Mutant Allele Frequency Research Articles

REMARRY and PURSUIT trials: liquid biopsy-guided rechallenge with anti-epidermal growth factor receptor (EGFR) therapy with panitumumab plus irinotecan for patients with plasma RAS wild-type metastatic colorectal cancer

BackgroundPrevious clinical trials have demonstrated the potential efficacy of rechallenge with anti- epidermal growth factor receptor (EGFR) monoclonal antibodies (mAbs) for patients with RAS/BRAF V600E wild-type metastatic colorectal cancer (mCRC). Moreover, post hoc biomarker analyses of clinical trials has suggested that RAS status in circulating tumor DNA (ctDNA) has a high probability to select patients who could benefit from anti-EGFR mAb rechallenge.MethodsThis trial is composed of 2 phases: a monitoring phase (REMARRY) and a trial phase (PURSUIT). A monitoring phase, the REMARRY study, aims to evaluate the dynamics of plasma RAS status during the subsequent treatments after refractory to anti-EGFR therapy in patients with mCRC with RAS/BRAF V600E wild-type tumors who have progressed after a response to previous anti-EGFR therapy, using a highly sensitive digital polymerase chain reaction OncoBEAM RAS CRC kit in a central laboratory (Sysmex, Japan). A trial phase, the PURSUIT trial, is a multicenter, single-arm phase II trial to assess the efficacy and safety of rechallenge therapy with panitumumab plus irinotecan in patients without RAS mutations in ctDNA (plasma RAS negative) in the REMARRY study. Key eligibility criteria of the PURSUIT trial include RAS/BRAF V600E wild-type mCRC in tumor tissue refractory or intolerant to fluoropyrimidine, oxaliplatin, and irinotecan; progression after complete or partial response to previous anti-EGFR therapy; plasma RAS negative (defined as plasma mutant allele frequencies [MAF] of all RAS ≤ 0.1%) within 28 days prior to enrollment; 4 months or more between the last administration of previous anti-EGFR mAb and the start of protocol treatment; and Eastern Cooperative Oncology Group (ECOG) Performance Status (PS) ≤ 1. The primary endpoint is the confirmed objective response rate (ORR). The target sample size of the PURSUIT trial is 50 patients. Biomarker analyses will be performed in parallel using the OncoBEAM RAS CRC kit and a next-generation sequencing-based ctDNA analysis (Guardant360).DiscussionOur trial aims to confirm the clinical benefit of anti-EGFR mAb rechallenge therapy in patients with plasma RAS negative. Moreover, through biomarker analyses, our trial will shed light on which patients would benefit from rechallenge in addition to being plasma RAS negative.Trial registrationThe REMARRY study: UMIN, UMIN000036424. Registered date: April 5, 2019. The PURSUIT trial: jRCT, jRCTs031190096. Registered date: October 1, 2019.

PCR-RFLP molecular confirmation of color dilution alopecia in dogs in Brazil.

Color dilution alopecia (CDA) is a dermatopathy observed exclusively in animals having a diluted coat color. In dogs, color dilution occurs as a result of a single-nucleotide variation (SNV) c.-22G > A in the melanophilin gene. We standardized a PCR-restriction-fragment length polymorphism (PCR-RFLP) technique to identify this mutation and determine its frequency in dogs in Brazil. The standardized PCR-RFLP technique could efficiently identify the SNV c.-22G > A in the melanophilin gene, with mutated allele frequencies of 0.1, 0.1, and 0.0875 in Dachshund, Miniature Pinscher, and Yorkshire Terrier breeds, respectively, with no statistical difference among the breeds (p = 0.252). The mutation was identified in 2 homozygous Dachshund dogs with alopecia, confirming the clinical characteristic of CDA. The standardization of a simpler and more accessible molecular technique for recognition of the SNV c.-22G > A in the melanophilin gene allows identification of heterozygous (phenotypically normal) dogs that can be excluded from reproduction, to avoid the birth of dogs with diluted coat color and consequently CDA.

Genetic and expression deregulation of immunoregulatory genes in rheumatoid arthritis.

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases globally, and is an important public health concern, associating with early death and systemic complications. Although key development in RA treatment has already been made still RA affected individuals face comorbidity and disability. Therefore, there is a need to discover new risk factors in helping early diagnosis and treatment of RA. The present study is designed to assess the variations of Vitamin D receptor (VDR) and interleukin -6 (IL-6) in RA patients. Polymorphisms of said genes were calculated in 300 RA patients and 300 controls, using Tetra-ARMS polymerase chain reaction. Secondly, expression levels of selected genes were checked using the quantitative PCR (qPCR) and obtained results were evaluated using a different statistical test. Logistic regression analysis showed that frequency of mutant allele of VDR gene polymorphisms (rs11168268, OR = 4.84; 95% CI = 2.94-7.97; p = 0.0001; rs2248098, OR = 1.65; 95% CI = 1.07-2.54; p = 0.02) and IL-6 gene polymorphisms (rs184229712, OR = 2.47; 95% CI = 1.56-3.92, p = 0.0001; rs36215814, OR = 2.14; 95% CI = 1.30-3.53; p = 0.002) was observed significantly higher in RA patients vs controls. Expression analysis showed the significant upregulation of IL-6 (p < 0.0001) and downregulation of VDR gene (p < 0.0001) in RA cases vs controls. ROC curve analysis showed that downregulation of IL-6 (AUC = 0.86, p < 0.001) and upregulation of VDR (AUC = 0.77, p < 0.001) was act as the good diagnostic marker for detection/diagnosis of arthritis. In conclusions, data from the present study showed the significant involvement of VDR and IL-6 gene variations in RA pathogenesis.

