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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. [ABSTRACT FROM AUTHOR]

Due to their physiological role in removing damaged cells, natural killer (NK) cells represent ideal candidates for cellular immunotherapy in the treatment of cancer. Thereby, the cytotoxicity of NK cells is regulated by signals on both, the NK cells as well as the targeted tumor cells, and the interplay and balance of these signals determine the killing capacity of NK cells. One promising avenue in cancer treatment is therefore the combination of NK cell therapy with agents that either help to increase the killing capacity of NK cells or sensitize tumor cells to an NK cell-mediated attack. In this mini-review, we present different strategies that can be explored to unleash the potential of NK cell immunotherapy. In particular, we summarize how modulation of apoptosis signaling within tumor cells can be exploited to sensitize tumor cells to NK cell-mediated cytotoxicity. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cellular therapy has entered the daily clinical life with the approval of CAR T cell therapeutics and dendritic cell (DCs) vaccines in the US and the EU. In addition, numerous other adoptive cellular products, including natural killer (NK) cells, are currently evaluated in early phase I/ II clinical trials for the treatment of cancer patients. Despite these promising accomplishments, various challenges remain to be mastered in order to ensure sustained therapeutic success. These include the identification of strategies by which tumor cells escape the immune system or establish an immunosuppressive tumor microenvironment (TME). As part of the innate immune system, DCs and NK cells are both present within the TME of various tumor entities. While NK cells are well known for their intrinsic anti-tumor activity by their cytotoxicity capacities and the secretion of pro-inflammatory cytokines, the role of DCs within the TME is a double-edged sword as different DC subsets have been described with either tumor-promoting or -inhibiting characteristics. In this review, we will discuss recent findings on the interaction of DCs and NK cells under physiological conditions and within the TME. One focus is the crosstalk of various DC subsets with NK cells and their impact on the progression or inhibition of tumor growth. In addition, we will provide suggestions to overcome the immunosuppressive outcome of the interaction of DCs and NK cells within the TME. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Significantly improved survival rates in children and adolescents with cancer have put fertility preservation high on the pediatric oncology agenda. Here we report a retrospective single-center study of 13 years experience of ovarian tissue cryopreservation (OTC) before sterilizing treatment in order to define the safety/benefits of OTC and study clinical/hormonal outcomes in girls. From 2000 to 2013, OTC was performed in 36 girls: eight had non-malignant disease and 28 had malignant disease. Laparoscopy was used to collect a third of each ovary that was frozen by a slow cooling protocol. Indications for OTC were 13 auto-, 19 allo-stem-cell-transplantation and 4 sterilizing chemotherapy. Ovarian tissue harvested by intraumbilical laparoscopy led to no major postoperative complications and did not delay chemotherapy. Histological analysis of ovarian tissue showed an average of 9 primordial follicles/mm2[0–83] and no malignant cells were identified. Median post-harvest follow-up was 36 months [1–112]: 26 girls were alive in complete remission and 10 had died. Hormonal results were evaluable for 27 patients (median age 17 yrs [5–26]): 16 patients were in premature ovarian insufficiency. OTC sampling one third of each ovary appears to be an appropriate approach to preserve fertility in children without consequences on subsequent therapeutic program. [ABSTRACT FROM AUTHOR]

Background: 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. Methods: 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. Results: Cardiorespiratory fitness was significantly lower in leukemia survivors, with lower relative VO2 peaks (p < 0.001), lower heart rate values (p = 0.02), and lower exercise power (p = 0.012), whereas rest metabolism and body mass index did not differ between the two groups. During exercise, upward of heart rate relative to VO2 peak was significantly higher (p < 0.001) in childhood leukemia survivors. We found lower carbohydrate and fat oxidation rates (p = 0.07) in leukemia survivors compared with healthy children, and also a significantly lower relative maximal fat oxidation rate (p = 0.014). Conclusion: 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. [ABSTRACT FROM AUTHOR]

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: NCT 02400970. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]