B-cell acute lymphoblastic leukemia (B-ALL) is the commonest childhood cancer and the prognosis of children with relapsed or therapy refractory disease remains a challenge. Treatment with chimeric ...antigen receptor-modified T cells targeting the CD19 antigen (CART-19 therapy) has been presented as a promising approach toward improving the outcome of relapsed or refractory disease. However, 10%-20% of the patients suffer another relapse. Epitope-loss under therapy pressure has been suggested as a mechanism of tumor cells to escape the recognition from CART-19 therapy. In this work, we analyzed the expression of CD19 isoforms in a cohort of 14 children with CD19 B-ALL and 6 nonleukemia donors. We showed that an alternatively spliced CD19 mRNA isoform lacking exon 2, and therefore the CART-19 epitope, but not isoforms lacking the transmembrane and cytosolic domains are expressed in leukemic blasts at diagnosis in children and in the bone marrow of nonleukemia donors. Furthermore, we clarified the sequence of a further isoform lacking the epitope recognized by CART-19 therapy and disclosed the presence of new isoforms. In comparison with the children, we showed that alternatively spliced CD19 mRNA isoforms affecting exon 2 are also expressed in 6 adult patients with CD19 B-ALL. On top of that, one of the adults expressed an isoform lacking the CD19 transmembrane and cytosolic domains. In conclusion, we proved that some of the CD19 isoforms contributing to CART-19 escape already preexist at diagnosis and could evolve as a dominant clone during CART-19 therapy suggesting the application of combined treatment approaches.
H3K27M mutant (mut) diffuse midline glioma (DMG) is a lethal cancer with no effective cure. The glycosphingolipids (GSL) metabolism is altered in these tumors and could be exploited to develop new ...therapies. We tested the effect of the glucosylceramide synthase inhibitors (GSI) miglustat and eliglustat on cell proliferation, alone or in combination with temozolomide or ionizing radiation. Miglustat was included in the therapy protocol of two pediatric patients. The effect of H3.3K27 trimethylation on GSL composition was analyzed in ependymoma. GSI reduced the expression of the ganglioside GD2 in a concentration and time-dependent manner and increased the expression of ceramide, ceramide 1-phosphate, sphingosine, and sphingomyelin but not of sphingosine 1-phosphate. Miglustat significantly increased the efficacy of irradiation. Treatment with miglustat according to dose recommendations for patients with Niemann-Pick disease was well tolerated with manageable toxicities. One patient showed a mixed response. In ependymoma, a high concentration of GD2 was found only in the presence of the loss of H3.3K27 trimethylation. In conclusion, treatment with miglustat and, in general, targeting GSL metabolism may offer a new therapeutic opportunity and can be administered in close proximity to radiation therapy. Alterations in H3K27 could be useful to identify patients with a deregulated GSL metabolism.
(1)
The high-grade neuroepithelial tumor of the central nervous system with BCOR alteration (HGNET-BCOR) is a highly malignant tumor. Preclinical models and molecular targets are urgently required ...for this cancer. Previous data suggest a potential role of insulin-like growth factor (IGF) signaling in HGNET-BCOR. (2)
The primary HGNET-BCOR cells PhKh1 were characterized by western blot, copy number variation, and methylation analysis and by electron microscopy. The expression of
and
was assessed by qRT-PCR. The effect of chemotherapeutics and IGF1R inhibitors on PhKh1 proliferation was tested. The phosphorylation of IGF1R and downstream molecules was assessed by western blot. (3)
Phkh1 cells showed a DNA methylation profile compatible with the DNA methylation class "HGNET-BCOR" and morphologic features of cellular cannibalism.
and
were highly expressed by three HGNET-BCOR tumor samples and PhKh1 cells. PhKh1 cells were particularly sensitive to vincristine, vinblastine, actinomycin D (IC
< 10 nM for all drugs), and ceritinib (IC
= 310 nM). Ceritinib was able to abrogate the proliferation of PhKh1 cells and blocked the phosphorylation of IGF1R and AKT. (4)
IGF1R is as an attractive target for the development of new therapy protocols for HGNET-BCOR patients, which may include ceritinib and vinblastine.
