic lesions were significantly relieved according to bone scintigraphy images. To date, the treatment has lasted 16 months, and the patient is still in follow-up showing sustained response to crizotinib. Conclusion: The study identified a novel MPRIP-ROS1 fusion that was sensitive to crizotinib, which provided a new driver of lung adenocarcinoma and potential therapeutic target for crizotinib. It also expanded NSCLC treatment of ROS1 rearrangement and highlighted the importance of genetic testing for precise treatment decision-making.

Background: NRG1 fusions are rare oncogenic drivers in solid tumors, and the incidence of NRG1 fusions in non-small cell lung cancer (NSCLC) was 0.26%. It is essential to explore potential therapeutic strategies and efficacy predictors for NRG1 fusion-positive cancers. Case presentation: We report an advanced lung adenocarcinoma patient harboring a novel NPTN-NRG1 fusion identified by RNA-based next-generation sequencing (NGS), which was not detected by DNA-based NGS at initial diagnosis. Transcriptomics data of the tissue biopsy showed NRG1α isoform accounted for 30% of total NRG1 reads, and NRG1β isoform was undetectable. The patient received afatinib as fourth-line treatment and received a progression-free survival (PFS) of 14 months. Conclusions: This report supports afatinib can provide potential benefit for NRG1 fusion patients, and RNA-based NGS is an accurate and cost-effective strategy for fusion detection and isoform identification.

Objective: Synchronous multiple primary lung cancer (SMPLC) has a reported occurrence from 0.5% to 2% in lung cancer, and the surgical treatment and prognosis were quite diverse. With the discovery of driver mutations in lung adenocarcinoma (ADC), next-generation sequencing (NGS) would provide an explicit answer to the key question, whether individual tumors represent intrapulmonary metastases or independent tumors. Here, we reported a 64-year-old female diagnosed with a synchronous trilateral early-stage ADC with distinct driver alterations. Materials and Methods: NGS test targeting 31 cancer-relevant genes and amplification RNA sequencing (if gene fusion was found on DNA level) were performed on the surgical tumor tissue. Results: A 64-year-old Chinese female never smoker was found with one nodule in the right upper lobe and two nodules in the right middle lobe through chest computed tomography. The lesions were resected through video-assisted thoracic surgery and diagnosed with stage IA ADC, T1N0M0, in the postoperative pathology. NGS detected three independent driver mutations in three primary sites, respectively, EGFR 19del, EGFR 20ins and ROS1 fusion. Conclusion: This is the first report of a synchronous trilateral early-stage ADC with distinct driver alterations. All individual tumors were independent identified by NGS methodology, which had provided a clear answer to the key question of SMPLC in this case and should be used as a routine genetic test to explore fully pathological diagnosis and more comprehensive oncogenesis information in the early-stage ADC clinical prevention.

Non‐small‐cell lung cancer (NSCLC) is an important cause of cancer‐related death worldwide. The distant metastasis heterogeneity of gene tumor mutations in tumors of NSCLC patients brings critical challenges for treatment. We sequenced the primary tumors and metastatic tissues of 48 NSCLC patients through 363 tumor‐related gene panels to examine gene mutations in primary tumors and metastatic tissues, and screen candidate carcinogenic and metastatic‐related driver mutations. The patient group included 21 patients in the metastatic group and 27 patients in the non‐metastatic group. The patient's median age was 62 years and 54% (26/48) of patients were women. Approximately 75% (36/48) of patients were non‐smokers. The mutation spectrum results showed that epidermal growth factor receptor (EGFR) gene mutation was the most frequent mutation (68.75%), followed by TP53 mutation (45.83%); 19del accounted for the largest proportion of EGFR mutations. Copy number variation (CNV) mutation spectrum results showed that EGFR amplification was more common in the metastatic group than the non‐metastatic group. The mutant‐allele tumor heterogeneity value of the metastatic group was higher than that of the non‐metastatic group (p = 0.013). The progression‐free survival of the metastatic group was significantly shorter than that in the non‐metastatic group (p = 0.041). Single nucleotide variant difference analysis showed that the frequency of TP53 mutations was higher in the metastasis group. The number of subclonal mutations in the primary and metastatic lesions in the metastasis group was significantly different; the number of subclonal sites in metastatic lesions was higher than that in primary lesions. Our results suggested that the gene mutations of NSCLC in primary and metastatic lesions and identified specific mutations related to metastasis of NSCLC. Our research will help to clarify key differences between gene mutations between primary and metastatic NSCLC. These findings will help to provide new theoretical support for the future targeted therapy of metastatic NSCLC.