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Vertebrate hematopoietic stem cells are derived from vental mesoderm, which is postulated to migrate to both extra- and intraembryonic positions during gastrula and neurula stages. Extraembryonic migration has previously been documented, but the origin and migration of intraembryonic hematopoietic cells have not been visualized. The zebrafish and most other teleosts do not form yolk sac blood islands during early embryogenesis, but instead hematopoiesis occurs solely in a dorsal location known as the intermediate cell mass (IM) or Oellacher. In this report, we have isolated cDNAs encoding zebrafish homologs of the hematopoietic transcription factors GATA-1 and GATA-2 and have used these markers to determine that the IM is formed from mesodermal cells in a posterior-lateral position on the yolk syncytial layer of the gastrula yolk sac. Surprisingly, cells of the IM then migrate anteriorly through most of the body length prior to the onset of active circulation and exit onto the yolk sac. These findings support a hypothesis in which the hematopoietic program of vertebrates is established by variations in homologous migration pathways of extra- and intraembryonic progenitors.

Whole-genome copy number analysis platforms, such as array comparative genomic hybridization (aCGH) and single nucleotide polymorphism (SNP) arrays, are transformative research discovery tools. In cancer, the identification of genomic aberrations with these approaches has generated important diagnostic and prognostic markers, and critical therapeutic targets. While robust for basic research studies, reliable whole-genome copy number analysis has been unsuccessful in routine clinical practice due to a number of technical limitations. Most important, aCGH results have been suboptimal because of the poor integrity of DNA derived from formalin-fixed paraffin-embedded (FFPE) tissues. Using self-hybridizations of a single DNA sample we observed that aCGH performance is significantly improved by accurate DNA size determination and the matching of test and reference DNA samples so that both possess similar fragment sizes. Based on this observation, we developed a novel DNA fragmentation simulation method (FSM) that allows customized tailoring of the fragment sizes of test and reference samples, thereby lowering array failure rates. To validate our methods, we combined FSM with Universal Linkage System (ULS) labeling to study a cohort of 200 tumor samples using Agilent 1 M feature arrays. Results from FFPE samples were equivalent to results from fresh samples and those available through the glioblastoma Cancer Genome Atlas (TCGA). This study demonstrates that rigorous control of DNA fragment size improves aCGH performance. This methodological advance will permit the routine analysis of FFPE tumor samples for clinical trials and in daily clinical practice.

9519 Background: Hedgehog (Hh) signaling is crucial in the development and homeostasis of many human organs and tissues. Aberrant Hh signaling is involved in tumorigenesis through promotion of cell proliferation, survival, and differentiation in wide range of human cancers, including approximately 30% of MBs. LDE225 is a potent and selective inhibitor of Smo, a key positive regulator of Hh signaling. The phase I is exploring the safety and pharmacokinetics of LDE225 in pediatric patients with advanced solid tumors that are potentially dependent on Hh signaling. Preliminary data from the ongoing phase I are presented. Methods: Dose-escalation was performed according to a Bayesian design starting at 372 mg/m2 of continuous once daily oral LDE225. Safety and preliminary efficacy of LDE225 at the maximum tolerated dose will be assessed in pediatric and adult patients with recurrent MB in a phase II expansion part. Pharmacokinetic profiles were performed at Day 1 and 21. Tumor samples were analyzed for Hh pathway activation status using a 5-gene Hh signature assay. Results: Thirty-three patients (24 MB, 3 rhabdomyoscarcoma [RMS], 3 osteosarcoma, and 1 each of neuroblastoma, gliomatosis and oligoastrocytoma) with a median age of 13 years (range, 4–17 y) have enrolled. Dose-limiting toxicity of Grade 4 creatine phosphokinase elevation occurred in 1 RMS patient out of 7 patients treated at 372 mg/m2. No dose-limiting toxicity was observed at 233 and 425 mg /m2. LDE225 at 233 and 372 mg/m2 was absorbed with a median Tmax of 2 h (range, 1–24 h). Systemic exposures were comparable with adults. Two MB patients achieved a confirmed complete response (CR) at doses of 372 and 425 mg/m2. Analysis of 14 available MB tumor samples using the 5-gene Hh signature assay showed that the 2 CR patients have Hh‑activated tumor. The remaining 12 tumor samples were from patients who did not achieve response and were determined to be Hh pathway non-activated. Conclusions: LDE225 is well tolerated in pediatric patients with advanced malignancies. Preliminary data show promising efficacy in medulloblastoma patients and support the use of the 5-gene Hh signature assay as a pre-selection tool in future trials.

