Chromosomal Abnormalities in MDS Are Linked to Dysregulation of CDC20 and CEP55 Genes

Myelodysplastic syndrome (MDS) is a clonal hematopoietic disorder characterized by cytopenias and an increased risk of progression to acute myeloid leukemia (AML). Its pathogenesis is strictly linked to chromosomal instability, which in turn provides a valuable prognostic marker. Malignant cells develop alternative routes to escape mitosis checkpoints, overcoming the mitotic arrest imposed by Spindle Assembly Checkpoint (SAC), a process dependent on CDC20 inactivation. Abnormal levels of CDC20 can inhibit mitotic arrest, promoting premature exit from mitosis. Overexpression of CEP55 also facilitates the mitotic exit, resulting in polyploidy (an event called Mitotic Slippage). Since chromosomal abnormalities are one of the most important prognostic factors for patients with MDS, this study aimed to analyze the possible link between chromosomal abnormalities and CDC20 and CEP55 mRNA expression in MDS. We evaluated the bone marrow cells from 45 patients diagnosed as MDS according to 2016 WHO-classification (1 MDS-SLD, 15 MDS-RS-MLD, 5 MDS-MLD, 1 t-MDS, and 23 MDS-EB) and 5 bone marrow of healthy controls. Conventional Karyotyping was performed by G-banding of 20 metaphases whenever possible. TaqMan expression assays for CDC20 (Hs00426680_mH) and CEP55 (Hs01070181_m1) were performed in duplicate and the expression ratios were calculated using the 2−ΔCq method. Normality was evaluated by Shapiro-Wilk test. Outliers were removed. The Student's t-test or one-way ANOVA with Tukey/Games Howell post-hoc test was used to analyze the influence of relative expression regarding variables. Patients with MDS showed increased expression of CDC20 and CEP55 compared to healthy individuals (p<0.0001 and p<0.0001). Regarding karyotype, there was the overexpression of CDC20 and CEP55 in patients with altered karyotype and aneuploid karyotype when compared to patients with normal karyotype (p <0.0001 and p =0.001; p = 0.013 and p = 0.022, respectively) (Figure 1A-D). CDC20 and CEP55 have fundamental functions in controlling the progression of metaphase to anaphase and both, when upregulated, induce chromosomal instability. Additionally, patients with del(7q) and complex karyotype showed hyperexpression of CEP55 when compared with patients with normal karyotype (p = 0.005 and p = 0.019) (Figure 1E-F), while patients with deletion (5q) had an increased expression of CDC20 when compared with patients with normal karyotype (p <0.0001). Our group previously demonstrate that high CDC20 protein expression is associated with complex karyotype in MDS patients. Thus, we hypothesized that the deregulation of CDC20 and CEP55 expression induces chromosomal changes, each one in its way. Both can cause disturbances in crucial phases of mitosis (anaphase and cytokinesis, respectively). Finally, we detected a strong correlation between CDC20 and CEP55 (r = 0.646; p <0.0001), suggesting both genes may play a synergistic role during chromosomal abnormalities in MDS, creating possible new targets to be evaluated in MDS. Our data suggest CDC20 and CEP55 as possible new therapeutic targets in MDS. There is a need for further studies, validations and urgent in-depth investigations in cell lines/primary samples or murine models. Disclosures No relevant conflicts of interest to declare.

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Chromosomal Instability: A Probable Unfavorable Prognostic Factor For Patients Of Myeloidysplastic Syndromes

Blood ◽

10.1182/blood.v122.21.5243.5243 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5243-5243

Author(s):

Yajuan Xiao ◽

Yuanlu Huang ◽

Na Xu ◽

Rong Lin ◽

Xuan Zhou ◽

...

Keyword(s):

