Negative Bone Marrow Research Articles

Retroviral Ectopic Expression of a Signaling-Defective Thrombopoietin Receptor (Mpl) Induces a Systemic Loss of Hematopoietic Stem Cells in Mice,

Abstract 4175MPL signaling, induced by the binding of Thrombopoietin (THPO), regulates megakaryopoiesis and platelet development, and is essential for hematopoietic stem cell (HSC) maintenance. Murine lineage negative, Sca-1 and c-kit positive bone marrow (BM) cells (LSK cells) and human CD34 positive cells expressing MPL have long term BM reconstitution capacities. In human patients, deficiency of MPL due to inactivating mutations in the MPL gene causes severe thrombocytopenia and progressive aplastic anemia, which is lethal if not treated by BM transplantation (BMT). This rare genetic disorder is termed congenital amegakaryocytic thrombocytopenia (CAMT).With the final aim of developing gene therapy to treat CAMT, we explored the effects of ectopic Mpl expression in wild-type C57Bl/6 mice. The enforced overexpression of Mpl induced a rapid expansion of all three hematological cell lineages, resembling chronic myeloproliferative disease (CMPD) 2 months after BMT (n=25 mice). Surprisingly, mice subsequently developed life threatening pancytopenia with normal Thpo levels despite low platelet counts (167+/−52 × 103/μl). The BM showed reduced LSK cell numbers (0.01+/−0.01 % versus controls 0.05+/−0.015%, p=0.03). Non-transduced, co-transplanted BM cells could not rescue the disturbed hematopoiesis. Based on these observations, we hypothesized that the ectopic overexpression of Mpl in hematopoietic cells induces a dominant-negative effect, dysregulating the Thpo-Mpl balance.To test our hypothesis, we ectopically expressed a signaling-defective truncated dominant-negative Mpl (dnMpl) receptor, lacking the complete intracellular domain, in a BMT model in wild-type C57Bl/6 mice (n=23). Lineage negative cells were transduced with gammaretroviral vectors expressing dnMpl from the SFFV promoter in the LTRs and transplanted into lethally irradiated mice. We observed long term engraftment with 57.6+/−1.9% dnMpl positive mononuclear cells in the peripheral blood (controls 76.7+/−7.8%) after 19 weeks and severe thrombocytopenia (174 +/− 32 x103/μl) in the dnMpl expressing animals (controls 951+/−233×103/μl). After 23 weeks, we detected 15–34% dnMpl positive BM cells (controls 15–99%). To address the question of whether the BM of dnMpl treated mice contained fewer HSCs compared to control transplanted mice, we analyzed the LSK cell number and saw a ∼4.6-fold reduction (0.02+/− 0.005% versus control mice 0.07+/−0.012%; p=0.0016). As Thpo/Mpl signaling is known to induce HSC quiescence, we speculated that the loss of these signals induces HSC cycling. Cell cycle analysis of the LSK cells of dnMpl treated mice showed a transition into G1/S/G2, indicating a loss of quiescent HSCs (G0 3.5 +/−1.6% vs controls 29.8 +/−10.5%). This effect was even more pronounced in CD34 negative LSK cells (5.4+/−4.0% vs. 44.5+/−8.5%). Secondary transplantation of dnMpl BM showed severely reduced repopulation capacity with 5 out of 6 secondary recipients dying due to graft failure.In selected animals we transplanted another batch of BM after 16 weeks without preconditioning of the mice. Long term repopulation of the second graft was seen in 71–79% of dnMpl treated mice (controls 0.2–1.1%) and BM cells further engrafted in secondary recipients.To address changes in HSC gene expression, we sorted LSK cells from dnMpl BM (pool of 4–5 dnMpl mice for each sample) and also separated dnMpl positive and negative cells. Our controls were LSK cells from control transplanted mice (one per sample). RNA was analyzed on an Affymetrix Mouse Genome 430 2.0 array in triplicates. Unsupervised clustering and principle component analysis revealed that dnMpl positive and negative samples clustered, suggesting a systemic effect on HSCs. We tested more than 4000 gene sets from the MySigDB Database (Broad Institute, Cambridge, MA) for enrichment in either of the phenotypes and found a profound downregulation of gene sets containing “stemness” genes in the LSK cells of dnMpl treated mice, irrespective of cellular dnMpl expression.In summary, we show that ectopic expression of dnMpl in a subset of BM cells disturbs Thpo/Mpl-signaling inducing thrombocytopenia and a systemic loss of HSCs. Besides underlining the strict requirement for regulated Mpl expression in gene therapy for CAMT, these data create new hypotheses for the pathogenesis of aplastic anemia and the development of non-cytotoxic conditioning regimens in BMT. Disclosures:No relevant conflicts of interest to declare.

Increased Mutation Frequency Induced by Oxidative Stress in the NUP98-HOXD13 MDS Mouse Model

Abstract 1705The myelodysplastic syndromes (MDS) comprise a group of hematologic disorders characterized by ineffective hematopoiesis, dysplasia, and transformation to acute myeloid leukemia (AML) in a subset of cases. The causative agents for de novo or therapy-related MDS (t-MDS) include exposure to known genotoxins, such as anti-cancer chemotherapy, or ionizing radiation. It is known that MDS is associated with chromosomal aberrations including deletions, amplifications, inversions, and translocations. However, the molecular mechanisms that lead to progression of MDS to AML have not been completely elucidated. Recently, several studies have reported that oxidative stress contributes to disease progression of MDS. Reactive oxygen species (ROS) play a role in regulating several biologic phenomena including activation of signaling pathways in response to cytokines, and the gene expression pattern induced by this signaling. ROS is also known to induce oxidative DNA damage, which can lead to activation of proto-oncogenes or inactivation of tumor suppressor genes. A mouse model for MDS, generated by expression of a NUP98-HOXD13 (NHD13) fusion gene, was exploited to investigate ROS levels. Lineage negative (Linneg) bone marrow mononuclear cell (BMNC) from NHD13 mice with MDS had a 6.0 fold increased level of ROS compared with wild-type (WT) Linneg BMNC. In order to address potential mechanism(s) leading to this increased ROS, a Real-Time Quantitative PCR array, which profiles the expression of 84 genes related to oxidative stress, was utilized and the data demonstrated down regulation of Myeloperoxidase (Mpo; −9.9 fold), Eosinophil peroxidase (Epx; −8.8 fold) and Lactoperoxidase (Lpo; −5.2 fold) in the Linneg BMNC from NHD13 mice. The increased ROS level in NHD13 Linneg BMNC was associated with a 2 fold increase in G2/M phase cells along with increased expression of p16INK4A (43 fold) and p21 (10 fold), possibly to allow for DNA repair at cell cycle checkpoints. In order to investigate genomic DNA damage by ROS, immunocytochemistry for γH2AX foci was performed and demonstrated a 2 fold increase in γH2AX foci in the NHD13 BMNC compared to WT BMNC. To determine if mis-repair of the ROS-induced DNA damage led to mutations of genomic DNA in vivo, mutation frequencies (MF) were determined using the Big Blue® cII mutation detection assay. Big Blue mice are transgenic mice that have a λLIZ shuttle vector, which is derived from the coliphage lambda. Selection of mutation is based on the ability of the λ phage to multiply through either the lytic or lysogenic cycle in E. coli host cells. Big Blue mice were crossed to NHD13 mice, and NHD13/Big Blue® mice were compared with littermate controls that were positive for the BigBlue transgene but negative for the NHD13 transgene. BMNC from the NHD13/BigBlue mice showed a 1.8-fold increased MF compared to control BMNC (p value = 0.0371). Mutations were comfirmed by sequencing the cII target gene, and revealed that the NHD13/BigBlue mice had a 3-fold higher frequency of frameshift mutations compared to the control animals. These results suggest that the oxidative stress induced by the NHD13 fusion may contribute to disease progression of MDS to AML through DNA damage and mutation. Disclosures:No relevant conflicts of interest to declare.

