Ovarian tissue cryopreservation for fertility preservation before hematopoietic stem cell transplantation in patients with sickle cell disease: safety, ovarian function follow-up, and results of ovarian tissue transplantation.

PURPOSE: To describe the experience of performing ovarian tissue cryopreservation (OTC) before hematopoietic stem cell transplantation (HSCT), among girls/women with severe sickle cell disease (SCD)(SS or S/ß0-thalassemia) who are, besides the usual surgical risk, at risk of SCD-related complications during the fertility preservation procedure for improving their counseling and management. METHODS: This retrospective study included 75 patients (girls/women) with SCD who have had OTC before myeloablative conditioning regimen (MAC) for HSCT. Characteristics of patients and data on OTC, ovarian status follow-up, and results of ovarian tissue transplantation (OTT) were collected in medical records. RESULTS: At OTC, the median (IQR 25-75; range) age of the patients was 9.6 (6.9-14.1; 3.6-28.3) years, 56/75 were prepubertal, and no SCD or surgery-related complications occurred. The median follow-up post-HSCT was > 9 years. At the last follow-up, among prepubertal patients at HSCT, 26/56 were ≥ 15 years old and presented with a premature ovarian insufficiency (POI), except 2, including the patient who had received an OTT to induce puberty. Eight were 13-15 years old and presented for POI. The remaining 22 patients were under 13. Among the 19 patients who were menarche at HSCT, 2 died 6 months post-HSCT and we do not have ovarian function follow-up for the other 2 patients. All the remaining patients (n = 15) had POI. Five patients had OTT. All had a return of ovarian function. One patient gave birth to a healthy baby. CONCLUSION: OTC is a safe fertility preservation technique and could be offered before MAC independent of the patient's age.

Brain injury pathophysiology study by a multimodal approach in children with sickle cell anemia with no intra or extra cranial arteriopathy.

Despite its high prevalence in children with sickle cell anemia (SCA), the pathophysiology of silent cerebral infarcts (SCI) remains elusive. The main objective of this study was to explore the respective roles of major determinants of brain perfusion in SCA children with no past or current history of intracranial or extracranial vasculopathy. We used a multimodal approach based notably on perfusion imaging arterial spin labeling (ASL) magnetic resonance imaging (MRI) and near infra-red spectroscopy (NIRS), as well as biomarkers reflecting blood rheology and endothelial activation. Out of 59 SCA patients (mean age 11.4±3.9 yrs), eight (13%) had a total of 12 SCI. Children with SCI had a distinctive profile characterized by decreased blood pressure, impaired blood rheology, increased P-selectin levels, and marked anemia. Although ASL perfusion and oximetry values did not differ between groups, comparison of biological and clinical parameters according to the level of perfusion categorized in terciles showed an independent association between high perfusion and increased sP-selectin, decreased red blood cell deformability, low hemoglobin F level, increased blood viscosity and no a-thalassemia deletion. NIRS measurements did not yield additional novel results. Altogether, these findings argue for early MRI detection of SCI in children with no identified vasculopathy and suggest a potential role for ASL as an additional screening tool. Early treatment targeting hemolysis, anemia and endothelial dysfunction should reduce the risk of this under diagnosed and serious complication.

Improved stenosis outcome in stroke-free sickle cell anemia children after transplantation compared to chronic transfusion.

We report here the 3-year stenosis outcome in 60 stroke-free children with sickle cell anaemia (SCA) and an abnormal transcranial Doppler history, enrolled in the DREPAGREFFE trial, which compared stem cell transplantation (SCT) with standard-care (chronic transfusion for 1-year minimum). Twenty-eight patients with matched sibling donors were transplanted, while 32 remained on standard-care. Stenosis scores were calculated after performing cerebral/cervical 3D time-of-flight magnetic resonance angiography. Fourteen patients had stenosis at enrollment, but only five SCT versus 10 standard-care patients still had stenosis at 3 years. Stenosis scores remained stable on standard-care, but significantly improved after SCT (P = 0·006). No patient developed stenosis after SCT, while two on standard-care did, indicating better stenosis prevention and improved outcome after SCT.

Gene Therapy in Patients with Transfusion-Dependent ß-Thalassemia.

