Eradication and Reduction of the Abnormal Chromosome 7 Clone with Azacitidine (AZA C) Is Achievable for One-Third of Patients (pts) with Myelodysplastic Syndrome (MDS).

Abstract The two most frequent cytogenetic abnormalities in pts with MDS involve rearrangements of chromosomes 5 and 7. Monosomy 7 or deletion 7q, alone or in a complex karyotypes are poor-risk abnormalities and associated with a low response rate to conventional therapies. As a part of our comprehensive longitudinal study of 220 patients treated with AZA C, we asked the question whether pts with −7/del(7q) alone or as a part of the complex clone, may achieve hematological and/or cytogenetic response. A minimum of three follow-up cytogenetic and FISH analyses were required as an inclusion criterion. A normal karyotype was observed in 129 of 229 pts (56%) and 100 pts (44%) had an abnormal karyotype at baseline. Among the 100 pts with an abnormal karyotype 29 pts (29%) had chromosome 7 abnormalities prior to the AZA C treatment. In an additional 12 pts chromosome 7 abnormalities developed during the course of disease and AZA C therapy (range 4 months to 5 years). Response of the abnormal chromosome 7 clone to AZA C therapy was observed in 3 patterns: pts who had −7/del(7q) present at baseline without any further cytogenetic change during the AZA C treatment (20 pts; 69%). Eight of these pts had hematological improvement (HI); Pts who had either reduction or elimination (complete cytogenetic response=CCR) of the abnormal chromosome 7 clone as a results of treatment (9 pts, 31%). Four of 29 pts (14%) had a reduction in the size of the abnormal clone from 100% to a mean of 23% (range 8%–50%) as judged by conventional cytogenetics. The median time to achieve reduction was 6 months (range 2 to 9 months) with median duration of 10.5 months (range 6 to 24 months) during maintenance therapy with AZA C. CCR was observed in 5 of 29 pts (17%) and occurred within a mean of 4.2 months (range 3–6 months). Repeated cytogenetic studies showed a normal karyotype and the CCR lasted a mean of 5.2 months (range 3–9 months) during therapy. FISH studies showed 2–5% cells with −7/del(7q) during the CCR. Four of the 5 pts had HI and one pt had CR. These patients relapsed with the diagnostic −7/del(7q) clone without additional cytogenetic abnormalities. 3) Of the 12 pts who developed −7/del(7q) while on Aza C therapy, 4 pts had a normal karyotype at baseline and developed −7/del(7q) after a mean of 16 months (range 6–39 months) therapy. One had PR and 3 pts had a stable disease. The other 8 pts were cytogenetically abnormal at baseline and developed −7/del(7q) as a subclonal evolution during therapy in 4 pts (range 4 months to 5 years) and following disease progression in 4 pts. Six pts had HI and two had a stable disease. In conclusion, our longitudinal study allowed us to delineate 3 categories of AZA C response to abnormal chromosome 7 clone: 31% had cytogenetic response: 17% complete and 14% major and 69% had no change in the abnormal −7/del(7q) clone. In additional 5% (12/229) pts, −7/del(7q) emerged either as a new abnormal clone or as a subclonal evolution during therapy or at progression. AZA C-based therapy can either stabilize or reduce/eradicate the abnormal chromosome 7 clone as determined by both, cytogenetics and FISH.

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Clinical Relevance of Cytogenetics Abnormalities in Adult Patients with Acquired Aplastic Anaemia.

Blood ◽

10.1182/blood.v106.11.3748.3748 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3748-3748

Author(s):

Vikas Gupta ◽

Carol Brooker ◽

Jennifer A. Tooze ◽

Qi-long Yi ◽

Deborah Sage ◽

...

Keyword(s):

Aplastic Anaemia ◽

Clinical Relevance ◽

Cytogenetic Study ◽

Cumulative Risk ◽

Normal Karyotype ◽

Similar Response ◽

Trisomy 8 ◽

Abnormal Karyotype ◽

Monosomy 7 ◽

Poor Prognostic Factor

Abstract The clinical relevance of cytogenetics abnormalities in aplastic anaemia (AA) patients at time of diagnosis is unclear. We evaluated the clinical course of 81 AA patients with successful cytogenetics at diagnosis and treated with immunosuppressive therapy (IST) from January 1993 to March 2004. A cytogenetic study was considered to be successful if there were a minimum of 15 evaluable metaphases in the absence of a clonal abnormality. Response to IST, survival and later clonal complications in patients with an abnormal karyotype (n=10) was compared to those with a normal karyotype (n=71). The cytogenetic abnormalities at diagnosis consisted of trisomy 6 (n=2), trisomy 8 (n=2), trisomy 15 (n=2), monosomy 7 (n=1), add(10) (n=1), t(3;11) (n=1) and t(4;6) (n=1). Four out of five evaluable patients with a trisomy responded to a first or subsequent course of IST. One patient with monosomy 7 achieved a complete response and later developed haemolytic PNH but with no recurrence of the monosomy 7. None of the patients with a non-numerical karyotypic abnormality responded to IST. No significant differences in 4-year event-free survival (EFS) (54% vs. 30%, p=0.15), overall survival (OS) (84% vs. 80%, p=0.33) or later clonal disorders (PNH, MDS and AML) were observed between the patients with a normal karyotype and those with an abnormal karyotype. Advanced age (≥60 years) was the only independent poor prognostic factor for survival in a multivariate analysis. Among the patients with a normal karyotype (n=71), 6 patients later developed a clonal cytogenetic abnormality with a cumulative risk of 10% at 4 years. These abnormalities were trisomy 15 (n=2), trisomy 6(n=1), monosomy 7 (n=2) and t(13;15) (n=1). None of the three patients who acquired trisomies developed any clinically significant problem, while acquisition of monosomy 7 was associated with a transformation to MDS/AML. Our data show that AA patients with a trisomy cytogenetic clone at diagnosis show a similar response to IST, evolution to later clonal abnormalities and survival, compared to those AA patients with a normal karyotype.

