Peripheral Blood Karyotyping Is Superior To Bone Marrow and Identifies Most Frequent Structural Chromosomal Rearrangements In Myelofibrosis

Abstract In the past cytogenetic studies of patients with MF were hindered because marrow (BM) specimens were used for karyotyping but frequently could not be obtained due to advanced marrow fibrosis. Since MF is characterized by the constitutive mobilization of immature myeloid cells into the peripheral blood (PB), we compared unstimulated PB specimens with BM specimens to determine their utility in successfully detecting cytogenetic abnormalities in patients with MF. We also simultaneously performed interphase FISH (IFISH) studies in order to determine if IFISH identified additional genomic abnormalities in MF. We evaluated 183 patients who had had successful conventional and molecular cytogenetic analyses. Our myeloproliferative neoplasm ( MPN)-FISH panel consisted of twelve probes (EGR1 at 5q31, D5S23 at 5p15.3, D7Z1 at 7p11-q11, D7S522 at 7q31, D8Z2 at 8p11-q11, CDKN2A at 9p21, CEP9 at 9p11-q11, ATM at 11q22,1, Rb1 at 13q14, and D20S18 at 20q12, CKS1B at 1q21 and CDKN2C at 1p32). BM cytogenetics was studied in 60 pts (33%) and PB cytogenetics were evaluated in 123 pts (67%). Conventional cytogenetics was informative for 96% (123/128) of PB specimens and 97% (60/62) of BM samples. When conventional cytogenetic analysis was compared to IFISH, concordant results were observed in 154 patients (84%). Moreover, conventional karyotyping identified chromosomal abnormalities in an additional 18 patients (10%), which were not targeted by the 12 loci FISH panel. Among these patients, structural abnormalities of chromosome 12 were detected in 6 patients (33%) which represents the most frequent structural abnormality detected in MF and was associated with poor survival. Of the 63 patients with concordant abnormal cytogenetics and FISH the most frequent abnormalities included: del(20)(q11q13) (n=30, 48%), unbalanced 1q translocations resulting in trisomy 1q or duplication of 1q (n=17, 27%), gain of chromosome 8 (n=9, 14%), +8,+9( n=4, 6%), trisomy 9 (n=4, 6%), del(7q)/-7( n=7,11%), deletion 13q (n=9,14%) and complex karyotype (n=16, 25%). The remaining 11pts (6%) had discordant conventional and molecular cytogenetic results and were divided into two categories: A) those with normal cytogenetics (with or without non clonal abnormalities) and an abnormal MPN IFISH panel and B) those with abnormal clonal karyotype with normal lFISH. When compared to BM, PB specimens had a similar rate of abnormal karyotype: 51% in PB vs 48% in BM. Our results unequivocally demonstrated that conventional cytogenetics of MF can be successfully obtained from unstimulated PB specimens in 96% of patients and that analysis of BM does not reveal additional cytogenetic abnormalities. We conclude that FISH analysis has limited value in MF and is only informative for those patients who lack mitotic cells or who are cytogenetically normal, and in these patients, IFISH detects cryptic abnormalities in 4%. Use of PB karyotyping in MF is sufficient to effectively detects clonal hematopoiesis which contributes to prognostic risk stratification and influences therapeutic decision making. Disclosures: No relevant conflicts of interest to declare.

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FISH Reveals Abnormalities of Unsuspected Regions in Chromosomally Normal De Novo Myelodysplastic Syndromes (MDS).

Blood ◽

10.1182/blood.v114.22.2599.2599 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2599-2599

Author(s):

Paolo Bernasconi ◽

Irene Dambruoso ◽

Marina Boni ◽

Paola Maria Cavigliano ◽

Ilaria Giardini ◽

...

Keyword(s):

