Resistance Mutations In Patients Research Articles

HIV protease resistance mutations in patients receiving second-line antiretroviral therapy in Libreville, Gabon

IntroductionIn 2022, the WHO reported that 29.8 million people around the world were living with HIV (PLHIV) and receiving antiretroviral treatment (ART), including 25‌ 375 people in Gabon (54% of all those living with HIV in the country). The literature reports a frequency of therapeutic failure with first-line antiretrovirals (ARVs) of between 20% and 82%. Unfortunately, data relating to the failure of second-line ARVs are scarce in Gabon. This study aims to determine the profiles of HIV drug resistance mutations related to protease inhibitors in Gabon.MethodologyPlasma from 84 PLHIV receiving ARVs was collected from 2019 to 2021, followed by RNA extraction, amplification, and sequencing of the protease gene. ARV resistance profiles were generated using the Stanford interpretation algorithm version 8.9-1 (https://hivdb.stanford.edu) and statistical analyses were performed using EpiInfo software version 7.2.1.0 (CDC, USA).ResultsOf 84 HIV plasma samples collected from 45 men and 39 women, 342 mutations were detected. Of these, 43.3% (148/342) were associated with nucleoside reverse transcriptase inhibitors (NRTIs), 30.4% (104/342) with non-nucleoside reverse transcriptase inhibitors (NNRTIs), and 26.3% (90/342) with protease inhibitors (PIs). Most NRTI mutations were associated with thymidine analogues (TAMs) (50.7%; 75/148), including T215F/V (14.9%; 22/148), D67DN/E/G/N/T (10.1%; 15/148), M41L (9.5%; 14/148), and K70E/KN/S/R (9.5%; 14/148). Resistance mutations related to non-TAM NRTIs (33.1%; 49/148) were M184V (29.1%; 43/148), and L74I/V (8.1%; 12/148). NNRTI mutations were predominantly K103N/S (32.7%; 34/104), V108I (10.6%; 11/104), A98G (10.6%; 11/104), and P225H (9.6%; 10/104). Minor mutations associated with PIs (60.0%; 54/90) were predominantly K20I (15.6%; 14/90) and L10F/I/V (14.5%; 13/90). The major mutations associated with PIs (40.0%; 36/90) were M41L (12.2%; 11/90), I84V (6.7%; 06/90), and V82A (6.7%; 06/90). The four most prescribed therapeutic regimens were TDF + 3TC + LPV/r (20.3%; 17/84), ABC + DDI + LPV/r (17.9%; 15/84), TDF + FTC + LPV/r (11.9%; 10/84), and ABC + 3TC + LPV/r (11.9%; 10/84).ConclusionThis study revealed that HIV drug resistance mutations are common in Gabon. The major mutations associated with PIs were M41L, I84V, and V82A. There is a need for access to new NRTIs, NNRTIs, and PIs for a better therapeutic management of PLHIV in Gabon.

Abstract B102: Development of covalent inhibitors targeting Ras Q61 hotspot mutants

Abstract Mutations in the proto-oncogenes RAS are a key driver of various cancers, including pancreatic, colorectal, non–small cell lung cancer (NSCLC), melanoma, and thyroid cancer. Despite being considered undruggable for many years, recent efforts have led to the FDA approval of two drugs, Sotorasib and Adagrasib. They covalently target the K-Ras mutant isoform K-Ras(G12C), thereby inhibiting its oncogenic effect. However, the use of these drugs is inherently limited to cancers driven by the K-Ras(G12C) mutant, which is very common only in NSCLC. Although other covalent inhibitors targeting the K-Ras mutants K-Ras(G12S) and K-Ras(G12R) have been developed, it has so far not been possible to target the other frequently mutated amino acid Q61. Here we present our approach for the development of novel covalent inhibitors specifically targeting Q61 mutations in Ras proteins, which are prevalent in melanoma, multiple myeloma, and thyroid cancer. We demonstrate how covalent inhibitors specifically react with the mutant Q61C in both K and H-Ras proteins as proof-of-concept and show our efforts to extend this to the other Q61 mutant isoforms. We base our strategy on the modification of well-established switch-II pocket K-Ras inhibitors and explore multiple novel scaffolds specifically designed to target mutations of Q61. In addition to its occurrence in various cancers, Ras(Q61X) is also a common resistance mutation in patients treated with MAPK inhibitors. This work could further increase the number of patients potentially benefiting from Ras(Q61X) targeting drugs. Citation Format: Julius Pampel, Johannes Morstein, Qinheng Zheng, David M. Peacock, Kevan M. Shokat. Development of covalent inhibitors targeting Ras Q61 hotspot mutants [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B102.