Circulating tumor cells, circulating tumor DNA, and disease characteristics in young women with metastatic breast cancer.

Clinical and genomic data from patients with early-stage breast cancer suggest more aggressive disease in premenopausal women. However, the association between age, disease course, and molecular profile from liquid biopsy in metastatic breast cancer (MBC) is not well characterized. Patients were classified as premenopausal (<45years), perimenopausal (45-55years), or postmenopausal (> 55years). Cohort 1 consisted of patients with MBC who consented for prospective serial evaluation of circulating tumor cells (CTCs) using CellSearch™. Cohort 2 included patients who, as part of routine care, had circulating tumor DNA (ctDNA) sequenced by the Guardant360™ assay. Clinicopathologic data were collected from retrospective review to compare disease features between premenopausal and postmenopausal women. Premenopausal women represented 26% of 138 patients in Cohort 1 and 21% of 253 patients in Cohort 2. In Cohort 1, younger patients had a shorter time to metastases and a higher prevalence of lung and brain metastases. Overall, there were similar rates of ≥ 5 CTCs/7.5mL, HER2 + CTC expression, and CTC clusters between pre- and postmenopausal women. However, for those with triple negative breast cancer, premenopausal women had a higher proportion of ≥ 5 CTCs/7.5mL. In Cohort 2, premenopausal women had a higher incidence of FGFR1 (OR 2.75, p = 0.022) and CCND2 (OR 6.91, p = 0.024) alterations. There was no difference in the ctDNA mutant allele frequency or the number of detected alterations between these age groups. Our data reveal that premenopausal women diagnosed with MBC have unique clinical, pathologic, and molecular features when compared to their postmenopausal counterparts. Our results highlight FGFR1 inhibitors as potential therapeutics of particular interest among premenopausal women.

Detection of EGFR Mutations in Plasma cfDNA and Paired CTCs of NSCLC Patients before and after Osimertinib Therapy Using Crystal Digital PCR.

Simple SummaryLiquid biopsy is a useful tool during longitudinal monitoring of NSCLC patients and requires highly sensitive and reliable technologies for accurate detection of genomic alterations. We evaluated and used crystal digital PCR to detect and quantify EGFR mutations in plasma cfDNA and paired CTCs of NSCLC patients before treatment with osimertinib and at progression of disease.Circulating tumor DNA (ctDNA) analysis has clinical utility in EGFR mutant NSCLC. Circulating tumor cells (CTCs) consist a unique source of information at the cellular level. Digital PCR (dPCR) is a valuable tool for accurate and valid analysis of gene mutations in liquid biopsy analysis. In the present study we detected EGFR mutations in ctDNA and paired CTCs under osimertinib therapy at two time points using crystal dPCR and the naica® system (Stilla Technologies). We quantified mutation allele frequencies (MAF) of EGFR mutations in 91 plasma cfDNA samples of 48 EGFR mutant NSCLC patients and in 64 matched CTC-derived genomic DNA samples, and the FDA-cleared cobas® EGFR mutation test in 80 identical plasma samples. Direct comparison between crystal dPCR and the cobas EGFR assay revealed a high concordance for all EGFR mutations. Our comparison of crystal dPCR results in ctDNA with the corresponding primary tissue has shown a strong correlation. EGFR mutations analysis in paired CTC-derived gDNA revealed a high heterogeneity. Crystal dPCR offers the unique advantages of high analytical sensitivity, precision, and accuracy for detecting and quantifying multiple EGFR mutations in plasma cfDNA and CTCs of NSCLC patients.

Association of CYP2C19*2 and *17 genetic variants with hypertension in Pakistani population

Purpose: To investigate the association of *2 and *17 single nucleotide polymorphisms (SNPs) of CYP2C19 gene with hypertension in Pakistani population.&#x0D; Methods: The study was conducted on 527 hypertensive patients and 530 unrelated healthy controls from selected regions of Pakistan. DNA was extracted from leukocytes and all patients and controls were genotyped for two SNPs (rs4244285 and rs12248560) of CYP2C19 gene by allele specific polymerase chain reaction (AS-PCR).&#x0D; Results: Multi-allelic polymorphism in CYP2C19 identified four distinct phenotypes known as ultra-rapid metabolizer (UM), extensive metabolizer (EM), intermediate metabolizer (IM) and poor metabolizer (PM) in hypertensive patients and controls. For CYP2C19*2 polymorphisms, overall wild type and mutant allele frequency were 75 and 25 % in hypertensive patients, and 64.2 and 35.8 % in controls. For CYP2C19*17 polymorphisms, the overall wild type and mutant allele frequency were 66.6 and 33.4 % in hypertensive patients and 75.6 % and 24.4 % in controls. Significant difference in allele frequencies for CYP2C19*2 and *17 was demonstrated between hypertensive and non-hypertensive subjects.&#x0D; Conclusion: To the best of our knowledge, this is the first report on CYP2C19 frequencies in hypertensive Pakistani patients. The finds should help clinicians to determine a suitable optimal dosage of some drugs in order to reduce side effects.

Voltage-Gated Sodium Channel (Vgsc) Mutation-Based Pyrethroid Resistance in Aedes aegypti Populations of Three Endemic Dengue Risk Areas of Sri Lanka.

