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IntroductionMolecular aberrations involving the mixed lineage leukemia (MLL) gene on 11q23 are found in 5% to 10% of acute leukemia cases. 1 In B-cell precursor (BCP) acute lymphoblastic leukemia (ALL), these aberrations are largely restricted to the immature CD10 Ϫ immunophenotypes (pro-B and CD10 Ϫ pre-B). The translocation t(4;11)(q22;q23) with MLL-AF4 (MLL-AFF1) fusion is known to be the most prevalent MLL fusion gene in ALL, but precise and reliable data regarding the prevalence of the different MLL fusion partner genes, that is, the MLL "recombinome" in adult ALL are lacking. Knowledge of the MLL recombinome is warranted, since MLL fusions are of interest in detecting minimal residual disease in affected patients 2,3 and also because controversy exists over whether adult ALL patients with pro-B ALL immunophenotype with or without MLL aberration might have a different prognosis. [4][5][6] We report our experience within the framework of the German Multicenter Therapy Trials for Adult ALL (GMALL) between January 2001 and October 2007 at the central diagnostic laboratory of the GMALL study group.We investigated 184 patients with a CD10 Ϫ BCP immunophenotype by reverse transcription polymerase chain reactions (RT-PCRs) for different MLL fusion genes. Since the chromosomal breakpoints in the MLL gene cluster in a relatively restricted region between exons 8 and 13 (numbering according to Nilsson et al 7 ), encompassing approximately 8.2 kb, we additionally investigated all samples by a recently published long-distance inverse polymerase chain reaction (LDI-PCR) method that also allowed the identification of unknown MLL translocation partners at the DNA level. Methods Patient materialBone marrow (n ϭ 136) and peripheral blood (n ϭ 45) samples (n ϭ 3 samples unspecified) were obtained for diagnostic purposes within the framework of the GMALL therapy studies 6/99 and 7/03 between January 2001 and October 2007. A list of GMALL study participants appears in the Supplemental Materials and Methods (available on the Blood website; see the Supplemental Materials link at the top of the online article). All samples were taken at the time of primary diagnosis and had a high blast count, as revealed by flow cytometry. The genetic investigations were done retrospectively and prospectively on archived residual material. Preparation of samples, immunophenotyping, and all RT-PCR investigations were performed at the central diagnostic laboratory of the GMALL study group in Berlin. The samples were obtained within clinical studies that were approved by the institutional ethics committees of all participating institutions. The study design and our investigations were conducted in accordance with the Declaration of Helsinki. Nucleic acid isolation and reverse transcriptionTotal RNA was isolated using the TRIZOL method (Invitrogen, Carlsbad, CA) or the RNEasy kit (QIAGEN, Hilden, Germany). Genomic DNA was For personal use only. on May 12, 2018. by guest www.bloodjournal.org From isolated using the PureGene Kit (Gentra Systems, Mi...

BackgroundDevelopment of clinically relevant tumor model systems for glioblastoma multiforme (GBM) is important for advancement of basic and translational biology. High molecular heterogeneity of GBM tumors is well recognized, forming the rationale for molecular tests required before administration of several of the novel therapeutics rapidly entering the clinics. One model that has gained wide acceptance is the primary cell culture model. The laborious and time consuming process is rewarded with a relative high success rate (about 60%). We here describe and evaluate a very simple cryopreservation procedure for GBM tissue prior to model establishment that will considerably reduce the logistic complexity.MethodsTwenty-seven GBM samples collected ad hoc were prepared for primary cell culture freshly from surgery (#1) and after cryopreservation (#2).ResultsTake rates after cryopreservation (59%) were as satisfactory as from fresh tissue (63%; p = 1.000). We did not observe any relevant molecular or phenotypic differences between cell lines established from fresh or vitally frozen tissue. Further, sensitivity both towards standard chemotherapeutic agents (Temozolomide, BCNU and Vincristine) and novel agents like the receptor tyrosine kinase inhibitor Imatinib did not differ.ConclusionsOur simple cryopreservation procedure facilitates collection, long-time storage and propagation (modeling) of clinical GBM specimens (potentially also from distant centers) for basic research, (pre-) clinical studies of novel therapies and individual response prediction.

The potential impact of different radiological features of glioblastoma multiforme (GBM) on overall survival (OS) like tumor volume, peritumoral edema (PTE), necrosis volume, necrosis-tumor ratio (NTR) and edema-tumor ratio (ETR) is still very controversial. To determine the influence of volumetric data on OS und to compare different measuring techniques described in literature. We prospectively evaluated preoperative MR images from 30 patients harboring a primary supratentorial GBM. All patients received gross-total tumor resection followed by standard radiation and chemotherapy (temozolomide). By 3D semi-automated segmentation, we measured tumor volume, necrosis volume, PTE, postoperative residual tumor volume and calculated ETR, NTR and the extent of resection. After critical review of the existing literature we compared alternative measuring techniques with the gold standard of 3D segmentation. Statistical analysis showed a significant impact of the preoperative tumor and necrosis volumes on OS (p = 0.041, respectively p = 0.039). Furthermore, NTR also showed a significant association with OS (p = 0.005). Comparison of previously described measuring techniques and scorings with our results showed that no other technique is reliable and accurate enough as a predictive tool. The critical review of previously published studies revealed mainly inaccurate measurement techniques and patient selection as potential reasons for inconsistent results. Preoperatively measured necrosis volume and NTR are the most important radiological features of GBM with a strong influence on OS. No other measuring techniques are specific enough and comparable with 3D segmentation.