Potential antiviral drug resistance mutations in patients with treatment-naive chronic hepatitis B

Potential antiviral drug resistance mutations in patients with treatment-naive chronic hepatitis B

Detection of Salmonella DNA and drug-resistance mutation by PCR-based CRISPR-lbCas12a system

Salmonella is an important foodborne pathogen, which can cause serious public health problems. Rapid and accurate detection of Salmonella infection and drug resistance mutations in patients will provide timely guidance for clinical treatment and avoid disease progression and other related clinical problems. Here, we established a highly sensitive and quick method for Salmonella and drug resistance mutation detection based on polymerase chain reaction (PCR) and CRISPR-lbCas12a system and evaluated its practicability with clinical samples.Specific CRISPR RNAs (crRNAs) and primers are designed for Salmonella DNA and parC gene S80I mutation diagnosis. CrRNAs with and without phosphorylated modification and different crRNA preparation methods are used to assess the effect on the detection system. After optimization, we detected as low as one copy of Salmonella DNA and drug resistance mutation parC S80I with the Salmonella DNA standard. For 94 clinical samples, this method also showed high sensitivity (100%, 95% CI: 84.98–100%) and specificity (98.48%, 95% CI: 90.73–99.92%) with less time (3 h) than plate culture (16 h) and conventional antimicrobial susceptibility testing (over 16 h). Besides, one parC S80I mutant strain was detected, which is consistent with the result of DNA sequencing. Taken together, we established a highly sensitive and specific method for Salmonella infection and parC S80I drug resistance mutation detection with fewer reagents and ordinary instruments. This assay has wide application prospects for fast detection of pathogen (bacterium and virus) infection, drug resistance determination, and proper treatment guidance.

Detection of resistance mutations in patients with anaplastic lymphoma kinase-rearranged lung cancer through liquid biopsy.

Tyrosine kinase inhibitors (TKIs) significantly improve clinical outcomes in patients with non-small cell lung cancer due to anaplastic lymphoma kinase (ALK) gene rearrangement. However, the rate of relapse with TKIs is high owing to the development of resistance mutations during treatment. Repeated biopsies during disease progression are crucial for elucidating the molecular mechanisms underlying the development of resistance to ALK inhibitors. Analysis of cell-free DNA (cfDNA) obtained from plasma is a novel approach for tumor genotyping. In this mixed prospective and retrospective observational cohort study, we investigated the clinical feasibility of continuous quantitative monitoring of ALK-acquired mutations in plasma obtained from patients with ALK+ non-small cell lung cancer by using a highly sensitive and specific droplet digital polymerase chain reaction (ddPCR) assay. We enrolled nine patients, including three treatment-naïve patients recently diagnosed with ALK+ non-small cell lung cancer via tissue biopsy and expected to receive ALK TKIs and six patients already receiving ALK TKIs. Plasma samples were collected from these patients every 3 months. cfDNA was extracted from 66 samples during the study period, and 10 ALK mutations were simultaneously evaluated. The numbers of samples showing the G1202R, C1156Y, G1269A, F1174L, T1151ins, and I1171T mutations were 32, 16, 5, 4, 1, and 1, respectively. The L1196M, L1152R, V1180L, and S1206Y mutations were not detected. Correlation analyses between progression-free survival and the time from treatment initiation (or treatment modification) to the detection of resistance mutations revealed that although resistance mutations may occur before a drug change becomes necessary, there is a duration during which the disease does not progress. Our findings suggest that real-time quantitative monitoring of ALK resistance mutations during the response period could provide a time course of changes while acquiring resistance mutations. This information would be beneficial for designing an appropriate treatment strategy.

