Mutations In Tumor Tissue Research Articles

Fecal gene detection based on next generation sequencing for colorectal cancer diagnosis.

BACKGROUNDColorectal cancer (CRC) is one of the most common malignancies worldwide. Given its insidious onset, the condition often already progresses to advanced stage when symptoms occur. Thus, early diagnosis is of great significance for timely clinical intervention, efficacy enhancement, and prognostic improvement. Featuring high throughput, fastness, and rich information, next generation sequencing (NGS) can greatly shorten the detection time, which is a widely used detection technique at present.AIMTo screen specific genes or gene combinations in fecal DNA that are suitable for diagnosis and prognostic prediction of CRC, and to establish a technological platform for CRC screening, diagnosis, and efficacy monitoring through fecal DNA detection. METHODSNGS was used to sequence the stool DNA of patients with CRC, which were then compared with the genetic testing results of the stool samples of normal controls and patients with benign intestinal disease, as well as the tumor tissues of CRC patients. Specific genes or gene combinations in fecal DNA suitable for diagnosis and prognostic prediction of CRC were screened, and their significances in diagnosing CRC and predicting patients' prognosis were comprehensively evaluated. RESULTSHigh mutation frequencies of TP53, APC, and KRAS were detected in the stools and tumor tissues of CRC patients prior to surgery. Contrastively, no pathogenic mutations of the above three genes were noted in the postoperative stools, the normal controls, or the benign intestinal disease group. This indicates that tumor-specific DNA was detectable in the preoperative stools of CRC patients. The preoperative fecal expression of tumor-associated genes can reflect the gene mutations in tumor tissues to some extent. Compared to the postoperative stools and the stools in the two control groups, the pathogenic mutation frequencies of TP53 and KRAS were significantly higher for the preoperative stools (χ2 = 7.328, P < 0.05; χ2 = 4.219, P < 0.05), suggesting that fecal TP53 and KRAS genes can be used for CRC screening, diagnosis, and prognostic prediction. No significant difference in the pathogenic mutation frequency of the APC gene was found from the postoperative stools or the two control groups (χ2 = 0.878, P > 0.05), so further analysis with larger sample size is required. Among CRC patients, the pathogenic mutation sites of TP53 occurred in 16 of 27 preoperative stools, with a true positive rate of 59.26%, while the pathogenic mutation sites of KRAS occurred in 10 stools, with a true positive rate of 37.04%. The sensitivity and negative predictive values of the combined genetic testing of TP53 and KRAS were 66.67% (18/27) and 68.97%, respectively, both of which were higher than those of TP53 or KRAS mutation detection alone, suggesting that the combined genetic testing can improve the CRC detection rate. The mutation sites TP53 exon 4 A84G and EGFR exon 20 I821T (mutation start and stop positions were both 7579436 for the former, while 55249164 for the latter) were found in the preoperative stools and tumor tissues. These "undetected" mutation sites may be new types of mutations occurring during the CRC carcinogenesis and progression, which needs to be confirmed through further research. Some mutations of "unknown clinical significance" were found in such genes as TP53, PTEN, KRAS, BRAF, AKT1, and PIK3CA, whose clinical values is worthy of further exploration.CONCLUSIONNGS-based fecal genetic testing can be used as a complementary technique for the CRC diagnosis. Fecal TP53 and KRAS can be used as specific genes for the screening, diagnosis, prognostic prediction, and recurrence monitoring of CRC. Moreover, the combined testing of TP53 and KRAS genes can improve the CRC detection rate.

Efficacy and Safety of Limertinib (ASK120067) in Patients With Locally Advanced or Metastatic EGFR Thr790Met-Mutated NSCLC: A Multicenter, Single-Arm, Phase 2b Study

IntroductionLimertinib (ASK120067) is a newly developed third-generation EGFR tyrosine kinase inhibitor targeting both sensitizing EGFR and EGFR Thr790Met (T790M) mutations. This study aimed to evaluate the efficacy and safety of limertinib in patients with locally advanced or metastatic EGFR T790M-mutated NSCLC. MethodsThis is a single-arm, open-label, phase 2b study conducted at 62 hospitals across the People’s Republic of China. Patients with locally advanced or metastatic NSCLC with centrally confirmed EGFR T790M mutations in tumor tissue or blood plasma who progressed after first- or second-generation EGFR tyrosine kinase inhibitors or with primary EGFR T790M mutations were enrolled. Patients received limertinib 160 mg orally twice daily until disease progression or unacceptable toxicity. The primary end point was objective response rate (ORR) assessed by independent review committee per the Response Evaluation Criteria in Solid Tumors version 1.1. Secondary end points included disease control rate, progression-free survival (PFS), duration of response (DoR), overall survival, and safety. Safety was assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 4.03. ResultsFrom July 16, 2019, to March 10, 2021, a total of 301 patients were enrolled and started the treatment of limertinib. All patients entered the full analysis set and safety set. By the data cutoff date on September 9, 2021, 76 (25.2%) remained on treatment. The median follow-up time was 10.4 months (range: 0.3–26.3). On the basis of full analysis set, the independent review committee-assessed ORR was 68.8% (95% confidence interval [CI]: 63.2%–74.0%) and disease control rate was 92.4% (95% CI: 88.8%–95.1%). The median PFS was 11.0 months (95% CI: 9.7–12.4), median DoR was 11.1 months (95% CI: 9.6–13.8), and median OS was not reached (95% CI 19.7 months–not evaluable). Objective responses were achieved across all prespecified subgroups. For 99 patients (32.9%) with central nervous system (CNS) metastases, the ORR was 64.6% (95% CI: 54.4%–74.0%), median PFS was 9.7 months (95% CI: 5.9–11.6), and median DoR was 9.6 months (95% CI: 8.1–15.2). For 41 patients who had assessable CNS lesion, the confirmed CNS-ORR was 56.1% (95% CI: 39.7%–71.5%) and median CNS-PFS was 10.6 months (95% CI: 5.6–not evaluable). In safety set, 289 patients (96.0%) experienced at least one treatment-related adverse event (TRAE), with the most common being diarrhea (81.7%), anemia (32.6%), rash (29.9%), and anorexia (28.2%). Grade ≥3 TRAEs occurred in 104 patients (34.6%), with the most common including diarrhea (13.0%), hypokalemia (4.3%), anemia (4.0%), and rash (3.3%). TRAEs leading to dose interruption and dose discontinuation occurred in 24.6% and 2% of patients, respectively. No TRAE leading to death occurred. ConclusionsLimertinib (ASK120067) was found to have promising efficacy and an acceptable safety profile for the treatment of patients with locally advanced or metastatic EGFR T790M-mutated NSCLC. Clinical Trial information: NCT03502850.

