Mutations In Tumor Tissue Research Articles

Concordance of ctDNA and tissue genomic profiling in advanced biliary tract cancer

Background & AimsRecent advances in molecular profiling enable the identification of potential therapeutic targets for biliary tract cancer (BTC). However, in patients with BTC, molecular profiling is hindered by challenges in obtaining adequate tissue samples. Circulating tumor DNA (ctDNA) may offer an alternative to tissue-based analysis. Here we aimed to assess the concordance between ctDNA and tissue genomic profiling in a large cohort of Asian patients with advanced BTC, and to evaluate the feasibility of liquid biopsy in BTC treatment. MethodsThis study included patients with systemic treatment-naive advanced BTC, treated at CHA Bundang Medical Center between January 2019 and December 2022. We enrolled patients with available baseline tissue-based next-generation sequencing (NGS), and sufficient plasma samples for ctDNA analysis (AlphaLiquid®100 from IMBdx). ResultsAmong 102 enrolled patients, 49.0% had intrahepatic cholangiocarcinoma, 26.5% extrahepatic cholangiocarcinoma, and 24.5% gallbladder cancer. The concordance between intra-patient ctDNA and tumor tissue mutations revealed a sensitivity of 84.8%, and positive predictive value of 79.4%. ctDNA revealed targetable alterations in 34.3% of patients—including FGFR2 fusion, IDH1 mutation, microsatellite instability (MSI)-high, ERBB2 amplification, PIK3CA mutations, BRCA1/2 mutations, and MET amplification. Notably, a novel FGFR2-TNS1 fusion was identified in ctDNA, which was not targeted in the tissue NGS panel. A high maximum somatic variant allele frequency in ctDNA was associated with poor prognosis after gemcitabine/cisplatin-based chemotherapy, in terms of both overall survival (p = 6.9 × 10−6) and progression-free survival (p = 3.8 × 10−7). ConclusionsAmong patients with advanced BTC, ctDNA-based genotyping showed acceptable concordance with tissue genomic profiling. Liquid biopsy using ctDNA could be a valuable complement to tissue-based genomic analysis in BTC. Impact and implicationsOur study is the first large-scale investigation of the clinical utility of liquid biopsy, focusing on ctDNA, as an alternative to conventional tumor tissue analysis, among Asian patients with advanced BTC. The results demonstrated acceptable concordance between analysis of ctDNA versus tissue for identifying therapeutic targets and potentially actionable genetic alterations. This indicates that ctDNA analysis can provide critical insights regarding advanced BTC treatment, particularly in cases where it is challenging to obtain or analyze tumor tissue.

Comparing baseline VAF in circulating tumor DNA and tumor tissues predicting prognosis of patients with colorectal cancer liver metastases after curative resection

IntroductionThe prognostic value of baseline variant allele frequency (VAF) in circulating tumor DNA (ctDNA) of colorectal cancer liver metastases (CRLM) patients after curative resection was rarely investigated. MethodsA single-center prospective study was performed to investigate the prognostic impact of baseline VAF in ctDNA and matched tumor tissues of CRLM patients after curative resection between May 2019 and May 2021 by the Illumina NovoSeq 6000 platform. The relationship of the tumor burden score (TBS) and the VAF in ctDNA and matched tumor tissues was evaluated by the Pearson correlation method. The survival curves of recurrence-free survival (RFS) and overall survival (OS) were plotted. Factors associated with RFS were calculated using Cox regression analysis, and an integrated prognostic model using significant baseline variables was proposed. ResultsThere were 121 patients with baseline ctDNA and matched tumor tissues enrolled in the study. A total of 417 mutations spanning 20 genes were identified in baseline tumor tissues of 119/121 (98.3 %) cases. The overall mutations in tumor tissues were completely covered by ctDNA in 52 of 121(43.0 %) patients. Baseline VAF in ctDNA but not in tumor tissues was significantly correlated to TBS of CRLM (R = 0.36, p < 0.001). Significantly longer RFS but not OS was observed in patients with lower VAF in ctDNA compared to those with higher one (p < 0.001 and p = 0.33 respectively). Multivariate Cox regression analysis showed higher VAF in baseline ctDNA was an independent risk factor for RFS. An integrated prognostic model including baseline metastasis location and VAF in ctDNA outperformed the traditional CRS model in predicting RFS. ConclusionBaseline VAF in ctDNA but not in tumor tissues influenced RFS of CRLM patients after curative resection.