Prevalence of Genotypic Resistance Mutations in Patients with Chronic Hepatitis B (HBV) Treated with Entecavir and Tenofovir in Two Reference Centers in the North and Northeast of Brazil

Mutations of genotypic resistance to nucleotide analogues Entecavirand Tenofovirhave been described in patients undergoing treatment and virgins for hepatitis B virus. The present study demonstrated in a sample of 263 patients with chronic HBV from the North and Northeast of Brazil, a mutation rate of resistance to nucleotide analogues of 3.8% (10). Of the 10 patients who had genotypic resistance mutations, only 1 had no genotypic resistance mutation for the first line treatment for hepatitis B, entecavir and tenofovir.Due to the emergence of vaccine escape mutations and resistance mutations to antiviral treatment, and the severity of liver disease caused by HBV, screening for genetic mutations is important due to the impact on therapeutic management.

Prevalence and Spectrum of HIV-1 Resistance Mutations in the Siberian Federal District.

The Siberian Federal District is among the most affected regions with a high prevalence of HIV-infection and is characterized by high HIV-infection incidence rate and high mortality among the HIV-infected population. HIV drug resistance poses a major threat to public health and is associated with increased mortality, HIV incidence, and cost of epidemic control programs. A total of 1281 samples from HIV-infected patients were sequenced and analyzed with the DEONA and HIVdb Program to assess the prevalence of drug resistance mutations in patients in the Siberian Federal District in 2016–2018. The federal surveillance data obtained from 0.5% of HIV-infected patients during the long-term follow-up care in 2021 were also used. The incidence rate of HIV infection in the Siberian Federal District has declined since 2016: from 135.8 per 100 thousand population to 81.1 per 100 thousand population in 2021. Mutations associated with resistance to NRTI and NNRTI were found in 10.3% of the samples in 2016–2018 and in 28.4% of the samples in 2020. The rising prevalence of drug resistance in HIV-infected patients indicates that it is increasingly important to continuously monitor and improve the approaches to the use of effective treatment regimens.

953P EGFR mutation testing from pleural effusions of advanced lung adenocarcinoma patients in Serbia

In Serbia, EGFR mutation testing is performed from liquid biopsy of lung cancer patients only by qPCR. The aim of this study was to evaluate the efficacy of this approach in pleural effusions of patients at diagnosis and after progression on EGFR-TKIs. Mutation testing was performed from blood and pleural effusions of advanced lung adenocarcinoma patients (stage IIIB or IV, ECOG performance status 0, 1, 2) using the Cobas® EGFR Mutation Test. In the period from 2016-2021, 124 patients were tested at baseline (53 % males, 47 % females, age range 37-83 years, median 61), and 104 after progression on first-line EGFR-TKIs (41 % males, 59 % females, age range 34-81 years, median 60). At diagnosis, 9 mutated samples were detected (7.3 %) with a turnaround time of 2 working days, and a 99.2 % testing success rate. At progression, an accordance rate of only 67 % with the initial driver mutation was observed from blood. The T790M mutation was detected in 34 patients (49 % of the mutated samples, 33% of total) which rendered them eligible for third-generation EGFR-TKIs. In the same period, only 6 samples (4.8 %) of pleural effusions were sent to our Laboratory along with blood, all from patients at progression. The testing was performed with a turnaround time of 2 working days, a 100 % testing success rate, and an accordance rate of 100 % with the initial driver mutation. In 3 samples (50 %), the T790M mutation was detected while the corresponding blood sample showed only the presence of the driver mutation. EGFR mutation testing from pleural effusion has proven valuable as an alternative liquid biopsy sample to blood, for detecting the T790M acquired resistance mutation in patients who progressed on first-line EGFR-TKIs in Serbia.