Efficacy and safety of ASK120067 (limertinib) in patients with locally advanced or metastatic EGFR T790M-mutated non–small cell lung cancer: A multicenter, single-arm, phase IIb study.

9106 Background: ASK120067 (Limertinib) is a newly developed third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) targeting both sensitizing EGFR and EGFR T790M mutations. This study aimed to evaluate the efficacy and safety of ASK120067 in patients with locally advanced or metastatic EGFR T790M mutated non-small cell lung cancer (NSCLC). Methods: This study was a single-arm, open-label, phase 2b study conducted at 62 hospitals across China. Patients with locally advanced or metastatic NSCLC with centrally confirmed EGFR T790M mutations in tumor tissue or blood plasma who progressed after first or second generation EGFR TKIs or with primary EGFR T790M mutations were enrolled. Patients received ASK120067 160mg orally twice daily, until disease progression, or unacceptable toxicity. The primary endpoint was objective response rate (ORR) assessed by Independent Review Committee (IRC) per RECIST1.1. Secondary endpoints included disease control rate (DCR), progression-free survival (PFS), duration of response (DOR), overall survival (OS), and safety. Safety was assessed according to CTCAE 4.03. Results: Between June 24th, 2019 and Feb 25th, 2021 301 patients were enrolled and received ASK120067 treatment. All patients entered the full analysis set (FAS) and safety set (SS). A total of 99 (32.9%) patients had central nervous system (CNS) metastases at baseline. By the data cutoff date on Sep 9th, 2021, 76 (25.2%) remained on treatment. The median follow-up time was10.4 (range 0.3-26.3) months. Based on FAS, the IRC-assessed ORR was 68.8% (95%CI 63.2%-74.0%) and DCR was 92.4% (95%CI 88.8%-95.1%). The median PFS was 11.0 (95%CI 9.7-12.4) months, median DOR was 11.1 (95%CI 9.6-13.8) months, and median OS was not reached (NR) (95%CI 19.7 months-NR). Objective responses were achieved across all pre-specified subgroups. For 99 patients with CNS metastases, the ORR was 64.6% (95%CI 54.4%-74.0%), median PFS was 9.7 (95%CI 5.9-11.6) months, and median DOR was 9.6 (95%CI 8.1-15.2) months. For 41 patients who had evaluable CNS lesion, the confirmed CNS-ORR was 56.1% (95%CI, 39.7%-71.5%) and median CNS-PFS was 10.6 (95%CI 5.6-NE) months. In SS, 289 (96.0%) patients experienced at least one adverse drug reaction (ADR), with the most common being diarrhea (81.7%), anemia (32.6%), rash (29.9%) and appetite decrease (28.2%). Grade ≥3 ADRs occurred in 104 (34.6%) patients, and the most common included diarrhea (13.0%), hypokalemia (4.3%), anemia (4.0%) and rash (3.3%). ADRs leading to dose interruption and dose discontinuation occurred 24.6% and 2% of patients, respectively. No ADR leading to death occurred. Conclusions: ASK120067 demonstrated promising efficacy and an acceptable safety profile for the treatment of patients with locally advanced or metastatic EGFR T790M mutated NSCLC. Clinical trial information: NCT03502850.

Pralsetinib treatment for multiple RET fusions in lung adenocarcinoma: a case report

Despite recent advances in treatments and knowledge of biomarkers, patients with metastatic lung cancer have a 5-year survival rate of 5%. Rearranged during transfection (RET) fusions occur in 1% to 2% of lung cancer patients. Pralsetinib has been used to treat non-small cell lung cancer with a single RET fusion; however, there have been no reports regarding its use in patients with multiple RET fusions. Genetic mutations in tumor tissues were tested using Amplification Refractory Mutation System-PCR and next-generation sequencing (NGS). Pleural fluids obtained from a male patient with non-small cell lung cancer were also used to detect genetic aberrations by NGS. Pleural fluid-based NGS revealed three RET rearrangements: CCDC6-RET (C2:R12), RET-NRG3 (R11:N3), and CCDC6-RET (C1:R12). All three rearrangements were targeted by pralsetinib, a RET fusion inhibitor. Pralsetinib drastically improved the patient’s condition within 4 days, and a partial response was achieved 1 week after pralsetinib was administered. We report for the first time the important clinical observation of a patient with multiple RET fusions who was effectively treated with pralsetinib.