Medulloblastoma (MB), an invasive primitive neuroectodermal tumor of the posterior fossa, is the most common brain tumor in children, comprising ∼20% of childhood and Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4818. doi:1538-7445.AM2012-4818

9541 Background: Temsirolimus (TEMSR), an mTOR inhibitor, prolongs survival in adults with advanced renal cell carcinoma. In part 1 of a phase I/II study of TEMSR in children with advanced solid tumors, 75mg/m2 was determined to be the MTD (Spunt et al. ASPHO 2007). Part 2 of the study explored the safety and efficacy of 75mg/m2 TEMSR in children with neuroblastoma, high grade glioma or rhabdoymyosarcoma. Methods: Part 2 of this open-label study enrolled patients (pts) with a confirmed diagnosis of recurrent/refractory neuroblastoma, high-grade glioma, or rhabdomyosarcoma, with measurable disease. TEMSR 75 mg/m2 was given intravenously once weekly until disease progression or intolerance. Primary efficacy endpoint was objective response (OR; complete response + partial response [PR]) within first 12 weeks. If fewer than 2 ORs occurred after 12 evaluable pts (those who received ≥3 TEMSR doses) were enrolled in one of each tumor types, then enrollment in that tumor type would be stopped for lack of efficacy...

A rare vascular portohepatic anomaly was identified in a Down syndrome patient with a 47,XY,−2,+der(2), + der(21)t(2;21)(p13;q22.1) mat chromosomal complement. This vascular defect involves a direct communication between the right portal vein and the inferior vena cava (IVC). We discuss the possibility that this vascular defect is a rare manifestation in Down syndrome. Alternatively, the existence of these 2 rare events in the same patient raises the possibility that they are causally related. © Wiley-Liss, Inc.

1569 Background: There is preclinical evidence that continuous low-dose chemotherapy may inhibit tumor endothelial proliferation and prevent tumor growth (Browder et al Cancer Res 2000;60:1878). Methods: We conducted a phase II study of continuous low-dose etoposide (VP-16), alternating with cyclophosphamide (CP), in combination with thalidomide (T) and celecoxib (C) in adult patients with recurrent malignant gliomas. There was no limit on the number of prior therapies. Patients received VP-16 [35 mg/m2 (maximum 100 mg) daily for 21 days] alternating with CP [2 mg/kg (maximum 100 mg/day) for 21 days]. Thalidomide was started at 200 mg daily and increased by 100 mg weekly to a maximum of 1200 mg/day, as tolerated. Celecoxib was started at 200 mg twice daily and increased to 400 mg twice daily. MRIs were performed every 6 weeks. Patients were treated until tumor progression or development of unacceptable toxicity. Serum was collected for measurement of angiogenic peptides. Results: 48 patients were enrolled (15 female, 33 male). 28 had glioblastomas (GBM); 20 had anaplastic gliomas (AG). Median age was 53 years (range 33–74); median KPS was 70 (range 60–100). Patients had average of 2.1 prior chemotherapies; 33% had 3 or more prior chemotherapies. Toxicities included neutropenia (8 G3, 8 G4), leukopenia (13 G3, 8 G4), lymphopenia (26 G3), anemia (1 G3), thrombocytopenia (1 G3); nausea (1 G3), vomiting (3 G3), constipation (5 G3; 2 G4), colitis (2 G4), rash (1 G3), dizziness (1 G3); hypoxia (1 G3), and infection (2 G3). 2 patients had DVT and 6 had pulmonary emboli. There were no treatment related deaths. Fatigue was common but usually mild. 12% of patients had PR, 59% had SD, 29% progressed at their first scan. For GBM patients, median progression-free survival (PFS) was 11 weeks, 6 month-PFS was 9% and median survival was 21 weeks. For AG patients, median PFS was 14 weeks; 6 month-PFS was 26% and median survival was 41.5 weeks. Correlation of angiogenic peptide levels and response will be reported. Conclusions: Although there were some responders this regimen did not significantly improve survival in this heavily pretreated group of patients. However, further studies combining metronomic chemotherapy with more potent angiogenesis inhibitors such as lenalidomide or VEGFR inhibitors may be warranted. [Table: see text]

