Specific and Recurrent Cytogenomic Abnormalities Originate in the Spleens of Myelofibrosis Patients and Contribute to Clonal Diversity and Disease Progression

Abstract Hematopoietic stem cells located in spleens (SP) of patients with myelofibrosis (MF) have functional properties that differ from those present in peripheral blood (PB) (Wang et al, JCI,122: 3888, 2012 ). We hypothesized that the spleen might be a source of malignant stem cells in MF which ultimately lead to disease progression and leukemic transformation. To investigate the genetic diversity of MF SP cells, cytogenetic analyses of SP and peripheral blood (PB )/bone marrow (BM) cells from 13 MF patients were performed. Nine of 13 patients (69%) had concordant normal (n=5) and abnormal (n=4) cytogenetic and FISH analyses when comparing SP and PB/BM cells. Four (30%) patients had discordant results with specific chromosomal abnormalities present in either SP or PB cells. One patient had 3 cytogenetic abnormalities associated with an unfavorable prognosis: (1;7), del(12p), and i(17q) in SP cells that were not observed in PB cells. A second patient who had progressed to acute leukemia had pentasomy 21 in 45% of SP cells but not in PB cells and had del (20q) in PB cells but not SP cells, suggesting that del (20q) had arisen in the BM while pentasomy 21 had originated in the SP . In a third patent 20% of PB cells had +8 while only 1.6% of SP cells had +8, indicating that the +8 clone originated in the BM. Studies of BM and PB of the 4 th patient showed a normal karyotype in 2008 and 2011 while 2% of SP cells in 2011, at the time of progression to MF, possessed del 20q while the BM remained normal. In 2014, when the patient developed an accelerated phase of MF, 85% of BM cells possessed del (20q), indicating that del(20q) originated in the SP and migrated to the BM. We next hybridized SP DNA from 12 of the 13 patients to the Agilent 400k platform [355515 (CGH) + 59646 (SNP)] and performed CGH and SNP analysis. These studies revealed that all 12 patients had additional gains and losses of chromosomal regions as well as uniparental disomy (UPD) in SP cells. Two groups of acquired genomic changes were observed in SP cells: 1) four acquired regions were present in over 60% of pts and 2) three regions of acquired changes were observed in 25- 33% of patients. Irrespective of the patient’s karyotype, gains of 4 chromosomal regions: 1p13.2 (RHOC), 12q24.31 (NCOR2), 13q34 (RASA3) and 17q12 (TAF15) were detected in 83%, 83%, 75% and 67%, of patient’s SP cells, respectively. All four genes are known to be involved in leukemogenesis. Gains of these 4 chromosomal regions have not been observed in normal controls or PB CD34+ cells from 437 patients with MPNs including MF (Klampfl et al, Blood 167, 2011, Rumi et al Am J Hematol 974, 2011). Gains of an additional three regions, 18q21.31 (NEDD4L), 16q23.2 (WWOX) and 17q21.31 (WNT3) were detected in the SP cells of 25-33% of patients. These genes have also been implicated in leukemogenesis. The greater the complexity of the karyotype of the SP cells the greater the number of copy number genomic changes (up to 92) were observed. Although SNP analyses demonstrated 28 acquired UPD regions in 12 patients 9p13-p24 was the most common occurring in 25% of patients. SNP analyses also demonstrated triplication of 9q and quadruplication of 9p, suggesting that UPD of the entire chromosome 9 in SP cells can be associated with disease progression. Transplantation of SP CD34+ cells with a normal karyotype or with one chromosomal abnormality into NOD/SCID/IL2Rγ(null) mice resulted in a modest degree of donor cell chimerism (0.2%-4%), while transplantation of SP CD34+ cells with UPD of 9p, the entire chromosome 9 UPD, del20q originating in SP cells, complex karyotypes and gains of 4 chromosomal regions (1p,13, 12q24, 13q34 and 17q12 ) resulted in a higher degree of donor cell chimerism (18%-25%) indicating the association of these genetic events with altered stem cell function. Our findings indicate that some chromosomal abnormalities are acquired in MPNs initially in the SP sometimes occurring years prior to their appearance in the BM/PB. Other abnormalities, such as del(20q) may originate in either the BM or SP. All MF SP cells were characterized by additional diverse cytogenomic changes involving at least four regions, containing genes associated with leukemogenesis, leading to recurrent copy number gains in 67% to 83% of MF patients. We hypothesize that the SP provides a microenvironment in a subpopulation of MF patients which is associated with increased genomic diversity resulting in disease progression and leukemic transformation. Disclosures Mascarenhas: Incyte Corporation: Consultancy.