The landscape of ACVR2A mutations in Chinese solid tumor patients.

e14523 Background: ACVR2A (activin A receptor type 2A) encodes a receptor that mediates the functions of activins, and it contains two polyadenine (A8) microsatellite loci, which are located in exon 3 and exon 10. Recent studies have shown that ACVR2A mutant was associated with microsatellite instability-high (MSI-H) and high tumor mutational burden(TMB-H) in gastrointestinal cancers. However, this association remains unclear in other solid tumors. Methods: We retrospectively analyzed the ACVR2A mutations from comprehensive 539-gene profiling of 10434 Chinese patients with pan-cancer. Somatic mutations in tumor tissue were assessed. We screened out ACVR2A mutations, calculated the mutation frequency, TMB in different types of cancer. Results: ACVR2A mutants were detected in 340 (2.9%) samples. The top 5 frequently cancers were colorectal cancer (98, 28.8%), lung carcinoma (68, 20%), hepatocellular carcinoma (64, 18.8%), gastric cancer (48, 14.1%) and biliary tract cancers (19, 5.6%). And 12.7% were other cancers (e.g., pancreatic cancer, small bowel adenocarcinoma, brain cancer). ACVR2A variants included truncation (50%), splicing site variant (9.0%), and other mutant types(41.1%). Truncation was the most common type, recurring in a few hot spots (K437Rfs*5/ K437Rfs*19, D96Tfs*54/ D96Rfs*4, V433del, R438Efs*19). The TMB in ACVR2A mutation group was significantly higher than that in ACVR2A wild-type group (p &lt; 0.001). In addition, TMB ≥10 muts/Mb was seen in 76.7% tumors with ACVR2A truncation or splicing site mutant(INACT) and 20.6% tumors with other mutant types(VUS)(p = 0.002). The median TMB of INACT group and VUS group was 35.46 muts/Mb (0.74-344.12) and 6.62 muts/Mb(0.5-598.53), respectively. Conclusions: We analyzed the distribution of ACVR2A mutants in Chinese patients with solid tumors. Our data shows that ACVR2A truncation or splice site mutant type is significantly associated with TMB-H, and this may be potential molecular marker of immunotherapy.

Concordance analysis between liquid biopsy (ctDNA) and tumor DNA molecular profiles from panel-based next-generation sequencing

IntroductionAnalysis of circulating tumor DNA (ctDNA), also known as liquid biopsy, has been postulated to be a useful test in the prognostication, molecular profiling, and monitoring of cancer patients. In this series we aimed to analyze the concordance between the mutation status of formalin-fixed paraffin-embedded (FFPE) tumor samples and matched ctDNA, considering tumor molecular profiling as the gold standard technique. MethodsThis retrospective study included cancer patients with complete diagnostics and gene mutations detected in a previous FFPE tumor tissue Next-Generation Sequencing (NGS) study with a matched frozen plasma sample available for an NGS ctDNA assay. Results and discussionSixty patients were included, 24 with colorectal carcinoma (CRC) and 36 with non-small cell lung cancer (NSCLC). In 27.1% of ctDNA studies a new mutation not previously detected in the matched tumor was found. 11.9% of these ctDNA results had the potential to impact clinical management. Globally, the concordance rate between FFPE tumor samples and ctDNA was 44.4%. When tumors were stratified by stage, the concordance was 76.5%, 70%, 36.4%, and 0% in tumor stages IV, III, II, and I, respectively. ctDNA molecular profiles showed a good concordance rate in advanced stage tumors and identified undetected mutations in tumor tissues. In early tumor stages the concordance was low, casting doubt on the usefulness of ctDNA in these patients.

KRAS and NRAS mutational analysis in plasma ctDNA from patients with metastatic colorectal cancer by real-time PCR and digital PCR.

Mutations in the KRAS and NRAS (RAS) genes are negative predictors of response to anti-EGFR therapy in metastatic colorectal cancer (mCRC). The detection of mutations in circulating tumor DNA (ctDNA) has emerged as a less invasive strategy to assess the molecular profile of mCRC patients. We aimed to perform RAS mutational analysis in ctDNA from mCRC patients using BEAMing Digital PCR (OncoBEAM) and Idylla ctDNA qPCR and evaluate the concordance rate with RAS mutational status in tumor tissue and between these two methodologies with different limits of detection. Blood samples were collected from 47 mCRC patients previously tested for RAS mutations in tumor tissue. DNA was extracted from plasma using the QIAamp Circulating Nucleic Acid Kit, and RAS mutation analysis was conducted using OncoBEAM RAS CRC and Idylla ctRAS assays. The overall agreement between tumor tissue and ctDNA analyses was 83% and 78.7% using the OncoBEAM and Idylla assays, respectively, with the concordance being 96.2% and 88.5% in naive treatment patients. The overall agreement between OncoBEAM and Idylla ctDNA analyses was 91.7%. Analysis of ctDNA is a viable strategy for clinical management of mCRC patients. Although the OncoBEAM assay sensitivity is somewhat higher, the fully automated Idylla platform also has good performance, while being cheaper and much less labor-intensive, for the detection of RAS mutations in plasma, either at diagnosis or after progression when considering anti-EGFR treatment rechallenge.

Use of Liquid Biopsy to Detect PIK3CA Mutation in Metastatic Breast Cancer.