Background: SET-NUP214 fusion resulting from a recurrent cryptic deletion, del(9)(q34.11q34.13) has recently been described in T-cell acute lymphoblastic leukemia (T-ALL) and in one case of acute myeloid leukemia (AML). The fusion protein appears to promote elevated expression of HOXA cluster genes in T-ALL and may contribute to the pathogenesis of the disease. We screened a panel of ALL and AML cell lines for SET-NUP214 expression to find model systems that might help to elucidate the cellular function of this fusion gene.

Hodgkin/Reed-Sternberg (HRS) cells represent the malignant fraction of infiltrated lymph nodes in Hodgkin lymphoma (HL). Although HRS cells display multiple chromosomal aberrations, few are recurrent and the targeted genes unknown. However, understanding the pathology of HL and developing rational therapies may well require identifying putative deregulated genes. Here, we analyzed the karyotype of the well-defined HL cell line L-1236 by spectral karyotyping and identified multiple abnormalities, therein, notably t(4;8)(q27;q24) which includes two breakpoint regions previously highlighted in HL. Target genes at 4q27 and 8q24 were shortlisted by high density genomic arrays and fluorescence in situ hybridization. Expression analysis of candidate target genes revealed conspicuous activation of phosphodiesterase PDE5A at 4q27 and inhibition of homeobox gene ZHX2 at 8q24. Treatment of L-1236 with PDE5A-inhibitor sildenafil or with siRNA directed against PDE5A and concomitant stimulation with cyclic guanosine monophosphate (cGMP) resulted in enhanced apoptosis, indicating PDE5A as an oncogene. Expression profiling of L-1236 cells following siRNA-mediated knockdown of ZHX2 showed inhibition of genes regulating differentiation and apoptosis, suggesting tumor suppressor activity of ZHX2. Downstream genes included STAT1 and several STAT1-target genes, indicating activation of STAT1-signaling by ZHX2 as analyzed by RQ-PCR and western blot. Taken together, we have identified a novel aberration with recurrent breakpoints in HL, t(4;8)(q27;q24), which activate PDE5A and repress ZHX2, deregulating apoptosis, differentiation, and STAT1-signaling in HL cells.

BackgroundGlioblastoma multiforme (GBM) is the most common and lethal brain tumor in adults, highlighting the need for novel treatment strategies. Patient derived xenografts (PDX) represent a valuable tool to accomplish this task.MethodsPDX were established by implanting GBM tissue subcutaneously. Engraftment success was compared between NMRI Foxn1nu and NOD/SCID as well as between fresh and cryopreserved tissue. Established PDX were analyzed histologically and molecularly. Five PDX were experimentally treated with different drugs to assess their potential for preclinical drug testing.ResultsEstablishment of PDX was attempted for 36 consecutive GBM cases with an overall success rate of 22.2% in NMRI Foxn1nu mice. No difference was observed between fresh or cryopreserved (20–1057 days) tissue in direct comparison (n = 10 cases). Additionally, engraftment was better in NOD/SCID mice (38.8%) directly compared to NMRI Foxn1nu mice (27.7%) (n = 18 cases). Molecular data and histology of the PDX compare well to the primary GBM. The experimental treatment revealed individual differences in the sensitivity towards several clinically relevant drugs.ConclusionsThe use of vitally frozen GBM tissue allows a more convenient workflow without efficiency loss. NOD/SCID mice appear to be better suited for initial engraftment of tumor tissue compared to NMRI Foxn1nu mice.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-017-1128-5) contains supplementary material, which is available to authorized users.

Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO2) are the key enzymes of tryptophan (TRP) metabolism in the kynurenine pathway (KP). Both enzymes function as indicators of immunosuppression and poor survival in cancer patients. Direct or indirect targeting of either of these substances seems thus reasonable to improve therapy options for patients. In this study, glioblastoma multiforme (GBM) as well as head and neck squamous cell carcinomas (HNSCC) were examined because of their different mechanisms of spontaneous and treatment-induced immune escape. Effects on gene expression and protein levels were examined. Accompanying assessment of TRP metabolites from treated GBM cell culture supernatants was conducted. Our results show a heterogeneous and inversely correlated expression profile of TRP-metabolizing genes among GBM and HNSCC cells, with low, but inducible IDO1 expression upon IFNγ treatment. TDO2 expression was higher in GBM cells, while genes encoding kynurenine aminotransferases were mainly confined to HNSCC cells. These data indicate that the KP is active in both entities, with however different enzymes involved in TRP catabolism. Upon treatment with Temozolomide, the standard of care for GBM patients, IDO1 was upregulated. Comparable, although less pronounced effects were seen in HNSCC upon Cetuximab and conventional drugs (i.e., 5-fluorouracil, Gemcitabine). Here, IDO1 and additional genes of the KP (KYAT1, KYAT2, and KMO) were induced. Vice versa, the novel yet experimental cyclin-dependent kinase inhibitor Dinaciclib suppressed KP in both entities. Our comprehensive data imply inhibition of the TRP catabolism by Dinaciclib, while conventional chemotherapeutics tend to activate this pathway. These data point to limitations of conventional therapy and highlight the potential of targeted therapies to interfere with the cells' metabolism more than anticipated.