ALBERT

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: 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: 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: The pattern of adolescent/young adult Hodgkin lymphoma (YAHL) suggests causation by a relatively late infection with a common childhood virus, but no causal virus has been found. Susceptibility is heritable and linked to lower interleukin 12 (IL12) levels, which can also result from fewer fecal-oral microbial exposures early in life. We studied twin pairs discordant for YAHL to examine exposures capable of altering the IL12 response and T-helper type 1 (Th1)–Th2 balance. One hundred eighty-eight YAHL-discordant twin pairs from the International Twin Study returned questionnaires (70% response). Exposure history of YAHL case-twins was compared with that of their unaffected control-twins using conditional logistic regression for matched pairs to calculate odds ratios (ORs). Behaviors likely to produce oral exposure to microbes conveyed decreases in risk (univariable OR range = 0.2-0.5, P = .003-.11). Significant adjusted ORs were seen for appendectomy (OR = 4.3, P = .001), eczema (OR = 4.2, P = .025), smoking (OR = 2.2, P = .054), and relatively more frequent behaviors associated with oral exposures (OR = 0.1; P = .004). Kappa statistics for intrapair agreement were higher than 0.8 for each significant finding. Our observations support a protective role for increased early oral exposure to the microbiome, suggesting that factors associated with increased Th2 and decreased Th1 cytokines are etiologically relevant to YAHL.

Description: Nodular sclerosing Hodgkin lymphoma (NSHL) is a distinct, highly heritable Hodgkin lymphoma subtype. We undertook a genome-wide meta-analysis of 393 European-origin adolescent/young adult NSHL patients and 3315 controls using the Illumina Human610-Quad Beadchip and Affymetrix Genome-Wide Human SNP Array 6.0. We identified 3 single nucleotide polymorphisms (SNPs) on chromosome 6p21.32 that were significantly associated with NSHL risk: rs9268542 (P = 5.35 × 10−10), rs204999 (P = 1.44 × 10−9), and rs2858870 (P = 1.69 × 10−8). We also confirmed a previously reported association in the same region, rs6903608 (P = 3.52 × 10−10). rs204999 and rs2858870 were weakly correlated (r2 = 0.257), and the remaining pairs of SNPs were not correlated (r2 〈 0.1). In an independent set of 113 NSHL cases and 214 controls, 2 SNPs were significantly associated with NSHL and a third showed a comparable odds ratio (OR). These SNPs are found on 2 haplotypes associated with NSHL risk (rs204999-rs9268528-rs9268542-rs6903608-rs2858870; AGGCT, OR = 1.7, P = 1.71 × 10−6; GAATC, OR = 0.4, P = 1.16 × 10−4). All individuals with the GAATC haplotype also carried the HLA class II DRB1*0701 allele. In a separate analysis, the DRB1*0701 allele was associated with a decreased risk of NSHL (OR = 0.5, 95% confidence interval = 0.4, 0.7). These data support the importance of the HLA class II region in NSHL etiology.

Description: We examined the types of Epstein-Barr virus–associated nuclear antigen-1 (EBNA-1) gene carboxy (C)-terminal mutations occurring in Hodgkin’s disease (HD) and reactive tissues from two different geographic regions. Previously reported EBNA-1 C-terminal region amino acid sequence variants, based on the amino acid at codon 487, include Prototype (P)-ala, which is found in the B95.8-derived prototype virus, P-thr, Variant (V)-leu, V-val, and V-pro. Using polymerase chain reaction (PCR) to amplify portions of the EBNA-1 gene, followed by DNA sequencing, we found a single EBNA-1 gene sequence variant in each tissue, whether reactive or neoplastic and whether from Brazil or the United States. Variant EBNA-1 gene sequences were more common in both neoplastic and non-neoplastic tissues from different geographic areas than the so-called prototype sequence. In the 17 Brazilian HD cases, 4 cases had P-thr variants and 13 had V-leu variants. In the six reactive tissues from Brazil, one had a P-ala variant, two had P-thr variants, and three had V-leu variants. In the 12 American HD cases, 2 had P-ala variants, 6 had P-thr variants, and 4 had V-leu variants. The 11 American reactive tissues included 2 P-ala variants, 5 P-thr variants, and 4 V-leu variants. In both countries, there were similar variant EBNA-1 sequences present in normal tissues and HD cases. Compared with the P-ala and P-thr cases, the V-leu cases were more likely to have the 30-bp latent membrane protein 1 (LMP1) gene deletion (P = 0.0075). In addition, cases of HD with the V-leu were statistically associated with a substitution of asparagine for glutamine at codon 322 of the C-terminal portion of the LMP1 gene. Our results suggest that any variation in EBNA-1 gene sequence is caused by a polymorphism present in pre-existing viral strains in the underlying population, and not a mutation occurring during oncogenesis.