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Genetic and Epigenetic Silencing of Mesoderm Specific Transcript (MEST) In Acute Myelogenous Leukemia.

Blood ◽

10.1182/blood.v116.21.3639.3639 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3639-3639

Author(s):

Anna R Poetsch ◽

Rainer Claus ◽

Lars Bullinger ◽

Tania Witte ◽

Michael LÃ¼bbert ◽

...

Keyword(s):

Dna Methylation ◽

Tumor Suppressor ◽

Cpg Island ◽

Normal Karyotype ◽

Epigenetic Silencing ◽

Myelogenous Leukemia ◽

Chromosome 7 ◽

Special Importance ◽

Monosomy 7 ◽

Chromosomal Changes

Abstract Abstract 3639 About 10 % of patients with either myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) present either with deletions on the long arm of chromosome 7 or monosomy 7. These chromosomal aberrations are associated with a poor prognosis. Following biallelic inactivation as proposed by Knudsen, a “second hit” of the remaining allele might be required for loss of gene function. Epigenetic silencing might display such a hit in particular since no additional genetic hits could be identified so far. The role of epigenetic regulation might be of special importance in patients with -7/7q-, since several studies have shown that patients harbouring a chromosome 7 abberrations do more benefit from a therapy with demethylating agents as compared to patients with other chromosomal changes. To address the issue of epigenetic silencing in these AML cases we utilized DNA methylation profiling to identify the potential tumor suppressor genes on chromosome 7. We used MBD2 based enrichment of methylated DNA from 4 AML patient samples with monosomy 7 and from 23 patient samples with with other chromosomal changes including normal karyotype AML. For validation we analyzed regional DNA methylation using quantitative MassArray technology on DNA from 115 del(7q) AML or monosomy 7 patients as well as normal karyotype patients (n=20) and CD34 + cells from healthy individuals (n=5). We could identify the gene MEST to be silenced by hypermethylation (> 30 %) of a CpG island on the remaining allele in 20% of the patients with -7/7q- and 40% of patients with normal karyotype. Thus, preferentially in patients that do not have a deletion already. MEST is an imprinted gene located on 7q32.2. However, silencing is correlated with hypermethylation of a CpG island located at an alternative promoter of MEST, independent of the methylation status of the imprinting control region. In two patient samples with monosomy 7 and hypermethylation of MEST (> 80%), DNA methylation was erased after 5-Aza-2′-deoxycytidine (DAC) treatment to less than 10 %. In concordance, also AML cell lines with hypermethylation of the MEST locus loose methylation after sublethal DAC treatment which leads to a reexpression of the gene. Thus, we hypothesize that MEST functions as a tumor suppressor in AML and is genetically as well as epigenetically silenced AML. Reactivation of MEST by demethylating treatment may contribute to the mechanism by which demethylating drugs display their therapeutic potential in leukemia. Disclosures: No relevant conflicts of interest to declare.

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Molecular and Prognostic Correlates of Cytogenetic Abnormalities in Chronic Myelomonocytic Leukemia: A Mayo Clinic-French Consortium Study

Blood ◽

10.1182/blood.v124.21.412.412 ◽

2014 ◽

Vol 124 (21) ◽

pp. 412-412

Author(s):

Emnet A Wassie ◽

Raphael Itzykson ◽

Terra L Lasho ◽

Olivier Kosmider ◽

Christy Finke ◽

...

Keyword(s):

