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B-cell acute lymphoblastic leukemia (B-ALL) is the most common type of cancer found in children. Although the overall survival rates are now >80%, 15%-20% of pediatric patients relapse, with survival rates subsequently dropping to 5%-10%. Cmpd10357, 3-amino-5-arylamino-6-chloro-N- (diaminomethylene) pyrazine-2-carboximide, is a highly potent, cell-permeant compound recently shown to have cytotoxic effects on solid tumors, including human breast cancer and high-grade gliomas, independent of their proliferative status. Cmpd10357 demonstrated concentration-dependent cytotoxicity in two human B-ALL cell lines, JM1 and Reh, at half-maximal inhibitory concentrations (IC50) of 3.2 and 3.3 μM, respectively. Cmpd10357, at a dose of 5 mg/kg, significantly prolonged survival in our B-ALL xenograft mouse model, with a median survival time of 49.0 days compared with 45.5 days in the control group (p < 0.05). The cytotoxicity of Cmpd10357 demonstrated caspase-independent, nonapoptotic cancer cell demise associated with the nuclear translocation of apoptosis-inducing factor (AIF). The cytotoxicity of Cmpd10357 in B-ALL cells was inhibited by Necrostatin-1 but not by Necrosulfonamide. These studies suggest that an AIF-mediated, caspase-independent necrosis mechanism of Cmpd10357 in B-ALL could be used in combination with traditional apoptotic chemotherapeutic agents.

In leukemia, a distinct subpopulation of cancer-initiating cells called leukemia stem cells (LSCs) is believed to drive population expansion and tumor growth. Failing to eliminate LSCs may result in disease relapse regardless of the amount of non-LSCs destroyed. The first step in targeting and eliminating LSCs is to identify and characterize them. Acute precursor B lymphoblastic leukemia (B-ALL) cells derived from patients were incubated with fluorescent glucose analog 2-(N-(7-Nitrobenz-2-oxa-1, 3-diazol-4-yl) Amino)-2-Deoxyglucose (NBDG) and sorted based on NBDG uptake. Cell subpopulations defined by glucose uptake were then serially transplanted into mice and evaluated for leukemia initiating capacity. Gene expression profiles of these cells were characterized using RNA-Sequencing (RNA-Seq). A distinct population of NBDG-low cells was identified in patient B-ALL samples. These cells are a small population (1.92% of the entire leukemia population), have lower HLA expression, and are smaller in size (4.0 to 7.0 μm) than the rest of the leukemia population. All mice transplanted with NBDG-low cells developed leukemia between 5 and 14 weeks, while those transplanted with NBDG-high cells did not develop leukemia (p ≤ 0.0001-0.002). Serial transplantation of the NBDG-low mouse model resulted in successful leukemia development. NBDG-medium (NBDG-med) populations also developed leukemia. Interestingly, comprehensive molecular characterization of NBDG-low and NBDG-med cells from patient-derived xenograft (PDX) models using RNA-Seq revealed a distinct profile of 2,162 differentially-expressed transcripts (DETs) (pin vivo, showing leukemia initiating capacity. Our study provides insight into the biologic mechanisms of B-ALL initiation and survival.

Neuroblastoma (NB) is the most common extracranial solid tumor in children. The outcomes for aggressive forms of NB remain poor. The aim of this study was to develop a new molecular-targeted therapy for NB using an antisense oligonucleotide (ASO) and superparamagnetic iron oxide (SPIO) nanoparticles (NPs), as a delivery vehicle, targeting the transcription regulator MAX dimerization protein 3 (MXD3). We previously discovered that MXD3 was highly expressed in high-risk NB, acting as an anti-apoptotic factor; therefore, it can be a good therapeutic target. In this study, we developed two ASO-NP complexes using electrostatic conjugation to polyethylenimine-coated SPIO NPs and chemical conjugation to amphiphilic polymers on amine-functionalized SPIO NPs. Both ASO-NP complexes demonstrated MXD3 knockdown, which resulted in apoptosis in NB cells. ASO chemically-conjugated NP complexes have the potential to be used in the clinic as they showed great efficacy with minimum NP-associated cytotoxicity.

BackgroundNeuroblastoma is the second most common extracranial cancer in children. Current therapies for neuroblastoma, which use a combination of chemotherapy drugs, have limitations for high-risk subtypes and can cause significant long-term adverse effects in young patients. Therefore, a new therapy is needed. In this study, we investigated the transcription factor MXD3 as a potential therapeutic target in neuroblastoma.MethodsMXD3 expression was analyzed in five neuroblastoma cell lines by immunocytochemistry and quantitative real-time reverse transcription PCR, and in 18 primary patient tumor samples by immunohistochemistry. We developed nanocomplexes using siRNA and superparamagnetic iron oxide nanoparticles to target MXD3 in neuroblastoma cell lines in vitro as a single-agent therapeutic and in combination with doxorubicin, vincristine, cisplatin, or maphosphamide - common drugs used in current neuroblastoma treatment.ResultsMXD3 was highly expressed in neuroblastoma cell lines and in patient tumors that had high-risk features. Neuroblastoma cells treated in vitro with the MXD3 siRNA nanocomplexes showed MXD3 protein knockdown and resulted in cell apoptosis. Furthermore, on combining MXD3 siRNA nanocomplexes with each of the four drugs, all showed additive efficacy.ConclusionThese results indicate that MXD3 is a potential new target and that the use of MXD3 siRNA nanocomplexes is a novel therapeutic approach for neuroblastoma.

MXD3 is a transcription factor that plays an important role in proliferation of human DAOY medulloblastoma cells. Here, we demonstrate that MXD3 is highly enriched in human precursor B acute lymphoblastic leukemia (preB ALL) samples compared to mobilized peripheral blood mononuclear cells, bone marrow, or hematopoietic stem cells from healthy donors. MXD3 knock-down in the preB ALL cell line Reh resulted in decreased cell numbers with no change in G0/G1, S or G2/M populations but increased apoptosis compared to control cells. Our results suggest that MXD3 is important for survival of Reh preB ALL cells, possibly as an anti-apoptotic factor.

Precursor B-cell acute lymphoblastic leukemia (preB ALL) is the most common childhood cancer.[1,2] Despite improved overall progress in the treatment, patients with certain types of preB ALL have a...

Whole-exome sequencing identifies a novel somatic mutation in MMP8 associated with a t(1;22)-acute megakaryoblastic leukemia

Targeted therapies, such as those using imatinib and rituximab, have revolutionized the treatment of Philadelphia chromosome-positive and CD20-positive acute lymphoblastic leukemia (ALL) respectively, yet these therapies are effective in only a subset of patients and remission is generally not durable. The next generation of targeted therapies includes the use of antibodies conjugated to potent cytotoxic agents and are classified as antibody drug conjugates (ADC). For B-lineage ALL, CD22 is an ideal target for ADC therapy because it is expressed on the majority of B-lineage ALL cells and because antibody binding mediates receptor internalization. HB22.7-SAP is a conjugate of our anti-CD22 monoclonal antibody (mAb), HB22.7, and the ribosome inhibiting protein, saporin (SAP). In vitro, HB22.7-SAP effectively bound to CD22 on the surface of pre-B ALL cell lines and exhibited potent and specific cytotoxicity. In a NOD/SCID xenograft mouse model of pre-B ALL, when compared to the vehicle-treated control, HB22.7-SAP increased the median survival time from 20 days to over 50 days without significant toxicity.

