Background
Ovarian tissue cryopreservation (OTC) is the only option available to preserve fertility in prepubertal females with neuroblastoma (NB), a childhood solid tumor that can spread to the ...ovaries, with a risk of reintroducing malignant cells after an ovarian graft.
Procedure
We set out to determine whether the analysis of TH (tyrosine hydroxylase), PHOX2B (paired‐like homeobox 2b), and DCX (doublecortin) transcripts using quantitative reverse transcriptase polymerase chain reaction (RT‐qPCR) could be used to detect NB contamination in ovarian tissue. Analyses were performed on benign ovarian tissue from 20 healthy women between November 2014 and September 2015 at the University Hospital of Clermont‐Ferrand. Pericystic benign ovarian tissues were collected and contaminated with increasing numbers of human NB cells (cell lines IMR‐32 and SK‐N‐SH) before detection using RT‐qPCR.
Results
TH and DCX transcripts were detected in uncontaminated ovarian tissue from all the donors, hampering the detection of small numbers of tumor cells. By contrast, PHOX2B was not detected in any uncontaminated ovarian fragment. PHOX2B levels were significantly increased from 10 NB cells. Our study is the first to evaluate minimal residual disease detection using NB mRNAs in human ovarian tissue. Only PHOX2B was a reliable marker of NB cells contaminating ovarian tissue.
Conclusions
These results are encouraging and offer hope in the near future for grafting ovarian tissue in women who survive cancer, whose fertility has been jeopardized by treatment, and who could benefit from OTC without oncological risk.
Ewing sarcoma (EWS) is a common pediatric solid tumor with high metastatic potential. Due to toxic effects of treatments on reproductive functions, the cryopreservation of ovarian tissue (OT) or ...testicular tissue (TT) is recommended to preserve fertility. However, the risk of reintroducing residual metastatic tumor cells should be evaluated before fertility restoration. Our goal was to validate a sensitive and specific approach for EWS minimal residual disease (MRD) detection in frozen germinal tissues. Thawed OT (
= 12) and TT (
= 14) were contaminated with tumor RD-ES cells (10, 100, and 1000 cells) and EWS-FLI1 tumor-specific transcript was quantified with RT-qPCR. All contaminated samples were found to be positive, with a strong correlation between RD-ES cell numbers and EWS-FLI1 levels in OT (
= 0.93) and TT (
= 0.96) (
< 0.001). No transcript was detected in uncontaminated control samples. The invasive potential of Ewing cells was evaluated using co-culture techniques. After co-culturing, tumor cells were detected in OT/TT with histology, FISH, and RT-qPCR. In addition, four OT and four TT samples from children with metastatic EWS were tested, and no MRD was found using RT-qPCR and histology. We demonstrated the high sensitivity and specificity of RT-qPCR to detect EWS MRD in OT/TT samples. Clinical trial: NCT02400970.
Leukemia is the most common cancer in pediatrics, with many late effects such as higher risk of dyslipidemia, insulin resistance, obesity, and metabolic syndrome. The objective of this work was to ...investigate substrate oxidation during submaximal exercise in survivors of childhood acute leukemia.
A total of 20 leukemia survivors and 20 healthy children were matched by sex, age, and Tanner stage. They all took a submaximal incremental exercise test to determine fat and carbohydrate oxidation rates.
Cardiorespiratory fitness was significantly lower in leukemia survivors, with lower relative VO
peaks (
< 0.001), lower heart rate values (
= 0.02), and lower exercise power (
= 0.012), whereas rest metabolism and body mass index did not differ between the two groups. During exercise, upward of heart rate relative to VO
peak was significantly higher (
< 0.001) in childhood leukemia survivors. We found lower carbohydrate and fat oxidation rates (
= 0.07) in leukemia survivors compared with healthy children, and also a significantly lower relative maximal fat oxidation rate (
= 0.014).
Despite impaired physical fitness and metabolic response to exercise, childhood leukemia survivors remained sensitive to physical activity interventions, and could readily adapt to submaximal exercise intensity.