Pentostatin, Alemtuzumab, and Low Dose Rituximab Is Effective Therapy for Relapsed/Refractory Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma (CLL)

Abstract 1790CLL is incurable with conventional therapies and prognosis is poor in patients with relapsed or treatment refractory disease. Patients with purine analogue refractory disease and defective TP53 function have limited therapeutic options. Based on: 1) The ability of purine analogues to decrease tumor burden; 2) Higher response rates in patients treated with alemtuzumab and rituximab vs. alemtuzumab alone; and 3) Data showing that more frequent lower doses of rituximab could be more effective than standard therapy, we hypothesized that combination therapy with pentostatin, alemtuzumab and low dose rituximab (PAR) would be effective and tolerable therapy for patients with relapsed/refractory CLL and those with previously untreated progressive CLL who had 17p13-.This two stage phase II University of Iowa/Mayo Clinic Lymphoma SPORE clinical trial (NCT00669318) was approved by Mayo Clinic and the University of Iowa IRBs according to the principles of the Declaration of Helsinki. The aims are to assess: 1) Complete (CR) and overall responses; and 2) Progression-free survival (PFS), duration of response, and time to next treatment (TTT). Patients with progressive CLL (including the SLL variant) defined by standard criteria are eligible for this study if they had either previously treated CLL or previously untreated CLL with 17p13-. Cycle 1 of therapy is 5 weeks with rituximab 20 mg/m2/d IV Mon-Wed-Fri starting day 1, subQ alemtuzumab beginning days 3–5 with a dose escalation (3 mg–10 mg– 30 mg/d) and then 30 mg/d Mon-Wed-Fri, and pentostatin 2mg/m2/dose/IV days 8 & 22. Cycles 2–3 are each of 4 weeks duration and are the same as weeks 2–5 of cycle 1. Patients who achieve CR after cycle 2 with a negative CT scan and bone marrow study with no evidence of residual CLL cells after immunohistochemical staining, do not receive cycle 3 of therapy. All patients receive granulocyte growth factor support after pentostatin therapy as well as PCP and herpes virus prophylaxis. Patients are tested for CMV reactivation by PCR weekly during therapy and treated with valganciclovir if reactivation is detected.We report the results of the planned interim analysis performed after completion of therapy for the first 19 patients (July 2008 - October 2010). The median patient age was 63 years (range 47–78) with 74% males. Two patients with 17p13- were previously untreated. Of the 17 previously treated patients (median 2 prior regimens, range 1–6), 8 (47%) had purine analogue refractory CLL (disease progression < 6 months of treatment). Nine (47%) patients had advanced clinical stage (Rai III-IV). Adverse molecular prognostic factors were 17p13- (n=8, 42%, 2 also had 11q22-), 11q22- (n=1), unmutated IGHV (12/17, 71%), ZAP70+ (≥20%, 12/17, 71%) and CD38+ (≥30%, 6/18, 33%). The median beta-2-microglobulin level was 4.3 (range 2.4 – 12.6).Grade 3–4 treatment related toxicities were 9 non-hematological and 20 hematological events. Grade 3–4 infections occurred in 3 patients including one patient requiring hospitalization for CMV re-activation. Fourteen (74%) patients responded to treatment with 6 (32%) CR (includes 2 CRi) and 8 (42%) partial responses (PR). Five (26%) patients had stable disease (SD). Eleven (58%) patients completed 3 cycles of therapy. One patient achieved a CR with no residual disease after 2 cycles of therapy and received no further treatment per protocol. Median duration of response is 7 months (95% CI: 4–not reached (NR)). Among the 8 patients with 17p13- there were 3 CR/CRi, 4 PR, and 1 SD. Four patients proceeded to allogeneic transplant with reduced intensity conditioning (RIC) and were censored for TTT at the initiation of transplant related therapy. With a median of 16 (range 6 – 35) months of follow up, median PFS is 7 months (95% CI: 5–NR), median TTT is 27 months (95% CI: 5-NR) and the calculated median overall survival is 23 months (95% CI: 14–NR).We report that PAR is effective and tolerable therapy in this population of relapsed refractory patients with high to very-high risk CLL. In addition, the responses in patients with 17p13- were similar to that of the patients who did not have 17p13-. The treatment regimen was useful for disease control in 3 patients who proceeded to reduced intensity conditioning allogeneic transplantation. This study shows that PAR could play an important role in the treatment of recurrent and high risk CLL.Supported by the University of Iowa/Mayo Clinic Lymphoma SPORE CA097274 Disclosures:Zent:GlaxoSmithKline: Research Funding; Genentech: Research Funding; Genzyme: Research Funding.