The exponential rise in molecular and genomic data has generated a vast array of therapeutic targets. Oligonucleotide-based technologies to down regulate these molecular targets have promising therapeutic efficacy. However, there is relatively limited success in translating this into effective in vivo cancer therapeutics. The primary challenge is the lack of effective cancer cell-targeted delivery methods, particularly for a systemic disease such as leukemia. We developed a novel leukemiatargeting compound composed of a monoclonal antibody directly conjugated to an antisense oligonucleotide (ASO). Our compound uses an ASO that specifically targets the transcription factor MYC-associated factor X (MAX) dimerization protein 3 (MXD3), which was previously identified to be critical for precursor B-cell (preB) acute lymphoblastic leukemia (ALL) cell survival. The MXD3 ASO was conjugated to an anti-cluster of differentiation-22 (CD22) antibody (αCD22 Ab) that specifically targets most preB ALL. We demonstrated that the αCD22 Ab-ASO conjugate treatment showed MXD3 protein knockdown and leukemia cell apoptosis in vitro. We also demonstrated that the conjugate treatment showed cytotoxicity in normal B cells, but not in other hematopoietic cells, including hematopoietic stem cells. Furthermore, the conjugate treatment at the lowest dose tested (0.2 mg/kg Ab for 6 doses — twice a week for 3 wks) more than doubled the mouse survival time in both Reh (median survival time 20.5 versus 42.5 d, p < 0.001) and primary preB ALL (median survival time 29.3 versus 63 d, p < 0.001) xenograft models. Our conjugate that uses αCD22 Ab to target the novel molecule MXD3, which is highly expressed in preB ALL cells, appears to be a promising novel therapeutic approach.

Primary pleural rhabdomyosarcoma is an extremely rare intrathoracic malignancy. We present a case of a previously healthy 2-year-old male complaining of cough and shortness of breath. The plain film, CT and MRI descriptions of this pleural tumor are presented. This is a fast growing tumor that is indistinguishable radiographically from other large intrathoracic tumors such as pleuropulmonary blastoma.

Some reports published from 1967 to 1999 describe the use of ointments containing high doses (0.1 to 0.2%, w/w) C. asiataica herb extracts to enhance wound repair. Lower doses at which burn wound repair is enhanced by such topical applications have not been established yet. We found that the application of asiaticoside at low doses of 10(-8) to 10(-12)% (w/w) facilitated burn wound repair. To clarify the accelerating mechanisms of asiaticoside on burn wound repair, we examined the effects of asiaticoside on the levels of various cytokines produced at the site of the burn wound. The topical application of a low dose (10 pg, 1 ng, or 100 ng/wound area) of asiaticoside increased monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF), and interleukin (IL)-1beta levels in burn wound exudates. Asiaticoside (10 pg to 100 ng/ml) enhanced MCP-1 production in HaCaT cells, but it had no direct effect on VEGF production. Furthermore, asiaticoside (10 pg to 100 ng/ml) increased the IL-1beta production in THP-1 macrophages with MCP-1, but it had no effect on IL-1beta production without MCP-1 or with lipopolysaccharide (LPS). These findings suggest that the enhancement of burn wound healing by asiaticoside might be due to the promotion of angiogenesis during skin wound repair as a result of the stimulation of VEGF production caused by the increase in MCP-1 expression in keratinocytes and the increase in IL-1beta expression in macrophages induced cooperatively by asiaticoside plus MCP-1.

Field experiment on long-term application of chemical fertilizers and farmyard manure is ongoing in a floodplain soil of Bangladesh since 1978. The treatments are application of N, NP, NK, NPK, NS, NZn, NSZn, NPKSZn, and N+FYM (cowdung as farmyard manure), along with control (no addition), and have been executed in every cropping. Forty-five times of paddy-rice cropping was done before soil sampling in July 2002. Among chemical properties of soil, the organic matter and total N concentrations, clay mineralogical composition, and the heavy metal concentration were assessed. The organic matter and total N concentrations of surface soil were increased from those in the initial state, although this was ascribed to continuous cropping of paddy-rice rather than the direct effect of chemical fertilizers and farmyard manure. Clay mineralogical change, such as fixation of K in the vermiculite interlayer to be transformed into clay mica, was not detected. Accumulation of heavy metals in soil was not noticed.

Mucopolysaccharidosis I is a lysosomal storage disorder caused by mutations in the IDUA gene, resulting in deficiency of α-l-iduronidase and accumulation of glycosaminoglycans. Bone marrow transplantation has been the only available therapy, soon to be joined by enzyme replacement. We have tested retroviral gene therapy in a knockout mouse model of the disease. Bone marrow from Idua −/− male donor mice was transduced with human IDUA cDNA in an MND vector and transplanted into 6–8-week-old, lethally irradiated female Idua −/− mice. Sham-treated mice received Idua −/− bone marrow that was either unmodified or transduced with eGFP. Unmodified Idua +/+ (wild type) bone marrow was transplanted for comparison. Recipient mice were sacrificed 2–6 months after transplantation. Three biochemical parameters were used to gauge therapeutic success: appearance of α-l-iduronidase activity, reduction of β-hexosaminidase activity and reduction of soluble glycosaminoglycan accumulation. Transplantation of unmodified +/+ bone marrow was effective in reducing storage in liver and spleen, but not in kidney or brain. The level of α-l-iduronidase activity achieved by transplantation of IDUA -transduced bone marrow varied greatly between experiments. But even modest activity resulted in correction of pathology of kidney, bladder epithelium, fibrocartilage, choroid plexus, and thalamus, as seen by light microscopy, while electron microscopy showed the presence of some normal neurons in the cortex. The partial correction of brain pathology is attributed to migration of donor hematopoietic cells, demonstrated by the presence of the Y chromosome and of normal microglia in the brain of mice receiving IDUA cDNA.

We report a child with thrombotic thrombocytopenic purpura (TTP) secondary to systemic lupus erythematosus. The diagnosis was confirmed by low ADAMTS13 activity (5%) along with the presence of a low titer inhibitor. Her clinical course was complicated by systemic lupus erythematosus, immunosuppressant therapy, and septic shock. She responded to plasma exchange and ADAMTS13 activity levels recovered. This case illustrates the heterogeneity of TTP and the difficulty of making a diagnosis of TTP. ADAMTS13 activity assay can be useful in the differential diagnosis of diseases with clinical features of thrombotic microangiopathy in pediatric patients. However, treatment needs to be decided carefully case-by-case.

Conventional chemotherapy for precursor B-cell (preB) acute lymphoblastic leukaemia (ALL) has limitations that could be overcome by targeted therapy. Previously, we discovered a potential therapeutic molecular target, MDX3 (MAX dimerization protein 3), in preB ALL. In this study, we hypothesize that an effective siRNA therapy for preB ALL can be developed using antiCD22 antibody (αCD22 Ab) and nanoparticles. We composed nanocomplexes with super paramagnetic iron oxide nanoparticles (SPIO NPs), αCD22 Abs and MXD3 siRNA molecules based on physical interactions between the molecules. We demonstrated that the MXD3 siRNA-αCD22 Ab-SPIO NP complexes entered leukaemia cells and knocked down MXD3, leading the cells to undergo apoptosis and resulting in decreased live cell counts in the cell line Reh and in primary preB ALL samples in vitro. Furthermore, the cytotoxic effects of the MXD3 siRNA-αCD22 Ab-SPIO NP complexes were significantly enhanced by addition of the chemotherapy drugs vincristine or doxorubicin. We also ruled out potential cytotoxic effects of the MXD3 siRNA-αCD22 Ab-SPIO NP complexes on normal primary haematopoietic cells. Normal B cells were affected while CD34-positive haematopoietic stem cells and non-B cells were not. These data suggest that MXD3 siRNA-αCD22 Ab-SPIO NP complexes have the potential to be a new targeted therapy for preB ALL.