Background Pyrethroid insecticides are widely used in many countries for chemical-based control of Ae. aegypti. Regardless of their efficacy, the constant use of insecticides has induced insecticide resistance mechanisms, such as knockdown resistance (kdr) in mosquitoes. Sri Lankan Vector Controlling Entities (VCE) have been using a variety of pyrethroid insecticides as the primary approach for dengue control. However, development of any resistance among the Aedes mosquitoes has been limitedly studied in the country. Therefore, the current study was conducted to evaluate the prevalence of F1534C, V1016G, and S989P mutations among Ae. aegypti mosquito populations in three dengue endemic high-risk regions of Sri Lanka. Methodology. Immature (both pupae and larvae) stages of Ae. aegypti mosquitoes were collected from Colombo, Gampaha, and Kandy districts of Sri Lanka from February 2018 to December 2019. Polymerase Chain Reaction- (PCR-) based assay for molecular genotyping of mutations was performed to identify the prevalence of kdr mutations in collected Ae. aegypti populations, separately. The frequencies of the resistant and susceptible kdr alleles were determined by using the Hardy–Weinberg equilibrium. Results The Ae. aegypti populations from Colombo, Gampaha, and Kandy districts showed 46%, 42%, and 22% of F1534C mutation allele frequencies, along with 15%, 12%, and 6% of V1016G mutation allele frequencies, respectively. The mutation allele frequencies of S989 in Colombo, Gampaha, and Kandy districts were 9.5%, 8.5%, and 4.5%, respectively. The wild-type (PP) genotype remained predominant within all the three districts, whereas the homogenous (QQ) mutation genotype occurred only in minority. The abundance of Q allele frequency in Ae. aegypti mosquitoes was relatively higher for all the three mutations in Colombo. Conclusions The findings clearly indicate that long-term insecticide applications and multiple use of pyrethroids have led to the acquisition of kdr mutations, leading to the development of insecticide resistance among local Ae. aegypti populations, especially in the Colombo and Gampaha districts. Therefore, evaluation of the prevalence levels of these kdr mutations highlights the necessity for shifting towards novel vector control strategies.

Allelic Variation of Soybean Maturity Genes E1–E4 in the Huang-Huai-Hai River Valley and the Northwest China

Soybean is planted in a wide span of the world, and flowering and maturity time is an important trait determining soybean yield formation and adaptation. Maturity loci E1, E2, E3 and E4 were frequently reported as the most influential genetic loci for soybean flowering and maturity. To understand the allelic variation and assess the phenological traits of cultivars with different E allelic combinations in natural environments, 251 cultivars of maturity group (MG) I–V were field tested in 42 locations across four sub-regions in the Huang-Huai-Hai and Northwest region of China and genotyped with KASP markers for E1–E4 loci. The results indicated that mutant alleles were only found in the E1 and E2 locus, all of the cultivars carried functional alleles in the E3 and E4 loci in this area, with the frequency of mutant allele to be higher in early maturity groups (MGs) than late MGs. Among nine E allelic combinations in this area, one photoperiodic insensitive mutation in E2 loci (E1/e2-ns/E3-Ha/E4 and E1/e2-ns/E3-Mi/E4) made up the largest proportion (25.10 and 18.33%), while two photoperiodic insensitive mutations in both E1 and E2 loci (e1-as/e2-ns/E3-Ha/E4) (1.20%) occupied the lowest proportion in this panel. The major combinations of E locus for MGI, MGII and MG III in this area were E1/E2-dl/E3-Mi/E4, E1/e2-ns/E3-Mi/E4 and E1/e2-ns/E3-Ha/E4, respectively. Cultivars carrying e1-as/e2-ns/E3-Ha/E4 genotype flowered earliest (34 days) on average, 7.6 days earlier than the latest-flowering E haplotype (E1/e2-ns/E3-Ha/E4). This study provided an opportunity to detect the E allelic combinations in the Huang-Huai-Hai River Valley and the Northwest China, which would facilitate the improvement of soybean adaptation in the future.

Circulating tumor DNA (ctDNA) as a tool to help guide germline testing in patients with solid malignancies.

10602 Background: As the use of circulating tumor DNA (ctDNA) is more widely implemented, incidental identification of pathogenic variants reflecting germline alterations in cancer predisposition genes will occur more frequently. Such mutations are expected to have a high mutant allele frequency (MAF) or occur in genes typically associated with inherited syndromes. When a similar analysis was conducted by our group, we found that MAF of about 30% or greater in BRCA1/ 2 was associated with confirmed putative germline mutations in patients with breast cancer (Jacob et al &amp; Davis et al, SABCS 2020). In this study, we extended this analysis to non-breast malignancies. Methods: Patients with non-breast solid malignancies and ctDNA testing between 2015-2020 were retrospectively identified from Northwestern Medicine. All ctDNA was analyzed using Guardant 360 (Guardant Health, Inc. Redwood City, Ca). Patients with ctDNA samples with mutations at high MAF (&gt;30%) and those with BRCA1/2 mutations at any MAF were identified. We reviewed these charts for referral to genetic counselors and/or CLIA-approved germline testing. Descriptive analysis was reported for these findings. Genetic alterations were classified as pathogenic or of unknown significance based on OncoKB (Chakravarty et al, JCO PO 2017). Results: We identified ctDNA samples of 548 patients with non-breast solid malignancies, of whom 56 had gene mutations occurring at high MAF (&gt;30%). Predominant cancer subtypes were lung (48%), colorectal (21%), pancreatic (7%), ovarian (3.5%), prostate (3.5%), and gastroesophageal (3.5%). The most common gene mutations identified were TP53 (46%), BRCA1/2 (18%), EGFR (18%), APC (13%), and KRAS (9%). 87.5% were pathogenic and 12.5% were of unknown significance. 11 patients (19.6%) had germline testing of whom 6 tested positive. These germline mutations were in BRCA2 (n = 3), EGFR, APC, and TP53. In addition to the 10 patients with BRCA1/2 mutations at high MAF (&gt;30%), we identified 70 patients with BRCA1/2 mutations at low MAF ( &lt; 30%). 54% were pathogenic and 46% were of unknown significance. 11 patients (14%) had germline testing of whom 3 tested positive for BRCA2, all at high MAF. 1 patient with a BRCA2 mutation at low MAF of 1.4% tested positive for a different germline BRCA2 variant. Conclusions: In patients with advanced cancers, ctDNA analysis can reveal variants with MAF &gt;30% that are reflective of a germline mutation. Unfortunately the rate of genetic testing in these patients was low (20%). Future studies with germline testing in patients with high MAF variants would help understand the prevalence of germline variants. This can facilitate developing a more standardized approach for genetic counselor referral to identify families that may benefit from interventions for early detection or prevention of future cancers.

