Clonal Chromosomal Abnormalities Research Articles

Evidence-based genomic diagnosis characterized chromosomal and cryptic imbalances in 30 elderly patients with myelodysplastic syndrome and acute myeloid leukemia

BackgroundTo evaluate the clinical validity of genome-wide oligonucleotide array comparative genomic hybridization (aCGH) for detecting somatic abnormalities, we have applied this genomic analysis to 30 cases (13 MDS and 17 AML) with clonal chromosomal abnormalities detected in more than 50% of analyzed metaphase cells.ResultsThe aCGH detected all numerical chromosomal gains and losses from the mainline clones and 113 copy number alterations (CNAs) ranging from 0.257 to 102.519 megabases (Mb). Clinically significant recurrent deletions of 5q (involving the RPS14 gene), 12p12.3 (ETV6 gene), 17p13 (TP53 gene), 17q11.2 (NF1 gene) and 20q, double minutes containing the MYC gene and segmental amplification involving the MLL gene were further characterized with defined breakpoints and gene contents. Genomic features of microdeletions at 17q11.2 were confirmed by FISH using targeted BAC clones. The aCGH also defined break points in a derivative chromosome 6, der(6)t(3;6)(q21.3;p22.2), and an isodicentric X chromosome. However, chromosomally observed sideline clonal abnormalities in five cases were not detected by aCGH.ConclusionsOur data indicated that an integrated cytogenomic analysis will be a better diagnostic scheme to delineate genomic contents of chromosomal and cryptic abnormalities in patients with MDS and AML. An evidence-based approach to interpret somatic genomic findings was proposed.

Inflammatory myofibroblastic tumor

Inflammatory myofibroblastic tumor is an uncommon lesion of unknown cause. It encompasses a spectrum of myofibroblastic proliferation along with varying amount of inflammatory infiltrate. A number of terms have been applied to the lesion, namely, inflammatory pseudotumor, fibrous xanthoma, plasma cell granuloma, pseudosarcoma, lymphoid hamartoma, myxoid hamartoma, inflammatory myofibrohistiocytic proliferation, benign myofibroblatoma, and most recently, inflammatory myofibroblastic tumor. The diverse nomenclature is mostly descriptive and reflects the uncertainty regarding true biologic nature of these lesions. Recently, the concept of this lesion being reactive has been challenged based on the clinical demonstration of recurrences and metastasis and cytogenetic evidence of acquired clonal chromosomal abnormalities. We hereby report a case of inflammatory pseudotumor and review its inflammatory versus neoplastic behavior.

Cytogenetic and comparative genomic hybridization study of Indian myelodysplastic syndromes

Introduction: Myelodysplastic syndromes (MDSs) are clonal stem cell disorders characterized by cytopenias, dysplasia in one or more cell lineages and ineffective hematopoiesis and are associated with significant morbidity and mortality due to bone marrow failure or evolution to acute myeloid leukemia. Clonal chromosomal abnormalities are detected in 40–60% of patients. Multiple recurrent chromosomal aberrations have been identified by cytogenetics including fluorescence in situ hybridization (FISH) which is now widely recognized as one of the most important diagnostic and prognostic markers in MDS. Methods: Conventional cytogenetics by GTG-banding, FISH, comparative genomic hybridization (CGH) was done on 40 primary MDS subjects. Results: Among 40 subjects, 10 (25%) were abnormal and 30 (75%) showed apparently normal karyotypes with GTG banding and FISH. The various aberrations observed were del 5q−, del 7q−, 20q−, +8. DNA copy number changes including losses (30%) and gains (20%) were detected by CGH in 11 (36.6%) out of 30 karyotypically normal MDS. However chromosome 7 (37%) and 1 (25%) is frequently involved in current study population. Conclusions: This study confirms that the apart from non-random chromosome aberrations, other chromosome regions also involved in the MDS development. The occupational, environmental and geographical variations might be influencing the disease. Furthermore cytogenetic studies are warranted in larger groups of MDS cases to identify newly acquired chromosome aberrations that may aid in cloning new genes involved in the neoplastic process, ultimately helping in the development of targeted therapeutic drugs.

Numerical chromosomal changes and risk of development of myelodysplastic syndrome–acute myeloid leukemia in patients with Fanconi anemia

Fanconi Anemia (FA) is an inherited bone marrow failure syndrome characterized by congenital abnormalities, progressive marrow failure and predisposition to myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), and solid tumors. The most common acquired chromosomal aberrations in FA patients are trisomy of 1q and monosomy of chromosome 7; the latter is known to be associated with poor prognosis. A few reports also suggest that gains of 3q are associated with progression to MDS–AML and overall poor prognosis. It is not uncommon for patients with Fanconi anemia to have easily detectable (oligoclonal) chromosomal alterations in their still normal (nonmalignant) marrow, which makes it even more challenging to determine the import of such alterations. We conducted a retrospective longitudinal analysis of fluorescent in situ hybridization (FISH) analysis for gains in 1q and 3q and for monosomy 7 and 7q deletions on 212 bone marrow samples from 77 children with FA treated at our institution between 1987 and 2007. Given the baseline increased chromosomal instability and defective DNA repair in patients with FA, which leads to unbalanced chromosomal aberrations such as deletions, insertions, and translocations, for the purpose of this analysis an abnormal clone was defined as ≥10% abnormal cells. Chromosome 3 and 7 aberrations were associated with increased risk of developing MDS–AML ( P = 0.019 and P < 0.001 respectively), although the significance of chromosome 3 aberrations disappeared when different observation times were accounted for. Gain of 1q alone did not predict development of MDS–AML. In conclusion, children with FA should be followed closely with FISH analyses, because some of the clonal chromosomal abnormalities may be early indicators of progression toward MDS–AML and thus also of the need for hematopoietic stem cell transplantation.

