Christian Vokuhl

Mutations in the SIX1/2 Pathway and the DROSHA/DGCR8 miRNA Microprocessor Complex Underlie High-Risk Blastemal Type Wilms Tumors

Blastemal histology in chemotherapy-treated pediatric Wilms tumors (nephroblastoma) is associated with adverse prognosis. To uncover the underlying tumor biology and find therapeutic leads for this subgroup, we analyzed 58 blastemal type Wilms tumors by exome and transcriptome sequencing and validated our findings in a large replication cohort. Recurrent mutations included a hotspot mutation (Q177R) in the homeo-domain of SIX1 and SIX2 in tumors with high proliferative potential (18.1% of blastemal cases); mutations in the DROSHA/DGCR8 microprocessor genes (18.2% of blastemal cases); mutations in DICER1 and DIS3L2; and alterations in IGF2, MYCN, and TP53, the latter being strongly associated with dismal outcome. DROSHA and DGCR8 mutations strongly altered miRNA expression patterns in tumors, which was functionally validated in cell lines expressing mutant DROSHA.

Mer tyrosine kinase promotes the survival of t(1;19)-positive acute lymphoblastic leukemia (ALL) in the central nervous system (CNS)

Patients with t(1;19)-positive acute lymphoblastic leukemia (ALL) are prone to central nervous system (CNS) relapses, and expression of the TAM (Tyro3, Axl, and Mer) receptor Mer is upregulated in these leukemias. We examined the functional role of Mer in the CNS in preclinical models and performed correlative studies in 64 t(1;19)-positive and 93 control pediatric ALL patients. ALL cells were analyzed in coculture with human glioma cells and normal rat astrocytes: CNS coculture caused quiescence and protection from methotrexate toxicity in Mer(high) ALL cell lines, which was antagonized by short hairpin RNA-mediated knockdown of Mer. Mer expression was upregulated, prosurvival Akt and mitogen-activated protein kinase signaling were activated, and secretion of the Mer ligand Galectin-3 was stimulated. Mer(high) t(1;19) primary cells caused CNS involvement to a larger extent in murine xenografts than in their Mer(low) counterparts. Leukemic cells from Mer(high) xenografts showed enhanced survival in coculture. Treatment of Mer(high) patient cells with the Mer-specific inhibitor UNC-569 in vivo delayed leukemia onset, reduced CNS infiltration, and prolonged survival of mice. Finally, a correlation between high Mer expression and CNS positivity upon initial diagnosis was observed in t(1;19) patients. Our data provide evidence that Mer is associated with survival in the CNS in t(1;19)-positive ALL, suggesting a role as a diagnostic marker and therapeutic target.

Mutually exclusive BCOR internal tandem duplications and YWHAE‐NUTM2 fusions in clear cell sarcoma of kidney: not the full story

Internal tandem duplication within the BCOR gene sequence that encodes the PUFD domain, important in the formation of the non‐canonical or variant polycomb repressor complex 1 (v‐PRC1), was very recently described in 100% of 20 clear cell sarcomas of kidney (CCSKs). None of those 20 cases bore the YWHAE–NUTM2 transcript, previously described by us in CCSK, and which constitutes the only other recurrent genetic aberration observed in CCSK, prompting consideration that these mutations might be mutually exclusive in CCSK. We analysed a cohort of 159 CCSKs and can now not only confirm that there is indeed mutual exclusivity of these BCOR and YWHAE mutations, but also show that a substantial proportion (in this series 11.8%) of CCSKs bear neither mutation when tested by these assays, raising the possibility of distinct aetiologies for subsets of CCSK. Clinical differences observed between the subsets support this notion. As CCSK may show poor chemo‐responsiveness, and current treatment protocols mandate the use of doxorubicin with its associated side‐effects, advances in understanding the disease biology with a view to more targeted and personalized treatment is a pressing need. Copyright