BACKGROUND: Donor availability and transplantation-related risks limit the broad use of allogeneic hematopoietic-cell transplantation in patients with transfusion-dependent ß-thalassemia. After previously establishing that lentiviral transfer of a marked ß-globin (ßA-T87Q) gene could substitute for long-term red-cell transfusions in a patient with ß-thalassemia, we wanted to evaluate the safety and efficacy of such gene therapy in patients with transfusion-dependent ß-thalassemia. METHODS: In two phase 1-2 studies, we obtained mobilized autologous CD34+ cells from 22 patients (12 to 35 years of age) with transfusion-dependent ß-thalassemia and transduced the cells ex vivo with LentiGlobin BB305 vector, which encodes adult hemoglobin (HbA) with a T87Q amino acid substitution (HbAT87Q). The cells were then reinfused after the patients had undergone myeloablative busulfan conditioning. We subsequently monitored adverse events, vector integration, and levels of replication-competent lentivirus. Efficacy assessments included levels of total hemoglobin and HbAT87Q, transfusion requirements, and average vector copy number. RESULTS: At a median of 26 months (range, 15 to 42) after infusion of the gene-modified cells, all but 1 of the 13 patients who had a non-ß0/ß0 genotype had stopped receiving red-cell transfusions; the levels of HbAT87Q ranged from 3.4 to 10.0 g per deciliter, and the levels of total hemoglobin ranged from 8.2 to 13.7 g per deciliter. Correction of biologic markers of dyserythropoiesis was achieved in evaluated patients with hemoglobin levels near normal ranges. In 9 patients with a ß0/ß0 genotype or two copies of the IVS1-110 mutation, the median annualized transfusion volume was decreased by 73%, and red-cell transfusions were discontinued in 3 patients. Treatment-related adverse events were typical of those associated with autologous stem-cell transplantation. No clonal dominance related to vector integration was observed. CONCLUSIONS: Gene therapy with autologous CD34+ cells transduced with the BB305 vector reduced or eliminated the need for long-term red-cell transfusions in 22 patients with severe ß-thalassemia without serious adverse events related to the drug product. (Funded by Bluebird Bio and others; HGB-204 and HGB-205 ClinicalTrials.gov numbers, NCT01745120 and NCT02151526 .).

Pediatric Evans syndrome is associated with a high frequency of potentially damaging variants in immune genes.

Evans syndrome (ES) is a rare severe autoimmune disorder characterized by the combination of autoimmune hemolytic anemia and immune thrombocytopenia. In most cases, the underlying cause is unknown. We sought to identify genetic defects in pediatric ES (pES), based on a hypothesis of strong genetic determinism. In a national, prospective cohort of 203 patients with early-onset ES (median [range] age at last follow-up: 16.3 years ([1.2-41.0 years]) initiated in 2004, 80 nonselected consecutive individuals underwent genetic testing. The clinical data were analyzed as a function of the genetic findings. Fifty-two patients (65%) received a genetic diagnosis (the M+ group): 49 carried germline mutations and 3 carried somatic variants. Thirty-two (40%) had pathogenic mutations in 1 of 9 genes known to be involved in primary immunodeficiencies (TNFRSF6, CTLA4, STAT3, PIK3CD, CBL, ADAR1, LRBA, RAG1, and KRAS), whereas 20 patients (25%) carried probable pathogenic variants in 16 genes that had not previously been reported in the context of autoimmune disease. Lastly, no genetic abnormalities were found in the remaining 28 patients (35%, the M- group). The M+ group displayed more severe disease than the M- group, with a greater frequency of additional immunopathologic manifestations and a greater median number of lines of treatment. Six patients (all from the M+ group) died during the study. In conclusion, pES was potentially genetically determined in at least 65% of cases. Systematic, wide-ranging genetic screening should be offered in pES; the genetic findings have prognostic significance and may guide the choice of a targeted treatment.

Paediatric multisystem inflammatory syndrome temporally associated with SARS-CoV-2 mimicking Kawasaki disease (Kawa-COVID-19): a multicentre cohort.