Background: Alkylator resistance contributes to treatment failure in high-risk neuroblastoma. Buthionine sulfoximine (BSO) can deplete glutathione and synergistically enhance in vitro sensitivity to the alkylating agent melphalan (L-PAM) for many neuroblastoma cell lines, but optimal use of this combination needs to be defined because clinical responses have been less frequent and not durable. Patients and Methods: The authors established and characterized a neuroblastoma cell line (CHLA-171) from a patient who died of progressive disease after treatment with BSO and low-dose LPAM. Results: CHLA-171 lacks MYCN amplification, expresses PGP (P-glycoprotein) 9.5 RNA, and shows cell surface antigen expression (human leukocyte antigen class I weakly positive, but HSAN 1.2 (hybridoma, SAN 1.2) and anti-GD2 (anti-ganglioside GD2 antibody) strongly positive) characteristic of neuroblastoma cell lines. Twenty-four hours of BSO treatment (0–1,000 mol/L) maximally depleted CHLA-171 glutathione to 36% of baseline. The cytotoxic response of CHLA-171 to BSO and L-PAM, alone and in combination, was measured by digital image microscopy (DIMSCAN) over a range of drug concentrations and compared with drug levels obtained in the patient during BSO/L-PAM therapy. As single agents, CHLA-171 was highly resistant to LPAM (LD90 42 mol/L; peak plasma concentration in the patient equals 3.9 mol/L) and moderately resistant to BSO (LD90 509 mol/L; steady-state concentration in the patient equals 397 mol/L). Treatment with a 10:1 (BSO:L-PAM) fixed ratio combination synergistically overcame resistance (3–4 logs of cell kill, combination index

Neuroblastoma is the second most common solid tumor in children. Most tumors arise in the adrenal glands or paravertebral region. Rarely, patients present with metastatic disease but no primary site can be found despite extensive imaging. We report here a patient with a large periorbital bone metastasis and bone marrow involvement but with no known primary site.

Background Despite intensive-alkylator based regimens, >50% of patients with high-risk neuroblastoma (NB) die from recurrent disease that is probably due, in part, to acquired alkylator resistance. Procedure Using buthionine sulfoximine (BSO)-mediated, glutathione (GSH) depletion to modulate melphalan (L-PAM) resistance, we examined six NB cell lines established after progressive disease following either standard chemotherapy, BSO/L-PAM therapy, or myeloablative therapy and autologous hematopoietic stem cell transplant (AHSCT). Results Four of the six cell lines (three p53-nonfunctional and one p53-functional) showed high-level L-PAM resistance. Conclusions Fixed ratio analysis demonstrated BSO/L-PAM synergy (combination index >1) for all cell lines tested. In L-PAM-resistant cell lines, the minimal cytotoxicity observed for BSO combined with nonmyeloablative concentrations of L-PAM was markedly enhanced (>4 logs total cell kill) when BSO was combined with myeloablative concentrations of L-PAM. In alkylator-resistant NB, the optimal use of BSO may require dose escalation of L-PAM to levels requiring AHSCT.

Of 29 infants with acute myeloid leukemia (AML), 14 (48%) had various 11q23 translocations. MLL rearrangements were examined in 21 of the 29 patients, and 11 (52%) showed the rearrangements. 11q23 translocations and/or MLL rearrangements were found in 17 (58%) of the 29 patients. While all but one of the 17 patients with 11q23/MLL rearrangements had M4 or M5 type of the FAB classification, the 12 patients without such rearrangements had various FAB types, including M2, M4, M4EO, M6 and M7. Of the 12 patients with other chromosome abnormalities or normal karyotypes, two had inv(16) ort(16;16), one had t(1;22)(p13;q13), and two had a novel translocation, t(7;12)(q32;p13). The breakpoint on 12p of the t(7;12) was assigned to intron 1 or the region just upstream of exon 1 of the TEL/ETV6 gene by fluorescence in situ hybridization. The event-free survival at 5 years for the 17 patients with 11q23/MLL rearrangements was 42.2%, and that for the 12 patients without such rearrangements was 31.3% (P = 0.5544). 11q231MLL rearrangements have been frequently reported and a poor prognosis in infant acute lymphoblastic leukemia implied. Our study showed that while 11q23/MLL rearrangements were also common in infant AML, AML infants with such rearrangements had a clinical outcome similar to that of AML infants without such rearrangements.

Green fluorescent protein (GFP) is a widely used intracellular reporter molecule to assess gene transfer and expression. A potential use for GFP is as a co-expressed marker, to select and enrich gene-modified cells by flow cytometry. Processed peptides derived from GFP and presented by the major histocompatibility complex on the cell surface could potentially induce T cell immune responses against GFP+ cells. Thus, clinical application of GFP is premature, since in vivo studies on its immunogenicity are lacking. Therefore, we investigated immune responses against EGFP (enhanced-GFP) in two transplantable murine models: the BALB/c (H-2d) BM185 pre-B leukemia and the C57BL/6 (H-2b) EL-4 T cell lymphoma. BM185 and EL-4 cell lines modified to express high levels of EGFP showed drastic reduction of disease development when transplanted into immunocompetent mice. BM185/ EGFP did lead to rapid development of disease in immunodeficient Nu/Nu mice. Mice surviving BM185/EGFP leukemia challenge developed high cytotoxic T lymphocyte (CTL) responses against EGFP-expressing cells. Furthermore, immune stimulation against BM185/EGFP cells could also be induced by immunization with EGFP+ transduced dendritic cells. The effects of the co-expression of EGFP and immunomodulators (CD80 plus GM-CSF) were also investigated as an irradiated leukemia vaccine. EGFP co-expression by the vaccine did not interfere with the development of CTLs against the parental leukemia or with the anti-leukemia response in vivo. These results indicate that the immune response against EGFP may interfere with its applicability in gene insertion/replacement strategies but could potentially be employed for leukemia cell vaccines.

In a study of FHIT gene abnormalities by reverse transcription‐polymerase chain reaction (RT‐PCR) and sequence analysis of the PCR products, we found normal and abnormal PCR products in 11 osteosarcomas, one osteosarcoma cell line and 3 Ewing sarcomas, and a normal PCR product only in 5 osteosarcomas and 8 Ewing sarcomas. Sequence analysis of the abnormal PCR products revealed 7 osteosarcomas lacking exons 4 to 6, 7, 8 or 9, two lacking exons 5 to 7 or 10, and two lacking exons 6 to 8 or 10. In the aberrant transcripts of the 11 osteosarcomas, fusion had occurred in the exon/intron junctions in 2 tumors, between a segment within an exon and a complete exon in 3, and between segments within exons in 6. The 3 Ewing sarcomas had lost exon 4 or 5 to exon 6 or 10, and fusion had occurred in the exon/intron junction in one, and between segments within exons in 2. These findings suggest that both abnormal or variant splicing and other mechanisms such as genomic instabilities in the FHIT locus may have resulted in the expression of aberrant transcripts. One osteosarcoma and one cell line established from this osteosarcoma showed different abnormal FHIT transcripts, indicating that the tumor cells with the initial aberrant transcripts may not have had a selective advantage for proliferation. The FHIT abnormalities did not seem to be correlated with lung metastasis or a poor clinical outcome in our patients with osteosarcoma or Ewing sarcoma.

We report a case of a 20-year-old Japanese female with recurrent anaplastic large cell lymphoma (ALCL) associated with Sjögren's syndrome (SjS). She was first diagnosed to have ALCL presenting with axillary lymphadenopathy, which within a month underwent spontaneous remission, at the age of 12 years. Eight years later she developed left inguinal lymphadenopathy with clinical overt sicca symptoms associated with elevated serum IgG, interleukin (IL)-1beta and IL-6 levels. Lymph node biopsy was now diagnostic of ALCL characterized by large pleomorphic CD30+ blast cells with the specific chromosomal abnormality, t(2;5)(p23;q35). In contrast to this the salivary gland and renal biopsy revealed infiltration of small lymphocytes, morphologically and cytogenetically distinct from the ALCL cells. Interestingly, SjS symptomatology correlated with disease activity of ALCL and based on an association with elevated IgG and IL-6 levels, suggesting that the concurrence of these two diseases could be more than coincidental. To the best of our knowledge, this is the first reported case of ALCL presenting concurrently with SjS.

CBP, which is located on 16p13 and encodes a transcriptional adaptor/coactivator protein, has been shown to fuse by the t(8;16)(p11;p13) translocation to MOZ on 8p11 in acute myeloid leukemia. We found a t(11;16)(q23;p13) in a child with therapy-related chronic myelomonocytic leukemia. Subsequent reverse transcriptase-polymerase chain reaction and direct sequencing analyses revealed the MLL-CBP fusion transcript in CMML cells. Because 11q23 translocations involving MLL and t(8;16) involving MOZ and CBP have been reported in therapy-related leukemias, both the MLL and CBP genes may be targets for topoisomerase II inhibitors. Accordingly, we believe that most t(11;16)-associated leukemias may develop in patients who have been treated with cytotoxic chemotherapy for primary malignant diseases.

