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Sample identification error is a severe medical error in clinical molecular diagnostic laboratories, which can lead to reporting the wrong results for the patient involved. Sample contamination can also lead to incorrect test reports. Avoiding sample identification error and sample contamination could be life-saving. Sample switch and sample contamination could happen on laboratory bench works, especially when pipetting into multi-well plates. It is difficult to realize such errors during laboratory bench work. Laboratory staff may not be aware of such an error when it happens. DNA fingerprinting technology can be used to determine sample identity and subsequently identify sample switch and sample contamination in the laboratory. Our laboratory has explored the usage of this technology in our quality control process and successfully established that DNA fingerprinting can be used to monitor sample switch and sample contamination in next-generation sequencing and BCR/ABL1 real-time PCR bench work.
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Diagnostic errors occur in the preanalytic, analytic, and postanalytic phases of specimen processing. Correlating clinical and imaging information with gross and microscopic findings is crucial to limit errors and unnecessary treatment. Herein, we report the case of a 54-year-old woman who presented with left breast bloody nipple discharge and subsequently underwent central duct excision. Pathology revealed a high-grade sarcoma. The patient presented to our institution for further management. Upon secondary pathology review and DNA fingerprinting analysis, the correct interpretation was rendered. Our case demonstrates the importance of clinical correlation and review of pathology slides prior to definitive therapy.
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Neoplasias de la Mama , Fibrosarcoma , Glándulas Mamarias Humanas , Secreción del Pezón , Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/cirugía , Femenino , Humanos , Persona de Mediana Edad , PezonesRESUMEN
BACKGROUND: The detection of CEBPA and FLT3 mutations by next generation sequencing (NGS) is challenging due to high GC content and Internal Tandem Duplications (ITDs). Recent advances have been made to surmount these challenges. In this study, we compare three commercial kits and evaluate the performance of these more advanced hybrid-capture and AMP-chemistry based methods. METHODS: Amplicon-based TSM 54-Gene Panel (Illumina) was evaluated against hybridization-capture SOPHiA Genetics MSP, OGT SureSeq, and AMP chemistry-based VariantPlex (Archer) for wet-lab workflow and data-analysis pipelines. Standard kit directions and commercial analysis pipelines were followed. Seven CEBPA and 10 FLT3-positive cases were identified that previously were missed on an amplicon NGS assay. The average reads, coverage uniformity, and the detection of CEBPA or FLT3 mutations were compared. RESULTS: All three panels detected all 10 CEBPA mutations and all 10 FLT3 ITDs with 100% sensitivity. In addition, there was high concordance (100%) between all three panels detecting 47/47 confirmed variants in a set of core myeloid genes. CONCLUSIONS: The results show that the NGS assays are now able to reliably detect CEBPA mutations and FLT3 ITDs. These assays may allow foregoing additional orthogonal testing for CEBPA and FLT3.
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Secuenciación de Nucleótidos de Alto Rendimiento , Leucemia Mieloide Aguda , Humanos , Proteínas Potenciadoras de Unión a CCAAT/genética , Tirosina Quinasa 3 Similar a fms/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Leucemia Mieloide Aguda/genética , Mutación , Secuencias Repetidas en Tándem/genéticaRESUMEN
Chronic myelogenous leukemia (CML) is a hematopoietic stem cell malignancy that accounts for 15-20% of all cases of leukemia. CML is caused by a translocation between chromosomes 9 and 22 which creates an abnormal fusion gene, BCR::ABL1. The amount of BCR::ABL1 transcript RNA is a marker of disease progression and the effectiveness of tyrosine kinase inhibitor (TKI) treatment. This study determined the analytical and clinical performance of a droplet digital PCR based assay (QXDx BCR-ABL %IS Kit; Bio-Rad) for BCR::ABL1 quantification. The test has a limit of detection of MR4.7 (0.002%) and a linear range of MR0.3-4.7 (50-0.002%IS). Reproducibility of results across multiple sites, days, instruments, and users was evaluated using panels made from BCR::ABL1 positive patient samples. Clinical performance of the assay was evaluated on patient samples and compared to an existing FDA-cleared test. The reproducibility study noted negligible contributions to variance from site, instrument, day, and user for samples spanning from MR 0.7-4.2. The assay demonstrated excellent clinical correlation with the comparator test using a Deming regression with a Pearson R of 0.99, slope of 1.037 and intercept of 0.1084. This data establishes that the QXDx™ BCR-ABL %IS Kit is an accurate, precise, and sensitive system for the diagnosis and monitoring of CML.