Abstract P3110: Mechanism Of Cardiotoxicity Associated With Tyrosine Kinase Inhibitor Osimertinib

Background: Osimertinib is a third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) for non-small cell lung cancer (NSCLC) treatment. Osimertinib potently and selectively inhibits EGFR-T790M resistance mutations in patients with NSCLC harboring these mutations. Emerging evidence from clinical trials has identified potential cardiotoxic effects of osimertinib including heart failure, atrial fibrillation (AF), and QT interval prolongation. The mechanisms underlying osimertinib-induced cardiotoxicity, however, are poorly understood. Methods: To investigate potential mechanisms of osimertinib-induced cardiotoxicity, we tested the drug in both in vitro cultured cardiomyocytes (CMs) and wild-type C57BL/6 mice in vivo , corresponding to doses used in preclinical development. Echocardiography was performed in mice to assess the effect on cardiac function. In vivo electrophysiology studies were also performed to assess effects on osimertinib-induced AF and QT prolongation susceptibility. Results: Osimertinib (500 nM, 72 hours) reduced CM viability in NRVMs (p=0.0001), and induced apoptosis as determined by TUNEL assay (p=0.004) and upregulated Bax/Bcl-2 ratio (mRNA: 1.45-fold, p=0.001). In wild-type C57BL/6 mice, osimertinib (5 mg/Kg/day, 4 weeks) decreased ejection fraction (37.15±3.99% vs control: 51.04±2.23%, p=0.0001) and fractional shortening (17.69±2.18% vs control: 25.56±1.51%, p=0.0001). This was associated with an increase in cardiac BNP expression (mRNA: 1.72-fold, p=0.005) and apoptotic Bax/Bcl-2 ratio (protein: 1.85-fold, p=0.02). Electrophysiology studies also demonstrated 2:1 atrioventricular block (p=0.03) in osimertinib treated mice and increased AF susceptibility. A combination of in vitro and in vivo mechanistic analyses demonstrated that osimertinib significantly inhibits pro-survival ERK signaling pathway. Conclusions: Our results demonstrate that the cardiotoxicity seen with osimertinib in clinical trials is largely recapitulated in preclinical models, and that osimertinib can directly induce CM death. These models may provide a foundation for further investigating mechanism of osimertinib-induced cardiotoxicity and interventions to mitigate it.

Detection of Patient HIV-1 Drug Resistance Mutations in Russia's Northwestern Federal District in Patients with Treatment Failure.

Highly active antiretroviral therapy (HAART) is currently a combination of three (less frequently four) antiretroviral drugs; these target pathways involved in various stages of HIV replication in the body. Treatment failure is a problem facing doctors and patients using HAART. The most common cause of therapeutic failure is the development of HIV drug resistance. The emergence of resistance is associated with processes involving mutation occurring in the viral genome under the influence of evolutionary factors. Sequencing reactions were performed using the AmpliSens HIV Resist-Seq. Assembly of consensus sequences from fragments obtained during sequencing was carried out using Unipro UGENE softwar. Isolate genotyping was performed using the MEGA-X software with the Neighbor-joining algorithm. According to the analysis, 72.05% of patients had at least one significant mutation associated with drug resistance for the corresponding viral subtype. HIV-1 A6 remains the predominant HIV-1 genetic variant in Russia’s Northwestern Federal District. Among samples with drug resistance mutations, in all cases, mutations associated with pharmacological resistance to two or three drug groups were found. Given the high incidence of resistance mutations in patients on ineffective ART, surveillance of HIV-1 drug resistance, in both ART-receiving and ART-naive individuals, appears necessary. A lack of vigilance and control measures may lead to the spread of primary ART-resistant HIV strains.