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Prognostic implications of morphology and karyotype in primary myelodysplastic syndromes

Blood ◽

10.1182/blood.v67.6.1765.1765 ◽

1986 ◽

Vol 67 (6) ◽

pp. 1765-1772 ◽

Cited By ~ 193

Author(s):

RH Jacobs ◽

MA Cornbleet ◽

JW Vardiman ◽

RA Larson ◽

MM Le Beau ◽

...

Keyword(s):

Median Survival ◽

Myelodysplastic Syndromes ◽

Chromosomal Abnormalities ◽

Bone Marrow Cells ◽

Normal Karyotype ◽

Refractory Anemia ◽

Survival Times ◽

Trisomy 8 ◽

Leukemic Transformation ◽

Abnormal Karyotype

Abstract Forty-nine patients with primary myelodysplastic syndromes (MDS) were subclassified according to French-American-British (FAB) Cooperative Group criteria. Eight patients had acquired idiopathic sideroblastic anemia (AISA), ten had chronic myelomonocytic leukemia (CMMoL), 14 had refractory anemia (RA), nine had refractory anemia with excess blasts (RAEB), and five had refractory anemia with excess blasts in transformation (RAEB-T); three patients could not be subclassified. The actuarial median survival for patients with AISA or with RA had not been reached at 60 months of follow-up. The median survival times for patients with CMMoL, RAEB, and RAEB-T were 25, 21, and 16 months, respectively. The percentages of patients with each subtype who developed ANLL were none in AISA, 20% in CMMoL, 7% in RA, 56% in RAEB, and 40% in RAEB-T. Patients with CMMoL had a poor prognosis independent of transformation to acute nonlymphocytic leukemia (ANLL), whereas patients with RAEB and RAEB-T had a high incidence of transformation and short survival times. Clonal chromosomal abnormalities were present in bone marrow cells from 19 patients at the time of diagnosis, and two others developed an abnormal karyotype at the time of leukemic transformation. The most frequent abnormalities, including initial and evolutionary changes, were trisomy 8 (9 patients), deletion of 5q (4 patients), and deletion of 20q (4 patients). The median survival times were 32 months for patients with an abnormal karyotype, and 48 months for those with a normal karyotype (P = 0.2). Specific chromosomal abnormalities were not associated with particular histologic subtypes; however, a high percentage of patients with RAEB and RAEB-T had an abnormal clone (89% and 80%, respectively). The percentages of patients with clonal abnormalities were 13% for AISA, 20% for CMMoL, and 29% for RA. The MDS transformed to ANLL in 42% of patients with an abnormal karyotype, compared to 10% of those with an initially normal karyotype (P less than .01). Among patients with RA, RAEB, and RAEB-T, the risk of leukemic transformation was confined to those with an abnormal karyotype (P less than .01). Thus, in the present study, morphology and karyotype combined were the best indicators of outcome in patients with MDS.

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Centrosome Aberrations in Chronic Myeloid Leukemia (CML) Are Correlated with Disease Progression and Chromosome Instability.

Blood ◽

10.1182/blood.v104.11.2949.2949 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2949-2949

Author(s):