Despite massive improvements in the treatment of B-ALL through CART-19 immunotherapy, a large number of patients suffer a relapse due to loss of the targeted epitope. Mutations in the CD19 locus and ...aberrant splicing events are known to account for the absence of surface antigen. However, early molecular determinants suggesting therapy resistance as well as the time point when first signs of epitope loss appear to be detectable are not enlightened so far. By deep sequencing of the CD19 locus, we identified a blast-specific 2-nucleotide deletion in intron 2 that exists in 35% of B-ALL samples at initial diagnosis. This deletion overlaps with the binding site of RNA binding proteins (RBPs) including PTBP1 and might thereby affect CD19 splicing. Moreover, we could identify a number of other RBPs that are predicted to bind to the CD19 locus being deregulated in leukemic blasts, including NONO. Their expression is highly heterogeneous across B-ALL molecular subtypes as shown by analyzing 706 B-ALL samples accessed via the St. Jude Cloud. Mechanistically, we show that downregulation of PTBP1, but not of NONO, in 697 cells reduces CD19 total protein by increasing intron 2 retention. Isoform analysis in patient samples revealed that blasts, at diagnosis, express increased amounts of CD19 intron 2 retention compared to normal B cells. Our data suggest that loss of RBP functionality by mutations altering their binding motifs or by deregulated expression might harbor the potential for the disease-associated accumulation of therapy-resistant CD19 isoforms.
The insulin-like growth factor (IGF) pathway plays an important role in several brain tumor entities. However, the lack of inhibitors crossing the blood-brain barrier remains a significant obstacle ...for clinical translation. Here, we targeted the IGF pathway using ceritinib, an off-target inhibitor of the IGF1 receptor (IGF1R) and insulin receptor (INSR), in a pediatric patient with an unclassified brain tumor and a
(
) germline mutation. Pathway analysis of the tumor revealed activation of the sonic hedgehog (SHH), the wingless and integrated-1 (WNT), the IGF, and the Notch pathway. The proliferation of the patient tumor cells (225ZL) was inhibited by arsenic trioxide (ATO), which is an inhibitor of the SHH pathway, by linsitinib, which is an inhibitor of IGF1R and INSR, and by ceritinib. 225ZL expressed INSR but not IGF1R at the protein level, and ceritinib blocked the phosphorylation of INSR. Our first personalized treatment included ATO, but because of side effects, we switched to ceritinib. After 46 days, we achieved a concentration of 1.70 µM of ceritinib in the plasma, and after 58 days, MRI confirmed that there was a response to the treatment. Ceritinib accumulated in the tumor at a concentration of 2.72 µM. Our data suggest ceritinib as a promising drug for the treatment of IGF-driven brain tumors.
Osteosarcoma (OS) is the second most common cause of cancer-related death in pediatric patients. The insulin-like growth factor (IGF) pathway plays a relevant role in the biology of OS but no IGF ...targeted therapies have been successful as monotherapy so far. Here, we tested the effect of three IGF specific inhibitors and tested ceritinib as an off-target inhibitor, alone or in combination with dasatinib, on the proliferation of seven primary OS cells. Picropodophyllin, particularly in combination with dasatinib and the combination ceritinib/dasatinib were effective in abrogating the proliferation. The ceritinib/dasatinib combination was applied to the primary cells of a 16-year-old girl with a long history of lung metastases, and was more effective than cabozantinib and olaparib. Therefore, the combination was used to treat the patient. The treatment was well tolerated, with toxicity limited to skin rush and diarrhea. A histopathological evaluation of the tumor after three months of therapy indicated regions of high necrosis and extensive infiltration of macrophages. The extension of the necrosis was proportional to the concentration of dasatinib and ceritinib in the area, as analysed by an ultra performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS). After the cessation of the therapy, radiological analysis indicated a massive growth of the patient's liver metastases. In conclusion, these data indicate that the combination of ceritinib/dasatinib is safe and may be used to develop new therapy protocols.
