e16452 Background: Non-invasive tools with high sensitivity to distinguish pancreatic cancer from benign pancreatic lesions are limited. Cell-free DNA (cfDNA) offers promise for early detection but is constrained by low DNA input and separated workflows. We developed SPIRAL (Single-Portion Input Resourceful Assay for Liquid Biopsy), which enables simultaneous genomic and epigenetic library construction from a single cfDNA sample. We evaluated a methylation-based assay using SPIRAL platform in patients with pancreatic lesions. Methods: In the prospective DAYBREAK Study (NCT05495685), peripheral blood samples were collected from patients with pancreatic cancer (n = 67) and benign pancreatic lesions (n = 31). cfDNA methylation was analyzed using the STELLA algorithm. Sensitivity and specificity were assessed overall and by disease stage and histology. Results: Overall sensitivity was 70% (48/67) with a specificity of 87% (27/31). Sensitivity increased by stage: 63% in stage I (12/19), 70% in stage II (23/33), 80% in stage III (8/10), and 100% in stage IV (5/5). Sensitivity was 79% (41/52) for pancreatic ductal adenocarcinoma and 30% (3/10) for pancreatic neuroendocrine tumors. Conclusions: A methylation-based cfDNA assay using the SPIRAL platform distinguishes malignant from benign pancreatic lesions. Ongoing studies integrating mutation and methylation features and expanding sample size may improve the performance. Clinical trial information: NCT05495685 .

Combined immune checkpoint inhibitor (ICI) and chemotherapy is the standard first-line treatment for advanced non-small cell lung cancer (NSCLC) without targetable driver mutations. However, reliable biomarkers predictive of clinical benefit are lacking. We analyzed 54 patients with advanced NSCLC treated first-line with ICI plus chemotherapy. Pretreatment tumor biopsies underwent targeted DNA and RNA-seq. Plasma samples for circulating tumor DNA (ctDNA) profiling were collected at multiple timepoints. Associations between genomic, transcriptomic, and ctDNA features and objective response rate (ORR) and progression-free survival (PFS) were assessed. Genomic analysis revealed that LRP1B mutations predicted longer PFS (HR = 0.35, p = 0.034), whereas FBXW7 mutations were associated with shorter PFS (HR = 5.39, p = 0.001). Exploratory transcriptomic analysis identified high SPP1 and LHCGR expression as predictors of poor prognosis, while high IL24 expression correlated with longer PFS. CD8(+) effector memory T-cell infiltration was significantly higher in responders (p = 0.029). Longitudinal ctDNA analysis showed that positivity at C2D1 (HR = 5.18, p = 0.004), C3D1 (HR = 17.81, p < 0.001), and C4D1(HR = 3.91, p = 0.018) was associated with inferior PFS. This multi-omics analysis highlights the potential of integrating genomic, transcriptomic, and ctDNA biomarkers to optimize immunotherapy strategies in NSCLC.