PIK3CA is associated with tumor progression, and the prevalence of PIK3CA mutation is high in breast cancer. Liquid biopsy offers convenient, noninvasive, and real-time insight into genetic alteration. In this study, we used liquid biopsy to detect PIK3CA mutations in patients with breast cancer. We recruited patients with histologically confirmed breast cancer and distant metastases between April 2020 and September 2020. Circulating DNA was extracted from plasma (ctDNA) and exosomes (exoDNA). PIK3CA mutations (exons 9 and 20) were analyzed by droplet digital PCR. Of the 52 patients recruited, 16 had PIK3CA mutations in tumor tissue or blood: 9 had exon 9 mutations (E542K and E545K) and 8 had exon 20 mutations (H1047 L and H1047R). In 8 (15%) of the 52 patients, PIK3CA mutations were detected by liquid biopsies using ctDNA in 5 (9%), exoDNA in 6 (11%), and both ctDNA and exoDNA in 3 (6%). Of the 8 patients with PIK3CA mutations detected by liquid biopsies, 3 had no PIK3CA mutations in the primary tumors. PIK3CA mutations can be detected by liquid biopsy even in patients with no PIK3CA mutations in their primary tumors; thus, combination analysis using tissue and liquid biopsies can provide clinically useful information for patients with breast cancer.

Early dynamics of circulating tumor DNA predict clinical response to immune checkpoint inhibitors in metastatic renal cell carcinoma.

ObjectivesDetection of genomic alterations in circulating tumor deoxyribonucleic acid of peripheral blood can guide the selection of systemic therapy in cancer patients. The predictive significance of circulating tumor deoxyribonucleic acid in metastatic renal cell carcinoma remains unclear, especially for patients treated with immune checkpoint inhibitors.MethodsIn this study, we collected plasma samples before and 1 month after commencing nivolumab monotherapy or nivolumab plus ipilimumab therapy from 14 metastatic renal cell carcinoma patients. We performed circulating tumor deoxyribonucleic acid genomic profiling in plasma cell‐free deoxyribonucleic acid by next‐generation sequencing using a commercially available pan‐cancer panel (Guardant360 CDx). Additionally, we also performed whole exome sequencing of tumor tissues and compared the concordance of genomic profiles with circulating tumor deoxyribonucleic acid.ResultsNine patients had circulating tumor deoxyribonucleic acid in pretreatment plasma samples with a total of 20 mutations (15 single nucleotide variants, three insertions/deletions, and two copy number amplification). VHL (30.0%) was the most frequently mutated gene, followed by TP53 (20.0%), and 45.0% of circulating tumor deoxyribonucleic acid mutations were concordant with somatic mutations in tumor tissues. Patients with decreasing circulating tumor deoxyribonucleic acid mutant allele frequency had better progression free survival when compared to those with increasing mutant allele frequency (P = 0.0441).ConclusionsOur findings revealed that early circulating tumor deoxyribonucleic acid dynamics can serve as a predictive biomarker for response to immune checkpoint inhibitors in metastatic renal cell carcinoma patients.

Abstract P1-08-27: Dynamic monitoring of circulating tumor DNA can predict chemotherapy response and prognosis in metastatic triple-negative breast cancer patients

Abstract Background: Chemotherapy is the main treatment for metastatic triple-negative breast cancer (mTNBC). However, mTNBC patients (pts) is often associated with chemotherapy resistance and poor prognosis. Biomarkers that can effectively predict the efficacy of chemotherapy for mTNBC are currently lacking. Detection of circulating tumor DNA (ctDNA) in plasma by liquid biopsy is a minimally invasive and highly sensitive method, which has been wildly used in the clinic and plays an important role in tumor diagnosis, efficacy evaluation, residual and recurrent tumor detection, etc. But yet, the clinical application of ctDNA in mTNBC remains relatively scarce. Here, for the first time, we set to explore the possibility of ctDNA as a biomarker for predicting chemotherapy response and prognosis in mTNBC.Methods: From May 2018 to October 2020, 38 mTNBC pts who received less than 3rd line standard chemotherapy were prospectively included. Tumor tissues were obtained prior chemotherapy, and plasma for ctDNA were collected at baseline, a day before the 3rd cycle chemotherapy and at disease progression. Next-generation sequencing (NGS,457 genes panel) was performed on all samples for mutation detection. And the ctDNA fraction, maximum variant allele frequency (max-VAF), tumor mutation burden (TMB) and other variate were calculated, further combined with clinical data, chemotherapy response and survival of pts for statistical analysis.Results: Finally, 109 blood samples and 13 tissue samples were detected by NGS. A total of 214 mutation genes and 397 mutation sites were detected. The mutation types included missense mutation, nonsense mutation, non-/frameshift insertion, non-/frameshift deletion, non-/frameshift substitution, splicing mutation and so on. The genes with the top 5 mutation frequencies were TP53 (32/38, 84.21%), PIK3CA (14/38, 36.84%), KMT2C (8/38, 21.05%), PTEN (6/38, 15.79%), NOTCH4 (6/38, 15.79%), respectively. The same variants were identified in 12 of 13 pts in paired plasma and tissue, with a concordance rate of 92.3%. The mutation rate in plasma ctDNA was significantly lower than that in tissues (15.26% ± 12.52% vs. 28.88 ± 16.54%, P &amp;lt; 0.001) but was still positively correlated with that in tissues (r = 0.306, P = 0.049). The median progression-free survival (mPFS) of pts with GNAS mutation was shorter than GNAS wild-type pts (3.000 vs. 6.100 months, P = 0.014). The area under curve of ctDNA fraction (0.812, P = 0.043), max-VAF (0.817, P = 0.043) and TMB (plasma) (0.759, P = 0.053) in predicting chemotherapy resistance or disease progression were larger than CEA (0.491, P = 0.888), CA125 (0.574, P = 0.243) and CA153 (0.482, P = 0.778). CtDNA fraction (r = 0.482, P &amp;lt; 0.001), max-VAF (r = 0.489, P &amp;lt; 0.001), TMB (plasma). (r = 0.419, P &amp;lt; 0.001) were correlated with chemotherapy response measured by RECIST v1.1 in CT imaging, while CEA (r = -0.024, P = 0.808), CA125 (r = 0.111, P = 0.266) and CA153 (r = -0.017, P = 0.865) had no correlation with chemotherapy response. The mPFS of pts with ctDNA fraction ≤ 50% or max-VAF ≤ 0.4 at baseline was significantly longer than that of pts with ctDNA fraction &amp;gt; 50% (6.100 vs. 3.430 months, P = 0.006) or max-VAF &amp;gt; 0.4 (6.100 vs. 3.430 months, P = 0.047). The elimination of mutations or the decrease of mutation rate in plasma ctDNA after 2 cycles of chemotherapy showed better chemotherapy response, while recurrence of mutations, increase of mutation rate and emergence of new mutations showed chemotherapy resistance.Conclusions: Mutations in tumor tissues and plasma ctDNA of mTNBC pts detected by NGS have high consistency. And compared with CT imaging and traditional tumor markers, dynamic monitoring ctDNA can more aptly reflect the change of whole-body tumor burden, better predict the chemotherapy response and prognosis in mTNBC. Citation Format: Huihui Li, Yajing Chi, Sha Yin, Bo Yu, Mu Su, Baoxuan Zhang, Ling Qiang, Guohua Ren, Lihua Song, Bing Bu, Shu Fang, Mao Shang, Qiaorui Tan, Xiaochu Man. Dynamic monitoring of circulating tumor DNA can predict chemotherapy response and prognosis in metastatic triple-negative breast cancer patients [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-08-27.