Michelle Giehl ◽

Alice Fabarius ◽

Chun Zheng ◽

Oliver Frank ◽

Andreas Hochhaus ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

Disease Progression ◽

Myeloid Leukemia ◽

Blast Crisis ◽

Chromosome Instability ◽

Chronic Phase ◽

Normal Karyotype ◽

Benign Tumors ◽

Preneoplastic Lesions ◽

Cd34 Cells

Abstract Purpose: Numerical and structural centrosome abnormalities are hallmarks of a variety of cancers and have been implicated in chromosome missegregation, chromosomal instability, and aneuploidy. These phenomena already occur in preneoplastic lesions like oral leukoplakia, early cervical neoplasias, and small benign tumors of colon and breast. Moreover, deviations from normal karyotype seem to increase as tumors enlarge and become malignant. Genetic instability is a common feature in chronic myeloid leukemia (CML). We sought to establish a relationship between centrosome abnormalities and cytogenetic aberrations in CD34+ cells from CML patients at diagnosis (chronic phase - CP) and in blast crisis (BC). Methods: Diagnosis of CML was established by hematologic, cytogenetic and molecular parameters. Treatment was performed according to the protocols of the German CML study group (www.kompetenznetz-leukaemie.de). CD34+ cells from ten umbilical cord blood specimens served as negative controls. Centrosome number and morphology were analyzed by immunofluorescence microscopy. In brief, CD34+ cells from ficollized peripheral blood samples were concentrated by magnetic cell sorting (MACS) and cytospun onto coated slides. After methanol fixation cells were incubated with antibodies directed to centrosomal proteins Pericentrin and gamma-Tubulin. Antibody-antigen complexes were stained by incubation with FITC- and Cy3-conjugated secondary antibodies. Results: CML CP samples tested at initial diagnosis (n=20) already displayed numerical and structural centrosome aberrations (30.0% +/−2.3) as compared with corresponding normal control cells (n=10) (2.3% +/−1.1). In BC samples (n=10) an increase of centrosome aberrations was observed (58.0% +/−2.0). Conclusion: The findings suggest that centrosome defects in CML occur early and are already present at primary diagnosis. Centrosome defects may contribute to disease progression by generation of further chromosome instability leading to accumulation of alleles carrying pro-oncogenic mutations and loss of alleles containing normal tumor suppressor genes and thus accelerating complex genomic changes associated with CML BC.

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High-Resolution Genomic Scan for Cryptic Chromosomal Lesions in MDS and AML.

Blood ◽

10.1182/blood.v104.11.3427.3427 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3427-3427

Author(s):

Christine L. O’Keefe ◽

Evan Howe ◽

Matt E. Kalaycio ◽

Mikkael Sekeres ◽

Anjali Advani ◽

...

Keyword(s):

Copy Number ◽

Chromosomal Abnormalities ◽

Normal Karyotype ◽

Hematologic Malignancies ◽

Comparative Genomic ◽

Telomeric Region ◽

Fluorescent Intensity ◽

Novel Technologies ◽

Healthy Elderly ◽

Chromosomal Changes

Abstract Cytogenetic analysis is of eminent importance for the diagnosis and prognosis of hematologic malignancies. Due to limitations of traditional karyotyping, novel technologies which improve resolution and sensitivity are under development. In array-based comparative genomic hybridization (A-CGH), differentially labeled test and reference DNA samples are hybridized to genomic microarrays. Differences in sequence copy number between the samples are reflected in a shift of the fluorescent intensity. The resolution of A-CGH is limited solely by the number of clones; it is theoretically possible to achieve linear coverage of the chromosomes. The principle of the CGH techniques allows for detection of unbalanced chromosomal changes of the whole genome. These types of genomic aberrations are most common in MDS, but may exist and further subclassify malignancies with defined balanced translocations. In MDS, depending on the study, 40–60% of patients have a normal or non-informative karyotype by traditional methods. It is likely that this number may be reduced if the resolution and sensitivity level is increased. Additionally, diagnosis of patients with known chromosomal abnormalities can be further refined. We first applied A-CGH to the analysis of normal marrow (N=8) to establish whether it will detect chromosomal defects that may acquired and are compatible with normal hematopoiesis. Moreover, defects may be present in healthy elderly. We utilized arrays of up to 2621 clones with a maxium coverage of 1Mb (Vysis, Spectral Genomics). The results were verified by a dye-swap protocol on two arrays per sample. Four controls showed a normal array profile or only changes in clones previously identified as having a polymorphic copy number within the human genome. The remaining controls had changes including a loss of material on 6p (N=1), loss of 6p and 8q material (N=1) and a gain of 4p and loss of 9p sequences (N=1). These changes may reflect unidentified polymorphisms. In contrast, one control had gains of multiple contiguous clones on chromosomes 9, 15 and 22. We also studied the marrow of patients with advanced MDS (N=43) using A-CGH and traditional cytogenetics. The cohort included patients with known singular lesions (N=7) and complex karyotypes (N=1). The remaining patients had either normal or non-informative cytogenetics. For a del 5q patient and a trisomy 21 patient, A-CGH verified the karyotype without identifying further lesions, in a second del 5q patient was a gain of material on 19p, and a monosomy X patient had a gain of 1p36 by CGH. In 3 cases with partially clonal defects, A-CGH did not detect the abnormality. A normal genomic composition was confirmed in a patient with noninformative (N=1) and normal (N=1) karyotypes. Losses of material on 2q and 3q and gains of material on 22q and the 11p telomeric region were identified in a patient with normal cytogenetics, while another "normal" had gains on 2p, 14q and 21q. Additionally, one normal karyotype had loss of chromosome 16 material and one had loss of 6p sequences. This pilot study demonstrates the utility of A-CGH analysis to study chromosomal aberrations in MDS. A-CGH allows for the detection of cytogenetically undetected abnormalities. Analysis of a large number of samples may allow for the detection of consensus defects or global genomic instability with clinical implications.

