Development Of New Mutations Research Articles

EXPLORING THE ROLE OF NGS IN GENETIC PROFILING FOR UNDERSTANDING NON-VIOLENT SUDDEN CARDIAC DEATH IN ASTANA

Sudden cardiac death is an unexpected and often unpredictable event that can have serious consequences for the health and lives of individuals not only in middle age but also in young individuals. Mutations, particularly in genes associated with cardiovascular disease, may play a main role in the development of such events, but their exact role and impact require detailed study and prevention of the development of new mutations by using the Next-generation sequencing (NGS). To evaluate the diagnostic importance of next-generation sequencing (NGS) by using our developed cardiogenetic panel in molecular autopsy in sudden non-violent deaths. A prospective study was conducted from 2018 to 2023 focusing of young individuals aged 18 to 45 years resigning in Astana who died of non-violent CVD. The data were analyzed from RSCE "Forensic Expertise Centre of the Ministry of Justice of the Republic of Kazakhstan" "Research Institute of Forensic Expertise" in Astana, blood samples were obtained from SCD victims for further DNA extraction. We implemented a next-generation sequencing (NGS) using the customized panel to target enrichment of coding sequences of 96 candidate genes in molecular autopsies of young adults who died of SCD. All identified genetic variants were classified according to ACMG guidelines in cases with non-diagnostic and diagnostic cardiac abnormalities (ischemic heart disease postmortem). Targeted sequencing and stepwise filtering of annotated variants identified 251 unique variants in 86 genes out of 96 in the entire group of cases studied. In patients with SCD with ischemic changes in the heart postmortem, the most frequent mutations were identified in the TTN, MYBPC3, LAMA2, MYH6 and GAA genes. Pathogenic variants in the genes of ion channels KCNQ1, KCNJ2, SCN5A, RYR2 were detected in individuals with non-diagnostic structural anomalies of the heart. Genetic screening of individuals who died of SCD identified variants with likely pathogenic functional effects with high frequency in 63% and 35% of SCD cases with non-diagnostic and diagnostic cardiac abnormalities, respectively, showing the high diagnostic value of genetic screening using a panel of genes for targeted NGS. Data from our prospective study have shown that genetic testing using NGS in young Astana victims with SCD results in a significant number of mutations, consequently, may allow genetic counselling of relatives to prevent SCD due to coronary heart disease and arrhythmias and may support differential diagnosis postmortem. Supported by Committee of Science of the of Science and Higher Education of Republic of Kazakhstan (AP1486990) and Nazarbayev University CRP(211123CRP1608).

Ctrl+Alt+Delete in the name of COVID-19: When a reset leads to misrecognition

When the COVID-19 pandemic struck the world in March 2020, it impacted all areas of society. Most conspicuous were the lockdowns that were quickly imposed in many countries along with other restrictions. These interventions into the everyday life of ordinary citizens were, perhaps not surprisingly, often met with resistance by citizens and businesses that felt their rights were being trampled on by governments. In this paper, we analyse reactions towards the far-reaching measures taken by the Danish government to contain the spread of the COVID-19 virus in the fur industry and thereby prevent the development of new mutations of the virus: to cull all minks and temporarily ban mink production in Denmark. We argue that by studying this case, valuable lessons can be learned regarding how a business community reacts when faced with a great reset. Taking the current climate crisis into consideration, it must be expected that emission-heavy industries, like agriculture, will be faced with calls to radically change their mode of production in the near future. In this sense, we propose to view the Danish mink case as an early example of what a great reset could look like, how it is perceived by those who experience it first-hand, and how feelings of resentment and resistance can develop following a logic of (mis)recognition.

Prevalence of the A2058G mutation in 23S rRNA gene, which determines Treponema pallidum macrolide resistance in Russian population