Development and Validation of a Targeted 'Liquid' NGS Panel for Treatment Customization in Patients with Metastatic Colorectal Cancer.

The detection of actionable mutations in tumor tissue is a prerequisite for treatment customization in patients with metastatic colorectal cancer (mCRC). Analysis of circulating tumor DNA (ctDNA) for the identification of such mutations in patients’ plasma is an attractive alternative to invasive tissue biopsies. Despite having the high analytical sensitivity required for ctDNA analysis, digital polymerase chain reaction (dPCR) technologies can only detect a very limited number of hotspot mutations, whilst a broader mutation panel is currently needed for clinical decision making. Recent advances in next-generation sequencing (NGS) have led to high-sensitivity platforms that allow screening of multiple genes at a single assay. Our goal was to develop a small, cost- and time-effective NGS gene panel that could be easily integrated in the day-to-day clinical routine in the management of patients with mCRC. We designed a targeted panel comprising hotspots in six clinically relevant genes (KRAS, NRAS, MET, BRAF, ERBB2 and EGFR) and validated it in a total of 68 samples from 30 patients at diagnosis, first and second disease progression. Results from our NGS panel were compared against plasma testing with BEAMing dPCR regarding the RAS gene status. The overall percent of agreement was 83.6%, with a positive and negative percent agreement of 74.3% and 96.2%, respectively. Further comparison of plasma NGS with standard tissue testing used in the clinic showed an overall percent agreement of 86.7% for RAS status, with a positive and negative percent agreement of 81.2% and 92.8%, respectively. Thus, our study strongly supports the validity and efficiency of an affordable targeted NGS panel for the detection of clinically relevant mutations in patients with mCRC.

Perioperative ctDNA-Based Molecular Residual Disease Detection for Non-Small Cell Lung Cancer: A Prospective Multicenter Cohort Study (LUNGCA-1).

We assessed whether perioperative circulating tumor DNA (ctDNA) could be a biomarker for early detection of molecular residual disease (MRD) and prediction of postoperative relapse in resected non-small cell lung cancer (NSCLC). Based on our prospective, multicenter cohort on dynamic monitoring of ctDNA in lung cancer surgery patients (LUNGCA), we enrolled 950 plasma samples obtained at three perioperative time points (before surgery, 3 days and 1 month after surgery) of 330 stage I-III NSCLC patients (LUNGCA-1), as a part of the LUNGCA cohort. Using a customized 769-gene panel, somatic mutations in tumor tissues and plasma samples were identified with next-generation sequencing and utilized for ctDNA-based MRD analysis. Preoperative ctDNA positivity was associated with lower recurrence-free survival (RFS; HR = 4.2; P < 0.001). The presence of MRD (ctDNA positivity at postoperative 3 days and/or 1 month) was a strong predictor for disease relapse (HR = 11.1; P < 0.001). ctDNA-based MRD had a higher relative contribution to RFS prediction than all clinicopathologic variables such as the TNM stage. Furthermore, MRD-positive patients who received adjuvant therapies had improved RFS over those not receiving adjuvant therapy (HR = 0.3; P = 0.008), whereas MRD-negative patients receiving adjuvant therapies had lower RFS than their counterparts without adjuvant therapy (HR = 3.1; P < 0.001). After adjusting for clinicopathologic variables, whether receiving adjuvant therapies remained an independent factor for RFS in the MRD-positive population (P = 0.002) but not in the MRD-negative population (P = 0.283). Perioperative ctDNA analysis is effective in early detection of MRD and relapse risk stratification of NSCLC, and hence could benefit NSCLC patient management.

