ALBERT

Description: Abstract 3466 Donor cell leukemia (DCL) in the setting of bone marrow/hematopoietic stem cell transplant (HCT) has not been well characterized. We analyzed 9 cases of DCL and performed a literature review (table). The indications for transplant and subtypes of DCL are shown (table). The 6 myelodysplastic syndrome (MDS) cases included 1 case of refractory cytopenia with multilineage dysplasia (RCMD), 2 cases of refractory anemia and 3 cases which were unclassifiable. Conventional cytogenetic analysis was performed on all 9 cases of DCL (table). All 9 cases had engraftment studies performed either by short tandem repeat analysis (3) or FISH analysis for donor gonosomal complement (6) when DCL was diagnosed. Seven cases had either engraftment studies or cytogenetic analysis performed periodically after HCT to test the donor cell engraftment and engraftment was confirmed in all. FISH analysis for monosomy 7, del(7q) and del(5q) was retrospectively performed on preserved donor cells in 4 cases after DCL was diagnosed. A low level of abnormalities was observed in preserved donor cells for the cases with del(7q) (2.9%) and del(5q) (8.2%). The 2 cases of AML received chemotherapy. Of the MDS cases, 2 received donor cell infusion, 1 received 6 cycles of revlimid, and 3, along with the case of CLL, received either supportive therapy or were simply observed. Six cases have clinical follow up ≥ 5 months and of these, 1 died of disease (AML) while the other 5 are alive, including 4 MDS and the 1 CLL. The disproportionate detection of DCL in sex mismatched HCT suggests a probable under-detection in the sex-matched population. In our analysis, the interval between HCT and diagnosis of DCL (table) falls within the range of currently reported cases. When stratified by type of DCL, the T-LGL group demonstrates presentation significantly earlier than other groups (Fig. A), indicating pathogenesis of T-LGL may involve a distinct pathway. When stratified by types of primary disease, the interval of the neoplastic group is shorter than that of benign group (Fig. B), implying that pre-HCT treatment may play a role in the pathogenesis of DCL. When stratified by stem cell sources, UCB group shows shorter latency than the other sources (Fig. C), suggesting a higher risk of DCL in this cell source. The low level cytogenetic abnormalities of preserved donor cells in our series and the longer latency of the benign group suggest that donor cells with an intrinsic defect may be predisposed to evolve into DCL. Total cases (%) Reported cases (%) Current cases (%) Number of cases 83 74 9 Age (years)     Median/range 37.0/3~70 36.0/4~62 53.0/3~70 Gender     Male 43 (52.4) 38 (52.0) 5 (55.6)     Female 39 (47.6) 35 (48.0) 4 (44.4) Primary disease     Neoplasms 76 (91.6) 67 (90.5) 9 (100)     Non-neoplasms 7 (8.4) 7 (9.5) 0 (0.0) Donor     Related 59 (72.0) 54 (74.0) 5 (55.6)     Unrelated 23 (28.0) 19 (26.0) 4 (44.4)     Sex-matched 28 (34.6) 27 (37.5) 1 (11.1)     Sex-mismatched 53 (65.4) 45 (62.5) 8 (88.9) Donor cell source     BM 48 (63.2) 44 (65.7) 4 (44.4)     BHSC 16 (21.0) 13 (19.4) 3 (33.3)     UCB 12 (15.8) 10 (14.9) 2 (22.2) 2nd neoplasm (DCL)     AML 31 (37.4) 29 (39.2) 2 (22.2)     MDS/MPN* 27 (32.5) 21 (28.4) 6 (66.7)     ALL 20 (24.1) 20 (27.0) 0 (0.0)     T-LGL 4 (4.8) 4 (5.4) 0 (0.0)     CLL 1 (1.2) 0 (0.0) 1 (11.1) Interval (months)     Median/range 24.0/1~312 24.0/2~312 26.0/1~193 Cytogenetics     Normal 21 (28.0) 20 (30.3) 1 (11.1)     Abnormal 54 (72.0) 46 (69.7) 8 (88.9)     -7 or del(7q)** 15 (27.8) 10 (21.7) 5 (62.5)     +8** 2 (3.7) 2 (4.4) 0 (0.0)     Del(20)** 4 (7.4) 2 (4.4) 2 (25.0)     Del(5q)** 2 (3.7) 1 (2.2) 1 (12.5)     11q23** 3 (5.6) 3 (6.5) 0 (0.0) Other abnormalities** 28 (51.9) 28 (60.9) 0 (0.0) Follow up (months)     Median/range 8.5/1~108 9.0/1~108 6.0/1~68     Died of disease 28 (46.7) 27 (52.9) 1 (11.1) DCL, donor cell leukemia; BM, bone marrow; BHSC, blood hematopoietic stem cells; UCB, umbilical cord blood; AML, acute myeloid leukemia; MDS, myelodysplastic syndrome; ALL, acute lymphoblastic leukemia (including B-cell and T-cell ALL); T-LGL, T-cell large granular lymphocyte leukemia; CLL, chronic lymphocytic leukemia. All the numbers represent the cases with data available. * One case of myeloproliferative neoplasm is included in this category. ** The percentage is calculated using number of total cytogenetic abnormalities in each column as denominator. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 898 Burkitt Lymphoma (BL) is a highly proliferative form of non-Hodgkin lymphoma and is characterized by translocation of the C-MYC gene to the immunoglobulin gene loci resulting in deregulation. The role of collaborating gene mutations in BL is largely unknown. We performed whole exome sequencing and gene expression profiling of 57 Burkitt lymphoma and 94 DLBCL exomes. Mutational analysis revealed that ID3 is recurrently mutated in 38% of Burkitt lymphoma samples. ID3 mutations did not occur in any of the 94 DLBCL cases. ID3 gene expression was also found to be a distinguishing feature of Burkitt lymphomas (P