Chromosomal Instability ◽

Chromosomal Abnormalities ◽

Conflicts Of Interest ◽

Normal Karyotype ◽

Chromosome 7 ◽

Complex Karyotype ◽

Karyotypic Analysis ◽

Hematopoetic Stem Cell ◽

Significant Difference

Abstract Objective Myelodysplastic syndromes (MDS) are a group of heterogeneous hematopoetic stem cell clonal disorders with a high frequency of karyotypic abnormalities (40-60%). Among karyotypic abnormalities, abnormal chromosome numbers (aneuploidy) occurs frequently. In aneuploidy, chromosomal instability (CIN) is defined as persistent mis-segregation of whole chromosomes and is caused by defects during mitosis with an odd number of chromosomes. CIN is associated with tumor heterogenesis, multidrug resistance and aggressiveness in solid tumor. Hence, we performed a one-center study on MDS patients to uncover the role of CIN in MDS clinical development. Method A total of 104 cases , 62 male and 42 female, aged from 15 years to 89 years, were tested by fluorescent in situ hybridization (FISH) and karyotypic analysis before any therapeutic intervention. According to the cytogenetic analysis of those two technology they were separated into 5 groups including: CIN, normal karyotype, complex karyotype excluding CIN, deletion chromosome 7 abnormality and other chromosomal abnormalities. All cases were followed up for a median of 19.5 months. Results Karyotyping and FISH identified 70 (67.3%) patients with abnormal karyotypes containing 32 cases of CIN, 9 cases of deletion chromosome 7 abnormality and 5 cases of complex karyotype excluding CIN. The median survival for CIN group was 13 months (incredible interval:6-20 months) compared with 23 months (incredible interval :20-27 months) in all cases, 44months in normal karyotype, 23 months in deletion chromosome 7 abnormality and 13 months in complex karyotype excluding CIN group (P=0.001 for log rank method). In CIN group, 11 cases transformed into acute leukemia with a incidence of 34% with no significant difference with total cases. And the length of time for leukemia transformation shows no significant difference between CIN group and total cases. Conclusion Chromosomal instability in MDS patients of the study revealed worst prognosis compared with other groups. This may suggest that chromosomal instability in MDS chromosomal abnormality confer a significant independent adverse impact on patients survival. However this effect might have no relation to leukemia transformation. Disclosures: No relevant conflicts of interest to declare.

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Spectral Karyotyping (SKY) Reveals a New Subset of MDS Patients with Clonal Chromosomal Abnormalities Not Detected by G-Banding Analysis

Blood ◽

10.1182/blood.v118.21.1718.1718 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1718-1718

Author(s):

Fabio Morato Oliveira ◽

Maria do Carmo Favarin ◽

Rodrigo T Calado ◽

Ana Paula N Rodrigues Alves ◽

Cassia Godoi ◽

...

Keyword(s):

Bone Marrow ◽

Cytogenetic Analysis ◽

Chromosomal Abnormalities ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Calf Serum ◽

Fluorescent Labeling ◽

Refractory Anemia ◽

Spectral Karyotyping ◽

Banding Analysis

Abstract Abstract 1718 Cytogenetic findings in bone marrow cells of MDS patients are essential for a correct diagnosis and classification of the disease and constitute one of the most important independent prognostic factors. The classical cytogenetic analysis, however, often cannot be fully resolved by G-banding because of the presence of marker chromosomes, rings or unidentified material attached to chromosomes. Spectral karyotyping (SKY) has proven to be an important tool for the interpretation of complex karyotypes or identification of suitable abnormalities in hematological malignancies. By using SKY analysis in combination with G-banding were identified new clonal chromosomal abnormalities “masked” by the limited resolution of classical cytogenetic. As a consequence changes in IPSS score were observed. Bone marrow samples of 46 (forty-six) MDS patients were incubated in RPMI 1640 with 20% fetal calf serum for 72h at 37°C. Chromosome preparations were obtained by using standard procedures and the subsequent cytogenetic analysis and interpretation were made according to ISCN 2009. The patients studied were classified as refractory anemia (RA) and refractory anemia with ringed sideroblast (RARS), with less than 5% blast. Slides for SKY were prepared by using the same fixed chromosome preparations, stored at −20°C, as employed for G-banding analysis. Chromosome labeling was performed with the SKY fluorescent labeling kit (Applied Spectral Imaging, Migdal HaEmek, Israel) according to the manufacturer's protocol. A minimum of twenty metaphases were analyzed using the SkyView 5.5 software (ASI, Carlsbad, CA, USA). In a group of 46 subjects studied, the cytogenetic analysis (G-banding) showed chromosomal aberrations in 13 patients (54.2%) and normal karyotype was observed in 11 subjects (45.8%). The abnormalities observed were dup(1)(q21q32), inv(3)(q21q26), t(3;3)(q21;q26), +4, del(5)(q31), −7, del(7)(q22q36), +8, add(17)(p12), +i(17)(q10), del(20)(q11). The group with normal cytogenetic, SKY analysis revealed “masked” chromosomal abnormalities in 6 patients, being t(7;9)(q36;q34), ins(1;6)(q21;?), t(11;12)(p15;q24.1), ins(3;5)(p21;?), t(8;16)(q23;?) and ins(6;11)(q21;?). Among 13 cases studied with previous chromosomal abnormalities by G-banding analysis, SKY identified additional abnormalities in 8 patients. Some abnormalities found include t(6;9)(q27;q22), t(12;17)(p13;p12) and t(8;11)(p12;q12). For both groups with normal and altered karyotypes, the profile of masked chromosomal abnormalities seen were insertions and translocations involving small segments of chromosomes. In the majority of the cases the frequency of abnormal clones was less than 50%. However, in all patients the abnormalities identified by SKY were classified as clonal. All abnormalities identified were confirmed by FISH, by using a set of probes. SKY analysis has proved to be a promising and reliable method for identification of additional and complex chromosomal abnormalities usually present in a great number of human neoplasias. The contribution for the prognostic information of these new chromosomal abnormalities identified beyond the limited resolution of G-banding in MDS will require a detail analysis of the patients' evolution. Financial Support: FAPESP (Proc. 07/52462-7) Disclosures: No relevant conflicts of interest to declare.

