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

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.

Download Full-text

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.

Download Full-text

Distinctive Spectra of Cytogenetic Alterations in Over Five Hundred East Asian Patients with Acute Leukemia

Blood ◽

10.1182/blood.v118.21.4408.4408 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4408-4408

Author(s):

Hyun-Jung Choi ◽

Hye Ran Kim ◽

Myung-Geun Shin ◽

Jong-Hee Shin ◽

Bock-Hee Park ◽

...

Keyword(s):

Acute Leukemia ◽

Chromosomal Aberrations ◽

Molecular Detection ◽

Conflicts Of Interest ◽

Detection System ◽

East Asian ◽

Normal Karyotype ◽

Fusion Genes ◽

Rt Pcr ◽

Conventional Cytogenetics

Abstract Abstract 4408 Background: Racial difference of spectra of chromosomal aberrations in various hematological malignancies was previously reported between Asian and western countries. Therefore, characterization of the cytogenetic profile is still a matter of interests for identifying cytogenetic risk factors in the current risk-adapted chemotherapy. The aim of this study was to investigate the spectrum of chromosomal aberrations in East Asian (Korean) acute leukemia patients, and to propose a panel of leukemic fusion genes suitable for the development of new molecular detection systems. Patients and Methods: We prospectively analyzed bone marrow samples from 502 patients with acute leukemia referred for screening leukemic fusion genes by simultaneous analysis of conventional cytogenetics, FISH and multiplex RT-PCR system in Chonnam National University Hwasun Hospital (Hwasun, Korea) for last 5 years. Results: A total of 334 patients were diagnosed with AML, 155 with ALL and 13 with mixed phenotype acute leukemia. Twenty types in 28 fusion genes were detected in 42.4% of the total patients by multiplex RT-PCR system and in 33.9% by conventional cytogenetics including FISH. In the group of AML, the common fusion genes were 57 PML/RARa, 38 AML1/MGT8, 12 CBFb/MYH11, 11 MLL1, 5 BCR/ABL1, SET/CAN, PLZF/RARA and TLS/ERG in 2 cases each, and one of each AML1/MDS1, TEL/ABL and TEL/MN1. In the group of ALL, the following fusion genes were detected: 33 BCR/ABL1, 22 TEL/AML1, 10 MLL1, 7 E2A/PBX, 2 SET/CAN and SIL/TAL, one TEL/ABL. In addition, cytogenetically cryptic translocations were detected in 14.8% patients with normal karyotype or numerical aberrations by conventional cytogenetics. Among 288 patients with negative results by multiplex RT-PCR system, one child with early pre-B-ALL harbored MLL1/AF4, and 8 patients had 3 types of clinically significant chromosomal aberrations such as t(3;3)(q21;q26.2), t(8;14)(p24.1;q32) and i(17)(q10), which were all confirmed by conventional cytogenetics. Conclusions: The current study demonstrated the spectrum and frequency of chromosomal aberrations in Korean patients with acute leukemia, which are different from Caucasian data. Also these results may offer important implications for the development of new multiplex molecular detection system. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Results of Conventional Cytogenetics and Interphase FISH (I-FISH) Analyses in Patients with a Clinical and Morphologic Diagnosis of CML: Analysis of 52 Cases.

Blood ◽

10.1182/blood.v104.11.4423.4423 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4423-4423

Author(s):

Seema Gupta ◽

Ellin Berman ◽

Suresh Jhanwar

Keyword(s):