Description: A role for microRNA (miRNA) has been recognized in nearly every biologic system examined thus far. A complete delineation of their role must be preceded by the identification of all miRNAs present in any system. We elucidated the complete small RNA transcriptome of normal and malignant B cells through deep sequencing of 31 normal and malignant human B-cell samples that comprise the spectrum of B-cell differentiation and common malignant phenotypes. We identified the expression of 333 known miRNAs, which is more than twice the number previously recognized in any tissue type. We further identified the expression of 286 candidate novel miRNAs in normal and malignant B cells. These miRNAs were validated at a high rate (92%) using quantitative polymerase chain reaction, and we demonstrated their application in the distinction of clinically relevant subgroups of lymphoma. We further demonstrated that a novel miRNA cluster, previously annotated as a hypothetical gene LOC100130622, contains 6 novel miRNAs that regulate the transforming growth factor-β pathway. Thus, our work suggests that more than a third of the miRNAs present in most cellular types are currently unknown and that these miRNAs may regulate important cellular functions.

Description: Abstract 433 Burkitt lymphoma (BL) is a relatively uncommon lymphoma, but is clinically important because it is curable when diagnosed properly. BL is also an important model disease for studying cancer. Chromosomal translocations of the MYC gene are a defining feature of BL. Diffuse large B cell lymphoma (DLBCL) is the most common form of lymphoma in adults and demonstrates overlapping morphology, immunophenotype and clinical behavior with BL. The genetic causes and the role of specific mutations in BL are largely unknown. Th e decoding of the human genome and the advent of high-throughput sequencing have provided rich opportunities for the comprehensive identification of the genetic causes of cancer. We began by sequencing 2 complete lymphoma tumor genomes (and paired normal tissue) derived from DLBCL and BL respectively. The pattern of somatic base alterations in both DLBCL and BL genomes indicated a predominance of G→A/C→T and A→G/T→C transitions (P

Description: Background: Mature B cell differentiation provides an important mechanism for the acquisition of adaptive immunity. Malignancies derived from mature B cells are common and constitute the majority of leukemias and lymphomas. MicroRNAs are known to play a role in oncogenesis, lineage-selection, and immune cell function, including early B cell differentiation. However, the full extent and function of microRNA expression during mature B cell differentiation and in B cell malignancies are not known. Methods: From normal young patients undergoing tonsillectomies, we sorted the mature B cell subsets (naive, germinal center, memory and plasma) using FACS, based on their expression of CD19, CD38, IgD and CD27. These sorted B cells were profiled for microRNA expression using a highly sensitive multiplexed real-time PCR assay, as well as for gene expression at the whole genome level using Affymetrix U133plus microarrays. miRNA targets can be predicted based on seed sequence matching of their 2–8 nt to the 3′UTR of gene transcripts. For each B cell stage, we experimentally validated microRNA regulation of predicted target genes of interest, LMO2, MYBL1 and PRDM1, by microRNA over-expression experiments and luciferase assays. Results: We found that microRNAs have a characteristic expression pattern that defines each mature B cell stage. Examination of both microRNA and mRNA expression showed that in each B cell population, the target genes predicted based on seed matching were expressed at lower levels, results that were highly significant (P

