RESUMO
TP53-disrupted chronic lymphocytic leukaemia (CLL) patients show a suboptimal long-term response to ibrutinib. We hereby report that ibrutinib-induced in vitro apoptosis and proliferation inhibition were significantly lower in TP53-mutated (TP53-M) CLL cells compared to TP53 wild-type cells. Contrariwise, venetoclax effectively killed TP53-M cells. Gene expression profile analysis of TP53-M cells revealed a downmodulation of B-cell receptor (BCR)-related genes and an upmodulation of genes with anti-apoptotic/pro-survival activity, suggesting that the survival and proliferation of TP53-M cells are less dependent on the BCR pathway. These observations further support the use of drug combinations for the optimal management of TP53-M CLL patients.
Assuntos
Regulação para Baixo/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Mutação , Pirazóis/farmacologia , Pirimidinas/farmacologia , Receptores de Antígenos de Linfócitos B/biossíntese , Proteína Supressora de Tumor p53/metabolismo , Adenina/análogos & derivados , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , Piperidinas , Sulfonamidas/farmacologia , Proteína Supressora de Tumor p53/genéticaRESUMO
Whole exome sequencing and copy number aberration (CNA) analysis were performed on cells taken from peripheral blood (PB) and lymph nodes (LN) of patients with chronic lymphocytic leukaemia (CLL). Of 64 non-silent somatic mutations, 54 (84·4%) were clonal in both compartments, 3 (4·7%) were PB-specific and 7 (10·9%) were LN-specific. Most of the LN- or PB-specific mutations were subclonal in the other corresponding compartment (variant frequency 0·5-5·3%). Of 41 CNAs, 27 (65·8%) were shared by both compartments and 7 (17·1%) were LN- or PB-specific. Overall, 6 of 9 cases (66·7%) showed genomic differences between the compartments. At subsequent relapse, Case 10, with 6 LN-specific lesions, and Case 100, with 6 LN-specific and 8 PB-specific lesions, showed, in the PB, the clonal expansion of LN-derived lesions with an adverse impact: SF3B1 mutation, BIRC3 deletion, del8(p23·3-p11·1), del9(p24·3-p13·1) and gain 2(p25·3-p14). CLL shows an intra-patient clonal heterogeneity according to the disease compartment, with both LN and PB-specific mutations/CNAs. The LN microenvironment might contribute to the clonal selection of unfavourable lesions, as LN-derived mutations/CNAs can appear in the PB at relapse.
Assuntos
Leucemia Linfocítica Crônica de Células B/genética , Mutação , Adulto , Idoso , Biópsia , Evolução Clonal , Variações do Número de Cópias de DNA , DNA de Neoplasias/genética , Exoma/genética , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/patologia , Linfonodos/patologia , Masculino , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/patologia , RecidivaRESUMO
Fludarabine refractoriness (FR) represents an unsolved clinical problem of chronic lymphocytic leukemia (CLL) management. Although next-generation sequencing studies have led to the identification of a number of genes frequently mutated in FR-CLL, a comprehensive evaluation of the FR-CLL genome has not been reported. Toward this end, we studied 10 FR-CLLs by combining whole-exome sequencing and copy number aberration (CNA) analysis, which showed an average of 16.3 somatic mutations and 4 CNAs per sample. Screening of recurrently mutated genes in 48 additional FR-CLLs revealed that ~70% of FR-CLLs carry ≥1 mutation in genes previously associated with CLL clinical course, including TP53 (27.5%), NOTCH1 (24.1%), SF3B1 (18.9%), and BIRC3 (15.5%). In addition, this analysis showed that 10.3% of FR-CLL cases display mutations of the FAT1 gene, which encodes for a cadherin-like protein that negatively regulates Wnt signaling, consistent with a tumor suppressor role. The frequency of FAT1-mutated cases was significantly higher in FR-CLL than in unselected CLLs at diagnosis (10.3% vs 1.1%, P = .004), suggesting a role in the development of a high-risk phenotype. These findings have general implications for the mechanisms leading to FR and point to Wnt signaling as a potential therapeutic target in FR-CLL.
