ALBERT

Description: Key Points TP53 3′UTR variations demonstrate prognostic value in DLBCL.

Description: Essential thrombocythemia (ET) is a clonal myeloproliferative disease that involves primarily the megakaryocytic lineage. ET is characterized by sustained thrombocytosis in the blood and increased in numbers of large, mature megakaryocytes in the marrow and, occasionally, in the extramedullary organs. Currently, there is no known genetic or biologic marker specific for ET. The etiology and pathogenesis of ET remain largely unclear, partially due to a lack of suitable animal model for the disease. We reported here the development of a transgenic (TG) mouse model, in which most aged mice presented with thrombocytosis, marrow megakaryocytic and myeloid hyperplasia, splenomegaly with marked extramedullary hematopoiesis, features that closely mimic ET in humans. TG mice were generated via microinjection of a mammalian construct consisting of a mitochondrial isoform of human 8-oxoguanine-DNA glycosylase (hOGG) under the control of a mouse metallothionein-1 (mMT-1) promoter. A total of 11 founder mice were obtained and shown to successfully integrate the transgene in their genome, as verified by PCR. Two of the male founder mice successfully transmitted the transgene to their offspring at an expected frequency of 50%. Three founder mice at the age of 12 months and 5 F1 offspring at the age of 4 months were examined. All aged hOGG TG founder mice and their offspring expressed high levels of hOGG mRNA in their liver by RT-PCR and direct DNA sequencing. Upon histologic examination, 3 TG founder mice displayed moderate to severe splenomegaly. The spleen weights were 2-, 4- and 10 times respectively in the 3 TG founder mice as compared to wild type, age-matched mice. Microscopically, the red pulp in the enlarged spleens was markedly expanded with aggregates of large, mature but dysplastic-appearing megakaryocytes, focally disrupting the normal splenic structure. Immunostaining for myeloperoxidase highlighted multiple clusters of myeloid precursors in the spleen of aged hOGG TG mice but none in the spleen of wild type aged mice. Peripheral blood from these aged TG founder mice showed marked thrombocytosis and platelet clumping. In bone marrow, the aged TG founder mice displayed marked myeloid and megakaryocytic hyperplasia without marrow fibrosis, indicative of myeloid and megakaryocytic proliferation. Of note, none of the above phenotype was seen in younger hOGG TG mice (4 months), indicating that the full development of this pathologic process was dependent on age. In summary, TG mice overexpressing the mitochondrial isoform of human OGG gene developed a phenotype, closely mimicking ET in humans and the full manifestation of this phenotype was age-dependent. The molecular basis for this process is currently unclear and remains speculative. One plausible and attractive hypothesis is related to mitochondrial DNA damage with resultant mitochondrial dysfunction, in which overexpression of hOGG gene causes more active repair of free radical-induced 8-oxoguanine from DNA, leaving an increased number of abasic sites, which may generate mutation, inhibit transcription and, ultimately, leading to mitochondrial dysfunction and development of this myeloproliferative disorder. Extensive molecular characterization is currently underway to explore this possibility.