3590 Background: Immune checkpoint inhibitors (ICIs) targeting PD-1 have significantly improved outcomes in locally advanced rectal cancer (LARC). Circulating tumor DNA (ctDNA) has emerged as a promising biomarker for treatment response monitoring and surveillance, capable of predicting long-term clinical outcomes. However, whether ctDNA serves as a prognostic predictor in patients with proficient mismatch repair (pMMR) LARC receiving neoadjuvant chemoradiotherapy (CRT) combined with immunotherapy remains to be explored. Methods: This prospective single-arm phase II trial enrolled pMMR LARC patients (cT 3-4 N 0 M 0 and cT 1-4 N 1-2 M 0 ) with an intermediate or high immunoscore (IS B ) (NCT05450029). Treatment-naïve patients received radiotherapy (50 Gy/ 25 fractions), 6 cycles of mFOLFOX6, and 5 cycles of sintilimab, followed by total mesorectal excision (TME) 6-8 weeks post-radiotherapy. Next-generation sequencing was used to evaluate baseline tumor tissue DNA and serial ctDNA dynamic changes. Baseline (T0) maximal somatic variant allelic frequency (maxVAF) was assessed, along with its changes at the first (T1, two cycles after therapy) and second clinical evaluation (T2, four cycles after therapy). Results: Tumor somatic mutations and aligned ctDNA analyses were conducted in 43 patients. The pathologic complete response (pCR) rate was 65.2%, with an objective response rate (ORR) of 93.5%. The R0 resection rate was 97.8%. The 3-year event-free survival rate, and 3-year overall survival rate remain immature. For dynamic ctDNA analysis, 37 patients were assessed using a 950-gene panel relevant to cancer. A significant decline in maxVAF from T0 to T1 was observed in the pCR group. Among patients who reached follow-up endpoints, circulating tumor DNA analysis reveals that patients with a > 50% decline in maxVAF from T0 to T1 demonstrated longer survival outcomes and higher response rates compared to those without such decline. Conclusions: Serial ctDNA analysis demonstrates applicability in predicting treatment response to CRT combined with immunotherapy in pMMR LARC. Dynamic changes in circulating ctDNA may serve as a potential prognostic indicator in this population. Future studies should integrate ctDNA profiling of comprehensive cancer-related gene panels with large-scale clinical data to refine patient stratification and optimize therapeutic management. Clinical trial information: NCT05450029 .

e22511 Background: Early detection of breast cancer is critical for improving patient outcomes. Liquid biopsy using circulating free DNA (cfDNA) offers a promising non-invasive approach in early detection. However, in practice, low quantity of cfDNA in blood frequently restrict comprehensive multi-analyte profiling. The SPIRAL platform allows both genomic and DNA methylation sequencing libraries to be prepared from a single low-input cfDNA sample. Here, we report the initial clinical performance of methylation sequencing via SPIRAL platform in differentiating between benign and malignant breast lesions in PERCEIVE-BREAST (NCT06979921) study. Methods: We analyzed plasma from 93 prospectively collected samples from the PERCEIVE-BREAST study, including 78 patients with breast cancer (75 invasive, 3 in situ carcinomas) and 15 with benign diseases. All samples were tested using the SPIRAL platform. The STELLA methylation analysis method was employed to determine sensitivity and specificity. Performance was also analyzed by cancer stage and molecular subtype. Results: For breast cancer detection, the sensitivity was 62% (48/78) and the specificity was 100% (15/15). Sensitivity increased with disease stage: 43% (12/28) in Stage I, 57% (16/28) in Stage II, 92% (12/13) in Stage III, and 100% (6/6) in Stage IV. For Stage 0, the sensitivity was 67% (2/3). By molecular subtype, the sensitivities were 44% (20/45) for HR+/HER2−, 88% (21/24) for HER2+, and 86% (6/7) for triple-negative breast cancer (TNBC). Conclusions: The SPIRAL assay demonstrated high specificity and clinically relevant sensitivity for detecting breast cancer using cfDNA from plasma, with performance improving at more advanced stages. These data support the utility of the SPIRAL platform for multi-modal liquid biopsy. Future work will integrate mutation detection with methylation analysis to evaluate whether this combination improves sensitivity. We also plan to validate these findings in a larger cohort to assess the assay's potential for early detection and prognosis in breast cancer. Clinical trial information: NCT06979921 .