Description: Abstract 4604 Background: Chronic lymphocytic leukemia (CLL) has the highest incidence and prevalence of any leukemia in the United States. It has a characteristic immunophenotype, but variant forms (typically termed “atypical CLL”) are commonly encountered. It is not known if this immunophenotypic heterogeneity in CLL is clinically relevant. We hypothesized that the immunophenotype of CLL cells correlates with both biologic parameters and clinical outcomes. Methods: We performed a retrospective review of clinical, biologic, and flow cytometric data of patients undergoing an evaluation of an absolute lymphocytosis at the Duke University and Durham VA Medical Centers. Patients meeting immunophenotypic criteria for CLL based on cell surface expression of CD5, CD19, CD20, CD23, and surface immunoglobulin light chain (sIg) were included in this analysis. Atypical CLL was defined if any of the following were observed: (1) expression of CD5, CD20, CD23, or sIg that differed from a typical CLL immunophenotype (e.g. bright CD20 or low CD23; hereafter non-standard immunophenotype), (2) expression of T cell markers CD7 or CD8, or (3) expression of CD123. Through electronic chart review, clinical and biologic parameters were abstracted. These included age; gender; ethnicity; WBC, hemoglobin, platelet count, and Rai stage at diagnosis; time to first treatment; overall survival; CD38, ZAP70, IGHV mutation status, and interphase cytogenetics (FISH). A chi square test or Fisher’s exact test for small sample sizes was used to test association of categorical data. The central tendencies of continuous measurement were compared using the Wilcoxon rank sum test. Overall survival and time to treatment were calculated using the Kaplan-Meier product limit method. Survival curves were compared using the log-rank test. This study was approved by IRBs at Duke University and Durham VA Medical Centers. Results: We reviewed 189 patients, including 119 (63%) with typical CLL immunophenotype and 70 (37%) with atypical. Among patients with atypical CLL, 93% (65 of 70) had a non-standard immunophenotype, 33% (23 of 70) had expression of T cell markers, and 33% (14 of 43) had expression of CD123. We observed expression of T cell markers in 12% (23 of 189) and CD123 in 12% (14 of 114) of the entire cohort, respectively. At the time of diagnosis, there was no association between typical or atypical CLL immunophenotype and age, gender, race/ethnicity, WBC, hemoglobin, platelet count, or Rai stage (p〉0.05 for all comparisons). With regard to biologic parameters, there was no association of CLL immunophenotype with the proportion of patients with CD38 ≥30%; however, the central tendency of CD38 was higher in the atypical group when analyzed as a continuous variable (median value 38% vs. 10%, p=0.04). There was no difference in the proportion of ZAP70 positive cases between typical and atypical groups. Analysis of interphase cytogenetics (FISH) data showed that a higher percentage of typical than atypical immunophenotypes had del 13q as a sole abnormality and a lower percentage had trisomy 12 (p=0.02). Despite the increased frequency of del 13q as a sole abnormality, lower frequency of trisomy 12, and lower expression of CD38, there was no difference between groups in time to first treatment (median 5.3 years for typical vs. 2.1 years for atypical; p= 0.11) or in overall survival (median 13.6 yrs64 years for typical vs. 18.0 years for atypical; p=0.11) (Figure 1). However, when patients with non-standard CLL immunophenotype were compared to those with a standard immunophenotype, a significantly shorter time to first treatment was observed (median 6.0 years for standard immunophenotype vs. 2.2 years for non-standard, p=0.03). Conclusion: Different immunophenotypic subtypes are commonly encountered in the care of patients with CLL. These groups have different biological characteristics, including differences in expression of CD38 and cytogenetic abnormalities. Exploratory analysis also showed differences in clinical outcomes based on immunophenotypic parameters. Validation studies are currently underway. Disclosures: No relevant conflicts of interest to declare.