BACKGROUND: Current data suggest that COVID-19 is less frequent in children, with a milder course. However, over the past weeks, an increase in the number of children presenting to hospitals in the greater Paris region with a phenotype resembling Kawasaki disease (KD) has led to an alert by the French national health authorities. METHODS: Multicentre compilation of patients with KD in Paris region since April 2020, associated with the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ('Kawa-COVID-19'). A historical cohort of 'classical' KD served as a comparator. RESULTS: Sixteen patients were included (sex ratio=1, median age 10 years IQR (4·7 to 12.5)). SARS-CoV-2 was detected in 12 cases (69%), while a further three cases had documented recent contact with a quantitative PCR-positive individual (19%). Cardiac involvement included myocarditis in 44% (n=7). Factors prognostic for the development of severe disease (ie, requiring intensive care, n=7) were age over 5 years and ferritinaemia >1400 µg/L. Only five patients (31%) were successfully treated with a single intravenous immunoglobulin (IVIg) infusion, while 10 patients (62%) required a second line of treatment. The Kawa-COVID-19 cohort differed from a comparator group of 'classical' KD by older age at onset 10 vs 2 years (p<0.0001), lower platelet count (188 vs 383 G/L (p<0.0001)), a higher rate of myocarditis 7/16 vs 3/220 (p=0.0001) and resistance to first IVIg treatment 10/16 vs 45/220 (p=0.004). CONCLUSION: Kawa-COVID-19 likely represents a new systemic inflammatory syndrome temporally associated with SARS-CoV-2 infection in children. Further prospective international studies are necessary to confirm these findings and better understand the pathophysiology of Kawa-COVID-19. Trial registration number NCT02377245.

Congenital macrothrombocytopenia with focal myelofibrosis due to mutations in human G6b-B is rescued in humanized mice.

Unlike primary myelofibrosis (PMF) in adults, myelofibrosis in children is rare. Congenital (inherited) forms of myelofibrosis (cMF) have been described, but the underlying genetic mechanisms remain elusive. Here we describe 4 families with autosomal recessive inherited macrothrombocytopenia with focal myelofibrosis due to germ line loss-of-function mutations in the megakaryocyte-specific immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor G6b-B (G6b, C6orf25, or MPIG6B). Patients presented with a mild-to-moderate bleeding diathesis, macrothrombocytopenia, anemia, leukocytosis and atypical megakaryocytes associated with a distinctive, focal, perimegakaryocytic pattern of bone marrow fibrosis. In addition to identifying the responsible gene, the description of G6b-B as the mutated protein potentially implicates aberrant G6b-B megakaryocytic signaling and activation in the pathogenesis of myelofibrosis. Targeted insertion of human G6b in mice rescued the knockout phenotype and a copy number effect of human G6b-B expression was observed. Homozygous knockin mice expressed 25% of human G6b-B and exhibited a marginal reduction in platelet count and mild alterations in platelet function; these phenotypes were more severe in heterozygous mice that expressed only 12% of human G6b-B. This study establishes G6b-B as a critical regulator of platelet homeostasis in humans and mice. In addition, the humanized G6b mouse will provide an invaluable tool for further investigating the physiological functions of human G6b-B as well as testing the efficacy of drugs targeting this receptor.

Extensive multilineage analysis in patients with mixed chimerism after allogeneic transplantation for sickle cell disease: insight into hematopoiesis and engraftment thresholds for gene therapy.

Although studies of mixed chimerism following hematopoietic stem cell transplantation in patients with sickle cell disease (SCD) may provide insights into the engraftment needed to correct the disease and into immunological reconstitution, an extensive multilineage analysis is lacking. We analyzed chimerism simultaneously in peripheral erythroid and granulomonocytic precursors/progenitors, highly purified B and T lymphocytes, monocytes, granulocytes and red blood cells (RBC). Thirty-four patients with mixed chimerism and ≥12 months of follow-up were included. A selective advantage of donor RBC and their progenitors/precursors led to full chimerism in mature RBC (despite partial engraftment of other lineages), and resulted in the clinical control of the disease. Six patients with donor chimerism <50% had hemolysis (reticulocytosis) and higher HbS than their donor. Four of them had donor chimerism <30%, including a patient with AA donor (hemoglobin >10 g/dL) and three with AS donors (hemoglobin <10 g/dL). However, only one vaso-occlusive crisis occurred with 68.7% HbS. Except in the patients with the lowest chimerism, the donor engraftment was lower for T cells than for the other lineages. In a context of mixed chimerism after hematopoietic stem cell transplantation for SCD, myeloid (rather than T cell) engraftment was the key efficacy criterion. Results show that myeloid chimerism as low as 30% was sufficient to prevent a vaso-occlusive crisis in transplants from an AA donor but not constantly from an AS donor. However, the correction of hemolysis requires higher donor chimerism levels (i.e ≥50%) in both AA and AS recipients. In the future, this group of patients may need a different therapeutic approach.

