RESUMO
Quantitative assessment of nucleophosmin 1 (NPM1) mutation status is integral to evaluating measurable residual disease (MRD) in NPM1-mutated acute myeloid leukemia (AML) patients. In a retrospective study, leftover peripheral blood (PB) specimens (n = 40) which were collected for routine clinical diagnostic evaluations of AML disease burden were tested by both a novel automated RT-qPCR quantitative NPM1 assay (Xpert NPM1 mutation assay) and the NPM1 mutA, mutB&D MutaQuant kit. Based on a Deming regression analysis, there was a high correlation (slope = 0.92; intercept = 0.12; Pearson's r = 0.982) between the quantitative results of the Xpert NPM1 mutation assay and the NPM1 mutA, mutB&D MutaQuant kit. The Xpert test quantitative results are thus highly correlated with the comparator method and the former has potential as a useful alternative for the monitoring of AML patients with a known NPM1 mutation.
Assuntos
Leucemia Mieloide Aguda , Mutação , Proteínas Nucleares , Nucleofosmina , Reação em Cadeia da Polimerase em Tempo Real , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/diagnóstico , Proteínas Nucleares/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Estudos Retrospectivos , Neoplasia Residual/genética , Neoplasia Residual/diagnóstico , Masculino , Feminino , Pessoa de Meia-IdadeRESUMO
Bone marrow failure (BMF) related to hypoplasia of hematopoietic elements in the bone marrow is a heterogeneous clinical entity with a broad differential diagnosis including both inherited and acquired causes. Accurate diagnostic categorization is critical to optimal patient care and detection of genomic variants in these patients may provide this important diagnostic and prognostic information. We performed real-time, accredited (ISO15189) comprehensive genomic characterization including targeted sequencing and whole exome sequencing in 115 patients with BMF syndrome (median age 24 years, range 3 months - 81 years). In patients with clinical diagnoses of inherited BMF syndromes, acquired BMF syndromes or clinically unclassifiable BMF we detected variants in 52% (12/23), 53% (25/47) and 56% (25/45) respectively. Genomic characterization resulted in a change of diagnosis in 30/115 (26%) including the identification of germline causes for 3/47 and 16/45 cases with pre-test diagnoses of acquired and clinically unclassifiable BMF respectively. The observed clinical impact of accurate diagnostic categorization included choice to perform allogeneic stem cell transplantation, disease-specific targeted treatments, identification of at-risk family members and influence of sibling allogeneic stem cell donor choice. Multiple novel pathogenic variants and copy number changes were identified in our cohort including in TERT, FANCA, RPS7 and SAMD9. Whole exome sequence analysis facilitated the identification of variants in two genes not typically associated with a primary clinical manifestation of BMF but also demonstrated reduced sensitivity for detecting low level acquired variants. In conclusion, genomic characterization can improve diagnostic categorization of patients presenting with hypoplastic BMF syndromes and should be routinely performed in this group of patients.
Assuntos
Transtornos da Insuficiência da Medula Óssea , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Transtornos da Insuficiência da Medula Óssea/diagnóstico , Transtornos da Insuficiência da Medula Óssea/genética , Criança , Pré-Escolar , Genômica , Transplante de Células-Tronco Hematopoéticas , Humanos , Lactente , Pessoa de Meia-Idade , Adulto JovemAssuntos
Transplante de Células-Tronco Hematopoéticas , Leucemia Mieloide Aguda , Humanos , Tirosina Quinase 3 Semelhante a fms/genética , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Mutação , Neoplasia Residual/diagnóstico , Neoplasia Residual/genética , Reação em Cadeia da Polimerase , PrognósticoAssuntos
Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Taxa de Mutação , Proteínas Proto-Oncogênicas c-bcl-2/genética , Sulfonamidas/uso terapêutico , Substituição de Aminoácidos/genética , Antineoplásicos/uso terapêutico , Estudos de Coortes , Análise Mutacional de DNA , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/genética , Frequência do Gene , Glicina/genética , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Modelos Moleculares , Mutação , Proteínas Proto-Oncogênicas c-bcl-2/química , Células Tumorais Cultivadas , Valina/genéticaRESUMO
Mutations in CALR have recently been detected in JAK2-negative myeloproliferative neoplasms (MPNs) and are key pathological drivers in these diseases. CALR-mutated MPNs are shown to have numerous clinicopathological differences to JAK2-mutated MPNs. The basis of these differences is poorly understood. It is unknown whether these differences result directly from any differences in intracellular signalling abnormalities induced by JAK2/CALR mutations or whether they relate to other phenomena such as a differing spectrum of genetic lesions between the two groups. We aimed to review the clinicopathological and molecular features of CALR- and JAK2-mutated MPNs from samples referred for diagnostic testing using a custom-designed targeted next-generation sequencing (NGS) panel. Eighty-nine CALR-mutated cases were compared with 70 JAK2-mutated cases. CALR-mutated MPNs showed higher platelet counts and a female predominance as compared to JAK2-mutated MPNs in our cohort. We have also observed differences between CALR mutation subtypes in terms of disease phenotype, mutational frequency and allelic burden. Type 1 CALR mutations were found to be more common in myelofibrosis, associated with a higher frequency and number of additional mutations and a higher mutant allelic burden as compared to type 2 CALR mutations. Despite these biological differences, our molecular characterisation suggests that CALR- and JAK2-mutated MPNs are broadly similar in terms of the quantity, frequency and spectrum of co-occurring mutations and therefore observed biological differences are likely to not be heavily influenced by the nature and quantity of co-mutated genes.
