Early diagnosis of primary immunodeficiency disorders (PID) is vital and allows directed treatment, especially in syndromes with severe or profound combined immunodeficiency. In PID patients with perinatal CMV or other opportunistic, invasive infections (e.g., Pneumocystis or Aspergillus), multi-organ morbidity may already arise within the first months of life, before hematopoietic stem cell transplantation (HSCT) or gene therapy can be undertaken, compromising the definitive treatment and outcome. Deficiency of Wiskott-Aldrich syndrome (WAS) protein-interacting protein (WIP deficiency) causes an autosomal recessive, WAS-like syndrome with early-onset combined immunodeficiency that has been described in three pedigrees to date. While WAS typically includes combined immunodeficiency, microthrombocytopenia, and eczema, the clinical and laboratory phenotypes of WIP-deficient patients–including lymphocyte subsets, platelets, lymphocyte proliferation in vitro, and IgE—varied widely and did not entirely recapitulate WAS, impeding early diagnosis in the reported patients. To elucidate the phenotype of WIP deficiency, we provide a comprehensive synopsis of clinical and laboratory features of all hitherto-described patients (n = 6) and WIP negative mice. Furthermore, we summarize the treatment modalities and outcomes of these patients and review in detail the course of one of them who was successfully treated with serial, unconditioned, maternal, HLA-identical donor lymphocyte infusions (DLI) against life-threatening, invasive CMV infection, followed by a TCRαβ/CD19-depleted, treosulfan/melphalan-conditioned, peripheral blood HSCT and repetitive, secondary-prophylactic, CMV-specific DLI with 1-year post-HSCT follow-up. This strategy could be useful in other patients with substantial premorbidity, considered “too bad to transplant,” who have an HLA-identical family donor, to eliminate infections and bridge until definitive treatment.

BackgroundDespite recent developments, the role of sirolimus in the heterogeneous spectrum of vascular anomalies is yet to be defined, in terms of indication, dosage, and therapy duration, recognizing both its potential and limitations.MethodsWe retrospectively analyzed 16 children with vascular anomalies treated with sirolimus in two pediatric centers between 2014 and 2020 [male: n = 7, the median age at diagnosis: 4.6 months (range, 0–281.4)]. In addition, repetitive volumetric analyses of the vascular anomalies were performed when possible (11 cases).ResultsTen patients were diagnosed with vascular malformations and 6 with vascular tumors. The mean therapy duration was 27.2 months (range, 3.5–65). The mean sirolimus level was 8.52 ng/ml (range, 5.38–12.88). All patients except one with central conducting lymphatic anomaly responded to sirolimus, with the most noticeable volume reduction in the first 4–6 months. Additional administration of vincristine was needed in five patients with kaposiform hemangioendothelioma and yielded a response, even in cases, refractory to sirolimus monotherapy. As a single agent, sirolimus led to impressive improvement in a patient with another vascular tumor—advanced epithelioid hemangioendothelioma. Complicated vascular malformations required long-term sirolimus therapy. Side effects of sirolimus included mucositis and laboratory abnormalities. No major infectious episodes were recorded. An infant with COVID-19, diagnosed while on sirolimus therapy, presented with a mild course.ConclusionIn the current series, we reported limitations of sirolimus as monotherapy, addressing the need to redefine its indications, and explore combination regimens and multimodal treatment strategies. Tools for objective evaluation of response trends over time could serve as a basis for the establishment of future therapeutic algorithms.