Description: The histiocytoses are rare disorders characterized by the accumulation of macrophage, dendritic cell, or monocyte-derived cells in various tissues and organs of children and adults. More than 100 different subtypes have been described, with a wide range of clinical manifestations, presentations, and histologies. Since the first classification in 1987, a number of new findings regarding the cellular origins, molecular pathology, and clinical features of histiocytic disorders have been identified. We propose herein a revision of the classification of histiocytoses based on histology, phenotype, molecular alterations, and clinical and imaging characteristics. This revised classification system consists of 5 groups of diseases: (1) Langerhans-related, (2) cutaneous and mucocutaneous, and (3) malignant histiocytoses as well as (4) Rosai-Dorfman disease and (5) hemophagocytic lymphohistiocytosis and macrophage activation syndrome. Herein, we provide guidelines and recommendations for diagnoses of these disorders.

Description: The hallmark of Hodgkin’s Lymphoma (HL) is the presence of large Hodgkin’s and Reed/Sternberg (HRS) cells that comprise only ~1–3% of the cellular infiltrate. Given the paucity of genomic analyses of these cells, we sought to characterize their DNA copy number alterations (CNA) by array comparative genomic hybridization (aCGH) using DNA isolated from laser capture microdissected (LCM) CD30+ HRS cells. Primary paraffin-embedded diagnostic samples were obtained from 27 patients (pts), including 15 pts with chemotherapy responsive and 12 pts with primary refractory HL. From each sample, 150 HRS cells were isolated by LCM from 5-μm thick tissue sections, amplified using whole genome amplification (WGA), and hybridized to a minimal tiling 19K whole genome bacterial artificial chromosome (BAC) array (BAC center to center distance of 165 kb). To define thresholds for calling gains and loss and to control for WGA noise, 1.05 ng DNA (DNA equivalent of 150 cells) was obtained from six normal (3 males and 3 females) individuals, amplified and analyzed by aCGH. Triplicate samples from a single HL pt confirmed mean replicate correlation as approximately Pearson r = 0.90. DNA gains and losses observed in 〉35% of the HL samples were localized to 22 and12 chromosome regions, respectively. Region-specific FISH analyses confirmed the presence or absence of aCGH-defined CNAs in CD30+ HRS for 10 of these regions (3–5 pt samples/FISH probe). CNA gains in 〉60% of HL samples included genes associated with growth and proliferation (AHR, BAI1, BOP1, COMMD5, LY6E, PTP4A3, SLURP1, CBFA2T3, SLC7A5, NOTCH1, FOXF1, TRAF2, IRF8, S100B, MYH14), cell cycle (AKT1, CDK10, SUMO3), drug metabolism (CYP11B1, CYP11B2, SLC19A1), angiogenesis and cell adhesion (COL18A1, CDH4, ITGB2), apoptosis regulation (FOXC2, FOXF1, GPR132), immune and lymphatic development (CBFA2T3, IL17C, IRF8, CLEC11A, RXRA, SPIB, ICOSLG) and invasion, metastasis or cancer-relatedness (VAV2, PSCA PTP4A3, GINS2, FUT7, TUBB2C, KLK, POLD1, TFF2). Losses observed in 〉40% of HL samples included SPRY1, NELL1, SLC1A3, GDNF, IL7R, SKP2, GRIA1, ID4, PPARGC1A, and TXNIP. Different CNA patterns between sensitive and refractory HL were identified. Genomic differences observed either specifically or in 〉35% of the HL chemosensitive pts included ~25 CNA gains including genes known to regulate T-cell trafficking or NFkB activation (CCL22, CX3CL1, CCL17, DOK4 and IL10). The refractory signature showed a higher frequency of CNA gains for three genes involved in the H4 ubiquitin ligase complex that play a role in the cellular response to DNA damage (HDAC4, CUL4A, and DDB2), and gains of ILKAP, GAS6, MADD, SPI1, PVR, MTCH2 CCND3, GPCI, MAPK11 with CNA losses of ELAC2, IL2, GRIA1, SLC17A6, IL21, and MAP2K4 genes. Of interest, CCL22 and CX3CL1 have been associated with a more favorable outcome and a lower risk of recurrence in other cancers, whereas gains or overexpression of SKP2, MTCH2 and CCND3 have been associated with a poor prognosis and a more aggressive cancer phenotype. Moreover, genomewide discrimination analyses on CNAs revealed two distinct clusters that correlated closely with the favorable and unfavorable IPS scores for the 27 HL pts evaluated. Overall, our proof-of-principle, exploratory genomic analyses of HD show that genomic profiles of small numbers of HRS cells is possible, HL samples shared many CNAs but differences may reflect disease course, and highlights the potential to build a genetic CNA map for HL to potentially guide prognosis, therapy decisions and drug discovery.