Assuntos
Caderinas/genética , Resistencia a Medicamentos Antineoplásicos/genética , Leucemia Linfocítica Crônica de Células B/genética , Idoso , Análise Mutacional de DNA , Feminino , Genótipo , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Mutação , TranscriptomaRESUMO
To assess the involvement of microRNAs (miRNAs) in B-cell receptor (BCR) stimulation, we first evaluated miRNA profiling following IgM cross-linking in chronic lymphocytic leukemia (CLL) cells and in normal B lymphocytes. Second, we combined miRNA and gene expression data to identify putative miRNA functional networks. miRNA profiling showed distinctive patterns of regulation after stimulation in leukemic versus normal B lymphocytes and identified a differential responsiveness to BCR engagement in CLL subgroups according to the immunoglobulin heavy chain variable region mutational status and clinical outcome. The most significantly modulated miRNAs in stimulated CLL are miR-132 and miR-212. Notably, these miRNAs appeared regulated in progressive but not in stable CLL. Accordingly, gene profiling showed a significant transcriptional response to stimulation exclusively in progressive CLL. Based on these findings, we combined miRNA and gene expression data to investigate miR-132 and miR-212 candidate interactions in this CLL subgroup. Correlation analysis pointed to a link between these miRNAs and RB/E2F and TP53 cascades with proproliferative effects, as corroborated by functional analyses. Finally, basal levels of miR-132 and miR-212 were measured in an independent cohort of 20 unstimulated CLL cases and both showed lower expression in progressive compared to stable patients, suggesting an association between the expression of these molecules and disease prognosis. Overall, our results support a model involving miR-132 and miR-212 upregulation in sustaining disease progression in CLL. These miRNAs may therefore provide new valuable strategies for therapeutic intervention.
Assuntos
Imunoglobulina M/imunologia , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/imunologia , MicroRNAs/sangue , Regulação para Cima , Adulto , Idoso , Idoso de 80 Anos ou mais , Proliferação de Células , Feminino , Redes Reguladoras de Genes , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
To clarify whether karyotype aberrations (KA) involving regions not covered by the standard fluorescence in situ hybridization (FISH) panel have independent prognostic relevance, we evaluated KA by conventional cytogenetics in a learning cohort (LC; n = 166) and a validation cohort (VC; n = 250) of untreated chronic lymphocytic leukemia (CLL) patients. In the VC, novel mitogens were used to improve metaphase generation and TP53, NOTCH1, and SF3B1 mutations were assessed. KA undetected by FISH were found in 35 and 35% of the cases in the LC and VC, respectively. In addition to FISH, KA allowed reclassification of 23 and 26% of cases in the LC and VC, respectively, into a higher cytogenetic risk group. By multivariate analysis, both in the LC and VC, KA other than isolated 13q deletion correlated with a shorter time to first treatment (TFT; P < 0.001 and 0.003, respectively), while a complex karyotype predicted a worse overall survival (OS, P = 0.015 and 0.010, respectively). In the VC, where a comprehensive biologic assessment was performed, a shorter TFT was also predicted by stage (P < 0.001), IGHV mutational status (P = 0.05), and del(17p)/TP53 mutations (P = 0.033) while stage (P = 0.023) and del(17p)/TP53 mutations (P = 0.024) independently predicted a shorter OS. FISH results did not independently impact on TFT and OS, in the LC and VC cohorts; this was also the case for NOTCH1 and SF3B1 mutations in the VC. We suggest that in CLL, conventional karyotyping with novel mitogens could be more effective than FISH for the detection of KA allowing for a more precise refinement of prognosis.