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Proteínas de Fusión bcr-abl , Leucemia Mielógena Crónica BCR-ABL Positiva , Proteínas de Fusión bcr-abl/genética , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/diagnóstico , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Reacción en Cadena de la Polimerasa/métodos , Inhibidores de Proteínas Quinasas/uso terapéutico , Reproducibilidad de los Resultados , Estados Unidos , United States Food and Drug AdministrationRESUMEN
In the era of personalized medicine, information on molecular change at the gene level is important for patient care. Such information has been used for disease classification, diagnosis, prognosis, risk stratification, and treatment, which is especially important in cancer patient care. Many molecular tests exist and can be used to detect the molecular changes at gene level. These tests include, but are not limited to, karyotyping, endpoint polymerase chain reaction (PCR), real-time PCR, Sanger sequencing, pyrosequencing, next-generation sequencing, and so forth. How to use the right tests for the right patients at the right time is essential for optimal patient outcome. This review puts together some information on molecular testing for acute myeloid leukemia.
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We describe the clinical validation of a targeted DNA and RNA-based next-generation sequencing (NGS) assay at two clinical molecular diagnostic laboratories. This assay employs simultaneous DNA and RNA analysis of all coding exons to detect small variants (single-nucleotide variants, insertions, and deletions) in 148 genes, amplifications in 59 genes, and fusions and splice variants in 55 genes. During independent validations at two sites, 234 individual specimens were tested, including clinical formalin-fixed, paraffin-embedded (FFPE) tumor specimens, reference material, and cell lines. Samples were prepared using the Illumina TruSight Tumor 170 (TST170) kit, sequenced with Illumina sequencers, and the data were analyzed using the TST170 App. At both sites, TST170 had ≥98% success for ≥250× depth for ≥95% of covered positions. Variant calling was accurate and reproducible at allele frequencies ≥5%. Limit of detection studies determined that inputs of ≥50 ng of DNA (with ≥3.3 ng/µl) and ≥50 ng RNA (minimum of 7 copies/ng) were optimal for high analytical sensitivity. The TST170 assay results were highly concordant with prior results using different methods across all variant categories. Optimization of nucleic acid extraction and DNA shearing, and quality control following library preparation is recommended to maximize assay success rates. In summary, we describe the validation of comprehensive and simultaneous DNA and RNA-based NGS testing using TST170 at two clinical sites.
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We present a patient who developed severe lower urinary tract symptoms following resection of a Ta low grade bladder urothelial carcinoma with immediate post-resection instillation of mitomycin-C. Urine cultures were negative. Radiographic imaging demonstrated a bladder mass. On biopsy she was found to have eosinophilic cystitis. We report the clinical scenario and discuss treatment options. A review of reported cases in the English literature is provided.
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Antibióticos Antineoplásicos/efectos adversos , Cistitis/inducido químicamente , Eosinofilia/inducido químicamente , Mitomicina/efectos adversos , Administración Intravesical , Antibióticos Antineoplásicos/administración & dosificación , Femenino , Humanos , Persona de Mediana Edad , Mitomicina/administración & dosificación , Factores de TiempoRESUMEN
BACKGROUND: Patients with deficient microsatellite mismatch repair (dMMR) colorectal cancer (CRC) may respond to immune checkpoint inhibition (ICI), whereas patients with microsatellite-stable (MSS) CRC have not demonstrated response. However, a proportion of MSS tumors display histomorphologic features characteristic of dMMR tumors consistent with an increased antigenicity. Therefore, a subset of patients with CRC not currently receiving ICI treatment may derive benefit from ICI therapy. We review tumors in which the histologic features suggestive of dMMR were in disagreement with the DNA mismatch repair proteins obtained by immunohistochemistry (IHC). Possible causes of such disagreement are discussed. MATERIALS AND METHODS: Three patients with CRC suggestive of histomorphologic immunogenicity underwent evaluation by IHC staining for mismatch repair (MMR) status, next-generation sequencing assays, and/or polymerase chain reaction. RESULTS: Findings compatible with an immunogenic response were similarly observed in all patients. Case 1 highlighted the limiting factors inherent to IHC staining for MMR status: a biopsy initially interpreted as MSS was subsequently interpreted as being dMMR. Case 2 examined the challenges in reconciling histologic characteristics traditionally associated with dMMR CRCs but ultimately determined to be MSS. Case 3 examined the microsatellite instability of CRC resulting from MLH1-methylation and/or MSH6 mutation. CONCLUSIONS: We demonstrated the challenges in establishing MMR status when confronted with conflicting results from histology, IHC, polymerase chain reaction, and next-generation sequencing. Given that dMMR status has been shown to be a biomarker for ICI responsiveness, the importance of accurate identification is critical.
