Molecular and phenotypic diversity of CBL-mutated juvenile myelomonocytic leukemia.

Mutations in the gene CBL were first identified in adults with various myeloid malignancies. Some patients with juvenile myelomonocytic leukemia (JMML) were also noted to harbor mutations in CBL, but were found to have generally less aggressive disease courses compared to other forms of Ras pathway-mutant JMML. Importantly, and in contrast to most reports in adults, the majority of CBL mutations in JMML patients are germline with acquired uniparental disomy occurring in affected marrow cells. Here, we systematically studied a large cohort of 33 JMML patients with CBL mutations and found this disease to be highly diverse in presentation and overall outcome. Moreover, we discovered somatically-acquired CBL mutations in 15% of pediatric patients who presented with more aggressive disease. Neither clinical features nor methylation profiling were able to distinguish somatic CBL patients from germline CBL patients, highlighting the need for germline testing. Overall, we demonstrate that disease courses are quite heterogeneous even among germline CBL patients. Prospective clinical trials are warranted to find ideal treatment strategies for this diverse cohort of patients.

Clinical utilization of blinatumomab and inotuzumab immunotherapy in children with relapsed or refractory B-acute lymphoblastic leukemia.

BACKGROUND: The treatment paradigm for patients with relapsed/refractory B-cell acute lymphoblastic leukemia (rrALL) has been revolutionized given recent clinical trials demonstrating remarkable success of immunotherapies and leading to drug approvals by United States and European agencies. We report experience with commercial blinatumomab and inotuzumab use at two North American pediatric oncology centers in children and adolescents/young adults with B-ALL. PROCEDURE: Patients 0-25 years old treated with the CD19 × CD3 bispecific T cell-engaging antibody blinatumomab and/or the CD22 antibody-drug conjugate inotuzumab from January 1, 2010, to June 1, 2018, were eligible. Disease status included relapsed B-ALL in second or greater relapse, primary chemotherapy-refractory B-ALL, or B-ALL complicated by severe infection precluding delivery of conventional chemotherapy. RESULTS: We identified 27 patients who received blinatumomab and/or inotuzumab outside of clinical trials during the study period. Four of the 13 patients (31%) with relapsed disease achieved minimal residual disease (MRD)-negative remission, and five patients (39%) underwent hematopoietic stem cell transplant (HSCT). In the 12 patients with primary chemorefractory B-ALL treated with immunotherapy, 11 (92%) achieved MRD-negative remission as assessed by flow cytometry; 10 patients (83%) underwent subsequent HSCT. Two patients with B-ALL in MRD-negative remission received blinatumomab due to severe infection and remained in remission after chemotherapy continuation. CONCLUSIONS: Blinatumomab and inotuzumab can induce deep remissions in patients with rrALL and facilitate subsequent HSCT or other cellular therapies. Blinatumomab can also serve as an effective bridging therapy during severe infection. The optimal timing, choice of immunotherapeutic agent(s), and duration of responses require further investigation via larger-scale clinical trials.

NUP98-NSD1 Driven MDS/MPN in Childhood Masquerading as JMML.

Overlapping myelodysplastic/myeloproliferative neoplasms (MDS/MPN) are clonal hematopoietic disorders with features of myelodysplasia and myeloproliferation. The only well-characterized MDS/MPN in children is juvenile myelomonocytic leukemia, an aggressive disorder of infants and toddlers. The biochemical hallmark of this disease is hyperactivation of the Ras/MAPK signaling pathway caused by mutations in Ras pathway genes in more than 90% of patients. Translocations involving receptor tyrosine kinases have been identified in rare cases. Here, we report a 2-year-old patient who presented with MDS/MPN driven by a cytogenetically cryptic NUP98-NSD1 fusion, a translocation thought to exclusively occur in patients with acute myeloid leukemia.

KBG syndrome patient due to 16q24.3 microdeletion presenting with a paratesticular rhabdoid tumor: Coincidence or cancer predisposition?

