ALBERT

Description: Background: Patients (pts) with sAML are evaluated together in large AML trials regardless of whether their sAML arises from antecedent MDS vs. from MPD vs. t-AML. Prognostic factors and outcomes may differ among these subgroups, and a prognostic scoring system would be helpful in determining which patients would benefit from induction chemotherapy. Methods: We conducted a retrospective review of all pts with newly diagnosed, pathologically-confirmed AML at Cleveland Clinic between 1997 and 2007 to identify sAML pts treated with cytarabine-based induction chemotherapy. Data on known prognostic factors (age, white blood cell count (WBC) at diagnosis, cytogenetic risk groups (as defined by CALGB 8461), and AML etiology) were collected as baseline characteristics and controlled for in stepwise multivariable analyses. Complete response (CR) and overall survival (OS) were analyzed. A prognostic scoring system for OS was developed based on the number of poor prognostic features present, derived from significant multivariable factors. Pts received 1 point for adverse cytogenetics, 1 point for having 1-10% peripheral blasts, and 1 point for AML arising from MDS or MPD. Pts with 0 points were favorable, 1 point intermediate, 2 or more points unfavorable. Results: Of 584 AML pts identified, 361 were treated with remission induction therapy, of whom 90 had AML arising from MDS, MPD, or t-AML. Thirty-nine (43%) had antecedent MDS, 21 (23%) an MPD, and 30 (33%) had t-AML, and 47% were female. Pts with AML arising from MDS were older at AML diagnosis (median of 67 years) vs. from MPD (61 years) and t-AML (60 years) (p=.02) but a shorter time from antecedent diagnosis/event (7 months, vs. 47 and 37 months, respectively (p

Description: Abstract 292 Background: Allelic deficiency for the RPS14 gene impairs differentiation and survival of erythroid progenitors in del(5q) MDS (Nature 2008; 451:335). Nucleolar stress arising from disruption of ribosome assembly fosters MDM2 sequestration by free ribosome components resulting in p53 stabilization and erythroid hypoplasia (Nat Cell Biol 2009; 11:501). We recently reported that reduced gene dosage of the lenalidomide (LEN) inhibitable, haplodeficient phosphatases CDC25C and PP2Acα is a key determinant of drug sensitivity in del(5q) MDS (PNAS 2009; 106: 12974). We now show that shRNA suppression of these genes to levels commensurate with haplodeficiency reinforces p53 accumulation, and that treatment with LEN promotes MDM2-mediated p53 degradation to transition del(5q) clones to G2/M arrest. We hypothesized that emergence of resistance to LEN in del(5q) MDS arises from two possible mechanisms: (1) up-regulation of haplodeficient drug targets or compensatory isotypes, or (2) inactivating mutations of the TP53 or CDC25C genes. Methods: To investigate mechanisms of LEN resistance, we studied sequential bone marrow (BM) specimens obtained at baseline (BL), response to treatment (TR) and treatment failure (TF) from 12 LEN treated patients with Low/INT-1 risk, transfusion-dependent del(5q) MDS. Eleven patients achieved clonal suppression and transfusion independence; 7 patients developed clinical drug resistance with primary clonal recovery. Immunohistochemical (IHC) staining for cdc25-C, -A and -B; PP2A–Ca and p53 were performed using a biotin-streptavidin-horseradish peroxidase method and compared to 6 age-matched controls; intensity of cytoplasmic or nuclear staining in hematopoietic elements was recorded after blinded review. DNA and RNA were extracted from cryopreserved BM mononuclear cells (BM-MNC) or fixed paraffin blocks from BM clot and biopsy sections. Expression of CDC25C splice variants was assessed by RT-PCR and total gene expression by real time (QT)-PCR. Exonic DNA encoding the catalytic [exons 8–14] and nuclear export domains [exon 11] of CDC25C and the DNA-binding domain of TP53 [exons 4–9] was sequenced for gene mutation analysis. Differences in mean values were compared by paired t-test. Results: P53 immunostaining was significantly higher in del(5q) BL specimens compared to controls ( relative expression [RE] 9.6 vs. 0.25; P =0.007). An admixture of nuclear and cytoplasmic staining for p53 and each cdc25 isotype was observed at BL that was largely restricted to erythroid precursors, whereas at TR cdc25-C and -A expression was primarily cytoplasmic, consistent with drug-induced nuclear exclusion. At TR, RE of only cdc25C (BL, 75 vs. TR, 49; P=0.05) and PP2A (29.2 vs. 12.3; P=0.025) was significantly reduced; whereas at TF cdc25C (TR, 43 vs. TF, 166; P=0.003), cdc25A (42.4 vs. 150; P=0.006), PP2A (7.3 vs. 65.6; P=0.028) and