Molecular Heterogeneity in BRAF-Mutant Gliomas: Diagnostic, Prognostic, and Therapeutic Implications.

Over the last few decades, deciphering the alteration of molecular pathways in brain tumors has led to impressive changes in diagnostic refinement. Among the molecular abnormalities triggering and/or driving gliomas, alterations in the MAPK pathway reign supreme in the pediatric population, as it is encountered in almost all low-grade pediatric gliomas. Activating abnormalities in the MAPK pathway are also present in both pediatric and adult high-grade gliomas. Across those alterations, BRAF p.V600E mutations seem to define homogeneous groups of tumors in terms of prognosis. The recent development of small molecules inhibiting this pathway retains the attention of neurooncologists on BRAF-altered tumors, as conventional therapies showed no significant effect, nor prolonged efficiency on the high-grade or low-grade unresectable forms. Nevertheless, tumoral heterogeneity and especially molecular alteration(s) associated with MAPK-pathway abnormalities are not fully understood with respect to how they might lead to the specific dismal prognosis of those gliomas and/or affect their response to targeted therapies. This review is an attempt to provide comprehensive information regarding molecular alterations related to the aggressiveness modulation in BRAF-mutated gliomas and the current knowledge on how to use those targeted therapies in such situations.

Retrospective and integrative analyses of molecular characteristics and their specific imaging parameters in pediatric grade 1 gliomas.

BACKGROUND: Pediatric low-grade gliomas (PLGG) are the most common brain tumors diagnosed during childhood and represent a heterogeneous group associating variable molecular abnormalities. To go further and develop specific statistical patterns between tumor molecular background, imaging features, and patient outcome, a retrospective study was performed in a group of non-neurofibromatosis type 1 (non-NF1) grade 1 PLGGs. PATIENTS AND METHODS: Seventy-eight children, followed from 2004 to 2017, were retrospectively reported. In this population, we analyzed radiological and molecular parameters. Their therapeutic management comprised surgery or surgery plus chemotherapies. RESULTS: Considering all 78 patients, 59 had only a surgical removal and 19 patients were treated with postoperative chemotherapy. Twelve progressions were reported in the partially resected and chemotherapeutic groups, whereas four deaths occurred only in the highly treated patients. As expected, in the global cohort, PLGG with BRAF p.V600E and/or CDKN2A loss exhibited poor outcomes and we evidenced significant associations between those molecular characteristics and their imaging presentation. In the chemo-treated patients, when associating initial and 6-month magnetic resonance imaging (MRI) parameters to the molecular features, the good risk situations were significantly linked to the presence of a large tumor cyst at diagnosis and the appearance during treatment of a higher cystic proportion that we called cystic conversion. CONCLUSION: So, additionally to the presence of BRAF p.V600E or CDKN2A deletion in grade 1 PLGGs, the absence on diagnostic MRI of cystic parts and/or cystic conversion at 6-month chemotherapy were significantly linked to a worst prognosis and response to treatment. These imaging features should be considered as prognostic markers in future PLGG studies.

Unrelated cord blood transplantation in patients with idiopathic refractory severe aplastic anemia: a nationwide phase 2 study.

Outcomes remain poor for refractory severe aplastic anemia (SAA) patients. Alternative donor transplantation may be considered, but results from previous studies are not encouraging. We conducted a prospective nationwide phase 2 study to assess unrelated cord blood (CB) transplantation (CBT) efficacy and safety in refractory SAA patients (Aplastic Anemia and Cord Blood Transplantation protocol). To demonstrate a significant difference in 1-year survival from 20% (null hypothesis) to 50% (alternative hypothesis), we needed to include 25 transplanted patients and therefore included 26 (median age, 16 years). Eligibility criteria required 1 or 2 unrelated CB units, containing separately or together >4 × 107 frozen nucleated cells (NCs) per kilogram of recipient body weight. Conditioning regimen comprised fludarabine (FLU), cyclophosphamide (CY), antithymocyte globulin (ATG), and 2-Gy total body irradiation (TBI). With a median follow-up of 38.8 months, engraftment occurred in 23 patients (88%); cumulative incidences of grade II-IV acute and chronic graft-versus-host disease were 45.8% and 36%, respectively. Twenty-three patients were alive at 1 year, with an 88.5% overall survival (OS) rate, differing significantly from the expected 20% (P < .0001; 84% OS at 2 years). CBT with units containing ≥4 × 107 frozen NCs per kilogram is therefore a valuable curative option for young adults with refractory SAA and no available matched unrelated donors. This trial was registered at www.clinicaltrials.gov as #NCT01343953.