Results of Radiation Therapy for Primary Extranodal Lymphoma of the Head and Neck: A Report of Case Series

Abstract 4761 Purpose:Radiation therapy is an important treatment modality for the extra-nodal lymphoma (ENL) of the head and neck. Intensity Modulated radiation therapy (IMRT) has been shown for head and neck cancers to be associated with decreased late side effects like Xerostomia compared with conventional RT techniques (Conv.RT). The purpose of the study is to determine the clinico-pathological features, treatment outcomes and late toxicities in patients (pts) with ENL of head and neck treated with different RT techniques. Methods:Retrospective review of records from 2007–2010 identified 14 pts with ENL of the head and neck treated with RT at Fox Chase Cancer Center. Eight were treated with IMRT and 6 with conventional RT technique. Thirteen had CD20+ non-Hodgkin lymphoma [NHL; 7 diffuse large B cell (DLBCL), 3 MALT, 3 others], and one Hodgkin’s lymphoma (HL; classic). All pts underwent routine staging work-up with negative bone marrow biopsy for all. Pre and post-treatment PET scans were done in 7 pts. Initial chemotherapy (R-CHOP × 4–6 courses for DLBCL, ABVD × 4 for HL), or no chemotherapy (MALT) was followed by involved field radiation therapy (median RT dose: 36 Gy for NHL, 30 Gy HL, 150– 180 cGy per fraction in 20 fractions over 4 weeks). Results:The median follow up was 28 months (range: 1– 52). The median age was 60 years (range: 42–95; 7 males and 7 female). Nine had stage IEA disease, 3 had stage II and 2 stage IV. Five had oropharynx involvement, two had paranasal sinus (DLBCL) 2 nasopharynx (1 DLBCL, 1 MALT) and 1 vallecula (HL). The overall response rate after combined modality treatment was 100%. There were no FDG avid lesion noted on post-treatment PET. There were no local or neck node relapses at last follow up. The 2-year actuarial survival rate was 80%. One DLBCL patient had systemic relapse involving stomach and bilateral testes, was treated with second-line chemotherapy and radiation therapy. At the last follow up, 12/14 were alive. RTOG grade 2 acute skin changes were noted in 6 pts (3 by each RT technique), grade 2 mucositis in 5 pts (3 with Conv.RT) and grade 2 xerostomia in 6 pts. None of the pts developed grade 3 xerostomia (both acute and late). Late grade 2 Xerostomia was seen in 5 pts treated with Conv.RT but not with IMRT. Conclusions:RT following chemotherapy or used alone in ENL of head and neck was associated with local and distant disease control in the majority of patients, with a toxicity profile that appears favorable for IMRT compared with conventional radiotherapy techniques. Larger studies are required to confirm that IMRT is as effective as conventional involved-field RT. Disclosures:Pro:Celgene: Consultancy, Honoraria.

The Serum Free Light Chain Assay Allows Earlier Detection of Relapse/Progression of Multiple Myeloma After Autologous Hematopoietic Cell Transplantation

Abstract 4466 The International Myeloma Working Group (IMWG) guidelines use serum free light chain (SFLC) response criteria only in multiple myeloma (MM) patients without measurable M-protein in serum or urine and to satisfy stringent complete remission (sCR) criteria. Current response criteria use changes in intact immunoglobulin measured by serum protein electrophoresis and immunofixation (SIF). The role of the SFLC assay during follow up of MM patients with measurable intact immunoglobulin after autologous hematopoietic cell transplantation (auto-HCT) is not well defined. In order to assess the role of the SFLC assay (Freelite; The Binding Site Group Ltd, Birmingham, UK) during the follow up of MM patients (with measurable M protein in serum or urine), and its role in detecting relapse compared to SIF test, a retrospective analysis of 20 MM patients after melphalan 200 mg/m2 conditioned first auto-HCT was conducted. Patients were followed for a median 15 months (range 3–36 months) between 2008 and 2011. Each patient had both the SFLC assay and SIF performed every 3 months at each routine clinic visit. No patient had renal failure (defined as serum creatinine 2 mg/dl or more). No patient deaths occurred during the follow up period. Two groups were identified. 10 patients without relapse had the following characteristics: median age at HCT 55 years (range 46–68 years); median time from diagnosis to auto-HCT 6.5 months (3–18 months); Durie-Salmon stage IIIA (n=7), stage IA (n=3); IgG kappa (n=5), IgG lambda (n=5); response to HCT very good partial remission VGPR 1 (n=5), CR1 (n=3), and sCR1 (n=2). All patients without relapse maintained a normal SFLC ratio (0.26–1.65) after HCT with negative SIF (n=5), or stable SIF (n=3) and in 2 patients (who were on lenalidomide maintenance), the SIF became negative 12 months and 18 months after HCT. 10 MM patients relapsed after auto-HCT: median age at HCT 55 years (range 46–68 yrs); Durie-Salmon stage IIIA (n=9), IA (n=1); IgG kappa (n=4), IgG lambda (n=2), kappa (n=2), lambda (n=1), IgA kappa (n=1); median time from diagnosis to HCT 8 months (range 4–28 months); response to HCT VGPR1 (n=7), CR1 (n=3). In all the patients who relapsed, the SFLC ratio became abnormal (&lt;0.26 or &gt;1.65) at a median of 3 months (range 3–12 months) before any other test detected relapse, (including SIF, bone marrow biopsy) and the SFLC ratio remained abnormal and continued to worsen over the follow up period. Salvage chemotherapy was begun at a median of 3 months (range 3–12 months) after the SFLC ratio became abnormal after confirmation of relapse by current approved methods (elevation of SIF or bone marrow biopsy). In follow up after HCT, 5 patients who relapsed developed an abnormal SFLC ratio (&lt;0.26 or &gt;1.65) without a concomitant increase in SIF and these patients had bone marrow biopsy confirmation of relapse (25% increase of plasma cells from baseline). In one patient, the SFLC ratio became abnormal at 3 months post HCT with negative SIF, and negative bone marrow biopsy at 3 months after HCT and multiple extramedullary plasmacytomas were found at 4 months after HCT accounting for the relapse. Conclusion: Abnormal SFLC ratio allowed earlier detection of relapse/progression of MM after auto-HCT even in those with measurable M protein in serum and urine and not only in light chain MM but also in those with intact heavy chain immunoglobulins compared to SIF and this can be critical for earlier intervention with salvage strategies. There are currently no guidelines for use of the SFLC assay in follow up of MM patients with measurable serum and urine M protein. The serum free light chain assay should be used concomitantly with serum protein electrophoresis and immunofixation in the follow up of MM patients after auto-HCT. Disclosures: No relevant conflicts of interest to declare.