Long-term event-free survival, chimerism and fertility outcomes in 234 patients with sickle-cell anemia younger than 30 years after myeloablative conditioning and matched-sibling transplantation in France.

Allogeneic stem cell transplantation remains the only curative treatment for sickle cell anemia (SCA), but the place of myeloablative conditioning in the procedure remains to be defined. The aim of the present study was to analyze long-term outcomes, including chimerism, SCA-related events and biological data (hemoglobin, reticulocytes, HbS%), and fertility in a French series of 234 SCA patients under 30 years of age who, from 1988 to 2012, received a matched-sibling-donor stem cell transplantation following standardized myeloablative conditioning [busulfan, cyclophosphamide and rabbit antithymocyte globulin (ATG)]. Since the first report of the series (1988-2004), 151 new consecutive patients with SCA have been similarly transplanted. Considering death, non-engraftment or rejection (donor cells <5%) as events, the 5-year event-free survival was 97.9% (95% confidence interval: 95.5-100%), confirming, since the year 2000, an at least 95% chance of cure. In the overall cohort (n=234, median follow up 7.9 years), event-free survival was not associated with age, but chronic-graft-versus-host disease (cGvHD) was independently associated with recipient's age >15 years (hazard ratio=4.37; P=0.002) and lower (5-15 vs 20 mg/kg) ATG dose (hazard ratio=4.55; P=0.001). At one year, 44% of patients had mixed chimerism (5-95% donor cells), but those prepared with ATG had no graft rejection. No events related to SCA occurred in patients with mixed chimerism, even those with 15-20% donor cells, but hemolytic anemia stigmata were observed with donor cells <50%. Myeloablative transplantation with matched-sibling donor currently has a higher event-free survival (98%) in patients under 30 years of age than that reported for non-myeloablative conditioning (88%). Nevertheless, the risk of cGvHD in older patients and the need to preserve fertility might be indications for a non-myeloablative conditioning.

Appropriate thresholds for accurate screening for ß-thalassemias in the newborn period: results from a French center for newborn screening.

Objectives: Newborn screening (NBS) for ß-thalassemia is based on measuring the expression of the hemoglobin A (HbA) fraction. An absence or very low level of HbA at birth may indicate ß-thalassemia. The difficulty is that the HbA fraction at birth is correlated with gestational age (GA) and highly variable between individuals. We used HbA expressed in multiples of the normal (MoM) to evaluate relevant thresholds for NBS of ß-thalassemia. Methods: The chosen threshold (HbA≤0.25 MoM) was prospectively applied for 32 months in our regional NBS program for sickle cell disease, for all tests performed, to identify patients at risk of ß-thalassemia. Reliability of this threshold was evaluated at the end of the study. Results: In all, 343,036 newborns were tested, and 84 suspected cases of ß-thalassemia were detected by applying the threshold of HbA≤0.25 MoM. Among the n=64 cases with confirmatory tests, 14 were confirmed using molecular analysis as ß-thalassemia diseases, 37 were confirmed as ß-thalassemia trait and 13 were false-positive. Determination of the optimum threshold for ß-thalassemia screening showed that HbA≤0.16 MoM had a sensitivity of 100% and a specificity of 95.3%, whatever the GA. Conclusions: NBS for ß-thalassemia diseases is effective, regardless of the birth term, using the single robust threshold of HbA≤0.16 MoM. A higher threshold would also allow screening for carriers, which could be interesting when ß-thalassemia constitutes a public health problem.

Sexual health of French adolescents with sickle cell disease.