Mayo Clinic ◽

Risk Model ◽

Normal Karyotype ◽

Chronic Myelomonocytic Leukemia ◽

Older Age ◽

World Health ◽

Abnormal Karyotype ◽

Cytogenetic Abnormalities ◽

Myelomonocytic Leukemia ◽

Monosomal Karyotype

Abstract Background: The prognostic significance of cytogenetic abnormalities in chronic myelomonocytic leukemia (CMML) was recently revisited (AJH, 89; 813-818, 2014 and Blood April, 2013). Using a large Mayo Clinic-French Consortium database, we analyzed the molecular and prognostic correlates of cytogenetic abnormalities in CMML. Methods: CMML diagnosis was according to World Health Organization criteria. Cytogenetic analysis and reporting was done according to the International System for Human Cytogenetic Nomenclature. Statistical analyses considered clinical and laboratory parameters obtained at time of cytogenetic studies. Results: Spectrum and frequency of cytogenetic abnormalities: A total of 409 patients participated in this study including, 268 (66%) from the Mayo Clinic and 141 (34%) from the French CMML consortium. Of these, 396 (97%) had ≥20 metaphases and 13 (3%) had ten to 19, analyzed. One hundred and fifteen (30%) patients displayed an abnormal karyotype, including 82 (71%) sole, 20 (17%) two and 13 (11%) complex abnormalities. The most common abnormalities were; +8 (23%), -Y (20%), -7/7q- (14%), 20q- (8%), +21 (8%) and der (3q) (8%). Other cytogenetic abnormalities included 5q-, 12p-, 13q- and i(17q), present at a much lower frequency (0.9-4%). Phenotypic correlates: Abnormal vs normal karyotype was associated with older age (p=0.03), hemoglobin<10 g/dL (p=0.0009), white blood cell count (WBC) >15 x 109/L (p=0.02), absolute neutrophil count (ANC) >10 x 109/L (p=0.03), absolute lymphocyte count (ALC) >2.5 x109/L ( p=0.04), peripheral blood (PB) blast ≥1% (p<0.0001), bone marrow (BM) blast ≥10% (p<0.0001) and circulating immature myeloid cells (IMC) (p=0.0003). +8 (p=0.01), +21 (p=0.03) and der (3q) (p=0.03) were associated with hemoglobin <10 g/dL. -Y was associated with older age (p=0.04), lower PB (p=0.04) and BM (p=0.02) blasts. -7/7q was associated with leukocytosis (p=0.005), neutrophilia (p=0.04), and higher PB blasts (p=0.004). 20q- was associated with thrombocytopenia (p=0.04). Molecular correlates: ASXL1 mutations were associated with abnormal karyotype (p=0.04) and SRSF2 with normal karyotype (p=0.02). In comparison to other abnormal karyotypes, the incidence of ASXL1 mutations was lower in –Y (P=0.04) and der(3q) (p=0.03). U2AF1 mutations were associated with monosomal karyotype (p=0.03) and SF3B1 with der (3q) (p<0.0001). Prognostic relevance : Median follow-up was 1.8 years with 244 (60%) deaths and 79 leukemic transformations (19%). A step-wise survival analysis resulted in three distinct cytogenetic risk categories (Figure 1): high (complex and monosomal karyotype), intermediate (all abnormalities not in high or low risk) and low (normal, sole -Y and sole der (3q)); the corresponding median survivals were 0.2 (HR 8.1, 95% CI 4.6-14.2), 1.7 (HR 1.7, 95% CI 1.2-2.3). In multivariable analysis, the particular cytogenetic risk stratification remained significant in the context of Mayo molecular model (p<0.0001), MDAPS (p<0.0001), and the GFM risk model (P<0.0001). The Mayo-French cytogenetic risk model was also effective in predicting leukemic transformation with HR of 10.9 (95% CI 4.2-27.8) for high and 2.2 (95% CI 1.3-3.7) for intermediate risk groups. Conclusion: Cytogenetic abnormalities are seen in approximately 30% of patients with CMML and display significant associations with certain molecular and phenotypic characteristics. We describe a novel cytogenetic prognostic model for both over-all and leukemia free survival in CMML. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Cytogenetics in Essential Thrombocythemia: Phenotype and Molecular Correlates and Prognostic Relevance in 818 Informative Cases

Blood ◽

10.1182/blood-2021-152549 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3629-3629

Author(s):

Naseema Gangat ◽

Jaya Kittur ◽

Yamna Jadoon ◽

Natasha Szuber ◽

Curtis A. Hanson ◽

...

Keyword(s):

Overall Survival ◽

Arterial Thrombosis ◽

Normal Karyotype ◽

Male Gender ◽

Hemoglobin Level ◽

Leukemic Transformation ◽

Abnormal Karyotype ◽

Cytogenetic Abnormalities ◽

Trisomy 9 ◽

History Of

Abstract Background Cytogenetic abnormalities at diagnosis are relatively uncommon in essential thrombocythemia (ET). In the current study of 818 consecutive patients with ET who were fully annotated for karyotype, we describe the spectrum and prevalence of cytogenetic abnormalities at diagnosis, followed by a comprehensive assessment of phenotypic and molecular correlates and prognostic relevance. Methods The study cohort consisted of 818 consecutive patients with ET that were diagnosed according to the World health Organization 2016 criteria and underwent evaluation between 1967-2021. In order to minimize the inadvertent inclusion of patients with masked polycythemia vera, JAK2 mutated cases with hemoglobin (Hb) level >16 g/dL in women and 16.5 g/dL in men were excluded; similarly, cases with anemia defined by sex adjusted Hb level of <11 g/dL in women and <12.5 g/dL in men were also excluded, in order to avoid inadvertent inclusion of patients with prefibrotic myelofibrosis. Cytogenetic studies were performed either at or within one year of diagnosis and reported according to the International System for Human Cytogenetic Nomenclature. Disease status and survival information was updated in May 2021. JMP Pro 16.0.0 software package, SAS Institute, Cary, NC was utilized for all analyses. Results Prevalence and spectrum of cytogenetic abnormalities Karyotype was normal in 755 patients (92%), showed loss of Y chromosome (-Y) in 16 (2%), and showed abnormalities other than -Y in 47 (5.7%); most common abnormalities included del(20q) (n=10, 21%), trisomy 9 (n=8, 17%), trisomy 8 (n=2, 4%), del(5q) (n=2, 4%), and del(3p) (n=2, 4%). Other sole cytogenetic abnormalities were identified in 18 (38%) patients. Phenotypic and molecular correlates Abnormal karyotype, other than -Y, in comparison with normal karyotype was associated with older age (median age; 63 vs 58 years, p=0.02), lower hemoglobin level (p=0.003), and a higher incidence of arterial thrombosis prior to/at diagnosis (25% vs 13%; p=0.03). 603 patients were annotated for driver mutations; abnormal/normal/-Y frequencies were 78%/60%/71% for JAK2, 22%/26%/14% CALR, 0%/3%/0% MPL and 0%/10% /14% triple negative (p=0.31). NGS information was available in 226 patients and showed absence of ASXL1 mutation in all patients with abnormal karyotype vs 8/211 (4%) with normal karyotype vs 2/4 (50%) with -Y (p<0.0001). Disease transformation and overall-survival. At a median follow-up of 9.6 years (range; 0.01-49.4 years), a total of 96 patients (12%) underwent fibrotic transformation: 6 (13%) with abnormal karyotype, 89 (12%) with normal karyotype and 1 (6%) with -Y (p=0.77). Leukemic transformation rates were also similar with respective frequencies of 4%, 3% and 0% (p=0.71). Abnormal karyotype and -Y were associated with inferior survival with median of 12 years (range; 0.1-34) and 9 years (range; 0.01- 19.9), respectively, compared to 21 years (range; 0.01-49.4) for normal karyotype (p<0.0001) (Figure). In univariate analysis, risk factors for overall survival included abnormal karyotype (p=0.001), - Y (p=0.004), age >60 years (p<0.0001), leukocytosis >11 x10 9/L (p<0.0001), male gender (p=0.0003), and history of thrombosis (p=0.001). During multivariable analysis, abnormal karyotype other than -Y (p=0.003), age >60 years (p<0.0001), leukocytosis >11 x10 9/L (p=0.001), and male gender (p=0.01) remained significant. Additional analysis suggested individual prognostic impact for del(20q) (p=0.04) and also for trisomy 9 (p=0.09) and other abnormalities (p=0.07), with borderline significance. Conclusion The current study confirms the association of abnormal karyotype in ET with older age, lower hemoglobin level, and history of arterial thrombosis, and its mutual exclusivity with ASXL1 mutations. Our observation regarding the independent adverse impact of abnormal karyotype other than -Y, on overall survival, in the absence of association with fibrotic or leukemic transformation, requires clarification from additional studies, which should also investigate the effect of specific abnormalities. Figure 1 Figure 1. Disclosures Szuber: Novartis: Honoraria.

