ABSTRACT
Ataxia-telangiectasia (A-T) is an autosomal-recessive disorder caused by pathogenic variants (PVs) of the ATM gene. Children with A-T are predisposed to hematological malignancies. We aimed to investigate their characteristics and outcomes in order to generate data-based treatment recommendations. In this multinational, observational study we report 202 patients aged ≤25 years with A-T and hematological malignancies from 25 countries. Ninety-one patients (45%) presented with mature B-cell lymphomas, 82 (41%) with acute lymphoblastic leukemia/lymphoma, 21(10%) with Hodgkin lymphoma and eight (4%) with other hematological malignancies. Four-year overall survival and event-free survival (EFS) were 50.8% (95% CI 43.6-59.1) and 47.9% (95% CI 40.8-56.2), respectively. Cure rates have not significantly improved over the last four decades (p=.76). The major cause of treatment failure was treatment-related mortality (TRM) with a four-year cumulative incidence of 25.9% (95% CI 19.5-32.4). Germline ATM PVs were categorized as null or hypomorphic and patients with available genetic data (n=110) were classified as having absent (n=81) or residual (n=29) ATM kinase activity. Four-year EFS was 39.4% (95% CI 29-53.3) vs 78.7% (95% CI 63.7-97.2), (p<.001), and TRM rates were 37.6% (95% CI 26.4-48.7) vs 4.0% (95% CI 0-11.8), (p=.017), for those with absent and residual ATM kinase activity, respectively. Absence of ATM kinase activity was independently associated with decreased EFS (HR=0.362, 95% CI 0.16-0.82; p=.009) and increased TRM (HR=14.11, 95% CI 1.36-146.31; p=.029). Patients with A-T and leukemia/lymphoma may benefit from de-escalated therapy for patients with absent ATM kinase activity and near-standard therapy regimens for those with residual kinase activity.
ABSTRACT
Philadelphia chromosome-positive B-cell precursor acute lymphoblastic leukemia (Ph+ BCPALL) is a high-risk acute lymphoblastic leukemia subtype characterized by the presence of BCR::ABL1 fusion gene. Tyrosine kinase inhibitors (TKIs) combined with chemotherapy are established as the first-line treatment. Additionally, rituximab (RTX), an anti-CD20 monoclonal antibody (mAb) is administered in adult BCP-ALL patients with ≥20% of CD20+ blasts. In this study, we observed a marked prevalence of CD20 expression in patients diagnosed with Ph+ BCP-ALL, indicating a potential widespread clinical application of RTX in combination with TKIs. Consequently, we examined the influence of TKIs on the antitumor effectiveness of anti-CD20 mAbs by evaluating CD20 surface levels and conducting in vitro functional assays. All tested TKIs were found to uniformly downregulate CD20 on leukemic cells, diminishing the efficacy of RTX-mediated complement-dependent cytotoxicity. Interestingly, these TKIs displayed varied effects on NK cell-mediated antibody-dependent cytotoxicity and macrophage phagocytic function. While asciminib demonstrated no inhibition of effector cell functions, dasatinib notably suppressed the anti-CD20-mAb-mediated NK cell cytotoxicity and macrophage phagocytosis of BCP-ALL cells. Dasatinib and ponatinib also decreased NK cell degranulation in vitro. Importantly, oral administration of dasatinib, but not asciminib, compromised NK cell activity within patients' blood, determined by ex vivo degranulation assay. Our results indicate that asciminib might be preferred over other TKIs for combination therapy with anti-CD20 mAbs.