Detection of antiviral drug resistance in patients with congenital cytomegalovirus infection using long-read sequencing: a retrospective observational study

BackgroundCongenital human cytomegalovirus (cCMV) infection can cause sensorineural hearing loss and neurodevelopmental disabilities in children. Ganciclovir and valganciclovir (GCV/VGCV) improve long-term audiologic and neurodevelopmental outcomes for patients with cCMV infection; however, antiviral drug resistance has been documented in some cases. Long-read sequencing can be used for the detection of drug resistance mutations. The objective of this study was to develop full-length analysis of UL97 and UL54, target genes with mutations that confer GCV/VGCV resistance using long-read sequencing, and investigate drug resistance mutation in patients with cCMV infection.MethodsDrug resistance mutation analysis was retrospectively performed in 11 patients with cCMV infection treated with GCV/VGCV. UL97 and UL54 genes were amplified using blood DNA. The amplicons were sequenced using a long-read sequencer and aligned with the reference gene. Single nucleotide variants were detected and replaced with the reference sequence. The replaced sequence was submitted to a mutation resistance analyzer, which is an open platform for drug resistance mutations.ResultsTwo drug resistance mutations (UL54 V823A and UL97 A594V) were found in one patient. Both mutations emerged after 6 months of therapy, where viral load increased. Mutation rates subsided after cessation of GCV/VGCV treatment.ConclusionsAntiviral drug resistance can emerge in patients with cCMV receiving long-term therapy. Full-length analysis of UL97 and UL54 via long-read sequencing enabled the rapid and comprehensive detection of drug resistance mutations.

Abstract P5-13-29: Analytical and clinical validation of a ctDNA assay for detecting copy number loss and structural rearrangement variants contributing to homologous recombination and repair (HRR) deficiency

Abstract Background: Inactivating HRR gene mutations can lead to HRR deficiency (HRD) and predict response to PARPi therapy in patients with breast cancer. Copy number loss and large genomic rearrangements (LGR) can result in HRD but are challenging to detect in ctDNA. Here, we present the analytical validation of homozygous deletions, loss of heterozygosity (LoH) and LGR detection on the Guardant360 (G360) liquid biopsy panel, previously validated for detection of small variants, copy number amplifications, and fusions. We present real-world outcomes of BRCA1/2-mutant PARPi-treated patients to demonstrate the clinical validity of the detected variants. Methods: Analytical validation was performed using the G360 83-gene ctDNA panel. Cell line DNA and clinical patient cfDNA were titrated into matched normal cell line DNA or healthy donor cfDNA to establish the limit of detection (LoD) and precision for copy number loss and LGRs, respectively. Accuracy results were compared to those from an orthogonal, externally validated tissue and ctDNA panel. De-identified, longitudinal, claims data were linked to the cancer genomic profiles in Guardant INFORM, a clinical-genomics database. Advanced PARPi treated breast cancer patients with an inactivating or reversion BRCA1/2 mutation detected by G360 were assessed. Results: The analytical sensitivity (95% LoD) for detecting homozygous and LoH deletions for deletion sizes >10MB was established at tumor fractions (TF) of 12.5% and 25%, respectively. The 95% LoD for LGRs was 0.2% variant allele fraction (VAF). The per-sample false positive rate for copy number loss and LGRs was <0.5%. Prevalence of BRCA1/2 homozygous deletions, LoH and LGRs in >1000 advanced breast cancer patients was 1.8%, 16.6% and 0.25% respectively, compared to 2.4%, 56.7% and 0.3% in TCGA. To verify the clinical impact of cfDNA-detected HRR alterations, overall survival was determined for PARPi-treated patients with >1 BRCA1/2 germline or somatic SNV, indel or LGR reversion mutation to be 23.2 months [16.4, 30, CI, n=75] compared to 54.4 [28, NA, CI, n=14] months for BRCA1/2-mutant patients without a reversion (p-value=0.049). Conclusion:. This analytical validation demonstrates that G360 detection of inactivating mutations, including copy number loss and LGRs, is highly sensitive, reliable and robust. Real-world evidence analysis confirmed worse survival outcomes in PARPi treated patients harboring a BRCA1/2 reversion compared to BRCA1/2-mutant patients with no reversion. This data further supports ctDNA as a compelling non-invasive means to identify potential PARPi sensitizing and resistance mutations in patients with advanced breast cancer. Citation Format: Jennifer Yen, Leylah Drusbosky, Caroline Weipert, Nicole Zhang, David Hanna, Catalin Barbacioru, Hao Wang, Alex Artyomenko, Arielle Yablonovitch, Yu Fu, Aaron Hardin, Nagesh Alla, Robert Foley, Max Maligska, Bhargavi Panchangam, Phil Yen, Jane Meisel, Neelima Vidula, Massimo Cristofanilli, Jeremy Force, Michael Dorschner, Martina Lefterova, Elena Helman, Becky Nagy, Darya Chudova, AmirAli Talasaz. Analytical and clinical validation of a ctDNA assay for detecting copy number loss and structural rearrangement variants contributing to homologous recombination and repair (HRR) deficiency [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 P5-13-29.