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Chromosomal Abnormalities in the Early Stem Cell Population of CML Patients in Complete Cytogenetic Remission.

Blood ◽

10.1182/blood.v110.11.36.36 ◽

2007 ◽

Vol 110 (11) ◽

pp. 36-36 ◽

Cited By ~ 1

Author(s):

Thomas G.P. Bumm ◽

Amy Hanlon Newell ◽

Jay Oost ◽

Jonathan VanDyke ◽

Susan B. Olson ◽

...

Keyword(s):

Cell Population ◽

Mononuclear Cells ◽

Normal Karyotype ◽

Cytogenetic Response ◽

Chromosome 8 ◽

Chromosome 7 ◽

Stem Cell Population ◽

Fish Analysis ◽

Trisomy 8 ◽

Cd 34

Abstract Clonal cytogenetic abnormalities, most commonly involving chromosomes 7 and 8, are detectable by conventional karyotyping in Ph-negative metaphases of some chronic myeloid leukemia (CML) patients with a major cytogenetic response (MCyR) to imatinib. It is unknown whether these abnormalities involve the primitive progenitor cell compartment, and whether their frequency in this compartment may exceed the frequency detected by karyotyping. To answer these questions we analyzed lineage-negative CD34+/CD38− and CD34+/CD38+ cells from CML patients in complete cytogenetic response (CCyR), using by fluorescent in situ hybridization (FISH) for chromosome 7 and 8 abnormalities and BCR-ABL. Methods: Mononuclear cells (MNC) were selected from the bone marrow of patients with CCR by Ficoll-Hypaque density gradient centrifugation and enriched for lineage-negative cells using an immunomagnetic column. Lineage-negative cells were further sorted into CD34+/38− and CD34+/38+ cells by multicolor FACS. Interphase FISH analysis was performed using 7 LSI D7S522 Spectrum Orange / CEP7 Spectrum Green (chromosome 7), CEP8 Spectrum Aqua (chromosome 8) and LSI BCR/ABL +9q34 TriColor Dual Fusion Probe. Thus far, 5 CML patients with CCR (3 with a normal karyotype and 2 with trisomy 8) and 1 normal control have been analyzed. Results: Of the three CML patients in CCR with normal karyotype, one had 9% deletion 7q (internal cut off of 0.5%) and the second 1.2% trisomy 8 (just over the internal cut off of 1%) cells in the CD-34+/38− population, while the CD-34+/38+ cell population did not show abnormalities. The third CCR patient had no abnormalities in the CD-34+/38− and CD-34+/38+ cell populations. Two CML patients in CCR with 45% trisomy 8 abnormal cells by conventional cytogenetics had 44% and 60% trisomy 8 positive cells in the CD34+/38+ population (two few CD34+/CD38− cells were available for analysis). No BCR-ABL signal was detected in any cell. In the healthy control, the CD34+/38+ cells were normal, but 4.1% of CD34+/38− showed a deletion of 7q. Conclusion: Clonal chromosomal of chromosomes 7 and 8 in Ph-negative primitive hematopoietic progenitor cells may be more common than suggested by conventional karyotyping. A larger cohort of CML patients in CCR and healthy individuals is under study to determine if this phenomenon is indeed related to CML or occurs also in normals. Results will have implications for the interpretation of karyotypes in patients with hematologic malignancies.

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Analysis of Clonal Cytogenetic Abnormalities in 143 Patients with Secondary AML/MDS

Blood ◽

10.1182/blood.v118.21.1473.1473 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1473-1473

Author(s):

Elena V Domracheva ◽

Elena A Aseeva ◽

Galina A Alimova ◽

Olga S Kremenetskaya ◽

Liubov A Shishigina ◽

...

