Ex Vivo Model to Assess the Exposure of Patients to Plasticizers from Medical Devices during Pre-CAR-T Cells' Apheresis.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Peripheral blood stem cell collection in children with extremely low body weight (≤8 kg). What have we learned over the past 25 years and where are the limits?

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.

Sensitive and Specific Detection of Ewing Sarcoma Minimal Residual Disease in Ovarian and Testicular Tissues in an In Vitro Model.

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 (n = 12) and TT (n = 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 (r = 0.93) and TT (r = 0.96) (p < 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.

RT-qPCR for PHOX2B mRNA is a highly specific and sensitive method to assess neuroblastoma minimal residual disease in testicular tissue.

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 (TH), paired-like homeobox 2b (PHOX2B) and doublecortin (DCX) mRNA expression in frozen/thawed testicular tissues of patients with non-obstructive azoospermia (NOA) contaminated (in vitro 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 in vitro model, TH and DCX transcripts were detected in uncontaminated testicular tissues, whereas PHOX2B mRNA was not detected. There was a strong positive association between the number of NB cells used for contamination and PHOX2B transcript levels. For IMR-32 and SK-N-SH NB cell lines, specificity and sensitivity rates of detection were 100% for PHOX2B following in vitro contamination with 10 tumor cells. In testicular samples from pre-pubertal males with and without NB, PHOX2B mRNA expression was not observed, but high expression levels of TH and DCX mRNA were detected, which were similar to expression detected in the in vitro model. Among the markers used in blood and bone marrow for NB MRD studies, the detection of PHOX2B transcripts by RT-qPCR may provide an accurate assessment of NB cells in testicular tissues from males who require fertility preservation.

Highly sensitive assessment of neuroblastoma minimal residual disease in ovarian tissue using RT-qPCR-A strategy for improving the safety of fertility restoration.

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.

Mobilization of hematopoietic stem cells by plerixafor alone in children: a sequential Bayesian trial.

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 8a.m. (h0). Collection by apheresis began at h5 provided that CD34+count exceeded 10 × 10(6)/L. Our main evaluation criterion was percent of children in which at least 5 × 10(6) 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 × 10(6) CD34+cells/L post-injection and so all were eligible for apheresis. Peak CD34+cell values were ranged from 11 to 44 × 10(6)/L and were reached in 4h to 6h. No side-effects were observed. Median number of CD34+cells collected per patient BW was 1.62 × 10(6)[0.47-3.5]. In 3 of the 5 patients, collection was>1.5 × 10(6) 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.

Hematologic, immunologic reconstitution, and outcome of 342 autologous peripheral blood stem cell transplantations after cryopreservation in a -80°C mechanical freezer and preserved less than 6 months.

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.

NK cell immunotherapy for high-risk neuroblastoma relapse after haploidentical HSCT.

Little is known on strategies to prevent or to treat relapses occurring after haploidentical stem cell transplantation (haplo-HSCT) performed for the high-risk neuroblastoma (NB). We describe a 6-year-old male with refractory NB who relapsed 22 months after haplo-HSCT. A complete remission was obtained with a combination of immuno-chemotherapy based on donor NK cells transplants, IL2 infusions and temozolomide/topotecan. This case is an incentive to explore both the immediate therapeutic effect of haplo-graft provided via haplo-NK cells and the immunogenic platform that haplo-HSCT offers for future treatment. Our post-relapse strategy shows that chemo- and bio-treatment should be viewed as complementary therapeutic options.

Use of cryopreserved autologous cells for extracorporeal photochemotherapy: clinical applications.

BACKGROUND: Using autologous cryopreserved mononuclear cells (MNCs) for extracorporeal photochemotherapy (ECP) offers several advantages, such as treating patients from geographically distant care centers or maintaining ECP schedule while dramatically reducing number of apheresis sessions. We previously reported that cryopreserved cells retain their immunomodulatory properties when exposed to UVA and psoralen. To date, there are no clinical data on the use of cryopreserved MNCs for ECP ("cryo-ECP"). CASE REPORTS: Three patients were treated by cryo-ECP for refractory dermatomyositis, juvenile localized scleroderma, and acute graft-versus-host disease. For the first two patients, cryo-ECP aimed to reduce the number of apheresis sessions. Each cell product was split into three equal fractions: one was infused, and the other two were frozen for later infusion. The third patient was referred to our center from a hospital 700 km away. Fifteen apheresis procedures were performed during his stay: 12 were immediately treated and infused while three were cryopreserved. After discharge, the three cryopreserved bags were thawed, ECP-treated, and then sent back to the patient. CONCLUSION: In all three patients, cryo-ECP was safe and feasible. These cases illustrate promising clinical applications of the technique, opening perspectives for making ECP much more acceptable to patients while extending its indications.

CD34+ immunoselection of autologous grafts for the treatment of high-risk neuroblastoma.