Medulloblastoma is the most common malignant brain tumor in children. Immunotherapy is yet to demonstrate dramatic results in medulloblastoma, one reason being the low rate of mutations creating new ...antigens in this entity. In tumors with low mutational burden, gene fusions may represent a source of tumor-specific neoantigens. Here, we reviewed the landscape of fusions in medulloblastoma and analyzed their predicted immunogenicity. Furthermore, we described a new in-frame fusion protein identified by RNA-Seq. The fusion involved two genes on chromosome 2 coding for the enhancer of polycomb homolog 2 (EPC2) and GULP PTB domain containing engulfment adaptor 1 (GULP1) respectively. By qRT-PCR analysis, the fusion was detected in 3 out of 11 medulloblastoma samples, whereby 2 samples were from the same patients obtained at 2 different time points (initial diagnosis and relapse), but not in other pediatric brain tumor entities. Cloning of the full-length sequence indicated that the fusion protein contains the N-terminal enhancer of polycomb-like domain A (EPcA) of EPC2 and the coiled-coil domain of GULP1. In silico analyses predicted binding of the neoantigen-derived peptide to HLA-A*0201. A total of 50% of the fusions described in the literature were also predicted to produce an immunogenic peptide. The EPC2-GULP1 fusion peptide was able to induce a de novo T cell response characterized by interferon gamma release of CD8+ cytotoxic T cells in vitro. While the functional relevance of this fusion in medulloblastoma biology remains to be clarified, our data support an immunotherapeutic approach for pediatric medulloblastoma patients carrying the EPC2-GULP1 fusion and other immunogenic fusions.
Background
Considering the improved outcome, a better understanding of the late effects in Wilms tumor survivors (WT-S) is needed. This study was aimed at evaluating renal function and determining ...the prevalence of clinical and subclinical renal dysfunction in a cohort of WT-S using a multimodal diagnostic approach.
Methods
Thirty-seven WT-S were included in this prospective cross-sectional single center study. To evaluate renal function, glomerular filtration rate (GFR) and urinary protein excretion were assessed. Additionally, kidney sonomorphology and blood pressure were analyzed.
Results
All examined WT-S (mean age 28.7 years, mean follow-up 24.8 years) had been treated with a combination of surgery and chemotherapy; 59.5% had received adjuvant radiotherapy. Impaired glomerular renal function was detected in a considerable proportion of WT-S, with age-adjusted cystatin-based GFR estimation below age norm in 55.9%. A lower cystatin-based estimated GFR (eGFR) correlated with longer follow-up time and higher irradiation dose. In 5 patients (13.5%) albuminuria was identified. Analysis of sonomorphology detected compensatory contralateral renal hypertrophy in 83.3% of WT-S. Chronic kidney disease (CKD) ≥ stage II was present in 55.9% of WT-S. Blood pressure measurements revealed arterial hypertension in 15 (40.5%) WT-S (newly diagnosed
n
=10). In 24.3% both CKD ≥ stage II and arterial hypertension were determined.
Conclusion
Even though WT-S are believed to carry a low risk for end-stage renal disease, in this study, a remarkable number of WT-S presented with previously unidentified subclinical signs of renal function impairment and secondary morbidity. Therefore, it is important to continue regular follow-up, especially after transition into adulthood.
Hemophagocytic Lymphohistiocytosis (HLH) is characterized by pathologic immune activation which occurs either as a familial disorder or as an acquired condition. The diagnosis of HLH requires the ...presence of five out of nine criteria: fever, splenomegaly, pancytopenia, hypertriglyceridemia, hypofibrinogenemia, hemophagocytosis in bone marrow, hyperferritinemia, low or absent natural killer cell activity and high level of soluble interleukin-2 receptor.