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Complex Aberrant Karyotype in Patients with Post-Transplant Relapses of Acute Myeloid and Lymphoid Leukemias Evaluated By Serial Cytogenetic Assays, Including mFISH

Blood ◽

10.1182/blood.v124.21.5313.5313 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5313-5313

Author(s):

Tatiana L Gindina ◽

Nikolay N Mamaev ◽

Sergey N Bondarenko ◽

Elena V Semyenova ◽

Natalia V Stancheva ◽

...

Keyword(s):

Bone Marrow ◽

Chromosomal Abnormalities ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Clinical Status ◽

Multicolor Fish ◽

General Group ◽

Post Transplant ◽

Active Disease ◽

Acute Myeloid

Abstract Background. Prognosis of acute myeloid leukemia (AML) with complex aberrant karyotype (CAK) is poor. These patients (pts) are often refractory to both chemotherapy and allogeneic HSCT (alloHSCT). At diagnosis the proportion of these pts is about 6-8 %, but their frequency essentially increases in relapses after chemotherapy and alloHSCT. Mechanism of CAK development and its effect on relapses have been studied insufficiently. Material and methods. Serial cytogenetic assays, including multicolor FISH, were performed on bone marrow cells from 99 patients with post-transplant relapses (PTR) AML (n=61) and ALL (n=38). Median ages for AML and ALL pts at HSCT were 23 and 17 years (range, 2-60 and 0.8-51 years), respectively. Results. Aberrant karyotypes were found in 90 % AML and 97 % ALL pts, respectively (Table 1). Of note, the proportion of CAK in general group of ALL patients, as well as in that with more 4 chromosomal abnormalities was significantly higher, compared to AML (66% vs. 36%, P=0.003 and 61% vs. 33%, P=0.006, respectively). Table 1. Frequency and characteristics CAKs in AML and ALL patients with the PTRs Leukemia type AML ALL P Patients, n 61 38 Karyotype, n (%) Normal 6 (10 %) 1 (3 %) NS Abnormal 55 (90 %) 37 (97 %) NS Complex karyotype, n (%) 22 (36 %) 25 (66 %) 0.003 3 abnormalities 2 (3 %) 2 (5 %) NS ³4 abnormalities 20 (33 %) 23 (61%) 0.006 NS - not significant The above difference in frequency of CAK in ALL and AML might be explained by higher frequency of myeloablative conditioning regimes in the ALL group, compared to AML (55% vs. 36 %, respectively; P=0.09). Of notice also, that fraction of CAK with 4 and more chromosomal abnormality in the group of children from 1 to 18 years was significantly higher in patients with ALL, as compared with AML (60% vs. 30%, respectively; P=0.03). Furthermore, a similar tendency was revealed also in the group of patients aged 19-40 years (difference insignificant; P=0.08) (Table 2). Table 2. The incidence of CAKs with ³4 chromosomal abnormalities in PTRs in different age groups. Patients, n (%) Age (years) AML ALL P 1-18 7/23 (30 %) 15/25 (60 %) 0.03 19-40 8/25 (32 %) 7/11 (64 %) 0.08 41-60 5/13 (39 %) 1/2 (50 %) 0.5 Finally, our data demonstrate, the proportion of CAK to be significantly higher in PTR of ALL pts compared with those of AML, when they were transplanted in active disease phase (70% vs. 32%; P=0.007; Table 3). Table 3. The incidence of CAKs with ³4 chromosomal abnormalities in PTRs, depending on the clinical status at alloHSCT. Patients, n (%) P Clinical status at HSCT AML ALL CR 1 3/13 (23 %) 2/7 (29 %) 0.6 CR ³2 6/14 (43 %) 7/11 (64 %) 0.2 Active disease 11/34 (32 %) 14/20 (70 %) 0.007 Discussion. Our study shows that number of karyotype abnormalities in acute leukemia with CAK+ is closely associated with previous chemotherapy and/or conditioning regimes. Despite it, only alloHSCT gives a hope for treatment of these pts. In order to improve overall results of treatment several modifications of alloHSCT and post-transplant treatment have been recently suggested. The main of them, called as early alloHSCT, includes modified FLAMSA-RIC conditioning regime, alloHSCT before obtaining results of standard courses chemotherapy followed by DLI in escalating doses at post-transplant period (Schmid et al., 2012). If such a possibility for early alloHSCT is lost, another variant of PTR prevention may be used. The latter can include the same DLI, hypomethylating agents, ATRA etc. As for PTR prevention in CAK+ ALL it may be different from AML. In our opinion, in these pts should be treated at the first with such target agents as tyrosine-kinase inhibitors, rituximab, ATRA etc. Conclusion. According to our data, the frequency of CAK in PTRs is high not only in AML, but ALL too. Mechanisms of CAK formation as well as treatment of CAK+ leukemia began to elucidate. In our opinion, the leading place in this treatment is to be given an early alloHSCT. Reference. Schmid C., et al. Early allo-SCT for AML with a complex aberrant karyotype – results from prospective pilot study. Bone Marrow Transplantation 2012; 47: 46-53. Disclosures No relevant conflicts of interest to declare.