Hematopathologic Evaluation of Myelodysplastic Features of Patients with CP-CML on Therapy with Clonal Cytogenetic Abnormalities In Ph Negative Clones

AbstractAbstract 1224 Background:Cytogenetic clonal aberrations in CML are a well recognized indicator of transition to blast phase. However, it is unclear how to interpret such changes when they occur in cells not harboring a Philadelphia Chromosome (Ph-), considering that some of these changes are often seen in patients with MDS/AML and isolated cytopenias can also be seen in CML patients in long term therapy with TKIs. Methods:We retrospectively reviewed 341 CML cases from January 2000 to July 2010, and determined the frequency, onset, character, and general course among CML patients harboring clonal chromosomal abnormalities (ChA) in Ph- cells during treatment with tyrosine kinase inhibitors (TKI's). In addition, we also performed a comprehensive hematopathology review on the bone marrow biopsy specimens of each case and categorized them based on findings comparable to bone marrow features found in MDS. Finally we will study the presence of TCR clonality and LGL expansions in relation with these findings. Results:In this review, we were able to identify 41 ChA among 36 patients (10.5% of cohort), with most cases demonstrating trisomy 8 (47% cohort) and loss/gain of chromosome Y (22% cohort). The median age was 62 years (range 33 – 78), with a male to female ratio of 7:2. The median number of treatments per patient was 2 (1-3), with a median follow-up of 77.5 months (18-196 mo). On hematopathologic evaluation of this cohort of 36 patients, only 3 patients (8% of cohort) had persistence of bone marrow features comparable to findings in MDS. Two cases demonstrated megaloblastoid erythroid features on the bone marrow, and another exhibited occasional hypogranulated granulocytes and nuclear irregularity in the erythroid precursors. Of note, 1 patient had concomitant mantle cell lymphoma at the time of diagnosis and residual MCL was found on review of several subsequent bone marrow biopsies. Post-treatment with TKIs 25 patients (69%) had normal cellularity or hypocellularity on bone marrow. The average percent cellularity on initial bone marrow post-treatment was 18.7%, reflective of the frequent cytopenias seen post-therapy. The more pronounced cytopenias were found in patients with persistent ChA. On peripheral blood evaluation the mean hemoglobin was 11.8g/dL, hematocrit 34%, and platelets 171,000/μL on presentation, with the majority of patients being transfusion independent. Of the 41 ChA, there were 2 patients who had marked hyperdiploidy with several ChA. One patient had a total of 7 different trisomies, in addition to a chromosome 17 deletion and t (7; 8). Post-therapy this patient's hyperdiploidy improved with an initial chromosomal number of 53 pre-treatment to 46 post-treatment, with the deletion 17 and t(7;8) remaining persistent throughout. The other patient, exhibiting the second highest hyperdiploid state, also had t (7; 8); interestingly, this chromosomal aberrancy emerged post-therapy, along with 1q32 and 3q12 ChA. Overall, none of these hematopathologic findings were however strongly consistent with the morphological findings typical for MDS. Conclusions:In summary, while clonal cytogenetic changes in Philadelphia Chromosome positive cells are a well recognized marker of disease progression, their presence in cells lacking this translocation does not seem to reflect a similar disposition. Additionally, on closer hematopathological review, the majority of the cytopenias seen in these patients are not consistent with definitive changes consistent with MDS. In this review of 341 patients, 36 patients with Ph- ChA, none of our cases had findings sufficient for diagnosis of MDS, suggesting the ChA post-therapy with TKIs are likely a benign feature. However, long term follow-up is still needed to confirm these data. Further relations with TCR clonality and LGL expansions will be presented. Disclosures:No relevant conflicts of interest to declare.

Multiple Mutations of CEBPA Contribute to Leukemogenesis: The Donor Origin of Leukemia Relapse After Allogeneic Hematopoietic Cell Transplantation.

AbstractAbstract 1303 Background:Leukemia relapse arising in cells of donor origin in the transplant recipient, called donor cell leukemia (DCL), is a rare disease entity after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The precise etiological mechanisms of DCL are unravelled and almost all the reported cases do not suggest a common mechanism. Careful analyses of the mechanisms with respect to the oncogenic transformation of donor-derived cells might provide a valuable insight into understanding of leukemogenesis.We aimed to assess whether those genetic mutations implicated in the development of common forms of AML contribute to the “leukemization” of donor cells in DCL. Methods:(1) A 36-year-old male was diagnosed with AML-M4. Cytogenetic evaluation demonstrated an abnormal clone 46 XY, del (9) (q11 q34) in 10/10 cells. The patient underwent 'allogeneic peripheral blood stem cell transplantation (allo-PBSCT) from a HLA-identical sister. Short tandem repeats (STR) analyses on day +28 showed complete donor chimera. Thirteen months after SCT, bone marrow aspiration revealed leukemia relapse. STR analyses showed still absolute donor chimera. The karyotype of bone marrow cells of the patient showed a new clonal chromosome abnormality: 45,XX, der(15;22) (q10;q10) in 10/10 cells, which was completely identical to the karyotype of the donor. Molecular evaluation suggested the patient developed DCL from a HLA-identical sibling. The examination of the donor's bone marrow showed normal and no malignant clone was detected by flow cytometry and fluorescence in-situ hybridization analyses. The donor remains healthy during a 25-month follow-up. (2) A series of archival stained bone marrow slides of the patient, including at the times of diagnosis, CR after one course of induction chemotherapy, lasting CR, before SCT, 1 month, 9 months and 12 months after SCT, samples of mononuclear-cell-enriched bone marrow at the times of relapse and CR after relapse, and buccal mucosal swab specimen during remission were available. (3) Buccal mucosal swab specimen and samples of mononuclear-cell-enriched peripheral blood and bone marrow were available from the donor. (4) Genomic DNA was extracted and analyzed for mutations in fms-related tyrosine kinase 3 gene (FLT3), neuroblastoma RAS viral oncogene homolog gene (NRAS), the CCAAT enhancer-binding proteinα gene (CEBPA), myeloid-lymphoid or mixed-lineage leukemia gene (MLL), and nucleophosmin gene (NPM1). Results:(1) DNA obtained from the patient during diagnosis was found to exhibit three different mutations in CEBPA, the gene encoding the crucial granulocytic differentiation factor C/EBPα. The mutation1 was the duplication of a cytosine residue at nucleotide 247 (247dupC) which resulted in the corresponding protein terminating prematurely at codon106 (Gln83fsX106). The mutation2 was a 6-bp duplication comprised nucleotides 584 to 589 (584—589dup) which resulted in an internal tandem duplication of amino acids 195 to 196 (His195—Pro196dup) in the protein. The mutation3 was a 3-bp duplication comprised nucleotides 914 to 916 (914—916dup) which resulted in the duplication of amino acids 305 (Gln305dup) in the protein. The 584—589dup mutation was remain found in DNA from the patient during lasting remission before SCT and from his buccal swab specimen, indicating this mutation should be germ-line mutation. In contrast, other two mutations were not found in the patient during remission and buccal swab specimen, indicating these two mutations should be somatically acquired. (2) The 584—589dup germ-line mutation was also found in DNA from peripheral blood cells, bone marrow cells and buccal swab specimen from the donor. (3) Donor-derived cells in the patient at the times of 1 month, 9 months and 12 months after SCT only exhibited the single germ-line mutation. When the patient relapsed with DCL, donor-derived leukemic cells were found to develop two somatic mutations in CEBPA in the patient microenvironment, which were completely identical to those observed in the patient with de novo leukemia. No other mutations were identified in FLT3, NRAS, MLL and NPM1 genes. Conclusions:Our findings suggest that multiple mutations of CEPBA impairing C/EBPα function may be sufficient to induce AML and a single mutation of CEBPA is not leukemogenic, but is the first step and predisposed to development of other mutations, ultimately inducing AML. Disclosures:No relevant conflicts of interest to declare.