Keyword(s):

Bone Marrow ◽

De Novo ◽

Conflicts Of Interest ◽

Chromosome 7 ◽

Secondary Neoplasms ◽

Monosomy 7 ◽

Cytogenetic Abnormalities ◽

De Novo Aml ◽

Secondary Mds ◽

Balanced Translocations

Abstract Abstract 1473 The incidence of the secondary neoplasms has increased because of the rising numbers of long-term survivors of tumours. Secondary leukemias (sL) and secondary MDS (sMDS) are among the most common types of secondary tumours. Until recently prognosis in cases of sL and sMDS was considered less favorable than in leukemias de novo. Age at presentation and identified clonal cytogenetic abnormalities are among the most important independent prognostic factors in adult patients with leukemias. It is obvious today that the presence of t(15;17)(q22;q12), t(8;21)(q22;q22), inv(16)(p13;q22)/t(16,16)(p13;q22) predicts a relatively favorable outcome, and in contrast the presence of inv(3)(q21q26)/t(3,3)(q21;q26), del(5q), −5, −7 or a complex karyotype (CK) with 3 or more abnormalities generally suggests a very poor prognosis. The monosomal karyotype (MK) defined as two or more distinct autosomal chromosome monosomies or one single autosomal monosomy in combination with at least one structural chromosomal abnormality is also considered as an adverse prognostic factor according to Breems D.A. et al., 2008; Medeiros B.C. et al., 2010 4-year overall survival in AML patients with MK is very low – 3–4%. Therefore, the purpose of our analysis was to determine the frequency of “unfavorable” and “highly unfavorable” (according to Breems D.A. et al.) clonal cytogenetic abnormalities, identified in our laboratory in bone marrow samples of 143 patients with sL/sMDS and to compare it with the frequency of MK in leukemias and MDS de novo according to a published multicenter study (Haase D. et al., 2007; Medeiros B.C. et al., 2010; Grimwade D. et al., 2010). All examined patients with sL/sMDS had solid tumors or lymphomas in anamnesis, for which they received chemotherapy and/or radiotherapy. sMDS was identified in 81 patients (54 patients – ≤5% blasts in bone marrow; in 27 patients – >5%). sAML was identified in 56 patients, sALL – in 1 patient, sCML – in 5 patients. Abnormal karyotypes were observed in 42 (52%) sMDS patients, in 37 (66%) sAML patients, in all 5 sCML patients, in the only sALL patient. The most frequent abnormality in sMDS was isolated monosomy 7: it was observed in 24.4% of the tested abnormal karyotypes. CK and MK are considerably more frequent in sMDS than in de novo MDS. CK occurred in 12 (30.9%) sMDS patients with abnormal karyotypes. Monosomies or deletions of the long arm of chromosome 7 were detected in 8 of 12 identified CK. Balanced translocations in sMDS were detected in only 9 (21%) of 42 karyotypes; no rearrangements involving 3q26, rather frequently occurred in de novo MDS, were registered. Very rare for de novo MDS t(1;7)(q10;q10) was found in 5 of these 9 cases. In general, chromosome 7 abnormalities (translocations, monosomies and/or deletions) were observed in 58.5% of sMDS cases with abnormal karyotypes. In de novo MDS chromosome 7 abnormalities were detected only in 21% of cases. On the contrary, del(5q) occurred more frequently in de novo MDS than in sMDS (30% versus 12.2%). Monosomic karyotypes occurred in 23.8% of sMDS patients with abnormal karyotypes. “Favorable” anomalies were presented in 5 of 37 sAML cases (13,5%) abnormal karyotypes. t(15;17), as a single anomaly, was detected in 3 patients; t(8;21) was detected in 2 cases. “Unfavorable” abnormalities, such as inv(3)(q21;q26)/t(3;3)(q21;q26) in complex karyotypes were observed in 4 cases. Chromosome 5 deletions in complex karyotypes were found in 5 cases, and only in 1 case - as a single anomaly. Other deletions, del(11)(q23), del(12)(p11), del(13)(q12), were found only as isolated anomalies. Complex karyotypes in sAML were observed in 40% (15 of 37) of cases with abnormal karyotypes, whereas in de novo AML CK occurred only in 18% of patients with abnormal karyotypes. Monosomic karyotypes occurred more often in patients with sAML - 27% compared to 13% of cases in de novo AML. In conclusion, prognostically “unfavorable” and “highly unfavorable” cytogenetic abnormalities account for 60% and 25% of all cases with karyotype abnormalities in sAML/sMDS. Thus, our study shows that “unfavorable” and “highly unfavorable” cytogenetic abnormalities in leukemic clone occur more often in sAML/sMDS than in de novo AML/MDS. Disclosures: No relevant conflicts of interest to declare.

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Cytogenetics in Hairy Cell Leukemia at Diagnosis and Relapse: The Rate of Therapy-Related Myeloid Malignancies Is Low

Blood ◽

10.1182/blood.v126.23.2644.2644 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2644-2644

Author(s):

Bartlomiej Getta ◽

Omar Abdel-Wahab ◽

Jae H. Park ◽

Martin S Tallman

Keyword(s):