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A Cytoplasmic FANCA-FANCC Complex Interacts and Stabilizes the Leukemic NPMc Protein.

Blood ◽

10.1182/blood.v114.22.3098.3098 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3098-3098

Author(s):

Wei Du ◽

Suzette Maynard ◽

Jared Sipple ◽

Jie Li ◽

Qishen Pang

Keyword(s):

Bone Marrow ◽

Nuclear Export ◽

Biological Function ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Nuclear Export Signal ◽

Normal Karyotype ◽

Present Evidence ◽

Normal Human ◽

Export Signal

Abstract Abstract 3098 Poster Board III-35 In a subset of AML with a normal karyotype, a frame-shift mutation in the extreme C-terminal of the nucleophosmin (NPM) gene results in the creation of a nuclear export signal, generating a mutant NPM protein (NPMc) that is permanently dislocated in the cytoplasm. In the present study, we have analyzed the interaction between NPMc and a cytoplasmic subcomplex of Fanconi anemia (FA) proteins. Sequence analysis of bone marrow samples from 46 FA patients shows that NPMc mutations were excluded from FA genome. NPMc was degraded more rapidly in AML bone marrow cells from FA patients (t1/2 < 30 min) than in AML cell line HL60 (t1/2 > 90 min). Further analysis revealed that inducible knockdown of FANCA or FANCC in leukemic OCI/AML3 cells carrying the NPMc mutation induced degradation of the cytoplasmic NPMc protein. Forced localization of FANCC to the nucleus also caused rapid NPMc degradation. We also show that NPMc degradation was mediated by the proteasome and that correction of mutant lymphoblasts from FA-A or FA-C patients with a functional FANCA or FANCC protein prevented NPMc ubiquitination and consequently degradation. Moreover, we demonstrate that the cytoplasmic FANCA and FANCC interacted with NPMc in the cytosolic fractions of normal human lymphoblaststic cells and that the acidic domains of NPM were required for the cytoplasmic FA-NPMc complex formation. Finally, using patient-derived FANCC mutant, a nuclearized FANCC and a NOG/SGM3 xenotansplant model, we present evidence that the cytoplasmic FANCA-FANCC complex was essential for NPMc stability and biological function. Thus, these findings reveal the potential molecular mechanism involved in the cytoplasmic retention of the leukemic NPMc. Disclosures No relevant conflicts of interest to declare.

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Study On the Clonal Origin of Dysplastic Cells by Fluorescence in Situ Hybridization in Myelodysplastic Syndromes.

Blood ◽

10.1182/blood.v114.22.4858.4858 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4858-4858

Author(s):

Jun Zhang ◽

Yongquan Xue ◽

Jinlan Pan ◽

Yafang Wu ◽

Juan Shen ◽

...

Keyword(s):

Bone Marrow ◽

In Situ Hybridization ◽

Fluorescence In Situ Hybridization ◽

Myelodysplastic Syndromes ◽

Chromosomal Abnormalities ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Banding Technique ◽

Clonal Origin

Abstract Abstract 4858 Objective To determining the clonal origin of dysplatic cells in Myelodysplastic syndromes (MDS) . Methods Karyotypic analyses of bone marrow cells using R-banding technique were carried out to determine the chromosomal abnormalities. Interphase fluorescence in situ hybridization (FISH) and morphologic analysis of bone marrow aspirates were performed in the same cells to investigate the clonal origin of dysplatic cells in 8 MDS patients. Result All patients had clonal karyotypic abnormalities: simple abnormality in 1 patient, complex abnormalities in 6 patients, coexistent of two unrelated clones in 1 patient. Most of dysplastic cells in 7 of 8 MDS patients derived from neoplasia clone while 1 patient had a reverse result,no matter what cell lineage was involved. Some of non-dysplastic cells of all patients derived from malignant clone; in 7 patients, the proportion of dysplastic cells in malignant clone were significantly higher than that of non- malignant clone. Conclusion Most of dysplastic cells in MDS derived from malignant clones, while the minority of them derived from non-malignant clones. Thus, it is reasonable to expect that in most cases myelodysplasia is present in malignant clone and can be taken as an important diagnostic evidence for MDS. Disclosures No relevant conflicts of interest to declare.