BCR-ABL Fusion Transcript Do Not Significantly Influence the Outcome of Chronic Myeloid Leukemia Patients In Early Chronic Phase Treated with Imatinib Mesylate: a GIMEMA CML WP Analysis.

AbstractAbstract 1230 Background:Chronic myeloid leukemia (CML) is characterized by the presence of the BCR-ABL fusion gene. Different types of BCR-ABL transcripts can be found, due to different genomic breakpoints and alternative splicing. The most frequent transcripts are the e13a2 (b2a2) and the e14a2 (b3a2), that codify for a p210 protein. Occasionally, both transcripts may be present. In the imatinib (IM) era, few data about the prognostic significance of the transcript type are available, particularly in the setting of early chronic phase (ECP): one study suggested that patients with the b2a2 transcript may be more sensitive to IM (de Lemos et al. Genet Mol Res 2005), while two larger studies suggested that patients with b3a2 transcript may have better responses to IM (Vega-Ruiz et al. ASH 2007; Lucas et al. Haematologica 2009). No systematic evaluations in the context of large prospective clinical trials have been performed. AIM: To investigate the influence of the type of BCR-ABL fusion transcript on the responses and the outcome of CML patients treated with IM in ECP. Methods:We performed an analysis of 3 concurrent clinical trials of the GIMEMA CML Working Party (Clin Trials Gov. NCT00514488, NCT00510926 and the observational trial CML/023). Response monitoring was based on conventional cytogenetic examination (bone marrow) and quantitative molecular (Q-PCR) evaluation (peripheral blood). Definitions: Complete Cytogenetic Response (CCgR): 0% Ph+; Major Molecular Response (MMR): BCR-ABL/ABL ratio <0.1% (International Scale); failures: according to the revised European LeukemiaNet criteria (Baccarani et al. J Clin Oncol 2009). Results:559 consecutive CML patients in early CP have been enrolled from January, 2004 to January, 2007. Patients expressing rare transcript types (e1a2 and e19a2) and patients with the presence of both b2a2 and b3a2 transcripts were excluded: 493 out of 559 patients were evaluable, 203 (41%) with a b2a2 transcript and 290 with a b3a2 transcript (59%). The 2 groups were comparable (no significant difference in sex, age, Sokal and Hasford scores, clonal chromosomal abnormalities in Ph+ cells before IM and imatinib dose). The median observation time is currently 60 (extremes 2–80) months. In patients with b2a2 and b3a2 transcript, the observed 12-months CCgR rates were 77% and 80%, respectively; the cumulative incidence of CCgR was 89% and 88%, respectively (no significant differences). The time to MMR was significantly shorter for patients with b3a2 transcript (fig.1), but the cumulative incidence of MMR was not significantly different (81% and 86% in patients with b2a2 and b3a2 transcript, respectively). The probabilities of Failure-Free Survival, Progression-Free Survival and Overall Survival were 69% and 75%, 83% and 89%, 87% and 92% in patients with b2a2 and b3a2 transcript, respectively (fig. 1); no difference was statistically significant. Conclusions:In our experience, based on 493 early CP CML patients treated frontline with IM, the type of BCR-ABL fusion transcript had no relevant prognostic impact and no outcome differences have been observed so far. Acknowledgments:Work supported by European LeukemiaNet, COFIN, University of Bologna and BolognAIL. [Display omitted] Disclosures:Castagnetti:Novartis: Honoraria; Bristol Myers Squibb: Honoraria. Gugliotta:Novartis: Honoraria. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy. Saglio:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Baccarani:Novartis: Consultancy, Research Funding, Speakers Bureau; Bristol Myers Squibb: Consultancy, Research Funding; Wyeth: Consultancy, Research Funding. Rosti:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol Myers Squibb: Honoraria, Speakers Bureau; Roche: Speakers Bureau.