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A Novel Assay for Development of Chromosomal Lesions in Human CD34+ Cells Following Exposure to Ionizing Radiation Reveals Increased Susceptibility To Acquire Unstable Chromosomal Lesions in CML CD34+ Cells.

Blood ◽

10.1182/blood.v110.11.997.997 ◽

2007 ◽

Vol 110 (11) ◽

pp. 997-997

Author(s):

Sujata Chakraborty ◽

Arjun Sehgal ◽

Stephen Forman ◽

Smita Bhatia ◽

Ravi Bhatia

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Cell Division ◽

Radiation Exposure ◽

Disease Progression ◽

Chromosome Painting ◽

Chromosomal Abnormalities ◽

Disease Evolution ◽

Cell Divisions ◽

Cd34 Cells

Abstract Acquisition of chromosomal lesions likely plays an important role in pathogenesis of primary and therapy-related leukemia, as well as in disease evolution in myeloproliferative and myelodysplastic disorders. However the process of acquisition of chromosomal lesions in hematopoietic stem cells (HSC) is not well understood. Development of persistent chromosomal abnormalities may involve several factors including acquisition of DNA lesions, repair of DNA damage, sensing of nonrepaired or misrepaired lesions and activation of cell cycle checkpoint and apoptotic pathways, and clonal growth advantage conferred by the lesions. In the current study we assessed the frequency, nature and kinetics of chromosomal lesions following exposure to genotoxic agents in normal human HSC and determined whether these were altered in CML, a prototypic HSC malignancy associated with genetic instability and acquisition of new chromosomal abnormalities during disease progression. CD34+ cells were selected from 4 normal donors and 4 newly diagnosed, untreated chronic phase CML patients. Cells were exposed to increasing doses of γ-radiation, cultured with growth factors and metaphase spreads assessed for development of chromosomal lesions. Chromosome painting was performed using chromosomes 1, 3, 5, 7, 11 and 21 probes, representing 32% of genomic DNA, with >100 metaphases scored per dose per time. Radiation exposure resulted in induction of chromosomal lesions in normal CD34+ cells in a dose-dependent manner. Chromosomal lesions were not seen in cells cultured without radiation exposure. The frequency of aberrant metaphases after 72 h culture (shown by cell cycle analysis to represent the first cell division for normal CD34+ cells) was 2.7% with 0.5 Gy, 4.8% with 1.0 Gy, 9.9% with 2.0 Gy and 23.5% with 4.0 Gy exposures. Chromosome aberrations observed at first division included both stable (translocations, insertions) and unstable (excess fragments, dicentrics) lesions. Subsequent results are shown for exposure to 2.0 Gy. The frequency of aberrant metaphases dropped to 5.3% at 144 h (3–4 cell divisions); only stable lesions persisted at this time. In contrast to normal CD34+ cells, first cell division in CML CD34+ cells was seen at 24 h, at which time 15.3% of the metaphases showed aberrations, whereas 11.2% of metaphases showed aberrations after 72 h (3–4 divisions) and 11.2% after 144 h (6–7 divisions). A striking difference between normal and CML cells was persistence of unstable aberrations after several cell divisions in the latter (42.9% unstable lesions present even after 6–7 divisions). These observations suggest impaired ability to sense and eliminate cells with chromosomal lesions or continued generation of such lesions after initial radiation exposure in CML cells. In conclusion we have developed a novel chromosome painting based assay for evaluation of acquisition of chromosomal lesions in primitive hematopoietic cells. We have demonstrated an inherent chromosomal instability that may contribute to clonal evolution and disease progression in CML CD34+ cells. This assay will provide a useful platform for: i) assessment of mechanisms underlying development of chromosomal lesions in response to DNA damage; and ii) assessing susceptibility to genotoxic agents, and allow improved understanding of pathogenesis and disease evolution in myeloid malignancies.

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Genomic Changes Associated with Leukemic Transformation of Myeloproliferative Disorders.