BackgroundWhile various PD-1 inhibitors are approved for advanced non-small cell lung cancer (NSCLC), direct comparisons of their real-world efficacy and safety are limited. This retrospective study evaluates four PD-1 inhibitors (pembrolizumab, sintilimab, tislelizumab, and camrelizumab) in real-world clinical practice to supplement registered clinical trial (RCT) data.MethodsWe retrospectively screened 563 patients with stage IIIB-IV NSCLC who received pembrolizumab, sintilimab, tislelizumab, or camrelizumab (as monotherapy or combined with chemotherapy) between February 1, 2019, and December 31, 2022. The follow-up cutoff date was September 15, 2023.ResultsA total of 409 eligible patients were included (pembrolizumab: 136; sintilimab: 115; tislelizumab: 123; camrelizumab: 35). For squamous NSCLC, no significant differences in effectiveness were observed among the four inhibitors, regardless of the treatment line. For nonsquamous NSCLC, second or later-line camrelizumab was associated with a higher risk of disease progression (progression-free survival [PFS] hazard ratio [HR] 2.29; 95% CI 1.01-5.19) and mortality (overall survival [OS] HR 3.14; 95% CI 1.28-7.74) compared to pembrolizumab, despite similar objective response rates. Regarding safety, immune-related adverse events (irAEs) were consistent and manageable. Notably, sintilimab demonstrated a significantly lower incidence of irAEs than the other three inhibitors across any grade (P = 0.004) and grades 3-5 (P = 0.005).ConclusionsIn this real-world, single-center retrospective study, sintilimab and tislelizumab showed efficacy estimates broadly similar to pembrolizumab in advanced NSCLC within prespecified strata. Camrelizumab was associated with shorter PFS/OS in previously treated nonsquamous NSCLC; however, this finding is hypothesis-generating given small subgroup sizes, treatment heterogeneity, and potential residual confounding. Overall irAEs were manageable; differences in recorded incidence should be interpreted cautiously.

This consensus by the CSCO Pancreatic Cancer Expert Committee establishes evidence-based guidelines for molecular testing in pancreatic ductal adenocarcinoma. It details recommendations for biomarkers (e.g., KRAS, BRCA, MSI), liquid biopsy, and precision imaging to direct targeted therapies and immunotherapy, aiming to standardize diagnosis and optimize individualized patient care. Pancreatic ductal adenocarcinoma (PDAC) is the most common pathological type of primary pancreatic malignancy, accounting for ~95% of cases and generally referred to as pancreatic cancer [1]. Its prognosis is extremely poor and its incidence continues to rise [2]. According to the most recent global cancer statistics, the incidence of pancreatic cancer ranks 12th among all cancers, and its mortality ranks 6th, making it one of the deadliest malignancies worldwide [3]. Approximately 57% of patients have metastatic disease at diagnosis and require systemic therapy, for which chemotherapy remains the standard first-line option [1]. However, the overall response rate to currently available systemic regimens is low, and the 5-year survival rate for patients with metastatic disease remains below 5% [3]. Although most pancreatic cancers harbor canonical driver mutations, they exhibit marked heterogeneity at the molecular level. Whole-genome sequencing (WGS) and integrative genomic analyses have identified molecular subtypes of PDAC with potential clinical relevance [4-9]. With the increasing implementation of precision oncology, the Chinese Society of Clinical Oncology (CSCO) Guidelines for the Diagnosis and Treatment of Pancreatic Cancer give a level 1 recommendation to perform genetic and other molecular testing on tissue or cytologic specimens as part of the pathological diagnostic work-up, in order to guide individualized treatment, including targeted therapy and immunotherapy [10]. To further promote the use of genetic and molecular testing in the precision treatment of pancreatic cancer, the CSCO Pancreatic Cancer Expert Committee convened a multidisciplinary panel to develop the present Chinese Expert Consensus on Precision Testing and Molecular Diagnosis of Pancreatic Cancer (2025), aiming to provide clinicians with an authoritative reference for precision diagnostics and treatment decision-making.