ABSTRACT
B-cell precursor acute lymphoblastic leukemia (BCP-ALL) with chromosome translocations like KMT2A gene rearrangement (KMT2A-r) and BCR-ABL1 fusion gene have been recognized as crucial drivers in both BCP-ALL leukemogenesis and treatment management. Standard diagnostic protocols for proliferative diseases of the hematopoietic system, like KMT2A-r-ALL, are genetically based and strongly molecularly oriented. Therefore, an efficient diagnostic procedure requires not only experienced and multidisciplinary laboratory staff but also considerable instrumentation and material costs. In recent years, a Raman spectroscopy method has been increasingly used to detect subtle chemical changes in individual cells resulting from stress or disease. Therefore, the objective of this study was to identify Raman signatures for the molecular subtypes and to develop a classification method based on the unique spectroscopic profile of in vitro models that represent specific aberrations aimed at KMT2A-r (RS4;11, and SEM) and the BCR-ABL1 fusion gene (SUP-B15, BV-173, and SD-1). Data analysis was based on chemometric methods, i.e. principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and support vector machine (SVM). The PCA-based multivariate model was used for pattern recognition of each investigated group of cells while PLS-DA and SVM were used to build models for the discrimination of spectra from the studied BCP-ALL molecular subtypes. The results showed that the studied molecular subtypes of ALL have characteristic spectroscopic profiles reflecting their peculiar biochemical state. The content of lipids (1600 cm-1), nucleic acids (789 cm-1), and haemoproteins (754, 1130, and 1315 cm-1), which are crucial in cell metabolism, was indicated as the main source of differentiation between subtypes. Identification of spectroscopic markers of cells with BCR-ABL1 or KMT2A-r may be useful in pharmacological studies to monitor the effectiveness of chemotherapy and further to understand differences in molecular responses between leukemia primary cells and cell lines.
Subject(s)
Precursor B-Cell Lymphoblastic Leukemia-Lymphoma , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Humans , Precursor Cell Lymphoblastic Leukemia-Lymphoma/diagnosis , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Spectrum Analysis, Raman/methodsABSTRACT
Ataxia-telangiectasia (AT) is a multisystemic neurodegenerative inborn error of immunity (IEI) characterized by DNA repair defect, chromosomal instability, and hypersensitivity to ionizing radiation. Impaired DNA double-strand break repair determines a high risk of developing hematological malignancies, especially lymphoproliferative diseases. Poor response to treatment, excessive chemotherapy toxicities, and the need for avoiding exposure to ionizing radiation make the successful clinical management of patients with AT challenging for oncologists. We describe the favorable outcome of the LBCL with IRF4 rearrangement at stage III in a 7-year-old female patient diagnosed with AT. The patient was treated according to the B-HR arm of the INTER-B-NHL-COP 2010 protocol, including the administration of rituximab, cyclophosphamide, methotrexate, prednisone, etc. She presented excessive treatment toxicities despite individually reduced doses of methotrexate and cyclophosphamide. However, in the MRI there was no significant reduction in pathologic lymph nodes after three immunochemotherapy courses. Therefore, a lymph node biopsy was taken. Its subsequent histopathological examination revealed tuberculosis-like changes, though tuberculosis suspicion was excluded. After two following immunochemotherapy courses, PET-CT confirmed complete remission. From March 2022 onwards, the patient has remained in remission under the care of the outpatient children's oncology clinic.
Subject(s)
Ataxia Telangiectasia , Lymphoma, Large B-Cell, Diffuse , Female , Humans , Child , Methotrexate/therapeutic use , Positron Emission Tomography Computed Tomography , Rituximab/genetics , Lymphoma, Large B-Cell, Diffuse/drug therapy , Ataxia Telangiectasia/drug therapy , Ataxia Telangiectasia/genetics , Prednisone/therapeutic use , Cyclophosphamide/therapeutic use , Mutation , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Vincristine/therapeutic use , Doxorubicin/therapeutic use , Ataxia Telangiectasia Mutated Proteins/geneticsABSTRACT