De Novo ◽

Chromosomal Abnormalities ◽

Conflicts Of Interest ◽

Prognostic Significance ◽

Normal Karyotype ◽

Low Risk ◽

Fish Analysis ◽

Disease Evolution ◽

Therapeutic Decisions ◽

Conventional Cytogenetics

Abstract Abstract 2599 Poster Board II-575 In de novo MDS the chromosomal pattern is a mandatory step for an accurate diagnosis, predicts overall survival (OS) and the risk of MDS/AML evolution, guides therapeutic decisions. However, conventional cytogenetics (CC) studies show a normal un-informative chromosomal pattern in about half of MDS patients, especially in low-risk disease. FISH with probes pinpointing the chromosomal regions most frequently affected in MDS can increase the incidence of abnormal karyotypes up to 60%, but the percentage of normal karyotypes remains high and makes the search of novel cytogenetic/molecular markers a urgent need. A fundamental contribution to overcome CC and FISH shortcomings, has been recently provided by array CGH (aCGH) studies which have revealed that, independently of the cytogenetic pattern, MDS patients may harbour novel abnormalities involving unsuspected chromosomal regions. Based on this assumption, we decided to investigate whether FISH with probes already employed in aCGH studies can truly unmask cryptic lesions in chromosomally normal MDS patients, whether these defects are either chromosomal gains/losses or balanced rearrangements and whether these chromosomal abnormalities influence OS and disease evolution. FISH analyses were carried out in thirty-five patients examined between January 2005 and June 2008. There were thirteen females and twenty-two males, whose median age was 66 years (range 24–78). According to WHO classification, 6 patients were classified as RA, 13 as RAEB-1 and 16 as RAEB-2. According to IPSS score, 7 patients were considered low-risk, 14 intermediate-1 risk and 14 intermediate-2 risk. Median follow-up was nine months (range 1–46). At the time of the analyses no patients has died; 6 have progressed to RAEB-2 and 3 to AML. Probes for FISH analysis were chosen following two criteria: the frequency of their involvement in chromosomal abnormalities identified by aCGH studies and their Mb position on Human Mar. 2003 assembly according to the UCSC genome browser. All probes, obtained from BACPAC Resources Center at C.H.O.R.I. (Oakland, USA), were labelled and applied as previously reported. The following probes were applied: RP11-912d8 (19q13.2); RP11-196p12 (17q11.2); RP11-269c4 (14q12); RP11-351o1 (10q21.3); RP11-144g6 (10q11.2); RP11-122a11 (7q34); RP11-951k18 (5q13.1); RP11-100m20 (4p14); RP11-544h14 (2q33). The cut-off values for interphase FISH (i-FISH) were obtained from the analysis of 300 nuclei from ten normal samples and were fixed at 10%. An abnormal FISH pattern was revealed in eighteen patients (51.4%). It was observed in 3/6 RA patients, in 7/13 RAEB-1 and in 8/16 RAEB-2 and in 2/7 IPSS low-risk, in 7/14 intermediate-1 risk and in 9/14 intermediate-2 risk MDS patients. Seven presented a 19q13.2 deletion, three a 14q12 deletion, four an amplification of band 4p14, two a defect of band 10q21.3, two a potential amplification and one a deletion of band 10q11.2, two a deletion of band 5q13.1 and one a deletion of band 17q11.2. Cryptic defects were also revealed in six of the nine patients who experienced disease evolution on FISH analyses. This event occurred in 2/3 RA, in 2/7 RAEB-1 and in 2/8 RAEB-2 patients with an abnormal FISH pattern. Despite these data, the prognostic significance of an abnormal FISH pattern needs to be assessed on additional patients. In conclusion, our data show that i) FISH can truly reveal novel lesions involving unsuspected chromosomal regions in 51% of MDS patients with a normal karyotype; ii) most of these lesions consist of chromosomal gains/losses; iii) an abnormal FISH pattern seems to correlate with disease progression, but this correlation needs to be tested on additional patients. Disclosures: No relevant conflicts of interest to declare.

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Cytogenetics and spectral Karyotyping analysis in CLL Patients: Correlation with FISH and ZAP70 Expression

Blood ◽

10.1182/blood.v116.21.4844.4844 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4844-4844

Author(s):

Fabio Morato Oliveira ◽

Daniel Mazza Matos ◽

Lorena Lobo Figueiredo Pontes ◽

Belinda Pinto Simoes ◽

Eduardo M. Rego ◽

...

Keyword(s):

Peripheral Blood ◽

Cytogenetic Analysis ◽

Chromosomal Abnormalities ◽

Conflicts Of Interest ◽

Karyotype Analysis ◽

Calf Serum ◽

Normal Karyotype ◽

Fish Analysis ◽

Interphase Cell

Abstract Abstract 4844 Cytogenetic abnormalities play an important role as prognostic factors in CLL. However, due the low mitotic index of CLL B cells in vitro, analysis of a set of subjects for the most commonly known aberrations is usually done by FISH on interphase cell. The objectives of this investigation were the use of the oligonucleotide DSP30 in combination with IL-2, as a B-cell mitogen for cytogenetic investigation in CLL and correlation among the karyotype analysis obtained (G-banding + SKY), FISH profile from unstimulated cells, ZAP70 expression and stratification status for each patient. For metaphase induction, peripheral blood mononuclear cells were cultured in RPMI 1640 medium with 20% fetal calf serum in the presence of the immunostimulatory CpG-oligonucleotide DSP30 and IL-2. Additionally, one set of cell culture was performed for each patient without any stimulant agent, for FISH analysis. The FISH panel included probes for the detection of +12, and deletions of 11q22.3 (ATM), 13q14 (D13S25 and D13S319), and 17p13 (TP53). The cut off levels for trissomy 12 (>2%), del(13q) (>2.4%), del(11q23.3) (>2.5%), del(17p13.1) (>3%) were established according to the iFISH patterns observed in a group of 4 age and sex-matched normal control peripheral blood samples studied with the same probes. Spectral karyotype analysis (SKY) was performed, according manufactures' instruction. The ZAP70 profile was obtained by flow cytometry analysis. In concordance with literature, the cut off value adopted for ZAP70 was 20%. In a group of 64 subjects studied, the cytogenetic analysis showed chromosomal aberrations in 52 patients (81.25%). The profile of abnormalities observed were del(6)(q24), +8(x2), del(11)(q13~q23), +12, +15(x2), del(12)(p13), -17, +21, +19, +18, del(13)(q31), del(14)(q24), del(17)(p13), +21, +4, +5, +11, t(1;12)(q31;p13), t(11;13)(q23;q12), t(15;18)(q11.1;q11), t(1;10)(p22;p14), t(14;22)(q32;q11), t(17;18)(q10;q10), t(9;13)(q21;q22), t(10;13)(q26;q14), t(9;12)(q12;p11), t(X;12)(p11.2;q24). Twelve patients exhibited normal karyotype (18.75%). All subjects presenting chromosomal abnormalities, by using G-banding analysis, were confirmed by SKY. In patients with normal cytogenetic, SKY analysis did not identified any criptic abnormality. Cells without any stimulant agent showed concordance with the cytogenetic profile obtained (FISH analysis). The ZAP70 expression did not show any relationship between the group of patients with chromosomal abnormalities and the group with normal karyotype. The use of the immunostimulatory oligonucleotide DSP30 in combination with IL-2 showed to be effective to induce cell cycle progression of CLL cells in vitro than others mitogens. Cytogenetic aberrations detected by G-banding in addition to FISH analysis were heterogeneous. The limited spectrum of chromosomal abnormalities seen by FISH analysis may contribute to underestimate the prognostic value, where others abnormalities may be present in patient's karyotype. These results indicate that classical cytogenetic analysis can contribute to the stratification of different subsets of CLL patients with complex karyotype associated with poor prognosis. Financial support: FAPESP (Proc. 07/52462-7). Disclosures: No relevant conflicts of interest to declare.