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Elevation of Mitochondrial DNA Copy Number in Bone Marrow Cells Associated with Increased Risk of Childhood Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v120.21.1423.1423 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1423-1423

Author(s):

Yu-Na Kim ◽

Hye-Ran Kim ◽

Hwan-Young Kim ◽

Trang Nguyen Thi Dai ◽

Dong-Kyun Han ◽

...

Keyword(s):

Bone Marrow ◽

Copy Number ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Normal Karyotype ◽

Mtdna Copy Number ◽

Increased Risk ◽

Childhood Acute Myeloid Leukemia ◽

Childhood Aml ◽

Marrow Cells

Abstract Abstract 1423 Background: Alteration of mitochondrial DNA (mtDNA) is now implicated in tumorigenesis as well as poor clinical outcomes. mtDNA copy number is altered in patients with various hematological malignancies such as lymphocytic leukemia, lymphoma and other disorders. Moreover, recent studies has reported that mtDNA copy number is associated with increased risk of non-hodgkin lymphoma (Blood 2008;112:4247–9). Therefore, the present study was fist to validate the hypothesis that alteration of mtDNA copy number in bone marrow cell would be associated with risk of childhood acute myeloid leukemia (AML). Patients and Methods: Direct sequencing of mtDNA control region, analysis of mtDNA minisatellites and mtDNA copy number were performed in 48 patients with initially diagnosed childhood AML cases and 48 matched case controls from Environmental Health Center for Childhood Leukemia and Cancer cohort. Molecular analysis of mtDNA including quantitation of mtDNA copy number was determined using our published protocol (Int J Cancer 2012;131:1332–41). Results: Numerous sequence alterations of mtDNA were only found in patient's bone marrow cells. Instability of mtDNA minisatellites was mainly occurred in the 303 polyC and 16184 polyC minisatellite. mtDNA copy number was significantly elevated in patients with childhood AML compared with those of control group (p=0.000075, Fig. 1). There was a dose-response relationship between tertiles of mtDNA copy number and risk of childhood AML (odds ratio [OR], 95% confidence interval [CI]: 1.0; 1.145 [0.412–3.183]; and 6.818 [2.244–20.712], respectively; p(trend) =.000). The effect was most pronounced for childhood AML harboring normal karyotype (OR: 1.0; 1.75[0.369–8.302]; 9.00 [1.888–42.904]; p(trend) =.008) (Table 1). Conclusion: mtDNA minisatellite inability was frequently observed in bone marrow cells of childhood AML. Elevation of mtDNA copy number could be associated with increased risk of childhood AML, particularly normal karyotype childhood AML. Disclosures: No relevant conflicts of interest to declare.

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Complex Karyotype in Mantle Cell Lymphoma (MCL) Is Associated with Inferior Progression-Free Survival in Patients (pts) Receiving R-Hypercvad Followed By Autologous Stem Cell Transplant

Blood ◽

10.1182/blood.v126.23.5095.5095 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5095-5095

Author(s):

Michael Jinpyo Lee ◽

Jeffrey Switchenko ◽

Janeen S Thomas ◽

Jean L. Koff ◽

Loretta Nastoupil ◽

...

Keyword(s):