Blood ◽

10.1182/blood.v112.11.3371.3371 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3371-3371

Author(s):

Nils Heinrich Thoennissen ◽

Norihiko Kawamata ◽

Terra L Lasho ◽

Tamara Weiss ◽

Daniel Nowak ◽

...

Keyword(s):

Molecular Mechanisms ◽

Chromosomal Abnormalities ◽

Neutral Loss ◽

Uniparental Disomy ◽

Myeloproliferative Disorders ◽

Lymphoid Cells ◽

Leukemic Transformation ◽

Altered Expression ◽

Snp Chip ◽

Genomic Changes

Abstract Myeloproliferative disorders (MPD) are a group of heterogeneous diseases that include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). They are characterized by increased hematopoiesis leading to elevated numbers of non-lymphoid cells and/or platelets in the peripheral blood. Beside thrombotic and hemorrhagic complications, MPD may evolve into secondary acute myeloid leukemia (sAML). Recently characterized markers suggest an opportunity to diagnose and identify subpopulations of MPD patients. In particular, altered expression and point mutations of PRV-1, MPL and JAK2 were commonly found in MPD, as well as deletions on chromosome 20q (del20q). Acquired uniparental disomy (UPD) on chromosome 1p (1pUPD) and 9p (9pUPD) leading to copy-neutral loss of heterozygosity (LOH) is a further mechanism found in MPD which often leads to homozygous activated mutations of MPL and JAK2, respectively. However, the molecular mechanisms involved in the transformation process to sAML remains unclear. Using standard metaphase cytogenetics (MC), chromosomal abnormalities are found in only a proportion of patients with MPD. We hypothesized that with new precise methods more genomic lesions can be uncovered that may be associated with leukemic transformation. To address this issue, we used 250K single nucleotide polymorphisms (SNP) Chip arrays to study chromosomal lesions in 40 sAML samples from patients who evolved from MPD; 7 had preexisting PV, 25 PMF, and 8 ET. Moreover, 43 additional samples of MPD (10 PV, 17 ET, and 16 PMF) were included in this study. SNP-chip analysis showed major chromosomal changes in almost all the sAML samples including monosomy 16, deletions of 1q-, 3p-, 6p-, 5q-, 7q-, 9q-, 12p-, 6q-, 3q-, 17p-, 19q-, and 20q- as well as trisomy 2, 3, 8, 9, 12, 15, 19, 21, and 22. We validated these data by MC. However, numerous new genomic alterations which contained potentially interesting genes that might contribute to leukemic transformation were detected by SNP Chip Array in patient samples with normal karyotype. Moreover, UPD was very frequent: 44% (19/43) of MPD and 53 % (21/40) sAML samples. 1pUPD occurred in 5 patients with MPD (1PV, 4 PMF; 12 %) compared to 5 patients with sAML (1 PV, 4 PMF; 13 %). 9pUPD was found in 16 MPD patients (8 PV, 7 PMF, 1 ET; 37%) and 6 sAML patients (3 PV, 2 PMF, 1ET; 15 %). All patients with 9pUPD proved to be positive for the JAK2 V617F mutation seen by allele specific PCR. Interestingly, the MPD samples only had UPD on 1p, 9p, and 12q. In contrast, sAML samples showed additional UPD regions on 7q, 11q, 12q, 16p, 17p, 19q, and 21q. Beside the evaluation of the non-matching groups of patients with MPD and sAML, we also evaluated 4 patients during their PMF and sAML stages by SNP Chip. The sAML samples acquired additional genomic changes including trisomy 8, 10, 14, 19, duplication on 3q and 6p, and heterozygote deletion on 18q. In contrast, 1pUPD, 9pUPD, and 12qUPD were detected in both MPD and sAML matching samples, suggesting that these changes do not play an immediate role in causing transformation. In conclusion, we detected chromosomal regions possessing genes which may be involved in the leukemic transformation of MPD patients.

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Multiplex ligation-dependent probe amplification identifies copy number changes in normal and undetectable karyotype MDS patients

10.21203/rs.3.rs-32982/v2 ◽

2020 ◽

Author(s):

Jing Ma ◽

xiaofei Ai ◽

Jinhuan Wang ◽

Limin Xing ◽

Chen Tian ◽

...