Curative or pre-emptive adenovirus-specific T cell transfer from matched unrelated or third party haploidentical donors after HSCT, including UCB transplantations: a successful phase I/II multicenter clinical trial.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (HSCT), the most widely used potentially curable cellular immunotherapeutic approach in the treatment of hematological malignancies, is limited by life-threatening complications: graft versus host disease (GVHD) and infections especially viral infections refractory to antiviral drugs. Adoptive transfer of virus-specific T cells is becoming an alternative treatment for infections following HSCT. We report here the results of a phase I/II multicenter study which includes a series of adenovirus-specific T cell (ADV-VST) infusion either from the HSCT donor or from a third party haploidentical donor for patients transplanted with umbilical cord blood (UCB). METHODS: Fourteen patients were eligible and 11 patients received infusions of ADV-VST generated by interferon (IFN)-γ-based immunomagnetic isolation from a leukapheresis from their original donor (42.9%) or a third party haploidentical donor (57.1%). One patient resolved ADV infection before infusion, and ADV-VST could not reach release or infusion criteria for two patients. Two patients received cellular immunotherapy alone without antiviral drugs as a pre-emptive treatment. RESULTS: One patient with adenovirus infection and ten with adenovirus disease were infused with ADV-VST (mean 5.83 ± 8.23 × 103 CD3+IFN-γ+ cells/kg) up to 9 months after transplantation. The 11 patients showed in vivo expansion of specific T cells up to 60 days post-infusion, associated with adenovirus load clearance in ten of the patients (91%). Neither de novo GVHD nor side effects were observed during the first month post-infusion, but GVHD reactivations occurred in three patients, irrespective of the type of leukapheresis donor. For two of these patients, GVHD reactivation was controlled by immunosuppressive treatment. Four patients died during follow-up, one due to refractory ADV disease. CONCLUSIONS: Adoptive transfer of rapidly isolated ADV-VST is an effective therapeutic option for achieving in vivo expansion of specific T cells and clearance of viral load, even as a pre-emptive treatment. Our study highlights that third party haploidentical donors are of great interest for ADV-VST generation in the context of UCB transplantation. (N° Clinical trial.gov: NCT02851576, retrospectively registered).

Follow-up of post-transplant minimal residual disease and chimerism in childhood lymphoblastic leukaemia: 90 d to react.

Relapse after transplantation is a major cause of treatment failure in paediatric acute lymphoblastic leukaemia (ALL). Here, we report the findings of a prospective national study designed to investigate the feasibility of immune intervention in children in first or subsequent remission following myeloablative conditioning. This study included 133 children who received a transplant for ALL between 2005 and 2008. Minimal Residual Disease (MRD) based on T cell receptor/immunoglobulin gene rearrangements was measured on days -30, 30, 90 and 150 post-transplantation. Ciclosporin treatment was rapidly discontinued and donor lymphocyte infusions (DLI) were programmed for patients with a pre- or post-transplant MRD status ≥10(-3) . Only nine patients received DLI. Pre- and post-transplant MRD status, and the duration of ciclosporin were independently associated with 5-year overall survival (OS), which was 62·07% for the whole cohort. OS was substantially higher in patients cleared of MRD than in those with persistent MRD (52·3% vs. 14·3%, respectively). Only pre-transplant MRD status (Hazard Ratio 2·57, P = 0·04) and duration of ciclosporin treatment (P < 0·001) were independently associated with relapse. The kinetics of chimerism were not useful for predicting relapse, whereas MRD monitoring up to 90 d post-transplantation was a valuable prognostic tool to guide therapeutic intervention.

Allogeneic hematopoietic stem cell transplantation for T-prolymphocytic leukemia: a report from the French society for stem cell transplantation (SFGM-TC).