MEIS1 Is Required for the Maintenance of Long Term Hematopoietic Stem Cells Wherein It Regulates Quiescence

Abstract 551Normal hematopoiesis is maintained by long-term hematopoietic stem cells (LT-HSCs) that are defined by their extensive self-renewal and multipotency. Self-renewal of LT-HSCs in turn is regulated by a complex network of intrinsic and extrinsic factors. The transcription factor MEIS1 is highly expressed in hematopoietic stem and progenitor cells and also in several leukemias, suggesting that MEIS1 might be important in regulating self-renewal. However, the role of MEIS1 in normal hematopoiesis has not been defined. To determine the role of MEIS1 in hematopoiesis, we studied conditional knockout mice. We generated transgenic mice bearing loxp sites flanking the homeodomain of MEIS1. The MEIS1-floxed mice were then bred to Rosa26-CreERT2 mice, the latter expressing cre-recombinase ubiquitously, that can be activated by estrogen or its analog Tamoxifen (Tam). Efficient, complete recombination was achieved in vivo by treating MEIS1-f/f-Cre (homozygous for MEIS1-flox) mice with Tam and in vitro by treating bone marrow cells with 4-hydroxy tamoxifen. Loss of MEIS1 expression was detected by QRT-PCR and western blotting. To determine the role of MEIS1 in the maintenance of adult hematopoiesis, MEIS1-f/f-Cre and control mice were treated with Tam and MEIS1 deletion confirmed by PCR. At three weeks post deletion, bone marrow analysis showed a significant reduction in the number of LT-HSCs defined as lin-/c-Kit+/Sca1+/CD48−/CD150+ in the MEIS1-depleted mice compared to controls (0.012% compared to 0.037%, N=6, p<0.05, t-test). However, the progenitor populations were unaffected by MEIS1 deletion. Over a period of 12 weeks of observation, the mice did not show any signs of distress and the peripheral blood counts of the experimental and control mice remained normal, indicating that short term hematopoiesis was not affected. Cell cycle analysis of LT-HSCs showed that MEIS1 deletion resulted in a significant shift of cells from G0 to G1 phase (G0 and G1 proportions respectively, 81.75±3.25% and 9.40±3% for control and 56.10±0.873% and 31.17±1.5% for MEIS1-deleted). To determine the effects of MEIS1 loss on intrinsic hematopoietic stem cell function, we performed competitive repopulation assays. Bone marrow cells harvested from MEIS1-f/f-Cre or MEIS1-f/+-Cre (control) mice were combined with equal numbers of bone marrow cells from BoyJ mice and transplanted via tail vein injection into lethally irradiated BoyJ mice. Four weeks after transplant, recipients were treated with Tam or vehicle for 5 days and deletion of MEIS1 confirmed by PCR on peripheral blood. Peripheral blood of recipient mice was analyzed at 1, 4, 8, 12 and 16 weeks after treatment and relative chimerism assessed by flow cytometry. At 1 and 4 weeks after treatment, the chimerism in the MEIS1 deleted group (Tam treated MEIS1-f/f-CreER) and the control groups (Tam treated MEIS1-f/+-CReER and vehicle treated MEIS1-f/f-CreER) was comparable (41%, 40.5% and 41.5% respectively, average, N=5 to 8). However, by 8 weeks after treatment, the MEIS1 deleted group showed a significant decline in chimerism compared to controls (18.2% compared to 43.1% and 35.1% respectively, p<0.02, t-test) and at 16 weeks the chimerism in the MEIS1-deleted group declined further (11.1% compared to 40.2% and 35.0% respectively, p<0.001). Subpopulation analysis showed loss of chimerism in granulocytes and in B and T lymphocytes. The latency and breadth of the effect of MEIS1 loss suggested an effect on the hematopoietic stem cell population. Indeed, bone marrow analysis of transplant recipients showed near complete loss of LT-HSC chimerism (3% compared to 70.25% and 75.6% respectively, p<0.001). Finally, we performed gene expression profiling on lineage negative bone marrow cells with and without MEIS1 deletion. Results showed that loss of MEIS1 was associated with decreased expression of hypoxia-responsive genes. Collectively, these results indicate that MEIS1 is required for the maintenance of the pool of LT-HSCs. Loss of MEIS1 promotes cycling and exhaustion of LT-HSCs. Further, we propose that activation of the hypoxia-response pathway may be one of the mechanisms by which MEIS1 exerts its effects on hematopoietic stem cells. Disclosures:No relevant conflicts of interest to declare.

5.24 Pentostatin, Alemtuzumab, and Low-Dose Rituximab Is Effective Therapy for Relapsed/Refractory Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma

Abstract 1790 CLL is incurable with conventional therapies and prognosis is poor in patients with relapsed or treatment refractory disease. Patients with purine analogue refractory disease and defective TP53 function have limited therapeutic options. Based on: 1) The ability of purine analogues to decrease tumor burden; 2) Higher response rates in patients treated with alemtuzumab and rituximab vs. alemtuzumab alone; and 3) Data showing that more frequent lower doses of rituximab could be more effective than standard therapy, we hypothesized that combination therapy with pentostatin, alemtuzumab and low dose rituximab (PAR) would be effective and tolerable therapy for patients with relapsed/refractory CLL and those with previously untreated progressive CLL who had 17p13-. This two stage phase II University of Iowa/Mayo Clinic Lymphoma SPORE clinical trial ([NCT00669318][1]) was approved by Mayo Clinic and the University of Iowa IRBs according to the principles of the Declaration of Helsinki. The aims are to assess: 1) Complete (CR) and overall responses; and 2) Progression-free survival (PFS), duration of response, and time to next treatment (TTT). Patients with progressive CLL (including the SLL variant) defined by standard criteria are eligible for this study if they had either previously treated CLL or previously untreated CLL with 17p13-. Cycle 1 of therapy is 5 weeks with rituximab 20 mg/m2/d IV Mon-Wed-Fri starting day 1, subQ alemtuzumab beginning days 3–5 with a dose escalation (3 mg–10 mg– 30 mg/d) and then 30 mg/d Mon-Wed-Fri, and pentostatin 2mg/m2/dose/IV days 8 & 22. Cycles 2–3 are each of 4 weeks duration and are the same as weeks 2–5 of cycle 1. Patients who achieve CR after cycle 2 with a negative CT scan and bone marrow study with no evidence of residual CLL cells after immunohistochemical staining, do not receive cycle 3 of therapy. All patients receive granulocyte growth factor support after pentostatin therapy as well as PCP and herpes virus prophylaxis. Patients are tested for CMV reactivation by PCR weekly during therapy and treated with valganciclovir if reactivation is detected. We report the results of the planned interim analysis performed after completion of therapy for the first 19 patients (July 2008 - October 2010). The median patient age was 63 years (range 47–78) with 74% males. Two patients with 17p13- were previously untreated. Of the 17 previously treated patients (median 2 prior regimens, range 1–6), 8 (47%) had purine analogue refractory CLL (disease progression < 6 months of treatment). Nine (47%) patients had advanced clinical stage (Rai III-IV). Adverse molecular prognostic factors were 17p13- (n=8, 42%, 2 also had 11q22-), 11q22- (n=1), unmutated IGHV (12/17, 71%), ZAP70+ (≥20%, 12/17, 71%) and CD38+ (≥30%, 6/18, 33%). The median beta-2-microglobulin level was 4.3 (range 2.4 – 12.6). Grade 3–4 treatment related toxicities were 9 non-hematological and 20 hematological events. Grade 3–4 infections occurred in 3 patients including one patient requiring hospitalization for CMV re-activation. Fourteen (74%) patients responded to treatment with 6 (32%) CR (includes 2 CRi) and 8 (42%) partial responses (PR). Five (26%) patients had stable disease (SD). Eleven (58%) patients completed 3 cycles of therapy. One patient achieved a CR with no residual disease after 2 cycles of therapy and received no further treatment per protocol. Median duration of response is 7 months (95% CI: 4–not reached (NR)). Among the 8 patients with 17p13- there were 3 CR/CRi, 4 PR, and 1 SD. Four patients proceeded to allogeneic transplant with reduced intensity conditioning (RIC) and were censored for TTT at the initiation of transplant related therapy. With a median of 16 (range 6 – 35) months of follow up, median PFS is 7 months (95% CI: 5–NR), median TTT is 27 months (95% CI: 5-NR) and the calculated median overall survival is 23 months (95% CI: 14–NR). We report that PAR is effective and tolerable therapy in this population of relapsed refractory patients with high to very-high risk CLL. In addition, the responses in patients with 17p13- were similar to that of the patients who did not have 17p13-. The treatment regimen was useful for disease control in 3 patients who proceeded to reduced intensity conditioning allogeneic transplantation. This study shows that PAR could play an important role in the treatment of recurrent and high risk CLL. Supported by the University of Iowa/Mayo Clinic Lymphoma SPORE [CA097274][2] Disclosures: Zent: GlaxoSmithKline: Research Funding; Genentech: Research Funding; Genzyme: Research Funding. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00669318&atom=%2Fbloodjournal%2F118%2F21%2F1790.atom [2]: /lookup/external-ref?link_type=GEN&access_num=CA097274&atom=%2Fbloodjournal%2F118%2F21%2F1790.atom