Background
Testicular tissue freezing is proposed for fertility preservation to (pre)pubertal boys with cancer before highly gonadotoxic treatment. Studies accurately comparing human (pre)pubertal ...testicular tissue quality before freezing and after thawing are exceptional. No study has reported this approach in a systematic manner and routine care.
Objectives
To assess the impact of a control slow freezing protocol on testicular tissue architecture and integrity of (pre)pubertal boys after thawing.
Materials and methods
(Pre)pubertal boys (n = 87) with cancer from 8 Reproductive Biology Laboratories of the French CECOS network benefited from testicular tissue freezing before hematopoietic stem cell transplantation. Seminiferous tubule cryodamage was determined histologically by scoring morphological alterations and by quantifying intratubular spermatogonia and the expression of DNA replication and repair marker in frozen‐thawed testicular fragments.
Results
A significant increase in nuclear and epithelial score alterations was observed after thawing (p < 0.0001). The global lesional score remained lower than 1.5 and comparable to fresh testicular tissue. The number of intratubular spermatogonia and the expression of DNA replication and repair marker in spermatogonia and Sertoli cells did not vary significantly after thawing. These data showed the good preservation of the seminiferous tubule integrity and architecture after thawing, as previously reported in our studies performed in prepubertal mice and rats.
Discussion
The current study reports, for the first time, the development of a semi‐quantitative analysis of cryodamage in human (pre)pubertal testicular tissue, using a rapid and useful tool that can be proposed in routine care to develop an internal and external quality control for testicular tissue freezing. This tool can also be used when changing one or several parameters of the freezing‐thawing procedure.
Conclusion
Control slow freezing protocol without seeding maintains the seminiferous tubule architecture and integrity, the concentration of spermatogonia and the expression of DNA replication and repair marker in spermatogonia and Sertoli cells after thawing.
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.
Purpose
Childhood lymphoma survivors (CLSs) are at high risk of reduced daily activity. This work studied metabolic substrate use and cardiorespiratory function in response to exercise in CLSs.
...Methods
Twenty CLSs and 20 healthy adult controls matched for sex, age, and BMI took an incremental submaximal exercise test to determine fat/carbohydrate oxidation rates. Resting echocardiography and pulmonary functional tests were performed. Physical activity level, and blood metabolic and hormonal levels were measured.
Results
CLSs reported more physical activity than controls (6317 ± 3815 vs. 4268 ± 4354 MET-minutes/week,
p
= 0.013), had higher resting heart rate (83 ± 14 vs. 71 ± 13 bpm,
p
= 0.006), and showed altered global longitudinal strain (− 17.5 ± 2.1 vs. − 19.8 ± 1.6%,
p
= 0.003). We observed no difference in maximal fat oxidation between the groups, but it was reached at lower relative exercise intensities in CLSs (Fatmax 17.4 ± 6.0 vs. 20.1 ± 4.1 mL/kg,
p
= 0.021). At V̇O
2
peak, CLSs developed lower relative exercise power (3.2 ± 0.9 vs. 4.0 ± 0.7 W/kg,
p
= 0.012).
Conclusion
CLSs reported higher levels of physical activity but they attained maximal fat oxidation at lower relative oxygen uptake and applied lower relative power at V̇O
2
peak. CLSs may thus have lower muscular efficiency, causing greater fatigability in response to exercise, possibly related to chemotherapy exposure during adolescence and childhood. Long-term follow-up is essential and regular physical activity needs to be sustained.
Anticoagulant therapy in pediatric patients remains an issue and safer therapies, such as direct oral anticoagulants could overcome the limitations of conventional anticoagulant treatments in this ...population. Edoxaban, a factor Xa inhibitor, is used for the prevention and treatment of venous thromboembolism. Due to its pharmacokinetic characteristics, edoxaban is a promising candidate molecule for children. This study compared edoxaban in vitro effect in children and adults.