Prevalence of HIV Infection and Resistance Mutations in Patients Hospitalized for Febrile Illness in Indonesia.

HIV prevalence in Indonesia is increasing, and only 64% of infected individuals know their status. In a prospective cohort of 1,453 hospitalized patients with unexplained fever, 46 (3.2%) had HIV, including 15 (1.1%) patients without a prior HIV diagnosis. Among 31 subjects previously known to have HIV, 21 (68%) had been receiving combination antiretroviral therapy (cART) at the time of enrollment. Of 39 HIV cases with HIV RNA levels ≥ 100 copies/mL, sequencing for genotype analysis and resistance testing was successful in 30 (77%) subjects. The most common HIV subtypes were AE (90%) and B (10%). Five (16.7%) subjects had resistance mutations to nucleoside and non-nucleoside reverse transcriptase inhibitors, and all of them were on cART. No evidence of transmitted drug resistance was found in newly diagnosed individuals. Hospital-based screening may be an efficient method to expand HIV testing and identify a significant number of new cases. Access to care, close monitoring, expansion of anti-retroviral options, and ensuring availability of CD4 determinations, viral load testing, and genotyping are crucial to control of the epidemic in Indonesia.

16P Evaluation of the relationship between clinicopathological features at diagnosis and acquisition of T790M resistance mutation in patients with EGFR-mutant metastatic lung cancer

Data of the acquisition of T790M resistance mutation in patients with EGFR-mutant metastatic lung cancer is limited. This study aimed to assess the relationship between clinicopathological features at diagnosis, and acquisition of T790M mutation in patients with EGFR-mutant metastatic non-small cell lung cancer. We evaluated the EGFR-mutant metastatic lung cancer patients' data who progressed under first-line treatment with tyrosine kinase inhibitors and acquired T790M resistance mutation retrospectively. Survival analyses were assessed with Kaplan-Meier and Cox-regression methods. The relationship between the acquisition of T790M mutation and clinicopathological parameters was evaluated with logistic regression analysis. Fifty-two patients were included in the study. The median age was 58 (range, 33-78) years. The proportion of female patients was 53.2%. The proportions of exon 19, exon 21, and rare mutations were 68.7%, 23.5%, and 7.8%, respectively. Forty-five (86.5%) patients were de-novo metastatic. The proportion of patients who had one, two, and three or more metastatic sites at diagnosis were 25.5%, 41.3%, and 33.2%, respectively. The proportio of brain, liver and adrenal gland metastasis were 29.4%, 13.7%, and 7.8%, respectively. All patients received tyrosine kinase inhibitors. After the disease progressed, the acquisition of T790M mutation was detected with liquid (75.5%) or standard biopsies (24.5%). T790M mutations were detected in 33 (63.5%) patients. In logistic regression analysis, age, gender, de-novo metastatic disease, number of metastatic sites, primary tumor localization (left or right lung), and type of tyrosine kinase inhibitor was not statistically significant for the acquisition of T790M mutations. Due to rarity, the data on the acquisition of T790M mutations are limited. In this study, we showed that clinicopathological features were not related to the acquisition of T790M mutations.