We studied MLL rearrangements in five patients with myeloid hematologic malignancies with trisomy 11. Two had acute monocytic leukemia (AMoL), one had chronic myelomonocytic leukemia, one had refractory anemia, and the other had juvenile chronic myelogenous leukemia. Only one patient, a 15-year-old boy with AMoL and simple trisomy 11, showed rearrangement of MLL. He did not respond to chemotherapy, and successfully underwent bone marrow transplantation, but suffered a relapse 22 months later. Reverse transcription-polymerase chain reaction (RT-PCR) and sequencing analyses of bone marrow cells revealed a tandem duplication of MLL, and his relapse was predictable by sequential RT-PCR studies before it was clinically evident. Of 16 acute myeloid leukemia patients with trisomy 11 and rearrangement of MLL reported, our patient was the youngest in age and the only one with AMoL.

The EWS gene is fused in Ewing sarcoma-like tumors by a chromosomal translocation to one of the four ETS-family genes: FLI1, ERG, ETV1, and E1AF. The orientation of EWS and FLI1 on chromosomes 22 and 11, respectively, is 5' centromeric and 3' telomeric, whereas that of ERG on chromosome 21 is the reverse. Although 10% of Ewing-family tumors express the EWS-ERG fusion transcript, there have been no reports on tumors with t(21;22)(q22;q12) identified by banding cytogenetics. We found the karyotype 50, XY, +8, +8, +12, +mar in all metaphase cells from a tumor. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis performed on the tumor and direct sequencing of the products identified the EWS-ERG fusion transcript. Subsequent two-color fluorescence in situ hybridization (FISH) analysis with EWS and ERG clones showed the fused signals on the der(21) chromosome, but no ERG signals on the chromosome 22 homologs. Thus, our RT-PCR and FISH analyses indicated that the chromosome 22 fragment containing the 5' portion of EWS had been inverted and inserted into chromosome 21 and had fused to the 3' portion of ERG. This subtle chromosome aberration could not be identified by routine cytogenetics. A chromosomal inversion/insertion has also been described in acute leukemia with the MLL-AF10 fusion gene, and this may be a common pathway for producing fusion of reverse-oriented genes in leukemias and solid tumors.

We studied 116 patients (93 children and 23 adults) with acute lymphoblastic leukaemia (ALL) using fluorescence in situ hybridization (FISH) with the yeast artificial chromosome (YAC) clone, 964c10, which includes the recently described ETS-like gene, TEL, on 12p13. FISH revealed that nine of the patients had a t(12 ;21), which had not been previously detected. The nine patients were all children, seven boys and two girls, aged 1-10 years (median 3 years), had an early B immunophenotype, and achieved complete remission, although two of them experienced haematological relapse. In addition to the t(12 :21), FISH also revealed that three of the nine had a del(12p) in the other homolog of chromosome 12 or in the der(12) chromosome itself, and that two others had 12p translocations in the other chromosome 12 homolog. Although chromosomal rearrangements associated with the t(12 ;21) were heterogenous and complex, fusion of the sequences from chromosomes 12 and 21 on the der(21)t(12 ;21) chromosomes was consistent, suggesting that the TEL-AML1 gene fusion on the der(21) chromosome may be critical in leukaemogenesis and that FISH or reverse transcriptase-polymerase chain reaction (RT-PCR) targeted to the chimaeric sequences on the der(21) will be most useful in detecting the t(12 :21) or following a patient with the t(12 ;21), which is one of the most frequent chromosomal rearrangements in both Caucasian and Asian childhood ALL.

Summary. In a study of 23 patients with t(8;21)-associated acute myeloid leukaemia the AML1-MTG8 fusion transcript was present in the majority of serial samples obtained from 17 patients followed for up to 34 months after diagnosis, but was absent in samples from all six patients who had been in continuous complete remission for 61 months after allogeneic bone marrow transplantation (BMT), or for 52, 53, 123,182 and 198 months, respectively, after courses of intensive chemotherapy. Previous studies showed that the AML1-MTG8 fusion transcript was present in most patients with this type of translocation in long-term remission. Our results indicate that blood cells of patients with t(8;21) in remission of over 10 years may not show the AML1-MTG8 fusion transcript, and that those of patients who have undergone allogeneic BMT or intensive chemotherapy may become fusion transcript-negative much earlier. Our study suggests that leukaemic cells with the AML1-MTG8 fusion transcript may survive for some time after courses of chemotherapy or BMT, but that they may eventually be eradicated by immunologic and other antileukaemic mechanisms.

The goal of our project is to develop a new therap y for childhood malignancies using nanoformulated siRNA targeting Mxd3, a molecule in the Sonic Hedgehog signali ng pathway, which we belie ve is important for cell survival. We plan to use cancer-s pecific ligands and superparamagnetic iron oxide nanoparticles (SPIO NPs) to carry siRNA. This delivery system will be tested in mouse xenograft models that we developed with primary cancer tissues. Our current focus is acute lymphoblastic leukemia (ALL), the most common cancer in children. We report our progress to date. Keywords: siRNA, nanoparticles, acute ly mphoblastic leukemia, leukemia-speci fic ligand, targeted therapy 1. INTRODUCTION 1.1 Childhood acute lymphoblastic leukemia and current treatment Leukemia is the most common cancer in children, with B-cell type ALL being the most common type (Figure 1 ) [1,2]. Despite the progress in medicine, certain types of ALL continue to have poor outcomes, with survival rates as low as 30% [2]. Current standard treatments take 2 ½ to 3 years and include systemic chemotherapy or radiation therapy. These treatments ar e highly toxic to growing ch ildren, as they inevitably damage healthy cells along with the leukemia cells. These toxicities are not limited to short-term side effects, but also result in long-term irreversible side effects th at decrease the quality of life of children who survive. These long-term effects involve multi-organ systems leading to poor growth, osteoporosis, infertility, heart failure, and secondary cancers [3]. To overcome the limitations and significant side effects of current treatments, we need new approaches that specifically target leukemia cells. A few promising targeted therapies have been clinically tested in different le ukemias, such as a BCR-ABL ty rosine kinase inhibitor in chronic myeloid leukemia (and in BCR-ABL positive ALL) and all-trans retinoic acid in acute myeloid leukemia [7]. However, no targeted therapy h as been applied exclusively for childhood ALL.

The goal of our project is to develop a unique ligand-conjugated nanoparticle (NP) therapy against childhood acute lymphoblastic leukemia (ALL). LLP2A, discovered by Dr. Kit Lam, is a high-affinity and high-specificity peptidomimetic ligand against an activated α4β1 integrin. Our study using 11 fresh primary ALL samples (10 precursor B ALL and 1 T ALL) showed that childhood ALL cells expressed activated α4β1 integrin and bound to LLP2A. Normal hematopoietic cells such as activated lymphocytes and monocytes expressed activated α4β1 integrin; however, normal hematopoietic stem cells showed low expression of α4β1 integrin. Therefore, we believe that LLP2A can be used as a targeted therapy for childhood ALL. The Lam lab has developed novel telodendrimer-based nanoparticles (NPs) which can carry drugs efficiently. We have also developed a human leukemia mouse model using immunodeficient NOD/SCID/IL2Rγ null mice engrafted with primary childhood ALL cells from our patients. LLP2A-conjugated NPs will be evaluated both in vitro and in vivo using primary leukemia cells and this mouse model. NPs will be loaded first with DiD near infra-red dye, and then with the chemotherapeutic agents daunorubicin or vincristine. Both drugs are mainstays of current chemotherapy for childhood ALL. Targeting properties of LLP2A-conjugated NPs will be evaluated by fluorescent microscopy, flow cytometry, MTS assay, and mouse survival after treatment. We expect that LLP2A-conjugated NPs will be preferentially delivered and endocytosed to leukemia cells as an effective targeted therapy.