T-prolymphocytic leukemia (T-PLL), a rare aggressive mature T-cell disorder, remains frequently resistant to conventional chemotherapy. Studies have suggested that allogeneic hematopoietic stem cell transplantation (HSCT) might possibly serve to consolidate the response to initial chemotherapy. The current report summarizes the outcome of 27 T-PLL cases identified in the registry in French Society for stem cell transplantation (SFGM-TC). Prior to HSCT, 14 patients were in complete remission (CR), 10 in partial response, three refractory, or in progression. Following HSCT, 21 patients achieved CR as best response. With a median follow-up for surviving patients of 33 (range, 6-103) months, 10 patients are still alive in continuous CR. Overall survival and progression-free survival estimates at 3 yr were 36% (95% CI: 17-54%) and 26% (95% CI: 14-45%), respectively. The relapse incidence after HSCT was 47% occurring at a median of 11.7 (range, 2-24) months. Overall cumulative incidence of transplant-related mortality was 31% at 3 yr. These results suggest that HSCT may allow long-term survival in patients with T-PLL following induction treatment; however, it is associated with a significant rate of toxicity.

Combination of Epstein-Barr virus nuclear antigen 1, 3 and lytic antigen BZLF1 peptide pools allows fast and efficient stimulation of Epstein-Barr virus-specific T cells for adoptive immunotherapy.

BACKGROUND: Epstein-Barr virus (EBV) infection is a major cause of morbidity following hematopoietic stem cell transplantation. EBV-infected B cells may not respond to rituximab treatment and may lead to a life-threatening post-transplantation lymphoproliferative disorder. Adoptive cellular immunotherapy using EBV-lymphoblastoid cell lines (LCL) as stimulating antigen has proved effective in restoring specific immunity. However, EBV presents several immunodominant antigens, and developing a swift and effective clinical-grade immunotherapy relies on the definition of a Good Manufacturing Practices (GMP) universal stimulating antigen. METHODS: Peripheral blood mononuclear cells (PBMCs) from six donors with a cellular immune response against EBV were immunoselected after stimulation with a new EBV antigen associated with an EBNA3 peptide pool. RESULTS: After immunoselection, a mean of 0.53 ± 0.25 × 106 cells was recovered consisting of a mean of 24.77 ± 18.01% CD4⁺-secreting interferon (IFN)-γ and 51.42 ± 26.92% CD8⁺-secreting IFN-γ. The T memory stem cell sub-population was identified. EBV-specific T cells were expanded in vitro, and their ability to secrete IFN-γ and to proliferate after re-stimulation with EBV antigen was confirmed. A specific lysis was observed against autologous target cells pulsed with EBV peptide pools (57.6 ± 11.5%) and against autologous EBV-LCL (18.3 ± 7.3%). A mean decrease of 94.7 ± 3.3% in alloreactivity against third-party donor mononuclear cells with EBV-specific T cells was observed compared with PBMCs before selection. CONCLUSIONS: Our results show that a combination of peptide pools including EBNA3 is needed to generate EBV-specific T cells with good specific cytotoxicity and devoid of alloreactivity, but as yet GMP grade is not fully achieved.

Human herpesvirus 6 infection after allogeneic stem cell transplantation: incidence, outcome, and factors associated with HHV-6 reactivation.

BACKGROUND: Human herpesvirus 6 (HHV-6) is susceptible to latency and reactivation in hematopoietic stem cell transplant (HSCT) recipients. We investigated the incidence of HHV-6 DNAemia and factors related to HHV-6 DNAemia and death after allogeneic stem cell transplantations. We also explored the relationship between HHV-6 viral load and the presence of clinical signs. METHODS: Data concerning age, sex, transplantation conditions, graft-versus-host disease (GVHD), treatments, clinical signs, outcome, HHV-6, and other infections were collected for a historical cohort of 390 HSCT performed between 1999 and 2008 in the Transplant Unit of Nancy University Hospital Center. Univariate analysis was used to evaluate influences between the different parameters. RESULTS: The study included 220 of the 390 allogeneic HSCTs. For the analyzed period, 44 patients (n=44/220, 20%) presented HHV-6 DNAemia in whole blood, including three integrated forms. Fifteen percent (7/41) of HHV-6-positive patients presented clinical signs not related to higher viral load (P=0.164). The factors associated with HHV-6 DNAemia were as follows: cord blood transplantation (P<0.001), conditioning regimen (P=0.030), acute GVHD (P=0.003), and the type of prophylactic treatment for GVHD (P=0.001). HHV-6 DNAemia was not significantly associated with cytomegalovirus infection (P=0.937). HHV-6 DNAemia was not associated with death (P=0.151). CONCLUSIONS: HHV-6 DNAemia was not so frequent after allogeneic transplantation. Factors associated with HHV-6 DNAemia were similar to those for other infections. No abnormally high death rate was observed in the HHV-6 positive population. The presence of clinical signs did not appear to be statistically related to HHV-6 viral load.