Simultaneous Detection of Genomic Rearrangements In Myelodysplastic Syndromes (MDS) with the Multiplex Ligation-Dependent Probe Amplification (MLPA) Assay

AbstractAbstract 1863 Background:Clonal chromosome abnormalities are present in the marrow cells in about 50% of patients with myelodysplastic syndromes (MDS) at the time of presentation. Cytogenetic analysis of the bone marrow is not only indicated in MDS for diagnostic purposes, but also to assess the individual prognosis according to IPSS scoring guidelines and plan tailored therapy. Conventional cytogenetics (CC) analysis is performed in clinical practice to detect chromosomal abnormalities. It has been reported that fluorescence in situ hybridization (FISH) is a more sensitive approach, but this analysis is limited to detection of the more frequent abnormalities on chromosomes 5, 7, 8, 11, and 20, and reports from the literature provide contradictory data. A new method has recently been described for the measurement of gene/chromosome copy number using genomic DNA: Multiplex Ligation-dependent Probe Amplification (MLPA). Aims:The purpose of this study was to perform the MLPA assay in a series of 29 MDS patients (M: 21, F: 8, median age 71 years, range 44–84), and to compare the results obtained with CC data. According to the WHO classification, 7 cases were classified as RA, 7 as refractory cytopenia with multilineage dysplasia, 2 as RAEB-1, 8 as RAEB-2, 1 as MDS_U, and 4 as CMML. According to the IPSS score, 8 were considered low risk, 11 intermediate-1 risk, 5 intermediate-2 risk, and 5 high risk. Methods:The MLPA assay was performed for all samples in two independent reactions, one for each probe mix (SALSA Probe-Mix P144 and P145) according to the manufacturer's recommendations (MRC-Holland). This mix contains 61 target sequences specific for different chromosome regions commonly involved in MDS: 5q (9 probes) + 5p (1 probe), 7q (8 probes) + 7p (2 probes), 8q (8 probes) + 8p (2 probes), 11q (8 probes), 12p (6 probes), 17q (2 probes) + 17p (4 probes), 20q (5 probes) + 20p (1 probe) and 21q (5 probes). The Probe mixes also include 21 reference probes selected from chromosomal regions that appear to be “quiet” in MDS. Data were analyzed with Coffalyser Software (MRC-Holland) using DNA from ten healthy donors as controls. The CC study was performed following standard protocols and at least 20 metaphases were analyzed. Results and Conclusions:Our study showed a good correlation between the MLPA and CC results, as shown in Table I, since most of the alterations were detected by both techniques. Discrepancies were found in 5 (17%) samples. MLPA analysis did not detect: in sample n°5 the presence of a chromosomal (chr.) translocation; in sample n°12 a chr. deletion and a chr. translocation; in sample n°17 a chr. deletion; in sample n°22 several chr. translocations and deletions; in sample n°28 a chr. gain. In fact, MLPA is not able to detect chr. translocations because it can reveal only chr. loss or gain; it can only analyse the chr. regions commonly involved in MDS (5, 7, 8, 11, 12, 17, 20 and 21); it can reveal chr. abnormalities only if the percentage of cells carrying the alterations is about 30–35% and do not show mosaicism. On the other hand, with CC we observed a karyotype failure (no metaphases) in 3 samples. MLPA proved to be rapid, cost effective, relatively easy to perform, had high throughput and enabled simultaneous analysis of many samples by automated data processing. MLPA and CC result complementary techniques, and MLPA is particularly useful in MDS cases with Karyotype failure.Table ISampleMLPAKaryotype1No anomalies46, XX2No anomalies46, XY3No anomalies46, XY4No anomalies46, XX5Del 5qDel 5q, t (1;12)6Del 7Del 77No anomalies46, XX8No anomalies46, XY9No anomalies46, XY10No anomalies46, XY11Del 5qDel 5q12Del 5q, Del 11q2345, XY, Del 11q23, Del 15, der 5 t(5;15) [80%]/46, XY [20%]13No anomalies46, XX14No anomalies46, XY15Trisomy 847, XY, Trisomy 816Del 7, Del 12p, Del 20qNo metaphases17Del 11q2346, XX, Del 11q23 [5]/46, XX, Del 9q22-23 Del 11q23 [15]18No anomalies46, XY19No anomalies46, XY20No anomalies46, XY21No anomalies46, XY22Del 5q, Del 12p, Del 17p1342~47, XX, Del 1p34 [3], Del 3 [6], t(4;18) [10], Del 5q [18], der 9 [3], Del 12 [10], t(13q;17q) [3], der 15 [4], Del 20 [3], Del 21 [2], Trisomy 22 [3], +m [10] [cp18]/46, XX [2]23Del 20q, Trisomy 21q, Trisomy 19p, Trisomy 8p48, XY, Trisomy 19, Del 20q, Trisomy 21 [12]/48, XY, Trisomy 8, Trisomy 19, Del 20q [8]24No anomalies46, XY25No anomalies46, XX26No anomaliesNo metaphases27No anomalies46, XX28No anomalies46, XY, add 11p15 [10]/46, XY [10]29No anomaliesNo metaphases Disclosures:No relevant conflicts of interest to declare.

Telomerase Activity Is High In Essential Thrombocythemia and Polycythemia Vera, but Not In Myelofibrosis – a Comprehensive Analysis on Telomerase Activity and Cytogenetics In Myeloproliferative Neoplasm and Myelodysplastic Syndromes