Development of custom amplicon-based test systems for detecting KRAS, NRAS, and BRAF gene mutations in colorectal cancer using next-generation sequencing (NGS).

e15061 Background: Polymerase chain reaction (PCR)-based mutation diagnostics in colorectal cancer is widely available. However, PCR has limitations in identifying rare mutations and precise mutation coordinates. Targeted sequencing, though promising, often incurs high costs and longer research durations, demanding higher DNA sample quality. Our study demonstrates the benefits of using a customized amplicon test system for diagnosing somatic mutations in tumor tissue, including detecting rare mutations not captured by PCR, requiring less input biomaterial, and similar research costs. Methods: A targeted NGS assay system was developed using region-specific primers for KRAS (exons 2, 3, and 4), NRAS (exons 2, 3, and 4), and BRAF (exon 15). Library preparation involved a three-step PCR process: Target PCR incorporating Illumina overhang adapter sequences, Index PCR introducing multiplexing indices and sequencing adapters, and PCR cleanup using magnetic beads. Sequencing utilized MiSeq v2 reagents with paired 300‐bp reads, generating high-quality, full-length reads. Validation included re-testing known samples. Tumor cell percentages in FFPE specimens were determined, and DNA was extracted using the QIAamp DNA FFPE Tissue Kit. Data analysis utilized proprietary software. Results: Out of 1234 samples, 1133 were tested, with mutations detected in 685 samples (60.45%). KRAS mutations were found in 548 (48.36%), NRAS mutations in 40 (3.5%), and BRAF mutations in 88 (7.7%) samples. Double mutations were observed in 9 samples (0.79%). Data on rare and double mutations are presented in the table. Conclusions: The developed test system accurately identifies essential mutations in colorectal cancer, including rare and potentially clinically significant variants. The required DNA amount ranged from 10 to 30 ng in 5 μl, nearly ten times less than well-known commercial PCR test systems like COBAS (Roche) and therascreen (QIAGEN). The cost of the study is comparable to PCR with a minimum study duration of 4 days. [Table: see text]

Abstract A001: Urine-based liquid biopsy test for the detection of residual bladder cancer in radical cystectomy patients

Abstract One commonly employed management strategy for muscle-invasive bladder cancer (MIBC) is neoadjuvant chemotherapy followed by radical cystectomy (RC) and urinary diversion. Approximately 35% of patients achieve a pathologic complete response (pCR) when evaluated from RC specimens. RC with urinary diversion is a life-altering operation with high complication rates and therefore determining who are complete responders would improve quality of life and could decrease costs and still achieve a high cure rate via RC avoidance. Unfortunately, current methods cannot accurately identify complete responders. Our lab has developed a urine-based liquid biopsy test in which DNA isolated from urine and germline is isolated and subjected to panel exome sequencing for enumeration of somatic variants. This occurs under the premise that tumor DNA identified (as mutant alleles) is a marker of residual disease while the absence of tumor DNA (meaning the absence of mutant alleles) can indicate a pCR. A 60 gene panel was designed to be used for exonic sequencing of urinary DNA (uDNA) to identify mutations indicative of bladder cancer or therapeutic targets. We have shown that most mutations in tumor tissue (Mt) are detectable as mutations in urine (Mu). We also showed that the presence or absence of residual Mu after completion of chemotherapy strongly associates with residual disease or pCR at the time of RC, respectively. Therefore, this test could be used after neoadjuvant therapy to better identify patients for RC avoidance. We optimized this test by studying pre-analytical factors affecting the yield, purity, and integrity of uDNA and the quality of sequencing data using prospectively collected samples from patients with MIBC. We are also in the process of testing commercial and novel preservatives to optimize DNA integrity, yield, and mutation detection accuracy to enable temporary ambient temperature storage without compromising mutation detection, permitting shipping/centralized testing. Lastly, we are comparing two library preparation approaches. Study of these preanalytical factors has increased the number of Mu that can be detected and were applied to a cohort of patients who underwent bladder sparing curative-intent treatment in a prospective clinical trial. Studies described herein will enable liquid biopsy tests to accurately identify patients with pCR after neoadjuvant therapy and might safely avoid radical cystectomy. Citation Format: Philip H. Abbosh, Henkel Valentine, Rhea Arya. Urine-based liquid biopsy test for the detection of residual bladder cancer in radical cystectomy patients [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A001.

Abstract 3701: Urine-based liquid biopsy test for the detection of residual bladder cancer in radical cystectomy patients