We report a new function for platelets: selective sequestration of tumor-derived angiogenesis regulatory proteins above the concentration of these molecules in plasma. Iodinated VEGF in a Matrigel pellet (from 100 to 600 ng/100 microl), implanted subcutaneously in mice, accumulates almost exclusively in platelets in a dose-dependent manner over a period as long as 2–3 weeks, without raising plasma levels of VEGF. Similarly, platelet VEGF increases in the presence of a single microscopic VEGF-secreting human tumor of up to only 1 mm3 in SCID mice without any increase of VEGF in plasma. In addition to VEGF, other factors such as bFGF, PDGF, BDNF, endostatin and other regulators of angiogenesis are taken up by platelets in a selective and quantifiable manner which is dependent on tumor generation of these molecules. Our data show that these proteins are not simply associated with the platelet surface, but are internalized. Furthermore, they are protected from degradation within the platelet, and are not released by classical degranulating agents, such as thrombin, ADP or epinephrine. Incubation of human platelets with endostatin at above physiological levels results in decrease of the majority of platelet-associated VEGF and bFGF in a concentration-dependent manner. Using SELDI-ToF mass spectroscopy of platelet extracts, we have found that this novel property of platelets enables the detection of microscopic tumors that undetectable by any presently available diagnostic method. The platelet angiogenic profile is more inclusive than a single biomarker because it can detect a wide range of tumor types and tumor sizes. Relative changes in the platelet angiogenic profile permit the tracking of a tumor throughout its development, beginning from an early in situ cancer. Conclusions: (i) While the half-life of mouse platelets is approximately 3 days, the platelet angiogenic profile persists for as long as the tumor (or Matrigel pellet) is present. This indicates that platelets may continuously scavenge proteins which regulate angiogenesis. (ii) The fact that the presence of a human tumor can now be detected at microscopic size, suggests that it may not be necessary to know the type and location of a tumor before initiating treatment, especially since it is feasible to use anti-cancer therapies of little or no toxicity. Figure Figure

SummarySmoothened (SMO) inhibitors recently entered clinical trials for sonic-hedgehog-driven medulloblastoma (SHH-MB). Clinical response is highly variable. To understand the mechanism(s) of primary resistance and identify pathways cooperating with aberrant SHH signaling, we sequenced and profiled a large cohort of SHH-MBs (n = 133). SHH pathway mutations involved PTCH1 (across all age groups), SUFU (infants, including germline), and SMO (adults). Children >3 years old harbored an excess of downstream MYCN and GLI2 amplifications and frequent TP53 mutations, often in the germline, all of which were rare in infants and adults. Functional assays in different SHH-MB xenograft models demonstrated that SHH-MBs harboring a PTCH1 mutation were responsive to SMO inhibition, whereas tumors harboring an SUFU mutation or MYCN amplification were primarily resistant.

In vertebrates, hematopoietic and vascular progenitors develop from ventral mesoderm. The first primitive wave of hematopoiesis yields embryonic red blood cells, whereas progenitor cells of subsequent definitive waves form all hematopoietic cell lineages. In this report we examine the development of hematopoietic and vasculogenic cells in normal zebrafish and characterize defects in cloche and spadetail mutant embryos. The zebrafish homologs of lmo2, c-myb, fli1, flk1, and flt4 have been cloned and characterized in this study. Expression of these genes identifies embryonic regions that contain hematopoietic and vascular progenitor cells. The expression of c-myb also identifies definitive hematopoietic cells in the ventral wall of the dorsal aorta. Analysis of b316 mutant embryos that carry a deletion of the c-myb gene demonstrates that c-myb is not required for primitive erythropoiesis in zebrafish even though it is expressed in these cells. Both cloche and spadetail mutant embryos have defects in primitive hematopoiesis and definitive hematopoiesis. The cloche mutants also have significant decreases in vascular gene expression, whereas spadetail mutants expressed normal levels of these genes. These studies demonstrate that the molecular mechanisms that regulate hematopoiesis and vasculogenesis have been conserved throughout vertebrate evolution and the clo and spt genes are key regulators of these programs.

Using gel retardation and in vitro "footprinting", we have analyzed the interactions between nuclear proteins derived from various mouse cells and the enhancer and interferon response sequences of the H-2Kb gene. We have found that a protein factor binds a site in the enhancer sequence that partially overlaps the interferon response sequence. This factor also binds to a similar sequence lying in the opposite orientation in the promoter of the mouse beta 2-microglobulin gene, suggesting a common regulatory mechanism. Transfection competition experiments indicate that this factor acts as a positive element in the expression of H-2 and beta 2-microglobulin genes.