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Biomarcadores de Tumor/genética , Colectomía , Neoplasias Colorrectales/diagnóstico , Reparación de la Incompatibilidad de ADN , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Anciano , Biomarcadores de Tumor/análisis , Biopsia , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/terapia , Análisis Mutacional de ADN , Femenino , Humanos , Inmunohistoquímica , Masculino , Inestabilidad de Microsatélites , Persona de Mediana Edad , Mutación , Selección de PacienteRESUMEN
OBJECTIVES: To compare the clinical significance of SF3B1/DNMT3A Comutations with SF3B1 or DNMT3A mutation alone in myelodysplastic syndrome (MDS) and clonal cytopenia of undetermined significance (CCUS). METHODS: We identified and compared 31 patients with only DNMT3A mutation, 48 patients with only SF3B1 mutation, and 16 patients with only SF3B1/DNMT3A comutations. RESULTS: SF3B1/DNMT3A comutations were found to be more common in MDS, whereas DNMT3A mutation alone was more common in CCUS. The patients with SF3B1/DNMT3A comutations were less likely to have poor cytogenetics than patients with DNMT3A mutation alone. Patients with SF3B1/DNMT3A comutations showed significantly longer median survival time and better overall survival than patients with DNMT3A mutation alone. CONCLUSIONS: Patients with SF3B1/DNMT3A comutations appear to have better clinical outcomes than patients with isolated DNMT3A mutation. These findings suggest that the favorable prognosis of SF3B1 mutation in is not abrogated by the concurrent presence of a DNMT3A mutation.
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Enfermedades de la Médula Ósea/genética , ADN (Citosina-5-)-Metiltransferasas/genética , Síndromes Mielodisplásicos/genética , Fosfoproteínas/genética , Factores de Empalme de ARN/genética , Anciano , Enfermedades de la Médula Ósea/mortalidad , ADN Metiltransferasa 3A , Femenino , Humanos , Masculino , Mutación , Síndromes Mielodisplásicos/mortalidadRESUMEN
Studies comparing the prognostic role of RUNX1 mutations (RUNX1mut) in acute myeloid leukemia (AML) and acute myeloid leukemia-with myelodysplasia-related changes (AML-MRC) are limited. Our study examines the genetic profile of 118 RUNX1mut AML patients including 57 AML with RUNX1mut and 61 AML-MRC with RUNX1mut and 100 AML, NOS patients with wild type RUNX1 (RUNX1wt). Results revealed that AML-MRC patients with RUNX1mut had shorter median overall survival (OS) (11 ± 3.3 months) when compared to AML with RUNX1mut (19 ± 7.1 months) and AML, NOS with RUNX1wt (not reached) (p = .001). The most common concurrent mutations observed in AML-MRC with RUNX1mut patients were DNMT3A, SRSF2, ASXL1, and IDH2 while in AML with RUNX1mut patients were ASXL1, SRSF2, TET2, IDH2, and DNMT3A. ASXL1 and TET2 mutations appeared to adversely affect OS in AML-MRC, but not in AML with RUNX1mut. Concurrent RUNX1/DNMT3A mutations, in contrast had negative impact on OS in AML with RUNX1mut, but not in AML-MRC with RUNX1mut.