AbstractAbstract 4462 IntroductionTelomere maintains genetic stability and cell replication by capping and protecting the end of the chromosomes. Telomere is synthesized by an enzyme called telomerase which contains a catalytic subunit, the human telomerase reverse transcriptase (hTERT). In this study, we compared the telomerase activity in two disease groups of distinctive pathophysiology - myeloproliferative neoplasm (MPN) and myelodysplastic syndromes (MDS) characterized by abnormally increased effective hematopoiesis and ineffective hematopoiesis resulting in intramedullary apoptosis, respectively. The result was also analyzed in association with the number of metaphase cells in conventional cytogenetic analysis. Materials and MethodsReal time PCR to measure telomerase activity was performed using Quantitative Telomerase Detection kit (Allied Biotech, Inc., Ijamsville, MD) as per manufacturer¡&hibar;s instruction. The study included bone marrow cells of 99 normal controls, 114 with MPN (72 at initial diagnosis and 42 at follow up) and 73 with MDS patients (50 at initial diagnosis and 23 at follow up). MPN patients at initial diagnosis consisted of 22 CML in chronic phase, 18 ET, 11 PV, 12 PMF and 8 MPN, unclassifiable. MDS patients at initial diagnosis consisted of 19 RA or RCMD, 10 RAEB-1 and 13 RAEB-2. ResultsA trend of decreasing telomerase activity was observed as age increases in normal controls: 8239.19 (molecules/reaction) in age under 70yrs vs. 6669.46 in age over 70yrs (p>.05). Telomerase activity was higher in those with clonal chromosomal abnormalities detected in conventional cytogenetic analysis than those without clonal abnormalities (47600.82 vs. 11729.21, p>.05). In those with clonal abnormalities, patients with clonal abnormalities in >50.0% of metaphase cells analyzed showed significantly higher telomerase activity than patients with clonal abnormalities in <50.0% of metaphase cells (19183.06 vs. 6077.40, p=.014). Telomerase activity in MPN was significantly higher than that in normal controls (12990.12 vs. 8033.06, p=.027). A decreasing order of telomerase activity was observed in ET, PV and CML (20112.08, 17196.70 and 9835.25), and PMF showed significantly low telomerase activity than other subtypes (p=.005). In contrast, MDS showed slightly lower telomerase activity than normal controls, however, the difference was not statistically significant (7170.66 vs. 8033.06, p>.05). Telomerase activity was the highest in RAEB-2 and the lowest in RAEB-1 in between, without statistical significance (10532.24 and 6963.78, p=.441). ConclusionHigh telomerase activity in patients with high clonal cell proportion in conventional cytogenetic analysis could be explained by the hypothesis that cells with high proliferative activity form metaphase cells easily. High telomerase activity in MPN and low telomerase activity in MDS reflect the abnormally increased hematopoiesis and the ineffective hematopoiesis in each disease. ET and PV showed higher telomerase activity than CML, which could be explained by further analysis in line with clinical findings of the patients other than disease subtype. Low telomerase activity in PMF might result from the scarcity of normal hematopoietic cells and the high apoptotic fraction of megakaryocytes compared with anti-apoptotic profile of ET cells. Our result suggests that the potential of telomerase inhibitors should be investigated in patients with high telomerase activity. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Cytogenetic Evolution (CE) In Patients (pts) with Low and Intermediate Risk Myelodysplastic Syndromes (MDS) Is Associated with Poor Prognosis

AbstractAbstract 2941 Introduction:MDS is a spectrum of abnormalities in the proliferation and differentiation of hematopoietic stem cells that result in peripheral cytopenias, bone marrow dysplasia and increased risk of transformation to acute myelogenous leukemia (AML). Cytogenetic abnormalities occur in more than 50% of patients (pts) and have an impact on survival and risk of transformation to AML. CE, or acquisition of additional clonal chromosomal abnormalities, has been reported to occur in 30 to 50% of primary MDS pts. Their impact on prognosis and transformation into AML among pts with low and intermediate risk MDS is not known. In this study, we analyzed the impact of CE on prognosis in lower risk MDS. Methods:we reviewed 722 pts clinic records of low and intermediate risk MDS pts at MD Anderson Cancer Center (MDACC) from 2000–2010 and conducted a retrospective analysis of all MDS pts with at least two consecutive cytogenetic analysis (365 patients, 50.6%) and compared the cytogenetic evolution group (CE group) with the group without cytogenetic changes (no CE group). Cytogenetic analysis was performed in the Cytogenetics Laboratory at MDACC. Results:CE was detected in 200 pts (55%). Characteristics of patients with CE are: median age 65 years (23-91), IPSS int-1 79%, diploid CG 42%, excess blasts 25%. Pts with CE were more frequently female (p=0.005), and had more frequently abnormalities of chromosome 5 and 7 (p<0.001) at baseline. There were no statistically significant difference between these two groups (p>0.05) regarding age, WBC, platelet, hgb, ANC, BM blasts percent, diagnosis (RA or RAEB), and IPSS score. There were more chr.-5/-7, insufficient metaphases, and other abnormalities, but less diploid cases in CE group compared with no CE group (p<0.001). History of malignancy (p=0.001) and prior chemotherapy exposure were also associated with CE (p=0.001), but this was not as strong for radiation exposure (p=0.066). Also, more CE patients required therapy for MDS compared to no CE patients (p=0.039). Progression free survival was significantly extended in no CE patients (p=0.02). Overall survival was a longer in no CE (34.1months), compared with CE group (26.2 months), although this was not statistically significant. Conclusion:CE is more commonly observed among pts with high-risk features, and is usually associated with disease progression and resistance. Also, prior malignancy and chemotherapy exposure were associated with CE in this study. This data indicates that genomic instability has a role in disease progression in MDS. Further analysis of CE in MDS is needed. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Cholestatic Hepatitis Due to Hepatosplenic Lymphoma