Assuntos
Calreticulina/genética , Janus Quinase 2/genética , Mutação , Transtornos Mieloproliferativos/diagnóstico , Transtornos Mieloproliferativos/genética , Fenótipo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Biomarcadores , Análise por Conglomerados , Estudos de Coortes , Análise Mutacional de DNA , Feminino , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
External quality assessment programs (EQAP) for molecular haematology generally only assess the analytical phase of laboratory testing or provide limited evaluation of post-analytical components. We incorporated comprehensive post-analytical evaluation into an existing national inter-laboratory sample exchange program for molecular haematology due to the increasing complexity of diagnostic molecular testing and interpretation. We report key findings from four years of longitudinal data using this approach. Eighteen participating laboratories enrolled in an annual reciprocal sample exchange program from 2019-2022, which covered conventional and next-generation sequencing (NGS) assays. Participants submitted results on their laboratory information system-generated reports which then underwent central review. Reports were assessed according to consensus values and relevant national and international reporting standards and guidelines. A total of 680 reports were received. Laboratories had high concordance in the analytical phase of testing, with incorrect variant detection observed in a total of six of 680 (0.9%) reports. In contrast, post-analytical concordance was much lower, with at least one discordance observed in 28.9-57.6% of all conventional reports and 33.3-100% NGS reports. The most frequent post-analytical discordances were: (1) not including key technical information on reports (total 41.9% conventional, 47.2% NGS); (2) not using standard gene and variant nomenclature (total 28.2% conventional, 25.6% NGS). NGS reports also demonstrated discrepancies in variant classification (total 20.4%) and interpretation (total 10.2%). The rate of discrepancies generally improved year-on-year. Inter-laboratory concordance for molecular haematology testing is high in the analytical phase, however opportunities exist for improvement in the post-analytical phase. Given that result interpretation is crucial for clinical decision-making and that molecular testing is a complex and evolving field, we suggest that EQAPs should comprehensively evaluate both analytical and post-analytical components of laboratory performance in order to harmonise reporting and to support the accurate interpretation of molecular haematology tests.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Laboratórios Clínicos , Hematologia/normas , Garantia da Qualidade dos Cuidados de Saúde , Técnicas de Diagnóstico MolecularAssuntos
DNA Tumoral Circulante/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Transtornos Linfoproliferativos/diagnóstico , Adulto , Idoso , Linfócitos B/patologia , Linfoma de Burkitt/diagnóstico , Linfoma de Burkitt/genética , Feminino , Genômica , Humanos , Transtornos Linfoproliferativos/genética , Masculino , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/genética , Hibridização de Ácido NucleicoRESUMO
BACKGROUND: The JAK2 V617F mutation is the most frequent somatic change in myeloproliferative neoplasms, making it an important tumour-specific marker for diagnostic purposes and for the detection of minimal residual disease. Sensitive quantitative assays are required for both applications, particularly for the monitoring of minimal residual disease, which requires not only high sensitivity but also very high specificity. METHODS: We developed a highly sensitive probe-free quantitative mutant-allele detection method, Quantitative Threefold Allele-Specific PCR (QuanTAS-PCR), that is performed in a closed-tube system, thus eliminating the manipulation of PCR products. QuantTAS-PCR uses a threefold approach to ensure allele-specific amplification of the mutant sequence: (i) a mutant allele-specific primer, (ii) a 3'dideoxy blocker to suppress false-positive amplification from the wild-type template and (iii) a PCR specificity enhancer, also to suppress false-positive amplification from the wild-type template. Mutant alleles were quantified relative to exon 9 of JAK2. RESULTS: We showed that the addition of the 3'dideoxy blocker suppressed but did not eliminate false-positive amplification from the wild-type template. However, the addition of the PCR specificity enhancer near eliminated false-positive amplification from the wild-type allele. Further discrimination between true and false positives was enabled by using the quantification cycle (Cq) value of a single mutant template as a cut-off point, thus enabling robust distinction between true and false positives. As 10,000 JAK2 templates were used per replicate, the assay had a sensitivity of 1/10(-4) per replicate. Greater sensitivity could be reached by increasing the number of replicates analysed. Variation in replicates when low mutant-allele templates were present necessitated the use of a statistics-based approach to estimate the load of mutant JAK2 copies. QuanTAS-PCR showed comparable quantitative results when validated against a commercial assay. CONCLUSIONS: QuanTAS-PCR is a simple, cost-efficient, closed-tube method for JAK2 V617F mutation quantification that can detect very low levels of the mutant allele, thus enabling analysis of minimal residual disease. The approach can be extended to the detection of other recurrent single nucleotide somatic changes in cancer.
