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[Figure][1] A 45-year-old man with a history of gout for 6 years was maintained with allopurinol. One and a half years prior to admission, he was diagnosed with diffuse large B-cell lymphoma (germinal center type) with primary orbital presentation. He failed combination chemotherapy with

Chronic myelocytic leukemia (CML) is a common neoplasm of hematopoietic pluripotent stem cells. Although the evolution from chronic phase to blast crisis (BC) in CML patients is an inevitable clinical feature, little is understood about the mechanisms responsible for the transformation. We have previously performed allelotype analysis in CML BC and have detected frequent loss of heterozygosity (LOH) on the short arm of chromosome 1. To know the common region of LOH where a putative tumor suppressor gene may reside, deletional mapping was performed using 33 microsatellite markers spanning chromosome 1 in 30 patients with CML BC (21 myeloid and 9 lymphoid). DNA was extracted from slides of bone marrow smears or from bone marrow mononuclear cells. In each patient, DNA from chronic phase was analyzed alongside DNA from either their BC or accelerated phase. Allelic loss on 1p was observed in 14 of the 30 individuals (47%): 10 of the 21 myeloid and 4 of the 9 lymphoid BC cases. Serial cytogenetic information was available in 10 cases with LOH on 1p; interestingly, deletions in this region were not detected. Two samples showed LOH at all informative loci on 1p, whereas the other 12 samples showed LOH on at least one but not all loci on 1p. The common region of LOH resided proximal to D1S508 and distal to D1S507 (1p36). Our results suggest that a tumor suppressor gene that frequently plays an important role in the evolution to BC resides on 1p36 in CML. © 1998 by The American Society of Hematology.

C/EBPepsilon (C/EBPε) knock-out (KO) mice have defects in granulocytic development and increased susceptibility to infection. We also observed a unique phenotype of C/EBPε KO mice showing prolonged lower grade, mild skin problems such as hair loss that progressed to severe wound problems; abdominal wall perforation to the peritoneum as well as mandibular skin perforation to the oral cavity, without significant clinical symptoms. The observation suggests inappropriate response to inflammation by loss of C/EBPε. To understand the functional defects of these granulocytes, we performed a systematic approach with ChIP-seq and microarray of bone marrow cells from C/EBPε KO mice. Duplicate ChIP-seq libraries were prepared from bone marrow cells of wild type and C/EBPε KO mice using two different C/EBPε specific antibodies. Analysis of pooled data identified 14,253 C/EBPε binding sites, and 12,865 of these binding sites overlapped with the two antibodies. Sixteen percent of C/EBPε binding sites were in the promoter regions within 1 kb of the transcriptional start sites. Direct canonical C/EBPε target genes were defined by 1.5 log2 fold or greater increase in expression in the Gr-1+/Mac-1+ cells of wild type bone marrow cells compared to those of the KO mice. 2,224 genes met this criteria including previously identified target genes, such as ltf, camp, ngp, of which 988 genes were bound by C/EBPε. C/EBPε bound to the promoter sites of 150 genes and the enhancer region of 838 genes. The other 1,236 up-regulated genes did not have binding of C/EBPε, suggesting their up-regulation was a secondary event during terminal granulocytic differentiation. Electromobility shift assay followed by antibody supershift confirmed that C/EBPε was able to bind to upstream regions of target genes such as lft, ngp, il-1b. Trem-1 is an amplifier of inflammatory response. Increased expression/activation of Trem-1 on neutrophil membranes takes part in cytokine production, phagocytosis, apoptosis, oxidative burst by cooperating with TLR4 in response to environmental conditions. Soluble TREM1 levels increase even in neutopenic patients, suggesting it is associated with emergent granulopoiesis in response to infection or inflammation. The expression of Trem-1 is regulated by NF-kB and PU.1. Trem-1 KO mice have reduced inflammation without defects in clearance of pathogens in mice. Considering previous reports demonstrating TLR4 increases transcription of C/EBP and potentiates transcriptional activity of C/EBP via NF-kB, regulatory loops between TLR4/NF-kB/C/EBP/Trem-1 may exist and regulate inflammatory response. ChIP-seq illustrated a C/EBPε binding site located 92 bp upstream from transcription start site of Trem-1 (GTTGTGAAAC). Microarray comparison of Trem-1 expression in Gr-1+/Mac-1+ bone marrow cells showed 5.3 log2 fold decreased gene expression in C/EBPε KO versus wild type mice. To confirm microarray data, quantitative PCR were performed with sorted Gr-1+ bone marrow cells from wild type and KO mice. The qPCR demonstrated that trem-1 expression of C/EBPε KO cells was 60% of wild type (p=0.001). A pGL3 basic luciferase vector with the first 291bp upstream of trem-1containing a putative C/EBPε binding site was cloned and co-transfected with 2 ug of C/EBPε expressing vector into 293T cells. The dual luciferase assay, normalized by renilla luciferase, showed 8 fold increase by C/EBPε (p In conclusion, we identified trem-1 as a downstream target of C/EBPε, which completes a positive regulatory loop of TLR4-NF-kB-C/EBPε-Trem1-TLR4 in response to inflammatory signal. Therefore, C/EBPε KO mice have an inappropriate inflammatory response by loss of the positive amplificatory loop, in addition to previously known defective clearance of pathogen by their inability to produce secondary granules. Disclosures No relevant conflicts of interest to declare.