The strongest predictors of outcome in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) are minimal residual disease (MRD) and specific molecular abnormalities. One unfavorable prognostic factor is the presence of IKZF1 gene aberrations, particularly when co-occurring with high MRD level at the end of induction treatment. The present study determines the predictive value of a recently-defined IKZF1-plus (IKZF1plus ) microdeletion profile in 373 children with BCP-ALL treated according to the ALL-intercontinental Berlin-Frankfurt-Munster protocol 2009 protocol. IKZF1-wild type (IKZF1wt ) patients demonstrated lower leukemic burden parameters than those carrying IKZF1 deletion (IKZF1del [n = 26, 7.0%]) or IKZF1plus pattern (n = 34, 9.1%): (i) median blast percentage at diagnosis (78.0% vs. 86.9% vs. 86.0%; p = 0.021); (ii) median MRD level at day 15 of induction protocol (0.3% vs. 2.1% vs. 0.8%; p = 0.011); (iii) poor steroid response (7.6% vs. 26.5% vs. 12.5%; p = 0.010). Minimal residual disease level at day 33 (MRD33) exceeding 10-4 was more frequently observed in both the IKZF1del and IKZF1plus subgroups than in IKZF1wt patients (n = 9 [36.0%] vs. n = 13 [41.9%] vs. n = 70 [24.0%], p = 0.051). IKZF1plus individuals showed a tendency for a lower MRD reduction between day 15 and 33 compared to IKZF1del patients (p = 0.124). IKZF1del and IKZF1plus patients showed decreased relapse-free survival (HR [95%CI] for IKZF1wt as reference = 2.72 [1.21-6.11] and 2.00 [0.87-4.49], respectively, p = 0.023). Both genetic markers including IKZF1del and IKZF1plus microdeletion profile provide additional predictive value of treatment outcome in childhood BCP-ALL and may contribute to more efficient patient stratification; the same is true in MRD guided protocols, which are based on flow cytometric measurements on day 15 of induction protocol.
Subject(s)
Ikaros Transcription Factor , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma , Child , Humans , Ikaros Transcription Factor/genetics , Neoplasm, Residual/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/diagnosis , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/therapy , Prognosis , Treatment OutcomeABSTRACT
Microdeletions of 7p12.1 encompassing the IKZF1 gene locus are rare, with few cases reported. The common phenotype includes intellectual disability, overgrowth, and facial dysmorphism accompanied, albeit rarely, by congenital anomalies. Haploinsufficiency of IKZF1 predisposes individuals to childhood acute lymphoblastic leukemia (ALL). In this study, we comprehensively analyzed the frequency of 7p12.1 deletions among 4581 Polish individuals who underwent chromosomal microarray testing for unexplained developmental delay, intellectual disability, and/or congenital anomalies. Two unrelated individuals (0.04%) with a de novo interstitial 7p12.1 microdeletion encompassing IKZF1 were identified. One developed ALL. Analysis of the incidence and the phenotype of constitutional 7p12.1 microdeletion, which based on the previously annotated patients data in public databases and literature reports, revealed 21 cases including five patients diagnosed with ALL.
Subject(s)
Chromosome Deletion , Chromosomes, Human, Pair 7/genetics , Craniofacial Abnormalities/genetics , Developmental Disabilities/genetics , Ikaros Transcription Factor/genetics , Phenotype , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Child , Child, Preschool , Craniofacial Abnormalities/pathology , Developmental Disabilities/pathology , Female , Humans , Male , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathologyABSTRACT
The germline variant at rs3824662 in GATA3 is a risk locus for Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL), the biological subtype of B-cell precursor (BCP)-ALL defined by a distinct gene expression profile and the presence of specific somatic aberrations including rearrangements of CRLF2. In this study, we investigated whether rs3824662 in GATA3 associates with CRLF2 expression in leukemic cells and predicts prognosis in pediatric BCP-ALL patients treated according to the ALL Intercontinental Berlin-Frankfurt-Münster (IC BFM) 2009 (n = 645) and the ALL IC BFM 2002 (n = 216) protocols. High expression of CRLF2 was observed at both protein and mRNA levels (fourfold higher in AA than in CA + CC) among GATA3 AA variant carriers, independent of the presence of P2RY8-CRLF2 fusion. Additionally, the AA variant at rs3824662 was a significant factor affecting minimal residual disease level at the end of induction phase and overall survival regardless of the risk group and the protocol. The germline variant at rs3824662 in GATA3 is a prognostic factor which associates with CRLF2 expression in leukemic cells supporting the hypothesis that GATA3 may have a regulatory effect on the CRLF2 pathway in pediatric BCP-ALL.