Clinical, functional and genetic analysis of twenty-four patients with chronic granulomatous disease - identification of eight novel mutations in CYBB and NCF2 genes.

Chronic granulomatous disease is an inherited disorder in which phagocytes lack a functional NADPH oxidase and cannot produce superoxide anions. The most common form is caused by mutations in CYBB encoding gp91phox. We investigated 24 CGD patients and their families. Twenty-one mutations in CYBB were classified as X91(0), X91(+) or X91(-) variants according to cytochrome b (558) expression. Point mutations in encoding regions represented 50 % of the mutations found in CYBB, splice site mutations 27 %, deletions and insertions 23 %. Eight mutations in CYBB were novel leading to X91(0)CGD cases. Two of these were point mutations: c493G>T and a double mutation c625C>G in exon 6 and c1510C>T in exon 12 leading to a premature stop codon at Gly165 in gp91phox and missense mutations His209Arg/Thr503Ile respectively. Two novel splice mutations in 5'intronic regions of introns 1 and 6 were found. A novel deletion/insertion c1024_1026delCTG/insT results in a frameshift introducing a stop codon at position 346 in gp91phox. The last novel mutation was the insertion of a T at c1373 leading to a frameshift and a premature stop codon at position 484 in gp91phox. For the first time the precise size of two large mutations in CYBB was determined by array-comparative genomic hybridization and carriers' status were evaluated by multiplex ligation-dependent probe amplification assay. No clear correlation between clinical severity and CYBB mutations could be established. Of three mutations in CYBA, NCF1 and NCF2 leading to rare autosomal recessive CGD, one nonsense mutation c29G>A in exon 1 of NCF2 was new.

Protective effect of IgM against colonization of the respiratory tract by nontypeable Haemophilus influenzae in patients with hypogammaglobulinemia.

BACKGROUND: Primary immunoglobulin deficiencies lead to recurrent bacterial infections of the respiratory tract and bronchiectasis, even with adequate immunoglobulin replacement therapy. It is not known whether patients able to secrete IgM (eg, those with hyper-IgM [HIgM] syndrome) are as susceptible to these infections as patients who lack IgM production (eg, those with panhypogammaglobulinemia [PHG]). OBJECTIVE: This study is aimed at identifying specific microbiological and clinical (infections) characteristics that distinguish immunoglobulin-substituted patients with PHG from patients with HIgM syndrome. METHODS: A cohort of patients with HIgM syndrome (n = 25) and a cohort of patients with PHG (n = 86) were monitored prospectively for 2 years while receiving similar polyvalent immunoglobulin replacement therapies. Regular bacterial analyses of nasal swabs and sputum were performed, and clinical events were recorded. In parallel, serum and saliva IgM antibody concentrations were measured. RESULTS: When compared with patients with PHG, patients with HIgM syndrome were found to have a significantly lower risk of nontypeable Haemophilus influenzae carriage in particular (relative risk, 0.39; 95% CI, 0.21-0.63). Moreover, patients with HIgM syndrome (including those unable to generate somatic hypermutations of immunoglobulin genes) displayed anti-nontypeable H influenzae IgM antibodies in their serum and saliva. Also, patients with HIgM syndrome had a lower incidence of acute respiratory tract infections. CONCLUSIONS: IgM antibodies appear to be microbiologically and clinically protective and might thus attenuate the infectious consequences of a lack of production of other immunoglobulin isotypes in patients with HIgM syndrome. Polyvalent IgG replacement therapy might not fully compensate for IgM deficiency. It might thus be worth adapting long-term antimicrobial prophylactic regimens according to the underlying B-cell immunodeficiency phenotype.

The clinical value of concomitant Epstein Barr virus (EBV)-DNA load and specific immune reconstitution monitoring after allogeneic hematopoietic stem cell transplantation.