Hairy Cell Leukemia ◽

Disease Type ◽

Chromosome 7 ◽

Hairy Cell ◽

Disease Relapse ◽

Cell Leukemia ◽

Abnormal Karyotype ◽

Cytogenetic Abnormalities ◽

Myeloid Malignancies

Abstract Cytogenetic analysis of a large cohort of hairy cell leukemia (HCL) patients (pts) has never been reported. The influence on outcome of cytogenetic abnormalities is unknown. We retrospectively reviewed records of pts with HCL treated at Memorial Sloan Kettering Cancer Center between January 1973 and December 2013. 501 pts were identified and 331 were confirmed to have a diagnosis of HCL by histopathology or immunophenotyping. 170 patients were excluded, either because their final diagnosis was an alternate disease or there was inaccessible clinical data. Descriptive statistics were used to evaluate the cohort. 260/331 (79%) were men, overall median age at diagnosis was 53 years (range 20-84 years) and median follow-up was 61 months. 241 had classical HCL and 18 had variant disease based on absent expression of CD25 by immunophenotyping or immunohistochemistry with median diagnostic WBC of 3.2x10^9/L and 8.4x10^9/L respectively. BRAF mutation testing was performed infrequently (<10% pts). In 72 pts we were unable to review immunophenotyping data to confirm disease type. 64 pts never received treatment, 156 received one line and 111 received at least 2 lines of therapy during follow up. The most common initial treatment was cladribine (n=215, 80%). Assessment of bone marrow cytogenetics was available in 105 of 331 patients (32%) at diagnosis with 92 cases of classical HCL, 8 variant HCL and 5 with unconfirmed disease type. Karyotype was normal in 95 patients (90.5%) and abnormal in 10 (9.5%). Abnormal karyotype was more common in patients with variant than classical disease, seen in 3/8 versus 7/92 respectively (p=0.007). 9/10 with abnormal karyotype had multiple clones identified including at least one cytogenetically normal metaphase. Sex chromosome loss was present in 4/10 pts (3 with loss of Y and 1 female with loss of X) while loss of genetic material from chromosome 7 was the most common autosomal abnormality (3/10 patients). The presence of an abnormal karyotype at diagnosis did not worsen survival compared to patients with a normal karyotype with an estimated 5 year OS of 100% and 97%, respectively. 30/111 pts (24 classical and 6 variant HCL) who received two or more lines of therapy had cytogenetics evaluated at some point during disease relapse and 17/30 had cytogenetic abnormalities. 6 of these had cytogenetics at diagnosis and 4/6 were normal. Pts with an abnormal karyotype received more lines of therapy prior to identification of a cytogenetic abnormality than those with normal cytogenetics with a median of 3 and 1 lines respectively (p=0.008). Pts with variant HCL were more likely to have abnormal cytogenetics at relapse (6/6) than those with classical disease (11/24) (p=0.017). Cytogenetic abnormalities of all evaluable pts with relapsed HCL were reviewed. The most common abnormalities seen at relapse were monosomies or partial chromosome deletions seen in 14/17 cases. The most common abnormalities were loss of material from chromosome 7 (6/17 pts) and chromosomes 13 and 17 (4/17 pts each). Multiple abnormalities were present in 11/17 pts. No abnormality appeared to be specific for classical or variant disease. In particular loss of material from chromosome 7 was seen in both disease types. Only 2 of the 331 pts developed a therapy-related acute myeloid leukemia (t-AML) both had classical HCL. The first developed t-AML with a complex monosomal karyotype after prior treatment with cladribine, pentostatin, fludarabine and rituximab. The second had t-AML with monosomy 7 following 4 treatments with cladribine. There were no cases of myelodysplasia. In summary we identified cytogenetic abnormalities in 10% of newly diagnosed HCL pts and found that this did not adversely affect survival. Pts with variant HCL are more likely to have abnormal cytogenetics at diagnosis and at disease relapse compared to those with classical disease. Cytogenetic changes appear to be more common in those who received more lines of cytotoxic chemotherapy. Loss of material from chromosome 7 is the most common cytogenetic change seen at diagnosis and at disease progression. The rate of therapy-related myeloid malignancies is low and is not increased in HCL variant. Identification of new molecular mutations arising during disease relapse may be insightful. Disclosures Park: Actinium Pharmaceuticals, Inc.: Research Funding; Juno Therapeutics: Consultancy.

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High-Resolution Genomic Arrays Facilitate Detection of Novel Cryptic Chromosomal Lesions in MDS.

Blood ◽

10.1182/blood.v106.11.370.370 ◽

2005 ◽

Vol 106 (11) ◽

pp. 370-370

Author(s):

Christine L. O’Keefe ◽

Ramon Tiu ◽

Lukasz Gondek ◽

Aaron Viny ◽

Karl Theil ◽

...

Keyword(s):

High Resolution ◽

Chromosomal Abnormalities ◽

Bone Marrow Cells ◽

Genomic Analysis ◽

Normal Karyotype ◽

Chromosome 7 ◽

Comparative Genomic ◽

Stem Loop ◽

Monosomy 7 ◽

Normal Cytogenetics

Abstract The evolution of abnormal hematopoietic clones characterized by acquired chromosomal abnormalities is the central event in the pathogenesis of MDS. Defective chromosomes have significant clinical implications in the management of MDS and suggest the presence of an inherent chromosomal instability. As karyotypic lesions are not found in all MDS patients, it is possible that in some the dysplastic clone may evolve without a chromosomal defect or, more likely, the resolution of routine metaphase cytogenetics is not sufficient to detect smaller lesions; in many instances lack of growth precludes the analysis. Array-based comparative genomic hybridization (A-CGH) allows for a high-resolution genomic scan that circumvents some of the limitations associated with the use of conventional cytogenetics. We hypothesized that high-resolution genomic analysis of genetic gains and losses by A-CGH may detect cryptic lesions, particularly in patients with negative/non-informative cytogenetics that may be of clinical/scientific significance. We examined bone marrow cells from 39 MDS patients (18 RA/RARS, 11 RAEB-t, 6 CMML and 4 secondary AML) and 11 controls using a 2632 BAC microarray and CGH. Dye swapping on duplicate arrays assured reproducibility of the CGH results, confirmed globally by a high resolution 50K SNP microarray in 4 patients and by microsatellite analysis in others. By traditional cytogenetics 19 patients had chromosomal lesions, 18 were normal and 2 tests non-informative. When A-CGH was applied, a normal karyotype was found in only 15% of patients in comparison to 46% by metaphase cytogenetics. Of note is that both cases with uninformative cytogenetics showed an abnormal CGH result and in several patients (N=11) with an abnormal karyotype additional lesions were found. Karyotypic results were confirmed in 7 cases; discordant analysis may be due to a lower proportion of dysplastic cells in marrow. Irrespective of the genomic area affected, when we studied the raw number of lesions more advanced forms of MDS (RAEB-t/AML) were evenly distributed between patients subdivided on sheer number of lesions (0, 1–17, >17). Many hotspots of genomic instability shared between patients were identified. For example, 1p26.3, 10q26 and 4p16 lesions were found in 2 or more patients. Interestingly, these regions contain genes of potential pathologic significance, including tubulin gamma complex associated protein 2 (TUBGCR2) and histone stem-loop binding protein (SLBP). Cryptic lesions on chromosome 7 (e.g. 7p21, 7q31) were identified in 5 patients with normal cytogenetics. These patients suffered from severe cytopenias, consistent with the prognosis of monosomy 7 and highlighting a consensus defect on chromosome 7. Certain chromosomes were rarely or never affected, implying that a more targeted array might be designed for clinical use. A-CGH Cytogenetics Unsuccessful Normal Abnormal Unsuccessful (N=2) 0 0 2 Normal (N=18) 0 3 15 Abnormal (n=19) 0 3 16 In summary, our study highlights the superior level of resolution of A-CGH as compared to metaphase analysis in the diagnosis of MDS. A prospective analysis is underway to determine the prognostic value of CGH-detected lesions and their pathophysiologic significance.