Overall survival and exploratory subgroup analyses from the phase 2 CodeBreaK 100 trial evaluating sotorasib in pretreated KRAS p.G12C mutated non-small cell lung cancer.

9003 Background: In the registrational phase 2 CodeBreaK 100 trial, sotorasib demonstrated an objective response rate (ORR) of 37.1% (95% Cl: 28.6, 46.2) and a median progression-free survival (PFS) of 6.8 months (95% Cl: 5.1, 8.2) in patients with pretreated KRAS p.G12C mutated non-small cell lung cancer (NSCLC). Tumor response was observed in patients bearing co-mutations in STK11, a driver of poor clinical outcomes with standard of care. Here, we report efficacy across an extended set of patient subgroups by key baseline characteristics and biomarkers. Methods: Sotorasib was given orally at 960 mg once daily to eligible patients who had advanced NSCLC harboring KRAS p.G12C and received prior standard therapies. Primary endpoint was ORR assessed by central review. Key secondary endpoints included PFS, overall survival, and safety. KRAS p.G12C mutant allele frequency (MAF) and tumor mutational burden (TMB) were analyzed by next-generation sequencing (NGS) using tissue samples. Mutational status of individual genes was determined by NGS using tissue and/or plasma samples. Correlations between response and KRAS p.G12C MAF, TMB, or co-mutations were analyzed in subsets of patients who had available respective results. Association between MAF and response was reported by odds ratio (95% CI), from a logistic regression with dependent variable of log odds of being a responder and an independent variable of MAF in a unit of 10%. Results: ORR across subgroups is presented in the Table. Response was independent of KRAS p.G12C MAF in the study population (odds ratio [95% CI]: 1.11 [0.88, 1.39]). OS remained immature. Conclusions: In the exploratory analyses of the phase 2 CodeBreaK 100 trial, the clinical benefit of sotorasib was observed across patient subgroups. Overall survival and updated exploratory analyses will be presented. Clinical trial information: NCT03600883. [Table: see text]

A first-in-human phase 1a/b trial of LY3484356, an oral selective estrogen receptor (ER) degrader (SERD) in ER+ advanced breast cancer (aBC) and endometrial endometrioid cancer (EEC): Results from the EMBER study.

1050 Background: Novel degraders and antagonists of ER are under evaluation in aBC, to overcome both ER mediated resistance and the bioavailability and dosing limitations of fulvestrant, the only approved SERD. ER is also overexpressed in ̃80% of EEC and endocrine therapy (ET) is utilized for these patients (pts). LY3484356, a novel, orally bioavailable SERD with pure antagonistic properties results in sustained inhibition of ER-dependent gene transcription and cell growth. Preclinically, LY3484356 shows favorable efficacy and pharmacokinetic (PK) properties, including antitumor activity in ESR1 mutants. Here we present the initial clinical data from EMBER, an ongoing first-in-human phase 1a/b trial of this novel agent. Methods: Phase 1a evaluated LY3484356 dose escalation (i3+3 design) in women with ER+, HER2- aBC (≤3 prior therapies for aBC following protocol amendment; prior ET sensitivity) and ER+ EEC (prior platinum therapy). Premenopausal women received a concomitant GnRH agonist. Key endpoints included determination of the recommended phase 2 dose, safety and tolerability, PK, and objective response rate and clinical benefit rate per RECIST v1.1. Results: As of the data cut (November 9, 2020), 28 pts (n = 24 aBC, n = 4 EEC) were enrolled at doses ranging from 200-1200 mg QD. Median age was 59 years (range, 35-80). Median number of prior therapies for aBC was 2 (range, 1-8; 6 pts enrolled prior to protocol amendment had received ≥4 prior therapies), including prior fulvestrant (46%), a CDK4/6 inhibitor (83%), and chemotherapy (33%). No dose-limiting toxicities were observed. Treatment-emergent adverse events (TEAEs) were mostly grade 1-2, including nausea (32%), fatigue (25%), and diarrhea (18%). The only grade 3 treatment-related AE was diarrhea (n = 1). TEAEs of bradycardia and QTc prolongation were not observed despite intensive central ECG monitoring. Dose-proportional increases in LY3484356 exposures were observed across all evaluated doses and t1/2 was 25-30 hours. At the starting dose level (200 mg QD), unbound LY3484356 exposures exceeded those achieved with fulvestrant. 16 of 28 pts were efficacy evaluable, with the remaining 12 pts ongoing prior to first scan. Among 16 evaluable pts, 11 (8 aBC, 3 EEC) had stable disease (10 pts ongoing), and 5 had progressive disease. RECIST responses were observed after the data cut and will be detailed at the meeting. Plasma ctDNA analysis indicated decreases in mutant allele frequencies, including mutant ESR1 in 9/12 (75%) evaluable pts across all dose levels. Conclusions: LY3484356 QD dosing shows favorable safety and PK properties, along with preliminary efficacy in pts with heavily pretreated ER+ aBC and EEC. Updated data will be presented at the meeting. Clinical trial information: NCT04188548 .