Description: Abstract 3960 Poster Board III-896 Introduction Rare cases of histiocytic sarcoma (HS) have been reported in association with indolent small B-cell neoplasms, either concurring with or following a small B-cell lymphoma. The biologic relationship between these two morphologically and immunophenotypically distinct neoplasms in same patients remains unclear, though recent data suggest a possible “transdifferentiation” from follicular lymphoma (FL) to HS. Patients and Methods We investigate the clonal relationship in two cases of B-cell lymphoma with subsequent HS, using immunohistochemical stains, PCR-based immunoglobulin heavy chain (IGH) gene rearrangement/sequencing analysis and interphase FISH study on formalin fixed, paraffin-imbedded tissue sections. Results Case 1 is a 62-year-old female with splenic marginal zone lymphoma (SMZL) who developed HS in a groin lymph node one year after the diagnosis of SMZL. PCR/sequence analysis of IGH gene showed a monoclonal rearrangement carrying identical DNA sequences of PCR products from the spleen with SMZL and the lymph node with HS. Case 2 is a 61-year-old female with a remote history of FL who developed supraclavicular lymphadenopathy and multiple other infiltrating foci. A supraclavicular lymph node biopsy demonstrated HS. PCR analysis detected a monoclonal rearrangement of the IGH gene and interphase FISH analysis revealed IGH/BCL-2 fusion, a genetic hallmark for FL. Although negative for other B-cell associated antigen markers, HSs show partial retention of primary neoplasms B-cell lymphomas' immunoprofiles, including expression of Oct-2 in both cases, and expression of bcl-6 and enhanced expression of bcl-2 in case 2. Conclusion The data provide a genotypic evidence of common clonal origin between mature B-cell lymphoma and subsequent HS in the same patients, suggesting “transdifferentiation” to HS could occur in other small B-cell lymphoma, in addition to FL. The transformed HSs might have incompletely inherited primary neoplasms' expression signatures by retaining B-cell lymphomas' characteristic immunoprofile to certain extent. The exact mechanism governing the conversion of mature B-cell lymphoma to HS is largely a mystery and remains to be elucidated by more scientific studies, though a few pathways have been proposed for the process, including transdifferentiation, dedifferentiation and common progenitor models. Disclosures: No relevant conflicts of interest to declare.

Description: Alterations of nuclear genes in human disease or tumors have been well investigated in past several decades and unequivocally established a predominant role in the pathogenesis. However, the relationship of mitochondrial genome alteration or dysfunction and human disease or tumor remains large unknown. Mitochondria are dynamic organelles involved in oxidative phosphorylation and production of reactive oxygen species (ROS). Accumulated evidence supports that mitochondrial DNA damage and dysfunction play vital roles in the development of a wide array of mitochondria-related diseases, such as obesity, diabetes, infertility, neurodegenerative disorders and malignant tumors in human. We previously described the development of a transgenic (TG) mouse model for mitochondrial damage by overexpressing human mitochondrial isoform of 8-oxoguanine DNA Glycosylase 1 (hOGG1) gene (Blood108:A 2246, 2006). The TG mice developed early onset obesity, female infertility, very high frequencies of B-cell lymphomas and human essential thrombocythemia like myeloproliferative disorders. We now reported here that major mitochondrial DNA deletions were frequently identified in a variety of organs in these hOGG1 TG mice and these deletions may largely contribute to the biologic phenotypes of the TG mice. The development and characterization of hOGG1 TG mice have been described previously. In the current study, mitochondrial DNA samples were extracted from various organs and tumor tissues of hOGG1 TG and age-matched non-TG control animals and subjected to PCR analysis using 8 specific primer sets franking the breakpoints of 7 major mitochondrial DNA deletions. Six deletions (3.7, 3.82, 3.86, 4.2, 4.9 and 5.2 kilobase in length) have been previously reported in the literatures. One novel deletion of 15.kilobase was identified in hOGG1 TG mouse in our laboratory. Among 7 major mitochondrial DNA deletion analyzed, Five (3.7, 3.86, 4.2, 5.2 and 15 kilobase in length) deletions were detected in higher frequency in various organs of hOGG1 TG but not in non-TG control mice, suggesting that those deletions might be resulted from overexpression of the transgene hOGG1. Notably, 3 deletions (del3729, del3868, and del15139) were identified in significantly higher in TG mouse spleen with myeloproliferative disorders or TG mouse spleen with diffuse large B-cell lymphoma, in comparison to the spleen of the age-matched wild type animals (P

