Marie A. Neu

CD19 Isoforms Enabling Resistance to CART-19 Immunotherapy Are Expressed in B-ALL Patients at Initial Diagnosis

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.

Burden of cardiovascular risk factors and cardiovascular disease in childhood cancer survivors: data from the German CVSS-study

Aims The cardiac and vascular late sequelae in long-term survivors of childhood cancer (CVSS)-study aimed to quantify the prevalence of cardiovascular risk factors (CVRF) and cardiovascular disease (CVD) in German childhood cancer survivors (CCS). Methods and results In the CVSS-study (NCT02181049), 1002 CCS (age range 23-48 years) diagnosed with neoplasia prior to 15 years of age between 1980 and 1990 prospectively underwent a systematic, standardized clinical and laboratory cardiovascular screening, identical to the population-based Gutenberg Health Study (GHS) cohort. For 951 individuals, prevalences of CVRF and CVD were primarily compared to the GHS sample and to two further German population-based cohorts. Using log-binomial regression models, an increased risk for occurrence of arterial hypertension [relative risk (RR) 1.38, 95% confidence interval (95% CI 1.21-1.57)] and dyslipidaemia [RR 1.26 (95% CI 1.12-1.42)] was found. This indicates a premature occurrence compared to the general population of approximately 6 and 8 years, respectively [rate advancement period estimator, RAPhypertension 5.75 (95% CI 3.5-8.0) and RAPdyslipidaemia 8.16 (95% CI 4.4-11.9)]. Overall, no differences were observed for obesity and diabetes. Overt CVD was present in 4.5% (95% CI 3.0-6.6%) of CCS [RR 1.89 (95% CI 1.34-2.66), RAPCVD 7.9 (95% CI 4.1-11.7)], of which the most frequent entities were congestive heart failure and venous thromboembolism. Prevalences of CVRF and CVD increased with age without reaching a plateau over time. Conclusion This large CCS screening examination revealed consistently in comparison to three population samples a considerably increased risk for premature CVD. The findings in these young adult CCS indicate a high burden of cardiovascular morbidity and mortality in the long term. Clinicaltrials. gov-Nr NCT02181049.

Next-generation sequencing reveals germline mutations in an infant with synchronous occurrence of nephro- and neuroblastoma

ABSTRACT Although neuro- and nephroblastoma are common solid tumors in children, the simultaneous occurrence is very rare and is often associated with syndromes. Here, we present a unique case of synchronous occurrence of neuro- and nephroblastoma in an infant with no signs of congenital anomalies or a syndrome. We performed genetic testing for possible candidate genes as underlying mutation using the next-generation sequencing (NGS) approach to target 94 genes and 284 single-nucleotide polymorphisms (SNPs) involved in cancer. We uncovered a novel heterozygous germline missense mutation p.F58L (c.172T→C) in the anaplastic lymphoma kinase (ALK) gene and one novel heterozygous rearrangement Q418Hfs*11 (c.1254_1264delins TTACTTAGTACAAGAACTG) in the Fanconi anemia gene FANCD2 leading to a truncated protein. Besides, several SNPs associated with the occurrence of neuroblastoma and/or nephroblastoma or multiple primary tumors were identified. The next-generation sequencing approach might in the future be useful not only in understanding tumor etiology but also in recognizing new genetic markers and targets for future personalized therapy.

Activation of the basal cell carcinoma pathway in a patient with CNS HGNET-BCOR diagnosis: consequences for personalized targeted therapy

High grade neuroepithelial tumor of the central nervous system with BCOR alteration (CNS HGNET-BCOR) is a recently described new tumor entity with a dismal prognosis. The objective of this study was to identify and validate pathways deregulated in CNS HGNET-BCOR as basis for targeted therapy approaches. We characterized the BCOR alteration in a pediatric patient with CNS HGNET-BCOR diagnosis by Sanger sequencing and demonstrated an elevated BCOR expression by qRT-PCR and western blot. By whole transcriptome sequencing and Ingenuity Pathway Analysis, we identified the activation of the Sonic Hedgehog (SHH) and of the WNT signaling pathway in two different regions of the primary tumor and of one inoculation metastasis compared to normal brain. We validated the activation of the SHH and of the WNT pathway by qRT-PCR analysis of GLI1 and AXIN2 respectively. GLI1 and AXIN2 were upregulated in the primary tumor and in two inoculation metastases compared to normal brain. Mutational analysis of SMO, PTCH1 and SUFU, three key components of the SHH pathway, revealed a Single Nucleotide Polymorphism (SNP) in PTCH1 (rs357564). We tested the effect of the GLI-inhibitor arsenic trioxide (ATO) on a short-term cell culture isolated from the metastasis. ATO was able to reduce the viability of the cells with an IC50 of 1.3 μM. In summary, these results provide functional evidence of altered BCOR expression and homogeneous coactivation of both the SHH and WNT signaling pathways, building the basis for potential novel therapeutic approaches for patients with a CNS HGNET-BCOR diagnosis.