Subject(s)
GATA3 Transcription Factor/genetics , Polymorphism, Single Nucleotide , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Receptors, Cytokine/genetics , Cells, Cultured , Child , Child, Preschool , Female , Germ-Line Mutation , Humans , Male , Oncogene Fusion , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Receptors, Cytokine/metabolism , Receptors, Purinergic P2Y/genetics , Survival AnalysisSubject(s)
Anemia, Aplastic , Hemoglobinuria, Paroxysmal , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma , Child , Humans , Anemia, Aplastic/pathology , Anemia, Aplastic/complications , Hemoglobinuria, Paroxysmal/complications , Hemoglobinuria, Paroxysmal/pathology , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/complicationsABSTRACT
AIM OF THE STUDY: To characterise expression of mTOR (mammalian target of rapamycin) in childhood B-cell acute lymphoblastic leukaemia (ALL), and to evaluate a possible link between mTOR and clinical characteristics. MATERIAL AND METHODS: The examined group consisted of 21 consecutive patients, aged 1-18 years, diagnosed with B-cell ALL in 2010, and 10 relapsed B-cell ALL patients diagnosed for the first time between 2009 and 2011, who developed relapse before 2014. All subjects were treated in the Department of Paediatric Haematology and Oncology of the Medical University of Warsaw according to the ALL-IC BFM 2002 Protocol. We evaluated mTOR and phospho-mTOR expression by immunohistochemistry using rabbit monoclonal antibodies. RESULTS: mTOR expression was found to be significantly associated with the risk of relapse and was more frequent in ALL recurrence. No significant relationship was detected between mTOR expression and other features of high-risk disease in paediatric ALL. CONCLUSIONS: mTOR activity could be considered a high-risk feature in paediatric B-cell ALL. Expression of mTOR kinase is observed remarkably more frequently in disease recurrence than at first diagnosis, indicating higher proliferative and survival potential of leukaemic cells in relapse. Routine analysis of mTOR activity could be performed to select patients that may potentially benefit from mTOR inhibitors (MTI) treatment.
ABSTRACT
Early recognition of children with chronic phase chronic myeloid leukaemia (CML-CP) at risk for developing a lymphoid blast crisis (LyBC) is desirable, because therapy options in CML-LyBC are limited. We used Multiplex Ligation-dependent Probe Amplification to determine whether B-cell lymphoid leukaemia-specific copy number alterations (CNAs) (e.g. IKZF1, PAX5, CDKN2A deletions) could be detected in CML-CP and may be used to predict disease progression to LyBC. CNAs were detected in all patients with CML-LyBC, but in none of the 77 patients with CML-CP. Based on this study we conclude that CNAs remain a hallmark of disease progression.
Subject(s)
Biomarkers, Tumor/genetics , DNA Copy Number Variations/genetics , DNA, Neoplasm/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Blast Crisis/genetics , Child , Disease Progression , Early Diagnosis , Humans , Ikaros Transcription Factor/genetics , Molecular Sequence Data , Multiplex Polymerase Chain Reaction/methods , Neoplasm Proteins/genetics , Point Mutation , PrognosisABSTRACT
Nijmegen breakage syndrome (NBS) is an autosomal recessive chromosomal instability disorder characterized by a high incidence of pediatric hematologic malignancies. Majority of patients affected are of Slavic origin and share the same founder mutation of 657del5 within the NBN gene encoding protein involved in DNA double-strand breaks (DSB) repair. We report a case of a pediatric patient with NBS, who developed t(9;11)/AF9-MLL-positive AML as a second malignancy after successful treatment of T-NHL. The coexistence of NBN and MLL mutations suggests that the profound dysfunction of NBN may promote alterations of MLL that is mediated by error-prone non-homologous end joining pathway particularly in patients treated with DNA topoisomerase II inhibitors.