Bone Marrow ◽

Research Funding ◽

Chromosomal Abnormalities ◽

Normal Karyotype ◽

Progression Free Survival ◽

Complex Karyotype ◽

Abnormal Karyotype ◽

Free Survival ◽

The Impact

Abstract Introduction: While cytogenetic assessments are frequently utilized in many hematologic malignancies as prognostic markers, their role in MCL is less well defined. Prior studies have identified a complex karyotype (≥3 chromosomal abnormalities) in 40 - 59.2 % of patients (pts) with MCL, and it has been associated with inferior progression-free survival (PFS) in those series (Cohen et al, 2015; Sarkozy et al, 2014). Our group recently published outcomes for MCL pts at our institution, including 35 pts who received R-HyperCVAD followed by autologous stem cell transplantation (ASCT) who had an overall survival (OS) at 5-years of 74% (Nastoupil et al, 2014). We conducted an analysis of the impact of pretreatment cytogenetics on outcomes for MCL. Methods: We included all pts diagnosed with MCL at Winship Cancer Institute of Emory University between 2002 and 2014 who had available pretreatment conventional cytogenetic and clinical data. Baseline demographic, clinical, laboratory, and therapeutic variables of interest were assessed. Univariate Cox proportional hazards models were fit for each covariate, and hazard ratios are reported. Overall survival (OS) was defined as time to death or last follow-up from diagnosis, and PFS was defined as time to progression, death, or last follow-up from diagnosis. These curves were estimated via the Kaplan-Meier method, and the groups were compared using log-rank tests. We performed a similar analysis for the subset of pts initially treated with HyperCVAD and ASCT. Statistical significance was defined as α=0.05. Results: Among 184 MCL pts, 52 had pretreatment cytogenetics data available. The median age at diagnosis was 64 years (range: 32-81), 36 (69%) were male, and 37 (86%) had stage IV disease. At diagnosis, 36 pts (97%) had ECOG performance status of 0 or 1, 16 (34%) had B-symptoms, 43 (86%) had bone marrow involvement, 8 (16%) had gastrointestinal tract involvement, and 29 (71%) had splenomegaly. Initial induction therapies included R-HyperCVAD (n=27), R-CHOP (n=10), R-bendamustine (n=6), and other/unknown (n=9). Thirty pts (58%) underwent ASCT in first complete or partial remission. Five pts (10%) met criteria for a complex karyotype, 11 (21%) had 1 or 2 detectable chromosomal abnormalities, and 36 (69%) pts had a normal karyotype. Cytogenetics were assessed on nodal samples (n=7) or bone marrow/peripheral blood (n=45). Among all assessed covariates, only elevated WBC count was associated with cytogenetic abnormalities (p=0.04). Overall, there was no significant association of PFS or OS with the presence of a complex karyotype or an abnormal karyotype with < 3 abnormalities when compared to a normal karyotype. Among all assessed variables, only splenomegaly was associated with worsened PFS (p=0.02). We conducted an exploratory analysis of the 24 pts who received R-HyperCVAD followed by ASCT, of which 2 had a complex karyotype. In that subset, median PFS for pts with a complex karyotype was 35.3 months, compared to 77.9 months for those with a non-complex abnormal karyotype or a normal karyotype. Conclusion: Contrary to prior reports, a smaller percentage of pts had complex karyotype (10%) in this cohort, and pretreatment cytogenetics did not impact PFS or OS among the entire heterogeneously treated sample. However, there appears to be inferior PFS for pts with a complex karyotype in the subset who received R-HyperCVAD followed by ASCT, and a larger, multi-center series, may illuminate the impact of a complex karyotype on this subset of aggressively treated patients. Disclosures Nastoupil: AbbVie: Research Funding; Genentech: Honoraria; Celgene: Honoraria; TG Therapeutics: Research Funding; Janssen: Research Funding. Flowers:Infinity Pharmaceuticals: Research Funding; Spectrum: Research Funding; Acerta: Research Funding; Janssen: Research Funding; Infinity Pharmaceuticals: Research Funding; Onyx Pharmaceuticals: Research Funding; Seattle Genetics: Consultancy; Gilead Sciences: Research Funding; Millennium/Takeda: Research Funding; Pharmacyclics: Research Funding; Janssen: Research Funding; Gilead Sciences: Research Funding; Acerta: Research Funding; AbbVie: Research Funding; Pharmacyclics: Research Funding; Onyx Pharmaceuticals: Research Funding; Millennium/Takeda: Research Funding; OptumRx: Consultancy; AbbVie: Research Funding; Seattle Genetics: Consultancy; Genentech: Research Funding; OptumRx: Consultancy; Genentech: Research Funding; Celegene: Other: Unpaid consultant, Research Funding; Celegene: Other: Unpaid consultant, Research Funding; Spectrum: Research Funding. Cohen:Celgene, Pharmacyclics, Millennium, Seattle Genetics: Consultancy; BMS, Janssen: Research Funding.

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Expression of JAK2V617I Does Not Result in Overt Myeloproliferative Disorder, but Results in Cytokine Hypersensitivity

Blood ◽

10.1182/blood.v124.21.3218.3218 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3218-3218

Author(s):

Stefan Brooks ◽

Samuel B Luty ◽

Hew Yeng Lai ◽

Lacey R Royer ◽

Sarah J Morse ◽

...