Personalized circulating tumor DNA in patients with hepatocellular carcinoma: a pilot study.

Mutational analysis of circulating tumor DNA (ctDNA) can potentially be used for early detection of recurrence after resection for hepatocellular carcinoma (HCC). Mutations from tumor may be identified in plasma as an early sign of recurrence. We conducted a pilot study investigating if somatic mutations could be detected in plasma in patients undergoing liver resection for HCC and in patients with advanced non-resectable HCC. We prospectively included patients undergoing curative liver resection for HCC. Tumor tissue was investigated with whole exome sequencing and preoperative blood samples were evaluated for ctDNA using targeted next-generation sequencing (NGS) with TruSight Oncology 500 including 523 cancer-associated genes. Subsequently, the method was evaluated in patients with advanced HCC. We included eight patients curatively resected for HCC, where tumor tissue mutations were identified in seven patients. However, only in one patient tumor specific mutations were found in the preoperative blood sample. In all three patients with advanced HCC, tumor mutations were detected in the blood. In patients with resectable HCC, ctDNA could not be reliably detected using the applied targeted NGS method. In contrast, ctDNA was detected in all patients with advanced HCC. Small tumors, tumor heterogeneity and limited sequencing coverage may explain the lack of detectable ctDNA.

PIK3CA mutations in plasma circulating tumor DNA predict survival and treatment outcomes in patients with advanced cancers

BackgroundOncogenic mutations in PIK3CA are prevalent in diverse cancers and can be targeted with inhibitors of the phosphoinositide-3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway. Analysis of circulating tumor DNA (ctDNA) provides a minimally invasive approach to detect clinically actionable PIK3CA mutations.Patients and methodsWe analyzed PIK3CA hotspot mutation frequency by droplet digital PCR (QX 200; BioRad) using 16 ng of unamplified plasma-derived cell-free DNA from 68 patients with advanced solid tumors (breast cancer, n = 41; colorectal cancer, n = 13; other tumor types, n = 14). Results quantified as variant allele frequencies (VAFs) were compared with previous testing of archival tumor tissue and with patient outcomes.ResultsOf 68 patients, 58 (85%) had PIK3CA mutations in tumor tissue and 43 (74%) PIK3CA mutations in ctDNA with an overall concordance of 72% (49/68, κ = 0.38). In a subset analysis, which excluded samples from 26 patients known not to have disease progression at the time of sample collection, we found an overall concordance of 91% (38/42; κ = 0.74). PIK3CA-mutated ctDNA VAF of ≤8.5% (5% trimmed mean) showed a longer median survival compared with patients with a higher VAF (15.9 versus 9.4 months; 95% confidence interval 6.7-17.1 months; P = 0.014). Longitudinal analysis of ctDNA in 18 patients with serial plasma collections (range 2-22 time points, median 5) showed that those with a decrease in PIK3CA VAF had a longer time to treatment failure (TTF) compared with patients with an increase or no change (10.7 versus 2.6 months; P = 0.048).ConclusionsDetection of PIK3CA mutations in ctDNA is concordant with testing of archival tumor tissue. Low quantity of PIK3CA-mutant ctDNA is associated with longer survival and a decrease in PIK3CA-mutant ctDNA on therapy is associated with longer TTF.

Abstract 580: The broad surveillance and unbiased detection of circulating tumor DNA using next-generation sequencing without a priori knowledge of somatic mutations is a prognostically significant biomarker in pancreatic ductal adenocarcinoma

Abstract Background. Detection of low frequency tumor-derived variants in circulating cell-free DNA (ccfDNA; i.e., circulating tumor DNA, ctDNA) without a priori knowledge of mutations in the matched solid tumor is confounded by next-generation sequencing (NGS) noise and constrained by theoretical and practical limitations associated with low DNA yield from plasma. Here, we leverage technical replicates (two libraries prepared and sequenced independently from each extracted ccfDNA sample) to simultaneously suppress NGS-associated errors and improve detection of ctDNA derived from pancreatic ductal adenocarcinomas (PDACs). Methods. Pre-operative ccfDNA and tumor DNA were acquired from 14 patients with PDAC (78.6% stage II-III) undergoing surgery aimed at complete total resection. For 11 patients, ccfDNA was also collected post-operatively within 100 days of surgery. Two technical replicates of ccfDNA libraries using molecular barcodes were independently generated using 2 mL plasma equivalents of ccfDNA for each library. Subsequently, libraries were capture-enriched using a 118 gene panel (~124 kb) and underwent NGS (paired-end, 125x2; HiSeq 2500). Consensus sequence determination and position-specific error modeling were coupled with restricting variant identification to nonreference alleles present in both patient replicates that corresponded to COSMIC-reported pathogenic mutations in PDAC. Results. PDAC-associated pathogenic mutations were detected in pre-operative ccfDNA in four genes (KRAS, TP53, SMAD4, ALK) from five patients (35.7%) with a variant allele frequency ranging from 0.08% to 1.59% (median: 0.25%). Of the nine ctDNA variants identified, five (55.6%) had a corresponding mutation in tumor DNA. The estimated median overall survival in patients with and without detection of pre-operative ctDNA was significantly different (312 vs. 826 days, respectively; χ2=5.3, P=0.0207). Using mutations identified in tumor tissue DNA to guide detection of pre-operative ctDNA yielded a similar survival analysis (369 vs 1,011 days, respectively; χ2=5.6, P=0.018). Detection of ctDNA in post-operative ccfDNA with or without guidance from tumor tissue mutations was not associated with outcomes. Applying the noise reduction strategy used to detect ctDNA in PDAC patients eliminated false positives from healthy controls. Conclusions. Acquisition of NGS data in replicate coupled with in silico error suppression strategies enabled detection of ctDNA without a priori knowledge of tumor variants, which overcomes a major obstacle limiting ccfDNA applications. Pre-operative ctDNA detected during a broad and untargeted survey of ccfDNA with NGS is a non-invasive, prognostic biomarker to help guide PDAC patient management. Citation Format: Kajsa E. Affolter, Sabine Hellwig, David A. Nix, Mary P. Bronner, Alun Thomas, Carrie L. Fuertes, Cindy L. Hamil, Ignacio Garrido-Laguna, Courtney L. Scaife, Sean J. Mulvihill, Hunter R. Underhill. The broad surveillance and unbiased detection of circulating tumor DNA using next-generation sequencing without a priori knowledge of somatic mutations is a prognostically significant biomarker in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 580.

