•OGM can be performed on cell cultures from prenatal samples.•OGM detects all cytogenetic abnormalities identified by karyotype and chromosomal microarray.•OGM identifies small additional structural ...variations that may be clinically significant.•The combined use of OGM and exome sequencing will significantly increase the diagnostic yield in the prenatal setting.
Cytogenetic analysis provides important information for prenatal decision-making and genetic counseling. Optical genome mapping (OGM) has demonstrated its performances in retrospective studies. In our prospective study, we assessed the quality of DNA obtained from cultures of amniotic fluid (AF) and chorionic villi (CV) and evaluated the ability of OGM to detect all clinically relevant aberrations identified by standard methods.
A total of 37 prenatal samples from pregnancies with a fetal anomaly on ultrasound were analyzed prospectively by OGM between January 1, 2021 and June 31, 2022. OGM results were interpreted blindly and compared to the results obtained by standard techniques.
OGM results were interpretable in 92% of samples. We observed 100% concordance between OGM and karyotype and/or chromosomal microarray results. In addition, OGM identified a median of 30 small (<100 kb) structural variations per case with the involvement of 12 OMIM genes, of which 3 were OMIM morbid genes.
This prospective study showed OGM performed well in detecting genomic alterations in cell cultures from prenatal samples. The place of OGM in relation to CMA or exome sequencing remains to be defined in order to optimize the prenatal diagnostic procedure.
Summary
Assessing minimal residual disease (MRD) in B‐cell precursor acute lymphoblastic leukaemia (BCP‐ALL) is essential for adjusting therapeutic strategies and predicting relapse. Quantitative ...polymerase chain reaction (qPCR) is the gold standard for MRD. Alternatively, flow cytometry is a quicker and cost‐effective method that typically uses leukaemia‐associated immunophenotype (LAIP) or different‐from‐normal (DFN) approaches for MRD assessment. This study describes an optimized 12‐colour flow cytometry antibody panel designed for BCP‐ALL diagnosis and MRD monitoring in a single tube. This method robustly differentiated hematogones and BCP‐ALL cells using two specific markers: CD43 and CD81. These and other markers (e.g. CD73, CD66c and CD49f) enhanced the specificity of BCP‐ALL cell detection. This innovative approach, based on a dual DFN/LAIP strategy with a principal component analysis method, can be used for all patients and enables MRD analysis even in the absence of a diagnostic sample. The robustness of our method for MRD monitoring was confirmed by the strong correlation (r = 0.87) with the qPCR results. Moreover, it simplifies and accelerates the preanalytical process through the use of a stain/lysis/wash method within a single tube (<2 h). Our flow cytometry‐based methodology improves the BCP‐ALL diagnosis efficiency and MRD management, offering a complementary method with considerable benefits for clinical laboratories.
This study presents an optimized 12‐colour flow cytometry panel for BCP‐ALL diagnosis and MRD monitoring, combining LAIP/DFN strategies with principal component analysis in a single tube. This approach enhances the specificity of differentiating hematogones and BCP‐ALL cells using specific markers: CD43 and CD81. Our method shows strong correlation with qPCR (r = 0.87) and simplifies the process (<2 h), offering a quicker, cost‐effective alternative for clinical labs.
In high‐risk neuroblastoma (HR‐NB), the clinical significance of long‐term minimal residual disease (MRD) monitoring using quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) for ...neuroblastoma mRNAs has not been investigated. We report long‐term MRD follow‐ups of four patients with HR‐NB throughout the disease (diagnosis, remission, and relapse) and treatment course (chemotherapy, autologous and allogeneic stem cell transplantation, and donor lymphocyte and natural killer cell infusions). The results showed the stability of mRNA marker expression after different treatments and demonstrated their validity to predict relapse and assess therapeutic response. This opens up the possibility of investigating the utility of long‐term molecular monitoring of MRD in prospective multicenter studies.
To evaluate the hypothesis that detection of neuroblastoma mRNAs by reverse transcriptase quantitative polymerase chain reaction (RTqPCR) in peripheral blood (PB) and bone marrow aspirates (BM) from ...children with stage 4 neuroblastoma are clinically useful biomarkers of risk.
RTqPCR for paired-like homeobox 2b (PHOX2B), tyrosine hydroxylase (TH), and doublecortin (DCX) mRNA in PB and BM of children enrolled onto the High-Risk Neuroblastoma Trial-1 of the European Society of Pediatric Oncology Neuroblastoma Group (HR-NBL1/SIOPEN) was performed at diagnosis and after induction therapy.