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We have identified a novel fusion gene in an 18-month old child with juvenile myelomonocytic leukemia (JMML) displaying a reciprocal chromosomal translocation t(5;7)(q33;p11.2). Molecular investigation at diagnosis revealed absence of mutations in KRAS, NRAS, PTPN11, or cCBL, but FISH analysis identified a rearrangement involving the PDGFRB gene located on chromosome 5q33. After temporary responses to imatinib (IM) and subsequently nilotinib (NIL) treatment, resistance associated with disease relapses was observed. Employment of the 5’-RACE technique facilitated identification of the PDGFRB fusion partner on chromosome 7p11.2, the NDEL1 gene encoding the nudE neurodevelopmental protein 1-like 1. The NDEL1 gene has not been implicated in any other reciprocal translocation to date, and it is conceivable that its ability to form dimers could drive permanent kinase activation of PDGFRβ. The chimeric mRNA contains the 5´exons 1-5 of NDEL1 fused in frame to the PDGFRB exons 10-22 containing the transmembrane and tyrosine kinase domains. To assess the oncogenicity of the fusion protein, Ba/F3 cells were transduced with the NDEL1-PDGFRB gene construct. The observation of IL3-independent growth confirmed the oncogenic potential of the novel fusion gene. The observed clinical resistance to IM and NIL prompted us to analyze the entire PDGFRB kinase domain for the presence of mutations by Sanger sequencing of overlapping amplicons. A point mutation in the activation (A) loop converting aspartate at the position 850 into glutamate (D850E) was detected in peripheral blood specimens from the time of first and second relapses, but not in the diagnostic sample. The crystal structure of the PDGFRβ TKD is not available, but protein modelling suggested that the mutation D850E destabilizes the inactive confirmation of the A-loop. This notion was in line with the observed clinical resistance to IM and NIL, but suggested sensitivity of the mutant to dasatinib (DAS). To test the predicted TKI responses, Ba/F3 cells transduced with wild type or mutant NDEL1-PDGFRB were tested in MTT assays against a panel of TKIs: Ba/F3-NDEL1-PDGFRBWT cells were sensitive to IM (IC50 = 60 nM), NIL (100 nM), DAS (5 nM), sorafenib (SOR; 20 nM), and ponatinib (PON; 10 nM), but insensitive to bosutinib (BOS; >2500 nM). Conversely, Ba/F3-NDEL1-PDGFRBD850E cells exhibited high resistance to IM (>2500), a 10-fold higher IC50 for NIL (1000 nM) and a 100-fold higher IC50 for SOR (2500 nM), but retained sensitivity to PON (15 nM) and DAS (15 nM). Mutations in the A-loop of different tyrosine kinases such as PDGFRα (D842V) or c-Kit (D816V) associated with resistance to IM have already been described in different tumor entities. However, the mutation D850E in the PDGFRβ TKD with apparent insensitivity to IM, NIL, and SOR revealed a completely different pattern of resistance than the same amino acid exchange at the corresponding site of PDGFRα (D842E). The latter mutation was previously shown to be sensitive to IM, NIL, and SOR with IC50 values of 4, 12.5, and 0.25 nM, respectively. This difference is intriguing because the exchange of a negatively charged amino acid, aspartate, to an amino acid with the same physical properties, glutamate, is not known to exert a major structural effect on the protein conformation, as observed for the D842E mutation in PDGFRα. We speculate that the great difference between the presence of the same amino acid exchange at corresponding positions in PDGFRα and PDGFRβ is the main interaction amino acid partner residue of aspartate at the position +3 which may influence the stability of the A-loop in its inactive conformation. In PDGFRα, it is histidine whose physical interaction with aspartate might not be affected by the change to glutamate. By contrast, the electrostatic bonds between arginine as the +3 residue in PDGFRβ might be greatly weakened by the elongation of the side chain in glutamate in comparison with aspartate, thus destabilizing the inactive conformation of the A-loop resulting in resistance to type II TKIs. To our knowledge, this is the first observation of an exchange between two negatively charged amino acids in a tyrosine kinase associated with a major change in responsiveness to TKI treatment. This finding is currently under further investigation, and may extend our understanding of structural interactions leading to TKI resistance. (Supported by the FWF SFB grant F4705-B20). Disclosures Valent: Novartis: Consultancy, Honoraria, Research Funding.