Purpose: A 25 y/o male presented with a one month history of pharyngitis, 15 lbs weight loss and jaundice for one week. He denied other complaints or history of liver disease in self or family. He was on no medications and had no prior medical or surgical history. On physical examination he was icteric, had splenomegaly, but no hepatomegaly or lymphadenopathy (LAP). Laboratory tests: WBC 1.7 K/mm3 with 3% segs, 9% bands, 44% lymph, 21% monos, Hgb 12.8 g/dL, PLT 146 K/mm3, INR 1.3, alb 3 g/dL, AST 132 U/L, ALT 210 U/L, alk phos 162 U/L, bilirubin 5.7mg/dL, LDH 200U/L. Tests for hepatitis A, B, C, HIV, CMV, EBV, Wilson's disease, autoimmune hepatitis, and iron overload were negative. CT scan showed no adenopathy, a normal size liver and an enlarged spleen. Liver biopsy revealed atypical lymphocytes within the hepatic sinusoids positive for CD3 and CD7 and negative for CD 4, 5, 8, 20 and 56 and for EBV consistent with hepatosplenic Tcell lymphoma (HSTL). Bone marrow biopsy was consistent with non-Hodgkin's T cell lymphoma (NHL). Cytogenetics by flow cytometry was negative for clonal chromosomal abnormalities. He is currently undergoing chemotherapy and being evaluated for allogenic bone marrow transplant. Primary HSTL is a rare malignancy that constitutes only 0.016% of all cases of NHL, however, late secondary hepatic involvement is common. HSTL affects mainly young males in the second or third decades of life, and usually is associated with immunosupression as in transplant recipients, SLE, and HIV patients, or in those with a history of malaria or previous malignancy. More recently it has been reported in Crohn's patients who have been treated with biologics or immunosuppressive therapy. Patients present with fatigue, fever, nausea, and weight loss, and have significant cytopenias, and minimal or absent LAP. All patients have splenomegaly and the majority have hepatomegaly. Bone marrow involvement may be subtle but is virtually universally present. HSTL is thought to arise from γδT cells, a subset of epithelial tissue and red splenic pulp associated cytotoxic Tcells with direct antigen recognition capability, acting at the interface between innate and adaptive immunity. There is an association with the isochromosome arm 7q cytogenetic abnormality. The majority of patients initially respond to chemotherapy, but relapses are common and long-term prognosis remains poor with median survival 16 months. HSTL is a rare malignancy that can present as cholestatic liver disease due to malignant cell infiltration. It should be considered in the differential diagnosis in patients with features of cholestasis and pancytopenia. More studies are needed for better understanding and treatment of this lethal condition.

Clonal chromosomal abnormalities in CD34+/CD38− hematopoietic cells from cytogenetically normal chronic myeloid leukemia patients with a complete cytogenetic response to tyrosine kinase inhibitors

Letter to the Editor Clonal chromosomal abnormalities in Ph-negative cells (CCA/Ph-) have been identified in 3-15% of chronic myeloid leukemia (CML) patients with a partial or complete cytogenetic response (CCyR) to imatinib(1). Trisomy 8 and deletions of chromosome 7 [either monosomy 7 or del 7(q)] account for the majority of cases, although a range of other karyotypic changes were seen at lower frequency. A recent study reported CCA/Ph- in patients on dasatinib, indicating that the phenomenon is not limited to imatinib therapy. In some patients, CCA/Ph is associated with a myelodysplastic syndrome (MDS), and progression to acute myeloid leukemia has been reported(1, 2). A single center report suggested inferior survival in newly diagnosed patients who develop CCA/Ph-(3). However, a larger study found that CCA/Ph- does not adversely affect the prognosis of patients with a major cytogenetic response (MCyR) to imatinib, indicating that this is a mostly benign condition(4). The reported incidence of CCA/Ph- is based on metaphase karyotyping, which is limited by the small number of cells analyzed and by the fact that only cells are assayed that can be induced to divide within the culture period. We thus hypothesized that conventional karyotyoping may underestimate the incidence of CCA/Ph- and decided to screen CD34+/CD38− cells from a cohort of CML patients with a CCyR to tyrosine kinase inhibitor (TKI) therapy for abnormalities of chromosomes 7 and 8. This primitive cell compartment is known to be enriched for hematopoietic progenitor and stem cells(5). We find CCA/Ph- in CD34+/CD38− cells from 4/19 patients, suggesting CCA/Ph- is more common than previously appreciated.

Phenomenon of evolution of clonal chromosomal abnormalities in childhood acute myeloid leukemia

Analysis of chromosomal abnormalities in bone marrow cells in 116 children with diagnosis of acute myeloid leukemia (AML) was performed. Frequency of evolution of clonal chromosome abnormalities in AML constituted 42,3%. The most abundant among them were numerical abnormalities of chromosomes 8, 9, and 21 as well as secondary structural abnormalities in region 12p12, 9p22, 9q22, 9q34, 11q14-23, and 16q22. Numerical abnormalities were registered in 26,7% cases. The basic mechanism of leukemic clone evolution was trisomy, deletion and monosomy. The frequency of evolution was 7 times higher in the age group up to 2 years and twice higher in the age group up to 5 years. The high frequency of evolution was established at t(15;17)(q22;q22) and the absence at inv(16)(p13q22). The patients with clonal evolution died earlier, before reaching remission, that can be connected with heavy initial state and high frequency of relapse. Conception of abnormality clone evolution was proposed at some stages: I--appearance of balanced rearrangement; II--trisomy; III--lose of chromosomal material. Appearance of unbalanced genome in evolution possess an advantage in proliferate activity and can be connected with the answer on chemotherapy. Identity of abnormal chromosome structure at diagnosis and relapse of disease can be an evidence of the influence of chemical agent on establishment of some types of evolution of chromosome abnormalities in leukemic cells in AML in children.

Myelodysplastic Syndrome Mimicking Idiopathic Thrombocytopenic Purpura

In patients with isolated thrombocytopenia, but without significant dysplasia, diagnosis of idiopathic thrombocytopenic purpura (ITP) rather than myelodysplastic syndrome (MDS) may be taken into account. It is important to make an accurate diagnosis because different treatments are used for ITP and MDS. The purpose of this study was to investigate the clinical and hematologic features of patients who were initially diagnosed as ITP but had cytogenetic abnormalities. We retrospectively reviewed cytogenetic studies of 100 patients who were diagnosed as ITP from 2004 to 2009 at Mokdong Hospital of Ewha Womans University based on clinical features and hematologic studies. Bone marrow pathology was re-evaluated based on 2008 WHO classification. Cytogenetic analysis was performed by 24-48 hr culture of bone marrow aspirates without using mitogens and 20 metaphases were analyzed. Of the 100 patients diagnosed as ITP initially, three patients (3%) had cytogenetic abnormalities. They had no thrombocytopenia-related symptoms and thrombocytopenia was found accidentally. The numbers of megakaryocytes in bone marrow were increased and dysplasia was not found in megakaryocyte, erythroid, and myeloid cell lineages. The proportion of blasts was within normal limits. Clonal chromosomal abnormalities found were der(1;7)(q10;p10), add(9)(q12), or t(7;11)(p22;q12). Presumptive diagnosis of MDS or diagnosis of idiopathic cytopenia of undetermined significance (ICUS) was made according to 2008 WHO classification. During the follow up, disease progression was not found. In patients with suspected ITP, cytogenetic analysis should be done. If specific clonal chromosomal abnormality is found, presumptive diagnosis of MDS has to be considered and close follow up is needed.