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IKZF1 deletions Are Detected In 40.3% Of 270 Adult B-Precursor-ALL and Are Independently Associated With BCR-ABL1 Positivity and Inferior Outcome

Blood ◽

10.1182/blood.v122.21.1372.1372 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1372-1372

Author(s):

Annette Fasan ◽

Claudia Haferlach ◽

Madlen Ulke ◽

Wolfgang Kern ◽

Torsten Haferlach ◽

...

Keyword(s):

Multiplex Pcr ◽

Normal Karyotype ◽

Chromosome 7 ◽

Employment Equity ◽

Gene Deletions ◽

Exon 2 ◽

Monosomy 7 ◽

Significant Difference ◽

Equity Ownership ◽

Exon 4

Abstract Introduction Total or intragenic deletions of or within the IKZF1 (Ikaros) gene on chromosome 7 have been shown to be frequent in B-precursor-ALL (B-ALL), highly associated with BCR-ABL1 positive (BCR-ABL1pos) B-ALL (Mullighan et al., Nature 2008), but also present in BCR-ABL1 negative (BCR-ABL1neg) B-ALL (Mullighan et al., N Engl J Med 2009). IKZF1 deletion pattern is known to be heterogenous and deletions involving exon 4, 7 or 8 are likely to have the same impact as whole gene deletions. To analyse intragenic IKZF1 deletions, we used a multiplex-PCR approach, which represents the standard detection method. In addition, we used multiplex ligation-dependent probe amplification (MLPA), which allows the detection of whole IKZF1 deletions. Aim We aimed at characterization of IKZF1 deletions pattern and correlation to clinical features in a cohort of 270 adult B-ALL cases. Patients and Methods IKZF1 deletion status was analyzed in blood or bone marrow samples from 270 adult B-ALL cases (subtypes as diagnosed by immunophenotyping: c-ALL: n=197; Pro-B ALL: n=51; Pre-B ALL: n=5). Additionally, 17 mature B-ALL cases were analyzed. The cohort consisted of 137 females and 133 males, median age was 58.3 years (range: 18.1-91.4 years). The cohort was classified into seven subgroups according to the following cytogenetics: 1) t(9;22)(q34;q11) (n=97), 2) 11q23/MLL rearrangements (n=24), 3) MYC rearrangements (n=14), 4) hypodiploidy (n=21), 5) hyperdiploidy (n=32), 6) normal karyotype (n=38), 7) other cytogenetic aberrations (n=42). In one case no cytogenetic data was available. In all 270 cases intragenic IKZF1 deletions were investigated by breakpoint-specific fluorescent multiplex PCR (according to Caye et al., Hematologica 2012). In 206 cases we additionally used MLPA (P335 SALSA MLPA kit IKZF1, MCR Holland, The Netherlands) to identify whole IKZF1 deletions not detectable by multiplex-PCR. Results In total, 132 IKZF1 alterations were identified in 109/270 cases (40.3%). With regard to ALL subtypes in c-ALL 47.7% (94/197) IKZF1 mutations were detected, in Pro-B ALL 23.5% (12/51) and in three of five Pre-B ALL cases. No IKZF1 deletions were detected in mature B-ALL and thus were mutually exclusive with MYC rearrangements. There was no significant difference in age, sex, leukocyte count, hemoglobin level or platelet count between patients with or without IKZF1 deletions, respectively. With regard to immunophenotype cases with IKZF1 deletions had a stronger expression of CD13 (38±28% vs. 27±25% positive cells; p=0.004), CD33 (24±23% vs. 17±21%; p=0.008), CD34 (72±25% vs. 43±35%; p<0.001%) and TdT (61±28% vs. 43±32%; p<0.001). In 86/109 cases (78.9%) one (monoallelic) intragenic IKZF1 deletion was detected by multiplex-PCR and MLPA. In detail, 38 patients (34.9%) showed deletions of exon 4-7, 15 cases (13.7%) deletions of exon 2-7, 5 cases (4.6%) deletions of exon 4-8 and 3 cases (1.8%) deletions of exon 2-8. In one case, a deletion of IKZF1 exons 2 and 3 was detected by MLPA. 23/109 cases (21.1%) showed two (biallelic) IKZF1 deletions. Whole IKZF1 gene deletions were detected by MLPA in 24/109 cases (22.0%) and were associated with cytogenetic abnormalities of the short arm of chromosome 7 in 21/40 cases (52.5%) including: monosomy 7 (n=14), i(7)(q10) (n=2), dicentric chromosomes 7 (n=1), and unbalanced translocations involving chromosome 7 (n=4). Four of 24 cases with MLL-rearrangements harbored IKZF1 deletions, three of these being whole IKZF1 gene deletions due to monosomy 7. IKZF1 deletions were highly associated with BCR-ABL1 positivity: 72/97 BCR-ABL1pos (74.2%) versus 37/172 BCR-ABL1neg cases (21.5%) (p<0.001). 61/85 cases (71.8%) with intragenic IKZF1 deletions were BCR-ABL1pos compared to 11/24 (45.8%) cases with whole IKZF1 deletions (p=0.018). In 97 BCR-ABL1pos cases, the additional presence of IKZF1 deletions was correlated to inferior survival (p=0.070). Conclusions 1) IKZF1 deletions were detected in 40.3% in adult B-ALL by using muliplex-PCR and MLPA. 2) Whole but also intragenic IKZF1 deletions are highly associated with BCR-ABL1 in adult B-ALL. 3) As 22% of IKZF1 deletions were whole gene deletions not detectable by standard multiplex-PCR, at least two molecular methods (multiplex-PCR and MLPA) are required to fully detect the whole spectrum of IKZF1 deletion patterns in adult B-ALL. Disclosures: Fasan: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Ulke:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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A Compendium of Cytogenetic Abnormalities in Myelofibrosis: Molecular and Phenotypic Correlates in 826 Patients