Description: Abstract 949 Introduction: Diffuse large B cell lymphoma (DLBCL) has a highly variable outcome, and individual risk assessment is largely based on clinical features. Gene expression profiling (GEP) stratifies patients into those with germinal center B-cell (GCB) and activated B-cell subtype (ABC) subtype with different prognoses. These groups have been shown to predict prognosis in patients treated with CHOP or R-CHOP. Conversely, the role of other recognized prognostic markers, such as BCL2 gene abnormalities or Bcl2 expression has been questioned in the new therapeutic era. Materials and Methods: In 438 patients treated with R-CHOP for de novo DLBCL, we analyzed the tumors by immunohistochemistry for Bcl2 protein expression and by interphase fluorescence in situ hybridization (FISH) for BCL2 translocation and other abnormalities. All cases were successfully studied by GEP. The cutoff for Bcl2 protein expression, 60%, used as prognostic factor was determined using receiver operating characteristic curves. Progression-free survival (PFS) and overall survival (OS) were assessed. Results: The t(14;18)(q32;q21) was detected in 82 cases (18.7%) and BCL2 gains occurred in 63 cases (14.3%). Both t(14;18) and BCL2 gains strongly correlated with higher levels of Bcl2 protein expression (p

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: Alterations of nuclear genes in human lymphoma and leukemias have been well investigated in past several decades and established a predominant role in the pathogenesis. However, the relationship of mitochondrial genome alteration or dysfunction and human lymphoma and leukemias remains large unknown. Mitochondria are dynamic organelles that play critical roles in oxidative phosphorylation, energy metabolism, cell growth and apoptosis. We have successfully generated a novel transgenic (TG) mouse model of mitochondrial disorder by overexpressing human hOGG1, a base excision DNA repair gene, in the mitochondria of a wide variety of tissues in mice. We reported here the high frequency of essential thrombocythemia (ET)-like myeloproliferative disorder and lymphoma in the TG mice. TG mice were generated via pronuclear microinjection of a mammalian expression construct of a mitochondrial isoform of hOGG under the control of a mouse metallothionein-1 promoter. Peripheral blood smears were prepared from TG and non-TG control mice for platelet counts and morphologic evaluation. TG mice were sacrificed and various organs were harvested for histologic, biochemical and molecular studies. Two of the male founder mice successfully transmitted the transgene to their offspring at an expected frequency of 50%. All the female mice failed to reproduction. All TG mice expressed high levels of hOGG mRNA in their liver by RT-PCR and direct DNA sequencing. Over-expression of this gene produced a wide range of adverse biological phenotype, manifesting early-onset obesity, metabolic disturbance, female infertility and high frequency of ET-like myeloproliferative disorder and lymphoma (〉90%) in nodal and extranodal sites. Eight TG mice (ranging from 1 to 2 years) become moribund and subsequently sacrificed. Upon histologic examination, the TG mice displayed moderate to severe splenomegaly. The spleen weights were 2-, 4- and 10 times respectively in the 3 TG founder mice as compared to wild type, age-matched mice. Extensive abdominal lymphadenopathy and numerous enlarged nodules involving liver, spleen, peritoneum, lung and diaphragm were identified. Microscopically, the red pulp in the enlarged spleens is markedly expanded with aggregates of large, mature but dysplastic-appearing megakaryocytes, focally disrupting the normal splenic structure. Various lymphomas ranged from low-grade lymphoma to high-grade Burkitt-like or lymphoblastic lymphomas were seen. Peripheral blood from these aged TG mice showed marked thrombocytosis and platelet clumping. In bone marrow, the aged TG mice displayed marked myeloid and megakaryocytic hyperplasia with relative erythroid hypoplasia in the absence of marrow fibrosis, indicative of myeloid and megakaryocytic proliferation. Bone marrow involvement by B-cell lymphoma was also seen. Of note, none of the above phenotype was seen in younger TG mice (4 months), indicating that the full development of this pathologic process is age-dependent. The molecular basis for this process is currently under investigation. Our preliminary data showed that mitochondrial DNA deletion or decrease in DNA copy numbers due to overexpressed hOGG1 and imbalance of base excision repair resulted in defects in mitochondrial respiration and increased ROS production. We thus hypothesize that oxidative stress caused by mitochondrial malfunction may play important part in the development of hematopoietic malignancies.