Objective. To investigate prevalence of the A2058G mutation in the Russian population of T. pallidum and its association with molecular subtypes. Materials and Methods. We analyzed DNA isolated from 325 samples of clinical material obtained from patients of dermatovenereological treatment and prophylactic institutions in 6 federal districts of the Russia in the period from 2014 to 2021. Patients were diagnosed with primary syphilis of the genital organs, primary syphilis of other sites, or secondary syphilis of the skin and mucous membranes. DNA was isolated using the Proba-NK reagent kit (DNA-technology, Russia) according to the manufacturer’s instructions. The presence of T. pallidum genetic material was confirmed by PCR with primers for the species-specific polA gene. Molecular typing was performed based on the analysis of polymorphic regions of species-specific T. pallidum genes. The primary decoding of the nucleotide sequences of the 23S rRNA gene fragment was carried out using the Sequencing Analysis 5.3.1 program. The analyzed fragments were aligned using the Mega 5 program. Results. Eight molecular subtypes of T. pallidum – 14d/f, 14d/g, 14b/f, 14c/f, 14i/f, 9d/f, 14b/g and 14e/f with stable dominance of subtype 14d/f – were identified in the Russian Federation in the interval 2014–2021. Three subtypes, 14d/g, 14b/g and 14b/f, carrying the A2058G mutation associated with azithromycin resistance, were identified. Conclusions. Studies on molecular typing of T. pallidum strains in the Russia have shown significant population heterogeneity. Three sublines containing the A2058G mutation were shown to exist, one of which – 14b/f – is described as rare. The obtained data confirm the relevance of continuous monitoring of the emergence of resistant strains and the development of new mutations.

Molecular Evolution of Infectious Bronchitis Virus and the Emergence of Variant Viruses Circulating in the United States.

Infectious bronchitis virus (IBV) is a highly infectious and transmissible gammacoronavirus that is nearly impossible to control through biosecurity. Coronaviruses are RNA viruses with an enormous capacity for rapid replication and high rates of mutation, leading to a tremendous amount of genetic diversity. Viral evolution occurs when selection working on genetic diversity leads to new mutations being fixed in the population over time. For IBV, the emergence of variant viruses is likely due to a combination of selection acting on existing genetic diversity, as well as on newly created mutations as the virus replicates, or genetic drift. Immunity against IBV creates a strong selection pressure; however, immunity can also reduce the viral load, decreasing replication and the development of new mutations. Examining the balance between immunity reducing infection, replication, and genetic diversity, and immune pressure selecting for new variants, is extremely difficult at best. Nonetheless, vaccination and immunity do play a role in the emergence of new antigenic variants of IBV. To complicate the situation even more, coronaviruses can undergo recombination, and several studies in the literature report recombination between IBV vaccines and field viruses. However, to our knowledge, unlike genetic drift, recombination alone has not been shown to result in a new antigenic and pathogenic IBV type emerging to cause widespread disease in poultry. Vaccines against IBV that result in an immune population can reduce transmission (basic reproductive number R0 less than 1), making vaccines for IBV the best control strategy available. However, IBV control remains extremely challenging because of the high number of antigenic variants causing disease in poultry and a limited number of vaccines that mostly provide only partial protection against infection and replication of those variants. Currently, there is one major variant IBV circulating in all sectors of US commercial poultry production: DMV/1639/11. This virus was initially detected in 2011, but only began causing significant disease in 2014/2015. Since then, it has affected all three sectors of poultry production (layers, breeders, broilers) and continues to predominate in certain regions of the United States. Additionally, a previously classified variant IBV, which is no longer considered a variant virus, GA08, is highly prevalent. This is attributed to heavy GA08-type IBV vaccine usage because disease caused by the GA08-type virus is rare. Interestingly, the major IBV detected in poultry for several decades, ArkDPI, is no longer among the most detected viruses in the United States. This change corresponds to the shift away from ArkDPI vaccine usage in the broiler sector as GA08 vaccine usage has increased and highlights the role IBV vaccines play in influencing viral populations in commercial chickens.

Management of pleural empyema in a 12-year-old obese patient with COVID-19: a pediatric case report

BackgroundWith the ongoing coronavirus disease (COVID-19) pandemic, along with the development of new mutations of the virus and an increase in the number of cases among pediatrics, physicians should be aware and alerted on the atypical presentations of the disease, especially in less expected individuals.Case presentationHere we present a 12-year-old obese boy (BMI = 37.5 kg/m2) who presented with empyema, which was following SARS-CoV-2 infection. The patient had no history of fever. Due to the onset of dyspnea, a chest tube was inserted for him which was later altered to a pleural drainage needle catheter.ConclusionOur case is the first report of COVID-19 presenting as empyema among pediatrics. Pleural empyema should be considered as a rare complication of COVID-19. Since there is still no guideline in the management of empyema in the context of COVID-19, delay in diagnosis and intervention may cause morbidity and mortality in children.

The Bayesian reconstruction and the evolutionary history of Salivirus type 1 and type 2: the worldwide spreading.