Keyword(s):

Copy Number ◽

Chromosome Banding ◽

Chromosomal Abnormalities ◽

Normal Karyotype ◽

Prognostic Information ◽

Significant Difference ◽

Banding Analysis ◽

Chromosome Banding Analysis ◽

Copy Number Changes ◽

Dependent Probe

Abstract Background Chromosomal abnormalities play an important role in classification and prognostication of myelodysplastic syndromes (MDS) patients. However, more than 50% low risk MDS patients harbor a normal karyotype. Recently, multiplex ligation-dependent probe amplification (MLPA) has emerged as an effective and robust method for the detection of cytogenetic aberrations in MDS patients. Methods To characterize the subset of MDS with normal karyotype or failed chromosome banding analysis, we analyzed 144 patient samples with normal karyotype or undetectable through regular chromosome banding, which were subjected to parallel comparison via fluorescence in situ hybridization (FISH) and MLPA. Results MLPA identifies copy number changes in 16.7% of 144 MDS patients and we observed a significant difference in overall survival (OS) (median OS: undefined vs 27 months, p=0.0071) in patients with normal karyotype proved by MLPA, versus aberrant karyotype cohort as determined by MLPA. Interestingly, patients with undetectable karyotype via regular chromosome banding indicated inferior outcome. Conclusion Collectively, MDS patients with normal or undetectable karyotype via chromosome banding analysis can be further clarified by MLPA, providing more prognostic information that benefit for individualized therapy.

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Hidden abnormalities and novel classification of t(15;17) acute promyelocytic leukemia (APL) based on genomic alterations

Blood ◽

10.1182/blood-2007-12-130260 ◽

2009 ◽

Vol 113 (8) ◽

pp. 1741-1748 ◽

Cited By ~ 40

Author(s):

Tadayuki Akagi ◽

Lee-Yung Shih ◽

Motohiro Kato ◽

Norihiko Kawamata ◽

Go Yamamoto ◽

...

Keyword(s):

Acute Promyelocytic Leukemia ◽

Copy Number ◽

Chromosomal Abnormalities ◽

Neutral Loss ◽

Chromosomal Translocation ◽

Promyelocytic Leukemia ◽

Genomic Alteration ◽

Trisomy 8 ◽

Genomic Changes ◽

Normal Copy Number

Abstract Acute promyelocytic leukemia (APL) is a hematopoietic malignant disease characterized by the chromosomal translocation t(15;17), resulting in the formation of the PML-RARA gene. Here, 47 t(15;17) APL samples were analyzed with high-density single-nucleotide polymorphism microarray (50-K and 250-K SNP-chips) using the new algorithm AsCNAR (allele-specific copy-number analysis using anonymous references). Copy-number-neutral loss of heterozygosity (CNN-LOH) was identified at chromosomes 10q (3 cases), 11p (3 cases), and 19q (1 case). Twenty-eight samples (60%) did not have an obvious alteration (normal-copy-number [NC] group). Nineteen samples (40%) showed either one or more genomic abnormalities: 8 samples (17%) had trisomy 8 either with or without an additional duplication, deletion, or CNN-LOH (+8 group); and 11 samples (23%) had genomic abnormalities without trisomy 8 (other abnormalities group). These chromosomal abnormalities were acquired somatic mutations. Interestingly, FLT3-ITD mutations (11/47 cases) occurred only in the group with no genomic alteration (NC group). Taken together, these results suggest that the pathway of development of APL differs in each group: FLT3-ITD, trisomy 8, and other genomic changes. Here, we showed for the first time hidden abnormalities and novel disease-related genomic changes in t(15;17) APL.

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High Mobility Group A1 Chromatin Remodeling Proteins Amplify Inflammatory Networks to Drive Leukemic Transformation in Chronic Myeloproliferative Neoplasia in Humans and JAK2V617F Transgenic Mouse Models

Blood ◽

10.1182/blood-2018-99-119549 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 102-102

Author(s):

Linda Resar ◽

Donna Marie Williams ◽

Lingling Xian ◽

Wenyan Lu ◽

Briyana Chisholm ◽

...