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Spleen Weight ◽

Hematopoietic Stem ◽

Gm Csf ◽

Empty Vector ◽

Increased Risk ◽

Germline Expression

Abstract BACKGROUND: A germline JAK2V617I mutation has recently been identified in a family with hereditary thrombocytosis (Mead et al, NEJM 2012). Like acquired MPN, family members with JAK2V617I have thrombocytosis and megakaryocytic hyperplasia in the marrow with increased risk of thrombosis. But unlike acquired MPN, individuals with this germline mutation do not develop a fibrotic bone marrow, splenomegaly, or transform to acute leukemia. Why germline JAK2V617I recapitulates some aspects but not others of the MPN phenotype in humans is unclear. To delineate the differences between JAK2V617F and JAK2V617I we compared the phenotype of mice with hematopoietic cells expressing JAK2V617F or JAK2V617I. METHODS AND RESULTS: Lethally irradiated C57B/6 mice were transplanted with bone marrow cells infected with retrovirus expressing JAK2V617F, JAK2V617I, or empty MSCV-IRES-GFP (MIG) vector. As expected, mice transplanted with JAK2V617F-expressing cells developed erythrocytosis and leukocytosis, whereas mice transplanted with JAK2V617I-expressing cells had peripheral blood counts similar to empty vector mice. Humans with germline JAK2V617I do not display constitutive activation of the kinase, but they do demonstrate cytokine hyper-responsiveness as evidenced by increased phosphorylation of STATs at low concentrations of ligand. We compared phosphorylated STAT5 in peripheral blood of mice transplanted with JAK2V617I, JAK2V617F, and MIG empty vector following stimulation with increasing concentrations of GM-CSF. At all concentrations of GM-CSF tested JAK2V617I and JAK2V617F-expressing cells had exaggerated phosphorylation of STAT5 as compared to MIG empty vector mice. We also measured phospho-STAT3 and STAT5 in unstimulated bone marrow and spleen from each mouse at time of euthanasia, there was no difference between JAK2V617I and MIG empty vector mice. JAK2V617F mice did demonstrate phosphorylation of STAT3 and STAT5 in the absence of GM-CSF, confirming the ability of JAK2V617F but not JAK2V617I to constitutively activate downstream signaling pathways. Next, to evaluate for histologic evidence of MPN and assess spleen size, all mice were euthanized at 120 days post-transplant. JAK2V617F mice had splenomegaly as expected, spleens from JAK2V617I mice appeared larger than empty vector mice, but spleen weight was not statistically different (p>0.05). While JAKV617I mice had increased cellularity of their marrow with increased numbers of megakaryocytes as compared to empty vector mice, this was not nearly to the extent of JAK2V617F mice. Mild fibrosis was seen in JAK2V617I mice, JAK2V617F mice had severe reticulin fibrosis in the marrow as expected. In the spleen architecture was preserved in the JAK2V617I mice, whereas in the JAK2V617F mice splenic architecture was disrupted by invasion of myeloid cells including megakaryocytes. To identify whether JAK2V617I affects the frequency of stem and progenitor cells or expands mature myeloid lineage cells we measured the frequency of hematopoietic stem cells, myeloid progenitors, and mature myeloid populations in JAK2V617F, JAK2V617I, and MIG empty vector. The bone marrow of JAK2V617I mice contained an increased percentage of GMP and MEP populations as compared to both the MIG empty vector and the JAK2V617F mice. Mature granulocyte (Gr-1+CD11b+) and erythroid (Ter119+) populations were expanded in the bone marrow and spleen of JAK2V617F but not JAK2V617I mice. CONCLUSIONS: These data demonstrate that the JAK2V617I mouse model recapitulates the effect of germline expression of JAK2V617I seen in humans: it results in cytokine hyper-responsiveness without the ability to constitutively activate downstream signals in the absence of ligand. Why JAK2V617F is so exquisitely conserved in acquired MPN is still unknown. Disclosures No relevant conflicts of interest to declare.

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Clonal Heterogeneity in Patients with Myelodysplastic Syndromes (MDS) and Complex Karyotypes

Blood ◽

10.1182/blood.v124.21.859.859 ◽

2014 ◽

Vol 124 (21) ◽

pp. 859-859

Author(s):

Zuzana Zemanova ◽

Kyra Michalova ◽

Jana Brezinova ◽

Halka Lhotska ◽

Karla Svobodova ◽

...

Keyword(s):

Bone Marrow ◽

Overall Survival ◽

Genomic Instability ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Driving Mechanism ◽