Neurotrophic tyrosine receptor kinase (NTRK) fusions are crucial in tumorigenesis and in guiding targeted therapy with TRK inhibitors. However, their rarity, fusion heterogeneity, and limitations of conventional pan-TRK immunohistochemistry (IHC) impede accurate clinical detection. This multicenter retrospective study analyzed 374 next-generation sequencing/fluorescence in situ hybridization-validated samples (195 NTRK positive and 179 NTRK negative) collected from 12 Chinese centers to investigate fusion heterogeneity and refine the interpretation of pan-TRK IHC. We developed an amplification protocol by combining the traditional pan-TRK IHC (EPR17341) with the OptiView Amplification Kit and established new interpretation criteria. A total of 40 solid tumor types were included, and 23 unique fusion partners were identified. Papillary thyroid cancer was the most common NTRK-positive tumor (49.74%) and harbored all 3 NTRK subtypes. Among NTRK-positive samples, NTRK3 (74.87%) was the most prevalent subtype, followed by NTRK1 (23.59%). ETS variant transcription factor 6 (ETV6) was the most frequent fusion partner identified in 122 of 195 cases. It was uniquely shared across all 3 NTRK subtypes, with its fusion to NTRK1 being reported for the first time. NTRK1 and NTRK3 exhibited marked fusion partner specificity, with no overlap in their associated partners except for ETV6. The optimized pan-TRK IHC protocol significantly improved staining efficiency by enhancing intensity and clarity. Consequently, the newly established criteria (cytoplasmic intensity ≥1 in ≥50% of tumor cells or any nuclear intensity ≥1) exhibited outstanding detection performance, achieving an overall sensitivity of 94.36% and increasing specificity to 79.89% compared with 60.22% under the conventional protocol. Particularly, the detection sensitivity for NTRK3 fusions was significantly enhanced and reached 95.89%. This study contributes to clarifying NTRK fusion distribution in patients and validates a standardized, sensitive pan-TRK IHC strategy for clinical screening.

Abstract Introduction: Genomic and epigenomic alterations serve as key biomarkers for tumor detection, yet conventional approaches require separate workflows and DNA inputs for each signal type, which is impractical when sample quantity is limited. Because tumor initiation and progression arise from coordinated genomic and epigenomic dysregulation, simultaneous assessment of both layers is critical for accurate tumor classification, mechanistic insight, and clinical decision-making. Genomic profiling captures oncogenic drivers such as mutations, copy number alterations, and fusions, whereas epigenomic features—particularly DNA methylation—provide complementary information on histological subtype, gene regulation, prognosis, and tumor burden. To address the limitations of segregated assays, we developed SPIRAL (Single Portion Input Resourceful Assay of Liquid biopsy), an integrated multi-omics method that generates genomic and methylation libraries from the same DNA aliquot. Here, we present a preliminary evaluation of SPIRAL in a liquid biopsy setting, demonstrating its ability to concurrently profile genomic and epigenomic signals from a single blood-derived DNA sample. Results: Analytical validation of SPIRAL was conducted using both cell line-derived and clinical samples. For genomic detection, the observed LoD95 was 0.8% for SNVs/indels (0.3% for hotspot variants), 0.3% for fusions, 20% tumor fraction for CNAs at six copies, 30% tumor fraction for homozygous deletions, and 1% tumor fraction for MSI. For epigenomic analysis, the LoD95 for global methylation was 5×10−5 in cell lines and 1×10−4 in clinical samples, with a limit of quantitation of 0.05% and a specificity of 98% in clinical samples. The LoD for promoter methylation was 1%. Conclusion: SPIRAL is a liquid biopsy technology capable of simultaneously profiling genomic and epigenomic alterations from the same portion of cfDNA. In addition to comprehensive genomic profiling, it enables tissue-free MRD detection, therapy-response monitoring through tumor-fraction quantification, tumor subtyping, and broader phenotypic characterization. This integrated approach provides a robust platform for biomarker discovery and has the potential to substantially advance precision cancer management. Citation Format: Lei Wang, Peiru Liu, Chaoran Zheng, Shanshan Yu, Yalan Jin, Fujun Qiu, Bo Yang, Yuan Sun, Xiaotian Wang, Shuailai Wu, Zhihong Zhang.. Analytical validation of SPIRAL: An integrated genomic and epigenomic liquid biopsy assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5316.