Keyword(s):

Disease Progression ◽

Mouse Models ◽

Cell Lines ◽

Molecular Mechanisms ◽

Immune Cell ◽

Cell Trafficking ◽

Murine Models ◽

Rna Seq ◽

Leukemic Transformation ◽

Cd34 Cells

Abstract Introduction: Myeloproliferative neoplasms (MPN) are clonal hematopoietic stem cell (HSC) disorders characterized by overproduction of mature blood cells and increased risk of transformation to myelofibrosis (MF) and acute myeloid leukemia (AML), although molecular mechanisms driving disease progression remain elusive. While most patients who acquire a JAK2V617F mutation in CD34+ cells present with chronic, indolent Polycythemia Vera (PV), ~25% will progress to MF or AML. High Mobility Group A1/2 (HMGA1/2) genes encode oncogenic chromatin remodeling proteins which are overexpressed in aggressive leukemia where they portend adverse outcomes. In murine models, Hmga1/2 overexpression drives clonal expansion and uncontrolled proliferation. HMGA1/2 genes are also overexpressed in MPN with disease progression. We therefore sought to: 1) test the hypothesis that HMGA proteins are required for leukemic transformation and rational therapeutic targets in MPN progression, and, 2) identify mechanisms mediated by HMGA1/2 during disease progression. Methods: We measured HMGA1/2 in JAK2V617F mutant human AML cell lines from MPN patients (DAMI, SET-2), CD34+ cells from PV patients during chronic and transformation phases, and JAK2V617F transgenic murine models of PV (transgenic JAK2V617F) and PV-AML (transgenic JAK2V617F/MPLSV; Blood 2015;126:484). To elucidate HMGA1/2 function, we silenced HMGA1 or HMGA2 via short hairpin RNA in human MPN-AML cell lines (DAMI, SET-2) and assessed proliferation, colony formation, and leukemic engraftment in immunodeficient mice. To further assess Hmga1 function in vivo, we crossed mice with heterozygous Hmga1 deficiency onto murine models of PV and PV-AML. Finally, to dissect molecular mechanisms underlying HMGA1, we compared RNA-Seq from MPN-AML cell lines (DAMI, SET-2) after silencing HMGA1/2 to that of controls and applied Ingenuity Pathway Analysis. Results: HMGA1/2 mRNA are up-regulated in all JAK2V617F-positive contexts, including primary human PV CD34+ cells and total bone marrow from JAK2V617F mouse models for PV compared to controls. Further, there is a marked up-regulation in both HMGA1/2 in CD34+ cells from PV patients after transformation to MF or AML and in leukemic blasts from our PV-AML mouse model compared to PV mice. Overexpression of HMGA1/2 also correlates with clonal dominance of human JAK2V617F-homozygous stem cells and additional mutations of epigenetic regulators (EZH2, SETBP1). Silencing HMGA1 or HMGA2 in human MPN-AML cell lines (DAMI, SET-2) dramatically halts proliferation, disrupts clonogenicity, and prevents leukemic engraftment in mice. Further, heterozygous Hmga1 deficiency decreases splenic enlargement in PV mouse models with advancing age. Moreover, heterozygous Hmga1 deficiency prolongs survival in the transgenic PV-AML murine model with fulminant leukemia and early mortality. PV-AML mice survived a median of 5 weeks whereas PV-AML mice with heterozygous Hmga1 deficiency survive a median of 12 weeks (P< 0.002). The leukemic burden was also decreased in mice with Hmga1 deficiency. Preliminary RNA-Seq analyses from DAMI and SET-2 cells show that HMGA1 drives pathways involved in Th1/Th2 activation, chemotaxis, cell-cell signaling, myeloid cell accumulation and other immune cell trafficking, inflammation, and injury, suggesting that HMGA1 co-opts immune and inflammatory networks to drive tumor progression. Surprisingly, atherosclerosis pathways are also induced by HMGA1. Conclusions: HMGA1/2 genes are overexpressed in MPN with highest levels in more advanced disease (MF, AML) both in primary human tumors and murine models. Strikingly, silencing HMGA1 or HMGA2 halts proliferation and clonogenicity in vitro and prevents leukemic engraftment in vivo. Further, heterozygous Hmga1 deficiency prolongs survival in a murine model of fulminant MPN AML and decreases tumor burdens. Finally, preliminary RNA-Seq analyses suggest that HMGA1 amplifies transcriptional networks involved in immune cell trafficking and inflammation to drive tumor progression. Unexpectedly, HMGA1 also regulates pathways involved in atherosclerosis, implicating HMGA1 as a novel link between clonal hematopoiesis and cardiovascular disease. Our findings further highlight HMGA1/2 as a key molecular switch for leukemic transformation in MPN and opens the door to novel therapeutic approaches to prevent disease progression. Disclosures No relevant conflicts of interest to declare.

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Multiplex ligation-dependent probe amplification identifies copy number changes in normal and undetectable karyotype MDS patients

Annals of Hematology ◽

10.1007/s00277-021-04550-8 ◽

2021 ◽

Author(s):

Jing Ma ◽

Xiaofei Ai ◽

Jinhuan Wang ◽

Limin Xing ◽

Chen Tian ◽

...