p53 (0.92 vs. 25.4; P=0.024) RE significantly increased. Nuclear localization of cdc25C and p53 but not cdc25A predominated at TF, consistent with escape from cdc25C inhibition. QT-PCR confirmed transcriptional up-regulation of CDC25C at TF with a mean 8.8-fold increase in gene expression vs. BL. DNA sequencing revealed no acquisition of somatic mutations within the CDC25C and TP53 exons studied [n=5]. Conclusions: Secondary resistance to LEN in del(5q) MDS is associated with over-expression and activation of the haplodeficient drug-inhibitable phosphatases, cdc25C and PP2A, with consequent restoration of wt-p53 activation. Absence of gene mutations within the coding exons analyzed suggests that transcriptional compensation alone is responsible for drug resistance. Novel agents targeting transcriptional repression of CDC25C may restore LEN sensitivity and merit investigation in drug resistant del(5q) MDS. Disclosures: List: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Komrokji:Celgene: Research Funding, Speakers Bureau. Lancet:Celgene: Research Funding. Maciejewski:Esai: Membership on an entity's Board of Directors or advisory committees; Celgene: Speakers Bureau. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Description: Dysfunction of the DNA repair machinery may be one of the potential mechanisms leading to DNA damage and subsequent evolution of chromosomal abnormalities in MDS. However, the complexity of the possible mechanisms that could result in increased propensity to chromosomal breaks constitutes one of the obstacles in the rational targeting of deficient repair pathways for future investigations. Our study involved 37 patients with MDS (19 RA/RARS, 16 RAEB/AML, 2 CMML) and 10 controls. As expression arrays constitute a suitable hypothesis-forming tool, we have applied a specialized expression array to interrogate the most important DNA repair mechanisms in highly purified CD34+ progenitor cells from controls (N=10) and MDS patients (N=12). Based on the expression cut off value of (〉1.5 x background) we detected 22/113 genes present on the array in all samples tested. 16/22 DNA repair genes were concordantly upregulated (〉 2 fold above the control values established in pooled control reference). As a defective base pair excision repair (BER) pathway could play a role in the generation of chromosomal breaks, we focused our investigation on enzymes involved in this pathway. Expression array analysis showed that the MPG gene was significantly overexpressed, and we have validated this finding using TaqMan PCR in original samples and additional cohort of patients; 21/37 cases showed significantly elevated MPG transcripts. Overexpression of MPG may be in response to increased alkylation of DNA or may serve as a marker of CpG island methylation. While the latter alternative has been the subject of intense study, increased level of MPG is consistent with the presence of chemically modified bases incorporated into DNA during a toxic insult. Polymerase b (POLB) is a downstream enzyme in BER pathway, and its upregulation could indicate increased activity of this repair mechanism. Consequently, to test the integrity of the BER pathway, we set out to determine POLB expression levels by TaqMan PCR; two distinct subsets of patients (〉2 and ≤ 2 fold of the expression in controls) were identified (5/21 and 16/21). While 4/5 vs. 7/16 patients had chromosomal abnormalities in high vs. low POLB group respectively, these patient groups were otherwise clinically indistinguishable. Hence, we devised our next set of experiments that would discern the differences between these patients at a molecular level. Increased POLB would be consistent with the adequate BER activity. Here, we chose to further investigate the consequences of POLB upregulation in order to distinguish whether BER function results in appropriate DNA repair (upregulation of POLB and MPG) or whether it constitutes response to the deficiency of the AP endonuclease (upregulation of MPG and normal or downregulation of POLB) located immediately downstream of MPG. The content of apurinic sites per genome constitutes a suitable parameter to distinguish the aforementioned possibilities. An apurinic site-specific genomic ELISA assay clearly identified patients (6/19) in whom low POLB expression coincided with an elevated number of apurinic sites (〉0.50 apurinic sites per 1x105bp). We conclude that such a constellation is reflective of altered function of AP endonuclease or inadequate expression of MPG that could predispose to single and double-stranded breaks, and consequently lead to evolution of abnormal clone.