Background:Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) have achieved great success in the treatment of non-small cell lung cancer (NSCLC) with activating EGFR mutations. However, acquired resistance is a major obstacle to long-term disease remission in clinical practice. Mesenchymal-epithelial transition (MET) gene amplification has been identified as a key resistance mechanism to first- and second-generation EGFR-TKIs. Case Description:We report the case of a 65-year-old female patient with advanced lung adenocarcinoma (LUAD) who developed bone and adrenal gland metastases following treatment with gefitinib. Next-generation sequencing (NGS) of a biopsy specimen revealed the co-occurrence of MET amplification and EGFR exon 19 deletion mutation. The combined treatment of savolitinib and gefitinib effectively controlled the disease, resulting in a favorable long-term clinical outcome. With continued follow-up through April 2025, the patient has maintained progression-free survival (PFS) over 8 years. However, monitoring revealed the patient had grade 4 peripheral edema, and negative circulating tumor DNA (ctDNA), which necessitated a savolitinib dose reduction. Subsequent minimal residual disease (MRD) assessments and radiological scans revealed a remarkable therapeutic response with sustained efficacy. Conclusions:We report the first case of a LUAD patient with MET amplification and EGFR exon 19 deletion mutation who achieved a durable response with targeted therapy. ctDNA monitoring enabled precise dose modulation that balanced therapeutic efficacy with toxicity management. This case establishes a paradigm for chronic cancer management, demonstrating that integrating molecular diagnostics with dynamic treatment optimization can effectively convert aggressive malignancies into manageable chronic conditions while preserving the quality of life of patients.

Background:Immune checkpoint inhibitors (ICIs) have transformed cancer treatment but carry risks of rare, life-threatening immune-related adverse events, particularly myocarditis. Prognostic biomarkers and optimal management strategies for severe ICI-associated myocarditis remain poorly defined. Methods:This single-center retrospective cohort study analyzed 71 patients diagnosed with ICI-associated myocarditis among 7,157 ICI-treated individuals at a tertiary center (August 2018-August 2024). Myocarditis severity was graded per American Society of Clinical Oncology (ASCO) guidelines. Cardiac biomarkers, including NT-proBNP and troponin T, were assessed. Binary logistic regression identified predictors of mortality. Treatment protocols and immunotherapy rechallenge outcomes were evaluated. Results:Severe myocarditis (Grades 3-4) occurred in 33 patients (46.5%), with an overall mortality rate of 54.5% in this subgroup (18/33). NT-proBNP levels were significantly elevated in fatal cases versus survivors (median: 13,804 vs. 4,050 pg/mL; P < 0.001) and independently predicted mortality risk (odds ratio (OR) 4.3, 95% confidence interval (CI) [1.2-21.9]; P = 0.023). A multimodal regimen combining plasmapheresis with high-dose corticosteroids, intravenous immunoglobulin, and mycophenolate mofetil was associated with improved survival. Among nine patients rechallenged with immunotherapy, seven (77.8%) tolerated subsequent cycles without recurrent immune toxicity, while two with prior Grade 2 myocarditis experienced symptom recurrence. Discussion:Elevated NT-proBNP emerges as a critical prognostic marker for risk stratification in severe ICI-associated myocarditis. Immunotherapy rechallenge appears feasible in select patients but warrants caution in those with prior moderate-grade myocarditis. These findings advocate for biomarker-guided escalation of therapies and shared decision-making frameworks to balance oncologic efficacy with cardiovascular safety.