BACKGROUND: Monitoring of EBV DNAemia after allogeneic hematopoietic stem cell transplantation (HSCT) is necessary, but not sufficient, to identify patients at risk of EBV-induced post-transplantation lymphoproliferative disorders (PTLD). Combining this with quantifying EBV-specific cellular immunity was shown to be helpful. In this study, we evaluated the value of IFNγ-Elispot assay in monitoring EBV DNAemia after HSCT. METHODS: EBV-DNA load in whole blood was monitored at least weekly using real-time PCR in 40 recipients of HSCT. Quantitative and qualitative T-cell recoveries, including EBV-specific T-cell quantification by Elispot assay, were studied 60, 100, 180 and 360 days after HSCT. RESULTS: Among the 35 evaluable patients, 14 (35%) presented EBV DNAemia, only 2/14 (14%) needing pre-emptive treatment with rituximab. The greatest risk factor for EBV DNAemia was the presence of anti-thymocyte globulin (ATG) (p=0.005). EBV-specific cellular immune recovery was monitored by IFNγ-Elispot assay. Using multivariate analysis, four factors were found to significantly influence IFNγ-Elispot results at defined times post-HSCT: EBV DNAemia, young age, global T-cell recovery and severe acute GVHD. In those cases where EBV DNAemia occurred and cleared spontaneously, Elispot results gave more than 1000 spot-forming cells (SFC)/10(6)PBMC. CONCLUSION: Elispot assay may be usefully combined with EBV-DNA load monitoring to determine when a patient should receive pre-emptive treatment, or when the clinician should avoid Rituximab use which severely immunocompromises patients.

Emergence of disseminated infections due to Geosmithia argillacea in patients with chronic granulomatous disease receiving long-term azole antifungal prophylaxis.

We report two cases of invasive infections due to Geosmithia argillacea, an emerging mold, in patients with chronic granulomatous disease receiving prolonged azole antifungal prophylaxis. One patient died despite receiving a combination of four antifungals, and the other developed cerebral and medullary lesions under a combination of caspofungin, posaconazole, terbinafine, and gamma interferon.

Comparison of in-house real-time quantitative PCR to the Adenovirus R-Gene kit for determination of adenovirus load in clinical samples.

In the context of hematopoietic stem cell transplantation, adenovirus infections are associated with relevant mortality and morbidity. Detection of adenovirus DNA by quantitative PCR is the "gold standard" for these patients. A total of 150 samples, namely, 78 whole-blood, 22 cerebrospinal fluid, 24 digestive biopsy, and 26 stool samples, from 29 patients, including 24 hematopoietic stem cell transplant recipients, were tested for the detection of adenovirus using an in-house real-time quantitative PCR assay (A. Heim, C. Ebnet, G. Harste, and P. Pring-Akerblom, J. Med. Virol. 70:228-239, 2003) and the commercially available Adenovirus R-Gene kit. Adenovirus DNA was automatically isolated from whole-blood samples (Magna Pure LC system; Roche) or was manually extracted from other specimens (QIAamp; Qiagen) using the appropriate kit. The intra- and interassay reproducibilities and sensitivities were evaluated with cell culture supernatant dilutions. Of the 150 samples tested, 86 were found to be positive and 55 were found to be negative using both techniques. Nine (6%) discordant results were obtained. In most cases, discrepant results concerned samples with low viral loads. Quantitative results for all concordant positive samples were analyzed using the Spearman correlation test. A good correlation between the results of the in-house assay and those of the kit assay was obtained (r = 0.95; P < 0.001). Regarding the threshold cycle value for internal control spiked samples, none of the 150 samples tested contained a PCR inhibitor. In conclusion, a relevant correlation of results between the in-house assay and the kit assay, as well as the high-quality reproducibility and sensitivity of the kit assay, warranted its use for follow-up of hematopoietic stem cell transplantation recipients.

Rapid generation of full clinical-grade human antiadenovirus cytotoxic T cells for adoptive immunotherapy.