Subject(s)
Chromosomes, Human, Pair 11/genetics , Leukemia, Monocytic, Acute/etiology , Nijmegen Breakage Syndrome , Translocation, Genetic , Adolescent , Cell Cycle Proteins/genetics , Female , Histone-Lysine N-Methyltransferase , Humans , Leukemia, Monocytic, Acute/genetics , Myeloid-Lymphoid Leukemia Protein/genetics , Nijmegen Breakage Syndrome/complications , Nijmegen Breakage Syndrome/genetics , Nuclear Proteins/geneticsABSTRACT
Genetic variations in tumor necrosis factor (TNF) and interleukin-10 (IL-10) were reported to influence susceptibility to and outcome of patients with non-Hodgkin lymphoma. Therefore, we investigated whether single nucleotide polymorphisms in TNF and IL-10 may play a role in the clinical course of patients with chronic lymphocytic leukemia (CLL). TNF-308G>A, IL-10-3575T>A, and IL-10-1082A>G seem to be functionally relevant, were genotyped in 292 previously untreated patients with CLL. The control group consisted of 192 randomly selected blood donors. The patients carrying TNF-308GG and IL-10-1082AA genotypes presented a higher 3-year treatment-free survival (56.6 vs. 40.6%, P = 0.05) as well as a 10-year overall survival (OS) rates (92.3 vs. 57.6%, P = 0.005) than those with other TNF-308 and IL-10-1082 genotype combinations. Multivariate analysis demonstrated the Rai stage (P = 0.0002), IGHV mutation status (P = 0.01), TNF-308G>A (P = 0.03), and TNF/IL-10 polymorphism-based risk groups (P = 0.05) to be independent factors predicting OS. When the mutated IGHV patients were analyzed, the homozygotes TNF-308GG and IL-10-1082AA presented a higher 10-year OS rate than those carrying other TNF-308 and IL-10-1082 genotypes (100 vs. 67.7%, P = 0.01). In the unmutated IGHV patients, only the TNF-308G>A polymorphism influenced OS. The genetic variations in TNF and IL-10 genes work as independent predictors of survival and may play a role in the clinical course of CLL. It suggests inherited ability of the host to shift the balance between the Th1 and Th2 response, which in turn might contribute to the pathogenesis and prognosis of B-cell malignancies.
Subject(s)
Interleukin-10/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/mortality , Polymorphism, Single Nucleotide , Tumor Necrosis Factor-alpha/genetics , Alleles , Case-Control Studies , Genotype , Humans , Immunoglobulin Heavy Chains/genetics , Interleukin-10/blood , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Mutation , Prognosis , Tumor Necrosis Factor-alpha/bloodABSTRACT
Acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) are the two most common hematologic malignancies, challenging to treat and associated with high recurrence and mortality rates. This work aims to identify specific Raman biomarkers of ALL cells with the KMT2A gene rearrangement (KMT2A-r), representing a highly aggressive subtype of childhood leukemia with a poor prognosis. The proposed approach combines the sensitivity and specificity of Raman spectroscopy with machine learning and allows us to distinguish not only myelo- and lymphoblasts but also discriminate B-cell precursor (BCP) ALL with KMT2A-r from other blasts of BCP-ALL. We have found that KMT2A-r ALL cells fixed with 0.5% glutaraldehyde exhibit a unique spectroscopic profile that enables us to identify this subtype from other leukemias and normal cells. Therefore, a rapid and label-free method was developed to identify ALL blasts with KMT2A-r based on the ratio of the two Raman bands assigned to phenylalanine - 1040 and 1008 cm-1. This is the first time that a particular group of leukemic cells has been identified in a label-free way. The identified biomarker can be used as a screening method in diagnostic laboratories or non-reference medical centers.