Esophageal submucosal gland duct adenoma (ESGDA) is an extremely rare benign tumor originating from the esophageal ducts, resembling sialadenoma papilliferum (SP) of the minor salivary glands. However, the clinicopathological and molecular features of ESGDA remain incompletely characterized. We performed a multicenter, retrospective study aimed to elucidate the clinicopathological and molecular mechanisms underlying ESGDA. From 2018 to 2024, 10 cases of ESGDA were identified across two institutions. The cohort included 2 females and 8 males, aged 51–75 years (mean: 64.1 ± 8.56 years). Comprehensive clinicopathological and molecular analyses were performed. Histologically, the tumors exhibited tubulopapillary and cystic architectures, with ducts lined by a double epithelial layer. Four cases predominantly consisted of large cystic structures mimicking esophageal cysts, while two cases displayed florid papillary and micropapillary patterns resembling adenocarcinomas. Interstitial lymphoid infiltrates and micropapillary/serrated structures were observed in all cases. Genomic analysis identified BRAF V600E mutations in 8 of the 10 tumors (80

e12604 Background: In the neoadjuvant treatment (NAT) setting, dual HER2-targeted therapy is associated with higher rates of pathologic complete response (pCR) compared to each therapy administered individually but with considerable variation between different patients. There is a pressing need for biomarkers that can predict treatment response during NAT. This study aims to evaluate the association between circulating tumor DNA (ctDNA) and response to anti-HER2 targeted therapy. Methods: In this prospective, observational study (ClinicalTrials.gov identifier, NCT06479460), we enrolled patients with HER2-positive early breast cancer who underwent NAT. Serial plasma samples before NAT (T0), at mid-therapy (T1), before surgery (T2), after surgery (T3), and paired pretreatment tumor biopsies were collected. A personalized ctDNA test was designed to detect up to 20 patient-specific mutations (from targeted sequencing of paired pretreatment tumor) by ultra-deep sequencing. Results: A total of 47 patients with HER2-positive early breast cancer were enrolled. At the time of data cutoff (13 January 2025), 31 patients (66%, 31/47) had completed NAT treatment, with 19 patients had achieved pathological complete response (pCR) and 12 did not. The median number of characterized alterations per patient was 7 (range, 1-26 alterations) for tissue samples. At T0, 35 of 47 (75%) patients were ctDNA-positive, which decreased over time (T1: 25%; T2: 4%). Additionally, ctDNA-positive at T0 was associated with greater number of positive lymph nodes ( P = 0.02) but not with any of the other clinical or pathological features analyzed. Interestingly, patients who remained ctDNA-positive at T1 were significantly more likely to have residual disease after NAT (86% non-pCR) compared to those who cleared ctDNA (17% non-pCR; P = 0.003). After NAC, 15 patients who achieved pCR were ctDNA negative (n = 16, 94%). Conclusions: Our study demonstrated that ctDNA can be used to predict tumor response to NAT in HER2-positive early breast cancer, providing information to tailor an individual’s therapeutic regimen. Clinical trial information: NCT06479460 .

Non-small cell lung cancer (NSCLC) is highly heterogeneous, leading to varied treatment responses and immune-related adverse reactions (irAEs) among patients. Habitat radiomics allows non-invasive quantitative assessment of intratumor heterogeneity (ITH). Therefore, our objective is to employ habitat radiomics techniques to develop a robust approach for predicting the efficacy of Immune checkpoint inhibitors (ICIs) and the likelihood of irAEs in advanced NSCLC patients. In this retrospective two center study, two independent cohorts of patients with NSCLC were used to develop (n = 248) and validate signatures (n = 95). After applying four kinds of machine learning algorithms to select the key preoperative CT radiomic features, we used clinical, radiomics and habitat radiomic features to develop the clinical signature, radiomics signature and habitat radiomic signature for ICIs prognostics and irAEs prediction. By combining habitat radiomic features with corresponding clinicopathologic information, the nomogram signature was constructed in the training cohort. Next, the internal validation cohort (n = 75) of patients, and the external validation cohort (n = 20) of patients treated with ICIs were included to evaluate the predictive value of the four signatures, and their predictive performance was assessed by the area under operating characteristic curve (AUC). Our study introduces a radiomic nomogram model that integrates clinical and habitat radiomic features to identify patients who may benefit from ICIs or experience irAEs. The Radiomics Nomogram model exhibited superior predictive performance in the training, validation, and external validation sets, with AUCs of 0.923, 0.817, and 0.899, respectively. This model outperformed both the Whole-tumor Radiomics Signature model (AUCs of 0.870, 0.736, and 0.626) and the Habitat Signature model (AUCs of 0.900, 0.804, and 0.808). The radiomics model focusing on tumor sub-regional habitat showed better predictive performance than the model derived from the entire tumor. Decision Curve Analysis (DCA) and calibration curves confirmed the nomogram’s effectiveness. By leveraging machine learning to predict the outcomes of ICIs, we can move closer to achieving tailored ICIs for lung cancer. This advancement will assist physicians in selecting and managing subsequent treatment strategies, thereby facilitating clinical decision-making.