CCAAT/enhancer-binding proteins (C/EBPs) are critical transcriptional regulators of differentiation of hematopoietic cells. Previous studies have shown that targeted disruption of theC/ebpα gene results in a lack of granulocytic differentiation with an arrest at the stage of immature myeloblasts. By using a gene replacement strategy in which C/EBPβ was expressed from the C/ebpα gene locus of C/EBPα-null mice, we have evaluated the ability of C/EBPβ to function for C/EBPα in directing differentiation along the granulocytic pathway. We show that the morphology and the differential cell counts of the bone marrow and peripheral blood cells from C/EBPβ knockin mice are indistinguishable from those of their wild-type littermates, indicating that hematopoiesis occurs normally in these animals. Additionally, we analyzed expression of 21 myeloid-specific genes, including markers for distinct stages of granulocytic differentiation, and found no significant differences in their levels of expression in the bone marrow of C/EBPβ knockin and wild-type mice. These results imply that C/EBPβ can substitute for C/EBPα during hematopoiesis when expressed from the C/ebpα gene locus.

Background: Myelodysplastic syndromes (MDS) are a heterogeneous group of hematopoietic disorders, which can be difficult to diagnose based only on morphologic bone marrow examination. Karyotyping can be useful in diagnosing borderline cases. However, only 40% of patients with MDS have an abnormal karyotype. Flow cytometry(FCM) approaches have been described but not clearly defined in MDS due to use of different criteria. We devised a 10-parameter FCM panel, mainly including myeloid and erythroid maturation markers, to differentiate MDS marrows from normal marrows. Design: Bone marrow from 91 patients with cytopenia(s) or anemia were included in the study(10/2005–7/2006). Cases were divided into 3 groups: 1. normal, not morphologically suspicious for MDS, 46 cases, 2. equivocal, morphologically suggestive but not diagnostic of MDS, 20 cases, 3. morphologically diagnostic of MDS, 25 cases. 10 FCM paremeters were performed and scored: 1. hypogranularity, 2. aberrant expression of CD56, 3. lack of CD10 expression, 4. decreased CD64 expression, 5. & 6. lack of CD13 or CD33 expression, 7. & 8. abnormal CD13/CD16 or CD16/CD11b pattern, 9. increase of CD 34 expression gating on all cells excluding erythrocytes, 10. decreased expression of CD71/Glycophorin gating on erythroid precursors. Karyotypings were analyzed. Results: 1. and 2. (see Table 1 and 2). 3. Karyotyping were anlayzed in 86 cases. Cytogenetic abnormalities were found in 2.2% (1/44) of normal group, 5.3% (1/19) of equivocal group and 34.8% (8/43) of MDS group. In MDS group 7 of 8 patients (87.5%) who had both morphologic and cytogenetic diagnosis of MDS were scored >=3 of 8 FCM parameters. Conclusions: Study showed the 8-parameter FCM panel was more predictive of MDS than 10-parameters one. Both CD13/CD16 & CD16/CD11b patterns were considered to be non-specific. In 8-parameter panel, zero score tended to rule out MDS, while score >=3 suggested MDS. The most specific FCM parameters were hypogranularity, CD34, aberrant expression of CD56 or lack of CD10 expression by mature granulocytes. CD71/Glycophorin A might be useful in identifying dysplasia in erythroid lineage. | Parameters/Group | Normal Group(A)(n=46) | Equivocal Group(B)(n=20) | MDS Group(C)(n=25) | CHITEST(P value)(GRoup A vs C) | |:-------------------------------------------------------------------------------------------------------------- | --------------------- | ------------------------ | ------------------ | ------------------------------ | | * These two parameters were deleted in the 8-parameter panel due to their high frequency seen in normal group. | | Hypogranularity | 4 (8.7%) | 6 (30%) | 18 (72%) | 0.0001 | | CD56 | 0 (0.0%) | 1 (5%) | 6 (24%) | 0.0005 | | CD10 | 7 (15.2%) | 7 (35%) | 15 (60%) | 0.0001 | | CD64 | 6 (13.1%) | 2 (10%) | 6 (24%) | 0.2396 | | CD13 | 0 (0.0%) | 0 (0.0%) | 2 (8.0%) | 0.0516 | | CD33 | 0 (0.0%) | 2 (10 %) | 0 (0%) | | | CD13/CD16* | 23 (50.0%) | 7 (35%) | 21 (84%) | 0.0047 | | CD34 | 8 (17.4%) | 7 (35%) | 13 (52%) | 0.0026 | | CD71 | 2 (4.3%) | 5 (25%) | 12 (48%) | 0.0001 | | CD16/CD11b* | 27 (58.7%) | 8 (40%) | 22 (88%) | 0.0107 | Table 1. Comparison of FCM Parameters in Patients with/without MDS | Score/Group | Normal Group (A)* (n=46) | Equivocal Group (B)* (n=20) | MDS Group (C)* (n=25) | |:-------------------------------------------------------------------------------- | ------------------------ | --------------------------- | --------------------- | | * Fish Exact T Test: Group A vs C: p3 | 0 (0.0%) | 3 (15%) | 17 (68%) | | *Mean score +/− SD | 0.65+/−1.06 | 1.55+/−1.54 | 2.8+/−1.65 | Table 2. Scoring with 8 FCM Parameters in Patients with/without MDS