Primary Colorectal Lymphoma—Clinical Outcomes in a Population-Based Series

PurposeThe purpose of this study was to investigate the characteristics and the outcomes of primary colorectal lymphomas using a population-based registry. MethodsAll cases of colorectal lymphoma diagnosed between 1980 and 2007 were identified using a provincial cancer registry. Patients meeting Dawson’s criteria and having a negative bone marrow biopsy were included. ResultsOne hundred ten cases of colorectal lymphoma were identified, 43 met the inclusion criteria. The majority of patients was male (86%), and the median age at diagnosis was 62 (range 26–82) years. Tumors were mostly located in the cecum (51.1%) and rectum (20.9%). The 5-year overall survival rate calculated by the Kaplan–Meier method was 57%. Age under 60 was associated with a better median survival time (265 vs 54 months; p < 0.0001). The surgical treatment was associated with a better overall survival compared to medical treatment alone (110 vs 56 months; p = 0.083). Tumors located in the rectum were associated with a decreased overall survival (41 months vs 110 months; p = 0.065). ConclusionsPrimary colorectal lymphoma is a rare disease. The age at diagnosis is an important predictor of outcome. Surgical resection may be associated with improved survival. Rectal lymphoma appears to be associated with a worse outcome and may warrant more aggressive therapy.

The influence of removal of primary tumor on incidence and phenotype of circulating tumor cells in primary breast cancer

Recent studies have shown that the detection of circulating tumor cells (CTC) pre and postoperatively in the peripheral blood of primary breast cancer patients may be an indicator for poor survival. This study aimed to investigate the influence of removal of the primary tumor on incidence and phenotype of circulating tumor cells in primary breast cancer. 209 primary breast cancer patients could be included into this analysis. Blood sampling was performed both pre and postoperatively. The blood specimens were immunomagnetically enriched using AdnaTest BreastCancerSelect within 4 h after blood withdrawal, followed by RNA isolation and subsequent gene expression analysis by reverse transcription and multiplex PCR using AdnaTest BreastCancerDetect. Three breast cancer-associated tumor markers and two hormone receptor genes were amplified: GA733-2, Muc-1, Her-2, ER, PR. In addition, bone marrow (BM) status was intraoperatively determined. Forty-three of 209 patients (21%) had pre and/or postoperatively circulating tumor cells. The positivity rates after surgery were higher but did not differ significantly (12% pre and 16% postoperatively, P = 0.264). Disseminated tumor cells in BM were seen in 32 of 209 cases (15%). Patients with positive BM status had significantly higher CTC positivity rates both pre and postoperatively compared to those with negative BM status. The most common CTC phenotype was triple negative (24 patients) followed by HER2+/ER-/PR- subtype (10) and ER and/or PR positive (9). Interestingly, 41 of 43 primary tumors (95%) were ER and PR positive. Removal of the primary tumor did not alter the phenotype of CTC. Surgery does not significantly influence the tumor cell load in the blood stream. CTC phenotype before and after the surgery generally remains identical but may differ from that of the primary tumor.

47 Chromosomal complexity in primary myelodysplastic syndromes (MDS) in Nagasaki atomic bomb survivors

null linage negative bone marrow cells, from the inducible pLVXpTuner-Green lentiviral vector, restored the erythroid differentiation to the level seen in wild type. Similar results were observed in mouse hematopoietic progenitor cell line (EML) engineered to express p15Ink4b using the inducible pTuner system. Notably, induction of p15Ink4b protein for 24 hours was sufficient to increase commitment of the EML cell line to erythroid lineage, assayed by primitive BFU-E colony formation and increased expression of erythroid linage specific genes. Of interest, increased expression of p15Ink4b was associated with increased expression of EpoR and Gata1 mRNA and protein, hypophosphorylated Rb protein, and decreased expression of myeloid lineage specific transcription factor PU.1 protein. Furthermore, p15Ink4b null mice show delayed recovery from chemically induced anemia assayed by colony formation in methylcellulose based media and flow-cytometric analysis of all hematopoietic progenitors frequency. In summary, our results implicated a novel function of p15Ink4b in the bifurcation of erythroid and myeloid differentiation, thereby contributing to the development of MDS and AML.

