ABSTRACT
Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant multisystemic vascular dysplasia, characterized by arteriovenous malformations (AVMs), mucocutaneous telangiectasia and nosebleeds. HHT is caused by a heterozygous null allele in ACVRL1, ENG, or SMAD4, which encode proteins mediating bone morphogenetic protein (BMP) signaling. Several missense and stop-gain variants identified in GDF2 (encoding BMP9) have been reported to cause a vascular anomaly syndrome similar to HHT, however none of these patients met diagnostic criteria for HHT. HHT families from UK NHS Genomic Medicine Centres were recruited to the Genomics England 100,000 Genomes Project. Whole genome sequencing and tiering protocols identified a novel, heterozygous GDF2 sequence variant in all three affected members of one HHT family who had previously screened negative for ACVRL1, ENG, and SMAD4. All three had nosebleeds and typical HHT telangiectasia, and the proband also had severe pulmonary AVMs from childhood. In vitro studies showed the mutant construct expressed the proprotein but lacked active mature BMP9 dimer, suggesting the mutation disrupts correct cleavage of the protein. Plasma BMP9 levels in the patients were significantly lower than controls. In conclusion, we propose that this heterozygous GDF2 variant is a rare cause of HHT associated with pulmonary AVMs.
Subject(s)
Arteriovenous Malformations , Telangiectasia, Hereditary Hemorrhagic , Activin Receptors, Type II/genetics , Arteriovenous Fistula , Arteriovenous Malformations/diagnosis , Arteriovenous Malformations/genetics , Child , Endoglin/genetics , Endoglin/metabolism , Epistaxis , Growth Differentiation Factor 2/genetics , Humans , Mutation , Pulmonary Artery/abnormalities , Pulmonary Veins/abnormalities , Telangiectasia, Hereditary Hemorrhagic/diagnosis , Telangiectasia, Hereditary Hemorrhagic/genetics , Telangiectasia, Hereditary Hemorrhagic/pathologyABSTRACT
Recently, host whole blood gene expression signatures have been identified for diagnosis of tuberculosis (TB). Absolute quantification of the concentrations of signature transcripts in blood have not been reported, but would facilitate diagnostic test development. To identify minimal transcript signatures, we applied a transcript selection procedure to microarray data from African adults comprising 536 patients with TB, other diseases (OD) and latent TB (LTBI), divided into training and test sets. Signatures were further investigated using reverse transcriptase (RT)-digital PCR (dPCR). A four-transcript signature (GBP6, TMCC1, PRDM1, and ARG1) measured using RT-dPCR distinguished TB patients from those with OD (area under the curve (AUC) 93.8% (CI95% 82.2-100%). A three-transcript signature (FCGR1A, ZNF296, and C1QB) differentiated TB from LTBI (AUC 97.3%, CI95%: 93.3-100%), regardless of HIV. These signatures have been validated across platforms and across samples offering strong, quantitative support for their use as diagnostic biomarkers for TB.
Subject(s)
Carrier Proteins/blood , Latent Tuberculosis/diagnosis , Mitochondrial Proteins/blood , Receptors, IgG/blood , Transcriptome/genetics , Tuberculosis, Pulmonary/diagnosis , Zinc Fingers/physiology , Adult , Aged , Biomarkers/blood , Carrier Proteins/genetics , Diagnostic Tests, Routine , Female , Gene Expression Profiling , Humans , Male , Middle Aged , Mitochondrial Proteins/genetics , Mycobacterium tuberculosis/genetics , Protein Array Analysis , RNA, Messenger/blood , RNA, Messenger/genetics , Receptors, IgG/genetics , Reverse Transcriptase Polymerase Chain Reaction , South Africa , Young AdultABSTRACT
Advances in technology have led to a massive expansion in the capacity for genomic analysis, with a commensurate fall in costs. The clinical indications for genomic testing have evolved markedly; the volume of clinical sequencing has increased dramatically; and the range of clinical professionals involved in the process has broadened. There is general acceptance that our early dichotomous paradigms of variants being pathogenic-high risk and benign-no risk are overly simplistic. There is increasing recognition that the clinical interpretation of genomic data requires significant expertise in disease-gene-variant associations specific to each disease area. Inaccurate interpretation can lead to clinical mismanagement, inconsistent information within families and misdirection of resources. It is for this reason that 'national subspecialist multidisciplinary meetings' (MDMs) for genomic interpretation have been articulated as key for the new NHS Genomic Medicine Service, of which Cancer Variant Interpretation Group UK (CanVIG-UK) is an early exemplar. CanVIG-UK was established in 2017 and now has >100 UK members, including at least one clinical diagnostic scientist and one clinical cancer geneticist from each of the 25 regional molecular genetics laboratories of the UK and Ireland. Through CanVIG-UK, we have established national consensus around variant interpretation for cancer susceptibility genes via monthly national teleconferenced MDMs and collaborative data sharing using a secure online portal. We describe here the activities of CanVIG-UK, including exemplar outputs and feedback from the membership.