Assuntos
Deleção Cromossômica , Interleucina-2/farmacologia , Cariotipagem , Leucemia Linfocítica Crônica de Células B/genética , Mitógenos/farmacologia , Oligonucleotídeos/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Transtornos Cromossômicos/genética , Cromossomos Humanos Par 13/genética , Cromossomos Humanos Par 17/genética , Citogenética , Feminino , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Mutação , Fosfoproteínas/genética , Prognóstico , Fatores de Processamento de RNA , Receptor Notch1/genética , Proteínas Recombinantes/farmacologia , Ribonucleoproteína Nuclear Pequena U2/genética , Tempo para o Tratamento , Proteína Supressora de Tumor p53/genéticaRESUMO
Accurate genomic characterization requires sufficient amounts of optimal quality DNA. An approach for increasing the DNA amount is the whole-genome amplification (WGA) method. We applied WGA to the molecular quantification and minimal residual disease (MRD) evaluation of acute lymphoblastic leukaemia (ALL), aiming to compare the results obtained from genomic DNA and amplified DNA with WGA, and to evaluate the applicability and the reliability of WGA-DNA. Twenty paired samples from adult ALL patients were sequenced to identify the functional germline V-D-J segment at diagnosis; real-time quantitative polymerase chain reaction (RQ-PCR) quantitative analysis was performed both at diagnosis and follow-up. Genomic DNA and WGA-DNA screening identified equivalent 87 rearrangements. At diagnosis, the quantitative evaluation of genomic DNA samples showed 1 logarithm difference to WGA-DNA samples; these levels are comparable, being within the degree of acceptability and confidence. In the follow-up samples, RQ-PCR analysis on genomic DNA and WGA showed concordant MRD results in 16/18 samples, while 2/18 were MRD-positive outside the quantitative range by RQ-PCR (i.e. <5 × 10(-5)) on genomic DNA and MRD-negative on WGA-DNA. WGA-DNA enables: (i) the design of accurate targets for MRD evaluation in ALL patients, (ii) accurate disease quantification at diagnosis, (iii) MRD quantification comparable to genomic DNA.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Receptores de Antígenos de Linfócitos T/genética , Adolescente , Adulto , Feminino , Genes de Imunoglobulinas , Genoma Humano , Humanos , Masculino , Neoplasia Residual , Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Adulto JovemRESUMO
Minimal residual disease (MRD) is becoming increasingly important in chronic lymphocytic leukaemia (CLL) as treatment strategies are progressively improving. The primary objective of this study was to compare the applicability of three different flow cytometric approaches: basic 4-colour analysis, European Research Initiative in CLL (ERIC) consensus method and 8-colour analysis. Secondly, we investigated the sensitivity and specificity of flow cytometry (FC) compared to molecular analyses for MRD detection. A total of 462 CLL samples were evaluated by basic FC; in 143, ERIC consensus method was also performed and all three FC methodologies were applied in a subgroup of 10 cases. No discordance in defining MRD-positive/negative samples was observed between the FC methods; within positive samples, the ERIC consensus method and 8-colour analysis showed the most accurate results. MRD was analysed by FC and polymerase chain reaction (PCR) in 243 cases: concordant results were obtained in 199/243 samples (81·9%); 42/243 were FC-/PCR+. Overall, the sensitivity and specificity of FC compared to PCR was 96·5% and 77·2%, respectively. Both FC and PCR proved suitable for the detection of MRD and prediction of progression-free survival, which was significantly reduced in MRD-positive patients, regardless of the methodology. These results offer the rationale for a strategy to monitor MRD in CLL patients.
Assuntos
Leucemia Linfocítica Crônica de Células B/diagnóstico , Neoplasia Residual/diagnóstico , Alelos , Citometria de Fluxo , Seguimentos , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Imunofenotipagem , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/mortalidade , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e EspecificidadeAssuntos
Proteína 3 com Repetições IAP de Baculovírus/genética , Cromossomos Humanos Par 11/ultraestrutura , Leucemia Linfocítica Crônica de Células B/genética , Deleção de Sequência , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Aberrações Cromossômicas , Cromossomos Humanos Par 11/genética , Análise Mutacional de DNA , Feminino , Seguimentos , Humanos , Hibridização in Situ Fluorescente , Leucemia Linfocítica Crônica de Células B/mortalidade , Masculino , Pessoa de Meia-Idade , PrognósticoRESUMO
The genetic lesions identified to date do not fully recapitulate the molecular pathogenesis of chronic lymphocytic leukemia (CLL) and do not entirely explain the development of severe complications such as chemorefractoriness. In the present study, BIRC3, a negative regulator of noncanonical NF-κB signaling, was investigated in different CLL clinical phases. BIRC3 lesions were absent in monoclonal B-cell lymphocytosis (0 of 63) and were rare in CLL at diagnosis (13 of 306, 4%). Conversely, BIRC3 disruption selectively affected 12 of 49 (24%) fludarabine-refractory CLL cases by inactivating mutations and/or gene deletions that distributed in a mutually exclusive fashion with TP53 abnormalities. In contrast to fludarabine-refractory CLL, progressive but fludarabine-sensitive patients were consistently devoid of BIRC3 abnormalities, suggesting that BIRC3 genetic lesions associate specifically with a chemorefractory phenotype. By actuarial analysis in newly diagnosed CLL (n = 306), BIRC3 disruption identified patients with a poor outcome similar to that associated with TP53 abnormalities and exerted a prognostic role that was independent of widely accepted clinical and genetic risk factors. Consistent with the role of BIRC3 as a negative regulator of NF-κB, biochemical studies revealed the presence of constitutive noncanonical NF-κB activation in fludarabine-refractory CLL patients harboring molecular lesions of BIRC3. These data identify BIRC3 disruption as a recurrent genetic lesion of high-risk CLL devoid of TP53 abnormalities.