Circulating tumor DNA for comprehensive noninvasive monitoring of lymphoma treated with ibrutinib plus nivolumab

AbstractTo advance the use of circulating tumor DNA (ctDNA) applications, their broad clinical validity must be tested in different treatment settings, including targeted therapies. Using the prespecified longitudinal systematic collection of plasma samples in the phase 1/2a LYM1002 trial (registered on www.clinicaltrials.gov as NCT02329847), we tested the clinical validity of ctDNA for baseline mutation profiling, residual tumor load quantification, and acquisition of resistance mutations in patients with lymphoma treated with ibrutinib+nivolumab. Inclusion criterion for this ancillary biological study was the availability of blood collected at baseline and cycle 3, day 1. Overall, 172 ctDNA samples from 67 patients were analyzed by the LyV4.0 ctDNA Cancer Personalized Profiling Deep Sequencing Assay. Among baseline variants in ctDNA, only TP53 mutations (detected in 25.4% of patients) were associated with shorter progression-free survival; clones harboring baseline TP53 mutations did not disappear during treatment. Molecular response, defined as a >2-log reduction in ctDNA levels after 2 cycles of therapy (28 days), was achieved in 28.6% of patients with relapsed diffuse large B-cell lymphoma who had ≥1 baseline variant and was associated with best response and improved progression-free survival. Clonal evolution occurred frequently during treatment, and 10.3% new mutations were identified after 2 treatment cycles in nonresponders. PLCG2 was the topmost among genes that acquired new mutations. No patients acquired the C481S BTK mutation implicated in resistance to ibrutinib in CLL. Collectively, our results provide the proof of concept that ctDNA is useful for noninvasive monitoring of lymphoma treated with targeted agents in the clinical trial setting.

1267P Clinical utility of ctDNA for detection of EGFR, ALK, BRAFV600E alterations and resistance mutations in patients with NSCLC at failure to targeted therapy

The comprehensive genomic profile by next generation sequencing (NGS) of circulating tumour DNA (ctDNA) can identify a wide spectrum of genomic alterations (GAs) in non-small cell lung cancer (NSCLC). We aimed to assess the clinical utility of amplicon-based ctDNA NGS for detecting GAs and resistance mutations at time of progression (PD).

A Pan-Canadian Validation Study for the Detection of EGFR T790M Mutation Using Circulating Tumor DNA From Peripheral Blood

IntroductionGenotyping circulating tumor DNA (ctDNA) is a promising noninvasive clinical tool to identify the EGFR T790M resistance mutation in patients with advanced NSCLC with resistance to EGFR inhibitors. To facilitate standardization and clinical adoption of ctDNA testing across Canada, we developed a 2-phase multicenter study to standardize T790M mutation detection using plasma ctDNA testing. MethodsIn phase 1, commercial reference standards were distributed to participating clinical laboratories, to use their existing platforms for mutation detection. Baseline performance characteristics were established using known and blinded engineered plasma samples spiked with predetermined concentrations of T790M, L858R, and exon 19 deletion variants. In phase II, peripheral blood collected from local patients with known EGFR activating mutations and progressing on treatment were assayed for the presence of EGFR variants and concordance with a clinically validated test at the reference laboratory. ResultsAll laboratories in phase 1 detected the variants at 0.5 % and 5.0 % allele frequencies, with no false positives. In phase 2, the concordance with the reference laboratory for detection of both the primary and resistance mutation was high, with next-generation sequencing and droplet digital polymerase chain reaction exhibiting the best overall concordance. Data also suggested that the ability to detect mutations at clinically relevant limits of detection is generally not platform-specific, but rather impacted by laboratory-specific practices. ConclusionsDiscrepancies among sending laboratories using the same assay suggest that laboratory-specific practices may impact performance. In addition, a negative or inconclusive ctDNA test should be followed by tumor testing when possible.