Salivirus (SalV) represents an emerging problem in public health especially during the recent years. In this study, the Bayesian evolutionary history and the spread of the virus through the different countries have been reported. a database of 81 sequences of SalV structural VP1 fragment were downloaded from GenBank, aligned and manually edited by Bioedit Software. ModelTest v. 3.7 software was used to estimate the simplest evolutionary model fitting the sequence dataset. A Maximum-Likelihood tree has been generated using MEGA-X to test the "clockliness" signal using TempEst 1.5.1. The Bayesian phylogenetic tree was built by BEAST. Homology modelling was performed by SWISS-Model and protein variability evaluated by ConSurf server. the phylogenetic tree showed a clade of SalV A2 and three main clades of SalV A1, revealing several infections in humans in South Korea, India, Tunisia, China, Nigeria, Ethiopia and USA. The Bayesian maximum clade credibility tree and the time of the most common recent ancestor dated back the root of the tree to the year 1788 with the probable origin in USA. Selective pressure analysis revealed two positive selection sites, His at 100th and Leu at 116th positions that at the homology modelling resulted important to guarantee protein stability and variability. This could contribute to the development of new mutations modifying the clinical features of this evolving virus. Bayesian phylogenetic and phylodynamic represented a useful tool to follow the transmission dynamic of SalV and to prevent new epidemics worldwide.

Mechanisms of Azacitidine Chemotherapy Resistance in AML and MDS and New Therapy Options

BACKGROUND:TET2 (Ten-eleven-translocation-2) mutations found in 10-30% of MDS and AML patients are actionable for Azacitidine (AZA) treatment (PMID: 21828143). Although response rate of AZA in older AML patients is ~47%, rate of relapse is high due to evolution of disease clones and development of new mutations during treatment cycle. Currently, there is no standard of care for AZA-resistant MDS and AML, and treating these individuals is challenging. Thus, there is an urgent need to identify mechanisms for resistance to AZA for TET2-mutant cancers.AIM: The objective of the study was to predict response to AZA in OCI-M2 disease model and its AZA-resistant (AZA-R) sub-clones using computational biology modeling (CBM) to determine mechanisms of resistance to AZA and identify new therapy options for AZA chemoresistance.METHODS: Parental OCI-M2 cells, representing an AML transformed from MDS, were treated with 8 mM AZA added to media every 2 days (IC50=2µM) for a period of 6 weeks to generate OCI-M2 AZA resistant subclones. AZA-R subclones together with parental AZA-sensitive cells were profiled using cytogenetics and NGS (TruSight Myeloid Sequencing, Illumina). Genomic data were analyzed via CBM system (Cellworks Group), which generates disease-specific protein network maps and enables digital drug simulations. Impact of digital drug treatments were quantified by measuring predicted effects on AML cell growth score, which is a composite of cell proliferation, viability and apoptosis.RESULTS: CBM correctly predicted the response of parental OCI-M2 AML cells to be sensitive to AZA and all its AZA-resistant subclones to be resistant to AZA treatment. Genomic analysis using CBM showed that OCI-M2 AML model harboured TET2mutation (Q1084P), KAT6A amplification and DNMT3B amplification, which are putative determinants of AZA sensitivity as the AZA-resistant subclones lacked cells harbouring these mutations (TET2, KAT6Aamp and DNMT3Bamp). Instead, AZA treatment selected clones with frameshift or nonsense mutations in the following genes: ASXL1, EZH2, CUX1, ATRX, BCOR, PTPN11, FLT3 along with preserved TET2 in all the clones. Mutations in ASXL1 and EZH2 were previously shown to reduce the importance of methylation in driving disease, thus potentially leading to subclone proliferation regardless of AZA. Also 4 of 6 AZA-resistant subclones had DNMT3A frameshift mutations which may enhance AZA resistance.CBM identified new drug combinations based on overlapping as well as unique mutations in AZA-resistant subclones. These combinations include Ruxolitinib + Venetoclax, Nelfinavir + Ruxolitinib or Venetoclax, Sorafenib or Dasatinib + Venetoclax. Some of these drugs were shown to effectively substitute AZA on the parental cell lines (Venetoclax IC=0.9µM, Ruxolitinib IC50=1.7µM, Sorafenib IC50=5.3µM). Whether AZA enhanced sensitivity to these drugs in AZA-R clones is currently under investigation.Conclusions: CBM-based genomic analysis identified secondary mutations in ASXL1, EZH2, CUX1, ATRX, BCOR, PTPN11 and FLT3 that associated with AZA chemoresistance. Digital drug screening identified new drug combinations including Venetoclax for validation testing in AZA-resistant MDS and AML. DisclosuresAbbasi:Cell Works Group Inc.: Employment. Singh:Cellworks Research India Private Limited: Employment. Kumar:Cellworks Research India Private Limited: Employment. Suseela:Cellworks Research India Private Limited: Employment. Patil:Cellworks Research India Private Limited: Employment. Dattatraya:Cellworks Research India Private Limited: Employment. Tiwari:Cellworks Research India Private Limited: Employment. Vali:Cell Works Group Inc.: Employment. Cogle:Celgene: Other: Steering Committee Member of Connect MDS/AML Registry.