Abstract The gold standard therapy for muscle-invasive bladder cancer (MIBC) is neoadjuvant chemotherapy followed by radical cystectomy (RC) and urinary diversion. Some patients may have their disease completely eradicated by chemotherapy as ~35% of patients achieve a pathologic complete response (pCR) when evaluated from RC specimens. RC with urinary diversion is a life-altering operation with high complication rates and therefore determining who are complete responders would improve quality of life and could decrease costs and still achieve a high cure rate via RC avoidance. Unfortunately, current methods cannot accurately identify complete responders. Our lab has developed a urine-based liquid biopsy test in which DNA isolated from urine and germline is isolated and subjected to panel exome sequencing for enumeration of somatic variants. This occurs under the premise that tumor DNA identified (as mutant alleles) is a marker of residual disease while the absence of tumor DNA (meaning the absence of mutant alleles) can indicate a pCR. A 55 gene panel was designed to be used for exonic sequencing of urinary DNA (uDNA) to identify mutations indicative of bladder cancer or therapeutic targets. We have shown that most mutations in tumor tissue are detectable as mutations in urine (Mu). We also showed that the presence or absence of residual Mu after completion of chemotherapy strongly associates with residual disease or pCR at the time of RC, respectively. Therefore, this test could be used after neoadjuvant therapy to better identify patients for RC avoidance. To optimize this test, we are currently evaluating pre-analytical factors affecting the yield, purity, and integrity of uDNA and the quality of sequencing data using prospectively collected samples from patients with MIBC. We are also testing commercial and novel preservatives to identify one that maintains cell-free DNA integrity, yield, and mutation detection accuracy to enable temporary ambient temperature storage without compromising mutation detection, permitting shipping/centralized testing. Lastly, we are comparing two library preparation approaches: classical hybrid capture-based (Agilent SureSelect XTHS) and amplicon+hybrid capture-based (Agilent HaloPlexHS) to determine which performs the best for identifying the variants in a sample. Our results showed that both sequencing depth and the quality bases mapped was higher when utilizing the SureSelect XTHS kit as compared to HaloPlexHS using approximately the same number of reads. This improvement may also increase the power to detect rare alleles. Studies described herein will enable liquid biopsy tests to accurately identify patients with pCR after neoadjuvant therapy and might safely avoid radical cystectomy. Citation Format: Rhea Arya, Henkel Valentine, Uttam Satyal, Raju Chelluri, Mengmeng Li, Philip Abbosh. Urine-based liquid biopsy test for the detection of residual bladder cancer in radical cystectomy patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3701.

Phase II study investigating the efficacy and safety of glesatinib (MGCD265) in patients with advanced NSCLC containing MET activating alterations

ObjectivesDysregulated signaling by mesenchymal epithelial transition factor (MET) and heightened AXL activation are implicated in the pathogenesis of non-small cell lung cancer (NSCLC). Glesatinib (MGCD265) is an investigational, oral inhibitor of MET and AXL. Materials and methodsThis open-label, Phase II study investigated glesatinib (free-base suspension [FBS] capsule 1050 mg BID or spray-dried dispersion [SDD] tablet 750 mg BID) in patients with advanced, previously treated NSCLC across four cohorts grouped according to presence of MET activating mutations or amplification in tumor or ctDNA. The primary endpoint was objective response rate (ORR). ResultsSixty-eight patients were enrolled: n = 28 and n = 8 with MET exon 14 skipping mutations in tumor tissue and ctDNA, respectively, and n = 20 and n = 12 with MET gene amplification in tumor tissue and ctDNA, respectively. Overall, ORR was 11.8 %, median progression-free survival was 4.0 months, and median overall survival was 7.0 months. Among patients with MET activating mutations, ORR was 10.7 % with tumor testing and 25.0 % with ctDNA testing. For MET amplification, responses were observed only in patients enrolled by tumor testing (ORR 15.0 %). Diarrhea (82.4 %), nausea (50.0 %), increased alanine aminotransferase (41.2 %), fatigue (38.2 %), and increased aspartate aminotransferase (36.8 %) were the most frequent adverse events assessed as related to study medication. Glesatinib exposure was similar with the SDD tablet and FBS capsule formulations. The study was terminated early by the sponsor due to modest clinical activity. ConclusionsGlesatinib had an acceptable safety profile in patients with advanced, pre-treated NSCLC with MET activating alterations. Modest clinical activity was observed, which likely reflects suboptimal drug bioavailability suggested by previously reported Phase I data, and pharmacodynamic findings of lower than anticipated increases in circulating soluble shed MET ectodomain (s-MET).

Plasma ctDNA liquid biopsy of IDH1, TERTp, and EGFRvIII mutations in glioma.

Circulating tumor DNA has emerging clinical applications in several cancers; however, previous studies have shown low sensitivity in glioma. We investigated if 3 key glioma gene mutations IDH1, TERTp, and EGFRvIII could be reliably detected in plasma by droplet digital polymerase chain reaction (ddPCR) thereby demonstrating the potential of this technique for glioma liquid biopsy. We analyzed 110 glioma patients from our biobank with a total of 359 plasma samples (median 4 samples per patient). DNA was isolated from plasma and analyzed for IDH1, TERTp, and EGFRvIII mutations using ddPCR. Total cfDNA was significantly associated with tumor grade, tumor volume, and both overall and progression-free survival for all gliomas as well as the grade 4 glioblastoma subgroup, but was not reliably associated with changes in tumor volume/progression during the patients' postoperative time course. IDH1 mutation was detected with 84% overall sensitivity across all plasma samples and 77% in the preoperative samples alone; however, IDH1 mutation plasma levels were not associated with tumor progression or survival. IDH1m plasma levels were not associated with pre- or postsurgery progression or survival. The TERTp C228T mutation was detected in the plasma ctDNA in 88% but the C250T variant in only 49% of samples. The EGFRvIII mutation was detected in plasma in 5 out of 7 patients (71%) with tissue EGFRvIII mutations in tumor tissue. Plasma ctDNA mutations detected with ddPCR provide excellent diagnostic sensitivity for IDH1, TERTp-C228T, and EGFRvIII mutations in glioma patients. Total cfDNA may also assist with prognostic information. Further studies are needed to validate these findings and the clinical role of ctDNA in glioma.