Factors associated with the emergence of integrase resistance mutations in patients failing dual or triple integrase inhibitor-based regimens in a French national survey.

Successful 2-drug regimens (2DRs) for HIV were made possible by the availability of drugs combining potency and tolerability with a high genetic barrier to resistance. How these deal with resistance development/re-emergence, compared with 3DRs, is thus of paramount importance. A national survey including patients who were either naive or experienced with any 2DR or 3DR but failing integrase strand transfer inhibitor (INSTI)-containing regimens [two consecutive plasma viral load (VL) values >50 copies/mL] was conducted between 2014 and 2019. Genotypic resistance tests were interpreted with the v28 ANRS algorithm. Overall, 1104 patients failing any INSTI-containing regimen (2DRs, n = 207; 3DRs, n = 897) were analysed. Five hundred and seventy-seven (52.3%) patients were infected with a B subtype and 527 (47.3%) with non-B subtypes. Overall, 644 (58%) patients showed no known integrase resistance mutations at failure. In multivariate analysis, factors associated with the emergence of at least one integrase mutation were: high VL at failure (OR = 1.24 per 1 log10 copies/mL increase); non-B versus B subtype (OR = 1.75); low genotypic sensitivity score (GSS) (OR = 0.10 for GSS = 2 versus GSS = 0-0.5); and dolutegravir versus raltegravir (OR = 0.46). Although 3DRs versus 2DRs reached statistical significance in univariate analysis (OR = 0.59, P = 0.007), the variable is not retained in the final model. This study is one of the largest studies characterizing integrase resistance in patients failing any INSTI-containing 2DR or 3DR in routine clinical care and reveals factors associated with emergence of integrase resistance that should be taken into consideration in clinical management. No difference was evidenced between patients receiving a 2DR or a 3DR.

Actionability of on-target ALK Resistance Mutations in Patients With Non-Small Cell Lung Cancer: Local Experience and Review of the Literature

IntroductionNon-small cell lung cancer (NSCLC) patients with Anaplastic Lymphoma Kinase (ALK) gene fusions respond well to ALK inhibitors but commonly develop on-target resistance mutations. The objective of this study is to collect clinical evidence for subsequent treatment with ALK inhibitors. Patients and MethodsLocal experience with on-target ALK resistance mutations and review of the literature identified 387 patients with ALK inhibitor resistance mutations. Clinical benefit of mutation-inhibitor combinations was assessed based on reported response, progression-free survival and duration of treatment. Furthermore, this clinical evidence was compared to previously reported in vitro sensitivity of mutations to the inhibitors. ResultsOf the pooled population of 387 patients in this analysis, 239 (62%) received at least 1 additional line of ALK inhibition after developing on-target resistance to ALK inhibitor therapy. Clinical benefit was reported for 177 (68%) patients, but differed for each mutation-inhibitor combination. Agreement between in vitro predicted sensitivity of 6 published models and observed clinical benefit ranged from 69% to 89%. The observed clinical evidence for highest probability of response in the context of specific on-target ALK inhibitor resistance mutations is presented. ConclusionMolecular diagnostics performed on tissue samples that are refractive to ALK inhibitor therapy can reveal new options for targeted therapy for NSCLC patients. Our comprehensive overview of clinical evidence of drug actionability of ALK on-target resistance mechanisms may serve as a practical guide to select the most optimal drug for individual patients.

Evaluation of the Impact of Minimal Residual Disease, FLT3 Allelic Ratio, and FLT3 Mutation Status on Overall Survival in FLT3 Mutation-Positive Patients with Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML) in the Chrysalis Phase 1/2 Study

Evaluation of the Impact of Minimal Residual Disease, FLT3 Allelic Ratio, and FLT3 Mutation Status on Overall Survival in FLT3 Mutation-Positive Patients with Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML) in the Chrysalis Phase 1/2 Study