The degree of erythroid chimaerism from chickens made chimaeric following stem cell infusion as embryos has been determined using a sensitive and simple procedure with monoclonal antibodies (MAbs) specific for erythroid differentiation (B-G) antigens. Highly labelled fluorescent latex beads coupled to specific MAbs have allowed for the detection of very low frequency chimaeric cell subpopulations. A direct correlation between the chimaerism of the bone marrow cells, from which mature RBCs had been eliminated, and the percentage RBC chimaerism in the blood was found. This comprises additional evidence that the B-G bearing bone marrow cells are precursors for mature RBCs.

The major transplantation antigens encoded by class I genes of the major histocompatibility complex (MHC) play a key role in a number of immunological processes, particularly in the recognition of foreign antigens by cytotoxic T cells of the host (Zinkernagel and Doherty 1980). The MHC class I heavy chain (37–45 kd) are non covalently associated on the cell surface with the non-polymorphic beta-2-microglobulin (beta-2m) light chain (12 kd).

Many malignant tumors demonstrate a definite propensity for metastasis to specific organs despite the fact that tumor cells with the potential for metastasis may circulate randomly throughout the body. Current concepts of organ specific metastasis (OSM) center around the generation of tumor cell variants with enhanced capacity for metastasis to specific organs. At present three hypotheses, mechanical, seed and soil, and specific tumor cell adherence (STCA), stand out as possible explanations for OSM. These possible mechanisms of OSM are by no means mutually exclusive. Recent efforts to understand OSM have included the selection of organ-specific metastasizing variants from tumor cell lines and an examination of their surface and metastatic properties. OSM-selected cell lines from many different tumor systems have been used to examine the relative contributions of the three mechanisms. While examples of each mechanism have been reported, the relative contributions of each for different tumor systems may differ substantially. Therefore, generalizations about the behavior of tumors based on studies with just a few tumor lines and systems may not be valid. There is substantial evidence that cell surface molecules are important in the process of OSM and homing of lymphocytes to specific lymph nodes. Monoclonal antibodies have been produced against putative cell surface receptors and initial biochemical characterization has begun. There is much evidence that cell surface glycoconjugates can serve as specific recognition structures on normal cells and in addition, may play important roles in OSM. The role of these carbohydrates is discussed. The chick embryo as a model system is discussed as it offers several advantages for the study of metastasis in general and OSM in particular. A variety of human and murine tumors, including some freshly isolated, have been shown to grow and metastasize in these embryos. Furthermore, cell lines which have been selected for OSM in adults show similar patterns of metastasis in chick embryos indicating that this system may be an especially attractive one for the analysis of OSM.

The pattern of reactivity of 10 monoclonal antibodies (MCA) developed against human lymphoid leukemia cells and tested with human T-cells, B-cells, as well T-lymphoma and B-lymphoma cell lines suggested that six of them detect la-like determinants, three detect HLA-like determinants, and the remaining one detects a non-Ia B-cell antigen. With the use of three binding assays, the six MCA that appeared to detect Ia-like determinants reacted strongly with the human colorectal cancer cell (CCC) line LoVo, and none of the three MCA that reacted with HLA-like determinants reacted with this cell line. Immunoprecipitation and polyacrylamide gel electrophoresis analysis confirmed the apparent specificities of the MCA for Ia-like and HLA molecules, demonstrated the presence of Ia-like molecules in LoVo, and failed to detect HLA in these cells. The cellular enzyme-linked immunosorbent assay was used for the testing of our anti-Ia and anti-HLA MCA with 15 other CCC lines. Marked heterogeneity was found in the expression of different Ia-like and HLA determinants defined by different or overlapping subsets of MCA, which suggested that these determinants might be present on different molecules or that different conformations of the same molecules exist in various CCC lines. Analysis of the surface phenotype of subclones of LoVo cells revealed the presence of stable variant cell subpopulations, which lost reactivity with four out of six of the anti-Ia-like MCA but retained at least one Ia-like molecule recognized by two of our MCA. All of the subclones maintained the HLA-negative phenotype. The possible immunologic and diagnostic consequences of the presence or absence of Ia-like or HLA markers on nonlymphoid tumor cells are discussed.