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Spectral Karyotyping (SKY) ANALYSIS APPLIED to CHRONIC LYMPHOCYTIC LEUKEMIA CELLS Immunostimulated with the COMBINATION DSP30/IL-2.

Blood ◽

10.1182/blood.v114.22.4692.4692 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4692-4692

Author(s):

Fabio Morato Oliveira ◽

Daniel Mazza Matos ◽

Lorena Lobo Figueiredo-Pontes ◽

Belinda Simoes ◽

Eduardo M. Rego ◽

...

Keyword(s):

Peripheral Blood ◽

Mononuclear Cells ◽

Chromosomal Abnormalities ◽

Conflicts Of Interest ◽

Calf Serum ◽

Normal Karyotype ◽

Lymphocytic Leukemia ◽

Fish Analysis ◽

Spectral Karyotyping ◽

Cytogenetic Profile

Abstract Abstract 4692 Cytogenetic abnormalities play an important role as prognostic factors in CLL. The immunostimulatory oligonucleotide DSP30 in combination with IL-2 is an easy and efficient stimulus in metaphase generation for chromosomal banding. This technique allows a more comprehensive chromosome analysis compared to FISH. On the other hand, spectral karyotyping (SKY) analysis, a recent molecular cytogenetic tool for the screening of the entire genome, has been shown to provide additional chromosome information. By using a combination of molecular cytogenetics strategies, the goal of this investigation was to use the SKY to identify masked chromosomal abnormalities in CLL cells stimulated by the combination of DSP30 and IL-2. In addition we compared the cytogenetic profile obtained (DSP30/IL-2) with FISH analysis from unstimulated cells and ZAP70 expression for each patient. For metaphase induction, peripheral blood mononuclear cells were cultured in RPMI 1640 medium with 20% fetal calf serum in the presence of the immunostimulatory CpG-oligonucleotide DSP30 and IL-2. One extra set of cell culture without any stimulant agent for iFISH analysis was performed for each patient. The iFISH panel included probes for the detection of +12, and deletions of 11q22.3 (ATM), 13q14 (D13S25 and D13S319), and 17p13 (TP53). The cut off levels for trissomy 12 (>2%), del(13q) (>2.4%), del(11q23.3) (>.5%), del(17p13.1) (>3%) were established according to the iFISH patterns observed in a group of 4 age and sex-matched normal control peripheral blood samples studied with the same probes. Spectral karyotype analysis (SKY) was performed, according manufactures' instruction, in all patients. The ZAP70 expression was determined by flow cytometry analysis and the cut off value was 20%. In a group of 35 subjects studied, the cytogenetic analysis with DSP30/IL-2 showed chromosomal aberrations in 27. The following abnormalities were observed: +4, +5, +8(x2), +11, +12, +15(x2), -17, +18, +19, +21, del(6)(q24), del(11)(q13∼q23), del(12)(p13), del(13)(q31), del(14)(q24), del(17)(p13), t(1;12)(q31;p13), t(11;13)(q23;q12), t(15;18)(q11.1;q11), t(1;10)(p22;p14), t(14;22)(q32;q11), t(17;18)(q10;q10), t(9;13)(q21;q22), t(10;13)(q26;q14), t(9;12)(q12;p11), t(X;12)(p11.2;q24). Eight patients exhibited normal karyotype. The SKY analysis confirmed the abnormalities previously seen by G-banding (DSP30/IL-2), however, did not identify any new abnormality in subjects with normal karyotype. The iFISH analysis agreed with the cytogenetic profile obtained with DSP30/IL-2. The ZAP70 expression did not show any relationship between the group of patients with chromosomal abnormalities and the group with normal karyotype. The use of the immunostimulatory oligonucleotide DSP30 in combination with IL-2 showed to be effective to induce cell cycle progression of CLL. Cytogenetic aberrations detected by G-banding in addition to FISH were heterogeneous. The limited panel used for iFISH analysis may contribute to underestimate the prognostic value, since others abnormalities may be present in patient's karyotype. In conclusion, SKY analysis did not reveal any masked abnormality beyond those showed by G-banding resolution. These results indicate that G-banding analysis (DSP30/IL-2) can contribute to the stratification of different subsets of CLL patients with complex karyotype associated with poor prognosis. Disclosures: No relevant conflicts of interest to declare.