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Leucemia Mieloide Aguda , Síndromes Mielodisplásicos , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Mutación , Síndromes Mielodisplásicos/genética , PronósticoRESUMEN
A risk mitigation strategy was implemented to determine if a higher prophylactic voriconazole dosage in patients with CYP2C19 rapid metabolizer neutropenic acute myeloid leukemia (AML) reduces the incidence of subtherapeutic trough concentrations. Patients with AML (n = 263) were preemptively genotyped for CYP2C19*2, *3, and *17 alleles as part of a single-center prospective, interventional, quality improvement study. CYP2C19 rapid metabolizers (CYP2C19*1/*17) were recommended to receive interventional voriconazole 300 mg twice daily, ultrarapid metabolizers (CYP2C19*17/*17) were recommended to avoid voriconazole, and all others received the standard prophylactic dosage of 200 mg twice daily. In this real-world setting, 202 patients (76.8%) were prescribed prophylactic voriconazole, and of these patients 176 (87.1%) received CYP2C19-guided prophylactic dosing. Voriconazole trough concentrations were obtained for 41 of the 58 (70.7%) CYP2C19 rapid metabolizers prescribed prophylactic voriconazole. Interventional voriconazole resulted in higher plasma trough concentrations (median 2.7 µg/mL) compared with the standard prophylactic dosage (median 0.6 µg/mL; P = 0.001). Subtherapeutic concentrations were avoided in 83.8% of CYP2C19 rapid metabolizers receiving interventional dosage compared to 46.2% receiving standard dosage (P = 0.02). CYP2C19 genotyping to preemptively guide prophylactic voriconazole dosing is feasible and may be a potential strategy for reducing the risk of subtherapeutic trough concentrations that potentiate breakthrough fungal infections.
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Antifúngicos/administración & dosificación , Citocromo P-450 CYP2C19/genética , Leucemia Mieloide Aguda/complicaciones , Micosis/prevención & control , Voriconazol/administración & dosificación , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Antifúngicos/farmacocinética , Relación Dosis-Respuesta a Droga , Femenino , Genotipo , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Neutropenia/etiología , Estudios Prospectivos , Gestión de Riesgos , Voriconazol/farmacocinética , Adulto JovenRESUMEN
BACKGROUND: Inverted papilloma (IP) is an uncommon sinonasal tumor. Squamous cell carcinoma (SCC) is associated with IP in about 7% of cases. To date, there has been no pooled analysis to formulate a survival outcome associated with this rare condition. PATIENTS AND METHODS: We retrospectively reviewed the medical records of patients with IP and SCC treated at our institution during 1999-2007. Including our series, a systematic review of literature on Medline database and pooled analysis were performed to establish a survival estimate. RESULTS: Six patients were identified. Squamous cell carcinoma was metachronous to the initial diagnosis of IP in 1 case and synchronous in 5 cases. Of 5 patients who had completed therapy at the time of this report, only 1 remained disease-free at 74 months. The median overall survival in our series was 33 months. Three patients developed distant metastases in brain, lung, bone, and liver. Literature review and pooled survival analysis consisting of 76 cases indicated a median overall survival of 126 months with 3- and 5-year survival rates of 63% and 61%, respectively. CONCLUSION: Although the survival outcome of SCC arising from IP seems comparable with sinonasal SCCs, some patients with this disease do have a highly aggressive disease, including hematogenous distant metastasis. Overall, about 40% of patients will die of the disease within the first 3 years.
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Carcinoma de Células Escamosas/mortalidad , Carcinoma de Células Escamosas/terapia , Papiloma Invertido/mortalidad , Papiloma Invertido/terapia , Neoplasias de los Senos Paranasales/mortalidad , Neoplasias de los Senos Paranasales/terapia , Adulto , Anciano , Biopsia con Aguja , Carcinoma de Células Escamosas/patología , Quimioterapia Adyuvante , Terapia Combinada , Femenino , Estudios de Seguimiento , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Procedimientos Quirúrgicos Otorrinolaringológicos/métodos , Papiloma Invertido/patología , Neoplasias de los Senos Paranasales/patología , Radioterapia Adyuvante , Muestreo , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
BACKGROUND/AIM: To report a case of mixed medullary/mucinous adenocarcinoma with unusual mutational gene profile. PATIENTS AND METHODS: A 79-year-old female was diagnosed with a colorectal carcinoma of the transverse colon. The diagnostic work-up of this case included thorough clinicopathological evaluation, immunohistochemistry and next generation Sequencing. RESULTS: The clinicopathological evaluation showed a tumor with morphological features of both medullary and mucinous colorectal carcinoma. Immunohistochemistry revealed the loss of DNA mismatch repair proteins. NGS showed that the medullary component of this tumor had a novel STK11 p.G270W mutation, which was not present in the mucinous component. Both the medullary and mucinous components also had BRAF V600E and AKT1 (pE17K) mutations. CONCLUSION: We report a novel mutation STK11 (p.G270W), in medullary carcinoma of the colon with an associated mucinous component.