Brentuximab vedotin (BV) is an antibody-drug conjugate that combines the CD30 monoclonal antibody with the microtubule-disrupting agent, monomethyl auristatin E, which induces apoptosis in the tumor cell upon its release from the conjugate. The safety and efficacy of BV have been assessed in several studies in patients with T- and B-cell lymphomas. This article reviews the currently available data on the distribution of CD30 expression in T- and B-cell lymphomas, as well as the various levels of CD30 positivity cutoff used in the literature. It also analyzes the relationship between CD30 expression levels and the clinical response to BV in clinical trials for both T- and B-cell lymphomas and investigates BV efficacy in patients with low or undetectable levels of CD30 and examines potential mechanisms by which BV exerts its effect on these patients. This review contributes to the growing evidence suggesting that CD30 expression levels do not predict the clinical benefit of BV as the drug demonstrated substantial efficacy in patients across a wide range of CD30 expression levels while suggesting that the antitumor activity was not associated with CD30 expression levels. Furthermore, the potential of BV as a targeted approach along with its mechanism of action is also summarized to explain its key role in the future treatments of lymphomas, especially for CD30-expressing lymphomas.

Background:Rearranged during transfection (RET) fusion is a key driver in non-small cell lung cancer (NSCLC). Accurate detection is essential for targeted therapeutic strategies, and its reliability varies with the diagnostic methods employed. This study systematically compares the concordance of various molecular profiling techniques for identifying RET fusions in early-stage NSCLC. Methods:This retrospective study included 40 NSCLC patients with RET fusions (RET+) identified by DNA sequencing (DNA-seq). Comprehensive detection was conducted using fluorescence in situ hybridization (FISH) and RNA sequencing (RNA-seq), incorporating both targeted RNA-seq and whole-transcriptome sequencing (WTS). Clinical and molecular features were evaluated for associations with fusion types. Results:Patients were predominantly female (67.5%) and never-smokers (87.5%), with a median age of 53 years. All patients underwent FISH, while 39 cases underwent RNA-seq, with one excluded due to RNA quality control failure. The most common fusions were KIF5B::RET and CCDC6::RET (89.7%), alongside noncanonical fusion partners such as ERC1 (5.0%) and CCDC186 (2.5%). WTS achieved a 79.5% (31/39) RET+ detection rate. Targeted RNA-seq uncovered an additional five RET+ cases missed by WTS. Concordance rates for fusion detection were 92.3% between DNA-seq and RNA-seq, 84.6% between RNA-seq and FISH, and 82.5% between DNA-seq and FISH. Interestingly, patients exhibiting nonreciprocal RET translocations were significantly younger (P=0.03) and presented a lower Ki67 proliferation index (P=0.03). Conclusions:Our findings underscore the complexity of RET fusion characterization and the necessity for a comprehensive diagnostic approach, which enhances the identification of both canonical and noncanonical alterations. This supports precision oncology initiatives aimed at optimizing treatment outcomes for RET+ NSCLC patients.