Assuntos
Antineoplásicos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas Inibidoras de Apoptose/genética , Leucemia Linfocítica Crônica de Células B/genética , Proteína Supressora de Tumor p53/genética , Vidarabina/análogos & derivados , Idoso , Proteína 3 com Repetições IAP de Baculovírus , Western Blotting , Análise Mutacional de DNA , Feminino , Humanos , Hibridização in Situ Fluorescente , Proteínas Inibidoras de Apoptose/metabolismo , Estimativa de Kaplan-Meier , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/mortalidade , Masculino , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Prognóstico , Modelos de Riscos Proporcionais , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ubiquitina-Proteína Ligases , Vidarabina/uso terapêuticoRESUMO
Chronic lymphocytic leukemia (CLL) with stereotyped B-cell receptor (BCR) belonging to subset #1 (IGHV1-5-7/ IGKV1-39) display a poor outcome. To characterize their genetic and genomic features and BCR function, we selected 20 subset #1 CLL from a series of 579 cases. Subset #1 CLL, all showing unmutated IGHV, were associated with the presence of del(11q) (50%) in comparison with unmutated CLL, unmutated stereotyped CLL other than subset #1 and with cases using the same IGHV genes but a heterogeneous VH CDR3 (non-subset #1 CLL). There were no distinctive features regarding CD38, ZAP-70, and TP53 disruption. NOTCH1, SF3B1, and BIRC3 were mutated in 15%, 0%, and 5% of cases, respectively, while BIRC3 was deleted in 22% of cases. Microarray unsupervised analysis on 80 unmutated/mutated/stereotyped/non-stereotyped CLL showed a tight clustering of subset #1 cases. Their genomic signature exhibited several differentially expressed transcripts involved in BCR signal transduction, apoptosis regulation, cell proliferation, and oxidative processes, regardless of del(11q). Accordingly, BCR ligation with anti-IgM revealed a significant higher proliferation of subset #1 versus unmutated non-subset #1 CLL, both at baseline and after 2448 hr stimulation. Subset #1 CLL represent a paradigmatic example of the direct link between BCR structure, function, and patients prognosis.