Phase II multicenter study of talazoparib for somatic BRCA1/2 mutant metastatic breast cancer.

TPS1110 Background: PARP inhibitors are approved for the treatment of HER2 negative metastatic breast cancer (MBC) with germline BRCA1/2 mutations, based on phase III studies demonstrating an improvement in progression-free survival (PFS) compared to chemotherapy in this population and better patient reported outcomes (Robson, NEJM, 2017; Litton, NEJM, 2018). However, germline BRCA1/2 mutations account for only 5-10% of breast cancer, limiting the current clinical applicability of PARP inhibitors. Somatic BRCA1/2 mutations are detectable in circulating cell-free DNA (cfDNA) in ̃13.5% of patients with MBC; in pre-clinical models, pathogenic somatic BRCA1/2 mutations have been shown to respond to PARP inhibition (Vidula, CCR, 2020). The purpose of this study is to evaluate the efficacy of talazoparib, a PARP inhibitor, in patients with MBC who have somatic BRCA1/2 mutations detectable in cfDNA, in the absence of a germline BRCA1/2 mutation, which we hypothesize will be effective in this setting. This study may help expand the population of patients with MBC who benefit from PARP inhibitors. Methods: This is an investigator initiated multicenter, single arm, phase II clinical trial studying the efficacy of talazoparib in 30 patients with MBC who have pathogenic somatic BRCA1/2 mutations detected in cfDNA. Patients with MBC who are found to have pathogenic somatic BRCA1/2 mutations detected in cfDNA in the absence of a germline BRCA1/2 mutation are eligible. Patients may have triple negative (with ≥ 1 prior chemotherapy), or hormone receptor positive/HER2 negative breast cancer (with ≥ 1 prior hormone therapy). Patients may have received any number of prior lines of chemotherapy, including a prior platinum (in the absence of progression). They must have adequate organ function and ECOG performance status ≤2, and should not have previously received a PARP inhibitor. Patients are treated with talazoparib 1 mg daily until disease progression or intolerability, with serial imaging using CT chest/abdomen/pelvis and bone scan performed at baseline and every 12 weeks, and cfDNA collection every 4 weeks. Primary endpoint is PFS by RECIST 1.1. Patients are being enrolled in a two-stage design with 80% power to demonstrate that the treatment is associated with “success” (PFS &gt; 12 weeks) in ≥53% patients (4% alpha). Secondary endpoints include objective response rate and safety (NCI CTCAE v 5.0). Exploratory analyses include studying serial changes in cfDNA BRCA1/2 mutant allelic frequency and comparing pre-and post-treatment cfDNA for the emergence of BRCA1/2 reversion and resistance mutations. This study is activated and open at Massachusetts General Hospital, where 2 patients are completing screening. It is also opening soon at 6 other academic centers (NCT03990896). Grant support includes a Pfizer ASPIRE award and 2020 Conquer Cancer Foundation of ASCO – Breast Cancer Research Foundation – Career Development Award. Clinical trial information: NCT03990896 .

Targeted ctDNA sequencing analysis reveals concurrent genomic alterations and its impact on TKI and immune checkpoint inhibitor therapy in advanced NSCLC from Asian population.

3042 Background: For advanced non-small cell lung cancer (NSCLC), liquid biopsy followed by targeted DNA sequencing is used in the clinical setting for driver-gene identification in circulating tumor DNA (ctDNA), tumor evolution and treatment monitoring. To understand the complexity and diversity of genetic alterations in advanced NSCLC, we examined the genetic landscape of late-stage NSCLC from an Asian population and its association with tyrosine kinase inhibitor (TKI) or immune checkpoint inhibitor (ICI) treatment. Methods: Plasma cell-free DNA (cfDNA) from 142 Asian patients with advanced NSCLC underwent real-world testing in a CAP-accredited and CLIA-certified central laboratory. We analyzed genetic alterations in cfDNA using an amplicon-based next-generation sequencing assay that covers 61 NSCLC related genes. 88 patients (62%) had received one or more lines of treatment. A similar cohort from Caucasian NSCLC was used for comparison (Blakely et al. Nat Genet 2017). Systematic co-mutation analysis, functional annotation and pathway enrichment analysis were performed. Results: The top 10 mutated genes in the Asian cohort were EGFR (66.2%), TP53 (48.6%), KRAS (11.3%), BRCA2 (7%), CTNNB1 (6.3%), PIK3CA (6.3%), SMAD4 (5.6%), BRCA1 (4.9%), MET (3.5%) and RB1 (3.5%). EGFR mutations occurred at hotspots in exons 18 – 21, with exon19 deletion, L858R, T790M, C797S, and G719A being the top-ranking alleles, similar to the Caucasian NSCLC cohort. Concurrent KRAS and EGFR mutations were higher in Asian cohort (8.5%) than in Caucasian cohort (0.56%). A rare PD-L1 structural variant (1.1%), reported to be related to immune evasion in other cancers (Kataoka et al. Nature 2016), was detected in the EGFR-mutated subgroup. Within the first line TKI-treated group, responders (CR+PR) harbored more alterations than non-responders (p &lt; 0.017). Among ICI-treated NSCLC patients (anti-PD-1/PD-L1 mono- or polytherapy), responders from 1st line or 2nd line ICI treatment harbored fewer mutations than non-responders, but the distribution of mutational allele frequency (MAF) in responders was shifted to the right (more clonal than subclonal mutations). Functional annotation suggests that concurrent mutated genes and copy number alterations in advanced EGFR-mutant NSCLC were enriched in cell cycle, DNA repair, PI3K-AKT signaling pathways. Conclusions: We characterized the ctDNA-derived genetic landscape of advanced NSCLC from an Asian population and dissected mutated genes with the outcome of TKI or ICI treatment. We also report a rare PD-L1 structural variant in the EGFR mutated subgroup, which could be associated with immune evasion. Our analyses support the occurrence of clonal and subclonal driver co-alterations in EGFR-driven NSCLC, underlining the clinical utility of ctDNA detection for NSCLC diagnosis and treatment selection.