Temporal changes of EGFR mutations and T790M levels in tumour and plasma DNA following AZD9291 treatment

AZD9291, a T790M specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), has demonstrated impressive response rates in tumours harbouring the EGFR T790M resistance mutation. Emergence of resistance to AZD9291 has been shown to occur through several different mechanisms including the development of new mutations (e.g. C797S) in the EGFR tyrosine kinase domain. We studied two patients with paired tumour biopsies and blood samples pre- and post-progression on AZD9291 to explore possible resistance mechanisms.Pre- and Post-AZD9291 tumour biopsies as well as serial plasma samples were collected from two patients on the AURA clinical study (AZD9291 First Time in Patients Ascending Dose study). Droplet digital PCR (ddPCR) assays were used to quantify T790M, the driver EGFR mutation, and the C797S mutation in genomic DNA from paired tumour biopsies and plasma cell-free DNA.In the first patient, both EGFR T790M and L858R became undetectable in the plasma within 1 month after treatment with AZD9291. However, the T790M and the original L858R mutation re-emerged with radiologically confirmed resistance to AZD9291. In patient two, the levels of T790M were undetectable at the time of radiological resistance to AZD9291 but increasing levels of the original EGFR exon 19 deletion was detected. MET amplification was detected in a biopsy performed on progression. The EGFR C797S mutation was not detected in either patient at the time of relapse.ddPCR of cell free DNA enables real time monitoring of patients on 3rd generation TKIs. As resistance mechanisms are variable, monitoring levels of the initial activating EGFR mutation may facilitate more reliable detection of progression.

Patients with Philadelphia-positive leukemia with Y253H or F359V mutation have a high risk of developing new mutations in the setting of dasatinib resistance

The treatment outcome and development of new mutations in patients with imatinib- and/or nilotinib-failure Philadelphia chromosome positive (Ph+) leukemia with highly nilotinib-resistant mutations (Y253H, E255K/V and F359V/C) were assessed on dasatinib therapy. A total of 111 patients with Ph+ leukemia were grouped into three cohorts by baseline BCR–ABL kinase domain mutation status: no mutation (n = 44), non-nilotinib-resistant mutations (n = 26) or nilotinib-resistant mutations (n = 41). The frequencies of hematological, cytogenetic and molecular responses and clinical resistance to dasatinib were similar among the three cohorts during dasatinib therapy. In dasatinib-resistant patients, new mutations were most frequently observed in the cohort with nilotinib-resistant mutations (p = 0.001). Multivariate analysis revealed that advanced disease and harboring Y253H or F359V mutation before dasatinib were independent predictors of developing new mutations. We conclude that patients with Ph+ leukemia with Y253H or F359V mutation have a high likelihood of developing new mutations in the setting of dasatinib resistance.

Philadelphia-Positive Leukemia Patients With The Y253H Or F359V Mutations Have a High Risk Of Developing New Mutations In The Setting Of Dasatinib Resistance