Adenovirus (ADV) infections are one of the major causes of morbidity and mortality after hematopoietic stem cell transplantation, despite new antiviral treatment strategies. We describe here a complete clinical-grade generation of human anti-ADV cytotoxic T cells to propose an adoptive immunotherapy. Peripheral blood mononuclear cells (PBMC) from 7 healthy donors, known for their good cellular immunity against ADV, were stimulated for 6 hours with a synthetic peptide pool covering the ADV5 Hexon protein interferon-gamma (IFN-gamma) secreting cells were isolated on a clinical device. After immunoselection, a mean number of 1.01 +/- 0.84 x 10(6) total nucleated cells was obtained. The isolated ADV-specific T cells were mainly CD4+ (mean=56% +/- 20.8%, yield=51% +/- 32.4%) but also CD8+ (mean=42% +/- 27%, yield = 56% +/- 39.3%). Isolated T lymphocytes (CTL) were expanded to carry out functional tests. Ability of the expanded CTL to secrete IFN-gamma and to proliferate after restimulation with the ADV peptide pool was confirmed. A high cytotoxicity against autologous target cells loaded with ADV antigens was observed but not against nonloaded target cells. We observed a decrease of 1.27 log of the allogeneic reaction against non HLA identical healthy donor PBMC with CTL compared with the PBMC before selection. Clinical-grade generation of ADV-specific T cells was achieved with a synthetic antigen. This technology has the advantage of being fast, and is sufficiently reactive to be proposed for immunotherapy if antiviral treatment fails.

Contribution of human herpesvirus 6 (HHV-6) viral load in whole blood and serum to investigate integrated HHV-6 transmission after haematopoietic stem cell transplantation.

BACKGROUND: Human herpesvirus 6 (HHV-6) is susceptible to latency and recurrence. A less-frequent form of HHV-6 persistence is the integration of viral DNA into host chromosomes. OBJECTIVES: To investigate HHV-6 viral load after haematopoietic stem cell transplantation (HSCT) in whole blood (WB) and serum with regard to integrated HHV-6 transmission diagnosis. STUDY DESIGN: HHV-6 DNA quantitation in serum and WB was performed using quantitative polymerase chain reaction for the follow-up of a 16-year-old girl after HSCT. In whole blood, results were expressed as HHV-6 genomic equivalent copies (gec) per milliliter of WB or per million cells. RESULTS: HHV-6 viral load (undetectable before HSCT) increased up to 3.05 x 10(7)gec/10(6)cells. HHV-6 viral load in the donor sample (3.44 x 10(6)gec/10(6)cells) was in favor of viral transmission through HSCT. The correlation between viral load in WB and serum was significant (p=0.0005). Viral load results expressed as gec/10(6)cells in WB was more reliable than results expressed as gec/ml of whole blood. CONCLUSION: These findings indicate that HHV-6 may be transmitted during HSCT as integrated virus contained in the graft. This reiterates that in the setting of HSCT, HHV-6 viral load must be correctly interpreted. Using HHV-6 viral load expressed as gec/10(6) cells may be more suitable for the follow-up of patients with integrated HHV-6.

Immune constitution monitoring after PBMC transplantation in complete DiGeorge syndrome: an eight-year follow-up.

A young boy with a confirmed complete DiGeorge Syndrome (cDGS) underwent a peripheral blood mononuclear cell transplantation (PBMCT) from his HLA-identical sister at 4.5 years of age, without a conditioning regimen. Eight years later, he is healthy with good immunological functions in the presence of a stable mixed T-cell chimerism. Absence of recent thymic emigrants is confirmed. We observe an inverted CD4+/CD8+ ratio, related to the CD8 subset expansion, a skewing of the TCR repertoire, especially on the CD8+ subset and a telomere loss on the CD8+ cells compared to the donor. However, these anomalies do not seem to have an impact on functional immunity. PBMCT in cDGS using an HLA-matched sibling donor provides good long-lasting immunity and is an easy alternative to bone marrow transplantation and to thymic transplantation.

Daclizumab, an efficient treatment for steroid-refractory acute graft-versus-host disease.

In a phase II study, daclizumab was given as single second-line agent to 62 patients with steroid-refractory acute graft-versus-host disease (aGVHD). Complete resolution of aGVHD was achieved in 68.8% of patients. This response rate was significantly associated with a lower number of involved organs and smaller extent of skin involvement. The 4-year event-free survival (EFS) was 54.6%. Grade > or =III aGVHD, > or =2 involved organs at baseline and patient age >18 years were independently associated with lower EFS. Daclizumab could be a suitable alternative treatment for aGVHD, particularly when limited to the skin or gastrointestinal tract.