Cytogenetic aberrations and the development of uterine leiomyomata

This study was conducted to define the cytogenetically critical regions of uterine leiomyomata, hoping to demonstrate the presence of possible genes involved in their evolution. It was carried out on 25 randomly selected uterine leiomyoma specimens obtained from 16 patients during hysterectomy or myomectomy operations. Successful tissue culture and karyotyping were performed in 19 specimens. There was no correlation between patient age, gravidity, or presenting symptom and the presence of chromosomal abnormality. A significant correlation was found between short culture turnaround time and the occurrence of chromosomal abnormality. Abnormal clonal karyotypes were present in 6 specimens, non-clonal abnormalities in 4 specimens and normal karyotypes were found in 9 specimens. Myomas with cross section >4 cm showed an increased incidence of abnormal karyotypes and a statistically significant higher incidence of clonal abnormalities. On the other hand, submucous myomas presented fewer clonal abnormalities than did intramural or subserosal myomas. Clonal chromosomal abnormalities involved 5 different chromosomes (2, 7, 8, 12, 22), which indicate genetic heterogeneity of such benign tumors and the need of molecular cytogenetic studies or molecular studies to characterize possible candidate genes at specific chromosomal breakpoints.

Incidence of malignancy and clonal chromosomal abnormalities in Fanconi anemia

Fanconi anemia (FA) is an autosomal recessive, cancer susceptibility disorder characterized by diverse clinical features, such as short stature, skeletal or skin abnormalities, progressive bone marrow (BM) failure, and increased risk of malignancies. Clonal chromosomal abnormalities are frequently reported in FA patients transformed to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). To study the incidence of malignancy and clonal chromosomal abnormalities in FA patients. Thirty-eight clinically diagnosed FA patients were studied at the time of diagnosis and the patients were followed-up for a maximum of 28 months at 3-month intervals. The median duration of follow-up of these patients was 19.8 months. Chromosomal breakage investigation using mitomycin C (MMC)- and diepoxybutane (DEB)-induced peripheral blood cultures were stimulated with phytohemagglutinin. Cytogenetic study was done on the BM cells to detect clonal chromosomal aberrations. Eleven (28.95%) out of 38 patients developed malignancies, including 6 (54.54%) MDS, 4 (36.36%) AML, and 1 (2.63%) squamous cell carcinoma. The clonal chromosomal abnormalities were detected in 5 (45.45%) FA patients who developed malignancies and the type of chromosomal abnormality detected were monosomies 5, 7, trisomy 10, dup(1)(q12-q24), and inv(7)(p11pter). The FA patients have a high risk of developing malignancies, and clonal chromosomal abnormalities play an important role in the prognosis of the disease. Therefore, FA patients need to be followed-up at regular intervals for early diagnosis and optimal management of the disease.

Diagnosis of Myelodysplastic Syndrome Among a Cohort of 119 Patients With Fanconi Anemia

Predisposition to myelodysplastic syndrome (MDS) and acute leukemia is a hallmark of Fanconi anemia (FA). Morphologic criteria for MDS in FA are not well established, nor is the significance of clonal chromosomal abnormalities. We reviewed bone marrow samples of 119 FA patients: 23 had MDS, with the most common subtype refractory cytopenia with multilineage dysplasia. The presence of MDS was highly correlated with the presence of clonal abnormalities. Neutrophil dysplasia and increased blasts were always associated with the presence of a clone, in contrast with dyserythropoiesis. The most frequent clones had gains of 1q and 3q and/or loss of 7. Karyotype complexity also correlated with MDS. One third of patients with 3q as a sole abnormality had no MDS; patients with 3q and an additional abnormality all had MDS. The data provide a rationale for integrating cytogenetic findings with independently evaluated morphologic findings for monitoring bone marrow status in FA.

Cytogenetic evolution correlates with poor prognosis in myelodysplastic syndrome

Clonal chromosomal abnormalities are observed in 30–50% of primary myelodysplastic syndrome (MDS) patients. Although the prognostic relevance of cytogenetics is generally appreciated, the prognostic value of cytogenetic evolution has rarely been evaluated. In this study, we retrospectively analyzed cytogenetic features at diagnosis and during follow-up in 85 patients with primary MDS. Cytogenetic evolution occurred in 18 of the 85 patients (21%), with chromosomes 8, 5, and 1 most often involved. Patients with higher levels of marrow blasts ( P = 0.034), more advanced stages of World Health Organization (WHO) subtypes (44% vs. 16%, P = 0.035), and higher risk International Prognostic Scoring System (IPSS) subgroups (47% vs. 16%, P = 0.021) had higher incidences of developing cytogenetic evolution. Furthermore, the median survival of patients in the group with cytogenetic evolution was 25.8 months, compared with 45.4 months for patients in the group without cytogenetic evolution ( P = 0.01). The same result was also found for time to progression: patients with cytogenetic evolution progressed more rapidly than those without cytogenetic evolution ( P = 0.007). Knowledge of cytogenetic evolution offers useful information for clinicians to make more accurate prognostic assessments for patients with MDS.

Evaluation of Conventional Cytogenetics Versus Fluorescence in Situ Hybridization Analysis in the Prognostic Scoring of Patients with Myelodysplastic Syndromes.