Subject(s)
Genetic Testing , Genetic Variation/genetics , Genomics , Neoplasms/genetics , Female , High-Throughput Nucleotide Sequencing , Humans , Ireland/epidemiology , Male , Neoplasms/epidemiology , Neoplasms/pathology , United Kingdom/epidemiologyABSTRACT
There are no validated molecular biomarkers to identify newly-diagnosed individuals with chronic-phase chronic myeloid leukemia likely to respond poorly to imatinib and who might benefit from first-line treatment with a more potent second-generation tyrosine kinase inhibitor. Our inability to predict these 'high-risk' individuals reflects the poorly understood heterogeneity of the disease. To investigate the potential of genetic variants in epigenetic modifiers as biomarkers at diagnosis, we used Ion Torrent next-generation sequencing of 71 candidate genes for predicting response to tyrosine kinase inhibitors and probability of disease progression. A total of 124 subjects with newly-diagnosed chronic-phase chronic myeloid leukemia began with imatinib (n=62) or second-generation tyrosine kinase inhibitors (n=62) and were classified as responders or non-responders based on the BCRABL1 transcript levels within the first year and the European LeukemiaNet criteria for failure. Somatic variants affecting 21 genes (e.g. ASXL1, IKZF1, DNMT3A, CREBBP) were detected in 30% of subjects, most of whom were non-responders (41% non-responders, 18% responders to imatinib, 38% non-responders, 25% responders to second-generation tyrosine kinase inhibitors). The presence of variants predicted the rate of achieving a major molecular response, event-free survival, progression-free survival and chronic myeloid leukemia-related survival in the imatinib but not the second-generation tyrosine kinase inhibitors cohort. Rare germline variants had no prognostic significance irrespective of treatment while some pre-leukemia variants suggest a multi-step development of chronic myeloid leukemia. Our data suggest that identification of somatic variants at diagnosis facilitates stratification into imatinib responders/non-responders, thereby allowing earlier use of second-generation tyrosine kinase inhibitors, which, in turn, may overcome the negative impact of such variants on disease progression.
Subject(s)
Biomarkers, Tumor/genetics , Epigenesis, Genetic , Gene Expression Regulation, Neoplastic/drug effects , Imatinib Mesylate/therapeutic use , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Mutation , Protein Kinase Inhibitors/therapeutic use , Adult , Aged , Aged, 80 and over , Female , Follow-Up Studies , Gene Expression Profiling , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Male , Middle Aged , Retrospective Studies , Survival Rate , Treatment Failure , Young AdultABSTRACT
Diamond-Blackfan anaemia (DBA) is a rare bone marrow failure syndrome characterised by anaemia, congenital anomalies and cancer predisposition. Although infections are the second leading cause of mortality in non-transplanted patients, immune function is largely unexplored. We identified quantitative deficits in serum immunoglobulins and/or circulating T, natural killer and B lymphocytes in 59 of 107 unselected patients (55·1%) attending our centre over a 7-year period. Immune abnormalities were independent of ribosomal protein genotype and arose in both steroid-treated and steroid-untreated patients. In summary, these data highlight the high prevalence and spectrum of infections and immune defects in DBA.