The MHC class I murine and beta-2-microglobulin genes are silent in embryonal carcinoma (EC) cells but are induced upon differentiation of these cells. We have previously shown that enhancer-like sequences located in the promoter of the H-2Kb gene are non-functional in F9 and PCC3 cells. We have previously purified a 48 kd protein (KBF1) from a mouse T cell line which binds to a palindromic sequence located in this enhancer and to a similar sequence in the promoter of the beta-2-microglobulin gene. We describe here the purification of a second protein (KBF2, 58 kd) which also binds to this sequence. While both activities are present in differentiated cells, KBF1 binding activity is absent in undifferentiated EC cells, where the palindromic sequence shows no enhancer activity. Upon differentiation, KBF1 binding activity is induced and the palindromic sequence becomes active as an enhancer. Thus, the absence of KBF1 activity in undifferentiated EC cells is at least in part responsible for the lack of expression of H-2 class I and beta-2-microglobulin genes in these cells and suggests that KBF1 activity is regulated during differentiation.

We have dissected the mouse H-2Kb gene promoter in order to define the sequences responsible for induction by tumour necrosis factor (TNF-alpha). An enhancer element (-187 to -158) composed of two imperfect direct palindromic repeats has been shown to be necessary and sufficient for TNF-alpha induction of a heterologous promoter. A multimer of either repeat is also responsive, while a single copy is not: this is the situation in the beta 2-microglobulin (beta 2-m) promoter which contains a single palindrome and does not respond to TNF-alpha. We had previously found that the two repeats can bind a factor named KBF1. We show here that in the uninduced state the transcription factor AP2 binds to the interpalindromic region, while in TNF-treated cells an NF kappa B-like activity is induced which displaces both KBF1 and AP2 and binds to the two palindromes. This strongly suggests that induction of an NF kappa B-like activity is responsible for TNF-alpha stimulation of mouse MHC class I genes.

PURPOSE: Progress has been made in the treatment of medulloblastoma, the most common childhood malignant brain tumor: However, many long-term survivors will have posttherapy growth hormone insufficiency with resultant linear growth retardation. Growth hormone replacement therapy (GHRT) may significantly improve growth, but there is often reluctance to initiate GHRT because of concerns of an increased likelihood of tumor relapse. PATIENTS AND METHODS: This study retrospectively reviewed the use of GHRT for survivors of medulloblastoma in 11 neuro-oncology centers in North America who received initial treatment for disease between 1980 and 1993 to determine its impact on disease control. A Landmark analysis was used to evaluate the relative risk of relapse in surviving patients. RESULTS: Five hundred forty-five consecutive patients less than 15 years of age at diagnosis were identified. Six-year progression-free survival (mean ± SD) was 40% ± 5% in children less than 3 years of age at diagnosis compared with 59% ± 3% for older patients. Older patients with total or near-total resections (P = .003) and localized disease at diagnosis (P < .0001) had the highest likelihood of survival. One hundred seventy patients (33% ± 3% of the cohort) received GHRT. GHRT use varied widely among institutions, ranging from 5% to 73%. GHRT was begun a mean of 3.9 years after diagnosis, later in children younger than 3 years at diagnosis (5.4 years). By Landmark analyses, for those surviving 2, 3, and 5 years after diagnosis, there was no evidence that GHRT increased the rate of disease relapse. CONCLUSION: This large retrospective review demonstrates that GHRT is underutilized in survivors of medulloblastoma and is used relatively late in the course of the illness. GHRT is not associated with an increased likelihood of disease relapse.

An enhancer binding factor, designated KBF1, has been purified from the nuclear extract of mouse BW5147 thymoma cells by five column chromatography steps including a sequence-specific DNA affinity column. Gel retardation and footprint analysis have shown that purified KBF1 has a binding activity specific for both H-2 and beta 2-microglobulin enhancer sequences. After SDS-polyacrylamide gel electrophoresis of the most purified preparation a 48-kd protein showed, after elution and renaturation, a binding activity to both enhancer sequences. These findings suggest that the expression of both H-2 and beta 2-microglobulin genes utilizes a common regulatory mechanism.

ConclusionsIn the present minireview we have discussed several types of model systems which have given valuable clues to the ways in which leukemia and lymphoma cells mediate organ specific metastasis. Most patent is that these types of malignancies probably utilize normal receptor-mediated mechanisms for organ specific recognition. The animal models which have been most extensively studied indicate that the following three recognition mechanisms can be employed by metastatic lymphoma variants:(1) Normal organ cells have receptors for determinants on metastatic variants 17(2) metastatic variants have receptors for determinants expressed on endothelial cells 2,19; and(3) an undefined interaction occurs between normal organ cells and metastatic variants 16.A great deal of evidence suggests that organ specific metastasis is at least partially mediated through carbohydrate determinants which are recognized by receptors. It is possible that these receptors may be part of a larger family of recognition molecule...