Establishment of a rhabdomyosarcoma xenograft model in human-adapted mice

The outcome of patients with advanced stage rhabdomyosarcoma (RMS) is still sobering. This outcome has not improved through conservative treatments. Therefore, novel treatment approaches such as immunotherapy need to be evaluated in human-adapted animal models. The aim of this study was to develop a humanized mouse model of childhood RMS as a basis for the study of immunotherapeutic approaches. Therefore, NOD/LtSz-scid IL2rgammanull-mice were used for all the experiments (n=19). The animals underwent sublethal irradiation on days 1 and 2 (1 x 300 cGy). After irradiation, the transplantation of human CD34+-cells (1,000,000 cells per animal i.v.) was carried out. Five animals served as the control and did not undergo stem cell transplantation. The engraftment of human cells was assessed in peripheral blood on days 21 and 55 by FACS analysis. Eight weeks after transplantation, the subcutaneous xenotransplantation of human alveolar and embryonal RMS cell lines was carried out. Tumor growth was monitored and tumors were resected 93 days after CD34+-transplantation. The tumor specimens were evaluated histologically. The successful engraftment of human cells with the establishment of a human immune system was observed in 12 out of 14 animals. B and T cells were mostly detected in the peripheral blood. There were only a few monocytes and almost no natural killer cells. The xenotransplantation of alveolar RMS resulting in subcutaneous tumor growth was feasible in 7 animals. The xenotransplantation of embryonal RMS was performed in 5 animals and led to tumor growth in 1 animal. A histological work up showed either alveolar or embryonal RMS cells with central necrosis. This is the first time a xenotransplantation model of human RMS has been developed in a humanized mouse model. The establishment of subcutaneous tumor xenografts was more effective in the alveolar subtype. This model offers a basic tool for further analyzing novel immunotherapeutic approaches in RMS, and could possibly be used in other solid pediatric tumors.

Central nervous system acute lymphoblastic leukemia: role of natural killer cells

Central nervous system acute lymphoblastic leukemia (CNS-ALL) is a major clinical problem. Prophylactic therapy is neurotoxic, and a third of the relapses involve the CNS. Increased expression of interleukin 15 (IL-15) in leukemic blasts is associated with increased risk for CNS-ALL. Using in vivo models for CNS leukemia caused by mouse T-ALL and human xenografts of ALL cells, we demonstrate that expression of IL-15 in leukemic cells is associated with the activation of natural killer (NK) cells. This activation limits the outgrowth of leukemic cells in the periphery, but less in the CNS because NK cells are excluded from the CNS. Depletion of NK cells in NOD/SCID mice enabled combined systemic and CNS leukemia of human pre-B-ALL. The killing of human leukemia lymphoblasts by NK cells depended on the expression of the NKG2D receptor. Analysis of bone marrow (BM) diagnostic samples derived from children with subsequent CNS-ALL revealed a significantly high expression of the NKG2D and NKp44 receptors. We suggest that the CNS may be an immunologic sanctuary protected from NK-cell activity. CNS prophylactic therapy may thus be needed with emerging NK cell-based therapies against hematopoietic malignancies.

Absence of BRAF mutation in pediatric and adolescent germ cell tumors indicate biological differences to adult tumors.

The V600E mutation of the BRAF gene has been reported to be associated with poor prognosis of germ cell tumors in adult patients. We analyzed the mutational status of the BRAF and KRAS gene as well as MLH1 and MSH6 expression as surrogate markers for microsatellite instability in 70 pediatric germ cell tumors. Neither BRAF and KRAS mutations nor loss of MLH1 and MSH6 expression were found. Our data provide further evidence for patient age related biological differences in germ cell tumors and demonstrate that prognostic biomarkers cannot necessarily be transferred from one age group to the other. Pediatr Blood Cancer 2012;59:732–735.

The role of Zeta-chain-associated protein kinase in acute lymphoblastic leukemia infiltration into the central nervous system