Diagnosis and treatment of Candida albicans endocarditis can be difficult. We report a case of this rare condition in which a patient on oral fluconazole presented with septic pulmonary emboli without initial echocardiographic evidence of vegetation. Rapid attainment of a tissue diagnosis, along with combined medical surgical treatment proved to be effective for this patient.

Precursor B cell acute lymphoblastic leukemia (preB ALL) is the most common childhood cancer, as well as the leading cause of childhood cancer-related mortality. Despite overall progress in treatment, certain types of patients with preB ALL have a dismal prognosis with an overall survival of 30%. In addition, current approaches predispose these young patients to late effects, including secondary malignancies. Therefore, more efficacious and less toxic approaches are needed. Targeted therapy for leukemia has the potential to reduce off-target effects, thus minimizing toxicity and late effects and improving efficacy. Monoclonal antibodies (mAbs) have proven utility in leukemia therapy based on their ability to specifically target the leukemic clone and minimize off target effects. However, mAbs are generally not adequate as single agents because of limited efficacy. Antibody drug conjugates (ADCs) provide a method to deliver a potent toxin to the interior of antigen-positive tumor cells. CD22 is an ideal target for ADC-mediated therapeutics for B-cell malignancies because 1) there is high CD22 expression (more than 90%) in B-cell type ALL and 2) CD22 undergoes rapid internalization upon mAb binding. In this study, we evaluated the anti-CD22 (aCD22) mAb as a vehicle for the targeted delivery of Monomethyl Auristatin E (mMAE), a derivative of the cytotoxic tubulin modifier auristatin E. Figure 1 Treatments and outcome of the animals Figure 1. Treatments and outcome of the animals First, we assessed the in vitro cytotoxicities of the aCD22 mAb-mMAE in preB ALL cell lines Reh and JM1. MTS assay showed that IC50 doses of aCD22 mAb-mMAE were 0.7nM and 1nM in Reh and JM1, respectively. Next, we assessed in vivo therapeutic efficacy of the aCD22 mAb-mMAE in a pre-clinical xenograft animal model of preB ALL, using a primary leukemia sample which was confirmed to be CD22 positive. Age matched female NOD/SCID/IL2Rg-/- (NSG) mice were randomly assigned to 4 treatment groups (n=8 per group): 1) PBS, 2) free mMAE (0.165mg/kg), 3) free aCD22 mAb (7.335mg/kg), and 4) aCD22 mAb-mMAE conjugate (7.5mg/kg). The dose of free aCD22 mAb and free mMAE was equivalent to those of each component in the aCD22 mAb-mMAE conjugate. Five million leukemia cells were inoculated per mouse via intra-bone marrow injection. Twenty four hours after leukemia inoculation, animals started receiving weekly iv treatments for 3 weeks. When compared to controls (PBS, free aCD22 mAb or mMAE treatments), the treatment with the aCD22 mAb-mMAE conjugate increased the median survival time of the mice by two fold (Figure. PBS vs. aCD22 mAb-mMAE p In conclusion, we demonstrated that aCD22 mAb-mMAE is efficacious in a preclinical preB ALL xenograft mouse model. Disclosures No relevant conflicts of interest to declare.

Precursor B cell (preB) acute lymphoblastic leukemia (ALL) is the most common cancer in children. Despite aggressive treatment, current approaches for preB ALL have significant limitations with a cure rate of only 30% in certain subtypes. In addition, long-term survivors are at risk for late effects that include development of secondary malignancies and toxicity to visceral organs. Therefore, there is a desperate need for more effective and less toxic therapy. Treatments and outcome of the animals. Dash-dots: PBS, dots: MXD3 ASO and aCD22 Ab unconjugated, gray solid: aCD22 Ab-ASO conjugates at 0.2mg of Ab/kg/dose, and black solid: aCD22 Ab-ASO conjugates at 1mg of Ab/kg/dose Treatments and outcome of the animals. Dash-dots: PBS, dots: MXD3 ASO and aCD22 Ab unconjugated, gray solid: aCD22 Ab-ASO conjugates at 0.2mg of Ab/kg/dose, and black solid: aCD22 Ab-ASO conjugates at 1mg of Ab/kg/dose Previously, we demonstrated that the transcription factor MXD3 is a critical regulator of preB ALL cell proliferation. Knockdown of MXD3 expression in preB ALL cells resulted in cell death as determined by annexin V and caspase assays (Satake et al., British Journal of Haematology, in press). We hypothesize that targeted delivery of an MXD3 therapeutic to preB ALL cells will increase therapy effectiveness and decrease off target effects and toxicity, thus increasing the quality of life. In the current study, we developed an antisense oligonucleotide (ASO) that specifically targets MXD3. Taking advantage of anti-CD22 antibody (aCD22 Ab), which specifically targets B cell ALL, we developed aCD22 Ab-MXD3 ASO conjugates as a potential combined phenotypic and molecularly targeted therapeutic. In in vitro tests, using the Reh ALL cell line, we confirmed successful delivery of the aCD22 Ab-MXD3 ASO compound to leukemia cells. This correlated with MXD3 knockdown at the protein level, and inhibition of leukemia cell growth. Cytotoxicity was tested on anonymized healthy donor normal blood cells, including CD34 positive hematopoietic stem cells (HSCs), B cells, and non-B cells from mobilized peripheral blood mononuclear cells. As expected, cytotoxicity was observed in normal B cells, but CD34 HSCs and non-B cells were unaffected. In combination with conventional preB ALL chemotherapy drugs (vincristine or doxorubin), we observed additive cytotoxic effects of aCD22 Ab-MXD3 ASO conjugates on cells in vitro. We determined the compound effectiveness in pre-clinical xenograft animal models of preB ALL, using both the Reh cell line as well as a primary leukemia sample. Age matched female NOD/SCID/IL2Rg-/- (NSG) mice were randomly assigned to 4 treatment groups: 1) PBS, 2) MXD3 ASO and aCD22 Ab unconjugated at the equivalent dose of the high dose group, 3) aCD22 Ab-ASO conjugates at 0.2mg of Ab/kg/dose, and 4) at 1mg of Ab/kg/dose. Five million leukemia cells were inoculated either intravenously (iv) (Reh) or intra-bone marrow (primary cells) to each mouse. Twenty-four hours following leukemia inoculation, animals started receiving twice weekly iv treatments for 3 weeks. All mice treated with PBS and unconjugated MXD3 ASO & aCD22 Ab, died of leukemia at approximately day 21 in the Reh model and at day 30 in the primary leukemia model (Figure). Animals treated with aCD22 Ab-MXD3 ASO conjugates, either at low or high dose, had significantly prolonged survival both in the Reh (p In conclusion, we have demonstrated the therapeutic efficacy of the aCD22 Ab-MXD3 ASO conjugates in preB ALL. This is the first study to demonstrate effective direct ASO-conjugated monoclonal Ab-mediated delivery for the treatment of leukemia or other malignancies. Future studies will focus on the further characterization of the compound, effectiveness of combination therapy with standard chemotherapeutic drugs and toxicological profile. Disclosures No relevant conflicts of interest to declare.