Blood ◽

10.1182/blood.v124.21.403.403 ◽

2014 ◽

Vol 124 (21) ◽

pp. 403-403

Author(s):

Emnet A Wassie ◽

Christy Finke ◽

Naseema Gangat ◽

Terra L Lasho ◽

Animesh Pardanani ◽

...

Keyword(s):

Conflicts Of Interest ◽

International System ◽

Normal Karyotype ◽

Primary Myelofibrosis ◽

World Health ◽

Trisomy 8 ◽

Abnormal Karyotype ◽

Cytogenetic Abnormalities ◽

Chromosome 1Q ◽

Favorable Prognosis

Abstract Background : Recent studies have suggested significant associations between karyotype and certain molecular or phenotypic features in primary myelofibrosis (PMF). In the current study of 835 consecutive patients, we examined the spectrum and prevalence of cytogenetic abnormalities in PMF and their molecular and phenotypic correlates. Methods : PMF diagnosis was according to World Health Organization criteria. Cytogenetic analysis and reporting was done according to the International System for Human Cytogenetic Nomenclature. Statistical analyses considered clinical and laboratory parameters obtained at time of cytogenetic studies. Spectrum and frequency of cytogenetic abnormalities : Analyzable metaphases were obtained in 826 (99%) of 835 patients studied; 681(82%) had ≥20 metaphases analyzed. 352 (42.6%) patients had abnormal karyotype, including 240 (68.2%) sole, 64 (18.2%) two and 48 (13.6%) complex; comparison of these groups revealed lower platelet count (p<0.01), higher DIPSS-plus score (p=0.03) and higher percentage of younger patients (p=0.04) with complex abnormalities. Monosomal karyotype was noted in 20 (5.7%) patients. Approximately 150 individual abnormalities were identified; most frequent were 20q- (23.3%), 13q- (18.2%), +8 (11.1%), +9 (9.9%), duplication of chromosome 1q (9.7%) and -7/7q- (7.1%). Other notable abnormalities including i(17q) (1.4%), 12p- (1.1%) and inv(3) (0.6%) were much less frequent. Trisomy 8 was the most frequent in the context of complex abnormality (25%). Among the 500 patients seen within one year of initial diagnosis, 179 (35.8%) had abnormal karyotype, which included 121 (67.6%) sole, 31 (17.3%) two and 27 (15.1%) complex abnormalities; the most common abnormalities were 20q- (24.6%), 13q- (15.1%), +8 (14%) and +9 (10%) whereas 11q- (1.7%), i(17q) (1.1%), inv(3) (0.6%), and 12p- (0.6%) were infrequent. Molecular correlates : 476 patients were annotated for JAK2, CALR and MPL mutations; abnormal karyotype frequencies were 43% in JAK2, 42% CALR, 33% MPL mutated and 34% triple-negative cases (p=0.3). 13q- was associated with mutant CALR (p=0.03) and +9 with mutant JAK2 (p=0.02). Subsets of patients were also screened for ASXL1, EZH2, IDH, SRSF2, U2AF1, and SF3B1 mutations; in all instances, mutational frequencies were higher in patients with normal karyotype, reaching significance with ASXL1 (p=0.02) and U2AF1 (p=0.01). Mutant SRSF2 was associated with 20q- (p=0.02). Phenotypic correlates : Phenotypic correlates included abnormal karyotype with anemia (p=0.02), leukopenia (p<0.01) and thrombocytopenia (p<0.01); complex karyotype with younger age (p=0.04) and thrombocytopenia (p<0.01); leukopenia with 20q-, +8 and -7/7q-; and thrombocytopenia with 20q- and -7/7q-. Cytopenias were less likely to occur with 13q- (p<0.01), which was instead associated with thrombocytosis (p<0.01). 20q- was associated with lower incidence of marked leukocytosis (p=0.02). Trisomy 8 was associated with lower incidences of constitutional symptoms (p<0.01) and marked splenomegaly (p<0.01). Conclusions : The association of 13q- with CALR mutations in PMF might underlie its association with both thrombocytosis and favorable prognosis. The association of +9 with JAK2 mutations might reflect selective clonal advantage through JAK2V617F dosage enhancement or mutation-induced chromosomal instability. The association of 20q- with mutant SRSF2 and thrombocytopenia warrant further clarification of its reported association with favorable prognosis. Disclosures No relevant conflicts of interest to declare.

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