Impact of Concordant and Discordant Bone Marrow Involvement on Outcome in Diffuse Large B-Cell Lymphoma Treated With R-CHOP

In diffuse large B-cell lymphoma (DLBCL), prior studies suggest that concordant bone marrow involvement with DLBCL portends a poorer prognosis, whereas discordant bone marrow involvement with small B-cell lymphoma does not. We examined the significance of bone marrow involvement in patients treated in the current era of therapy including rituximab. We performed a retrospective analysis of the prognostic impact of bone marrow involvement in an unselected population of patients with newly diagnosed DLBCL treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone in British Columbia and Auckland, New Zealand, with complete clinical information and evaluable staging bone marrow biopsies. In total, 795 patients were identified. Six hundred seventy (84.3%) of 795 had a negative bone marrow, 67 patients (8.4%) had concordant and 58 (7.3%) had discordant involvement. Median follow-up was 41 months (range, 1 to 115). Progression-free survival (PFS) was inferior in those with concordant (P < .001) and discordant (P = .019) involvement while overall survival (OS) was inferior in those with concordant involvement (P < .001) only. In a multivariate analysis controlling for the International Prognostic Index (IPI) score, concordant involvement remained an independent predictor of PFS (P < .001) and OS (P = .007). Discordant involvement was associated with older age, elevated lactate dehydrogenase, advanced stage, and increased number of extranodal sites and was not a negative prognostic factor independent of the IPI score. The negative prognostic impact of discordant involvement is adequately represented by the IPI score, while the risk with concordant involvement is greater than that encompassed by this predictor. The results emphasize the need for accurate staging assessment of bone marrow involvement in DLBCL.

Immunoglobulin D-Lambda Type Multiple Myeloma Presenting with FDG-PET/CT Negative Bone Marrow Involvement

We here report a rare case of a patient with IgD-lambda-positive multiple myeloma presenting with FDG-PET/CT negative bone marrow involvement. A 72-year-old man was admitted to our hospital for evaluation of a paravertebral tumor of the chest. Thoracotomy was performed and a histopathological evaluation of resected intrathoracic tumor demonstrated a plasmacytic neoplasma. Initially we thought that this case was a solitary plasmacytoma because there were no positive findings on postoperative FDG-PET/CT. However, bone marrow aspiration study demonstrated massive infiltration of myeloma cells (72%). It is necessary to recognize that IgD-lambda type myeloma cells may not be sufficiently metabolically active to form high uptake on FDG-PET/CT.

Iron-Deficiency Anemia in Castleman Disease: Implication of the Interleukin 6/Hepcidin Pathway

In addition to occasional autoimmune hemolytic anemia, unexplained iron-deficiency anemia has been reported in childhood Castleman disease (CD). The recent discovery of hepcidin has regenerated the research on iron metabolism. This hormone is a key regulator of iron homeostasis, mainly by inhibiting intestinal iron absorption. Liver expression of hepcidin increases in response to interleukin 6 (IL-6). With chronic overproduction of IL-6 as a hallmark, CD could be an interesting human model for studying the contribution of the IL-6/hepcidin pathway in the pathogenesis of anemia of chronic disease. We report here the case of a 16-year-old boy with chronic iron-deficiency anemia (plasma ferritin: 19 μg/L; plasma iron: 2.2 μmol/L; negative bone marrow Perls' Prussian blue stain), inflammatory syndrome (C-reactive protein: 108 mg/L), and growth retardation for the previous 2 years. Diagnostic workup revealed a large mesenteric mass corresponding to localized CD of mixed histologic type. Resection of the tumor resulted in complete resolution of iron-deficiency anemia and inflammatory syndrome. Parallel variations of plasma IL-6, C-reactive protein, and hepcidin concentrations, together with tumor immunohistochemistry, strongly suggested that IL-6 synthesized by the tumor caused both the inflammation and iron deficiency through enhancement of hepcidin production by the liver. The results of this unique case study (1) explain the mechanism of iron deficiency observed in some children with CD, (2) confirm in vivo the regulatory effect of IL-6 in human hepcidin production, and (3) suggest that iron deficiency is a causal link between IL-6 and anemia of chronic disease.

Bmi-1 Is Dispensable for the Development of Acute Myeloid Leukemia Mediated by MLL-AF9

AbstractAbstract 63The Polycomb group 1 gene Bmi1 has been shown to be required for several normal adult stem cell types, including hematopoietic stem cells. High expression of Bmi1 is correlated with adverse prognosis in human AML and MDS. Bmi1 null murine fetal liver cells transformed with HoxA9 and Meis1 give rise to a primary leukemia of expanding blasts that fails to expand in secondary recipient mice thus demonstrating a defect in leukemia self-renewal (Nature. 2003; 423:255). More recently, Bmi1 has also been shown to bind PML-RARA, and to be required for in vitro replating of PML-RARA immortalized murine colony forming cells (cfc).Our laboratory recently demonstrated that MLL-AF9, a recurrent translocation commonly found in human acute leukemia, more efficiently transforms murine granulocyte macrophage progenitors (GMP) compared to the combination of HoxA9 and Meis1. We decided to test if MLL-AF9 is able to bypass the requirement for Bmi1 previously reported in other murine leukemia models. Lineage marker negative (Lin-) bone marrow cells from WT and Bmi1 −/− mice were transduced with an MSCV-based ecotropic retroviral vector expressing MLL-AF9 and linked via an internal ribosomal entry site (IRES) the selectable marker, GFP. Cells were harvested and injected into 5 sublethally (200 cGy) irradiated NOD/SCID mice per group. All mice developed AML based on GFP expression, spleen size, forward/side scatter profile and surface marker staining (Mac1 positive/Gr1 positive, B220 negative, CD3 negative). Median latency in the WT group was 54 days, whereas mice receiving a graft consisting of Bmi1 −/− cells transduced with MLL-AF9 developed leukemia with a median latency of 89 days. The experiment was repeated with very similar results with median latencies of 50 days (WT group) and 70 days (Bmi1 −/−). To confirm sustained self-renewal, we then proceeded to secondary transplantation experiments. Secondary leukemia developed with similar latency in recipients of primary WT (31.5d) and Bmi1−/− (32d) leukemic bone marrow cells. The experiment was repeated with bone marrow from mice with primary leukemias from the second cohort and again similar latencies were observed (42.5 days in the WT group vs 29.5 days in the Bmi1 −/− group.). Finally we proceeded to tertiary transplantation experiments. All recipients of secondary AML cells developed AML with median latencies of 17.5 d (WT) and 21 d (KO).To compare our results with a system closer to the original studies of leukemogenesis in a Bmi1 −/− background, we chose to transduce lin- bone marrow from WT and Bmi1 −/− with retroviral vectors for HoxA9-IRES-GFP and Meis1-pgk-puro. Both, WT and HoxA9/Meis1 transduced cells replated in in vitro methylcellulose cultures for at least 4 rounds. In the first of 2 in vivo experiments, 5/5 recipients of wild type cells developed acute myeloid leukemia with a median latency of 88 days. In contrast, none of the five recipients of HoxA9/Meis1a transduced Bmi1−/− cells developed leukemia, and these mice were sacrificed 6 months after transplantation. In a second independent experiment, 5/5 recipients of WT HoxA9/Meis1 transduced cells developed AML with a median latency of 62 days. One recipient of Bmi1 −/− cells transduced with HoxA9/Meis1 developed leukemia. One mouse died from a spontaneous lymphoma. The remaining 3 mice in this cohort did not develop leukemia. These findings are consistent with the previously published data and suggest that MLL-AF9 is more efficient in generating AML in Bmi1−/− bone marrow compared to the combination of HoxA9 and Meis1.To further characterize the Bmi1 −/− leukemias, we analyzed expression of the primary Bmi1 target p16 by Western blot. In primary, secondary and tertiary AMLs from Bmi1 −/− mice, derepression of p16 could be demonstrated. We analyzed the cell cycle distribution of cultured tertiary leukemia cells. WT cells showed a S phase proportion of 44–45 percent (n=2), whereas the Bmi1 −/− cells demonstrated a S-phase proportion of 30–38% (n=2). These results demonstrate modest effects of p16 derepression on cell cycle without catastrophic failure to divide. This is mirrored by the in vivo development of secondary and tertiary leukemias in the presence of elevated p16 levels. Preliminary data suggest a similar but less pronounced derepression of p19. Future experiments will focus on the mechanisms that allow MLL-AF9 transduced cells to grow in the presence of derepressed p16 and p19. Disclosures:Armstrong:Epizyme, Inc: Consultancy.