Keyword(s):

Copy Number ◽

Chromosome Banding ◽

Chromosomal Abnormalities ◽

Normal Karyotype ◽

Prognostic Information ◽

Significant Difference ◽

Banding Analysis ◽

Chromosome Banding Analysis ◽

Copy Number Changes ◽

Dependent Probe

AbstractChromosomal abnormalities play an important role in classification and prognostication of myelodysplastic syndrome (MDS) patients. However, more than 50% of low-risk MDS patients harbor a normal karyotype. Recently, multiplex ligation-dependent probe amplification (MLPA) has emerged as an effective and robust method for the detection of cytogenetic aberrations in MDS patients. To characterize the subset of MDS with normal karyotype or failed chromosome banding analysis, we analyzed 144 patient samples with normal karyotype or undetectable through regular chromosome banding analysis, which were subjected to parallel comparison via fluorescence in situ hybridization (FISH) and MLPA. MLPA identifies copy number changes in 16.7% of 144 MDS patients, and we observed a significant difference in overall survival (OS) (median OS: undefined vs 27 months, p=0.0071) in patients with normal karyotype proved by MLPA versus aberrant karyotype cohort as determined by MLPA. Interestingly, patients with undetectable karyotype via regular chromosome banding indicated inferior outcome. Collectively, MDS patients with normal or undetectable karyotype via chromosome banding analysis can be further clarified by MLPA, providing more prognostic information that benefit for individualized therapy.

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Prognostic implications of morphology and karyotype in primary myelodysplastic syndromes

Blood ◽

10.1182/blood.v67.6.1765.bloodjournal6761765 ◽

1986 ◽

Vol 67 (6) ◽

pp. 1765-1772 ◽

Cited By ~ 2

Author(s):

RH Jacobs ◽

MA Cornbleet ◽

JW Vardiman ◽

RA Larson ◽

MM Le Beau ◽

...

Keyword(s):

Median Survival ◽

Myelodysplastic Syndromes ◽

Chromosomal Abnormalities ◽

Bone Marrow Cells ◽

Normal Karyotype ◽

Refractory Anemia ◽

Survival Times ◽

Trisomy 8 ◽

Leukemic Transformation ◽

Abnormal Karyotype

Forty-nine patients with primary myelodysplastic syndromes (MDS) were subclassified according to French-American-British (FAB) Cooperative Group criteria. Eight patients had acquired idiopathic sideroblastic anemia (AISA), ten had chronic myelomonocytic leukemia (CMMoL), 14 had refractory anemia (RA), nine had refractory anemia with excess blasts (RAEB), and five had refractory anemia with excess blasts in transformation (RAEB-T); three patients could not be subclassified. The actuarial median survival for patients with AISA or with RA had not been reached at 60 months of follow-up. The median survival times for patients with CMMoL, RAEB, and RAEB-T were 25, 21, and 16 months, respectively. The percentages of patients with each subtype who developed ANLL were none in AISA, 20% in CMMoL, 7% in RA, 56% in RAEB, and 40% in RAEB-T. Patients with CMMoL had a poor prognosis independent of transformation to acute nonlymphocytic leukemia (ANLL), whereas patients with RAEB and RAEB-T had a high incidence of transformation and short survival times. Clonal chromosomal abnormalities were present in bone marrow cells from 19 patients at the time of diagnosis, and two others developed an abnormal karyotype at the time of leukemic transformation. The most frequent abnormalities, including initial and evolutionary changes, were trisomy 8 (9 patients), deletion of 5q (4 patients), and deletion of 20q (4 patients). The median survival times were 32 months for patients with an abnormal karyotype, and 48 months for those with a normal karyotype (P = 0.2). Specific chromosomal abnormalities were not associated with particular histologic subtypes; however, a high percentage of patients with RAEB and RAEB-T had an abnormal clone (89% and 80%, respectively). The percentages of patients with clonal abnormalities were 13% for AISA, 20% for CMMoL, and 29% for RA. The MDS transformed to ANLL in 42% of patients with an abnormal karyotype, compared to 10% of those with an initially normal karyotype (P less than .01). Among patients with RA, RAEB, and RAEB-T, the risk of leukemic transformation was confined to those with an abnormal karyotype (P less than .01). Thus, in the present study, morphology and karyotype combined were the best indicators of outcome in patients with MDS.

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