Description: Background: Early MDS becomes more advanced as immature myeloid cells proliferate, angiogenesis increases, genetic lesions accumulate, and tumor suppressor genes become inactivated through hypermethylation. Progression to acute myeloid leukemia (AML) may be prevented by targeting these defects through combination therapy, using an immunomodulatory, anti-angiogenic agent, lenalidomide (LEN), and a hypomethylating drug, azacitidine (AZA). Methods: We conducted a multicenter, Phase I trial in patients (pts) with advanced MDS (IPSS score ≥1.5, or FAB or WHO classification with ≥5% myeloblasts) starting in 6/06, with results reported through 7/07. Pts were enrolled using a “3+3” design (See Table), and could not receive LEN or AZA previously. Cycles lasted 28 days, to a maximum of 7 cycles of therapy. The primary endpoint was to determine the maximum tolerated dose (MTD) and dose-limiting toxicities (DLTs, defined as Grade 3/4 non-hematologic toxicity or 〉50% neutrophil (ANC) or platelet (plt) drop without recovery by Day 56) of the combination. A secondary endpoint was response as defined by the Modified International Working Group. Dose Level AZA Schedule LEN Schedule 1 75 mg/m2 SC days 1–5 5 mg PO days 1–14 2 75 mg/m2 SC days 1–5 5 mg PO days 1–21 3 75 mg/m2 SC days 1–5 10 mg PO days 1–21 4 50 mg/m2 SC days 1–5, 8–12 5 mg PO days 1–14 5 50 mg/m2 SC days 1–5, 8–12 5 mg PO days 1–21 6 50 mg/m2 SC days 1–5, 8–12 10 mg PO days 1–21 Results: Seven patients have been enrolled, 6 are evaluable for toxicity data. Median age was 64 years (range 52–70), 1 pt was female, and median follow-up is 5.5 months (range 1.5–13). All pts had RAEB-2; IPSS scores were 1.5 (4), 2.0 (2), and 3.0 (1), with IPSS cytogenetic risk categories of poor (1), intermediate (1), and good (5). No pt had a del (5q) lesion. Median time from MDS diagnosis was 3.5 wks (range 2–106). No DLTs occurred in Dose Levels 1 or 2, and MTD has not yet been reached. Grade 1/2 non-hematologic toxicities (n=6) included fatigue (4), injection site reaction (6), rash (3), pruritis (3), constipation or diarrhea (6), dizziness (1), and mucositis (1). Grade 3/4 non-hematologic toxicities included febrile neutropenia (1). Median ANC drop was 16.4% and plt drop was 10.4%. Although one patient was delayed 1 week in starting cycle 2 for neutropenia, there were no dose-reductions for toxicities. Four pts are evaluable for response: 2 had a complete response, 1 an erythroid response, and 1 progressive disease. Conclusions: The combination of LEN and AZA is well-tolerated and early results suggest efficacy in advanced MDS. Responses and toxicity data from higher Dose Levels will be presented.

