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.
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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.
Papillary renal cell carcinoma (PRCC) is a rare entity in children with no established therapy protocols for advanced diseases. Immunotherapy is emerging as an important therapeutic tool for ...childhood cancer. Tumor cells can be recognized and killed by conventional and unconventional T cells. Unconventional T cells are able to recognize lipid antigens presented via CD1 molecules independently from major histocompatibility complex, which offers new alternatives for cancer immunotherapies. The nature of those lipids is largely unknown and α-galactosylceramide is currently used as a synthetic model antigen. In this work, we analyzed infiltrating lymphocytes of two pediatric PRCCs using flow cytometry, immunohistochemistry and qRT-PCR. Moreover, we analyzed the CD1d expression within both tumors. Tumor lipids of PRCC samples and three normal kidney samples were fractionated and the recognition of tumor own lipid fractions by unconventional T cells was analyzed in an
assay. We identified infiltrating lymphocytes including γδ T cells and iNKT cells, as well as CD1d expression in both samples. One lipid fraction, containing ceramides and monoacylglycerides amongst others, was able to induce the proliferation of iNKT cells isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors and of one matched PRCC patient. Furthermore, CD1d tetramer stainings revealed that a subset of iNKT cells is able to bind lipids being present in fraction 2 via CD1d. We conclude that PRCCs are infiltrated by conventional and unconventional T cells and express CD1d. Moreover, certain lipids, present in pediatric PRCC, are able to stimulate unconventional T cells. Manipulating these lipids and T cells may open new strategies for therapy of pediatric PRCCs.
The ganglioside GD2 is an important target in childhood cancer. Nevertheless, the only therapy targeting GD2 that is approved to date is the monoclonal antibody dinutuximab, which is used in the ...therapy of neuroblastoma. The relevance of GD2 as a target in other tumor entities remains to be elucidated. Here, we analyzed the expression of GD2 in different pediatric tumor entities by flow cytometry and tested two approaches for targeting GD2. H3K27M-mutant diffuse midline glioma (H3K27M-mutant DMG) samples showed the highest expression of GD2 with all cells strongly positive for the antigen. Ewing's sarcoma (ES) samples also showed high expression, but displayed intra- and intertumor heterogeneity. Osteosarcoma had low to intermediate expression with a high percentage of GD2-negative cells. Dinutuximab beta in combination with irinotecan and temozolomide was used to treat a five-year-old girl with refractory ES. Disease control lasted over 12 months until a single partially GD2-negative intracranial metastasis was detected. In order to target GD2 in H3K27M-mutant DMG, we blocked ganglioside synthesis via eliglustat, since dinutuximab cannot cross the blood-brain barrier. Eliglustat is an inhibitor of glucosylceramide synthase, and it is used for treating children with Gaucher's disease. Eliglustat completely inhibited the proliferation of primary H3K27M-mutant DMG cells in vitro. In summary, our data provide evidence that dinutuximab might be effective in tumors with high GD2 expression. Moreover, disrupting the ganglioside metabolism in H3K27M-mutant DMG could open up a new therapeutic option for this highly fatal cancer.
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.
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