ObjectiveThe clinical significance of individual mutations in Philadelphia-positive (Ph+) leukemia patients is not well-characterized on dasatinib therapy. The treatment outcomes and development of new mutations in imatinib- and/or nilotinib-failure Ph+ leukemia patients with highly nilotinib-resistant mutations (Y253H, E255K/V and F359V/C) were assessed during dasatinib therapy. MethodsOne hundred and eight Ph+ leukemia patients, including 36 with chronic myeloid leukemia in the chronic phase, 18 in the accelerated phase, 40 in the blastic phase and 14 with Ph+ acute lymphoblastic leukemia, who failed imatinib (n=79) or imatinib and nilotinib (n=29) were treated with dasatinib. Those harbored highly dasatinib-resistant mutations (T315I/A, V299L and F317L/V/I/C) were excluded. The patients were grouped into 3 cohorts by baseline BCR-ABL kinase domain (KD) mutation status: no mutation (n=42), non-nilotinib-resistant mutation (any mutation except highly nilotinib-resistant mutations; n=25), or nilotinib-resistant mutation (Y253H, E255K/V or F359V/C; n=41). BCR-ABL KD mutation analysis was detected by direct sequencing at the time of dasatinib resistance. ResultsWith a median follow-up of 6 months (range 2-60 months), the frequencies of hematological and cytogenetic responses and clinical resistance to dasatinib, failure-free survival, progression-free survival and alternative treatment-free survival were comparable by baseline BCR-ABL KD mutation status. Sixty of sixty-four patients (93.8%) underwent BCR-ABL KD mutation analysis at the time of developing clinical resistance to dasatinib. Among the 60 evaluated patients, 33 new mutations were first identified in 29 (48.3%) patients at a median of 3 months (range, 2-26 months) of dasatinib therapy, including 8 of 28 (28.6%) with no mutation, 4 of 9 (44.4%) with non-nilotinib-resistant mutations and 17 of 23 (73.9%) with nilotinib-resistant mutations at baseline. New mutations were most frequently observed in the cohort with nilotinib-resistant mutations (P=0.031). Specially, the subset with the Y253H (12/13, 92.3%) or F359V (5/5, 100%) in the cohort with nilotinib-resistant mutations was more likely to acquire new mutations. Multivariate analyses revealed that harboring the Y253H (HR=9.0, 95%CI: 2.7-30.5; P<0.001) or F359V (HR=7.6, 95%CI: 1.5-39.4; P=0.016) mutation at baseline and a lack of any hematologic response to dasatinib (HR=2.1, 95%CI: 1.4-3.1; P<0.001) were identified as independent predictors of developing new mutations during dasatinib therapy. ConclusionsOur study suggested that Ph+ leukemia patients with the Y253H or F359V mutation had a high likelihood of developing new mutations in the setting of dasatinib resistance. Our data highlight the importance of closer monitoring and mutation follow up for therapeutic choices, especially alternative therapies, which should be considered for patients with the Y253H or F359V mutation during dasatinib therapy. Larger studies are needed to assess the significance of various BCR-ABL KD mutations prior to and during sequential TKI therapy for Ph+ leukemia patients. Disclosures:No relevant conflicts of interest to declare.

Chronic Myelogenous Leukemia Patients with Highly Nilotinib-Resistant BCR-ABL Mutations Demonstrate Similar Efficacy to Dasatinib, but Have a Higher Likelihood of Developing New Mutations in the Event of Clinical Resistance