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Adenocarcinoma Mucinoso/genética , Carcinoma Medular/genética , Colon Transverso/patología , Anciano , Femenino , Humanos , MutaciónRESUMEN
One of the major quality assurance (QA) goals in many molecular laboratories is to avoid sample pipetting errors on the lab bench; especially when pipetting into multiwell plates. A pipetting error can cause a switch in patient samples, which can lead to recording the wrong results for the patient samples involved. Such pipetting errors are difficult to identify when it happens in lab bench work. DNA fingerprinting is a powerful tool in determining sample identities. Our laboratory has explored the usage of this technology in our QA process and successfully established that DNA fingerprinting can be used to monitor possible sample switch in gene rearrangement lab bench work. We use florescent light to quench the florescence in the gene rearrangement polymerase chain reaction products. After that, DNA fingerprinting technology is used to identify the sample DNA in the gene rearrangement polymerase chain reaction plate. The result is compared with the corresponding patient's blood sample DNA to determine whether there is a sample switch during the lab bench work.
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Técnicas de Laboratorio Clínico/métodos , Técnicas de Laboratorio Clínico/normas , Dermatoglifia del ADN , Control de Calidad , Humanos , Reacción en Cadena de la Polimerasa , Error Científico ExperimentalRESUMEN
AIM: To report the first case of a Richter syndrome where small lymphocytic lymphoma (SLL) progressed to a CD3+ diffuse large B-cell lymphoma (DLBCL). METHODS: Macrodissection of small and large cell lymphomatous components was performed. This was followed by flow cytometric analysis along with molecular B-cell immunoglobulin (heavy and light chains) and T-cell receptor (γ and ß chains) gene rearrangement studies to investigate a clonal relationship between the components. RESULTS: The immunophenotypic profile was similar between small and large cell components of the lymphoma by flow cytometry. Furthermore, shared clonal peaks were observed between both components based on molecular B-cell and T-cell receptor gene rearrangement studies, confirming a clonal relationship. CONCLUSIONS: Chronic lymphocytic leukaemia/SLL may rarely undergo Richter transformation to a DLBCL demonstrating lineage infidelity. This is a potentially important diagnostic pitfall and such cases should not be confused with a de novo T-cell lymphoma.
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Neoplasias de Cabeza y Cuello/patología , Leucemia Linfocítica Crónica de Células B/patología , Linfoma de Células B Grandes Difuso/patología , Anciano de 80 o más Años , Transformación Celular Neoplásica/patología , Citometría de Flujo , Humanos , Metástasis Linfática , MasculinoRESUMEN
OBJECTIVES: To compare the mutational profiles of patients with primary myelofibrosis (PMF), polycythemia vera (PV), and essential thrombocytosis (ET). METHODS: Next-generation sequencing results of 75 cases of PMF, 33 cases of PV, and 27 cases of ET were compared. RESULTS: Mutation rates of ASXL1 and SRSF2 were significantly higher in PMF than in PV or ET. ASXL1 mutations appeared to be more frequently associated with risk of transformation to acute myeloid leukemia than JAK2 or TET2 mutations. The most common mutation-cytogenetic combinations in myeloproliferative neoplasm (MPN) were mutations of JAK2 or ASXL1 with del(20q) and were more common in patients with PMF and PV than in patients with ET. Differences were also found between patients with PMF and PV. CONCLUSIONS: PMF, PV, and ET show different mutational profiles, which may be helpful in resolving the differential diagnosis between MPNs. Due to the relatively small number of cases and variable testing over time, larger controlled studies are necessary to confirm the findings.
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Policitemia Vera/genética , Mielofibrosis Primaria/genética , Trombocitemia Esencial/genética , Anciano , Análisis Mutacional de ADN , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Tasa de MutaciónRESUMEN
Microdissection is a useful method in tissue sampling prior to molecular testing. Tumor heterogeneity imposes new challenges for tissue sampling. Different microdissecting methods have been employed in face of such challenge. We improved our microdissection method by separately microdissecting the morphologically different tumor components. This improvement helped the pyrosequencing data analysis of two specimens. One specimen consisted of both adenocarcinoma and neuroendocrine components. When both tumor components were sequenced together for KRAS (Kirsten rat sarcoma viral oncogene homolog) gene mutations, the resulting pyrogram indicated that it was not a wild type, suggesting that it contained KRAS mutation. However, the pyrogram did not match any KRAS mutations and a conclusion could not be reached. After microdissecting and testing the adenocarcinoma and neuroendocrine components separately, it was found that the adenocarcinoma was positive for KRAS G12C mutation and the neuroendocrine component was positive for KRAS G12D mutation. The second specimen consisted of two morphologically different tumor nodules. When microdissected and sequenced separately, one nodule was positive for BRAF (v-raf murine sarcoma viral oncogene homolog B1) V600E and the other nodule was wild type at the BRAF codon 600. These examples demonstrate that it is necessary to microdissect morphologically different tumor components for pyrosequencing.