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Correlation between Immunoglobulin Gene Rearrangements and Chromosomal Abnormalities in Multiple Myeloma.

Blood ◽

10.1182/blood.v110.11.4771.4771 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4771-4771

Author(s):

Giovanna Piras ◽

Maria Monne ◽

Antonella Uras ◽

Laura Pilo ◽

Luciana Arca ◽

...

Keyword(s):

Multiple Myeloma ◽

Chromosomal Abnormalities ◽

Normal Karyotype ◽

Fish Analysis ◽

Immunoglobulin Gene ◽

Cytogenetic Aberrations ◽

Oncogenic Pathways ◽

Clonality Analysis ◽

Conventional Cytogenetics ◽

Clonogenic Cell

Abstract Background: Multiple Myeloma (MM) is characterized by frequent and complex genetic abnormalities that contribute to the pathogenesis and its prognostic eterogeneity. There is evidence for two oncogenic pathways in the early development of clonal plasma cell disorder: i) non-hyperdiploid carring translocation of the immunoglobulin heavy-chain locus and various oncogenes ii) hyperdiploid tumors with infrequent IgH translocation. The MM clonogenic cell is positively selected during the development and reaction of the germinal center. The immunoglobulin gene (IG) repertoire in MM follows a pattern similar to that of the normal repertoire. However, available data from analysis of IGH and IGK/L genes according to cytogenetic aberrations are limited. In the present study we investigated the frequency and characteristics of IGK and incomplete DJH as well as complete VDJH rearrangements in parallel with chromosomal abnormalities in a series of untreated MM patients. Materials and Methods. Bone marrow aspirates were collected from 53 MM patients with a mean age of 69.6 (range 48–84) between 2003–2007. The serum monoclonal component was IgG and IgA in the 77% and 22% patients respectively; 1 patient presented with IgD k MM. Cytogenetics and FISH analysis were performed simultaneously in 37 MM. In 18 (50.5%) samples kariotype analysis was successful. Interphase FISH analysis was perfomed using a set of probes specific for RB-1 (13q14), D13S319 (13q14.3), IgH (14q32), and p53 (17p13.1) loci, t(4;14), t(14;16), t(11;14) and a multicolor probe set for detection of aneuploidy (Vysis, Downers Grove, IL, USA). Genomic DNA was isolated for clonality analysis. IGHV-J, IGHD-J, IGKV-J, IGKV-KDE, IGKJ-C-INTRON-KDE rearrangements were amplified by PCR and analyzed following the BIOMED-2 protocol. Results: Conventional cytogenetics allowed to detect 16 patients with a normal kariotype, 1 hyperdiploid kariotype with monosomy 13, 1 hyperdiploid kariotype with 3q21 deletion. FISH panel analysis resulted in 4 patients with hyperdiploid kariotype and 7 with abnormalities for RB-1 and/or D13S319. IGH rearrangements were detected in 3 patients and the t (4;14) was found in 1 case. The p53 deletion, t(11;14) and t(14;16) were not detected. The overall detection rate of clonality by amplifying VDJH and DJH rearrangements using family-specific primers was 90%. We found a high frequency (71.7%) of DJH rearrangements with DH3 segment under represented (4%). The DH7 segment was rearranged in the 15% of MM. Incomplete DJH and complete VDJH rearrangements were present at frequencies of 20% and 29.5%, respectively. IGK locus rearrangements were detected in 38 out of 53 MM and the 60% presented the non-productive IGKV-KDE and IGKJ-C-INTRON-KDE rearrangements. Parallel analysis of clonality pattern and chromosomal abnormalities showed that complete VDJH rearrangements were present in all hyperdiploid MM and in a small proportion (4/16) of the MM with normal karyotype. Conclusions: Our results confirm previous estimations about IgH repertoire usage. Despite the small numbers, our findings indicate that complete Ig rearrangements might be correlated with hyperdiploid MM. Combining cytogenetics and IgH clonality studies might help to identify distinct subgroups of MM and provide a framework for dissection of disease prognosis and clinical management. Research funded by Regione Autonoma Sardegna.

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Three Novel AML Cases Harboring the Semi-Cryptic t(7;21)(p22;q22) Translocation Expressing RUNX1-USP42 Fusion Transcripts Associated with Diploidy/Tetraploidy and/or 5q Alterations: a Probably Underestimated Combination

Blood ◽

10.1182/blood.v116.21.2708.2708 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2708-2708

Author(s):

Eric Jeandidier ◽

Carine Gervais ◽

Isabelle Radford-Weiss ◽

Catherine Gangneux ◽

Valerie Rimelen ◽

...