AbstractAbstract 3755 Objective:Assess the outcome of imatinib- and/or nilotinib-failure chronic myelogenous leukemia (CML) patients with pre-existing, highly nilotinib-resistant mutations (Y253H, E255K/V and F359V/C), during dasatinib therapy. Methods:Sixty-one CML patients who failed imatinib (n=44) or imatinib and nilotinib (n=17), including 20 in chronic phase (CP), 16 in accelerated phase (AP) and 25 in blast phase (BP), were switched to dasatinib, except for patients with highly dasatinib-resistant mutations (T315I/A, F317L/V/I/C and V299L). Patients were grouped into 3 cohorts: no mutation (n=22), no-specific mutation (any mutation except highly nilotinib-resistant mutations; n=15), or specific mutation (Y253H, E255K/V or F359V/C; n=24), according to baseline BCR-ABL mutation status. Cumulative incidence frequencies of hematological response (HR), complete hematological response (CHR), major cytogenetic response (MCR), complete cytogenetic response (CCR) and major molecular response (MMR) were compared. Probabilities of clinical resistance to dasatinib (according to European LeukemiaNet recommendations), progression-free survival (PFS) and overall survival (OS), and dynamics of BCR-ABL mutations during dasatinib therapy were also assessed. Results:Prior nilotinib was associated with a higher proportion of specific mutation versus no mutation (50.0% vs 13.6%, P=0.001) and non-specific mutation (50.0% vs 13.3%, P=0.02). Patients harboring specific mutation also had a higher frequency of advanced phase CML before dasatinib treatment than patients with no mutation (83.3% vs 50.0%, P=0.008), and a similar frequency to those with non-specific mutation (83.3% vs 66.7%, P=0.266). Response, PFS and OS rates, and follow-up duration were similar among the 3 cohorts by baseline mutation status, as shown in Table. Probability of clinical resistance to dasatinib in patients with specific mutation was not significantly different to those with no mutation (54.2% vs 77.3%, P=0.100) or non-specific mutation (54.2% vs 33.3%, P=0.204) despite that there was a significant difference between the cohort with no mutation and non-specific mutation (77.3% vs 33.3%, P=0.008). Among 32 patients who developed clinical resistance to dasatinib and were assessed for BCR-ABL mutation status, newly detectable mutations were identified in 14 patients and most frequently occurred in those already harboring specific mutation versus those with no mutation (76.9% vs 26.7%, P=0.008) and non-specific mutation (76.9% vs 0%, P=0.015) at baseline. T315I (9/14, 64.3%) was the most common newly acquired BCR-ABL mutation. Harboring specific mutation compared with other mutation states at baseline (76.9% vs 16.7%, P=0.001), and developing hematological resistance rather than cytogenetic resistance during dasatinib therapy (65.0% vs 8.1%, P=0.002), were identified as risk factors associated with the development of new mutations. Conclusions:Imatinib- and/or nilotinib-failure patients with highly nilotinib-resistant BCR-ABL mutations demonstrate similar efficacy to dasatinib as those with no or any other mutation. However, patients with highly nilotinib-resistant mutations had a higher likelihood of developing new mutations at the time of clinical resistance. More extensive studies are needed to assess the significance of various BCR-ABL mutations prior to and during tyrosine kinase inhibitor therapy for CML.Table:Response, survival and clinical resistance to dasatinib by baseline BCR-ABL mutation statusMutation statusP valueNo mutation n=22Non-specific mutation n=15Specific mutation n=24HR, n (%)16 (72.7)14 (93.3)21 (87.5)0.202CHR, n (%)16 (72.7)12 (80.0)19 (79.2)0.832MCR, n (%)a9 (69.2)8 (80.0)12 (70.6)0.826CCR, n (%)a8 (61.5)8 (80.0)10 (58.8)0.511MMR, n (%)a3 (23.1)5 (50.0)4 (23.5)0.2813-year PFS, %84.673.765.20.4693-year OS, %75.680.854.00.798Clinical resistance to dasatinib, n (%)17 (77.3)5 (33.3)13 (54.2)0.027Follow-up (months), median (range)31 (2..C48)24 (2..C48)7 (1..C48)0.277a40 patients were evaluated including 13 with no mutation,10 with non-specific mutation and 17 with specific mutation Disclosures:No relevant conflicts of interest to declare.

Resistance, Outcome and the Development of Mutations with Dasatinib in Patients with Chronic-Phase Chronic Myeloid Leukemia (CML-CP).