PURPOSE: Focussing on sickle cell disease (SCD), the objective of this study was to assess adolescents' sexual heath experience in the context of their chronic illness. MATERIALS AND METHODS: We included teenagers from 14 to 19 years old followed for SCD in a hospital located in Créteil (France) from March 2017 to February 2018. Their sexual health experience was assessed by a self-questionnaire with three key themes: contraceptive experience, awareness of sexuality with chronic disease and level of information on the genetic transmission of the disease. RESULTS: 99 questionnaires were analysed. Only six SCD adolescents (one girl and five boys) reported being sexually active. Despite a very regular follow-up of their illness, only 13% of the boys and girls had received information on contraception at the hospital. Most adolescents (85% of boys and 81% of girls) did not think that the disease would interfere with sexual intercourse. The genetic pattern was well known (85% of boys and 87% of girls). CONCLUSION: Young people with SCD need more information on contraception. Clinicians caring for them should be aware of the need for sexual health information in order to propose prevention actions adapted to these young people with chronic disease.

[Effects of a standardized musical intervention on the management of pain and anxiety-state of sickle-cell adolescents]. / Effets d'une intervention musicale standardisée sur la gestion de la douleur et de l'anxiété-état des adolescents drépanocytaires.

The effect of a standardized musical intervention for adolescents with sickle cell disease was studied. Two groups were evaluated using the visual analog scale of pain and the anxiety-state inventory before and after a standardized musical intervention or breathing intervention. A significant decrease in scores was observed, most notably for the group benefiting from the standardized musical intervention. This intervention could be integrated into the overall management of adolescents with sickle cell disease.

Ovarian tissue cryopreservation for fertility preservation in 418 girls and adolescents up to 15 years of age facing highly gonadotoxic treatment. Twenty years of experience at a single center.

INTRODUCTION: The preservation of fertility is an integral part of care of children requiring gonadotoxic treatments for cancer or non-malignant diseases. In France, the cryopreservation of ovarian tissue has been considered and has been offered as a clinical treatment since its inception. The aim of this study is to review 20 years of activity in fertility preservation by ovarian tissue cryopreservation (OTC) for children and the feasibility of oocyte isolation and cryopreservation from the ovarian tissue at a single center. MATERIAL AND METHODS: Retrospective study including patients aged 15 years or younger who underwent OTC, combined for some with oocyte cryopreservation of isolated oocytes, before a highly gonadotoxic treatment for malignant or non-malignant disease was initiated. We describe the evolution of activities in our program for fertility preservation and patient characteristics at the time of OTC and follow up. RESULTS: From April 1998 to December 2018, 418 girls and adolescents younger than 15 years of age underwent OTC, representing 40.5% of all females who have had ovarian tissue cryopreserved at our center. In all, 313 patients had malignant diseases and 105 had benign conditions. Between November 2009 and July 2013, oocytes were isolated and also cryopreserved in 50 cases. The mean age of patients was 6.9 years (range 0.3-15). The most frequent diagnoses in this cohort included neuroblastoma, acute leukemia and hemoglobinopathies; neuroblastoma being the most common diagnosis in very young patients. During follow up, three patients requested the use of their cryopreserved ovarian tissue. All had undergone ovarian tissue transplantation, one for puberty induction and the two others for restoring fertility. So far, no pregnancies have been achieved. Eighty-four patients who had OTC died. CONCLUSIONS: Ovarian tissue cryopreservation is the only available technique for preserving fertility of girls. To our knowledge this is the largest series of girls and adolescents younger than 15 years so far reported on procedures of OTC before highly gonadotoxic treatment in a single center.

Association of Matched Sibling Donor Hematopoietic Stem Cell Transplantation With Transcranial Doppler Velocities in Children With Sickle Cell Anemia.