Assuntos
Análise Mutacional de DNA/métodos , Janus Quinase 2/genética , Leucemia Eritroblástica Aguda/genética , Mutação Puntual , Reação em Cadeia da Polimerase em Tempo Real/métodos , Alelos , Primers do DNA , Éxons , Reações Falso-Positivas , Células HL-60 , Humanos , Neoplasia ResidualRESUMO
FLT3 internal tandem duplication (ITD) quantitation is key to prognostication in acute myeloid leukaemia (AML). One potential source of variability in the allelic ratio (AR) is the number of polymerase chain reaction (PCR) cycles used. Using 30 archived samples of varying ITD lengths and AR, we compared two FLT3-ITD assays (Huang and RATIFY), evaluated the effect of PCR cycle number on each assay, and examined the potential clinical consequences. Huang and RATIFY assays at 35 and 27 PCR cycles, respectively, were highly concordant. A progressive decrease in AR (median 47%) was observed with the RATIFY assay when the PCR cycles were increased from 27 to 35 cycles, potentially impacting risk categorisation in 29% of patients. In contrast, minimal change in AR was observed with the Huang assay. Hence, both FLT3-ITD assays were almost identical using respective standard conditions, but the effect of PCR cycle number is assay-dependent, which may impact risk stratification in AML.
Assuntos
Leucemia Mieloide Aguda , Humanos , Reação em Cadeia da Polimerase , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Tirosina Quinase 3 Semelhante a fms/genética , Mutação , PrognósticoRESUMO
BACKGROUND: Measurable residual disease (MRD) monitoring in acute lymphoblastic leukemia (ALL) is an important predictive factor for patient outcome and treatment intensification. Molecular monitoring, particularly with quantitative polymerase chain reaction (qPCR) to measure immunoglobin heavy or kappa chain (Ig) or T-cell receptor (TCR) gene rearrangements, offers high sensitivity but accessibility is limited by expertise, cost, and turnaround time. Flow cytometric assays are cheaper and more widely available, and sensitivity is improved with multi-parameter flow cytometry at eight or more colors. METHODS: We developed a 10-color single tube flow cytometry assay. Samples were subject to bulk ammonium chloride lysis to maximize cell yields with a target of 1 × 106 events. Once normal maturation patterns were established, patient samples were analyzed in parallel to standard molecular monitoring. RESULTS: Flow cytometry was performed on 114 samples. An informative immunophenotype was identifiable in all 22 patients who had a diagnostic sample. MRD analysis was performed on 87 samples. The median lower limits of detection and quantification were 0.004% (range 0.0005%-0.028%) and 0.01% (range 0.001%-0.07%) respectively. Sixty-five samples had concurrent molecular MRD testing, with good correlation (r = 0.83, p < 0.001). Results were concordant in 52 samples, and discordant in 13 samples, including one case where impending relapse was detected by flow cytometry but not Ig/TCR qPCR. CONCLUSIONS: Our 10-color flow cytometric MRD assay provided adequate sensitivity and good correlation with molecular assays. This technique offers rapid and affordable testing in B-ALL patients, including cases where a suitable molecular assay cannot be developed or has reduced sensitivity.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras , Doença Aguda , Adulto , Citometria de Fluxo/métodos , Humanos , Imunofenotipagem , Neoplasia Residual/diagnóstico , Neoplasia Residual/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Receptores de Antígenos de Linfócitos TRESUMO
Identification of patients at risk of initial & recurrent cytomegalovirus (CMV) reactivation following allogeneic stem cell transplant (alloSCT) may help guide prophylactic strategies. T-cell receptor beta (TRB) deep sequencing was used to identify and enumerate the T-cell repertoire harbouring TRB sequences with annotated specificity to CMV (pubCMVrep), as well as the overall T-cell receptor (TCR) repertoire diversity at day +30 & day +60 post-alloSCT for 65 patients. T-cells harbouring TRB sequences with annotated specificity for CMV were identifiable in all patients. 56% of patients required CMV treatment and 23% of the cohort developed recurrent CMV. PubCMVrep size at day +30 was not associated with reactivation, however amongst patients with antecedent CMV viremia a low day +60 pubCMVrep was associated with a greater incidence of recurrent CMV (75% vs. 21%, HR 6.16, 95% CI 1.29-29.40, P = 0.0008). Moreover, patients with high pubCMVrep only developed recurrent CMV in the setting of GVHD. Low TCR diversity at day +30 was associated with a greater incidence of initial CMV reactivation (71% vs. 22%, HR 5.39, 95% CI 1.70-17.09, p = 0.0002). pubCMVrep and TCR diversity are promising biomarkers to identify patients at risk of initial & recurrent CMV who may benefit from novel prophylactic strategies.