Subject(s)
Anemia, Diamond-Blackfan , Genotype , Immunity, Cellular , Immunity, Humoral , Adolescent , Anemia, Diamond-Blackfan/genetics , Anemia, Diamond-Blackfan/immunology , Anemia, Diamond-Blackfan/mortality , Anemia, Diamond-Blackfan/pathology , Child , Child, Preschool , Female , Humans , Infant , Male , Ribosomal Proteins/genetics , Ribosomal Proteins/immunology , United Kingdom/epidemiologyABSTRACT
It is currently unclear whether post-transplant diffuse large B-cell lymphomas (PT-DLBCL) display a similar genomic landscape as DLBCL in immunocompetent patients (IC-DLBCL). We investigated 50 post-transplant lymphoproliferative disorders (PTLDs) including 37 PT-DLBCL samples for somatic mutations frequently observed in IC-DLBCL. Targeted Next Generation Sequencing (NGS) using the Ion Torrent platform and a customized panel of 68 genes was performed on genomic DNA. Non-tumoural tissue was sequenced to exclude germline variants in cases where available. A control cohort of 76 IC-DLBCL was available for comparative analyses. In comparison to IC-DLBCLs, PT-DLBCL showed more frequent mutations of TP53 (P = 0·004), and absence of ATM and B2M mutations (P = 0·004 and P = 0·016, respectively). In comparison to IC-DLBCLs, Epstein-Barr virus (EBV)+ PT-DLBCL had fewer mutated genes (P = 0·007) and particularly fewer mutations in nuclear factor-κB pathway-related genes (P = 0·044). TP53 mutations were more frequent in EBV- PT-DLBCL as compared to IC-DLBCL (P = 0·001). Germinal centre B cell (GCB) subtype of PT-DLBCL had fewer mutations and mutated genes than GCB-IC-DLBCLs (P = 0·048 and 0·04 respectively). Polymorphic PTLD displayed fewer mutations as compared to PT-DLBCL (P = 0·001). PT-DLBCL differs from IC-DLBCL with respect to mutations in genes related to DNA damage control and immune-surveillance, and EBV association is likely to have a bearing on the mutational pattern.
Subject(s)
Lymphoproliferative Disorders/genetics , Mutation , Organ Transplantation/adverse effects , Adult , Aged , Cohort Studies , DNA, Neoplasm/genetics , Epstein-Barr Virus Infections/complications , Epstein-Barr Virus Infections/immunology , Female , Genes, p53/genetics , Humans , Immunocompromised Host , Lymphoma, Large B-Cell, Diffuse/etiology , Lymphoma, Large B-Cell, Diffuse/genetics , Lymphoma, Large B-Cell, Diffuse/immunology , Lymphoma, Large B-Cell, Diffuse/pathology , Lymphoproliferative Disorders/etiology , Lymphoproliferative Disorders/immunology , Lymphoproliferative Disorders/pathology , Male , Middle Aged , Young AdultABSTRACT
Chronic myeloid leukemia (CML) is the paradigm for targeted cancer therapy. RT-qPCR is the gold standard for monitoring response to tyrosine kinase-inhibitor (TKI) therapy based on the reduction of blood or bone marrow BCR-ABL1. Some patients with CML and very low or undetectable levels of BCR-ABL1 transcripts can stop TKI-therapy without CML recurrence. However, about 60 percent of patients discontinuing TKI-therapy have rapid leukaemia recurrence. This has increased the need for more sensitive and specific techniques to measure residual CML cells. The clinical challenge is to determine when it is safe to stop TKI-therapy. In this review we describe and critically evaluate the current state of CML clinical management, different technologies used to monitor measurable residual disease (MRD) focus on comparingRT-qPCR and new methods entering clinical practice. We discuss advantages and disadvantages of new methods.