Description: Mutation of PIG-A gene in the hematopoietic stem cell is the primary pathogenic lesion in paroxysmal nocturnal hemoglobinuria (PNH). However, the key element to the understanding of the disease evolution is the ability of the defective hematopoietic clone to outgrow their normal counterparts. It is believed that this growth advantage operates only in the pro-apoptotic milieu that exists in the context of immune-mediated bone marrow failure. The lack of glycosylphosphatidyl inositol-anchored proteins (GPI-AP) on the cell surface of affected cells is likely responsible for the ability of the PNH clone to evade immune attack and expand. GPI-AP are known to be associated with lipid raft microdomains (LRM). LRM contain a large number of receptors and regulatory proteins, whose function depend upon the presence of GPI-AP. Consequently, we hypothesized that GPI-AP deficiency can result in altered LRM which can either blunt proaptoptotic or inhibitory signals or possibly lead to exaggeration of prosurvival stimuli. We utilized paired GPI-AP-deficient (CD−) and wild type (CD+) EBV-transformed B cells derived from a PNH patient, as well as similarly paired lines derived from K562. LRM isolation was performed using sucrose density gradient centrifugation. PNH cells clearly displayed an altered protein repertoire in the LRM, based on analysis by 2D gel electrophoresis and SDS-PAGE, some of which is likely due to the loss of GPI-AP. Immunoblotting revealed lack of the GPI-AP CD55, while Lyn kinase, a known raft-associated protein was present in both PNH and WT LRM. However, an altered LRM function in PNH cells was illustrated via phosphotyrosine immunoblotting: rafts from PNH cell showed a distinct phosphorylation pattern from that observed in WT cells, suggesting distinct raft-dependent signaling in these paired cell lines. In subsequent experiments we have studied the functional consequences of an altered LRM structure in PNH. Two models were established. I) Based on the association of Fas with LRM we stipulated that it may not adequately transduce apoptotic signals in GPI-AP deficient rafts. While Fas was present in LRM of both CD+ and CD− cells as shown by Western blot, in GPI-AP deficient CD− cells, the apoptotic response to Fas agonist CH11 was attenuated in comparison to CD+ cells (23% vs. 60% of apoptotic cells). II) In the second model, we measured differential response of PNH cells to TNFa signaling, shown to be associated with LRM. As a readout, we used TNFa-induced p38 MAPK phosphorylation, which was shown to suppress hematopoiesis; PNH as compared to WT cells showed markedly reduced phosphorylation of p38 after TNF treatment, and exhibited increased basal phosphorylation of NFkB and AKT. These results suggest decreased inhibitory and increased survival signaling in PNH cells treated with TNF. Importantly, we observe increased activity of PDK1, the PI3K-dependent enzyme upstream of AKT, in LRM of PNH cells versus WT cells. Thus, our data show that lipid rafts in PNH cells orchestrate a skewed pro-survival signaling response that may play an important role in their ability to persist in the immune-mediated pro-apoptotic milieu that is a hallmark of bone marrow failure.

Description: Large granular lymphocyte (LGL) leukemia is a semiautonomous proliferation of cytotoxic T cells (CTL) often accompanied by lineage-restricted immune cytopenias, including neutropenia and anemia. Clonal expansion of CD57+ CTL in LGL leukemia may constitute an extreme pole of a spectrum of immune-mediated responses that range from polyclonal such as in aplastic anemia, likely with multiple clones and target antigens, oligoclonal as in some immune-mediated cytopenias, to clonally-restricted LGL lymphoproliferations. Consequently, LGL leukemia may be a clonally exaggerated response to chronic antigenic stimulation. NKG2D is present on reactive CTL, the normal counterparts of leukemic LGLs. Its ligand, MICA, is a tightly regulated, non-peptide-presenting HLA-related molecule induced during viral infections or cellular stress. As a mechanism of escape from anti-tumor cellular effector mechanisms, soluble MICA (sMICA) is secreted by various epithelial tumors. Similarly, herpes and adenoviruses have developed mechanisms for cellular MICA sequestration or secretion as means to evade immune-mediated elimination of infected cells. We thus hypothesized that NKG2D-MICA signaling plays a role in cytopenias associated with LGL leukemia. We studied a cohort of 47 well annotated patients with LGL leukemia. Flow cytometric analysis showed that while CD57 expression on CD8 cells was expectedly higher in LGL than controls (p