Description: Alterations of nuclear genes in human diseases including neoplasms have been well investigated in past several decades and unequivocally established their predominant role in the pathogenesis. However, the relationship of mitochondrial genome alteration with human diseases remains largely unknown. Mitochondria are dynamic organelles involved in oxidative phosphorylation and production of reactive oxygen species (ROS). Accumulated evidence supports that mitochondrial DNA damage and dysfunction play vital roles in the development of a wide array of mitochondria-related human diseases, such as obesity, diabetes, infertility, neurodegenerative disorders and malignant tumors. We previously described the development of a transgenic (TG) mouse model for mitochondrial DNA damage by overexpressing human mitochondrial isoform of 8-oxoguanine DNA Glycosylase 1 (hOGG1) gene. Over-expression of this gene produced a wide range of adverse biological phenotypes, manifesting early-onset obesity, metabolic disturbance, female infertility, high frequency of B-cell lymphoma and human essential thrombocythemia like myeloproliferative disorder, involving the lymph node, bone marrow, spleen, liver and other extranodal sites. Development of these hematopoietic neoplasms appeared to be age-dependent. In the current study, we focused on the pathogenesis of the hematopoietic neoplasms by characterization of the neoplasms via pathologic, biochemical and molecular approaches. While expression of mOGG1 was very similar in parallel organs from transgenic and wild-type control mice, the hOGG1 TG mice expressed very high levels of human OGG1 transgene mRNA, being 6.8- and 112-fold as high as the endogenous mouse OGG, in the spleen and bone marrow. By contrast, hOGG1 transgene mRNA were not detected at all in the above two organs from control mice, indicating that the transgene is highly expressed in the hematopoietic organs in TG mice. We then measured mitochondrial NADH dehydrogenase 1 (ND1) gene expression as an indirect measure of mitochondrial respiratory function. ND1 mRNA levels in the spleen (4) and lymphoma (4) of TG mice were 83% and 58% higher, respectively, than those in the spleen (4) of wild-type control mice (P 〈 0.01), indicative of increased mitochondrial respiration in the lymphoma and spleen of hOGG1 TG mice. We next measured the levels of intracellular H2O2 production in the lymphoma and spleen of hOGG1 transgenic (4) and the spleen from wild-type control (4) mice. The amount of H2O2 produced in the lymphoma and the spleen of hOGG1 transgenic mice was ~166% and ~66% higher, respectively, than that in the spleen from wild-type control mice (P 〈 0.001). The amount of H2O2 produced in the lymphoma was ~60% higher than that in the spleen from hOGG1 transgenic mice (P 〈 0.05). Finally, we examined mitochondrial DNA alterations in TG mice. Mitochondrial DNA samples were extracted from various organs and lymphoma tissues from hOGG1 TG and age-matched non-TG control animals and subjected to PCR analysis using specific primer sets franking the breakpoints of 7 major mitochondrial DNA deletions. Six deletions (3.7, 3.82, 3.86, 4.2, 4.9 and 5.2 kilobase in length) have been previously reported in the literatures. One novel deletion of 15.kilobase was identified in hOGG1 TG mouse in our laboratory. Among 7 major mitochondrial DNA deletion analyzed, Five (3.7, 3.86, 4.2, 5.2 and 15 kilobase in length) deletions were detected in higher frequency in various organs of hOGG1 TG but not in non-TG control mice, suggesting that those deletions might be resulted from overexpression of the transgene hOGG1. Notably, 3 deletions (del3729, del3868, and del15139) were present in significantly higher frequencies in spleen with myeloproliferative disorders or lymphoma from TG mice in comparison to the spleen of the age-matched wild type animals (P