ABSTRACT
BACKGROUND: Tyrosine kinase inhibitors (TKIs) are the cornerstone of successful clinical management of patients with chronic myeloid leukemia (CML). Quantitative monitoring of the percentage of the fusion transcript BCR-ABL1 (breakpoint cluster region-c-abl oncogene 1, non-receptor tyrosine kinase) BCR-ABL1IS (%BCR-ABL1IS) by reverse transcription-quantitative PCR (RT-qPCR) is the gold standard strategy for evaluating patient response to TKIs and classification into prognostic subgroups. However, this approach can be challenging to perform in a reproducible manner. Reverse-transcription digital PCR (RT-dPCR) is an adaptation of this method that could provide the robust and standardized workflow needed for truly standardized patient stratification. METHODS: BCR-ABL1 and ABL1 transcript copy numbers were quantified in a total of 102 samples; 70 CML patients undergoing TKI therapy and 32 non-CML individuals. 3 commercially available digital PCR platforms (QS3D, QX200 and Raindrop) were compared with the platform routinely used in the clinic for RT-qPCR using the EAC (Europe Against Cancer) assay. RESULTS: Measurements on all instruments correlated well when the %BCR-ABL1IS was ≥0.1%. In patients with residual disease below this level, greater variations were measured both within and between instruments limiting comparable performance to a 4 log dynamic range. CONCLUSIONS: RT-dPCR was able to quantify low-level BCR-ABL1 transcript copies but was unable to improve sensitivity below the level of detection achieved by RT-qPCR. However, RT-dPCR was able to perform these sensitive measurements without use of a calibration curve. Adaptions to the protocol to increase the amount of RNA measured are likely to be necessary to improve the analytical sensitivity of BCR-ABL testing on a dPCR platform.
Subject(s)
Fusion Proteins, bcr-abl/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Neoplasm, Residual/genetics , Proto-Oncogene Proteins c-abl/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/diagnosis , Neoplasm, Residual/diagnosisABSTRACT
Molecular monitoring of BCR-ABL1 expression in chronic myeloid leukaemia (CML) is well established. As the International Scale (IS) normalised BCR-ABL1/ABL1 ratio at 3 months post-treatment is now an important milestone in patients' treatment schedule, the reliable and reproducible measurement of BCR-ABL1 levels is therefore paramount. IS conversion factors (CF) are established via sample exchange and yearly ratification with an external reference laboratory. Since any change to an established IS CF could lead to discontinuity in longitudinal results, we wished to add an internal verification step as an additional safeguard. We used the Cepheid GeneXpert qPCR and IS calibrated Xpert BCR-ABL Monitor cartridge system, parallel to our in-house pipeline on 50 CML samples, over the period of one week to verify the CF for those samples and compare it to the externally provided CF. The median non-IS in-house BCR-ABL1/ABL1 values were significantly different than that from the IS GeneXpert, but they became non-significant when adjusted to CF provided by the CXM and by the current external CF, validating it. These metrics can help decide to accept or reject an updated CF value, especially where a significant change in CF might lead to a discontinuity in ongoing patient monitoring.
Subject(s)
Fusion Proteins, bcr-abl/analysis , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/diagnosis , Monitoring, Physiologic/methods , Humans , Monitoring, Physiologic/standards , Real-Time Polymerase Chain Reaction/methods , Reference StandardsABSTRACT
Identification and quantitative monitoring of mutant BCR-ABL1 subclones displaying resistance to tyrosine kinase inhibitors (TKIs) have become important tasks in patients with Ph-positive leukemias. Different technologies have been established for patient screening. Various next-generation sequencing (NGS) platforms facilitating sensitive detection and quantitative monitoring of mutations in the ABL1-kinase domain (KD) have been introduced recently, and are expected to become the preferred technology in the future. However, broad clinical implementation of NGS methods has been hampered by the limited accessibility at different centers and the current costs of analysis which may not be regarded as readily affordable for routine diagnostic monitoring. It is therefore of interest to determine whether NGS platforms can be adequately substituted by other methodological approaches. We have tested three different techniques including pyrosequencing, LD (ligation-dependent)-PCR and NGS in a series of peripheral blood specimens from chronic myeloid leukemia (CML) patients carrying single or multiple mutations in the BCR-ABL1 KD. The proliferation kinetics of mutant subclones in serial specimens obtained during the course of TKI-treatment revealed similar profiles via all technical approaches, but individual specimens showed statistically significant differences between NGS and the other methods tested. The observations indicate that different approaches to detection and quantification of mutant subclones may be applicable for the monitoring of clonal kinetics, but careful calibration of each method is required for accurate size assessment of mutant subclones at individual time points.