Abstract 1122Poster Board I-144Dasatinib is an FDA approved tyrosine kinase inhibitor (TKI) targeted at BCR-ABL for the treatment of CML after imatinib resistance or intolerance. A phase III dose-optimization study of dasatinib in CML-CP, where patients received either 100 mg or 140 mg of dasatinib on a once- or twice-daily schedule, indicated that 100 mg once daily dasatinib provided durable disease control with an estimated progression-free survival (PFS) of 73% at 36 months. We conducted a retrospective analysis to expand on these results to determine if earlier cytogenetic response (CyR) predicts superior outcomes within this dosing arm and also to quantify progression to advanced-phase CML and characterize the quality of BCR-ABL mutations associated with loss of response to dasatinib. Our landmark analysis demonstrated that 90% of patients receiving dasatinib 100 mg once daily who achieved complete cytogenetic response (CCyR) at 12 months were progression-free at 36 months, a considerable improvement over those without CCyR at 12 months (Table 1). Of the 164 patients receiving dasatinib 100 mg once daily, 59 had attained CCyR at 6 months of therapy. The rate of PFS after 36 months within this cohort was 93%, whereas those with partial CyR or those without major cytogenetic response (MCyR) at 6 months had 36-month PFS rates of 76% and 54%, respectively. A total of 36 subjects experienced progression events while receiving dasatinib 100 mg once daily: 8 due to death, 5 due to development of advanced phases of CML, 3 due to increases ≥30% in Ph+ metaphases, 9 due to increasing white blood-cell count, 4 due to loss of complete hematologic response, 4 due to loss of MCyR, and 3 for unknown reason. The majority (86%) of patients who do progress while receiving dasatinib remain in CP at 36 months.The development of mutations in BCR-ABL is a known mechanism of loss of response to dasatinib. In participants with loss of response to dasatinib who have available mutation data (n=61), the incidence of developing mutations during dasatinib therapy (at any dose) is 19% (5/27) in patients without baseline mutations, and 47% (16/34) in those with mutations at initiation of dasatinib. Of the patients who lost response to dasatinib and developed new mutations during therapy, 13 patients harbored T315I, 6 possessed F317L, 3 had V299L, and 1 acquired E255K. Three other patients developed mutations not associated with resistance to dasatinib (Table 2). New mutations that emerged in the 100 mg once daily arm and 70 mg twice daily arm were of similar frequency. In conclusion, patients who achieve early and complete CyRs to second-line dasatinib exhibit reduced rates of long-term progression versus those without CCyR at 6 months. Of the patients who do progress while receiving dasatinib, the majority remain in CP at 36 months. Of patients treated with 100 mg once daily, only 3% progressed to accelerated or blast phase with 36 months of follow-up. This transformation-free survival rate favors the use of dasatinib following imatinib failure in patients who have the option of pursuing allogeneic stem cell transplantation. Finally, the development of new mutations leading to resistance during dasatinib therapy is uncommon, and the lower total daily dose employed by the 100 mg once daily regimen does not appear to select for a greater variety of mutations than have been previously identified in patients treated with 70 mg twice daily. In patients who lose response to dasatinib and develop new mutations, a switch to an alternate TKI or stem-cell transplantation may be appropriate.Table 1Progression-free survival of subjects with or without cytogenetic responses receiving dasatinib 100 mg once dailyCytogenetic ResponsePFS (95% CI)12 monthsCCyR (n=55)90% (81–100)partial CyR (n=23)77% (60–95)minimal/minor/none (n=33)63% (43–83)6 monthsCCyR (n=59)93% (85–100)partial CyR (n=26)76% (59–93)minimal/minor/none (n=53)54% (36–73)Abbreviation: CI, confidence interval.Table 2Subjects developing new mutations during dasatinib by select dosing armsAll subjects* (n=61)100 mg once daily* (n=13)70 mg twice daily* (n=14)M244V100E255K101V299L321F311L101T315I1333F317L631M351T100*Subjects with loss of response to dasatinib and available baseline and progression/end-of-treatment mutation data. DisclosuresShah:Bristol-Myers Squibb: Consultancy; Novartis: Consultancy. Bahceci:Bristol-Myers Squibb: Employment. Lambert:Bristol-Myers Squibb: Employment. Ploughman:Bristol-Myers Squibb: Employment. Radich:Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

Analysis of Molecular Data and the Emergence of Mutations for Chronic-Phase Chronic Myelogenous Leukemia (CML-CP) Patients Treated with Dasatinib After Imatinib Failure.