Assuntos
Infecções por Citomegalovirus , Transplante de Células-Tronco Hematopoéticas , Citomegalovirus , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Receptores de Antígenos de Linfócitos T/genética , Transplante de Células-Tronco/efeitos adversos , Transplante Homólogo , Ativação ViralRESUMO
Context Detection of measurable residual disease after therapy is an important predictor of outcome in acute myeloid leukemia. Objective To investigate the feasibility of using next-generation sequencing (NGS) in the diagnostic laboratory to perform quantitative NPM1 mutation assessment using ultradeep (approximately 300 000×-500 000×) sequencing (NGS-q NPM1) as a method of assessing residual disease burden in patients with acute myeloid leukemia. Design A flexible NGS-based assay for the detection and quantitation of NPM1 mutations was developed by polymerase chain reaction amplification of target DNA sequences, sequencing on an Illumina (San Diego, California) MiSeq, and analyzing data with an in-house-designed bioinformatic pipeline. NGS-q NPM1 was compared with current NPM1 quantitation methods (real-time quantitative-polymerase chain reaction and multiparameter flow cytometry). Results The NGS-q NPM1 assay had a sensitivity of between 10-4 and 10-5 and showed high concordance and correlation with reference methodologies. Moreover, the NGS-q NPM1 assay was able to be integrated into the laboratory's existing, targeted amplicon-based sequencing workflow. Conclusions An NGS-based, quantitative NPM1-mutation assessment can be used to monitor patients with acute myeloid leukemia, and it has some practical advantages over existing modalities.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Leucemia Mieloide Aguda/diagnóstico , Proteínas Nucleares/genética , Biomarcadores Tumorais/análise , Análise Mutacional de DNA/métodos , Humanos , Mutação , Neoplasia Residual , Proteínas Nucleares/análise , Nucleofosmina , Sensibilidade e EspecificidadeRESUMO
Massively parallel sequencing (MPS) technology has become routinely available for diagnosis, prognostication and therapeutic decision-making in haematological malignancies. However, increased throughput and wider coverage of genes can have unintended consequences. Germline variants of potential clinical significance (GVPCSs) detected during cancer testing may have implications for patients and families beyond the biological evaluation of a specific tumour. 721 reports generated from MPS panels used in the routine testing of myeloid and lymphoid malignancies were reviewed and variants within genes of potential germline relevance (TP53, RUNX1, GATA2 and WT1 in all contexts and CBL, KRAS and NRAS in the setting of juvenile myelomonocytic leukaemia) were analysed. A variant allele fraction threshold of ≥33.09% for considering germline origin of variants within cancer samples was established. The detection rate of incidental, pathogenic germline variants was 0.42%. Patient education and confirmatory germline sample testing of GVPCSs in appropriate circumstances are recommended.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Hematológicas/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias/genética , Mutação em Linhagem Germinativa , Neoplasias Hematológicas/diagnóstico , HumanosAssuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Medula Óssea/patologia , Linfoma de Burkitt/genética , Segunda Neoplasia Primária/genética , Biópsia , Linfoma de Burkitt/diagnóstico , Linfoma de Burkitt/patologia , Genômica/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Segunda Neoplasia Primária/tratamento farmacológico , Segunda Neoplasia Primária/patologia , Proteínas de Fusão Oncogênica/genéticaRESUMO
De novo presentation of acute myeloid leukemia (AML) expressing the Philadelphia (Ph) chromosomal abnormality is rare and is associated with a dismal prognosis. To date, reported cases of Ph(+) AML have expressed either the e13a2 or e14a2 BCR-ABL fusion transcripts. We report a unique case of de novo AML expressing the e6a2 fusion transcript and describe disease sensitivity to both imatinib before allogeneic stem-cell transplantation and dasatinib for AML relapse after allogeneic stem-cell transplantation. Furthermore, we report that sustained molecular remission has been achieved despite withdrawal of tyrosine kinase inhibitor (TKI) therapy.