Subject(s)
DNA/analysis , Fusion Proteins, bcr-abl/genetics , High-Throughput Nucleotide Sequencing , Polymerase Chain Reaction , Sequence Analysis, DNA , Comparative Genomic Hybridization , DNA/genetics , DNA/metabolism , Fusion Proteins, bcr-abl/chemistry , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathologyABSTRACT
Recent studies indicate that 40% of chronic myeloid leukemia patients who achieve sustained undetectable BCR-ABL1 transcripts on tyrosine kinase inhibitor therapy remain disease-free after drug discontinuation. In contrast, 60% experience return of detectable disease and have to restart treatment, thus highlighting the need for an improved method of identifying patients with the lowest likelihood of relapse. Here we describe the validation of a personalized DNA-based digital PCR (dPCR) approach for quantifying very low levels of residual disease, which involves the rapid identification of t(9;22) fusion junctions using targeted next-generation sequencing coupled with the use of a dPCR platform. t(9;22) genomic breakpoints were successfully mapped in samples from 32 of 32 patients with early stage disease. Disease quantification by DNA-based dPCR was performed using the Fluidigm BioMark platform on 46 follow-up samples from 6 of the 32 patients, including 36 samples that were in deep molecular remission. dPCR detected persistent disease in 81% of molecular-remission samples, outperforming both RT-dPCR (25%) and DNA-based quantitative PCR (19%). We conclude that dPCR for BCR-ABL1 DNA is the most sensitive available method of residual-disease detection in chronic myeloid leukemia and may prove useful in the management of tyrosine kinase inhibitor withdrawal.
Subject(s)
Fusion Proteins, bcr-abl/genetics , High-Throughput Nucleotide Sequencing/methods , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Polymerase Chain Reaction/methods , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Neoplasm, Residual , Precision Medicine/methods , Sensitivity and SpecificityABSTRACT
The tyrosine kinase inhibitor (TKI) imatinib has revolutionized the management of chronic myeloid leukaemia (CML). However, around 25% of patients fail to sustain an adequate response. We sought to identify gene-expression biomarkers that could be used to predict imatinib response. The expression of 29 genes, previously implicated in CML pathogenesis, were measured by TaqMan Low Density Array in 73 CML patient samples. Patients were divided into low and high expression for each gene and imatinib failure (IF), probability of achieving CCyR, progression free survival and CML related OS were compared by Kaplan-Meier and log-rank. Results were validated in a second cohort of 56 patients, with a further technical validation using custom gene-expression assays in a conventional RT-qPCR in a sub-cohort of 37 patients. Patients with low PTCH1 expression showed a worse clinical response for all variables in all cohorts. PTCH1 was the most significant predictor in the multivariate analysis compared with Sokal, age and EUTOS. PTCH1 expression assay showed the adequate sensitivity, specificity and predictive values to predict for IF. Given the different treatments available for CML, measuring PTCH1 expression at diagnosis may help establish who will benefit best from imatinib and who is better selected for second generation TKI.