Central nervous system infiltration and relapse are poorly understood in childhood acute lymphoblastic leukemia. We examined the role of zeta-chain-associated protein kinase 70 in preclinical models of central nervous system leukemia and performed correlative studies in patients. Zeta-chain-associated protein kinase 70 expression in acute lymphoblastic leukemia cells was modulated using short hairpin ribonucleic acid-mediated knockdown or ectopic expression. We show that zeta-chain-associated protein kinase 70 regulates CCR7/CXCR4 via activation of extracellular signal-regulated kinases. High expression of zeta-chain-associated protein kinase 70 in acute lymphoblastic leukemia cells resulted in a higher proportion of central nervous system leukemia in xenografts as compared to zeta-chain-associated protein kinase 70 low expressing counterparts. High zeta-chain-associated protein kinase 70 also enhanced the migration potential towards CCL19/CXCL12 gradients in vitro. CCR7 blockade almost abrogated homing of acute lymphoblastic leukemia cells to the central nervous system in xenografts. In 130 B-cell precursor acute lymphoblastic leukemia and 117 T-cell acute lymphoblastic leukemia patients, zeta-chain-associated protein kinase 70 and CCR7/CXCR4 expression levels were significantly correlated. Zeta-chain-associated protein kinase 70 expression correlated with central nervous system disease in B-cell precursor acute lymphoblastic leukemia, and CCR7/CXCR4 correlated with central nervous system involvement in T-cell acute lymphoblastic leukemia patients. In multivariate analysis, zeta-chain-associated protein kinase 70 expression levels in the upper third and fourth quartiles were associated with central nervous system involvement in B-cell precursor acute lymphoblastic leukemia (odds ratio=7.48, 95% confidence interval, 2.06–27.17; odds ratio=6.86, 95% confidence interval, 1.86–25.26, respectively). CCR7 expression in the upper fourth quartile correlated with central nervous system positivity in T-cell acute lymphoblastic leukemia (odds ratio=11.00, 95% confidence interval, 2.00–60.62). We propose zeta-chain-associated protein kinase 70, CCR7 and CXCR4 as markers of central nervous system infiltration in acute lymphoblastic leukemia warranting prospective investigation.

Small cell undifferentiated (SCUD) hepatoblastomas: All malignant rhabdoid tumors?

Small cell undifferentiated (SCUD) hepatoblastoma is a rare variant of hepatoblastoma with poor outcome and loss of INI1 expression, sharing this with malignant rhabdoid tumors (MRT). We studied all tumors from the files of the Kiel Pediatric Tumor Registry (KTR) with the initial diagnosis of SCUD and MRT. After re‐review, we performed immunistochemistry, fluorescence in situ hybridization, and multiplex ligation dependent probe amplification for loss of expression and deletion of INI1/SMARCB1 in 23 tumors. Morphologically, 12 of the tumors had a small cell morphology, 9 showed the typical picture of MRT, and 2 were composed of both small cells and rhabdoid cells. All but 1 of the 23 tumors showed loss of INI1 protein expression by immunohistochemistry. Nineteen of the INI1 negative tumors were analyzed by FISH technique and all showed a deletion of the INI1/SMARCB1 gene (17 homozygous deletions, 2 heterozygous deletions). We investigated 14 of these cases by multiplex ligation dependent probe amplification and verified the deletions in all cases. In conclusion, we postulate that SCUD hepatoblastoma is not a hepatoblastoma but represents a malignant rhabdoid tumor of the liver.

ETV6–NTRK3 in congenital mesoblastic nephroma: A report of the SIOP/GPOH nephroblastoma study

Congenital mesoblastic nephroma (MN) is a rare pediatric renal tumor representing approximately 5% of all pediatric renal tumors. Three different types of MN are distinguished histologically: classical, cellular, and mixed. A frequent genetic alteration is the translocation t(12;15) resulting in a fusion of the ETV6 gene on 12p13 and the NTRK3 gene on 15p15 that occurs almost exclusively in cellular MN. The aim of this study was to determine translocation status of a large cohort of MN with respect to tumor subtype and outcome.

Small Cell Undifferentiated Hepatoblastomas (SCUD): All Malignant Rhabdoid Tumors?

Small cell undifferentiated (SCUD) hepatoblastoma is a rare variant of hepatoblastoma with poor outcome and loss of INI1 expression, sharing this with malignant rhabdoid tumors (MRT). We studied all tumors from the files of the Kiel Pediatric Tumor Registry (KTR) with the initial diagnosis of SCUD and MRT. After re‐review, we performed immunistochemistry, fluorescence in situ hybridization, and multiplex ligation dependent probe amplification for loss of expression and deletion of INI1/SMARCB1 in 23 tumors. Morphologically, 12 of the tumors had a small cell morphology, 9 showed the typical picture of MRT, and 2 were composed of both small cells and rhabdoid cells. All but 1 of the 23 tumors showed loss of INI1 protein expression by immunohistochemistry. Nineteen of the INI1 negative tumors were analyzed by FISH technique and all showed a deletion of the INI1/SMARCB1 gene (17 homozygous deletions, 2 heterozygous deletions). We investigated 14 of these cases by multiplex ligation dependent probe amplification and verified the deletions in all cases. In conclusion, we postulate that SCUD hepatoblastoma is not a hepatoblastoma but represents a malignant rhabdoid tumor of the liver.