Keyword(s):

Bone Marrow ◽

Chromosomal Abnormalities ◽

Conflicts Of Interest ◽

Fusion Transcript ◽

Fish Analysis ◽

Chimeric Transcript ◽

Rt Pcr ◽

Balanced Translocation ◽

Fusion Transcripts ◽

Cryptic Translocation

Abstract Abstract 2708 RUNX1 is implicated in numerous chromosomal abnormalities acquired in acute myeloid leukemia (AML). The most frequent one, the t(8;21) is associated with a particular morphology together with a favorable prognosis. This is not the case for other 21q abnormalities, that are much less frequent and for which the prognosis is quite different. Moreover, beside point mutations, conventional cytogenetics failed to detect some of chromosomal alterations involving RUNX1. Recently 3 cases of the rare and semi-cryptic t(7;21)(p22;q22) translocation expressing the RUNX1-USP42 fusion transcripts have been reported, demonstrating the recurrence of this abnormality in AML. We describe here 3 additional cases with the same translocation and fusion transcripts, associated to 5q alterations leading to EGR1 and CSF1R heterozygous losses. In all our patients, the t(7;21)(p22.1;q22.3) was initially detected by the systematic FISH evaluation of the blastic populations using ETO-AML1 Dual Fusion probe. Patient#1 bone marrow karyotype was characterized by a tetraploid clone (89,XXYY) with loss of chromosomes 15, 17 and 18 in addition to the t(7;21), and a unbalanced translocation der(5)t(5;13)(q23;q?) between long arms of chromosomes 5 and 13, resulting in a heterozygous loss of EGR1 and CSF1R. Patient #2 blood and bone marrow karyotypes revealed a diploid clone with a del(5)(q31q33) associated with the t(7;21). The FISH analysis confirmed EGR1 and CSF1R deletions. In patient #3, the bone marrow karyotype showed diploid/tetraploïd clones, both harboring the t(7;21)(p22;q22), confirmed by FISH experiments (WCP7, AML1 probes). In addition, a der(5)t(1;5)(q3?2;q21-23) was identified within the tetraploïd clone, resulting in the loss of EGR1 and CSF1R, confirmed by FISH. In all three cases a RUNX1-USP42 fusion transcript was detected using RT-PCR, as well as the reciprocal transcript. Sequence analysis of RT-PCR products showed that the breakpoints occurred exactly in the same introns of USP42 and RUNX1 as in the previously described cases. For patient #1 and #3 a chimeric transcript was found formed of the RUNX1 exon 7 fused to the USP42 exon 3. In patient #2, a shorter chimeric transcript arised from the fusion of the RUNX1 exon 5 to the exon 3 of USP42. As already noticed in the previous reports, an alternative splicing of the RUNX1 exon 6 has been detected in these three cases. The description of these 3 novel t(7;21) confirm the recurrence of this balanced translocation in AML, and shows that this chromosomal abnormality is often associated with diploid/tetraploid clones and/or 5q alterations. Special attention should be paid in karyotype analysis of AML with diploid or tetraploid clones harboring 5q alterations. In such cases RUNX1 rearrangements should be explored using FISH analysis, and RUNX1-USP42 fusion transcript should be searched by RT-PCR in positive cases. Prospective and retrospective studies of AML have now to be settled in order to assess the incidence and clinical relevance of this cryptic translocation. Disclosures: No relevant conflicts of interest to declare.

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Coexistance of Multiple Myeloma and Myelodysplastic Syndrome or Lymphoid Diseases At Diagnosis: Evidence for Two Clonal and Independent Chromose Abnormalities

Blood ◽

10.1182/blood.v118.21.5063.5063 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5063-5063

Author(s):

Hossein Mossafa ◽

Sabine Defasque ◽

Christine Fourcade ◽

JeanPierre Hurst ◽

Bertrand Joly

Keyword(s):