KBG syndrome is a rare autosomal dominant disorder caused by constitutive haploinsufficiency of the ankyrin repeat domain-containing protein 11 (ANKRD11) being the result of either loss-of-function gene variants or 16q24.3 microdeletions. The syndrome is characterized by a variable clinical phenotype comprising a distinct facial gestalt and variable neurological involvement. ANKRD11 is frequently affected by loss of heterozygosity in cancer. It influences the ligand-dependent transcriptional activation of nuclear receptors and tumor suppressive function of tumor protein TP53. ANKRD11 thus serves as a candidate tumor suppressor gene and it has been speculated that its haploinsufficiency may lead to an increased cancer risk in KBG syndrome patients. While no systematic data are available, we report here on the second KBG syndrome patient who developed a malignancy. At 17 years of age, the patient was diagnosed with a left-sided paratesticular extrarenal malignant rhabdoid tumor. Genetic investigations identified a somatic truncating gene variant in SMARCB1, which was not present in the germline, and a constitutional de novo 16q24.3 microdeletion leading to a loss of the entire ANKRD11 locus. Thus, KBG syndrome was diagnosed, which was in line with the clinical phenotype of the patient. At present, no specific measures for cancer surveillance can be recommended for KBG syndrome patients. However, a systematic follow-up and inclusion of KBG syndrome patients in registries (e.g., those currently established for cancer prone syndromes) will provide empiric data to support or deny an increased cancer risk in KBG syndrome in the future.

Serum with phospholipase A2 receptor autoantibodies interferes with podocyte adhesion to collagen.

BACKGROUND: The majority of sera from patients with primary membranous nephropathy have autoantibodies against the M-type phospholipase A2 receptor (PLA2R) which is expressed on human podocytes. The rabbit variant of PLA2R attaches to collagen type IV via the fibronectin type II domain, which is also present in the human variant of PLA2R. DESIGN: To assess whether the human PLA2R variant is also involved in attachment to collagen type IV, we conducted a cell adhesion assay on a collagen-coated surface using PLA2R-transfected and mock-transfected human embryonic kidney (HEK) cells. To test the hypothesis that sera from patients containing anti-PLA2R antibodies interfere with the adhesion of podocytes to collagen, we performed cell adhesion assays on a collagen type IV-coated surface using positive and negative serum samples from patients and cultured human podocytes in vitro expressing PLA2R. RESULTS: The HEK cell adhesion assay confirmed an enhanced attachment of PLA2R-transfected cells to collagen type IV. We confirmed diminished podocyte adhesion in the presence of serum with anti-PLA2R antibodies. The concentration of anti-PLA2R antibodies correlated with proteinuria and to the degree of diminished adhesion of podocytes. CONCLUSIONS: We demonstrated that serum of patients containing autoantibodies directed to PLA2R interferes with the ability of podocytes to attach to collagen type IV in vitro, providing evidence of a serum soluble pathogenic factor interfering with podocyte adhesion in membranous nephropathy.

Effective identification of cancer predisposition syndromes in children with cancer employing a questionnaire.

Approximately 10% of children with newly diagnosed cancer have a cancer predisposition syndrome (CPS). The optimal diagnostic approach to identify them among children diagnosed with cancer is unknown. OBJECTIVE: To determine whether the use of a one-page questionnaire can improve the CPS diagnosis among children with an oncologic condition. DESIGN: Comparative effectiveness research. SETTING: Referral center for children with cancer. RESULTS: 739 children diagnosed with an oncologic condition between 2012 and 2019. All children with a newly diagnosed oncologic condition presenting to Hannover Medical School between January 1st 2017 and December 31st 2019 were prospectively evaluated with a CPS questionnaire. Children in whom the questionnaire suggested the need of a genetic workup were further evaluated. All children diagnosed with an oncologic condition between January 1st 2012 and December 31st 2016 served as control. The CPS diagnoses established during both time periods were evaluated and compared. A CPS was diagnosed in 27 out of 287 children (9.4%) during the questionnaire period versus 24 out of 452 children (5.3%) during the control period (P = 0.032). CONCLUSION: The CPS questionnaire appears to significantly improve the diagnosis of children with CPS among children with a newly diagnosed oncologic condition.

Proportion of children with cancer that have an indication for genetic counseling and testing based on the cancer type irrespective of other features.

In children with cancer, specific clinical features such as physical anomalies, occurrence of cancer in young relatives, specific cancer histologies, and unique mutation/methylation signatures may indicate the presence of an underlying cancer predisposition syndrome (CPS). The proportion of children with a cancer type suggesting a CPS among all children with cancer is unknown. To determine the proportion of children with cancer types suggesting an underlying CPS among children with cancer. We evaluated the number of children with cancer types strongly associated with CPS diagnosed in Germany between 2007 and 2016. Data were obtained from various sources including two national pediatric pathology reference laboratories for brain and solid tumors, respectively, various childhood cancer trial offices as well as the German Childhood Cancer Registry. Among 21,127 children diagnosed with cancer between 2007 and 2016, 2554 (12.1%) had a cancer type strongly associated with a CPS. The most common diagnoses were myelodysplastic syndrome and juvenile myelomonocytic leukemia, retinoblastoma, malignant peripheral nerve sheath tumor, infantile myofibromatosis, medulloblastomaSHH, rhabdoid tumor as well as atypical teratoid/rhabdoid tumor. Based on cancer type only, 12.1% of all children with cancer have an indication for a genetic evaluation. Pediatric oncology patients require access to genetic counselling and testing.