Subject(s)
Benzamides/therapeutic use , Gene Expression , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Piperazines/therapeutic use , Protein Kinase Inhibitors/therapeutic use , Pyrimidines/therapeutic use , Receptors, Cell Surface/genetics , Cohort Studies , Disease-Free Survival , Drug Resistance, Neoplasm/genetics , Gene Expression Profiling , Humans , Imatinib Mesylate , Kaplan-Meier Estimate , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/diagnosis , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/mortality , Leukemia, Myeloid, Chronic-Phase/diagnosis , Leukemia, Myeloid, Chronic-Phase/drug therapy , Leukemia, Myeloid, Chronic-Phase/genetics , Leukemia, Myeloid, Chronic-Phase/mortality , Middle Aged , Patched Receptors , Patched-1 Receptor , Pilot Projects , Predictive Value of Tests , Prognosis , Sensitivity and Specificity , Transcriptome , Treatment FailureABSTRACT
Approximately one-third of patients with chronic myeloid leukaemia will fail to achieve or maintain responses to imatinib. Changes in solute carrier family 22 (organic cation transporter), member 1 (SLC22A1, also termed OCT1), the main transporter for imatinib, have been proposed as a possible predictive factor. We analysed SLC22A1 mRNA levels and single nucleotide polymorphisms (SNPs) located in exon 7 in 153 diagnostic whole blood samples from two patient cohorts. The level of SLC22A1 expression did not significantly correlate with imatinib failure or achievement of molecular remission. The SNP 408V>M (g.1222G>A) was present in 65% of patients and was associated in all cases with an eight base-pair insertion (8(+) allele) at the 3' end of exon 7. The latter generates an alternative splice site, leading to a premature stop codon. M420del was found in 33% of patients and never in cis with 8(+) (the 3(-) allele). Significantly longer times to 1% and 0·1% molecular responses (by quantitative reverse transcription polymerase chain reaction) were seen in patients with 8(+) 8(+) or 8(+) N compared to those with the remaining four genotypes (N = no insertion or deletion). Patients lacking 8(+) and 3(-) (NN, 18%) showed the best outcomes overall. Thus, while SLC22A1 expression does not appear to affect response, alterations in its splicing or amino acid sequence may do so.
Subject(s)
Antineoplastic Agents/therapeutic use , Benzamides/therapeutic use , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Organic Cation Transporter 1/genetics , Piperazines/therapeutic use , Protein Kinase Inhibitors/therapeutic use , Pyrimidines/therapeutic use , Adult , Aged , Aged, 80 and over , Alleles , Alternative Splicing , Biological Transport , Codon, Nonsense , Drug Resistance/genetics , Exons/genetics , Female , Fusion Proteins, bcr-abl/blood , Genotype , Humans , Imatinib Mesylate , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Male , Middle Aged , Mutagenesis, Insertional , Polymorphism, Single Nucleotide , Protein Isoforms/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence DeletionABSTRACT
BCR-ABL1 compound mutations can confer high-level resistance to imatinib and other ABL1 tyrosine kinase inhibitors (TKIs). The third-generation ABL1 TKI ponatinib is effective against BCR-ABL1 point mutants individually, but remains vulnerable to certain BCR-ABL1 compound mutants. To determine the frequency of compound mutations among chronic myeloid leukemia patients on ABL1 TKI therapy, in the present study, we examined a collection of patient samples (N = 47) with clear evidence of 2 BCR-ABL1 kinase domain mutations by direct sequencing. Using a cloning and sequencing method, we found that 70% (33/47) of double mutations detected by direct sequencing were compound mutations. Sequential, branching, and parallel routes to compound mutations were common. In addition, our approach revealed individual and compound mutations not detectable by direct sequencing. The frequency of clones harboring compound mutations with more than 2 missense mutations was low (10%), whereas the likelihood of silent mutations increased disproportionately with the total number of mutations per clone, suggesting a limited tolerance for BCR-ABL1 kinase domain missense mutations. We conclude that compound mutations are common in patients with sequencing evidence for 2 BCR-ABL1 mutations and frequently reflect a highly complex clonal network, the evolution of which may be limited by the negative impact of missense mutations on kinase function.
Subject(s)
Fusion Proteins, bcr-abl/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Mutation, Missense/genetics , Piperazines/therapeutic use , Protein Kinase Inhibitors/therapeutic use , Pyrimidines/therapeutic use , Benzamides , Cloning, Molecular , DNA Mutational Analysis/methods , Drug Resistance, Neoplasm/genetics , Fusion Proteins, bcr-abl/chemistry , Gene Expression Regulation, Leukemic/genetics , Humans , Imatinib Mesylate , Protein Structure, TertiaryABSTRACT
Reverse transcription quantitative polymerase chain reaction (RTqPCR)is currently the most sensitive tool available for the routine monitoring of disease level in patients undergoing treatment for BCRABL1 associated malignancies. Considerable effort has been invested at both the local and international levels to standardise the methodology and reporting criteria used to assess this critical metric. In an effort to accommodate the demands of increasing sample throughput and greater standardization, we adapted the current best-practice guidelines to encompass automation platforms and improved multiplex RT-qPCR technology.