Abstract 3282Poster Board III-1The kinase inhibitor dasatinib has demonstrated efficacy in patients with CML-CP who fail imatinib due to resistance or intolerance. Imatinib failure is often associated with the acquisition of resistant mutations within BCR-ABL. Failure of dasatinib is associated with a limited spectrum of mutations, which are T315I/A, V299L, and F317L/I/V, suggesting these mutations are dasatinib resistant (DR). Only a fraction of patients with T315I respond to dasatinib, most patients with F317L have hematologic responses but few have cytogenetic responses, and V299L is uncommon in imatinib-resistant patients but is mostly seen after dasatinib failure. In vitro data suggests Q252H and E255K/V also confer a degree of dasatinib resistance. However, their clinical association with dasatinib resistance or inferior response is unclear. This retrospective analysis seeks to determine if the mutation status at the start of dasatinib impacts the initial molecular response, and to examine the type of mutations lost and gained during therapy. To assess the significance of intermediate sensitivity (DI) mutations (Q252H and E255K/V), BCR-ABL levels were measured against the International Scale (IS), and mutation status was assayed at initiation of dasatinib (baseline), at progression, or study termination or completion (progression/completion). Data were taken from 479 patients with CML-CP treated with dasatinib 70 mg twice daily, after imatinib failure for the phase II START-C or -R studies. Patients were grouped according to their baseline mutation status: DR, DI, dasatinib sensitive (DS, all other mutations not DR or DI), and no mutations. Patients with more than one mutation at baseline that qualified for more than one grouping were rare, and classified according to the most resistant mutation. By 6 months of dasatinib therapy, patients with DR mutations at baseline had the highest levels of BCR-ABL transcripts (Table 1). By comparison, those with DI, DS, or no mutations had a reduced proportion with high transcript levels. In addition, no patients with DR at baseline achieved a reduction of BCR-ABL by 6 months to IS ratio < 1, whereas significant reductions were identified in patients with DI, DS, or no mutations. A subset of 267 patients had mutation analysis performed at progression/completion. Of these, 115 had mutations at baseline and 152 had no mutations. The patients with baseline imatinib resistance were more likely than those with imatinib intolerance to acquire new mutations. Of the resistant patients with no mutations at baseline, 7% (9/124) developed new mutations and the majority (7/9) was DR. Among resistant patients with baseline mutations, 23% (26/112) had new mutations and the majority was DR (20/26). Although 36 patients gained new mutations (including 16 T315I/A, 8 F317L, and 6 V299L), many baseline mutations were no longer detectable at progression/completion. DS mutations were lost in 68/102 patients. In total, 76 patients had detectable mutations at progression/completion, with 51% (39/76) having DR mutations but only 5% (4/76) with DI mutations. Patients with baseline DR mutations were more likely to retain their DR mutations (11/13). Of 8 patients with DI mutations at baseline, only 3 retained their DI mutations and 3 gained DR mutations. Of 94 patients with only DS mutations at baseline, 36 (38%) retained their DS mutations, and 17 (18%) developed DR mutations. Patients with high levels of BCR-ABL at 3 months had the highest incidence of new DR mutations at progression/completion (18/145 evaluable patients; 12%) compared to patients with lower levels (5/79; 6%). In conclusion, the development of new mutations during second-line dasatinib is rare (36/267). Patients who harbored DI mutations at baseline were also rare, and an expanded prospective analysis of dasatinib efficacy in this cohort may be necessary to clarify their significance. However, from the current analysis there is no compelling clinical evidence to suggest that these patients have an inferior molecular response. Relative to DR mutations, the DI mutations (Q252H and E255K/V) were rarely present at progression/completion.Patients with baseline mutations and 6-month BCR-ABL transcript level after second-line dasatinibIS ratio ≤ 1.01 < IS ratio ' 10IS ratio > 10DR0% (0/13)23% (3/13)77% (10/13)DI56% (5/9)0% (0/9)44% (4/9)DS36% (46/128)20% (25/128)45% (57/128)No mutations42% (92/221)18% (40/221)40% (89/221) Disclosures:Branford:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Hochhaus:Bristol-Myers Squibb: Research Funding; Novartis: Research Funding. Bahceci:Bristol-Myers Squibb: Employment. Ploughman:Bristol-Myers Squibb: Employment. Mukhopadhyay:Bristol-Myers Squibb: Employment. Hughes:Bristol-Myers Squibb: Advisor, Honoraria, Research Funding; Novartis: Advisor, Honoraria, Research Funding.

Persistence of Genotypic Resistance to Nelfinavir among Women Exposed to Prophylactic Antiretroviral Therapy during Pregnancy

We assessed the development of drug resistance in women exposed to antiretroviral therapy (ART) for prevention of mother-to-child transmission (PMTCT) after 24 weeks postpartum in a prospective cohort of HIV-1-infected women. HIV-1-infected women, who received prophylactic ART during pregnancy, had genotypic resistance testing performed at the start (T1) of and 24 weeks after ART interruption (T2). The women had CD4 counts >250 cells/ml and no AIDS defining conditions. Of the 30 eligible women, the median age was 27 years [25-75% interquartile range (IQR): 21-32] and the median gestational age of ART initiation was 22 weeks (IQR: 19-27): 19 (63.3%) received zidovudine (ZDV) plus lamivudine (3TC) plus nelfinavir (NFV). At entry, most women (96.7%) were asymptomatic (CDC93 A1/A2), with a median CD4 count of 446 (IQR: 353-686) and median viral load (VL) of 8560 copies/ml (IQR: 3,252-19,515). No HIV-1 vertical transmission was observed. HIV subtype B was the most prevalent (70%). The development of new mutations associated with ART resistance was analyzed at T2. NFV resistance was observed in 4 out of 17 (23.5%) patients exposed to this drug: two major mutations D30N (1/17) and L90M (1/17) and minor mutations (N88S, 2/17). Mutations on positions 44, 69, and 118 (1/28) were present on reverse transcriptase (RT) analysis. No new nonnucleoside reverse transcriptase inhibitor (NNRTI)-associated mutation was observed. In this cohort, ART regimens were very efficient at blocking HIV vertical transmission. However, the high rate of NFV-resistant mutations observed in the postpartum period indicates the need for discussion of ART choices during pregnancy and the potential impact on future therapeutic options for these women. Women previously exposed to ART for PMTCT who will start HIV treatment should have genotypic resistance testing performed.