Description: The risk pattern of adolescent/young adult Hodgkin lymphoma (HL) is consistent with causation by a relatively late infection to a common childhood virus. However a causal virus has not yet been convincingly found. Susceptibility is known to be heritable, and a lower genetically determined interleukin-12 [IL-12] response is a risk factor. A lower IL-12 response can also be produced by diminished gastrointestinal exposure to the environmental microbiome, with resulting persistence of a Th2-skewed cytokine phenotype. We have therefore studied twin pairs discordant for HL to search for early life differences in sources of both viral and non-viral infection. HL-discordant twin pairs volunteered for the International Twin Study in ignorance of specific hypotheses. 70% of the questionnaires sent to individual twins were returned producing 188 informative pairs. The Kappa statistic was used to assess between-twin agreement. Designating the twin with HL as the case and the unaffected twin as the control, odds ratios (ORs) and 95% confidence intervals (CI) were calculated using conditional logistic regression for matched pairs. Tonsillectomy or appendectomy at least 5 years prior to diagnosis were associated respectively with a 2- and 3-fold statistically significant increase in risk. Infection with 3 or more childhood exanthems was associated with a 60% decreased risk (95% confidence intervals 0.2, 0.9). Behaviors likely to produce oral exposure to the environmental microbiome conveyed statistically significant decreases in risk (OR=0.2–0.5). A history of eczema increased risk (OR= 2.8, 95% CI= 1.0, 7.8). Kappa statistics were high (〉0.8) for significant findings. Our evidence supports a role for early exposure to various infections in the etiology of adolescent/young adult Hodgkin lymphoma. Table 1. History of infections, immune-related surgeries and other childhood experience at least 5 years prior to diagnosis and risk of adolescent-young adult Hodgkin Lymphoma diagnosed between 13–50 years of age in disease-discordant twin pairs from the International Twin International Twin Registry (n = 188 total pairs). Exposures Kappa1 Ratio of Exposure Discordant Pairs2 Odds Ratio3 95% CI4 1. Kappa calculated from the 93 double respondent twin pairs (pairs in whom both twins returned questionnaires) 2 Total number of twin pairs in which case-twin was exposed and the unaffected co-twin was unexposed/Total number of twin pairs in which unaffected co-twin was exposed and the case-twin was unexposed 3 Odds ratio estimated using conditional logistic regression using SAS Version 8.1 4 Confidence interval estimated using conditional logistic regression 3 or more childhood exanthems - 6/15 0.4 0.2, 0.9 Infectious mononucleosis 0.72 22/19 1.2 0.6, 2.1 Cold Sores (Herpes Simplex 1) 0.70 18/10 1.8 0.8, 3.9 Tonsillectomy 0.86 25/12 2.1 1.1, 4.1 Appendectomy 0.84 33/11 3.0 1.5, 5.9 Eczema 0.79 14/5 2.8 1.0, 7.8 Sucked pacifier/thumb/fingers more as an infant/young child? 0.87 18/34 0.5 0.3, 0.9 Put more things in mouth as an infant/young child? 0.86 10/30 0.3 0.2, 0.7

Description: Multiple myeloma (MM) is a terminal differentiated B cell chronic lymphoproliferative disorder characterized by the latent accumulation of plasma cells with a low proliferative index and an extended life span in bone marrow or extramedullary tissues. While studies on neoplastic cells isolated directly from MM patients or on MM-derived cell lines demonstrated that IL-6 is a major growth and survival factor for initiation of signaling in MM cells in an autocrine or paracrine manner, some MM cells independent on extracellular signaling such as IL-6 have been reported, suggesting other mechanisms may be involved in signal transduction pathways in MM cells. Accumulating evidence showed that IL-6 induces intracellular signaling through members of the signal transducers and activators of transcription (STAT) family of proteins by activating the janus kinase(JAK) family of protein tyrosine kinases, which subsequently phosphorylate and activate cytoplasmic Stat proteins. Activated Stat proteins dimerize and translocate to the nucleus, where they bind to specific DNA response elements and induce expression of Stat-regulated genes. Our previous studies demonstrated that one of the Stat family proteins, Stat3, was constitutively activated (phosphorylated) in the majority of MM patients, suggesting constitutive activation of JAK-2 kinase activities. However, other mechanisms might be also responsible JAK2 constitutive activation in MM cells, in particular in IL-6 independent MM cells. A specific point mutation of JAK-2 kinase gene has been identified recently showing its presence in higher frequency of certain types of chronic myeloproliferative disorders. The point mutation yields constitutive activation of JAK-2 kinase activity in the absence of extracellular signaling and plays an important role in the pathogenesis of those disorders. In this study, we try to determine whether JAK-2 (Val617Phe) point mutation was present in MM patients and if so, what kind of role it may play in the pathogenesis of MM. JAK-2 (Val617Phe) point mutation was analyzed by using an allele-specific polymerase chain reaction followed by separation and detection with capillary electrophoresis. The test has a detection sensitivity of up to 200 picogram mutated DNA. Bone marrow aspirates with various degrees of involvement by MM (ranged10–80%) were selected and all the cases have been verified by morphologic examination and demonstrated to have positive immunoglobulin heavy or kappa light gene rearrangements. Bone marrow specimens from total 59 MM patients were tested for JAK2 (Val617Phe) mutation and none of any MM cases with the specific point mutation were identified (0/59). The results indicated that JAK2 (Val617Phe) mutation was very infrequency or absence in MM patients, despite the fact that constitutional activation of JAK2 kinase as well as Stat3 activation was commonly seen in MM patients. The study suggests that other yet unknown mechanisms may also involve JAK2 activation, particularly in IL-6 independent MM cells.