Assuntos
Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/mortalidade , Receptores de Antígenos de Linfócitos B/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Proliferação de Células , Análise por Conglomerados , Feminino , Regulação Leucêmica da Expressão Gênica , Humanos , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Imunofenotipagem , Leucemia Linfocítica Crônica de Células B/metabolismo , Masculino , Pessoa de Meia-Idade , Prognóstico , Receptores de Antígenos de Linfócitos B/metabolismo , Reprodutibilidade dos Testes , Transdução de Sinais , TranscriptomaRESUMO
In a phase II trial, we evaluated chlorambucil and rituximab (CLB-R) as first-line induction treatment with or without R as maintenance for elderly chronic lymphocytic leukemia (CLL) patients. Treatment consisted of eight 28-day cycles of CLB (8 mg/m(2) /day, days 1-7) and R (day 1 of cycle 3, 375 mg/m(2) ; cycles 4-8, 500 mg/m(2) ). Responders were randomized to 12 8-week doses of R (375 mg/m(2) ) or observation. As per intention-to-treat analysis, 82.4% (95% CI, 74.25-90.46%) of 85 patients achieved an overall response (OR), 16.5% a complete response (CR), 2.4% a CR with incomplete bone marrow recovery. The OR was similar across Binet stages (A 86.4%, B 81.6%, and C 78.6%) and age categories (60-64 years, 92.3%; 65-69, 85.2%; 70-74, 75.0%; ≥75, 81.0%). CLB-R was well tolerated. After a median follow-up of 34.2 months, the median progression-free survival (PFS) was 34.7 months (95% CI, 33.1-39.5). TP53 abnormalities, complex karyotype, and low CD20 gene expression predicted lack of response; SF3B1 mutation and BIRC3 disruption low CR rates. IGHV mutations significantly predicted PFS. R maintenance tended towards a better PFS than observation and was safe and most beneficial for patients in partial response and for unmutated IGHV cases. CLB-R represents a promising option for elderly CLL patients.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais Murinos/administração & dosagem , Clorambucila/administração & dosagem , Intervalo Livre de Doença , Esquema de Medicação , Feminino , Humanos , Quimioterapia de Indução , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Rituximab , Análise de Sobrevida , Resultado do TratamentoRESUMO
We analyzed TP53 mutations in 483 chronic lymphocytic leukemia patients at different phases of the disease and found a higher incidence of mutations at the later phases and a distinctive mutation profile in each phase. p53 function evaluated by immunoblotting and flow cytometry after cell irradiation was impaired in 28 of 109 cases. Three phenotypically different dysfunctions were observed: type I, associated with heterozygous missense TP53 mutations (typically present at diagnosis) and partially resistant to radiation-induced killing; types II and III, with a higher incidence of microdeletions, nonsense mutations and bi-allelic TP53 defects (common in progressive and chemoresistant cases) and a complete radioresistance. Furthermore, in 4 of 28 patients, all chemoresistant, we found p53 dysfunctions without TP53 mutations. In chronic lymphocytic leukemia patients, a disease phase-specific variability in the p53 mutation profile and function takes place, and both analyses could be useful to guide treatment choices.
Assuntos
Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Apoptose/genética , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Éxons , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Mutação/efeitos da radiação , Estadiamento de NeoplasiasRESUMO
BACKGROUND: The genetic characterization of chronic lymphocytic leukemia cells correlates with the behavior, progression and response to treatment of the disease. DESIGN AND METHODS: Our aim was to investigate the role of ATM gene alterations, their biological consequences and their value in predicting disease progression. The ATM gene was analyzed by denaturing high performance liquid chromatography and multiplex ligation probe amplification in a series of patients at diagnosis. The results were correlated with immunoglobulin gene mutations, cytogenetic abnormalities, ZAP-70 and CD38 expression, TP53 mutations, gene expression profile and treatment-free interval. RESULTS: Mutational screening of the ATM gene identified point mutations in 8/57 cases (14%). Multiplex ligation probe amplification analysis identified six patients with 11q deletion: all of them had at least 20% of deleted cells, analyzed by fluorescent in situ hybridization. Overall, ATM point mutations and deletions were detected in 14/57 (24.6%) cases at presentation, representing the most common unfavorable genetic anomalies in chronic lymphocytic leukemia, also in stage A patients. Patients with deleted or mutated ATM had a significantly shorter treatment-free interval compared to patients without ATM alterations. ATM-mutated cases had a peculiar gene expression profile characterized by the deregulation of genes involved in apoptosis and DNA repair. Finally, definition of the structure of the ATM-mutated protein led to a hypothesis that functional abnormalities are responsible for the unfavorable clinical course of patients carrying these point mutations. CONCLUSIONS: ATM alterations are present at diagnosis in about 25% of individuals with chronic lymphocytic leukemia; these alterations are associated with a peculiar gene expression pattern and a shorter treatment-free interval.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Progressão da Doença , Perfilação da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Supressoras de Tumor/genética , Adulto , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/metabolismo , Análise por Conglomerados , Proteínas de Ligação a DNA/metabolismo , Feminino , Duplicação Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/mortalidade , Masculino , Pessoa de Meia-Idade , Mutação , Prognóstico , Proteínas Serina-Treonina Quinases/metabolismo , Reprodutibilidade dos Testes , Análise de Sobrevida , Proteínas Supressoras de Tumor/metabolismoRESUMO
Trisomy 12, the third most frequent chromosomal aberration in chronic lymphocytic leukemia (CLL), confers an intermediate prognosis. In our cohort of 104 untreated patients carrying +12, NOTCH1 mutations occurred in 24% of cases and were associated to unmutated IGHV genes (P=0.003) and +12 as a sole cytogenetic abnormality (P=0.008). NOTCH1 mutations in +12 CLL associated with an approximately 2.4 fold increase in the risk of death, a significant shortening of survival (P<0.01) and proved to be an independent predictor of survival in multivariate analysis. Analogous to +12 CLL with TP53 disruption or del(11q), NOTCH1 mutations in +12 CLL conferred a significantly worse survival compared to that of +12 CLL with del(13q) or +12 only. The overrepresentation of cell cycle/proliferation related genes of +12 CLL with NOTCH1 mutations suggests the biological contribution of NOTCH1 mutations to determine a poor outcome. NOTCH1 mutations refine the intermediate prognosis of +12 CLL.