Here we present a 6-month-old girl with parents from Southern Italy. She suffered from hepatosplenomegaly and a recurrent high fever for 3 months' duration. On admission, she showed neurological symptoms including irritability and neck stiffness. Blood analysis revealed bicytopenia, namely anemia and thrombocytopenia. The first bone marrow aspirate was indicative for neither malignancies nor HLH. At this time, additional investigations indicated macrophage activation syndrome with elevated ferritin value (11.741ng/ml), soluble interleukin-2 receptor (CD25) levels (16.018U/ml), hypertriglyceridemia (582mg/dl) supportive of the diagnosis of HLH. Because of the worsening of her clinical condition, a second bone marrow aspirate and biopsy was performed 5 days later when the child became pancytopenic. The second bone marrow aspirate still did not show morphological signs of HLH, but microorganisms were detected in at least one macrophage, which were consecutively diagnosed as Leishmania amastigotes within macrophages by positive Leishmanial polymerase chain reaction from bone marrow specimen. Consistent with the diagnosis of visceral Leishmaniasis Amphotericin B (liposomal) therapy was initiated which resulted in dramatic resolution of fever, splenomegaly, ferritin levels, and recovery of blood counts within 96 hours.
Familial hemophagocytic lymphohistiocytosis (FHL) manifests as acute illness with prolonged fever, cytopenias and hepatosplenomegaly. Onset typically occurs within the first months of life. Other findings include liver dysfunction and bone marrow hemophagocytosis. The median survival of children with typical FHL, without treatment, is less than two months. Acquired HLH is typically associated with T-/NK-cell lymphomas and autoimmune diseases but also with infections including EBV, malaria and Leishmaniasis. Even in non-endemic areas, our case of vertically transmitted Leishmaniasis highlights the importance of recognizing infectious etiologies of HLH compared with hereditary forms of HLH in early infancy to initiate appropriate therapy to prevent life-threatening complications.
Russo:Novo Nordisk: Other: conference fee, travel and accomodation support ; Octapharma: Other: conference fee, travel and accomodation support. Lammle:Baxter: Other: congress travel and accomodation support ; Ablynx: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: congress travel and accomodation support ; Siemens: Honoraria, Other: congress travel and accomodation support ; Bayer: Honoraria, Other: congress travel and accomodation support; Alexion: Other: congress travel and accomodation support .
Due to sensitive neuroimaging techniques, cerebrovascular complications such as cerebral microbleeds (CMB) and cerebral cavernous malformations (CCM) are increasingly recognized as considerable late ...effects after treatment for pediatric brain tumor. The aim of this study was to analyze CMB in a cohort of patients after cranial irradiation therapy for medulloblastoma or other pediatric brain tumors using susceptibility-weighted magnetic resonance imaging (SWI).
Forty former pediatric brain tumor patients were enrolled in this prospective cross-sectional study and examined by cranial MRI including SWI sequences. Cerebral microbleeds, clinical symptoms and disability were evaluated.
Thirty-six (90%) of the examined individuals (mean follow-up age 22.2 y; mean follow-up time 13.5 y) were affected by CMB. Longer follow-up time and higher craniospinal irradiation doses correlated with higher total lesion count (p < 0.01). Thirteen patients (32.5%) presented with clinical symptoms. Individuals with CMB were more severely disabled than patients without CMB (p < 0.05).
Cerebrovascular sequelae occur frequently after treatment for pediatric brain tumor. In this study, a remarkable part of pediatric brain tumor patients presents with CMB. As a sign of vascular damage, they can cause clinical symptoms and may correspond to neurocognitive decline. Further studies are needed to standardize MRI protocols and to improve quality of long-term follow-up.
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