Background: V617F mutation in Janus Kinase 2 (JAK2) gene has been found in chronic myeloproliferative disorders (MPD) including polycythemia vera (90%), essential thrombocythemia and chronic idiopathic myelofibrosis (30–50%), and occasionally in myelodysplastic syndromes (MDS). “5q- Syndrome” is a MDS that shares features with MPD and characterized by an atypical megakaryocytic hyperplasia in bone marrow and usually thrombocytosis in peripheral blood. The most common deleted region for this syndrome is 5q13.3q33.1. An interstitial deletion with variable proximal (5q12-14) and distal (5q31-33) breakpoints has been found in other MDS with/without additional chromosomal abnormalities beyond “5q- Syndrome”. To date, JAK2 mutation was detected in 6/97(6.2%) of patients having diagnosis of MDS with “5q- Syndrome”. Design: In our study 21 MDS patients (10 with “5q- Syndrome” and 11 MDS with isolated or complex 5q-) whose diagnosis by both bone marrow aspiration/biopsy and conventional chromosomal analysis were confirmed. Materials and Method: Primers were created to amplify a 460 bp fragment containing the site of JAK2 V617F mutation. Forty-five cycles of PCR were performed at an annealing temperature of 57°C. Resulting PCR product was digested with 2 U BsaXI for 16 hours and with an additional 2 U BsaXI for another 16 hours at 37°C, then analyzed on a 2% agarose gel. The mutant allele remained undigested whereas the wild-type allele was digested into 241 bp, 189 bp and 30 bp fragments. All experiments included a positive (HEL cells) and negative (K562 cells) control. Results: PCR results showed clear wild type PCR patterns in all 21 cases. Conclusion: No JAK2 mutations were detected in 21 patients either with “5q- Syndrome” or other 5q- associated MDS suggesting that JAK2 mutations are infrequent in these MDS patients.

We developed a mouse model of acute T/natural killer (NK) acute lymphoblastic leukemia (ALL) that is always preceded by polyclonal lymphocyte expansion to determine how aberrant promoter DNA methylation and consequent gene silencing might be contributing to leukemic transformation. We demonstrated the non-random nature of methylation in the transformation from benign polyclonal expansion to leukemia, and identified a novel tumor suppressor gene silenced in the majority of human leukemia but not solid tumors. To this, we utilized restriction landmark genomic scanning (RLGS) on eight IL-15 transgenic (tg) mice with T/NK ALL, and performed RLGS on an additional five spleens from IL-15tg mice without ALL, but with polyclonal T/NK expansion, and on four spleens from wild type (wt) mice. A total of 2447 RLGS fragments with good resolution on each RLGS profile were analyzed. Only 1–2 variable changes were detected in either wt or polyclonal T/NK expansion controls. In contrast, the eight spleens with clonal T/NK leukemia had 45 to 209 changes (1.8 to 8.5% of total fragments) consistent with aberrant DNA methylation. The association of RLGS fragment losses or reduced intensity with leukemic transformation versus polyclonal expansion was highly significant (P