Concordance of circulating tumor DNA and matched formalin-fixed paraffin-embedded tumor tissue in gastric cancer as a predictor of recurrence.

Combined analysis of the variant composition of circulating tumor DNA (ctDNA) from cell-free plasma and DNA from tumor tissue could provide insight into the implications of the genetic alterations responsible for the intratumoral and intertumoral heterogeneity of gastric cancer. We aimed to evaluate the usefulness of this approach in these patients. Cell-free plasma and formalin-fixed paraffin-embedded tumor tissue samples from 46 patients with gastric cancer were examined. Targeted deep sequencing was performed using a commercially available kit. The cell-free DNA (cfDNA) concentration was higher in stage II-IV versus stage I patients and in larger versus smaller tumors. Only 12 of the 36 (33.3%) alterations in the tumor tissue samples were in concordance with those in the ctDNA samples. Two variants were in concordance in stage I samples and 10 in stage II-IV samples. Actionable variants that were detected in concordance were in the stage II-IV samples. Preoperative ctDNA positivity of actionable variants was significantly associated with cfDNA concentration, lymphatic invasion, N stage, and TNM stage. Cancer recurrence was significantly associated with tumor size, lymphatic/vascular invasion, TNM stage, and ctDNA-tumor tissue variant concordance. Preoperative ctDNA genetic analysis using a multigene panel offers substantial clinical benefits when performed in conjunction with targeted deep sequencing of tumor tissue. Concordance between preoperative ctDNA and tumor tissue mutations may serve as a prognostic indicator in patients with gastric cancer.

Mutational Signature Changes in Patients With Metastatic Squamous Cell Carcinoma of the Anal Canal.

We examined the concordance of genetic mutations between pretreatment tumor tissue and posttreatment circulating tumor DNA (ctDNA) in patients with metastatic squamous cell carcinoma of the anal canal (SCCA) and assessed the impact of therapy on this concordance. We analyzed next-generation sequencing reports from pretreatment tumor tissue and posttreatment ctDNA in 11 patients with metastatic SCCA treated at Vanderbilt University Medical Center between 2017 and 2021. Among the mutations identified in posttreatment ctDNA, 34.5% were also found in pretreatment tumor tissue, while 47.6% of pretreatment tumor tissue mutations were found in posttreatment ctDNA. Four patients had preservation of potentially actionable mutations in both pretreatment tissue and posttreatment ctDNA, while 7 patients had newly identified mutations in posttreatment ctDNA that were not present in pretreatment tumor tissue. Patients with SCCA demonstrate a high degree of temporal mutational heterogeneity. This supports the hypothesis that ctDNA can serve as a real-time tracking mechanism for solid tumors' molecular evolution in response to therapy. Our findings highlight the potential of ctDNA in identifying emerging actionable mutations, supplementing information from tissue-based genomic assessments. Further research, ideally with larger and multi-institutional cohorts, is needed to validate our findings in this relatively rare tumor type.

Predicting therapeutic responses in head and neck squamous cell carcinoma from TP53 mutation detected by cell-free DNA.

Head and neck squamous cell carcinoma (HNSCC) is an epithelial malignant tumor originating from the oral cavity, oropharynx, nasal cavity, sinuses, nasopharynx, hypopharynx, or larynx. Mutations in TP53 are the most common of all somatic genomic changes in HNSCC, and TP53 mutations are associated with the response to immunotherapy and chemotherapy. Tumor-derived circulating cell-free DNA (cfDNA) is a minimally invasive method to determining genetic alterations in cancer. This study aimed to explore the therapeutic responses of patients with HNSCC with TP53 mutation and the accuracy of cfDNA for detecting TP53 mutation. Information on TP53 mutations, patient survival time, and clinical data in HNSCC were downloaded from The Cancer Genome Atlas database. The difference in immune infiltration between the TP53-mutant group and the wild-type group was compared. We applied the single-sample gene set enrichment analysis method on the transcriptome of HNSCC samples to assess the distribution of immune cell types between the two groups. The chemotherapy response was constructed using the R software package, "pRRophetic". Gene set enrichment analysis was performed based on the TP53 mutation. The next-generation sequencing was executed on cfDNA from nine patients with HNSCC to detect genetic alterations. Tumor biopsy (n=9) was sequenced using the same technique. TP53 was the most frequently mutated gene in HNSCC. The TP53 mutation was related to immune cells and the expression of immune-associated genes. The TP53 mutation group showed lower response to immunotherapy but high sensitivity to some chemotherapies compared with the wild-type group. TP53 was the most frequently mutated gene (6/9; 66.67%) in cfDNA. Only 27.27% of TP53 mutations in tumor tissue were detected outside of cfDNA. TP53 mutation could be used as a specific predictor of treatment response in patients with HNSCC. Using cfDNA to detect the TP53 mutations in patients with HSNCC is a feasible method. The results suggested that the therapeutic response in patients could be predicted by detecting TP53 mutations in cfDNA, and large-scale and prospective studies are needed to validate this hypothesis.