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Neoplasias Pulmonares/genética , Neoplasias Pulmonares/cirugía , Microdisección/métodos , Análisis de Secuencia de ADN/métodos , Temperatura , Adenocarcinoma/genética , Diferenciación Celular , Humanos , Mutación/genética , Sistemas Neurosecretores/patología , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/genéticaRESUMEN
Next-generation sequencing is becoming increasingly common in clinical laboratories worldwide and is revolutionizing clinical molecular testing. However, the large amounts of raw data produced by next-generation sequencing assays and the need for complex bioinformatics analyses present unique challenges. Proficiency testing in clinical laboratories has traditionally been designed to evaluate assays in their entirety; however, it can be alternatively applied to separate assay components. We developed and implemented a multi-institutional proficiency testing approach to directly assess custom bioinformatics and variant interpretation processes. Six clinical laboratories, all of which use the same commercial library preparation kit for next-generation sequencing analysis of tumor specimens, each submitted raw data (FASTQ files) from four samples. These 24 file sets were then deidentified and redistributed to five of the institutions for analysis and interpretation according to their clinically validated approach. Among the laboratories, there was a high rate of concordance in the calling of single-nucleotide variants, in particular those we considered clinically significant (100% concordance). However, there was significant discordance in the calling of clinically significant insertions/deletions, with only two of seven being called by all participating laboratories. Missed calls were addressed by each laboratory to improve their bioinformatics processes. Thus, through our alternative proficiency testing approach, we identified the bioinformatic detection of insertions/deletions as an area of particular concern for clinical laboratories performing next-generation sequencing testing.
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Biología Computacional/métodos , Biología Computacional/normas , Pruebas Genéticas/métodos , Pruebas Genéticas/normas , Secuenciación de Nucleótidos de Alto Rendimiento/normas , Ensayos de Aptitud de Laboratorios , Encuestas de Atención de la Salud , Humanos , Laboratorios/normasRESUMEN
AIMS: Assessment of peripheral blood tumour burden for staging of cutaneous T cells lymphoma is most often accomplished by flow cytometry (FC) using various non-standarised strategies. We report the results of calculating absolute Sezary cell counts (SCCs) by FC, based on the identification of aberrant T cell clusters on a virtual 6-dimensional space and independently of the expected immunophenotype (6D-FC SCC). METHODS: 6D-FC SCCs were calculated on 65 peripheral blood specimens from 28 patients with erythrodermic cutaneous T cells lymphoma (stage III or IV). Comparisons were made with recommended FC strategies and correlations with overall mortality were studied. RESULTS: At first visit, 17 of 28 patients (61%) had 6D-FC SCCs meeting current criteria for Stage IV disease (≥1000 SC/µL); while only 2 patients (7%) met Stage IV criteria on other tissues. As defined by comprehensive staging using clinicomorphological criteria and 6D-FC SCCs, Stage IV disease identified a subgroup of patients with worse overall survival (p=0.0227). Residual non-aberrant CD4 T cells were markedly decreased in Stage IV disease (p=0.018). Among 65 specimens, discrepancies were observed between 6D-FC SCCs and usual FC thresholds for Stage IV disease, namely a CD4:CD8 ratio ≥10:1 (9 discrepancies, 14%), and ≥40% aberrant CD4 T cells (4 discrepancies, 6%). Surprisingly, 8 cases (12%) from 6 patients exhibited two distinctively separate clusters of aberrant CD4 T cells with different CD7 and/or CD26 expression. CONCLUSIONS: Visual 6-dimensional identification of aberrant T cell clusters by FC allows for the calculation of clinically significant SCCs. Simplified gating strategies and relative quantitative values might underestimate the immunophenotypical complexity of Sezary cells.