Multiple Myeloma ◽

Plasma Cells ◽

Chromosomal Abnormalities ◽

Conflicts Of Interest ◽

Critical Role ◽

Chromosome 8 ◽

Trisomy 8 ◽

Hematopoietic Stem ◽

Monosomy 7 ◽

Trisomy 12

Abstract Abstract 5063 Introduction, We describe the simultaneous presentation of multiple myeloma (MM) and yeloproliferative disorders (MPD) or lymphoid diseases (LD) at diagnosis. Therapy-related myelodysplasia (tMDS) occurring during the course of MM is generally believed as a result from hematopoietic stem cell-toxic therapies, such as ionizing radiation and alkylating agent-based chemotherapies (melphalan, nitrosoureas).Patients and methods, We study a total of 342 patients (151 F, 191 M; median age 68.1 years; range 42 to 93 Years), diagnosed with MM based on the International Staging System. The basis for inclusion of patients in this study was with previous untreated MM ones. The study was performed in accordance with the declaration of Helsinki. To determine whether chemotherapies for MM factors play the critical role in the development of secondary disease, simultaneously two different cultures were processed, an unstimulated 96 hours culture (U96HC) on whole BM(WBM), a short-time 24 hours culture (ST24HC) after CD138+ plasma cells (PCs) depleted on negative fraction (CD138- cells) of BM and the FISH was investigated on purified CD138+.All samples were enriched in PCs by the Automated Magnetic Cell Sorter (Miltenyi technology)proceeded with anti-CD138 specific antibodies applied. The CD138+ PCs and the CD138- cells were collected in different tubes. The CD138− cells were used for a ST24HC. FISH was performed on the purified CD138+, PCs with a recommended FISH panel (MM International Working Group). Screening was performed systematically for the following unbalanced alterations and reciprocal rearrangements: del(13)(q14)(D13S25), del(17)(p13)(TP53),+3(D3Z), +9(D9Z1), +15(D15Z14), t(4;14)(p16;q32)/IGH-FGFR3, t(11;14)(q13;q32)/IGH-CCND1 (Abbott).After observing the results of U96HC on whole BM (CD138+ and CD138− cells), ST24HC (CD138− cells) and FISH for each patient, two clone cytogenetically were distinct and unrelated chromosomal abnormalities were found in 40 (11.7%) of the 342 MM patients (6 F, 34 M; median age 74 years; range 42 to 87 Years) 34 had a MPD and 6 had a LD. A second immunophenotyping analysis confirmed the presence of those LD/MM simultaneous haematological malignancy. In the cases of the patients with MM/ MPD, the frequency of cytogenetic abnormality unrelated to the myeloma clone was respectively; the 20q deletion, detected for 13 the 34 patients, the 20q- is a sole abnormality for 12 cases and associated with a complex caryotype in 1 case. The trisomy of chromosome +8 was observed in 7 cases, the del(7q) or monosomy 7 in 5 cases, loss of gonosome Y in 4 cases, del(11) for 2 cases, translocation t(9;22) in one case, 5q abnormality in one case and trisomy 9 with JAK2 V617F mutation in one case. For the patients with MM/LD, 5 patients had a trisomy +12 and or trisomy +18 like sole abnormality or associated with others cytogenetics abnormalities and one patient had 6q deletion. Discussion, Whereas in the literature the most common cytogenetic abnormalities typifying MPD after alkylator-based therapy include partial or complete deletions of chromosomes 5, 7, and 20 as well as trisomy 8. In our study we observed those abnormalities with the same frequency for the patients had simultaneous MPD associated in untreated MM at diagnosis. Six patients had simultaneous LD and MM. The marginal zone lymphoma was confirmed for 3 patients. The CC observed a trisomy +12 for those three patients associated with +18 and +19 for 2 cases and del(13) and trisomy 3 for one among them. We demonstrated in untreated MM patients the coexistence of MM and MPD or LD at diagnosis with MPD-type or LD-type chromosome abnormalities within MM signature karyotype. We hence recommend that CC studies, 96 hours WBM, 24 hours on negative fraction CD138− cells and FISH on purified CD138+ PCs, the three should be an integral part of the evaluation of patients with MM at diagnosis into clinical trials using HDT is warranted to determine whether patients who are predisposed to developing tMDS/sAML, they can be identified prospectively. Disclosures: No relevant conflicts of interest to declare.

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Genome Wide Profiling Of Chromosomal Abnormalities In 37 Patients With Monoclonal Gammopathy Of Undetermined Significance (MGUS)

Blood ◽

10.1182/blood.v122.21.1874.1874 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1874-1874

Author(s):

Aneta Mikulasova ◽

Vladimira Vallova ◽

Jan Smetana ◽

Henrieta Greslikova ◽

Renata Kupska ◽

...

Keyword(s):