Leukemia stem cells (LSCs) are the root of leukemia, and are responsible for drug resistance and disease relapse. However, LSCs have not yet been identified for acute lymphoblastic leukemia (ALL). Among many challenges, lack of phenotypic markers is one of the major problems in identifying ALL LSCs. In this study, we demonstrated a novel method to isolate LSCs from both T- and B- cell ALLs and further characterized their transcriptome profile at the single cell level. We have recently identified a novel method to isolate ALL LSCs based on cellular metabolic activities. We demonstrated that these isolated LSCs had in vivo leukemia-initiating capability (LIC). We have developed a series of primary ALL xenograft mouse models using patient samples and NOD/SCID/IL2Rg-/- (NSG) mice. Leukemia cells harvested from several generations of these mice were used in this study. We isolated LSCs and non-LSCs from 4 different B-cell type ALL samples and transplanted them separately into healthy NSG mice. Cell numbers used varied between 5, 10, and 50,000 per mouse, and the number of the animals varied between three and eight per group. All the animals transplanted with LSCs developed leukemia between 5-14 weeks, whereas those transplanted with non-LSCs did not develop the disease within the same timeframe or by the end of the study, which was more than 4 months after leukemia development in the LSC group. In order to characterize and identify potential therapeutic targets in the LSCs, we investigated the transcriptome profile of these cells. First, we performed genomewide microarray gene expression profiling of RNA isolated from the LSCs and non-LSCs using 4 ALL cell lines (Reh, JM1, Jurkat, and Molt4). There were 173 genes which showed at least 2-fold difference in gene expression between the LSCs and non-LSCs. Using a panel of primer sets for the 100 genes exhibiting the highest difference in expression, we performed qRT-PCR for these genes in the isolated LSCs and non-LSCs from 11 primary ALL samples (10 B-cell and 1 T-cell type) transplanted and harvested from our NSG xenograft mouse models at different generations. There was a distinct difference in the transcriptome profile between the LSCs and non-LSCs in these primary ALL samples. Overall gene expression of 93 LSC signature genes was much lower in the LSCs than in the non-LSCs. Recent advances in microfluidic technologies allowed us to investigate cells at single cell resolution. Growing evidence suggests that cancer stem cells consist of heterogeneous cell populations (subclones). Therefore, we further investigated whether these isolated LSCs have subclones using the Fluidigm C1 and Biomark system. Preliminary results using a primary ALL sample harvested from our xenograft mouse model, indicate that there are at least two distinct subclones in the LSCs based on principal component analysis of the single cell data. In summary, we 1) developed a novel method to isolate ALL LSCs which have in vivo LIC, 2) demonstrated that isolated LSCs have a distinct transcriptome profile, and 3) discovered that the LSCs seem to consist of subclones. Currently we are in the process of performing detailed comprehensive transcriptome analyses and additional single cell transcriptome assays. Disclosures No relevant conflicts of interest to declare.

Neuroblastoma is a cancer of the sympathetic nervous system and the most common extracranial solid tumor in children. Almost half of neuroblastoma patients have a high-risk phenotype at diagnosis: metastatic disease. Prognosis of these patients is very poor with only 30% survival despite the most intensive therapies currently available. In addition, patients who respond successfully to treatment suffer from side effects from chemo and radiation therapies, leading to life-long irreversible complications. Therefore, there is a desperate need for a neuroblastoma-targeted therapy that is more effective and has fewer side effects than conventional therapies. Our goal is to develop a molecular-targeted therapy that can be used in tandem with current treatments. In our previous study, we identified the transcription factor MXD3 as a novel molecular target for neuroblastoma (presented in the 2013 AACR annual meeting). In the current study, we demonstrated in vitro efficacy of an MXD3 targeted therapeutic using siRNA and nanoparticles. We have developed MXD3 targeting methods using MXD3 siRNA and superparamagnetic iron oxide nanoparticles (NPs). We assembled siRNA on the surface of NPs and introduced them to SK-N-DZ cells (neuroblastoma cell line with high MXD3 expression) in vitro. Upon a single treatment with the siRNA-NP-complexes, the siRNAs were successfully delivered into the cells and MXD3 knockdown was confirmed at the protein level as early as 4 hours post treatment. The cells treated with the MXD3 siRNA-NP-complexes showed significantly decreased proliferation compared to those treated with control siRNA-NP-complexes or untreated cells (p In conclusion, we have demonstrated novel in vitro delivery of the MXD3 siRNA-NP complexes into neuroblastoma cells. We have also shown that MXD3 knockdown induced apoptosis and inhibited cell proliferation. Furthermore, we showed additive effects of the MXD3 siRNA-NP complexes with doxorubicin and vincristine. Further work is ongoing to determine the therapeutic effects in other neuroblastoma cell lines with different MXD3 expression and side effects in normal blood cells. The future plan is to optimize our nanocomplexes for efficient in vivo delivery, which is the major hurdle in the development of siRNA-based therapeutics. Citation Format: Connie Duong, Cathy Chen, Sakiko Yoshida, Gustavo Barisone, Jan Nolta, Elva Diaz, Nitin Nitin, Noriko Satake. Novel targeted therapy for neuroblastoma: Silencing the MXD3 gene using siRNA. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5423. doi:10.1158/1538-7445.AM2014-5423

Cytogenetic and molecular-genetic characteristics in peripheral T cell lymphoma (PTL) have not been well defined, except for those in adult T cell leukemia/lymphoma (ATL/L). Translocations and inversions involving a chromosome band 14q32 were extremely common abnormalities reported in PTL and ATL/L. We studied the involvement of TCL1, a recently isolated gene located in 14q32.1, in tumor tissues from 20 patients with PTL including three with 14q32 translocations by two color fluorescent in situ hybridization (FISH) using two cosmid probes flanking the TCL1 gene. The two cosmid signals were separated in none of them, but much increased in number in one tumor without 14q32 translocation, indicating that the TCL1 genomic region was amplified in this tumor. Reverse transcription-polymerase chain reaction (RT-PCR), however, failed to detect the TCL1 transcript in the tumor. These findings suggest that an oncogene other than TCL1 may be located in 14q32.1, and its amplification may be involved in the neoplastic process of PTL.

The t(12;21) (p13;q22) is observed in approximately 20-25% of childhood B-lineage acute lymphoblastic leukemia (ALL) cases in both Asian and Caucasian populations. This translocation results in the fusion of TEL, a recently described ETS-like gene on 12p13, and AML1, which was shown to be involved in the formation of fusion genes with ETO and EVI1 in myeloid leukemias. Fluorescence in situ hybridization (FISH) and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis are useful in detecting this translocation which is not readily identified with routine cytogenetic techniques. The t(12;21) is associated with a distinct subgroup of patients characterized by an age between 1 and 10 years, an early B immunophenotype, and a good prognosis. A high incidence of the deletion of non-translocated TEL is another characteristic of leukemic cells with this translocation. TEL-AML1 hybrid protein thought to be critical in leukemogenesis possesses the HLH domain of TEL fused to almost the entire AML1 protein, although the detailed mechanisms of leukemogenesis remain obscure. RT-PCR combined with FISH analysis of posttreatment samples appears to be useful in detecting early relapse or minimal residual disease and thus, is expected to optimize the treatment strategy for patients with t(12;21).

Laser tweezers Raman spectroscopy was used to characterize the oxygenation response of single normal adult, sickle, and cord blood red blood cells (RBCs) to an applied mechanical force. Individual cells were subjected to different forces by varying the laser power of a single-beam optical trap, and the intensities of several oxygenation-specific Raman spectral peaks were monitored to determine the oxygenation state of the cells. For all three cell types, an increase in laser power (or mechanical force) induced a greater deoxygenation of the cell. However, sickle RBCs deoxygenated more readily than normal RBCs when subjected to the same optical forces. Conversely, cord blood RBCs were able to maintain their oxygenation better than normal RBCs. These results suggest that differences in the chemical or mechanical properties of fetal, normal, and sickle cells affect the degree to which applied mechanical forces can deoxygenate the cell. Populations of normal, sickle, and cord RBCs were identified and discriminated based on this mechanochemical phenomenon. This study demonstrates the potential application of laser tweezers Raman spectroscopy as a single-cell, label-free analytical tool to characterize the functional (e.g., mechanical deformability, oxygen binding) properties of normal and diseased RBCs.