Description: Background: Both CIMF and MDS/MPD are clonal hematopoietic disorders characterized by cytogenetic abnormalities and myelofibrosis with resultant organomegally. Arsenic Trioxide (ATO) has demonstrated efficacy in MDS in two multicenter trials, including patients with MDS/MPD. Ascorbic acid (AA) enhances the activity of ATO by depleting intracellular glutathione. Thalidomide (Thal), in combination with ATO or steroids, is efficacious in patients with MDS and in patients with CIMF. Methods: We conducted a multicenter trial of Thal (50mg/d PO x 2 weeks, then 100mg/d), ATO (0.25mg/kg IV d1-5 of week 1, then twice weekly weeks 2–12), Dexamethasone (4mg/d PO x 5d every 4 weeks), and AA (1000mg PO 1–2 hours prior to each ATO infusion) over a 12-week cycle in patients with CIMF or MDS/MPD, from 1/05 to 7/07. Patients continued on Thal for an additional 3 months. Bone marrow was assessed at baseline and after week 12, as was spleen size. The primary endpoint was response as defined by the Modified International Working Group (IWG) treatment response criteria for MDS, and the IWG criteria for myelofibrosis with myeloid metaplasia (MMM). Results: Twenty-eight patients have been enrolled; 27 were evaluable for response. The median age was 66.5 years; 23 patients (82%) had MDS/MPD: 8 with CMML and 15 with MDS/MPD-u, with cytogenetics of: normal (13), +22p (1), +8 (2), −Y (1), +9 (1), −12p (1), complex (2), unknown (2). Five patients (18%) had CIMF with cytogenetics of: normal (3), dup Chr6 (1), unknown (1); Of 14 patients tested, 5 had the JAK2V617F mutation: 1 with CIMF and 4 with MDS/MPD. No patient had the MPL515 mutation. Thirteen patients had splenomegally, and 15 received prior therapies, including erythropoietin agents (9), 5-azacytidine (2), hydroxyurea (1), anagrelide (2), investigational agents (1), and prednisone (2). Median baseline lactate dehydrogenase (LDH), leukocyte count, and platelet count were: 300U/L, 12.1k/L, and 126.5k/L, respectively. Reasons for starting therapy included anemia (8 (29%)), thrombocytopenia (9 (32%)), or disease-related symptoms (11 (39%)). Seven patients (25%) did not complete the entire 12 week course of therapy: 3 withdrew consent, 1 had a low platelet count, 2 due to an adverse event, and 1 due to no response. Responses occurred in 7 patients (26% of total, 35% of those completing 1 cycle of therapy): 6 (21%) by the IWG MMM criteria, 4 (14%) by the IWG MDS criteria, and 3 (11%) by both. Using the IWG MMM criteria, 1 patient with MDS/MPD had a partial remission and 5 had clinical improvement: 2 with MDS/MPD and 1 with CIMF had hemoglobin responses, and 2 had a spleen response. Using the modified IWG MDS criteria, 1 patient with CIMF and 2 with MDS/MPD had erythroid responses; 1 with MDS/MPD had a platelet response. No patient with a JAK2V617F mutation responded. Grade3/4 non-hematologic toxicities included edema (4), fatigue (4), pleural effusion (2), dyspnea (3), motor and sensory neuropathy (1), pericardial effusion (1), zoster (1), and hematoma (1). There were no thrombotic episodes. Conclusion: The TADA regimen is well-tolerated and yields clinical responses in patients with MDS/MPD or CIMF, even in the absence of the JAK2V617F mutation.

Description: The JAK2-STAT5 pathway plays an key role in chronic MPD (cMPD) with the JAK2 V617F mutation. However, JAK2 also initiates several second messenger pathways, including the Ras-ERK and PI3K-AKT pathways. Since these essential second messenger pathways are also regulated by SFK activity, and crosstalk between JAK and SFK has been demonstrated, we investigated the potential SFK-dependent regulation of STAT5, Ras-ERK and PI3K-AKT in primary cells from patients with cMPD and myeloid cell lines with the JAK2 V617F mutation (HEL) or Epo-dependent (UT-7/Epo). In primary cells from JAK2 V617F patients, we show that SFK activity is required for optimal JAK2 induced phospho (p)-STAT5, nuclear targeting of p-STAT5, and STAT5-DNA binding. Both endogenous and Epo-induced colony formation in methylcellulose by peripheral blood cells derived from polycythemia vera (PV) patients was markedly suppressed with the SFK inihibitor dasatinib; endogenous colony formation was abrogated while both the number and size of Epo-induced colonies was markedly reduced (〉 70% inhibition at 1 uM), suggesting that despite of the presence of activating JAK2 V617F in primary cells, SFK activity is required for optimal proliferation. Interestingly, a similar observation was also made in a PV patient lacking the JAK2 V617F mutation. When HEL or Epo-stimulated UT-7/Epo cells were treated with a JAK inhibitor (P6), there was significant inhibition of p-STAT5 induction and second messenger pathways, demonstrating the importance of this proximal kinase. Inhibition of JAK2 did not affect SFK activation however, suggesting that SFK may lie upstream of JAK2. Interestingly, when SFK activity was suppressed (PP2 or dasatinib), a marked inhibition of p-STAT5 (and STAT5 DNA-binding), p-AKT, p-ERK, p-90RSK, p-S6 ribosomal protein(rp), and global protein tyrosine phosphorylation was observed in cell lines and primary cells. These findings