Abstract 1757Poster Board I-783 BackgroundThe myelodysplastic syndromes (MDS) encompass a heterogeneous group of progressive bone marrow insufficiency disorders characterized by chronic cytopenias and a risk of progression to acute leukemias. Non-random clonal chromosomal abnormalities, seen in almost 50% of patients with MDS, are associated with clinically and biologically distinct forms of the disease with prognostic and therapeutic implications. Currently, conventional cytogenetics (CC) and fluorescence in situ hybridization (FISH) techniques are commonly used to detect the chromosomal abnormalities in MDS. In this retrospective study, an effort is made to evaluate the merit of concomitant CC and FISH analyses for prognostic scoring in MDS. MethodsA cohort of 86 MDS patients with both CC and FISH results were analyzed. The FISH technique focused on chromosomes 5, 7, 8 and 20. Data was collected on patient age, sex, WHO (World Health Organization) classification, CC and FISH findings, histologic transformation and transfusion dependence. For each patient, IPSS scores were calculated based on either CC (IPSS-CC) or FISH (IPSS-FISH). A weighted kappa statistic was calculated to measure agreement between the CC and FISH methods. Fisher's Exact tests were performed to measure the association between concordance/discordance in scores while survival time for concordant and discordant scores was calculated using Kaplan-Meier estimation and compared using a log-rank test. A significance test of 0.05 was assumed for all comparisons. ResultsA cohort of 86 MDS patients with 64% male and median age of 68.5 years at diagnosis was studied. According to the WHO classification, 34% of the patients had refractory anemia (RA), 11% had refractory anemia with ringed sideroblasts (RARS), 13% had refractory anemia with excess blasts –1 (RAEB-1), 20% had refractory anemia with excess blasts –2 (RAEB-2) and 20% had refractory cytopenia with multilineage dysplasia (RCMD).Of the 86 patients, 74 had concordant IPSS-CC and IPSS-FISH scores. The p-value for weighted kappa statistic was 0.87 with 95% confidence interval (0.80, 0.94) indicating that there is strong agreement beyond chance between the IPSS-CC and IPSS-FISH. Of the remaining 12 patients with discordant scores, 9 had IPSS-CC > IPSS-FISH while 3 patients (eliminated from comparative analysis for small size) had IPSS-FISH > IPSS-CC. Of the several variables compared between the concordant and discordant groups, only WHO subgroup turned out to be statistically significant. Concordant scores were significantly more likely to be in the RA (37%) and RCMD (23%) cohort, while all discordant patients were in the RAEB-1 (43%), RAEB-2 (29%), and RARS (14%) subgroups (p=0.004).The 9 discordant patients were compared to 27 patients with concordant scores belonging to WHO subgroups RAEB-1, RAEB-2 and RARS. The prognostic variables for comparison were patient survival, leukemic transformation and transfusion dependence. 33% of the discordant patients had leukemic transformation as compared to 11% of concordant patients (p=0.15). Although the difference was large, the sample size was too small to be statistically significant. ConclusionCurrently CC and FISH analyses are both being employed to detect the chromosomal abnormalities in MDS patients. This retrospective study indicates that there is statistically significant concordance between cytogenetic and FISH analyses. Conceivably, doing one or other test would most likely be sufficient for prognostic scoring of the vast majority of MDS patients especially those in RA and RCMD subclasses. The discordant scores are significantly more likely to occur in the RAEB-1, RAEB-2 and RARS classes hence justifying this subgroup of patients as potential candidates for both CC and FISH analyses. In our 9 patients with discordant scores (IPSS-CC > IPSS-FISH), a trend was observed towards higher leukemic transformation when compared to WHO subgroup matched concordant patients. While further validation of the current findings by larger prospective studies is awaited, this study unravels the MDS patient sub-population where CC and FISH could be complementary and not superfluous. DisclosuresNo relevant conflicts of interest to declare.

Cytogenetic abnormalities in mesenchymal stem cells in chronic lymphocytic leukemia (CLL) patients and normal subjects

e22002 Background: Mesenchymal stem cells (MSC) residing in the marrow support hematopoiesis and protect cancer cells from undergoing cell death induced by chemotherapy. Recent reports have described clonal cytogenetic abnormalities in the MSC of acute myeloid leukemia and myelodysplastic syndrome patients. To determine if cytogenetic abnormalities are present in MSC from CLL patients, we analyzed karyotypes of MSC from 13 CLL patients and 5 normal subjects. Methods: Stromal cells from marrow core biopsies of 13 CLL patients and 5 normal control subjects were isolated, cultured and confirmed as MSC based on their immunophenotype and capacity to differentiate into three lineages. After 3–4 non-stimulated cell culture passages, the karyotype was analyzed in 5–40 metaphase cells from each subject Abnormalities were considered clonal using the accepted convention of the same chromosomal gain or rearrangement in 2 or more cells or loss in at least 3 cells. For each CLL patient, interphase FISH analysis to detect the common CLL-associated abnormalities was performed on freshly isolated peripheral blood mononuclear cells (PBMC). Results: Clonal cytogenetic abnormalities of the cultured MSC were observed in 6 of 13 CLL patients (46%) and 3 of 5 control subjects (60%). The 6 CLL MSC had different clonal abnormalities than the PBMC CLL FISH studies. One CLL MSC exhibited trisomy 5, one exhibited trisomy 8, and one had monosomy X clone. One MSC had a balanced t(1;16). One MSC had a balanced t(3;12) and a del(3p), and one had a complex karyotype with 4 unbalanced rearrangements. The clonal aberrations observed in the 3 controls included a del(3p) in two cases, and in one case an inv(5) and a del(10q). There was no correlation of the chromosomal abnormalities in CLL MSC with clinical stage. Conclusions: Marrow MSC derived from CLL patients and normal subjects do show an array of cytogenetic abnormalities including clonal chromosomal abnormalities. However the genetic abnormalities found in both CLL and normal MSC could represent acquired genomic instability associated with advanced age, rather than oncogenesis associated with CLL. [Table: see text]