Neuroblastoma is the most common extra-cranial tumor in children, with around 700 new diagnoses each year. High-risk patients have poor outcomes and only 30% survive despite the most intensive therapies available, which can also harm healthy cells. Thus, we are developing novel targeted therapies for neuroblastoma to increase treatment efficacy and limit damage to healthy cells. We utilized the One-Bead One-Compound (OBOC) combinatorial split-mix method developed by Kit Lam (Lam et al., 1991) to identify unique ligands against neuroblastoma cells for therapeutic delivery. The OBOC libraries consist of hundreds to millions of 90 μm Tentagel beads, and each bead displays 1013 copies of a peptide on its surface. Thus, each bead has one unique compound on its exterior. Twelve random OBOC libraries with varying peptide lengths (5 to 16 amino acids) were screened against three human neuroblastoma cell lines (SK-N-BE, SK-N-DZ, and IMR-32) to determine a library for more stringent screening of ligands. 37,500 beads from each library were screened against each cell line and the libraries averaged 2 positive candidates per 18,750 beads within 2 hours of incubation. The results also suggested preference for longer peptide libraries, but an intermediate length random decamer library was chosen for further screening due to both a good yield of positive beads and convenient sequencing. Each neuroblastoma line was screened against 150,000 beads from the decamer library to identify potential ligands. Positive beads were selected, retested three times, and decoded using Edman degradation. Several cancer cell lines (A549 lung cancer, and Reh and Jurkat leukemia cell lines) were used as negative controls. Ten beads per cell line displaying high binding affinity were selected. Candidate beads with positive binding to all three neuroblastoma cell lines are currently being sequenced and the ligands will be synthesized. We plan to characterize the ligands for high affinity and specificity, as well as internalization capacity, and to identify their receptors. Currently, there are very few targeted therapies for neuroblastoma. We believe our ligand discoveries can be developed for future novel targeted therapies in neuroblastoma. Citation Format: Andy Iskandar, Connie Duong, Cathy Chen, Eduardo Sanchez, David Olivos, Kit Lam, Noriko Satake. Identifying neuroblastoma-specific ligands for cancer targeted therapy using a one-bead one-compound combinatorial approach. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4545. doi:10.1158/1538-7445.AM2013-4545

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Up to 45% of the cases are high risk at diagnosis, representing very aggressive, usually metastasized tumors that are refractory or recurrent after all available therapies. Prognosis for these patients is very poor with only 30% survival. MXD3 is a transcription factor previously shown in our lab to be a novel member of the Hedgehog (Hh) pathway (Yun, Rust, Ishimaru, Diaz, Mol Cell Biol, 2007) in granule neuron precursors. In these cells, overexpression of MXD3 results in higher MYCN expression, suggesting a functional interaction between the two transcription factors. MXD3 phenocopies MYCN expression in mouse models of medulloblastoma; we have also shown that it is expressed in human medulloblastoma and that its knockdown reduces proliferation of human medulloblastoma cell lines (Barisone et al., PLoS One, 2012). More than 20% of NBs have MYCN amplification, which is considered a marker of poor prognosis. In the current study, we investigated a possible role for MXD3 in NB cell proliferation. Using qRT-PCR we observed high levels of MXD3 mRNA expression in 5 NB cell lines: SK-N-DZ, IMR-32, SH-SY5Y, SK-N-BE and SK-N-SH. Immunoblot analysis with anti-MXD3 monoclonal antibodies confirmed that the protein was present in these cells. Furthermore, immunohistochemistry analysis showed strong immunoreactivity in a cohort of 16 primary NB tissue specimens. We investigated the role of MXD3 in NB cell proliferation by silencing MXD3 in the SK-N-DZ cell line. We used lentiviral delivery to knockdown MXD3 using an RNA interference approach. Upon transduction by the viral vector, MXD3 knockdown was confirmed at both the RNA and protein level. Within 48 hours, MXD3 protein levels were reduced >90% in cells infected with the shMXD3 virus but not with the control virus. MXD3 knockdown resulted in decreased proliferation in NB cells, supporting our hypothesis that it may be involved in the maintenance of NB. We analyzed cell cycle progression after knockdown using flow cytometry. We observed no significant differences in the G0/G1, S or G2/M populations between experimental and control samples. Our results suggest that MXD3 is important for NB cell proliferation, possibly by acting as an anti-apoptotic factor. Therefore, MXD3 is a potential candidate for targeted therapies against neuroblastoma. Citation Format: Gustavo Barisone, Noriko Satake, Carly Lewis, Connie Duong, Cathy Chan, Kit Lam, Jan Nolta, Elva Diaz. Mxd3 is required for proliferation of human neuroblastoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3128. doi:10.1158/1538-7445.AM2013-3128

Abstract 3523 Disregulation of the Hedgehog (Hh) signaling pathway is known to drive proliferation in several cancers, including hematopoietic malignancies. The role of the Hh pathway in precursor B cell (preB) acute lymphoblastic leukemia (ALL), the most common leukemia in children, is unclear; however, recent reports suggest that Hh signaling is involved in the development of B cell precursors as well as the self-renewal and survival of B cell malignancies. MXD3 is a transcription factor previously shown in our lab to be a novel member of the Hh pathway (Yun, Rust, Ishimaru, Diaz, Mol Bio Cell, 2007). We have previously shown that MXD3 is expressed in medulloblastoma (the most common brain tumor in children) and that its knockdown reduces proliferation of human medulloblastoma cell lines (Barisone et al., PLoS, 2012). In the current study, we investigated a possible role for MXD3 in preB ALL cell proliferation. Using quantitative real-time reverse-transcription PCR (qRT-PCR) we observed high levels of MXD3 mRNA expression in 8 primary preB ALL samples as well as in the preB ALL cell lines Reh and JM1. However, MXD3 levels were very low in mobilized peripheral blood mononuclear cells from healthy donors, mouse bone marrow and spleen. Indeed, MXD3 levels in the primary ALL cells and cell lines ranged between 13 and 35 fold increase when compared to the normal cells. Immunoblot analysis with anti-MXD3 monoclonal antibodies confirmed that the protein was present in the ALL samples but not in normal cells. We investigated the role of MXD3 in cell proliferation and survival, by silencing MXD3 in the Reh cell line. We used lentiviral delivery to knockdown MXD3 using an RNA interference approach. Briefly, lentiviruses were produced carrying a short hairpin RNA sequence specific for MXD3 (shMXD3) or a negative control sequence (shCTRL). Upon transduction by the viral vector, MXD3 knockdown was confirmed at both the RNA and protein level. Within 48 hours after transduction, MXD3 protein levels were reduced >90% in cells infected with the shMXD3 virus but not with the control virus. Interestingly, MXD3 knockdown resulted in decreased proliferation in Reh cells, supporting our hypothesis that it may be involved in the maintenance of this malignancy. As a first step toward understanding the mechanisms by which MXD3 exerts its function in leukemia cells, we analyzed cell cycle progression and apoptosis levels after knockdown using flow cytometry. We observed no significant differences in the G0/G1, S or G2/M populations between experimental and control samples. However, samples where MXD3 had been knocked down showed higher levels of apoptosis when compared to controls. Taken together, our results suggest that MXD3 is important for preB ALL cell proliferation, possibly by acting as an anti-apoptotic factor. Therefore, MXD3 may represent a suitable candidate for future efforts in developing targeted therapies against preB ALL. Disclosures: No relevant conflicts of interest to declare.