TP53 Targeted Deep Sequencing of Cell-Free DNA in Esophageal Squamous Cell Carcinoma Using Low-Quality Serum: Concordance with Tumor Mutation.

Circulating cell-free DNA (cfDNA) is emerging as a potential tumor biomarker. CfDNA-based biomarkers may be applicable in tumors without an available non-invasive screening method among at-risk populations. Esophageal squamous cell carcinoma (ESCC) and residents of the Asian cancer belt are examples of those malignancies and populations. Previous epidemiological studies using cfDNA have pointed to the need for high volumes of good quality plasma (i.e., >1 mL plasma with 0 or 1 cycles of freeze-thaw) rather than archival serum, which is often the main available source of cfDNA in retrospective studies. Here, we have investigated the concordance of TP53 mutations in tumor tissue and cfDNA extracted from archival serum left-over from 42 cases and 39 matched controls (age, gender, residence) in a high-risk area of Northern Iran (Golestan). Deep sequencing of TP53 coding regions was complemented with a specialized variant caller (Needlestack). Overall, 23% to 31% of mutations were concordantly detected in tumor and serum cfDNA (based on two false discovery rate thresholds). Concordance was positively correlated with high cfDNA concentration, smoking history (p-value = 0.02) and mutations with a high potential of neoantigen formation (OR; 95%CI = 1.9 (1.11–3.29)), suggesting that tumor DNA release in the bloodstream might reflect the effects of immune and inflammatory context on tumor cell turnover. We identified TP53 mutations in five controls, one of whom was subsequently diagnosed with ESCC. Overall, the results showed that cfDNA mutations can be reliably identified by deep sequencing of archival serum, with a rate of success comparable to plasma. Nonetheless, 70% non-identifiable mutations among cancer patients and 12% mutation detection in controls are the main challenges in applying cfDNA to detect tumor-related variants when blindly targeting whole coding regions of the TP53 gene in ESCC.

The real-world BRCA1/2 germline mutations in Chinese solid tumors.

e22500 Background: Breast cancer susceptibility genes BRCA1 and 2 are tumor suppressor genes and they play an important role in DNA damage response and repair during homologous recombination. Hereditary breast and ovarian cancer (HBOC) syndrome is an autosomal dominant disease due to BRCA1 and 2 germline mutation. Germline mutations of BRCA1 and 2 genes significantly increase the risk of developing breast cancer, ovarian cancer, prostate cancer and a broad range of cancers. PARP inhibitor (PARPi) monotherapy and combinations have shown promising efficacy against a variety of cancer types with BRCA mutations. Nevertheless, the knowledge of incidence of BRCA1 and 2 germline mutations in solid tumor remains poorly understood. Herein, next generation sequencing of 539-gene profiling was performed to explore the incidence of BRCA1 and 2 germline mutations in Chinese solid tumors. Methods: We retrospectively analyzed the BRCA1 and BRCA2 germline mutations from a comprehensive 539-gene profiling of 8535 Chinese patients with pan-cancer. 539-gene profiling contains the somatic mutations in tumor tissue or blood ctDNA and the germline mutations in blood leukocyte. We screened out the pathogenic and likely pathogenic mutations in BRCA germline mutations, and calculated the mutation frequency and the median age in every cancer type. Results: In 8535 patients with pan-cancer, 110 patients were found pathogenic or likely pathogenic germline mutations in BRCA gene and the mutation frequency was 1.29%, of which 40 BRCA1 mutations and 70 BRCA2 mutations were found in patients, respectively. The total median age was 58.15. Eliminated a few types of cancers that had a smaller number (less than 50), the higher frequency of mutations contained ovarian cancer (14.78%, media age 58), prostatic cancer (6%, media age 58.33), breast cancer (5.2% media age 57.33). The details of the top 8 cancer types with mutation frequency and media age were shown in Table. Conclusions: This was the first report of the incidence of BRCA1 and 2 germline mutations in Chinese solid tumors, which expanded the understanding of BRCA1/2 and provided a direction for clinical trial design of PARPi monotherapy and combinations.[Table: see text]

ESR1 Gene Mutation in Hormone Receptor-Positive HER2-Negative Metastatic Breast Cancer Patients: Concordance Between Tumor Tissue and Circulating Tumor DNA Analysis.