The combination of EGFR-TKIs and anlotinib as a first-line therapy for EGFR-mutant advanced non-small cell lung cancer: A multicenter, single-arm, phase II clinical trial.

9030 Background: Currently,EGFR-TKIs are widely accepted as the standard treatment for EGFR- mutant advanced non-small-cell lung cancer (NSCLC); however, acquired resistance is inevitable. Combination therapy is considered as a strategy to overcome the resistance to EGFR-TKIs. Anlotinib, a novel multi-targeting, small-molecule TKI, has shown active to suppress tumor angiogenesis and growth. However, there is still a lack of evidence supporting the use of EGFR-TKIs in combination with anlotinib for the treatment of NSCLC until now. A multi-center, single-arm, phase II clinical trial was therefore designed to examine the efficacy and safety of EGFR-TKIs combined with anlotinib for treatment-naïve, advanced NSCLC patients, and unravel the possible mechanisms. Methods: This study was conducted in 14 research centers in Fujian, China. The main eligibility criteria were stage IV or relapsed nonsquamous NSCLC with EGFR mutations (exon 19 deletion,, and L858R), ECOG score 0-2,and age 20 to 75 years and no previous systemic treatment. Patients with asymptomatic brain metastases were admitted.Eligible patients were given gefitinib (250 mg QD) or icotinib (125 mg TID) in combination with anlotinib (10 mg per day, on days 1‒14; 21 days per cycle) until disease progression. The primary endpoint is progression-free survival (PFS) and safety, and the secondary endpoint is overall survival (OS), objective response rate (ORR) and disease control rate (DCR).Peripheral blood was sampled pre-treatment, once every two months during treatment and after disease progression, and T790M mutation was detected in plasma ctDNA using a droplet digital PCR (ddPCR) assay. Results: Of 60 patients enrolled (August 2, 2018 to May 28, 2020). As of February 1, 2021, 37 patients (61.7%) experienced PFS events and 10 (16.7%) died. The ORR was 78.3%, and the DCR was100%.Median PFS was 13.0 months (95%CI,10.7-15.3).The 5 most common treatment-related adverse events included rash (63.3%), fatigue (55.0%), hypertension (48.3%), diarrhea (33.3%) and hand-foot syndrome (30.0%), and grade 3 adverse events included hypertension (5.0%), rash (1.67%), hypertriglyceridemia (1.67%), vomiting (1.67%) and elevated ALT (1.67%); no grade 4 adverse events or drug-related deaths were observed. Peripheral blood samples were collected from 36 patients pre-treatment, and 30.6% were identified with low-frequency de novo T790M mutations, with the mutation-allele frequency (MAF) ranging from 0.01% to 0.28%. Conclusions: The combination of the first-generation EGFR-TKIs and anlotinib shows impressive ORR and DCR, and acceptable toxicity in treatment-naïve advanced NSCLC patients with activating EGFR mutations, and we observed a high proportion of patients harboring de novo EGFR T790M mutations in this study. Clinical trial information: NCT03720873.

IMerge: A phase 3 study to evaluate imetelstat in transfusion-dependent subjects with IPSS low or intermediate-1 risk myelodysplastic syndromes that are relapsed/refractory to erythropoiesis-stimulating agent treatment.

TPS7056 Background: Current treatment options for red blood cell (RBC) transfusion-dependent (TD) patients (pts) with lower risk (LR) myelodysplastic syndromes (MDS) relapsed after or refractory to erythropoiesis-stimulating agents (ESAs) have limited efficacy and durability; new approaches are needed. Imetelstat is a first-in-class telomerase inhibitor that targets cells with short telomeres and active telomerase, characteristics observed in MDS pts across all disease stages. IMerge (MDS3001) is a Phase 2/3 global study of imetelstat for TD pts with non-del(5q) LR MDS post ESA therapy. The results from Phase 2 part indicated that imetelstat achieved durable RBC transfusion independence (RBC-TI) and the most frequently reported adverse events were manageable and reversible grade ≥3 cytopenias. Among 38 pts with median follow-up of 24 months, 8-week, 24-week and 1-year TI rates were 42%, 32% and 29%, respectively; these responses were seen across different LR MDS subtypes. Median TI duration was 20 months and the longest TI was 2.7 years. A high and durable hematologic improvement-erythroid (HI-E) rate of 68% for a median duration of 21 months were also achieved. Reduction of variant allele frequency of mutations by imetelstat treatment was observed in some pts and correlated with clinical benefits (Platzbecker et al EHA 2020; Steensma et al JCO 2020). These results support the Phase 3 part of the trial. Methods: IMerge is two-part, Phase 2/3 study (ClinicalTrials.gov: NCT02598661). The Phase 3 part of the study is open for enrollment to adult pts with International Prognostic Scoring System (IPSS) low or intermediate-1 risk, non-del(5q) MDS who are TD, are relapsed after or refractory to ESAs, and have not received treatment with lenalidomide or hypomethylating agents. The study is a randomized (2:1) double-blind, placebo-controlled trial to compare efficacy of imetelstat vs placebo that will enroll approximately 170 pts and will be conducted at approximately 120 centers in North America, Europe, Asia and Middle East. Imetelstat is administered as 2-hour IV infusion every 4 weeks at 7.5 mg/kg. The primary endpoint of the study is to assess the rate of RBC-TI lasting ≥8 weeks. Secondary endpoints include safety, rate of RBC-TI ≥24 weeks, time to RBC-TI start, RBC-TI duration, rate of HI-E, the amount and relative change in RBC transfusions, rate of CR or PR, overall survival, progression of MDS, pharmacokinetics, and quality of life. Biomarkers relevant to the mechanism of action of imetelstat will be assessed to demonstrate target inhibition and their association with clinical responses. Cytogenetics and mutation analyses will be performed to evaluate the impact of imetelstat on reduction/depletion of malignant clones leading to disease modification. The study is currently recruiting pts. Clinical trial information: NCT02598661.