Structural Changes ◽

Plasma Cells ◽

Array Cgh ◽

Chromosomal Abnormalities ◽

Conflicts Of Interest ◽

Multiple Displacement Amplification ◽

Chromosome 8 ◽

Genome Wide ◽

Technique Optimization ◽

Chromosomal Changes

Abstract Introduction The incidence of clonal DNA copy number alterations (CNAs) in plasma cells (PCs) is considered as one of the most important and independent prognostic factors in patients with multiple myeloma (MM). Also in the premalignancy MGUS, there are specific chromosomal changes in PCs such as del(13)(q14), IGHtranslocation, gain(1)(q21) and hyperdiploidy. However, not much is known about their significance in relation to malignant transformation. Also, MGUS research is further complicated by small number of malignant cells that could be obtained from patients. Aim Array-CGH technique optimization and DNA CNAs analysis in relation to prognosis at genome-wide level in MGUS patients. Material and methods We have analysed 37 MGUS patients (22M/15F; median age 62) using array-CGH, Agilent platforms “Human Genome CGH, 4×44K” (n=10) and “SurePrint G3 CGH+SNP, 4×180K” (n=27). DNA was isolated from separated PCs (using CD138, CD19 and CD56 markers) and amplified by multiple displacement amplification (MDA). Results CNAs were observed in 57% (21/37) MGUS patients. Numerical and structural CNAs were found in 46% (17/37) and 41% (15/37) MGUS patients, respectively. We distinguished two genetic subgroups similar to MM patients: hyperdiploid and non-hyperdiploid. Hyperdiploidy was present in 32% (12/37) MGUS patients. The most frequent whole-chromosome gains were: 9 (83%, 10/12), 19 (83%, 10/12), 3 (75%, 9/12), 11 (67%, 8/12) and 15 (58%, 7/12). Non-hyperdiploidy was detected in 68% (25/37) MGUS patients. In both, hyperdiploid and non-hyperdiploid subgroups we have found loss of chromosomes 13 (25% vs. 20%) and Y (17% vs. 4%). Loss of chromosome 8 was detected only in hyperdiploid subgroup (8%) and losses of chromosomes 21, 22 and X only in non-hyperdiploid subgroup (8%, 4% and 8%, respectively). Structural changes were significantly more often present in hyperdiploid than in non-hyperdiploid patients (67% vs. 28%, p<0.05). We have identified whole-arm chromosome changes 1q gain and 16q loss in both hyperdiploid and non-hyperdiploid patients (17% vs. 12%, 8% vs. 8%, respectively), but 16p gain was seen only in hyperdiploid patients (8%). Segmental chromosome changes were also present in both hyperdiploid and non-hyperdiploid patients (58% vs. 24%). Interestingly, we have detected more segmental losses (15 vs. 8) and gains (6 vs. 2) in hyperdiploid than in non-hyperdiploid patients, but size median of these losses (3.33 Mb vs. 19.6 Mb) and gains (36.1 Mb vs. 63.5 Mb) was smaller in hyperdiploid than in non-hyperdiploid patients. Moreover, we found one MGUS patient who has progressed to MM and required therapy after 6 months from MGUS diagnosis. Genome-wide profile of MGUS patient was especially unique by high number of structural changes (n=11) compared to other 15 MGUS patients with structural CNAs (median 1; 1 – 6). MGUS patient’s profile showed hyperdiploidy (gains of chromosomes 3, 5, 9, 11, 15 and 19), losses of chromosomes 8, 13 and Y, 7 segmental losses in areas 1p34.2-p13.1, 6p23, 6q12-q27, 7q36.3, 12p12.1-p11.23, 12q12, 12q21.2-q23.3 and 4 segmental gains in areas 6p25.3-p23, 6p23-p11.1, 6q11.1-q12, Xq21.33-q28. In addition, gains and losses, which included whole or large parts of chromosomes, showed unusual profile associated with other alterations. These findings have suggested a complex karyotype. Summary In our study we have optimized protocol of array-CGH analysis from amplified DNA and we have used it in first 37 MGUS patients. We found there are various chromosomal changes (numerical, whole-arm and segmental) in more than half of MGUS patients. Similar to MM, hyperdiploid and non-hyperdiploid genetic subgroups were identified. We also described one MGUS case with unique genome-wide profile indicating unfavourable prognosis. Support NT13492, NT13190, NT11154, OPVK CZ.1.07/2.3.00/20.0183, MSM0021622434, GAP304/10/1395 Disclosures: No relevant conflicts of interest to declare.

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Myelodysplastic Syndromes: A Multidisciplinary Integrated Diagnostic Work-up for Patients' Risk Stratification

Blood ◽

10.1182/blood.v124.21.5579.5579 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5579-5579

Author(s):

Elena Ciabatti ◽

Maria Immacolata Ferreri ◽

Angelo Valetto ◽

Alice Guazzelli ◽

Veronica Bertini ◽

...

Keyword(s):

Myelodysplastic Syndromes ◽

Mutational Analysis ◽

Conflicts Of Interest ◽

Normal Karyotype ◽

Diagnostic Workup ◽

Fish Analysis ◽

Rt Pcr ◽

Risk Scoring ◽

Conventional Cytogenetics ◽

Work Up

Abstract Conventional cytogenetics continues to have a fundamental role in the classification and risk scoring of myelodysplastic syndromes (MDS). Nevertheless, non-informative karyotypes represent up to 20% of cases. Some different molecular methods, not included in the routinary diagnostic workup, such as aCGH or mutational analysis, could be able to detect new abnormalities and improve the subtyping of MDS. The aim of this study was to propose a new diagnostic workup to determine the eventual adjunctive value offered by FISH, aCGH, and somatic mutation assays in respect of the the conventional cytogenetics only. In this study, we analyzed 50 patients: 29% female and 71% male, median age 71 (range 30-88 years), 66% at low/int1 IPSS risk, 54% at very-low/low R-IPSS risk, 33% with RCMD, 15% with RA, 14% with RARS, 14% with RAEB, 8% 5q-, and 16% with MMCL. We assessed these new MDS cases by different techniques: i) conventional cytogenetics; ii) FISH for chromosome 5, 7, PDGFRa, and PDGFRb; iii) aCGH, and iiii) specific RT-PCR for ASXL1, EZH2, TP53, and TET2 mutations. Conventional cytogenetics showed 42% of patients with at least one chromosomal aberration, including +8, del(11), del(7), del(5), -Y, +6, del(13), +14, del(20), and complex karyotypes (6% of cases). After FISH analysis, we were able to correctly classify as affected by the 5q- syndrome 2 cases who then received lenalidomide. The aGCH allowed to detect quantitative chromosomal aberrations in 44% of cases (del(13), -7, del(12), del(16), del(17), del(11), del(8), dupl(14), 5q-), including 10 cases (20%) showing a normal karyotype. After the RT-PCR, 32% of patients resulted mutated, with highest frequency for TP53 (22%). Four of these TP53-mutated patients showed normal karyotype, and resulted unmutated also by FISH and aCGH; in a case TP53 mutated we added treatment with steroid. Other 3 patients TP53-mutated did not respond to azacitidine Four low-risk patients (8%) showed ASXL1 gene mutation, three of them not earlier detected by cytogenetics or aCGH. One of these patients died after progression into acute leukemia. The identification of TP53 or ASXL1 mutations after RT-PCR and of dupl(14) by the aCGH prompted us to strictly follow patients at high risk of transformation. Only one case showed TET2 mutation; although TET2 mutations have been related to a better survival in patients receiving 5-azacitidine, this patient resulted not-responsive after 9 cycles. In conclusion, these results sustain the necessity of an integrated work-up for the diagnosis and the correct risk scoring of MDS patients. Disclosures No relevant conflicts of interest to declare.