Description: To minimize toxicity and monitor immune recovery without interference of long term use of immunosuppressive agents, we have investigated T-cell depleted, nonmyeloablative allogeneic therapy using matched family member donors. Methods: Seventy five patients who were not candidates for ablative therapy due to age or comorbid diseases received fludarabine 30mg/sq m and cyclophosphamide 500mg/sq m IV qd x 4 with alemtuzumab 20mg IV qd x 5 followed by stem cell infusion. No other post transplant immunomodulation was provided. Results: Patient diagnoses included lymphoma/myeloma (20), leukemias/MDS (30), myelofibrosis/aplasia (7) and metastatic solid tumours (18). The median age was 50 (range 18–17) with a median follow up of 18 months. The median CD34+ cell dose collected was 13.4 x 106/kg. Engraftment occurred in 100% of patients with a median of 97% donor cells responsible for patient hematopoiesis by 3 months. Forty six also had a DLI (range 106–108 CD3+ cells/kg). Overall, Grade III–IV acute GVHD occurred in only 5/75 (7%) and 18 (29%) had grade II–IV. Four patients developed chronic GVHD. The transplant regimen was well tolerated with 4% 100 day treatment related mortality. In those with hematologic malignancies, only 7% started in remission, though 45 (60%) attained a CR. The most common cause of death in this group was progressive disease (28%), followed by infections (5%), and GVHD (5%). A subgroup of 21 of these older, more infirm patients who had high risk AML in 1–2nd CR or PR or ≥2 chronic stable phase CML, partially responding lymphoma, or severe myelofibrosis have been followed for at least 1 year. At 1 year, 13/21 (62%) remain alive and in continuing remission. Phenotypic analysis of lymphocyte subsets (measured by flow) revealed recovery at 6 months. Figure Figure T cell VBeta family recovery (spectratype analysis using PCR) revealed robust recovery by 6 months as well (first figure pre and second is 6 months post transplant), despite T depletion. Figure Figure TRECs analysis reveals little, if any, recovery in these patients for at least 12–18 months. Conclusions: Nonablative allogeneic transplantation using alemtuzumab for T cell depletion results in reliable engraftment with little acute GVHD or TRM. Immune recovery assays reveal that despite T cell purging, T cell subset and VBeta families have significant recovery by 6 months and TRECS results show the recovery is primarily from peripheral expansion of transplanted donor cells, not education of new T cells. These data possibly reflect the advantage of low dose donor lymphocyte boosts without planned long term use of immunosuppressive agents. The future challenge will to be to develop strategies to further improve immune recovery in this setting.

Description: Background: Burkitt lymphoma (BL) is a highly aggressive lymphoma that can be cured in up to 80% of patients when treated with intensive multi-agent chemotherapy. The distinction between BL and diffuse large B-cell lymphoma (DLBCL) is critical because there are important differences in their clinical management. However, the distinction can be difficult because of an overlap between DLBCL and BL in morphology, immunophenotype and cytogenetics. Previous work has shown that gene expression profiling can distinguish these entities with a high degree of certainty. Our previous work has demonstrated that microRNAs play a direct role in regulating key transcription factors in normal and malignant B cells. We investigated whether microRNA expression could be used to reliably distinguish BL from DLBCL. Methods: Biopsy samples were collected from 104 patients with a diagnosis of either sporadic BL (N=25) or DLBCL (N=79). All cases were reviewed for pathology diagnosis and profiled for microRNA expression using microarrays. Using the 30 most highly differentially expressed microRNAs with the highest t-statistic, we applied singular value decomposition to identify the 10 most predictive microRNAs. Using those 10 microRNAs, we constructed a Bayesian predictor to distinguish BL from DLBCL. The predictor performance was tested using leave-one-out cross-validation. We further applied gene expression profiling to 52 cases of DLBCL to identify the molecular subsets of DLBCL: activated B cell type and germinal center B cell type DLBCL. We constructed a Bayesian predictor to distinguish these molecular subsets based upon their gene expression. A separate predictor was created from the microRNA profiles of these DLBCL subsets and tested using leave-one-out cross-validation. In order to understand the effects of lineage-specific microRNAs in B cell lymphomas, we applied FACS-sorting to isolate mature B cell subsets including naïve B cells, germinal center B cells, plasma cells and memory cells. We compared the microRNAs involved in germinal center differentiation to those expressed highly in Burkitt lymphoma. Results: The predictor constructed based on microRNA expression was 90% accurate in distinguishing Burkitt lymphoma from DLBCL, using pathology diagnosis as the standard. The microRNA-based predictor was also over 90% accurate in the distinction of the molecular subsets of DLBCL, compared to the gold standard of gene expression-profiling. Further, we found that the Burkitt lymphoma cases consistently expressed microRNAs related to normal germinal center B cell differentiation, suggesting that they also maintain expression of B cell stage-specific microRNAs. Conclusion: This study demonstrates that the microRNA expression profiles can be used to accurately distinguish Burkitt lymphoma from DLBCL. The ability of the predictor to identify the molecular subsets of patients with DLBCL and those with BL suggests that it will be useful in the diagnosis and management of patients with Burkitt lymphoma. Further, the patterns of microRNA expression and their target genes suggests a role for microRNAs in the pathophysiology of these tumors.