Molecular characteristics and successful management of a respiratory syncytial virus outbreak among pediatric patients with hemato-oncological disease.

Background: Respiratory syncytial virus (RSV) is responsible for upper and lower respiratory tract infection in adults and children. Especially immunocompromised patients are at high risk for a severe course of infection, and mortality is increased. Moreover RSV can spread in healthcare settings and can cause outbreaks. Herein we demonstrate the successful control and characteristics of a RSV outbreak that included 8 patients in our Department of Pediatric Hematology and Oncology. Methods: We performed an epidemiologic investigation and a molecular analysis of the outbreak strains. Moreover we present the outbreak control bundle and our concept for RSV screening in the winter season. Results: RSV A and B strains caused the outbreak. RSV B strains affected 3 patients, 2 of whom were co-infected with RSV A. Exactly this RSV A strain was detected in another 5 patients. Our multimodal infection control bundle including prophylactic RSV screening was able to rapidly stop the outbreak. Conclusion: An infection control bundle in RSV outbreaks should address all potential transmission pathways. In pediatric settings the restriction of social activities might have a temporal negative impact on quality of life but helps to limit transmission opportunities. Molecular analysis allows better understanding of RSV outbreaks and, if done in a timely manner, might be helpful for guidance of infection control measures.

Antiphospholipase A2 receptor autoantibodies: a comparison of three different immunoassays for the diagnosis of idiopathic membranous nephropathy.

BACKGROUND: The recent identification of circulating autoantibodies directed towards the M-type phospholipase A2 receptor (PLA2R) has been a major advancement in the serological diagnosis of idiopathic membranous nephropathy (IMN), a common cause of nephrotic syndrome in adults. The goal of this study was to compare the performance characteristics of two commercial assays as well as the first addressable laser bead immunoassay (ALBIA) developed for the detection of anti-PLA2R antibodies. METHODS: Serum samples of 157 IMN patients and 142 controls were studied. Samples were tested by a cell based immunofluorescence assay (CBA-IFA, Euroimmun, Germany), by ELISA (Euroimmun), and by a novel ALBIA employing an in vivo expressed recombinant human PLA2R. RESULTS: Overall, the three assays showed significant qualitative and quantitative correlation. As revealed by receiver operating characteristic analysis, the ALBIA correlated better with the CBA-IFA than the ELISA (P = 0.0003). The clinical sensitivities/specificities for IMN were 60.0% (51.0-68.5%)/98.6% (95.0-99.8%) and 56.2% (47.2-64.8%)/100.0% (97.4-100.0%) for ALBIA and CBA-IFA, respectively. CONCLUSION: The ALBIA represents a promising assay for the detection of anti-PLA2R antibodies showing similar performance to the CBA-IFA and the advantage of ease of use and suitability for high throughput, rapid turnaround times, and multiplexing.

An anti-phospholipase A2 receptor quantitative immunoassay and epitope analysis in membranous nephropathy reveals different antigenic domains of the receptor.

The phospholipase A2 receptor (PLA2R) was recently discovered as a target autoantigen in patients with idiopathic membranous nephropathy (IMN). Published evidence suggests that the autoantibodies directed towards a conformation dependent epitope are currently effectively detected by a cell based assay (CBA) utilizing indirect immunofluorescence (IIF) on tissue culture cells transfected with the PLA2R cDNA. Limitations of such IIF-CBA assays include observer dependent subjective evaluation of semi-quantitative test results and the protocols are not amenable to high throughput diagnostic testing. We developed a quantitative, observer independent, high throughput capture immunoassay for detecting PLA2R autoantibodies on an addressable laser bead immunoassay (ALBIA) platform. Since reactive domains of PLA2R (i.e. epitopes) could be used to improve diagnostic tests by using small peptides in various high throughput diagnostic platforms, we identified PLA2R epitopes that bound autoantibodies of IMN patients. These studies confirmed that inter-molecular epitope spreading occurs in IMN but use of the cognate synthetic peptides in immunoassays was unable to conclusively distinguish between IMN patients and normal controls. However, combinations of these peptides were able to effectively absorb anti-PLA2R reactivity in IIF-CBA and an immunoassay that employed a lysate derived from HEK cells tranfected with and overexpressing PLA2R. While we provide evidence of intermolecular epitope spreading, our data indicates that in addition to conformational epitopes, human anti-PLA2R reactivity in a commercially available CBA and an addressable laser bead immunoassay is significantly absorbed by peptides representing epitopes of PLA2R.