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Acute Mixed Lineage Leukemia With an inv(8)(p11q13) Resulting in Fusion of the Genes for MOZ and TIF2

Blood ◽

10.1182/blood.v92.6.2118.418k09_2118_2122 ◽

1998 ◽

Vol 92 (6) ◽

pp. 2118-2122 ◽

Cited By ~ 7

Author(s):

Jian Liang ◽

Leonard Prouty ◽

B. Jill Williams ◽

Mark A. Dayton ◽

Kerry L. Blanchard

Keyword(s):

Acute Leukemia ◽

Chromosomal Abnormalities ◽

Fusion Gene ◽

Mixed Lineage Leukemia ◽

Chromosome 8 ◽

Acute Monocytic Leukemia ◽

Fish Analysis ◽

Creb Binding Protein ◽

Monocytic Leukemia ◽

Interaction Domain

Chromosomal abnormalities in acute leukemia have led to the discovery of many genes involved in normal hematopoiesis and in malignant transformation. We have identified the fusion partners in an inv(8)(p11q13) from a patient with acute mixed lineage leukemia. We show by fluorescence in situ hybridization (FISH) analysis, Southern blotting, and reverse transcriptase-polymerase chain reaction (RT-PCR) that the genes for MOZ, monocytic leukemiazinc finger protein, and TIF2,transcriptional intermediary factor 2, are involved in the inv(8)(p11q13). We demonstrate that the inversion creates a fusion between the 5′ end of MOZ mRNA and the 3′ end of TIF2 mRNA maintaining the translational frame of the protein. The predicted fusion protein contains the zinc finger domains, the nuclear localization domains, the histone acetyltransferase (HAT) domain, and a portion of the acidic domain ofMOZ, coupled to the CREB-binding protein (CBP) interaction domain and the activation domains of TIF2. The breakpoint is distinct from the breakpoint in the t(8;16)(p11;p13) translocation in acute monocytic leukemia with erythrophagocytosis that fuses MOZ with CBP. The reciprocalTIF2-MOZ fusion gene is not expressed, perhaps as a result of a deletion near the chromosome 8 centromere. TheMOZ-TIF2 fusion is one of a new family of chromosomal rearrangements that associate HAT activity, transcriptional coactivation, and acute leukemia. © 1998 by The American Society of Hematology.

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Atypical Chronic Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-110189 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5455-5455

Author(s):

Chandralekha Ashangari ◽

Praveen K. Tumula

Keyword(s):

Chronic Myeloid Leukemia ◽

Peripheral Blood ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Philadelphia Chromosome ◽

Myeloproliferative Neoplasm ◽

Peripheral Blood Flow ◽

Current Standard ◽

Atypical Chronic Myeloid Leukemia ◽

Wbc Count

Abstract Introduction: Atypical chronic myeloid leukemia (aCML), BCR-ABL1 negative is a rare myelodysplastic syndromes (MDS)/myeloproliferative neoplasm (MPN) for which no current standard of care exists. We present one of the rare presentations of aCML in an elderly patient. Case: A 76 year old male presented to the Hematology clinic for consultation after discharge from local hospital for elevated WBC count. Past medical history was significant for COPD, acid reflux, peripheral arterial disease and hypertension. Physical exam was unremarkable. Initial labs were significant for leukocytosis of 30 k/cu mm, anemia with Hb 10 gm/dl, thrombocytosis 695,000 with neutrophilia of ANC 25,200. Peripheral blood was negative for JAK2 V617F and BCR-ABL. Peripheral blood flow cytometry showed granulocytic left shift with 1.5% myeloblasts. Bone marrow biopsy suggestive of hypercellular marrow (100%) with myeloid predominance, atypical megakaryocytes, increased ring sideroblasts (49% of NRBC), increased blasts (5%) and dysgranulopoeisis over all suggestive of Myelodyplastic Syndrome/Chronic Myeloproliferative Disorder (MDS/MPD). Cytogenetics were positive for U2AF1 positive, CSF3R T6181, CSF3R Q776 pathognomonicof atypical CML and negative for BCR-ABL, FLT3. He was considered transplant ineligible. He was started on Azacitadine and is currently receiving 2nd cycle therapy. He is also receiving darbepoeitin periodically to avoid frequent transfusions. He is currently transfusion independent. Discussion: Increased WBC count (e.g., cutoffs of >40×109/L or 50×109/L), increased percentage of peripheral blood myeloid precursors, female sex, and older age are adverse prognostic factors for overall survival or leukemia-free survival in aCML. aCML cases lack in Philadelphia chromosome. Overall 50-65% of patients show cytogenetic abnormalities. The most frequent is +8 (25%). Other changes such as -7 and del(12p) have also been recurrently observed. Patients with aCML have an estimated median survival between 14 and 30 months. aCML tends to exhibit a more aggressive clinical course than other MDS/MPN subtypes. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

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