Gliomas, which generally have a poor prognosis, are the most common primary malignant brain tumors in adults. Recent genome-wide association studies have demonstrated that inherited susceptibility plays a role in the development of glioma. Although first-degree relatives of patients exhibit a two-fold increased risk of glioma, the search for susceptibility loci in familial forms of the disease has been challenging because the disease is relatively rare, fatal, and heterogeneous, making it difficult to collect sufficient biosamples from families for statistical power. To address this challenge, the Genetic Epidemiology of Glioma International Consortium (Gliogene) was formed to collect DNA samples from families with two or more cases of histologically confirmed glioma. In this study, we present results obtained from 46 U.S. families in which multipoint linkage analyses were undertaken using nonparametric (model-free) methods. After removal of high linkage disequilibrium SNPs, we obtained a maximum nonparametric linkage score (NPL) of 3.39 (P=0.0005) at 17q12–21.32 and the Z-score of 4.20 (P=0.000007). To replicate our findings, we genotyped 29 independent U.S. families and obtained a maximum NPL score of 1.26 (P=0.008) and the Z-score of 1.47 (P=0.035). Accounting for the genetic heterogeneity using the ordered subset analysis approach, the combined analyses of 75 families resulted in a maximum NPL score of 3.81 (P=0.00001). The genomic regions we have implicated in this study may offer novel insights into glioma susceptibility, focusing future work to identify genes that cause familial glioma.
Chromosomal structural abnormalities in ALL are powerful independent predictors of prognosis, and directly impact choice of therapy. Currently, clinical detection of these abnormalities is based on karyotype and fluorescent in-situ hybridization (FISH), but these methods have limitations. Under optimal circumstances, structural abnormalities are detectable in well over 90% of ALL cases, but in actuality, typical cytogenetic laboratories demonstrate only a 50–60% abnormality detection rate. Karyotype may fail due to unsuccessful cell growth in culture and/or relative overgrowth of normal lymphocytes. FISH is limited by the expense and labor intensity of performing a separate assay for each probe used. Array comparative genomic hybridization (CGH) may have clinical utility as a complementary diagnostic tool in pediatric ALL. Its advantages include the ability to detect copy number changes in regions too small to be identifiable by karyotype; to identify novel abnormalities for which specific FISH probes do not exist in current diagnostic laboratories; and to provide information in as many as 50% of cases which show a failed or normal karyotype. In addition to its clinical utility, array CGH provides a wealth of information which may be mined for discovery of new pathways in leukemogenesis and additional prognostic factors within existing disease subgroups. The main limitation of array CGH is its inability to detected balanced translocations. We evaluated the diagnostic utility of a bacterial artificial chromosome (BAC) array CGH platform, the SpectralChip 2600, with an average resolution of 1.0 MB across the genome. We analyzed 50 pediatric ALL bone marrow specimens obtained at diagnosis, and compared the findings to the clinical results based on karyotype and standard 5-probe FISH panel. The cases ranged from 1–15 years (mean 5 years), with marrow containing between 33–94% leukemic blasts (mean 77%). Each sample was hybridized to the chip with a healthy control of the opposite gender. The sensitivity of array CGH in detecting abnormalities identified by karyotype and FISH was approximately 88%. Several of the abnormalities “missed” by CGH, which lowered the sensitivity score, were subsequently found to be erroneous karyotype calls when followed up with specific FISH probes. In addition, array CGH detected numerous additional areas of amplification and deletion which were subsequently validated by FISH, including in 10 cases for which cytogenetics was either normal or unsuccessful. Loss of 1p31, loss of 7p21, and gain of 16p13 were aberrations that were each noted to occur in three or more different cases, and hence may be worthy of further study. In the future, development of a customized ALL chip which is enriched for probes at sites of known amplification and deletion could further heighten diagnostic sensitivity, obviate the need for performance of multiple FISH tests, and provide valuable information in the substantial number of cases with a normal or failed karyotype analysis. Balanced translocations would still require testing via a multiplex PCR assay or a combination of available FISH probes.