Personalized therapy: CNS HGNET-BCOR responsiveness to arsenic trioxide combined with radiotherapy

High-grade neuroepithelial tumor of the central nervous system with BCOR alteration (HGNET-BCOR) is a rare, highly malignant tumor. At the time of this publication, no standard protocol exists to treat this tumor entity. In this work, we tested the responsiveness of the primary culture PhKh1 derived from tumor tissue from a pediatric HGNET-BCOR patient (P1) to inhibitors of the Sonic hedgehog pathway combined with radiation. The SMO inhibitors vismodegib and itraconazole had low effect on the proliferation of the PhKh1 cells. However, the GLI inhibitor arsenic trioxide reduced the expression of GLI target genes in the PhKh1 cells and in combination with radiotherapy significantly decreased their clonogenic potential. PhKh1 cells resistant to arsenic trioxide were characterized by the overexpression of molecular chaperones. We combined arsenic trioxide and radiation in the relapse therapy protocol of P1, achieving complete remission after seven weeks. Clinical remission lasted for six months, when P1 developed systemic metastases. Meanwhile, an increase in the concentration of circulating tumor DNA carrying a BCOR internal tandem duplication was observed. Molecular characterization of a second patient (P2) was also performed. In P2, we detected a larger tandem duplication and greater activation of the Sonic hedgehog pathway than in P1. These findings suggest that combining arsenic trioxide with radiotherapy may represent a new therapeutic approach. Moreover, peripheral blood analysis for circulating tumor DNA could help in the early detection of systemic metastases.

Susceptibility-weighted magnetic resonance imaging of cerebrovascular sequelae after radiotherapy for pediatric brain tumors

BACKGROUND AND PURPOSE 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). MATERIALS AND METHODS 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. RESULTS 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). CONCLUSIONS 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.

Cardiovascular risk factors are important determinants of platelet-dependent thrombin generation in adult survivors of childhood cancer

Cardiovascular disease is the most frequent non-malignant cause of morbidity and mortality in adult survivors of childhood or adolescent cancer. Thrombin generation (TG) analysis gives insight in hypercoagulability as an important mechanism linked to cardiovascular risk factors (CVRFs). In 200 individuals, from the cardiac and vascular late sequelae in long-term survivors of childhood cancer study, TG in platelet-rich plasma (PRP) and platelet-free plasma (PFP) at 1pM tissue factor was investigated. Endogenous thrombin potential (ETP) and peak height were the analysed parameters of a TG curve. Sex-specific multivariable linear regression analysis adjusted for age and CVRFs was used to assess the clinical determinants of TG. Females presented with higher ETP and peak height compared to males, both in PRP and PFP. Hypertension (beta estimate, ß: 184.8 [90.7; 278.8]), obesity (ß: 161.9 [63.9; 259.5]), and HbA1c (ß: 715.6 [97.4; 1333.8]) were associated with higher ETP in PRP only. ETP in PRP was positively associated with obesity and HbA1c in both males and females and with dyslipidemia (ß: 253.07 [72.92; 433.22]) and systolic hypertension (ß: 436.7 [119.02; 754.39]) in females only. CVRFs showed no association with TG variables in PFP. In conclusion, this study presents an important relation between traditional CVRFs and TG in the presence of platelets only. Sex-specific differences in TG with females presenting with higher TG, particularly those with dyslipidemia and systolic hypertension, were demonstrated. These results highlight the potential of the platelet-coagulant function in identifying cancer survivors at higher risk for adverse cardiovascular events.

Prospective analysis of long-term renal function in survivors of childhood Wilms tumor

BackgroundConsidering 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.MethodsThirty-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.ResultsAll 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.ConclusionEven 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.

Novel loss of function mutation in KRIT1/CCM1 is associated with distinctly progressive cerebral and spinal cavernous malformations after radiochemotherapy for intracranial malignant germ cell tumor

PurposeCerebrospinal cavernous malformations (CCMs) are vascular lesions characterized by dilated and leaky capillary caverns. CCMs can cause seizures, focal neurological deficits or acute intracranial hemorrhage; however, most patients are asymptomatic. CCMs occur either sporadically or as a familial autosomal-dominant disorder. We present a clinical and molecular study of a patient with distinctive cerebral and spinal cavernous malformations following radiochemotherapy for a malignant brain tumor.MethodsThe patient had multiple magnet resonance imaging (MRI) examinations of his brain and spine following radiochemotherapy for a primary intracranial germ cell tumor (GCT), as part of his oncologic follow-up. The MRI sequences included susceptibility-weighted imaging (SWI). The coding exons and their flanking intronic regions of KRIT1/CCM1 gene were analyzed for mutations by polymerase chain reaction (PCR) and direct sequencing.ResultsMRI revealed numerous cerebral and spinal microhemorrhages and pronounced cavernous malformations that progressed with subsequent follow-up imaging. Genetic analysis demonstrated a novel heterozygous KRIT1/CCM1 two base pair deletion (c.1535_1536delTG) in exon 14. This deletion leads to a frameshift with a premature stop codon at nucleotide position 1553 and a highly likely loss of function of the KRIT1 protein.ConclusionWe describe a patient with a novel heterozygous germ line loss of function mutation in KRIT1, which is associated with rapid-onset and highly progressive CCMs after radiochemotherapy for a malignant brain tumor.