High Resolution Genome-Wide Copy Number Analysis of Pediatric Burkitt Lymphoma Identifies Copy Number Alterations In the Majority of Patients

AbstractAbstract 3123Although cure rates are high for pediatric Burkitt lymphoma (BL), a subset of patients relapses and succumbs to the disease. BL is characterized by translocation of the MYC gene with an immunoglobulin gene, but secondary changes including gain of 1q, gain of 13q, loss of 13q, loss of 17p, and others have been described by both conventional cytogenetic and oligo array CGH approaches. Secondary changes may contribute to the clinical heterogeneity of BL as evidenced by the fact that loss of 13q is associated with a worse prognosis in pediatric BL (Poirel et al., Leukemia 23:323, 2009). However, high resolution, genome-wide copy number analysis has not yet been reported in pediatric BL. The objective of this study was to identify copy number alterations (CNAs) in pediatric BL using a genome-wide approach, and to examine the relationship between CNAs and clinical parameters including outcome. After institutional review board approval, we identified 30 pediatric BL patients treated at Primary Children's Medical Center (n=25, Salt Lake City, UT) and Penn State Hershey Medical Center (n=5, Hershey, PA) with available formalin-fixed, paraffin-embedded (FFPE) diagnostic biopsy specimens. Age, site, and gender data were available for all specimens, and 22/24 of the Utah patients were treated according to the COG 5961 protocol with full clinical information and follow-up available. DNA was isolated from FFPE biopsies containing at least 80% tumor. In addition, germline DNA was isolated from negative staging bone marrow clot sections on Utah patients (n=25) to serve as a pooled normal reference and to provide germline copy number variation data on individual patients. Tumor and paired normal DNA was submitted for Molecular Inversion Probe (MIP) assay (330K Cancer Panel, Affymetrix, Santa Clara, CA). The Nexus Copy Number (BioDiscovery, El Segundo, CA) software package was used to analyze the MIP data with the following stringent call criteria: SNPRank segmentation, 5.0E-4 significance threshold, 1000 kb maximum contiguous probe spacing, minimum of 5 probes per segment, gains ≥ 2.7 copy number value, and loss ≤ 1.3 copy number value. Patients included 23 males and 7 females with a mean age of 8.0 years (range 2 – 18). At presentation, lactate dehydrogenase (LDH) levels ranged from 443 – 13851 U/L and uric acid levels ranged from 1.5 – 13 mg/dL. Patients were Murphy stage I (n=1), II (n=9), III (n=13), and IV (n=2). A total of three patients relapsed (one stage II patient and two stage III patients) and three died (the two stage III patients that relapsed and a different stage II patient who did not have a complete clinical response). 27 of the 30 tumor samples and 22/25 paired normals had adequate DNA for MIP analysis. The 3 tumor samples without adequate DNA were clinically similar to the others. We identified a total of 103 CNAs (defined as change seen in the same cytoband in 1 or more patients), which included 63 gains and 40 copy number losses. 23/27 cases (85%) had at least one gain or loss. We identified 21 recurrent CNAs (same cytoband affected in 2 or more patients), which included 14 gains and 7 losses. We found gains of 1q in 10/27 patients (37%), gains of 13q in 4/27 patients (15%) and losses of 17p in 3/27 patients (11%). Despite a relatively small sample size, deletion of 17p13 was significantly associated with relapse (p=0.041). To our knowledge, this is the first report of high-resolution genome-wide copy number analysis of pediatric BL. Furthermore, we show for the first time that FFPE archived materials can be used for high resolution gene copy number analysis in lymphomas. We identified CNAs in 85% of the pediatric BL cases, which include previously described changes of 1q, 13q, and 17p. Although sample size limited statistical power, deletion of the 17p13 locus was associated with relapse. We plan to extend these studies in a larger sample of patients to evaluate the potential prognostic significance of both the 17p13 locus deletion as well as additional recurrent CNAs. Disclosures:No relevant conflicts of interest to declare.

The Specificity of Immunophenotypic Alterations in Blasts in Nonacute Myeloid Disorders

Data regarding flow cytometry (FC) in nonacute myeloid disorders is confounded by variable gating strategies and controls limited to normal bone marrow (BM) samples. Blasts in diagnostic BM samples of myelodysplastic syndromes (MDSs), myeloproliferative neoplasms (MPNs), and chronic myelomonocytic leukemias (CMMLs) were compared with 20 nonneoplastic cytopenias/cytoses (CCs) and negative staging BM samples using 4-color FC. Blasts in 10 of 20 CCs showed immunophenotypic differences vs control samples. Immunophenotypic alterations were identified in 18 of 21 MDSs, 11 of 14 MPNs, and 7 of 7 CMMLs vs control samples and 13 (62%) of 21 MDSs, 7 (50%) of 14 MPNs, and 3 (43%) of 7 CMMLs vs CCs. Neoplastic-specific blast immunophenotypic changes included expression of CD7, CD11b, CD15, CD36, and CD56; CD34 overexpression; HLA-DR variability; lack of CD13 and CD33; underexpression of CD13, CD33, CD45, and HLA-DR; and partial loss of CD13, CD33, CD38, and CD117. In all cases, blasts were CD34+. Several blast immunophenotypic alterations are shared in neoplastic and nonneoplastic BM samples. Approximately 40% to 60% of neoplastic BM samples exhibited aberrancies not seen in reactive BM samples.