Molecular profiling by NGS upon progression disease in advanced stage NSCLC patients.

e21196 Background: Non-small cells lung cancer (NSCLC) patients treated with Tyrosine Kinase Inhibitors (TKI) at first-line ultimately develop disease progression after 10 to 14 months. Mechanisms underlying TKI-resistance are not complete understood. Tumor re-biopsy is often unfeasible for NSCLC patients and liquid biopsy is a suitable alternative in identify mechanism by which tumors progress. Methods: 113 plasma samples were collected from advanced stage EGFR positive NSCLC patients upon disease progression according to RECIST V1.1. cfDNA NGS profiling was carried out using the Oncomine™ Pan-Cancer Cell-Free Assay and sequenced on an Ion GeneStudio S5 Plus System. Variant calling and annotation were performed with Torrent Suite and Ion Reporter (v5.16) software, respectively. In addition, we developed a bioinformatic pipeline, using RStudio for variant filtering. Results: Overall, 344 somatic variants were detected with an average number of variants per patient of 2.32 and a median Mutant Allele Frequency (MAF) of 0.83%. Most mutated genes were EGFR (63.72%), TP53 (60.18%), APC (16.81%), MAP2K1 (11.50%), PIK3CA (10.62%), FGFR3 (7.08%), KRAS (7.08%) and BRAF (6.19%). As expected, SNPs were the most frequent mutation type (72.67%), following by INDELs (24.41%) and CNVs (2.91%). We found high co-occurrence between MYC-GNA11, MYC-CCND2, HRAS-AR and EGFR-TP53 genes using Jaccard’s score (0.5, 0.5, 0.5 and 0.46, respectively). Within EGFR-mutated patients, 40% had T790M resistance mutation. Finally, we found KRAS mutations and missense PIK3CA mutations were mutually exclusive. Conclusions: Molecular profiling of liquid biopsies collected upon disease progression using NGS help to identify molecular mechanisms underlying treatment failure.

Ultrasensitive Detection of BRAF Mutations in Circulating Tumor DNA of Patients With Metastatic Thyroid Cancer

Background: Liquid biopsy is a promising technology that can offer various advantages for molecular testing over tissue-based approaches. Most studies trying to address the utility of liquid biopsy in thyroid cancer have failed so far to achieve satisfactory rates of detection of relevant mutations. In this study, we examined a newly developed approach for ultrasensitive detection of oncogenic mutations in thyroid cancer using BRAF mutation as a proof-of-concept. In an exploratory analysis, we also correlated our findings with clinical outcomes and with levels of standard of care biomarkers. Methods: We included a group of patients with metastatic thyroid carcinoma. Cell free DNA (cfDNA) was isolated from an average of 2 ml of plasma and from matched formaldehyde fixed paraffin tissue blocks (FFPB) that were obtained from prior surgery. Extracted DNA was subject to preamplification of mutant copies using Q5 High-Fidelity PCR kit. Digital droplet PCR was performed on pre-amplified purified DNA where BRAF mutated allele frequencies (AF) were measured using BioRad ddPCR Qx200. Results: Thirty-three patients were included in our study with a median age at diagnosis of 62. Our method achieved a sensitivity of detection of 47.6% and a specificity of 80%. Mutant BRAF V600E was detected in cfDNA of 54.5% of patients (n=18) compared to 80.8% in isolated DNA from matched FFPB. Median overall survival (OS) was shorter in patients with wild type (WT) BRAF in both ctDNA and tissue (127m vs 218m, p=0.015; 116m vs 223m, p=0.004). Thyroglobulin (Tg) levels did not correlate with BRAF mutations either quantitatively or qualitatively. In the papillary thyroid carcinoma-classic variant cohort (n=20), however, patients with cfDNA mutant BRAF were more likely to have elevated Tg (90.9% versus 44.4% respectively, p=0.05). Conclusions: Our study provided a proof of concept for a newly developed technique to provide high sensitivity of mutation detection in thyroid cancer. The achieved sensitivity of detection is the highest to date using liquid biopsy in this tumor type. While we addressed only BRAF mutations in our study, the same approach can potentially be used for other mutations as well, likely changing the paradigm for use of liquid biopsy in thyroid cancer.

Longitudinal Relationship between Idylla Plasma ctBRAF V600 Mutation Detection and Tumor Burden in Patients with Metastatic Melanoma.