suggest that SFK activity is involved in the most proximal aspects of Epo signaling and facilitates JAK2 activation of key second messenger pathways essential for proliferation, differentiation, and survival. Since JAK2 and SFK coordinately regulate multiple second messenger pathways, we next examined the relative importance of each by specifically targeting either the Ras-ERK pathway (MEK inhibition) or the PI3K-AKT pathway (PI3K and mTOR inhibition) downstream from JAK2 and SFK in both primary cells and cell lines. Using intracellular phospho-flow cytometry and immunoblotting, we show that the SFK-dependent Ras-ERK-RSK axis can directly induce p-S6rp, a key component in translational control and cell cycle progression. To determine whether this finding can be reproduced in vivo, we examined p-S6rp expression in bone marrow biopsies from PV patients (n=16); a marked hyperphosphorylation S6rp was consistently seen in patients’ megakaryocytes as compared to control specimens obtained from reactive processes, lymphoma staging, CMML, or even a case of hypoxia-induced polycythemia (all showing low/absent p-S6rp). Simultaneous staining for p-mTOR revealed that the level of p-mTOR was not increased. In sum, our findings suggest that SFK activation is necessary for optimal signal transduction along of the JAK2-STAT5 axis. In addition, the Ras-ERK-RSK pathway, requiring both JAK and SFK, may play a key role in S6rp hyperphosphorylation leading to increased cell cycling. Both findings point towards potential therapeutic targets, along with JAK2, for treating cMPD.

Description: Aplastic anemia (AA) and myelodysplastic syndrome (MDS) show pathophysiologic and clinical overlap; e.g., MDS can present with hypocellular marrow and a substantial proportion of AA patients develop MDS. A majority of AA patients respond to immunosuppression (IS), also effective in some patients with MDS. Patients with MDS show expansion of single hematopoietic clone frequently characterized by chromosomal aberrations while AA is oligoclonal, likely due to a decrease in available stem cells. Owing to the low resolution of metaphase cytogenetics and its dependence on cell growth in vitro, the ability to detect clonal chromosomal defects is limited in AA and the test is often negative or non-informative in MDS. High-density SNP arrays (SNP-A) can be used for precise identification of unbalanced genomic lesions. We hypothesize that cryptic clonal genomic aberrations do exist in some patients with AA. Their detection may have important clinical implications; it could help to distinguish patients unresponsive to IS, indicate propensity to MDS evolution and point towards pathogenetic genetic lesions. We have applied 50K and 250K SNP-A to analyze bone marrow (N=17), blood (N=49) or both (N=3) of patients with AA (N=26), PNH (N=33) and hypoplastic MDS (N=10). The results were analyzed using CNAG and Exemplar software. Defects identified in marrow of healthy controls (N=36) were used to define the minimal pathologic lesion (only 1 lesion was identified). In AA and PNH, clonal chromosomal abnormalities were found in 5/10 marrow and 15/52 blood samples, respectively, and in 4/10 bone marrows from hypocellular MDS. Of these lesions, known chromosomal defects (following progression to MDS) were confirmed in 3 AA patients and most significantly we also found previously cryptic abnormalities in 17 patients with AA (4 marrow, 13 blood) and normal metaphase cytogenetics. The new lesions involved segmental uniparental disomy (UPD) involving portions of chromosomes 1,2,6,7,8, 9,10,13 and 14. For example, we found LOH due to UPD at 7q (q21.11, q31.33, N=3). The results of 50K SNP-A were validated by 250K scan. LOH due to deletions (monoploid) or UDP (diploid) were confirmed by microsatellite and Taqman PCR copy number analysis, respectively. The lesions were somatic and not present in nonclonal lymphocytes. Overlapping UDP at 2p21.1–p22.1 (2AA, 1PNH) and 2p16.1 (2AA, 1hypoMDS) was detected. Examples of genes linked to SNP in deleted lesions include FANCL, LOC151445 and VRK2. Moreover, LOH due to UDP resulted in homozygosity for very minor alleles of SNPs linked to KIA1607 or NHMT (control population frequency 20%, 19%) and NRX1 or SMEK3 (14%, 27%) in 2q21 and 2p16, respectively. Hematologic response to IS was seen in 7/12 (58%) AA patients in whom lesions were found. However, only 5 of these patients were sampled prior to IS, 2 subsequently responded and 3 remained refractory. In contrast, 16/22 AA patients with normal chromosomes by SNP-A treated with IS showed a hematologic response (72%). Our study, representing the first application of high-density SNP-A for karyotyping of patients with AA, demonstrates that some patients with AA may harbor cryptic clonal defects possibly consistent with the diagnosis of MDS. It is likely that these lesions have clinical and prognostic significance.