Maintenance of HSC by Wnt5a secreting AGM-derived stromal cell line

The microenvironment wherein hematopoietic stem cells (HSC) reside orchestrates HSC self-renewal vs. differentiation decisions. Stromal cells derived from ontogenically divergent hematopoietic microenvironments can support HSC in vitro and have been used to decipher factors that influence HSC fate decisions. Employing stromal cell lines derived from the aorta-gonad-mesonephros and embryonic liver, we aim to identify secreted factors that maintain/expand HSC in vitro. We cultured murine lineage antigen-negative (Lin(-)) bone marrow cells in transwells above the UG26-1B6, urogenital ridge-, and EL08-1D2, embryonic liver-derived cell lines. We, also, performed real-time quantitative PCR analysis to identify differentially expressed genes from the Wnt family of proteins in ontogenically different stromal cell lines. Lin(-) murine bone marrow cells maintained for 3 weeks in transwells above UG26-1B6 but not EL08-1D2 cells contained competitive repopulating HSC. Addition of as few as 25% UG26-1B6 cells to EL08-1D2 feeders led to maintenance of HSC in noncontact cultures, validating soluble factors are secreted by the UG26-1B6 cells. As we found that Wnt5a was significantly higher expressed in UG26-1B6 than EL08-1D2 cells, we added Wnt5a to EL08-1D2 transwell cultures or an antibody against Wnt5a to UG26-1B6 transwell cultures. Addition of Wnt5a to EL08-1D2 transwell cultures restored maintenance of HSC, whereas addition of an anti-Wnt5a antibody to UG26-1B6 transwell cultures inhibited maintenance of competitive repopulating HSC. We demonstrate that stromal cell lines generated from embryonic microenvironments provide a tool to identify secreted proteins that play a role in the maintenance of HSC, and that at least one of the factors produced by UG26-1B6 cells responsible for preserving HSC is Wnt5a.

Surgical therapy of skeletal complications in multiple myeloma

Patients with multiple myeloma are often treated surgically as though they have bone metastases. Due to major differences in oncological therapy and comparatively long survival times these patients should be considered separately. Seventy-five multiple myeloma patients were treated surgically (83 interventions) for skeletal complications of the disease. Location and dissemination, symptoms, method of surgery, complications, recurrence and survival time were evaluated retrospectively. Most of the lesions were in the axial skeleton or the proximal extremities apart from one distal lesion of the fibula, and most surgery was performed in the spine (35 patients). The mean follow-up of patients was 5.4 years (range 1-25 years). Survival proved to be very favourable (37% at five years). Patients with a single bone lesion, a negative bone marrow biopsy, no paraproteinaemia in serum or a Salmon-Durie-stage I had a better survival probability. Surgical treatment in patients with multiple myeloma was mostly limited to a palliative approach but survival time was better (37% at five years) than in patients with metastatic bone disease which has to be considered in their surgical treatment.

Monitoring Residual Myeloma

Numerous methods exist for monitoring residual disease in multiple myeloma using peripheral blood, urine, and bone marrow (BM) techniques. This study was conducted in a cohort of 83 patients to compare residual disease detection using serum protein electrophoresis (SPE) and urine protein electrophoresis (UPE) with immunofixation with immunoperoxidase staining of BM tissue sections. Of the 83 patients, 49 had a positive SPE and/or UPE result and all had BM involvement shown by immunohistochemical analysis. The results for SPE and UPE were negative in 17 patients, and all 17 had a negative immunohistochemical BM result for a negative predictive value for SPE/UPE of 100%. In addition, SPE and UPE detected residual disease in 17 patients with negative BM biopsy studies. SPE and UPE can be used to screen patients for residual disease, and negative results for both can obviate the need for BM biopsy for the detection of residual disease by immunohistochemical analysis.

Minimal Disseminated Disease Is An Independent Poor Prognosis Marker Among High Risk Burkitt's Lymphoma Patients .

Abstract 2944Poster Board II-920 Introduction:The chromosomal translocation t(8;14)(q24;q32) represents a specific tumor marker in Burkitt's lymphoma (BL). This chromosomal aberration involves the MYC oncogene on chromosome 8 and the immunoglobulin heavy-chain (IgH) locus on chromosome 14. We have previously demonstrated that this genetic abnormality can be used as a marker of Minimal Disseminated Disease (MDD) in BL (Mussolin et al, JCO, 2007). The aim of the study was to assess of the prevalence of MDD in BL at diagnosis in children enrolled in the AIEOP LNH-97 clinical protocol and the evaluation of its impact on prognosis. Patients and Methods:We established a simplified long-distance PCR (LD-PCR) assay which can amplify up to 15-20 Kb DNA sequence making it possible to detect the t(8;14) at the genomic level with the sensitivity of 10-4. The assay was based on 4 separate PCR reactions in which one primer complementary to the first exon of the MYC gene is used with one of four primers for the IgH locus (1 for the JH region and 1 for each of the 3 constant regions). Results:LD-PCR was applied to prospectively study 124 BL biopsies and detected a specific PCR product in 88 of them (71%). Of the 88 positive BL patients we studied both the tumor and the bone marrow (BM) at diagnosis in 76: BM was positive by LD-PCR in 25 patients (33%), whereas only 10 (13%) were positive at the standard morphological and/or immunophenotypical analyses. Most of the MDD positive patients (88%) belonged to the R4 Risk Group according to BFM definition (stage III or stage IV according to St. Jude staging classification and LDH≥1000 U/l). The 3-year progression-free survival (PFS) was 68% (SE 10%) in MDD positive R4 patients compared with 96% (SE 4%) in MDD negative R4 patients (p= 0.02), whereas there was no difference in PFS between children with morphological involvement of BM at diagnosis versus those who had negative BM (PFS=62.5% (SE 17%) vs. PFS= 87% (SE 6%), respectively, p= 0.09). By multivariate analysis (including MDD, gender, LDH, CNS involvement) MDD was predictive of higher risk of failure (Hazard Ratio: 8.4 , p= 0.04). Conclusions:We demonstrated that MDD identifies a poor prognosis subgroup among high risk Burkitt's lymphoma patients. We suggest that a more effective risk-adapted therapy, possibly including anti-CD20 monoclonal antibody, should be considered in these patients. Disclosures:No relevant conflicts of interest to declare.