Circulating tumor DNA (ctDNA) may complement radiography for interim assessment of patients with cancer. Our objective was to explore the relationship between changes in plasma ctDNA versus radiographic imaging among patients with metastatic melanoma. Using the Idylla system, we measured B-Raf proto-oncogene (BRAF) V600 ctDNA in plasma from 15 patients with BRAF V600E/K-positive primary tumors undergoing standard-of-care monitoring, including cross-sectional computed tomography (CT) imaging. BRAF V600 mutant allele frequency (%MAF) was calculated from the Idylla Cq values and directly measured using droplet digital polymerase chain reaction (ddPCR). The Idylla ctDNA assay demonstrated 91% sensitivity, 96% specificity, 91% positive predictive value, and 96% negative predictive value for the presence of > 93 mm metastatic disease. Qualitative ctDNA results corresponded to changes in RECIST (Response Evaluation Criteria in Solid Tumors) 1.1 status determined by CT imaging in 11 of 15 subjects (73%). Calculated %MAF results correlated with ddPCR (R2 = 0.94) and provided evidence of progressive disease 55 and 97 days in advance of CT imaging for two subjects with persistently positive qualitative results. Overall, interim ctDNA results provided evidence of partial response or progressive disease an average of 82 days before radiography. This pilot study supports the feasibility of using the Idylla plasma BRAF V600 ctDNA assay as a complement to CT scanning for routine monitoring of therapeutic response. Somatic mutation quantification based on Cq values shows promise for identifying disease progression and warrants further validation.

TP53 mutation and MET amplification in circulating tumor DNA analysis predict disease progression in patients with advanced gastric cancer.

BackgroundGastric cancer (GC) is a heterogeneous disease that encompasses various molecular subtypes. The molecular mutation characteristics of circulating tumor DNA (ctDNA) in advanced gastric cancer (AGC), especially the clinical utility of TP53 mutation and MET amplification in ctDNA need to be further explored.ObjectivesThe aim of this study was mainly to assess the clinical utility of TP53 mutation and MET amplification in ctDNA as biomarkers for monitoring disease progression of AGC.Patients and MethodsWe used multigene NGS-panel technology to study the characteristics of ctDNA gene mutations and screen the key mutant genes in AGC patients. The Kaplan-Meier method was used to calculate the survival probability and log-rank test was used to compare the survival curves of TP53 mutation and MET amplification in ctDNA of AGC patients. The survival time was set from the blood test time to the follow-up time to observe the relationship between the monitoring index and tumor prognosis.ResultsWe performed mutation detection on ctDNA in 23 patients with AGC and identified the top 20 mutant genes. The five most frequently mutated genes were TP53 (55%), EGFR (20%), ERBB2 (20%), MET (15%) and APC (10%). TP53 was the most common mutated gene (55%) and MET had a higher frequency of mutations (15%) in our study. Kaplan-Meier analysis showed that patients with TP53 mutant in ctDNA had shorter overall survival (OS) than these with TP53 wild (P < 0.001). The Allele frequency (AF) of TP53 mutations in patient number 1 was higher in the second time (0.94%) than in the first time (0.36%); the AF of TP53 mutations in patient number 16 was from scratch (0∼0.26%). In addition, the AF of TP53 mutations in patients who survive was relatively low (P = 0.047). Simultaneously, Kaplan-Meier analysis showed that patients with MET amplification also had shorter OS than these with MET without amplification (P < 0.001).ConclusionTP53 and MET are the two common frequently mutant genes in ctDNA of AGC patients.TP53 mutation and MET amplification in ctDNA could predict disease progression of AGC patients.

High concordance of actionable genomic alterations identified between circulating tumor DNA-based and tissue-based next-generation sequencing testing in advanced non-small cell lung cancer: The Korean Lung Liquid Versus Invasive Biopsy Program.

Because of the growing number of actionable biomarkers in non-small cell lung cancer (NSCLC), sufficient tissue availability for testing is becoming a greater challenge. Liquid biopsy offers a potential solution by complementing standard tissue-based methods. In this study, the authors analyzed the concordance of actionable genomic alterations sequenced from circulating tumor DNA (ctDNA; Guardant360) and tissue (Oncomine Focus Assay). From September 2015 to May 2018, 421 paired plasma and tissue samples from patients with advanced NSCLC who had previously undergone tissue testing by standard methods were collected. Both types of samples were available for 287 patients (262 in cohort 1 [treatment-naive] and 25 in cohort 2 [treatment failure]), and only 1 sample type was available for 134 patients (50 in cohort 3 [plasma only] and 84 in cohort 4 [tissue only]). In cohort 1, 198 samples (77.6%) showed concordance between tissue and plasma next-generation sequencing (NGS). Among the discordant cases, plasma testing detected additional genomic alterations in 11 patients (4.2%). In 50 patients without tissue-based NGS results (cohort 3), the ctDNA-based test detected genomic alterations in 20 samples (40.0%). The median allele frequency (AF) of mutations identified with ctDNA-based NGS (0.74%) was lower than that identified with the tissue-based NGS test (13.90%). Clinical responses to matched targeted therapy occurred, regardless of the ctDNA AF. Upfront ctDNA-based testing identified 60.4% of patients with genomic alterations. In addition, ctDNA-based testing uncovered 12.0% more actionable alterations when it was performed after tissue-based NGS testing. The results indicate that a ctDNA-based test identifies additional patients with actionable genomic alterations and could, therefore, be used to complement traditional tissue-based testing for NSCLC. Circulating tumor DNA (ctDNA)-based next-generation sequencing (NGS) testing is becoming essential as the number of actionable genomic biomarker increases for the treatment selection of non-small cell lung cancer. This study demonstrates the additive value of ctDNA-based testing in addition to tissue-based NGS and standard of care-based biomarker testing for detecting additional patients with actionable genomic alterations. Clinical responses have also been observed in patients with a low allele frequency detected by ctDNA-based NGS testing.