Subject(s)
Fusion Proteins, bcr-abl/blood , High-Throughput Screening Assays , Automation, Laboratory , Biomarkers , Diffusion of Innovation , Fusion Proteins, bcr-abl/genetics , Fusion Proteins, bcr-abl/metabolism , High-Throughput Screening Assays/standards , Humans , Kinetics , Limit of Detection , Molecular Probes/metabolism , Multiplex Polymerase Chain Reaction , Neoplasm Proteins , Proto-Oncogene Proteins c-abl/blood , Proto-Oncogene Proteins c-abl/genetics , Proto-Oncogene Proteins c-abl/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The introduction of tyrosine kinase inhibitors (TKIs), starting with imatinib and followed by second and third generation TKIs, has significantly changed the clinical management of patients with chronic myeloid leukemia (CML). Despite their unprecedented clinical success, a proportion of patients fail to achieve complete cytogenetic remission by 12 months of treatment (primary resistance) while others experience progressive resistance after an initial response (secondary resistance). BCR-ABL1 kinase domain (KD) mutations have been detected in a proportion of patients at the time of treatment failure, and therefore their identification and monitoring plays an important role in therapeutic decisions particularly when switching TKIs. When monitoring KD mutations in a clinical laboratory, the choice of method should take into account turnaround time, cost, sensitivity, specificity, and ability to accurately quantify the size of the mutant clone. In this article, we describe in a "manual" style the methods most widely used in our laboratory to monitor KD mutations in patients with CML including direct sequencing, D-HPLC, and pyrosequencing. Advantages, disadvantages, interpretation of results, and their clinical applications are reviewed for each method.
Subject(s)
DNA Mutational Analysis/methods , Fusion Proteins, bcr-abl/genetics , Genes, abl , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Protein-Tyrosine Kinases/genetics , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Chromatography, High Pressure Liquid/methods , Drug Resistance, Neoplasm/genetics , Fusion Proteins, bcr-abl/antagonists & inhibitors , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/enzymology , Mutation , Polymerase Chain Reaction/methods , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Protein Structure, Tertiary/genetics , Protein-Tyrosine Kinases/antagonists & inhibitors , Quality Control , RNA, Messenger/genetics , RNA, Messenger/isolation & purification , RNA, Neoplasm/genetics , RNA, Neoplasm/isolation & purification , Sequence Analysis, DNA/methods , Specimen HandlingABSTRACT
Cyclin-dependent protein kinases (CDKs) are central to the appropriate regulation of cell proliferation, apoptosis, and gene expression. Abnormalities in CDK activity and regulation are common features of cancer, making CDK family members attractive targets for the development of anticancer drugs. Here, we report the identification of a pyrazolo[1,5-a]pyrimidine derived compound, 4k (BS-194), as a selective and potent CDK inhibitor, which inhibits CDK2, CDK1, CDK5, CDK7, and CDK9 (IC50= 3, 30, 30, 250, and 90 nmol/L, respectively). Cell-based studies showed inhibition of the phosphorylation of CDK substrates, Rb and the RNA polymerase II C-terminal domain, down-regulation of cyclins A, E, and D1, and cell cycle block in the S and G2/M phases. Consistent with these findings, 4k demonstrated potent antiproliferative activity in 60 cancer cell lines tested (mean GI50= 280 nmol/L). Pharmacokinetic studies showed that 4k is orally bioavailable, with an elimination half-life of 178 min following oral dosing in mice. When administered at a concentration of 25 mg/kg orally, 4k inhibited human tumor xenografts and suppressed CDK substrate phosphorylation. These findings identify 4k as a novel, potent CDK selective inhibitor with potential for oral delivery in cancer patients.