Abstract 4847 The number of individuals with sickle cell disease (SCD) in the U.S. is estimated to be over 70,000, and the average life span of a SCD patient is 39 years. Despite the high incidence of SCD in the U.S. and its significant negative effects on patient health, there are currently only limited treatment options for this disease. Also, although a single mutation in the globin gene is known to cause SCD, there is significant clinical diversity. As technology advances, many studies, including genome-wide analyses and proteomics, have been conducted to understand the disease mechanism and find better treatments. However, none has yet led to the development of novel therapies. We need a practical approach to study red blood cells (RBCs) to increase our knowledge of this heterogeneous disease. Laser tweezers Raman spectroscopy (LTRS) is a label-free, laser-based method for the biochemical analysis of single living cells. Based on the phenomenon of inelastic light scattering of photons by molecular bonds, Raman spectroscopy can provide detailed information, in the form of a Raman spectrum, about the molecular structure and conformations inside single living cells. Moreover, the integration of laser tweezers enables 1) simple, convenient analysis of single suspension cells such as RBCs and 2) a method to impart a mechanical force on a cell (via the optical forces) and simultaneously monitor the biochemical response of that cell to the applied force. Previous studies using optical tweezers have shown different RBC elasticity between samples from SCD patients treated with hydroxyurea and those that were not treated with hydroxyurea. Raman spectroscopy has been applied to both normal and pathological RBCs in previous studies, but the results were limited to the identification of characteristic signatures. Our integration of the two methods, Raman spectroscopy and laser tweezers, is a new biophotonic approach for studying the intricate relationship between the biochemical and mechanical properties of a single cell that was not previously possible and is not easily carried out with existing methods. As such, we believe that this recent emerging single cell-based technology can be developed and used as an RBC functional assay. We have previously shown that LTRS can detect a force dependent oxygenation state transition in individual RBCs trapped at different laser powers, presumably due to the optical forces stretching the cell and inducing changes in the hemoglobin-hemoglobin and hemoglobin-membrane interactions that change the oxygen content of the cell. We have also shown characteristic Raman signatures indicating different oxygen content in different RBC samples at specific applied forces. Signatures were different among RBCs from cord, normal adult, and SCD patient blood, suggesting laser tweezers Raman spectroscopy can be used to measure the different force dependent oxygen content in a single RBC. We are currently validating our system to determine whether Raman spectroscopy can be used to identify and distinguish gene-corrected RBCs from normal and sickle RBCs. Our preliminary results showed distinct Raman fingerprints associated with the membrane structure of RBCs. Spectral fingerprints were observed to be different between normal and sickle RBCs, and gene-corrected RBCs were observed to have Raman profiles similar to those of normal RBCs. We speculate that the signature overlap between normal and gene-corrected samples is due to partial gene correction. These results are encouraging and strongly suggest that Raman spectroscopy can not only identify molecular structures but also be used as a functional assay. Disclosures: No relevant conflicts of interest to declare.

Abstract 3576 Primary leukemia cells are known to be difficult to culture in vitro. Therefore, mouse xenograft models are commonly used to study human leukemia biology and to develop new therapeutics. Leukemia can be maintained and expanded through serial transplantations. Although many mouse models with different types of leukemia, mouse strains, and inoculation methods, have been used, there are very few studies validating the models. We have established acute lymphoblastic leukemia (ALL) mouse models using primary ALL samples and NOD/SCID/IL2Rg null (NSG) mice. In order to increase engraftment efficiency, we transplanted leukemia cells into healthy adult mice via intra-bone marrow (BM) injection (1 to 100 million cells per mouse to tibia bilaterally). To our knowledge, the model with primary ALL samples in NSG mice via intra-BM injection is novel. A total of 9 samples, 2 T cell ALL and 7 precursor B (preB) ALL, were transplanted into cohorts of 3 to 8 mice each. Of these, 1 T cell ALL and 6 preB ALL samples engrafted and 1 T cell and 1 preB ALL sample failed to engraft. Leukemia engraftment was observed between 10 to 33 weeks after transplantation. In each cohort, each mouse developed leukemia at approximately the same time after leukemia transplantation. Necropsy showed significant hepatosplenomegaly and white BM in all mice, indicating leukemia infiltration. Cells were harvested from BM, spleen and other leukemia-infiltrated organs, and confirmed to be human origin by flow cytometry using anti-HLA-ABC and anti- human CD45 antibodies. Nearly 100% of BM was replaced with human leukemia cells. One T cell and 1 preB ALL sample have been serially transplanted using the same method as the primary transplantation to quaternary and tertiary generations, respectively. We observed that the time to develop leukemia became shorter with each transplantation: 16 weeks for the first transplantation and 10 weeks for the tertiary transplantation in a T cell ALL sample. Some mice transplanted with a preB ALL sample developed chloroma. Interestingly, chloroma development was consistently observed with this sample, which was transferred through serial transplantation although the patient did not develop chloroma. Morphology and immunophenotype were similar to the original leukemia. There were some changes in CD expression patterns; however, immunophenotyping was consistent with the original leukemia. To help understand phenotype progression in transplanted leukemia samples, we are currently comparing the transcriptome profiles of leukemia samples obtained from the patient, primary and quaternary mice for T cell ALL, and primary and tertiary mice for preB ALL samples (preB ALL patient sample not available). Next-generation sequencing (NGS)-based RNA-Sequencing (RNA-Seq) is in progress for this analysis. In addition to obtaining digital expression profiles, and differential gene expression, RNA-Seq analysis will reveal the complete repertoire of splice variants, point mutations, and fusion transcripts in the ALL samples. Complete results of these analyses for our mouse models with both T cell and preB ALL will be presented. Thus, this technique will provide the most detailed transcriptomic analysis and no validation studies have yet been reported on ALL samples sequentially engrafted NSG mice using RNA-Seq. Disclosures: No relevant conflicts of interest to declare.

Laser tweezers Raman spectroscopy (LTRS) was used to demonstrate that a red blood cell (RBC) in a single beam optical trap transitions from an oxygenated to a partially deoxygenated state with increasing trapping power. Continuous switching between the two states is possible by repeatedly cycling between low and high trapping powers. Alterations in the hemoglobin conformation and interactions due to cell folding in the trap are proposed to be responsible for the transition. This study demonstrates that mechanically induced biochemical changes by optical forces need to be considered when applying single beam optical tweezers for cell analysis. LTRS holds promise as a functional assay to characterize normal and diseased RBCs based on their biochemical response to the forces of a single beam optical trap.

Renal medullary carcinoma (RMC) is a rare disease with only ∼100 cases having been reported to date with the majority of these patients also having the sickle cell disease or trait. Multiple therapies, including chemotherapy, radiation, and resection, have been tried; however, this disease is almost uniformly fatal. We developed new mouse models of RMC that were created using patient derived tumor xenografts in the NOD SCID IL2 receptor γ chain null (NSG) mouse. RMC was diagnosed in a 9 year old male with sickle cell trait and multiple metastases in liver and lungs. The patient was treated by resection of the renal tumor, followed by chemotherapy, including bortezomib, paclitaxel/carboplatin/ bevacizumab, doxil/bevacizumab, and sunitinib. The patient showed partial or no response to each regimen and developed malignant pleural effusion and died 9 months p=0.0043 from diagnosis. NSG xenograft models were formed from both the patient primary renal tumor specimen and malignant pleural effusion. Comparative microarray gene expression data collection, genomic SNP data collection for copy number variance (CNV), and histology analyses were performed with the original patient renal tumor specimen and the two xenograft models for determining predictive chemotherapy responses for therapeutic efficacy. Additionally, the xenograft tumors were used for chemotherapy efficacy studies for using these models for future predictive efficacy studies. Both primary renal tumor and the metastatic pleural effusion developed large mouse xenograft tumors (P0) that were passaged into secondary mouse passages (P1). H & E histology showed that both the patient renal tumor and the renal tumor-derived xenograft tumors were moderately differentiated and morphologically similar. The malignant pleural effusion xenograft tumors were highly differentiated and not morphologically similar to the primary patient renal tumor or the renal tumor-derived xenograft. Sunitinib and temsirolimus were evaluated in comparison to vehicle control for tumor growth delay in P1 malignant pleural effusion xenografted mice and sunitinib efficacy was established vs. vehicle controls. Sunitinib demonstrated was highly efficacious compared to vehicle control with 110% tumor growth delay (p=0.0043) and temsirolimus tumor growth delay was not efficacious (p>0.05). Since RMC is a rare disease for which there is currently no effective therapy, these new mouse models will be a useful for developing new treatments for this lethal disease. Furthermore, with a series of samples (normal, sickle cell trait kidney, RMC at diagnosis, and tumor cells from pleural effusion), we expect to understand molecular mechanisms underlying the disease progression pathway (pathogenesis). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1585. doi:10.1158/1538-7445.AM2011-1585

Three cases of X-linked auto-immune enteropathy with haemolytic anaemia and polyendocrinopathy are described from one related Japanese kindred. Two boys had died due to severe diarrhoea accompanied by total or subtotal intestinal villous atrophy. In contrast, although one patient showed the same symptoms and had circulating IgG antibodies against enterocytes, his condition improved dramatically and he developed well following the use of cyclosporin A (CSA). CSA may be beneficial in patients with this rare disorder. Auto-immune enteropathy should be considered as a cause of protracted diarrhoea with unknown aetiology.