Blood samples were prospectively collected from 87 adults and 97 children (n = 12: <2 year-old; n = 8: 2–4 year-old; n = 9: 5–7 year-old; n = 14: 8–9 year-old; n = 10: 10–13 year-old; n = 15: 14–15 year-old; and n = 29: 16–18 year-old). Plasma samples were supplemented in vitro with edoxaban to a final concentration of 50, 150 or 300 ng/mL, and then edoxaban effect on prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen (Clauss assay), specific anti-factor Xa activity and thrombin generation assay (TGA) (with 5pM tissue factor and 4 nM phospholipids) was evaluated.
PT, aPTT, and specific anti-Xa activity exhibited similar dose-dependent responses to edoxaban in the different age groups. The reduction of thrombin peak, the most edoxaban-sensitive TGA parameter, was similar in adults and children, but for the youngest group (<2 year-old) where the peak value reduction (median Q1–Q3) was higher than in adults (51% 44–59 versus 40% 32–46, p < 0.01; 74% 63–80 versus 65% 58–70, p < 0.05; and 84% 73–88 versus 76% 70–80, p < 0.05 for 50, 150 and 300 ng/mL edoxaban, respectively).
Edoxaban in vitro effect are comparable in children and adults except in the <2-year-old group.
•PT and aPTT exhibited similar dose-dependent responses to edoxaban regardless of age.•Thrombin generation is decreased in children under 8 years of age.•Edoxaban impact on thrombin generation was greater in child <2 year-age than adults.
Hematopoietic progenitor cells‐apheresis (HPC‐A) collection is now a routine procedure for autologous hematopoietic stem cell transplantation. Here we present our 25 years' experience of HPC‐A ...collection in children weighing 8 kg or less, with a focus on the evolution of our standard operating procedures, and the safety limits for these young patients, in the Pediatric Apheresis Unit of Clermont‐Ferrand University Hospital (France). Fifteen children weighing 8 kg or less underwent 26 HPC‐A collections over 25 years. Median CD34+ cell yield by leukapheresis was 4.4 106/kg. No procedure‐related complications were encountered during or after the collection. No patient had profound thrombocytopenia or anemia that needed post‐collection transfusions. Our experience in pediatric oncology patients who underwent HPC‐A collections shows that this procedure can be performed even in the smallest of children with no increase in toxicity provided all precautions are taken to ensure that the procedure is carried out under the ideal conditions.
Neuroblastoma (NB) is the most common type of extracranial solid tumor in children with a high prevalence in toddlers. For childhood cancer survivors, preservation of reproductive potential is an ...important factor for quality of life. The optimization of NB minimal residual disease (MRD) detection in testicular tissue is crucial to evaluate the risk of malignant cell reintroduction. The first step in the present study was to assess the accuracy of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect tyrosine hydroxylase (
), paired-like homeobox 2b (
) and doublecortin (
) mRNA expression in frozen/thawed testicular tissues of patients with non-obstructive azoospermia (NOA) contaminated (
model) with an increasing number of IMR-32 and SK-N-SH NB cells. Testicular tissues were frozen by slow or snap freezing. The second step was to determine the expression levels of these markers in testicular samples from 4 pre-pubertal males (2 with stage IV NB and 2 with non-NB malignancy). The yield of extracted RNA was similar in testicular samples frozen by slow or snap freezing. In the
model,
and
transcripts were detected in uncontaminated testicular tissues, whereas
mRNA was not detected. There was a strong positive association between the number of NB cells used for contamination and
transcript levels. For IMR-32 and SK-N-SH NB cell lines, specificity and sensitivity rates of detection were 100% for
following
contamination with 10 tumor cells. In testicular samples from pre-pubertal males with and without NB,
mRNA expression was not observed, but high expression levels of
and
mRNA were detected, which were similar to expression detected in the
model. Among the markers used in blood and bone marrow for NB MRD studies, the detection of
transcripts by RT-qPCR may provide an accurate assessment of NB cells in testicular tissues from males who require fertility preservation.
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