Chromosome 5 ◽

Clonal Heterogeneity ◽

Increased Risk ◽

Marrow Cells

Abstract Introduction: Complex chromosomal aberrations (CCAs) are seen in approximately 20% of patients with newly-diagnosed MDS and are associated with poor prognosis. Bone marrow cells of MDS patients with CCAs are characterized by a high degree of genomic instability, which is connected with an increased risk of formation of different subclones with additional aberrations. The phenomenon of clonal evolution can be observed either at diagnosis or during the disease progression. At diagnosis it is manifested by the presence of two or more related subclones derived from one founder clone. These subclones can obtain a proliferative advantage leading to the clonal expansion. The aim of the study was to perform detailed genome-wide analyses of bone marrow cells of previously untreated MDS patients with CCAs, to investigate the clonal heterogeneity and to assess the frequency and clinical significance of related cytogenetic subclones with complex karyotypes. Methods: A comprehensive molecular cytogenetic analysis of fixed bone-marrow cells from 182 adults with CCAs (³3 aberrations) identified with conventional G-banding in the diagnosis of MDS was performed. The CCAs were studied through FISH with Vysis DNA probes (Abbott, Des Plaines, IL) and mFISH/mBAND methods (MetaSystems, Altlussheim, Germany). Genomic imbalances were identified with CytoChip Cancer SNP 180K (BlueGnome, Cambridge, UK) or with Illumina Human CytoSNP-12 arrays (Illumina, San Diego, CA). Results: Cytogenetic subclones were detected in 143 of 183 patients with complex karyotypes (78.6%). Among them, 98/183 cases (53.8%) displayed a defined number of subclones, whereas in 45/183 patients (24.7%) a precise definition of the individual subclones was not possible due to the high number of heterogeneous findings. Therefore these patients were subsumed as having composite karyotype. In 138/143 cases with clonal heterogeneity (94.9%) subclones showed related karyotypes. Twelve patients had one clone with an interstitial 5q deletion as the sole abnormality and other subclones with additional changes. One of these 12 cases displayed a subclone with complex karyotype in which deleted chromosome 5 was involved in an unbalanced translocation. Five patients (5.1%) had a combination of related clones with del(5q) and additional changes and unrelated subclones without this aberration. Complex aberrations were often associated with the loss of heterozygosity (LOH) of 17p (44%), however a difference in the frequency of LOH 17p in groups with and without clonal heterogeneity was not ascertained (p = 0.782). Moreover, the overall survival (OS) of patients with clonal heterogeneity did not differ from that of patients with one homogeneous complex aberrant clone (p = 0.391). Median OS in both groups was three months only. Conclusions: The results of this study confirmed clonal heterogeneity at the cytogenetic level as a frequent finding in patients with MDS and complex karyotypes. Although clonal diversity has been implicated as a driving mechanism of tumor development and progression and is usually associated with more aggressive disease, in this cohort of MDS cases with complex karyotypes, the presence of different subclones did not affect patients’ overall survival. It seems that the dismal prognosis of patients with CCAs is generally correlated with overall genomic instability and the complexity of aberrations. This may be manifested at different genetic levels by various phenomena such as increasing number of aberrations, accumulation of gene mutations and/or losses of heterozygosity of tumor-suppressor genes (including LOH TP53), involvement of deleted chromosome 5 in unbalanced rearrangements, extensive chromosome shattering, and clonal heterogeneity as well. Supported by RVO-VFN64165, GACR P302/12/G157/1, PRVOUK-P27/LF1/1, and MHCR 00023736. Disclosures No relevant conflicts of interest to declare.

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Tissue methylation and demethylation influence translesion synthesis DNA polymerases (TLS) contributing to the genesis of chromosomal abnormalities in myelodysplastic syndrome

Journal of Clinical Pathology ◽

10.1136/jclinpath-2020-207131 ◽

2020 ◽

pp. jclinpath-2020-207131

Author(s):

Gabrielle Melo Cavalcante ◽

Daniela Paula Borges ◽

Roberta Taiane Germano de Oliveira ◽

Cristiana Libardi Miranda Furtado ◽

Ana Paula Negreiros Nunes Alves ◽

...

Keyword(s):

Dna Methylation ◽

Bone Marrow ◽

Myelodysplastic Syndrome ◽

Chromosomal Abnormalities ◽

Dna Polymerases ◽

Translesion Synthesis ◽

Normal Karyotype ◽

Dna Demethylation ◽

Repair System ◽

Low Expression

AimsDNA methylation has its distribution influenced by DNA demethylation processes with the catalytic conversion of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC). Myelodysplastic syndrome (MDS) has been associated with epigenetic dysregulation of genes related to DNA repair system, chronic immune response and cell cycle.MethodsWe evaluated the tissue DNA methylation/hydroxymethylation in bone marrow trephine biopsies of 73 patients with MDS, trying to correlate with the mRNA expression of 21 genes (POLH, POLL, REV3L, POLN, POLQ, POLI, POLK, IRF-1, IRF-2, IRF-3, IRF-4, IRF-5, IRF6, IRF-7, IRF-8,IRF-9, MAD2, CDC20, AURKA, AURKB and TPX2).ResultsThe M-score (5mC) was significantly higher in patients with chromosomal abnormalities than patients with normal karyotype (95% CI –27.127779 to –2.368020; p=0.022). We observed a higher 5mC/5hmC ratio in patients classified as high-risk subtypes compared with low-risk subtypes (95% CI –72.922115 to –1.855662; p=0.040) as well as patients with hypercellular bone marrow compared with patients with normocellular/hypocellular bone marrow (95% CI –69.189259 to –0.511828; p=0.047) and with the presence of dyserythropoiesis (95% CI 17.077703 to 51.331388; p=0.001). DNA pols with translesion activity are significantly influenced by methylation. As 5mC immunoexpression increases, the expressions of POLH (r=−0.816; r2 =0.665; p=0.000), POLQ (r=−0.790; r2=0.624; p=0.001), PCNA (r=−0.635; r2=0.403; p=0.020), POLK (r=−0.633; r2=0.400; p=0.036 and REV1 (r=−0.578; r2=0.334; p=0.049) decrease.ConclusionsOur results confirm that there is an imbalance in the DNA methylation in MDS, influencing the development of chromosomal abnormalities which may be associated with the low expression of DNA polymerases with translesion synthesis polymerases activity.

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