Subject(s)
Leukemia, Myeloid, Acute , Myeloid-Lymphoid Leukemia Protein , Humans , Myeloid-Lymphoid Leukemia Protein/genetics , Spectrum Analysis, Raman , Leukemia, Myeloid, Acute/diagnosis , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Biomarkers , Hematopoietic Stem CellsABSTRACT
Breakpoint cluster region-Abelson (BCR::ABL1) gene fusion is an essential oncogene in both chronic myeloid leukemia (CML) and Philadelphia-positive (Ph+) B-cell acute lymphoblastic leukemia (B-ALL). While tyrosine kinase inhibitors (TKIs) are effective in up to 95% of CML patients, 50% of Ph+ B-ALL cases do not respond to treatment or relapse. This calls for new therapeutic approaches for Ph+ B-ALL. Previous studies have shown that inhibitors of the thioredoxin (TXN) system exert antileukemic activity against B-ALL cells, particularly in combination with other drugs. Here, we present that peroxiredoxin-1 (PRDX1), one of the enzymes of the TXN system, is upregulated in Ph+ lymphoid as compared to Ph+ myeloid cells. PRDX1 knockout negatively affects the viability of Ph+ B-ALL cells and sensitizes them to TKIs. Analysis of global gene expression changes in imatinib-treated, PRDX1-deficient cells revealed that the nonhomologous end-joining (NHEJ) DNA repair is a novel vulnerability of Ph+ B-ALL cells. Accordingly, PRDX1-deficient Ph+ B-ALL cells were susceptible to NHEJ inhibitors. Finally, we demonstrated the potent efficacy of a novel combination of TKIs, TXN inhibitors, and NHEJ inhibitors against Ph+ B-ALL cell lines and primary cells, which can be further investigated as a potential therapeutic approach for the treatment of Ph+ B-ALL.
ABSTRACT
BACKGROUND: The increasing volume and intricacy of sequencing data, along with other clinical and diagnostic data, like drug responses and measurable residual disease, creates challenges for efficient clinical comprehension and interpretation. Using paediatric B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) as a use case, we present an artificial intelligence (AI)-assisted clinical framework clinALL that integrates genomic and clinical data into a user-friendly interface to support routine diagnostics and reveal translational insights for hematologic neoplasia. METHODS: We performed targeted RNA sequencing in 1365 cases with haematological neoplasms, primarily paediatric B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) from the AIEOP-BFM ALL study. We carried out fluorescence in situ hybridization (FISH), karyotyping and arrayCGH as part of the routine diagnostics. The analysis results of these assays as well as additional clinical information were integrated into an interactive web interface using Bokeh, where the main graph is based on Uniform Manifold Approximation and Projection (UMAP) analysis of the gene expression data. At the backend of the clinALL, we built both shallow machine learning models and a deep neural network using Scikit-learn and PyTorch respectively. FINDINGS: By applying clinALL, 78% of undetermined patients under the current diagnostic protocol were stratified, and ambiguous cases were investigated. Translational insights were discovered, including IKZF1plus status dependent subpopulations of BCR::ABL1 positive patients, and a subpopulation within ETV6::RUNX1 positive patients that has a high relapse frequency. Our best machine learning models, LDA and PASNET-like neural network models, achieve F1 scores above 97% in predicting patients' subgroups. INTERPRETATION: An AI-assisted clinical framework that integrates both genomic and clinical data can take full advantage of the available data, improve point-of-care decision-making and reveal clinically relevant insights promptly. Such a lightweight and easily transferable framework works for both whole transcriptome data as well as the cost-effective targeted RNA-seq, enabling efficient and equitable delivery of personalized medicine in small clinics in developing countries. FUNDING: German Ministry of Education and Research (BMBF), German Research Foundation (DFG) and Foundation for Polish Science.