Description: In MDS, cytogenetics has a major prognostic influence on the phenotype of the malignant clone and specific defects may point towards potential therapeutic targets. However, traditional metaphase cytogenetics (MC) has limited resolution and does not allow for detection of uniparental disomy (UPD). Defects on chromosome 5q have been studied using various methods to identify a minimal commonly deleted region (CDR). SNP-array karyoptyping (SNP-A) allows for precise detection of copy number changes as well as UPD. We hypothesized that SNP-A may reveal new lesions on chromosome 5 and allow for better definition of CDRs and pathogenic genes. Of 512 patients, 15% showed a 5q abnormality as a sole or associated aberration by MC. DNA was available in 189 patients and was subjected to 250K SNP-A. In 7 patients with normal/non-informative MC, a deletion on 5q was clearly detectable by SNP-A; in total, SNP-A identified 5q abnormalities in 14% patients in this group (vs. 11% by MC). UPD 5q was found in one patient with CMML. By SNP-A, 6/27 patients showed an isolated 5q deletion. SNP-A can also be used to construct precise cytogenetic maps. The commonly deleted region (CDR1,5q31.2, 137,472,900–139,451,900) was present in 24/27 patients. Significant overlap occurs with the CDR previously defined by Fairman, Zhao, Horrigan et al. This region includes important genes such as Cdc25C and EGR1. Of 24 patients with a deletion in CDR1, 21 had multilineage dysplasia predominantly in the megakaryocytic line (92%). While elevated platelet counts occurred in 3 patients, increased levels of megakaryocytes were common (83%). Previous studies by Bouldwood/Jaju suggested that the minimal CDR among patients with 5q- syndrome (CDR2, 5q33.1-33.2) differs slightly from that associated with secondary AML/MDS (CDR1). However, when patients (5/27) with classical 5q- syndrome were analyzed, all displayed single deletions spanning both CDR1 and CDR2. Other areas of partial overlaps were also identified (5q12.1; 5q13.3) more centromeric to CDR1 and present in 7/27 patients. 2 cases were particularly interesting: 1 with segmental UPD involving the CDR, the other showing a small deletion defining the CDR itself. In the latter patient, marked thrombocytosis was present and SNP-A demonstrated a complex chromosomal rearrangement. While MC revealed a segmental deletion of 5q and a concomitant duplication of this abnormal homolog, SNP-A showed that while the p arm portion had been duplicated, the q arm, with the exception of two small deletions (1.35 and 1.98Mb in length, confirmed by FISH), had a normal diploid set. SKY clarified that chr. 5 material had indeed been displaced to both chr. 3 and 7 with a reciprocal translocation of chr. 3 material occurring on the abnormal chr. 5. In sum, our studies demonstrate the utility of SNP-A as a karyotyping tool that can detect previously cryptic areas of LOH on chr. 5 and facilitate definition of shared 5q defects. We also show that our patients with 5q- syndrome had lesions spanning both 5q33 and the more proximal 5q31.2 area, making pathogenic distinction based on cytogenetics difficult.