Endocrine therapy represents the cornerstone of treatment in hormone receptor-positive (HR+), HER2-negative metastatic breast cancer (mBC). The natural course of this disease is marked by endocrine resistance, mainly due to Estrogen Receptor 1 (ESR1) acquired mutations. The aim of this study is to evaluate the concordance between ESR1 status in metastatic tumor specimens and matched circulating tumor DNA (ctDNA). Forty-three patients with HR+, HER2-negative mBC underwent both a metastatic tumor biopsy and a liquid biopsy at the time of disease progression. DNA extracted from formalin fixed paraffin embedded (FFPE) tumor specimens and ctDNA from matched plasma were analyzed by droplet digital (dd)PCR for the main ESR1 mutations (Y537S, Y537C, Y537N, D538G, E380Q). We observed a total mutation rate of 21%. We found six mutations on tissue biopsy: Y537S (1), D538G (2), Y537N (1), E380Q (2). Three patients with no mutations in tumor tissue had mutations detected in ctDNA. The total concordance rate between ESR1 status on tumor tissue and plasma was 91%. Our results confirm the potential role of liquid biopsy as a non-invasive alternative to tissue biopsy for ESR1 mutation assessment in mBC patients.

Abstract PS4-06: Comparison of liquid biopsy and tissue based detection of PIK3CA mutations in HR positive metastatic breast cancer patients

Abstract The treatment of hormone receptor positive metastatic breast cancer patients has changed dramatically over the last few years and combination strategies attempting to overcome resistance of the disease are gaining importance. After introducing CDK 4/6 inhibitors in the treatment one of the subsequent strategies is definitely targeting PI3 kinase pathway. Several drugs have been tested, but only recently, SOLAR-1 phase III trial demonstrated the benefit of adding alpelisib to fulvestrant, with acceptable tolerability. With this trial the importance of PIK3CA, testing was postulated. In the present study a comparison of liquid biopsy and tissue-based detection of PIK3CA mutations in HR positive metastatic breast cancer patients was performed. Materials and Methods: Plasma and the most recent tumor tissue were screened for PIK3CA hotspot mutations from 58 patients with metastatic hormone-receptor positive breast cancer. For plasma samples, SiMSen-Seq NGS sequencing was used covering 11 frequent PIK3CA mutations, which were applied for stratification in SOLAR-1. Only plasma samples with variant allele frequency (VAF) &amp;gt;1% were considered as positive. For tumor tissue samples, targeted Ion Torrent NGS was used. Matched tissue plasma samples were available from 40 patients.Results: PIK3CA mutations were detected in 25/50 (50.0%) tissue samples and in 18/48 (37.5%) plasma samples. VAF ranged from 4.4 to 72.9% with an average of 28.7% in tissue samples, and from 1.1% to 49.9% with an average of 8.2% in plasma samples. In tumor samples, the most frequent variant was H1047R (11/25, 44.0%), followed by E545K (8/25, 32.0%), E542K (4/25, 16.0) and H1047L (2/25, 8.0%). In plasma samples, the most frequent PIK3CA variant was H1047R (12/18, 66.7%), followed by E542K (6/18, 33.3%), and E545K (2/18, 11.1%). Double mutations in plasma samples occurred in two patients (both with H1047R + E542K). In 15/48 (31.3%) plasma samples, 19 low-level mutations (AF between 0.1% and 1.0 %) were detected, with an average of 0.49% (range 0.18-0.98).Matched tissue and plasma samples were available from 40/58 (68.9%) patients with a median time between matched tissue and plasma collection of 285 days (25th-75th percentile: 24-1833 days). The overall concordance rate between plasma and tissue samples was 62.5% (25/40 patients). In detail, 8/40 patients (20.0%) had the same PIK3CA mutation in plasma and tumor tissue, while in 17/40 patients (42.5%) were tested negative in both sample types Discordant results occurred in 15/40 samples (37.5%). Specifically, nine patients had a PIK3CA mutation in the tissue sample only, while four patients had a PIK3CA mutation in the plasma sample only. In addition, in two patients a different PIK3CA mutation was detected in the plasma sample compared to the tissue sample.Conclusion: SiMSen-Seq based detection of PIK3CA mutations from plasma demonstrated promising results for selection of candidate patients for Alpelisib treatment. However, these results will be validated in a larger cohort of patients. Our data align with FDA recommendation to initially carry out the mutation testing in ctDNA. Only if the test is negative for PIK3CA mutations in plasma, patients should undergo testing for PIK3CA mutations in tumor tissue. Citation Format: Christoph Suppan, Qing Zhou, Stephan Jahn, Ricarda Graf, Eva Valentina Klocker, Angelika Terbuch, Karl Kashofer, Florian Posch, Hanno Gerritsmann, Nadia Dandachi, Ellen Heitzer, Marija Balic. Comparison of liquid biopsy and tissue based detection of PIK3CA mutations in HR positive metastatic breast cancer patients [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-06.

Prognostic significance of longitudinal evaluation on egfr mutant ctdna during tki treatment in nsclc

Detection of EGFR mutations in tumor tissue is the gold standard for prescribing tyrosine kinase inhibitor therapy.However even in the same molecularly defined population effect of the treatment, response duration and survival might differ significantly. We did a prospective clinical trial to assess ctDNA-based EGFR mutations as a prognostic marker for patients with lung adenocarcinoma receiving TKI as first-line treatment.