Subject(s)
Artificial Intelligence , Translational Research, Biomedical , Humans , Hematologic Neoplasms/genetics , Hematologic Neoplasms/diagnosis , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/diagnosis , Computational Biology/methods , Child , In Situ Hybridization, Fluorescence/methods , Female , Male , Biomarkers, Tumor/genetics , Gene Expression Profiling/methodsSubject(s)
Cell Cycle Proteins/genetics , Central Nervous System Neoplasms/etiology , Germ-Line Mutation , Heterozygote , Neoplasm Recurrence, Local/etiology , Nuclear Proteins/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Adolescent , Central Nervous System Neoplasms/pathology , Child , Child, Preschool , Female , Follow-Up Studies , Founder Effect , Humans , Infant , Male , Neoplasm Recurrence, Local/pathology , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Prognosis , Retrospective Studies , Risk Factors , Survival RateABSTRACT
Leukemias are a remarkably diverse group of malignancies originating from abnormal progenitor cells in the bone marrow. Leukemia subtypes are classified according to the cell type that has undergone neoplastic transformation using demanding and time-consuming methods. Alternative is Raman imaging that can be used both for living and fixed cells. However, considering the diversity of leukemic cell types and normal leukocytes, and the availability of different sample preparation protocols, the main objective of this work was to verify them for leukemia and normal blood cell samples for Raman imaging. The effect of glutaraldehyde (GA) fixation in a concentration gradient (0.1 %, 0.5 %, and 2.5 % GA) on the molecular structure of T-cell acute lymphoblastic leukemia (T-ALL) and peripheral blood mononuclear cells (PBMCs) was verified. Changes in the secondary structure of proteins within cells were indicated as the main effect of fixation, as shown by an increase in band intensity at 1041 cm-1, characteristic for in-plane δ(CH) deformation in phenylalanine (Phe). Different sensitivity of mononuclear and leukemic cells to fixation was observed. While the 0.1 % concentration of GA was too low to preserve the cell structure for an extended period of time, a GA concentration of 0.5 % seemed optimal for both normal and malignant cells. Chemical changes in PBMCs samples stored for 11 days were also investigated, which manifested in numerous modifications in the secondary structure of proteins and the content of nucleic acids. The impact of cell preculturing for 72 h after unbanking was verified, and there was no significant effect on the molecular structure of cells fixed with 0.5 % GA. In summary, the developed protocol for the preparation of samples for Raman imaging allows for the effective differentiation of fixed normal leukocytes from malignant T lymphoblasts.
Subject(s)
Leukemia , Leukocytes, Mononuclear , Humans , Leukocytes , Leukemia/metabolism , Cell DifferentiationABSTRACT
We analyzed the pattern of whole-genome copy number alterations (CNAs) and their association with the kinetics of blast clearance during the induction treatment among 195 pediatric patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) who displayed intermediate or high levels of minimal residual disease (MRD). Using unsupervised hierarchical clustering of CNAs > 5 Mbp, we dissected three clusters of leukemic samples with distinct kinetics of blast clearance [A - early slow responders (n=105), B - patients with persistent leukemia (n=24), C - fast responders with the low but detectable disease at the end of induction (n=66)] that corresponded with the patients' clinical features, the microdeletion profile,the presence of gene fusions and patients survival. Low incidence of large CNAs and chromosomal numerical aberrations occurred in cluster A which included ALL samples showing recurrent microdeletions within the genes encoding transcription factors (i.e., IKZF1, PAX5, ETV6, and ERG), DNA repair genes (XRCC3 and TOX), or harboring chromothriptic pattern of CNAs. Low hyperdiploid karyotype with trisomy 8 or hypodiploidy was predominantly observed in cluster B. Whereas cluster C included almost exclusively high-hyperdiploid ALL samples with concomitant mutations in RAS pathway genes. The pattern of CNAs influences the kinetics of leukemic cell clearance and selected aberrations affecting DNA repair genes may contribute to BCP-ALL chemoresistance.
Subject(s)
Precursor B-Cell Lymphoblastic Leukemia-Lymphoma , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Child , Humans , DNA Copy Number Variations , Kinetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Neoplasm, Residual , Chromosome Aberrations , Transcription Factors/geneticsABSTRACT
Adrenocortical carcinoma (ACC) is a rare cancer in childhood. ACC is frequently associated with germline TP53 variants, with founder effects especially due to the p.Arg337His mutation. ACC leads to the secretion of adrenocortical hormones, resulting in endocrine syndromes, which is the usual trigger for establishing the diagnosis. We present a surprising ACC pathology in a non-secreting, ectopic retroperitoneal tumour in a 4-year-old boy, successfully controlled with chemotherapy and mitotane after microscopically incomplete tumour resection with spillage. Genomic analysis (gene panel sequencing and copy-number microarray) demonstrated a novel p.Phe338Leu tetramerisation domain (TD) TP53 variant in the proband and his cancer-free mother and a monoallelic deletion encompassing the TP53 locus in cancer tissue, consistent with cancer-predisposition syndrome. While the recurrent p.Arg337His variant translates into high ACC risk, residue 338 and, in general, TD domain variants drive heterogeneous clinical scenarios, despite generally being considered less disruptive than TP53 DNA-binding domain mutations.