Assuntos
Cromossomos Humanos Par 12 , Perfilação da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/genética , Mutação , Receptor Notch1/genética , Transcrição Gênica , Trissomia , Idoso , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/mortalidade , Masculino , Pessoa de Meia-Idade , Taxa de Mutação , Prognóstico , Análise de SobrevidaRESUMO
Given that TP53 alterations predict prognosis and response to therapy in chronic lymphocytic leukemia (CLL), screening for TP53 mutations has an increasing role in patient management. TP53 direct sequencing is a time-consuming method, while the AmpliChip p53 Research Test is a novel non time-consuming microarray-based resequencing assay and queries Exons 2-11. We evaluated the impact of TP53 mutations on clinical outcome by analyzing 98 untreated CLL using the AmpliChip p53 Research Test and direct sequencing and performed microarrays analysis on TP53 mutated and/or deleted cases. The AmpliChip p53 Research Test detected 17 mutations in 14 patients (17.3%); a significant association between TP53 mutations and del(17p) was recorded. From a clinical standpoint, a higher percentage of mutation was found in CLL with unfavorable outcome (17.2% vs. 7.1% in progressive vs. stable cases). Detection of TP53 mutations by the AmpliChip p53 Research Test was associated with a significantly worse survival (P = 0.0002). Comparison of the array and direct sequencing tests showed that the p53 Research Test detected more mutations, although it failed to identify two microdeletions. Finally, microarrays analysis showed a more distinctive signature associated with del(17p) than with TP53 mutations, likely due to a concomitant gene dosage effect. The AmpliChip p53 Research Test is a straightforward method that bears prognostic value. This study confirms a high percentage of TP53 mutations in CLL with unfavorable outcome and a significant association between TP53 aberrations and del(17p). Finally, specific gene expression profiles are recognized for TP53 alterations.
Assuntos
Genes p53/genética , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/mortalidade , Proteína Supressora de Tumor p53/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular , Deleção Cromossômica , Cromossomos Humanos Par 17/genética , Progressão da Doença , Feminino , Perfilação da Expressão Gênica/métodos , Instabilidade Genômica , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Mutação Puntual , Prognóstico , Deleção de Sequência/genéticaRESUMO
Wastewater-based viral surveillance was proposed as a promising approach to monitor the circulation of SARS-CoV-2 in the general population. The aim of this study was to develop an analytical method to detect SARS-CoV-2 RNA in urban wastewater, and apply it to follow the trends of epidemic in the framework of a surveillance network in the Lombardy region (Northern Italy). This area was the first hotspot of COVID-19 in Europe and was severely affected. Composite 24 h samples were collected weekly in eight cities from end-March to mid-June 2020 (first peak of the pandemic). The method developed and optimized, involved virus concentration using PEG centrifugation, and one-step real-time RT-PCR for analysis. SARS-CoV-2 RNA was identified in 65 (61%) out of 107 samples, and the viral concentrations (up to 2.1 E + 05 copies/L) were highest in March-April. By mid-June, wastewater samples tested negative in all the cities corresponding to the very low number of cases recorded in the same period. Viral loads were calculated considering the wastewater daily flow rate and the population served by each wastewater treatment plant, and were used for inter- city comparison. The highest viral loads were found in Brembate, Ranica and Lodi corresponding to the hotspots of the first peak of pandemic. The pattern of decrease of SARS-CoV-2 in wastewater was closely comparable to the decline of active COVID-19 cases in the population, reflecting the effect of lock-down. This study tested wastewater surveillance of SARS-CoV-2 to follow the pandemic trends in one of most affected areas worldwide, demonstrating that it can integrate ongoing virological surveillance of COVID-19, providing information from both symptomatic and asymptomatic individuals, and monitoring the effect of health interventions.