Dynamic Monitoring of Circulating Tumor DNA in Patients With Metastatic Colorectal Cancer.

Plasma circulating tumor DNA (ctDNA) is a valuable resource for tumor characterization and for monitoring of residual disease during treatment; however, it is not yet introduced in metastatic colorectal cancer (mCRC) routine clinical practice. In this retrospective exploratory study, we evaluated the role of ctDNA in patients with mCRC treated with chemotherapy plus bevacizumab. Fifty-three patients were characterized for RAS and BRAF status on tumor tissue before the start of treatment. Plasma was collected at baseline, at first clinical evaluation, and at disease progression. ctDNA analysis was performed using Oncomine Colon cfDNA Assay on the Ion S5 XL instrument. At baseline, from a plasma sample, RAS, BRAF, or PIK3CA mutations were detected in 44 patients. A high correspondence was observed between ctDNA and tumor tissue mutations (KRAS 100%, NRAS 97.9%, BRAF 97.9%, PIK3CA 90%). Low baseline variant allele frequency (VAF) was found to be associated with longer median progression-free survival (PFS) compared with those with high VAF (15.9 v 12.2 months, P = .02). A higher PFS {12.29 months (95% CI, 9.03 to 17.9) v 8.15 months (95% CI, 2.76 to not available [NA]), P = .04} and overall survival (34.1 months [95% CI, 21.68 to NA] v 11.1 months [95% CI, 3.71 to NA], P = .003) were observed in patients with large decline in VAF at first evaluation. ctDNA analysis is useful for molecular characterization and tumor response monitoring in patients with mCRC. Quantitative variations of released ctDNA are associated with clinical outcomes.

Detection and Monitoring of Tumor-Derived Mutations in Circulating Tumor DNA Using the UltraSEEK Lung Panel on the MassARRAY System in Metastatic Non-Small Cell Lung Cancer Patients.

Analysis of circulating tumor DNA (ctDNA) is a potential minimally invasive molecular tool to guide treatment decision-making and disease monitoring. A suitable diagnostic-grade platform is required for the detection of tumor-specific mutations with high sensitivity in the circulating cell-free DNA (ccfDNA) of cancer patients. In this multicenter study, the ccfDNA of 72 patients treated for advanced-stage non-small cell lung cancer (NSCLC) was evaluated using the UltraSEEK® Lung Panel on the MassARRAY® System, covering 73 hotspot mutations in EGFR, KRAS, BRAF, ERBB2, and PIK3CA against mutation-specific droplet digital PCR (ddPCR) and routine tumor tissue NGS. Variant detection accuracy at primary diagnosis and during disease progression, and ctDNA dynamics as a marker of treatment efficacy, were analyzed. A multicenter evaluation using reference material demonstrated an overall detection rate of over 90% for variant allele frequencies (VAFs) > 0.5%, irrespective of ccfDNA input. A comparison of UltraSEEK® and ddPCR analyses revealed a 90% concordance. An 80% concordance between therapeutically targetable mutations detected in tumor tissue NGS and ccfDNA UltraSEEK® analysis at baseline was observed. Nine of 84 (11%) tumor tissue mutations were not covered by UltraSEEK®. A decrease in ctDNA levels at 4-6 weeks after treatment initiation detected with UltraSEEK® correlated with prolonged median PFS (46 vs. 6 weeks; p < 0.05) and OS (145 vs. 30 weeks; p < 0.01). Using plasma-derived ccfDNA, the UltraSEEK® Lung Panel with a mid-density set of the most common predictive markers for NSCLC is an alternative tool to detect mutations both at diagnosis and during disease progression and to monitor treatment response.

Circulating Tumor DNA as a Minimal Residual Disease Assessment and Recurrence Risk in Patients Undergoing Curative-Intent Resection with or without Adjuvant Chemotherapy in Colorectal Cancer: A Systematic Review and Meta-Analysis.