Importance: In children with sickle cell anemia (SCA), high transcranial Doppler (TCD) velocities are associated with stroke risk, which is reduced by chronic transfusion. Whether matched sibling donor hematopoietic stem cell transplantation (MSD-HSCT) can reduce velocities in patients with SCA is unknown. Objective: To determine the association of MSD-HSCT with TCD velocities as a surrogate for the occurrence of ischemic stroke in children with SCA. Design, Setting, and Participants: Nonrandomized controlled intervention study conducted at 9 French centers. Patients with SCA were enrolled between December 2010 and June 2013, with 3-year follow-up ending in January 2017. Children with SCA were eligible if younger than 15 years, required chronic transfusions for persistently elevated TCD velocities, and had at least 1 sibling without SCA from the same 2 parents. Families agreed to HLA antigen typing and transplantation if a matched sibling donor was identified or to standard care in the absence of a matched sibling donor. Exposures: MSD-HSCT (n = 32), compared with standard care (n = 35) (transfusions for ≥1 year with potential switch to hydroxyurea thereafter), using propensity score matching. Main Outcomes and Measures: The primary outcome was the highest time-averaged mean of maximum velocities in 8 cerebral arteries, measured by TCD (TCD velocity) at 1 year. Twenty-five of 29 secondary outcomes were analyzed, including the highest TCD velocity at 3 years and normalization of velocities (<170 cm/s) and ferritin levels at 1 and 3 years. Results: Sixty-seven children with SCA (median age, 7.6 years; 35 girls [52%]) were enrolled (7 with stroke history). In the matched sample, highest TCD velocities at 1 year were significantly lower on average in the transplantation group (129.6 cm/s) vs the standard care group (170.4 cm/s; difference, -40.8 cm/s [95% CI, -62.9 to -18.6]; P < .001). Of the 25 analyzed secondary end points, 4 showed significant differences, including the highest TCD velocity at 3 years (112.4 cm/s in the transplantation group vs 156.7 cm/s in the standard care group; difference, -44.3 [95% CI, -71.9 to -21.1]; P = .001); normalization rate at 1 year (80.0% in the transplantation group vs 48.0% in the standard care group; difference, 32.0% [95% CI, 0.2% to 58.6%]; P = .045); and ferritin levels at 1 year (905 ng/mL in the transplantation group vs 2529 ng/mL in the standard care group; difference, -1624 [95% CI, -2370 to -879]; P < .001) and 3 years (382 ng/mL in the transplantation group vs 2170 ng/mL in the standard care group; difference, -1788 [95% CI, -2570 to -1006]; P < .001). Conclusions and Relevance: Among children with SCA requiring chronic transfusion because of persistently elevated TCD velocities, MSD-HSCT was significantly associated with lower TCD velocities at 1 year compared with standard care. Further research is warranted to assess the effects of MSD-HSCT on clinical outcomes and over longer follow-up. Trial Registration: ClinicalTrials.gov Identifier: NCT01340404.

Long-term treatment follow-up of children with sickle cell disease monitored with abnormal transcranial Doppler velocities.

Stroke risk in sickle cell anemia (SCA), predicted by high transcranial Doppler (TCD) velocities, is prevented by transfusions. We present the long-term follow-up of SCA children from the Créteil newborn cohort (1992-2012) detected at risk by TCD and placed on chronic transfusions. Patients with normalized velocities and no stenosis were treated with hydroxyurea, known to decrease anemia and hemolytic rate. Trimestrial Doppler was performed and transfusions restarted immediately in the case of reversion to abnormal velocities. Patients with a genoidentical donor underwent transplant. Abnormal time-averaged maximum mean velocities (TAMMV) ≥200 cm/s were detected in 92 SCA children at a mean age of 3.7 years (range, 1.3-8.3 years). No stroke occurred posttransfusion after a mean follow-up of 6.1 years. Normalization of velocities (TAMMV < 170 cm/s) was observed in 83.5% of patients. Stenosis, present in 27.5% of patients, was associated with the risk of non-normalization (P< .001). Switch from transfusions to hydroxyurea was prescribed for 45 patients, with a mean follow-up of 3.4 years. Reversion, predicted by baseline reticulocyte count ≥400 × 10(9)/L (P< .001), occurred in 28.9% (13/45) patients at the mean age of 7.1 years (range, 4.3-9.5 years). Transplant, performed in 24 patients, allowed transfusions to be safely stopped in all patients and velocities to be normalized in 4 patients who still had abnormal velocities on transfusions. This long-term cohort study shows that transfusions can be stopped not only in transplanted patients but also in a subset of patients switched to hydroxyurea, provided trimestrial Doppler follow-up and immediate restart of transfusions in the case of reversion.