BACKGROUND: Graft contamination has been blamed for causing relapse in children with high-risk neuroblastoma (HRNB) after autologous hematopoietic stem cell transplantation (HSCT). PROCEDURE: We report the long-term results of hematopoietic reconstitution, post-transplant complications, and clinical outcome of 44 children with HRNB treated with busulfan/melphalan high-dose chemotherapy followed by transplantation of purged CD34+ immunoselected autologous peripheral HSCT. Minimal residual disease (MRD) of grafts was evaluated by anti-GD2 immunofluorescence or tyrosine hydroxylase reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: Contaminating neuroblasts were found in 19/38 grafts (50%) before CD34+ positive selection, and none after (technique sensitivity of one cell in 10(5)). A median of 6.5 × 10(6) CD34+ cells/kg (range 0.8-23.7) were transplanted with only 2% of TRM. Neutrophils and platelet recovery occurred within a median of 12 days (range 9-47) and 44 days (range 12-259), respectively, without any secondary graft failure. Twenty-three percents of patients experienced a sepsis (10/44) and 14% a pyelonephritis (6/44). Recurrence of varicella zoster virus occurred in 21% of patients (9/44). Negative RT-PCR MRD within the leukapheresis product and cis-retinoic acid therapy were significantly and independently associated to a better survival (P < 0.05). Overall and event-free survivals at 5 years post-transplant were at 59.3% and 48.3% respectively. CONCLUSIONS: Besides high rates of manageable infections due to late immune recovery, transplantation with CD34+ immunoselected grafts in HRNB children was feasible and did not affect long-term hematopoiesis.

Photopheresis in pediatric graft-versus-host disease after allogeneic marrow transplantation: clinical practice guidelines based on field experience and review of the literature.

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.

Significance of molecular quantification of minimal residual disease in metastatic neuroblastoma.

Molecular detection of tumor cells is the most sensitive approach to study residual disease in bone marrow (BM), peripheral blood (PB), and peripheral blood stem cell (PBSC) autografts from children with metastatic neuroblastoma (NB). We have developed a real-time PCR assay that allows the quantification of tyrosine hydroxylase (TH) mRNA, a tissue-specific marker of neuroblasts. We investigated a total of 165 BM, PB, and PBSC samples from 30 children over 1 year of age with stage IV NB and correlated the findings with disease status and patient survival. The levels of TH mRNA agreed well with clinical status and were significantly different across the groups that included samples obtained from patients at diagnosis, after three cycles of chemotherapy, in complete or very good partial remission and at relapse. We found that overall survival was significantly worse for patients with >1000 TH copies in BM after initial chemotherapy (p=0.0075). In 57% of cases, autologous PBSC harvests were found to be contaminated by neuroblasts, the level of TH >500 copies being associated with a decreased survival (p=0.003). In addition, molecular quantification enabled an estimation of tumor depletion in contaminated autografts using CD34 selection (median, 3 logs). In conclusion, quantification of minimal residual disease in metastatic NB using real-time RT-PCR for TH mRNA appears to be of potential clinical value. Further studies are needed to ascertain prognostic implications of molecular analysis of residual disease.

Extracorporeal photochemotherapy for graft versus host disease in pediatric patients.

Although worldwide experience with extracorporeal photochemotherapy (ECP) for GvHD treatment has grown enormously over the past decade, only a few pediatric centers have experience with ECP. Studies reporting clinical outcome in children with GvHD treated by ECP comprises a very limited number of patients with only few information described. This review article remain focused on the efficacy and the safety aspect of ECP in pediatric patients to provide information about the steps that should be taken to overcome the difficulties with ECP use in children with GvHD. Data concerning 19 children with acute GvHD and 54 children with chronic GvHD treated with ECP and reported so far have been considered. The principal reasons for the restriction in the use of ECP in children such as: (1) technical difficulties of leukapheresis procedures (venous access, hemodynamic, metabolic and hematological tolerance); and (2) the necessity of a specially adapted pediatric patient approach to improve the psychological tolerance of this treatment are discussed. The data of this retrospective review demonstrate that ECP is beneficial and well tolerated in children with GvHD. It can be safely used even in young children with low body weight and a poor performance status when it was performed by a qualified pediatric team. The observations concerning the response rate and onset suggest that in children with acute GvHD, ECP should be started early in the course of disease and employed over a relatively short period of time. As far as chronic GvHD is concerned, despite the fact that it is preferable to begin ECP early as second line therapy, it may also be beneficial in patients with late-stage disease.

Successful extracorporeal photochemotherapy for chronic graft-versus-host disease in pediatric patients.

A total of 254 extracorporeal photochemotherapy (ECP) procedures were performed in 8 children (median age 10 years; range 5-15) with extensive resistant chronic graft-versus-host disease (GVHD). ECP was carried out in the pediatric environment using a Cobe Spectra separator and UV-MATIC irradiator. A peripheral venous with a single-lumen permanent central catheter access (69% of ECP-apheresis) or a dual-lumen permanent central catheter access (26% of ECP-apheresis) were used preferentially. A median platelet decrease of 17% (0-71) (p = 0.0001) and median hemoglobin level decrease of 15 g/L (0-31)(p = 0.0001) were noted following each ECP-apheresis. However, none of the patients had profound thrombocytopenia or anemia. Two minor episodes of catheter related-bacteriemia (Staphylococcus aureus) were noted (2310 catheter-days). A negative correlation was found between lymphocyte collection efficacy (median = 38%) and pre ECP-apheresis lymphocyte count (r = 0.4, p = 0.00001). The median of 5 x 10(7) lymphocytes/kg (0.1-50.10(7)/kg) was irradiated in each procedure. All patients are alive and well, and 7/8 experienced a dramatic improvement in their cutaneous status. Liver and gut disease resolved completely in 4/6 and 5/5 patients, respectively. In all patients, a concomitant immunosuppressive therapy was stopped (5/8) or considerably reduced (3/8). Five patients with more than 2 years follow-up after discontinuation of ECP are in remission with no immunosuppression treatment. They have normal growth rates and normal school and sport activity. Our study shows that ECP is beneficial, well tolerated, and can be safely used for chronic GVHD treatment even in young children with low body weight and a poor performance status. We believe that having a dedicated pediatric environment together with an experienced, motivated, and specifically pediatric team is of crucial importance for improving patient's acceptance of this long-term therapeutic program.