Despite aggressive chemotherapy, relapse occurs in almost half of children with acute myeloid leukemia (AML), with very dismal survival. Novel and mechanism-driven therapies are desperately needed to conquer chemotherapy resistance and leukemic stemness in pediatric AML. Within the bone marrow niche, stromal cells protect leukemia cells from chemotherapy, maintain leukemic stemness, and eventually lead to disease recurrence. We developed an in vitro AML cell-stromal cell co-culture model to mimic bone marrow microenvironment. Stroma-leukemia cell interaction leads to activation of various signaling molecules in AML cells that allow them to evade apoptosis. One such example is extracellular signal-regulated kinases 1/2 (ERK1/2), important pro-survival proteins. ERK1/2 are activated by the Ras/Raf/ mitogen-activated protein kinase kinase (MAPK/ERK kinase or MEK) pathway downstream of signals from the stroma. We recently showed that stromal co-culture activates ERK1/2 in pediatric AML samples, contributing to chemotherapy resistance (Long, et al, 2017, Oncotarget, 8:90037). To identify genes that are regulated in AML cells by ERK1/2 activation, 4 pediatric AML samples were cultured alone, or co-cultured with mOrange-expressing stroma for 24 hours, in the presence or absence of a selective MEK inhibitor, selumetinib (1 μM). Thereafter, cells were flow sorted to exclude mOrange+ stroma and CD45high/SSClow lymphocytes. Sorted AML cells underwent total RNA extraction for nCounter® PanCancer Pathways Panel (Nanostring Technologies) gene expression profiling study. We focused on the genes that were either up- or down-regulated by co-culture with stroma, and changed in the reverse direction by the addition of selumetinib. We chose a few genes among the list (BMP2, BNIP3, H2AFX, DUSP2, FZD3, BCL2L1, CHEK2) that are reported to be involved in oncogenesis, chemotherapy resistance, cell growth and survival. Using qRT-PCR, we confirmed bone morphogenic protein 2 (BMP2) to be upregulated in AML cells by stroma, and the effect of stroma was reversed by selumetinib. Further, we confirmed the same change of BMP2 at the protein level by FACS. Smad1, 5 and 8 are transcriptional factors immediately downstream from BMP receptors and play a central role in BMP signal transduction. Using FACS we discovered stroma-induced activation of Smad 1/8 in pediatric AML patient samples, which was partially alleviated by selumetinib and a selective BMP inhibitor, K02288 (10 μM). BMPs are growth factors that belong to the transforming growth factor beta (TGF-beta) superfamily and are thought to be involved in stem cell properties such as self-renewal. To determine if the BMP-Smad pathway plays a role in chemotherapy resistance, 4 pediatric AML patient samples were cultured on or off stromal cells, and treated with cytarabine (10 μM) with or without K02288 (10 μM) for 24h. Cells were analyzed for cytarabine-induced apoptosis with Annexin V staining by FACS, excluding stromal cells and lymphocytes. K02288 treatment did not alter cytarabine-induced apoptosis. We next tested the potential role of BMP-Smad pathway in leukemic self-renewal in pediatric AML samples. Pediatric AML patient samples were plated at 50,000/ml or 100,000/ml in Methocult medium (H-4535) to quantify stem and progenitor cells. Samples were treated with vehicle or K02288 (10 μM). Colonies and viable cells were counted and normalized to control 7-14 days after plating. Harvested cells were stained for CD34, CD14 and CD11b to evaluate differentiation by FACS. Remaining cells were replated at the same density for 2 more rounds. We found that K02288 reduced colony counts (e.g., 100±0% in vehicle control, v. 25±10% in K02288, n=5, p<0.002, post 2nd plating) and decreased the number of viable cells at 2nd and 3rd plating (Figure 1). The BMP inhibitor also promoted differentiation of leukemic stem cell in pediatric AML samples, as evidenced by increased CD14 and CD11b expression. In contrast, for normal pediatric bone marrow samples, K02288 did not change colony counts or the number of viable cells, and it did not promote differentiation. Those data suggested BMP-Smad is likely to contribute to leukemic stemness in pediatric AML without disrupting normal hematopoietic stem cells. Therefore, BMP-Smad pathway may be a promising therapeutic target to reduce leukemia burden and improve survival for children with AML. Disclosures No relevant conflicts of interest to declare.
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