Emerging data have suggested that circulating tumor DNA (ctDNA) can be a reliable biomarker for minimal residual disease (MRD) in CRC patients. Recent studies have shown that the ability to detect MRD using ctDNA assay after curative-intent surgery will change how to assess the recurrence risk and patient selection for adjuvant chemotherapy. We performed a meta-analysis of post-operative ctDNA in stage I-IV (oligometastatic) CRC patients after curative-intent resection. We included 23 studies representing 3568 patients with evaluable ctDNA in CRC patient post-curative-intent surgery. Data were extracted from each study to perform a meta-analysis using RevMan 5.4. software. Subsequent subgroup analysis was performed for stages I-III and oligometastatic stage IV CRC patients. Results showed that the pooled hazard ratio (HR) for recurrence-free survival (RFS) in post-surgical ctDNA-positive versus -negative patients in all stages was 7.27 (95% CI 5.49-9.62), p < 0.00001. Subgroup analysis revealed pooled HRs of 8.14 (95% CI 5.60-11.82) and 4.83 (95% CI 3.64-6.39) for stages I-III and IV CRC, respectively. The pooled HR for RFS in post-adjuvant chemotherapy ctDNA-positive versus -negative patients in all stages was 10.59 (95% CI 5.59-20.06), p < 0.00001. Circulating tumor DNA (ctDNA) analysis has revolutionized non-invasive cancer diagnostics and monitoring, with two primary forms of analysis emerging: tumor-informed techniques and tumor-agnostic or tumor-naive techniques. Tumor-informed methods involve the initial identification of somatic mutations in tumor tissue, followed by the targeted sequencing of plasma DNA using a personalized assay. In contrast, the tumor-agnostic approach performs ctDNA analysis without prior knowledge of the patient's tumor tissue molecular profile. This review highlights the distinctive features and implications of each approach. Tumor-informed techniques enable the precise monitoring of known tumor-specific mutations, leveraging the sensitivity and specificity of ctDNA detection. Conversely, the tumor-agnostic approach allows for a broader genetic and epigenetic analysis, potentially revealing novel alterations and enhancing our understanding of tumor heterogeneity. Both approaches have significant implications for personalized medicine and improved patient outcomes in the field of oncology. The subgroup analysis based on the ctDNA method showed pooled HRs of 8.66 (95% CI 6.38-11.75) and 3.76 (95% CI 2.58-5.48) for tumor-informed and tumor-agnostic, respectively. Our analysis emphasizes that post-operative ctDNA is a strong prognostic marker of RFS. Based on our results, ctDNA can be a significant and independent predictor of RFS. This real-time assessment of treatment benefits using ctDNA can be used as a surrogate endpoint for the development of novel drugs in the adjuvant setting.

The insights of mutations of gynecological oncology by liquid biopsy for Chinese patients.

e17501 Background: Liquid biopsy drew dramatic attention in the field of cancer research because of its attribution of nontraumatic, easy to obtain, providing overall state of tumor, allowing for real-time monitoring and so on. However, the transformation from research to application of liquid biopsy technique in clinical gynecological oncology management require more evidence and further investigation. Methods: Blood samples of 104 patients were collected, of whom 33 were diagnosed as cervical cancer, 20 were diagnosed as endometrial cancer, and 51 were diagnosed as ovarian cancer. cfDNA were extracted from blood and examined by next-generation sequencing technique with Haplox HapOnco panel. cfDNA mutations were analyzed and compared with genomic mutations in tumor tissues in publications. Results: In cfDNA, there were 120 genes mutated in cervical cancer patients, 150 genes mutated in endometrial cancer patients, and 58 genes mutated in ovarian cancer patients. Detecting by cfDNA, the average number of mutations was 6 (4.1 to 7.9, 95% CI) for each cervical cancer patient, 8.1 (2.1 to 14.1, 95% CI) for each endometrial cancer patient, and 5.1 (3.7 to 6.5, 95% CI) for each ovarian cancer patient. As the mutations in cfDNA compared with those in tumor tissues, the results revealed that there was no significant difference in mutation rate for 94.2% genes in blood and tumor tissues for cervical cancer patients, 93.3% for endometrial cancer patients, and 98.3% for ovarian cancer patients. Besides, there were no significant differences in microsatellite instability rates in blood and tumor tissues for cervical cancer patients and endometrial cancer patients. Conclusions: Most mutated genes in tumor tissues were detected in blood for gynecological oncology patients. The mutation rate for most genes and microsatellite instability rate had no significant difference in blood and tumor tissues, which suggested that liquid biopsy using cfDNA is helpful in gynecological oncology management.

Clinical and molecular Characterization of NSCLCs with MET exon 14 skipping mutation coexist MET amplification.

e20506 Background: MET exon 14 skipping mutation (METex14) and MET amplification are the most prevalent molecular alterations associated with MET carcinogenesis in non-small cell lung cancer, with respective frequencies of 3-4% and 1-5%. Recent research indicates that patients with METex14 may also carry MET amplification, and that MET-TKI therapy has a higher response rate in this patient population. We evaluated the clinical and molecular characteristics of patients with altered or co-altered MET using NGS data of non-small cell lung cancer cases from Jiangsu Simcere diagnostic Co., ltd. Methods: During 2019-2021, samples from 33074 NSCLC patients underwent comprehensive genomic profiling (CGP) using hybrid-capture technology at the Simcere diagnostic laboratory (Nanjing, China). To detect gene mutations in tumor tissue or blood samples of patients, next-generation sequencing (NGS) was performed. MET amplification was defined as MET copy number greater than 2.25. Results: MET exon 14 skipping was found in 325 pts (0.98%), MET amplification was found in 136 pts (0.41%), and the co-alter population consisted of only 36 pts (0.11%). Receptor, Donor, D1010, Y1003, and other METex14 subtypes represented 12.0%, 36.6%, 24.6%, 0.2%, and 26.2%, respectively, of the population. Then, we divided MET-altered NSCLC patients into three groups: MET14 (carrying only METex14), AMP (carrying only MET amplification), and MET14&amp;AMP (MET14 concomitant MET amplification). The differences in age, gender, TMB copy number, and MET copy number were then compared between these groups. Patients with AMP were considerably younger than those with METex14&amp;AMP or AMP. (MET14&amp;AMP: 69.0±9.27 vs AMP: 63.5±10.6, P = 0.032; MET14: 70.0±10.9, P &lt; 0.001). The AMP group contained significantly more male patients than the MET14 group (68.0% vs. 54.5%, P = 0.019), but there were no significant differences between the remaining groups. In contrast to tissue-based TMB, the AMP group was significantly higher than the other two (AMP:17.059.5 vs MET 14: 2.945.79; P0.001; MET14&amp;AMP: 3.650.63, P = 0.005). In spite of this, there was no statistically significant difference between the AMP and MET14 &amp; AMP groups in terms of MET copy number or percentage of MET CN &gt; = 10. Conclusions: In general, AMP pts are younger and have a higher TMB than co-alteration pts; however, there is no significant difference in MET CN between AMP and co-alteration pts; whether the coexisting group is sensitive to MET-TKI is unknown, and future research should focus on the treatment of co-alteration and AMP pts.