Assuntos
COVID-19 , Águas Residuárias , Controle de Doenças Transmissíveis , Humanos , Pandemias , RNA Viral , SARS-CoV-2 , Vigilância Epidemiológica Baseada em Águas ResiduáriasAssuntos
Linhagem da Célula/genética , Células Clonais/patologia , Leucemia Mieloide Aguda/genética , Mutação , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Adulto , Feminino , Rearranjo Gênico , Genes de Cadeia Pesada de Imunoglobulina , Humanos , Neoplasia Residual/genética , Receptores de Antígenos de Linfócitos T/genética , Análise de Sequência de DNAAssuntos
Genes de Cadeia Pesada de Imunoglobulina/genética , Leucemia de Células Pilosas/genética , Leucemia Linfocítica Crônica de Células B/genética , Proteínas Proto-Oncogênicas B-raf/genética , Células Clonais/patologia , Rearranjo Gênico , Humanos , Leucemia de Células Pilosas/complicações , Leucemia de Células Pilosas/patologia , Leucemia Linfocítica Crônica de Células B/complicações , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Pessoa de Meia-Idade , MutaçãoRESUMO
In chronic lymphocytic leukemia (CLL), spontaneous regressions are an exceptional phenomenon, whose biologic features are unknown. We describe 9 CLL patients who underwent a spontaneous clinical regression over an 11-year follow-up, despite a residual neoplastic clone detected by flow cytometry. CD38 and ZAP-70 were negative in all cases. Immunoglobulin heavy chain variable region (IgVH) genes, mutated in all 7 evaluable patients, were restricted to the VH3 family in 6, with the usage of V(H)3-30 gene in 2. The light chain variable region genes were mutated in 6 of 8 cases, with the use of V(kappa)4-1 gene in 3. Microarray analysis of CLL cells showed a distinctive genomic profile with an overrepresentation of BCR-related and ribosomal genes, regulators of signal transduction and transcription. The number of activated T lymphocytes expressing IFN-gamma, TNF-alpha, and IL-4 was similar between CLL in spontaneous regression and healthy persons. In conclusion, spontaneous clinical regressions can occur in CLL despite the persistence of the neoplastic clone, and the biologic features include negative CD38 and ZAP-70, mutated V(H)3-30 and V(kappa)4-1 genes. The peculiar gene profile suggests that BCR signaling may play an important role in this scenario as the most significant feature of the leukemic clone in regression.
Assuntos
Leucemia Linfocítica Crônica de Células B/patologia , Remissão Espontânea , ADP-Ribosil Ciclase 1/análise , Adulto , Idoso , Células Clonais , Feminino , Seguimentos , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Genes de Imunoglobulinas , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/imunologia , Masculino , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos B/genética , Proteína-Tirosina Quinase ZAP-70/análiseRESUMO
A comprehensive panel of clinical-biological parameters was prospectively evaluated at presentation in 112 patients with chronic lymphocytic leukemia (<65 years), to predict the risk of progression in early stage disease. Eighty-one percent were in Binet stage A, 19% in stages B/C. Treatment-free survival was evaluated as the time from diagnosis to first treatment, death or last follow up. In univariate analysis, advanced stage, hemoglobin, platelets, white blood cell, leukemic lymphocyte count, raised beta 2-microglobulin and LDH, unmutated immunoglobulin variable region genes, CD38, del(17p), del(11q) and +12, were significantly associated with a short treatment-free survival; the T/leukemic lymphocyte ratio was associated with a better outcome. Multivariate analysis of treatment-free survival in stage A patients selected a high white blood cell count and unmutated immunoglobulin variable region genes as unfavorable prognostic factors and a high T/leukemic lymphocyte ratio as a favorable one. At diagnosis, these parameters independently predict the risk of progression in stage A chronic lymphocytic leukemia patients.