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.
The treatment of relapsed or refractory leukemia remains a major problem. Among the new therapeutic approaches, the use of modified T lymphocytes, called chimeric antigen receptor T cells (CAR-T ...cells), seems promising. The first step of their preparation is leukapheresis, which involves the collection of mononuclear cells from the patient. This medical procedure requires numerous medical devices (MDs) made of plasticized polyvinylchloride (PVC). These compounds can leach out of the devices during contact with the patient's blood. The aim of our study was to evaluate the migration of the plasticizers contained in the MD during a simulated pre-CAR-T cell leukapheresis procedure, and to measure the patient's and their lymphocytes' exposure to them.
The qualitative and quantitative composition of the MD used for pre-CAR-T cell apheresis was determined by gas chromatography-mass spectrometry (GC-MS). Then, an ex vivo leukapheresis model using an ethanol/water simulant was performed to evaluate the plasticizers' migration under simulated clinical conditions of pre-CAR-T cells' cytapheresis. The plasticizers released into the simulant were quantified by GC-MS.
Diethylhexylphthalate (DEHP) was found in the apheresis kit, with amounts ranging from 25% to 59% (g/100 g of PVC). Bis(2-ethylhexyl) adipate was detected at trace levels. A total of 98.90 ± 11.42 mg of DEHP was released into the simulant, corresponding to an exposure dose of 1.4 mg/kg for a 70 kg patient.
Patients undergoing a pre-CAR-T cell apheresis are mainly exposed to DEHP, which can impact their health because of its endocrine disruption effect, but could also lead to a decrease in CAR-T cells' efficiency/quality.
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.
BACKGROUND: Controlled‐rate freezing and storage in nitrogen is the standard technique for cryopreservation of peripheral hematopoietic progenitor cells (PHPCs) but presents high cost and dimethyl ...sulfoxide (DMSO) toxicity. Cryopreservation at −80°C, by uncontrolled rate freezing with only 3.5% DMSO, preserves the functional capacities of PHPCs, produces successful engraftment, and reduces toxicity during infusion.
STUDY DESIGN AND METHODS: Long‐term hematopoietic and immunologic reconstitution for 342 autografts (311 adults, 31 children) after PHPCs were cryopreserved at −80°C was studied at 3, 6, and 12 months. The median (range) storage time of PHPCs cryopreserved was 1.7 (0.1‐5.99) months.
RESULTS: Hemoglobin (Hb), white blood cells, and platelets (PLTs) reach normal values to trilineage at 12 months for 39% patients. Multivariate analysis shows a significant impact on CD34+ infused and on conditioning regimen for PLTs. Hb was influenced by growth factor administration at 3 months. Long‐term recovery is also highly dependent on blood counts (Hb, PLT, and neutrophil) at start of high‐dose chemotherapy. Only 43% of patients had reached normal lymphocyte values at 12 months after transplant, and a profound CD4+ T‐lymphocyte deficit remained, as others reported.
CONCLUSION: Transplantation with PHPCs cryopreserved at −80°C for no more than 6 months is satisfactory for long‐term hematopoietic and immunologic reconstitution, even if a profound CD4+ T lymphocyte deficit persists at 1 year. This easier and cheaper cryopreservation method also leads to successful engraftment.
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.
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.
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.
BACKGROUND: Extracorporeal photochemotherapy (ECP) gives positive results in the management of graft‐versus‐host disease (GVHD), but in children, specific difficulties can outweigh this benefit. ...These difficulties must be taken into consideration when establishing a standardized reproducible procedure for implementation under a quality management plan.
STUDY DESIGN AND METHODS: Twenty‐seven children underwent ECP for severe acute GVHD (aGVHD) or chronic GVHD (cGVHD) after allogeneic marrow transplantation. Data were collected prospectively, with particular emphasis placed on technical, biologic, immunologic, clinical, and long‐term follow‐up issues.
RESULTS: The 27 children underwent a total of 750 sessions. Mononuclear cells were collected on a commercially available apheresis system (COBE Spectra, Gambro BCT). Overall survival was 73 percent, and ECP led to significant improvement in 21 of the 27 patients (11 with complete response and 10 with partial response, i.e., >50% of organ involvement). Tolerance was good overall, the main limiting factors being vascular access and the psychological impact of repeated apheresis procedures. Children weighing less than 25 kg were not more susceptible to side effects.
CONCLUSION: A specifically pediatric‐dedicated and ‐experienced team faces only limited difficulties when treating children with GVHD by ECP. Overall, ECP is efficient and well tolerated. Our experience was therefore pooled together with available pediatric data to establish clinical practice guidelines. These guidelines consider ECP as a first‐line therapy in Grade IV aGVHD (in association with conventional pharmacologic approaches) and limited cGVHD and as a second‐line therapy in steroid‐resistant Grades II to III aGVHD and extensive cGVHD.
Abstract Background The rapid kinetics of hematopoietic stem cells induced by Plerixafor (Mozobil®, Genzyme) should be of particular interest in children. We therefore conducted a prospective trial ...to determine whether a one-day mobilization by plerixafor alone was efficient enough in children with cancer. Methods Children with solid malignancies were consecutively recruited for this phase-IIA, Bayesian single-center prospective study. Mobilization consisted in one subcutaneous injection of 240 μg plerixafor/kg body weight at 8 a.m. (h0). Collection by apheresis began at h5 provided that CD34+ count exceeded 10 × 106 /L. Our main evaluation criterion was percent of children in which at least 5 × 106 CD34+/kg could be collected during the first apheresis. Results No patients fulfilled the success criterion, and so a stopping criterion was met after 5 patients. All patients reached the threshold value of 10 × 106 CD34+ cells/L post-injection and so all were eligible for apheresis. Peak CD34+ cell values were ranged from 11 to 44 × 106 /L and were reached in 4 h to 6 h. No side-effects were observed. Median number of CD34+ cells collected per patient BW was 1.62 × 106 0.47−3.5. In 3 of the 5 patients, collection was >1.5 × 106 CD34+/kg BW. Conclusion In children, a ‘one-day’ mobilization regimen consisting of one injection of 240 μg/kg plerixafor alone in hematological steady state provides a faster and shorter mobilization than in adults. This strategy may be an attractive option for completing an insufficient graft. More studies are warranted to optimize the use of plerixafor in children.
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