Correlation between MSI, TMB, and TGFBR2 gene mutation in solid tumors.

e14558 Background: TGFBR2 encodes a receptor kinase which signals to downstream effectors in the TGF-ß signaling pathway. Recent research has shown that truncation mutations of TGFBR2 in gastric cancer with high tumor mutational burden. However, the relationship remains unclear in other solid tumors. Methods: We retrospectively analyzed the TGFBR2 mutations of 19370 Chinese patients with pan-cancer during 2019-2022 (Simcere Diagnosis, Nanjing, China) Next-generation sequencing (NGS) assessed somatic mutations in tumor tissue. We figured out TGFBR2 mutation frequency, TMB and MSI in TGFBR2-mutant, TGFBR2 wild-type. Results: We have detected TGFBR2 mutants in 536 (2.8%) samples. The top 5 frequent cancers were colorectal cancer (132, 24.7%), lung cancer (118, 22%), gastric cancer (78, 14.5%), biliary tract tumors(52, 9.7%) liver cancer (33, 6.2%).TGFBR2 variants included missense/indel/insert (238, 44.4%), truncation (219, 40.9%), splicing site variant (23, 4.3%), and CNV (33, 6.2%).The inactive variants include missense, which occurs in the TGFBR2 serine/threonine kinase domain, truncation and splicing site variant. However, truncation was the most common type, recurring in a few hot spots (p.K153Sfs*35, p.K153Afs*3, p.R520* ). And we found that the TMB and MSI in the TGFBR2 mutation group was significantly higher than that in the TGFBR2 wild-type group [TMB-H (TMB ≥10 musts/Mb): TGFBR2-MUT vs TGFBR2-WT, 41.2% vs 9.5%, p &lt; 0.01; MSI-H: TGFBR2-MUT vsTGFBR2-WT, 27.4% vs1.7% p &lt; 0.01]. In the TGFBR2 mutation group, It was also observed that TMB and MSI in the TGFBR2 truncation or splicing site mutant (INACT) group was significantly higher than that in the other mutant types (VUS) group [TMB-H (TMB ≥10 musts/Mb): TGFBR2-INACT vs TGFBR2-VUS, 51.0% vs 27.9%, p &lt; 0.01; MSI-H: TGFBR2-INACT vs TGFBR2-VUS 39.4% vs 10.6% p &lt; 0.01]. And then, we separately analyzed the correlation between MSI, TMB and TGFBR2 gene mutation in the top 5 frequent cancers. Over 60% of patients with TGFBR2 mutations in colorectal cancers have high TMB and MSI values [TMB-H (TMB ≥10 musts/Mb): TGFBR2-MUT vs TGFBR2-WT, 71.3% vs 12.8%, p &lt; 0.01; MSI-H: TGFBR2-MUT vs TGFBR2-WT, 61.2% vs 3.0% p &lt; 0.01]. Conclusions: We analyzed the distribution of TGFBR2 mutants in Chinese patients with solid tumors. Our data showed that TGFBR2 mutant was significantly associated with TMB-H and MSI-H, and this may be a potential molecular marker of immunotherapy. And studies have shown that the combination of TGF-β inhibitors and immune checkpoint inhibitors can increase the killing effect of T cells on tumors. In short, detection of TGFBR2 mutation has certain clinical significance.