High levels of TH, PHOX2B, or DCX mRNA in PB or BM at diagnosis strongly predicted for worse event-free survival (EFS) and overall survival (OS) in a cohort of 290 children. After induction therapy, high levels of these mRNAs predicted worse EFS and OS in BM but not in PB. Combinations of mRNAs in BM did not add to the predictive power of any single mRNA. However, in the original (n = 182) and validation (n = 137) PB cohorts, high TH (log10TH > 0.8) or high PHOX2B (log10PHOX2B > 0.28) identify 19% of children as ultrahigh risk, with 5-year EFS and OS rates of 0%; OS rate was 25% (95% CI, 16% to 36%) and EFS rate was 38% (95% CI, 28% to 49%) in the remaining children. The magnitude of reduction in mRNA level between diagnosis and postinduction therapy in BM or PB was not of additional predictive value.
High levels of TH and PHOX2B mRNA in PB at diagnosis objectively identify children with ultrahigh-risk disease who may benefit from novel treatment approaches. The level of TH, PHOX2B, and DCX mRNA in BM and/or PB at diagnosis might contribute to an algorithm to improve stratification of children for treatment.
Abstract
Telomeres protect chromosome ends and control cell division and senescence. During organogenesis, telomeres need to be long enough to ensure the cell proliferation necessary at this stage of ...development. Previous studies have shown that telomere shortening is associated with growth retardation and congenital malformations. However, these studies were performed in newborns or postnatally, and data on telomere length (TL) during the prenatal period are still very limited. We measured TL using quantitative PCR in amniotic fluid (AF) and chorionic villi (CV) samples from 69 control fetuses with normal ultrasound (52 AF and 17 CV) and 213 fetuses (165 AF and 48 CV) with intrauterine growth retardation (IUGR) or congenital malformations diagnosed by ultrasound. The samples were collected by amniocentesis at the gestational age (GA) of 25.0 ± 5.4 weeks and by CV biopsy at 18.1 ± 6.3 weeks. In neither sample type was TL influenced by GA or fetal sex. In AF, a comparison of abnormal versus normal fetuses showed a significant telomere shortening in cases of IUGR (reduction of 34%, P < 10−6), single (29%, P < 10−6) and multiple (44%, P < 10−6) malformations. Similar TL shortening was also observed in CV from abnormal fetuses but to a lesser extent (25%, P = 0.0002; 18%, P = 0.016; 20%, P = 0.004, respectively). Telomere shortening was more pronounced in cases of multiple congenital anomalies than in fetuses with a single malformation, suggesting a correlation between TL and the severity of fetal phenotype. Thus, TL measurement in fetal samples during pregnancy could provide a novel predictive marker of pathological development.
The immunosurveillance mechanisms governing high-risk neuroblastoma (HR-NB), a major pediatric malignancy, have been elusive. We identify a potential role for natural killer (NK) cells, in particular ...the interaction between the NK receptor NKp30 and its ligand, B7-H6, in the metastatic progression and survival of HR-NB after myeloablative multimodal chemotherapy and stem cell transplantation. NB cells expressing the NKp30 ligand B7-H6 stimulated NK cells in an NKp30-dependent manner. Serum concentration of soluble B7-H6 correlated with the down-regulation of NKp30, bone marrow metastases, and chemoresistance, and soluble B7-H6 contained in the serum of HR-NB patients inhibited NK cell functions in vitro. The expression of distinct NKp30 isoforms affecting the polarization of NK cell functions correlated with 10-year event-free survival in three independent cohorts of HR-NB in remission from metastases after induction chemotherapy (n = 196, P < 0.001), adding prognostic value to known risk factors such as N-Myc amplification and age >18 months. We conclude that the interaction between NKp30 and B7-H6 may contribute to the fate of NB patients and that both the expression of NKp30 isoforms on circulating NK cells and the concentration of soluble B7-H6 in the serum may be clinically useful as biomarkers for risk stratification.
Cells from patients with the autosomal recessive disorder ataxia–telangiectasia (A–T) display accelerated telomere shortening upon culture in vitro. It has been suggested that A–T cells are in a ...chronic state of oxidative stress, which could contribute to their enhanced telomere shortening. In order to examine this hypothesis, we monitored the changes in telomere length in A–T homozygous, heterozygous and control fibroblasts cultured in vitro under various conditions of oxidative stress using quantitative fluorescent in situ hybridization. Compared with normal cells, the rate of telomere shortening was 1.5-fold increased under ‘normal’ levels of oxidative stress in A–T heterozygous cells and 2.4–3.2-fold in A–T homozygous cells. Mild chronic oxidative stress induced by hydrogen peroxide increased the rate of telomere shortening in A–T cells but not in normal fibroblasts and the telomere shortening rate decreased in both normal and A–T fibroblasts if cultures were supplemented with the anti-oxidant phenyl-butyl-nitrone. Increased telomere shortening upon oxidative stress in A–T cells was associated with a significant increase in the number of extra-chromosomal fragments of telomeric DNA and chromosome ends without detectable telomere repeats. We propose that the ATM (A–T mutated) protein has a role in the prevention or repair of oxidative damage to telomeric DNA and that enhanced sensitivity of telomeric DNA to oxidative damage in A–T cells results in accelerated telomere shortening and chromosomal instability.
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