Abstract 1047: Plasma KRAS mutations as predictive biomarkers for pancreatic cancer in high-risk group

Abstract Pancreatic cancer (PANC) has among solid malignancies one of the lowest 5-years survival rates - less than 10%. To improve the prognosis of PANC, it is necessary to develop tools that will enable earlier diagnosis. More than 90% of PANC cases are characterized by the presence of KRAS mutations in tumor tissue that play a critical role in the initiation of PANC. Recently, attention has been focused on liquid biopsy which can better reflect the whole genetic profile of the tumor and may enable early diagnosis. Our study aims to discover whether KRAS gene mutations can be detected in the plasma isolated from the PANC risk group patients - diabetes mellitus II (DMII) and chronic pancreatitis (CP). The KRAS mutations were analyzed in 34 PANC, 89 DMII, and 31 CP patients by droplet digital PCR. Plasma cell-free DNA was investigated for three mutations hotspots: KRAS p.G12D c.35G&amp;gt;A, p.G12V c.35G&amp;gt;T, p.G12R. Detailed results of the study are currently under evaluation and will be presented during the meeting. We believe that these results discover whether plasma analysis of KRAS mutation can serve as a biomarker for early diagnosis of malignant transformation in risk group patients. Supported by grant AZV NU-21-07-00247, by Czech science Foundation 22-05942S and by the project National Institute for Cancer Research (Programme EXCELES, ID Project No. LX22NPO5102). Citation Format: Klara Cervena. Plasma KRAS mutations as predictive biomarkers for pancreatic cancer in high-risk group [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1047.

Mutation Spectrum in Liquid Versus Solid Biopsies From Patients With Advanced Gastroenteropancreatic Neuroendocrine Carcinoma.

Gastroenteropancreatic neuroendocrine carcinomas (GEP-NEC) are rare and have a poor prognosis. Most GEP-NEC are diagnosed with metastatic disease, with only minor biopsies available for molecular diagnostics. We assessed the applicability of liquid biopsies for molecular profiling of GEP-NEC. We performed massive parallel sequencing of 76 cancer-related genes in circulating tumor DNA from 50 patients with advanced GEP-NEC and compared findings to previous analyses of solid tumor biopsies from the same patients. Plasma samples were collected before therapy, and the median time span between blood and tissue sampling was 25 days. We detected 178 somatic mutations in the liquid biopsies, 127 (71%) were also detected in the solid biopsies, whereas 51 (29%) were unique to the liquid biopsies. In the same 76 genes, we previously detected 199 somatic mutations (single nucleotide variants) in solid biopsies, of which 127 (64%) were also now detected in liquid biopsies. In exploratory subgroup assessments, concordance was higher in patients with liver metastases (P = 1.5 × 10-5) and increasing with level of liver involvement (P = 1.2 × 10-4). The concordance was similar between GEP-NEC with different primary sites, except being lower in esophageal cases (P = .001). Concordance was not associated with tumor mutation burden. Tumor tissue mutations also detected in liquid biopsies was lower for MSI (40%) versus MSS tumors (70%; P = 7.8 × 10-4). We identified potentially targetable mutations in plasma of 26 (52%) of patients with GEP-NEC; nine patients (18%) had potentially targetable mutation detected only in liquid biopsies. Liquid biopsy analyses may be an applicable alternative to solid biopsies in GEP-NEC. Liquid biopsies may add additional mutations compared with tumor biopsies alone and could be useful for biomarker assessment in clinical trials for these patients.

Localization of EGFR Mutations in Non-small-cell Lung Cancer Tissues Using Mutation-specific PNA-DNA Probes.

Epidermal growth factor receptor (EGFR) signaling inhibitors are potent therapeutic agents for EGFR-mutant non-small-cell lung cancer, but the effects of such inhibitors on the localization of EGFR mutations in tumor tissues remain to be elucidated. Thus, a simple and efficient technology for the detection of mutations in tumor tissue specimens needs to be developed. Using an EGFR mutation-specific peptide nucleic acid (PNA)-DNA probe, the EGFR mutation-positive part of whole NSCLC tissues was visualized by immunofluorescence. Formalin-fixed paraffin-embedded sections obtained from A549, NCI-H1975, HCC827 and PC-9 tumors transplanted into nude mice were subjected to staining using PNA-DNA probes specific for the mRNA sequences producing the L858R, del E746-A750 and T790M mutations. The probes for the L858R mutation showed intense positive staining in H1975 cells, and the probe for the del E746-A750 mutation exhibited positive staining specifically in HCC827 and PC-9 tumors. On the other hand, A549 tumors without EGFR mutation did not show any significant staining for any PNA-DNA probe. In combination staining, the addition of cytokeratin stain increased the positive staining rate of each PNA-DNA probe. In addition, the positive staining rate of the probes for the L858R mutation was comparable to that of the antibody to EGFR L858R mutated protein. PNA-DNA probes specific for EGFR mutations might be useful tools to detect heterogeneous mutant EGFR expression in cancer tissues and efficiently evaluate the effect of EGFR signaling inhibitors on tissues of EGFR-mutant cancer.

Personalized approach in the treatment of thyroid cancer: A review

The main goal is to improve treatment outcomes of disseminated inoperable thyroid cancer through a personalized choice of targeted agents. Considering gene mutations in drug choice will further improve the survival of patients with rare mutations. Despite the small sample of patients, mutation detection, regardless of localization, and basket studies will allow future results to be obtained much faster than in a dozen years and use these results in this rare category of patients. Basket studies allow patients with rare disorders to receive personalized therapy according to mutations in tumor tissue, regardless of the affected organ.