Dna Sequences ◽

Lymphoblastic Leukemia ◽

Normal Karyotype ◽

Genetic Alterations ◽

Fish Analysis ◽

Newly Diagnosed ◽

Rt Pcr ◽

Trisomy 8 ◽

Ph Chromosome ◽

Conventional Cytogenetics

Abstract The presence of the Philadelphia chromosome (Ph), derived from a balanced translocation between chromosomes 9q and 22q, serves as a hallmark for the diagnosis of CML or Ph-positive ALL. In recent years, I-FISH has become the method of choice to detect Ph-positive clones at diagnosis and during treatment. In addition, I-FISH analysis is also able to identify variant translocations and loss of DNA sequences, some of which are known to be predictors of poor clinical outcome. To determine the contribution of I-FISH in detecting genetic alterations at diagnosis and during therapy, we summarized our experience in a series of patients with CML and Ph+ ALL studied in a single institution between 2000–2004. A total of 52 patients had both cytogenetic and I-FISH analyses available for review. The 52 patients included 30 patients in chronic phase, 10 patients in accelerated phase, 9 patients in blastic phase, and 3 patients with acute lymphoblastic leukemia (ALL). Sixteen of the 52 patients had newly diagnosed CML, and the remaining 36 patients had studies performed while on imatinib. Conventional cytogenetics detected the Ph-chromosome in 44 patients (85%), whereas I-FISH detected Ph chromosome positivity in all 52 patients (100%). In three of the 16 patients with newly diagnosed disease, I-FISH identified variant translocations in the setting of a normal karyotype. Two of these 3 patients were found to be RT-PCR negative for bcr/abl transcripts. Nineteen of 52 patients (37%) had additional abnormalities detected by karyotype. Nine of these 19 patients (47%) had more than one additional cytogenetic abnormality. The most common abnormalities in the Ph+ clone included: 6 patients with 2 copies of the Ph chromosome, 4 patients with trisomy 21, 2 patients with deletion (20q), 2 patients with isochromosome 17, and 2 patients with trisomy 8. Independent clones with trisomy 8 were noted in an additional 2 patients on imatinib therapy. A total of 12 of the 52 patients (23%) had variant signal patterns by I-FISH; loss of DNA sequences on chromosomes 9 and 22 occurred in 4 and 3 cases respectively and a variant signal pattern involving additional chromosomes was seen in 5 patients. While in general we found a good correlation between the proportion of Ph-positive cells detected by conventional cytogenetics and I-FISH, differences between the two types of analyses were observed in 8 patients. Six patients had a normal karyotype (20 cells analyzed) with positive I-FISH analysis (range 3.2–99%). Two of the 8 patients had a small percentage of Ph-positive metaphases with negative I-FISH. These results underscore the significance of utilizing both conventional cytogenetics and I-FISH in CML patients at diagnosis as well as during therapy. The biologic and clinical implications of the disparity between conventional cytogenetics, I-FISH, and in some cases RT-PCR results are not yet fully realized.

Download Full-text

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.

Download Full-text

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.

Download Full-text

Absence of Mutations of the Histone Methyltransferase Gene EZH2 In Splenic B-Cell Marginal Zone Lymphoma

Blood ◽

10.1182/blood.v116.21.5086.5086 ◽

2010 ◽

Vol 116 (21) ◽

pp. 5086-5086

Author(s):

Luz Martínez-Avilés ◽

Marta Salido ◽

Beatriz Bellosillo ◽

Vera Adema ◽

Ana Ferrer ◽

...

Keyword(s):

B Cell ◽

Marginal Zone ◽

Mutational Analysis ◽

Conflicts Of Interest ◽

Direct Sequencing ◽

Normal Karyotype ◽

Marginal Zone Lymphoma ◽

Low Grade ◽

Splenic Marginal Zone Lymphoma ◽

7Q Deletion

Abstract Abstract 5086 Background Splenic marginal zone lymphoma (SMZL) is a rare low-grade B-cell lymphoproliferative disorder with characteristic clinical, cytological, histological and immunophenotypical features. The most common cytogenetic abnormality, present in 30–40% of the patients is the 7q deletion, that extends from 7q21 to 7q36. This aberration may represent a primary pathogenic event in SMZL. Recently, mutations in the EZH2 gene, located at 7q36.1, have been described in different hematological malignancies including B-cell lymphomas. However, the role of the EZH2 gene in SMZL has to be elucidated. Aim To determine the prevalence of EZH2 mutations in a cohort of SMZL patients. Patients and Methods Twenty-nine patients with SMZL were screened for mutations in the EZH2 gene. From the whole cohort, 11 patients presented 7q deletion (three of them as a single anomaly), 11 had a normal karyotype and 7 had other cytogenetic aberrations. The mutational analysis of the EZH2 gene was performed by direct sequencing using primers covering the whole exome of the gene. DNA was extracted from CD19 isolated B-cells from peripheral blood or from total lymphocytes if the percentage of pathologic B-cell was higher than 50%. Results From the whole cohort of 29 SMZL patients, no pathogenic mutations (frameshift or nonsense mutations) were detected in the EZH2 gene in any of the patients analyzed. Five patients harboured the missense mutation D185H in exon 6, that has been previously described as a single nucleotide polymorphism (SNP). Conclusions In conclusion, the EZH2 gene is not mutated in our series of SMZL patients suggesting that this gene is not involved in the pathogeny of this entity. Acknowledgments: Fellowship FI2008 (AGAUR) to LMA, This work was supported (in part) by grants from Instituto de Salud Carlos III FEDER; Red Temática de Investigación Cooperativa en Cáncer (RTICC, FEDER): RD06/0020/0031 and RD07/0020/2004; Ministerio de Sanidad y Consumo (Spain): PI07/0586. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Bone Marrow Failure with 13q Deletion: A Distinct Clinicopathological Entity with Immune Pathophysiology,

Blood ◽

10.1182/blood.v118.21.3420.3420 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3420-3420

Author(s):

Kohei Hosokawa ◽

Takamasa Katagiri ◽

Naomi Sugimori ◽

Ken Ishiyama ◽

Yumi Sasaki ◽

...

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Survival Rates ◽

Snp Array ◽

Normal Karyotype ◽

Genetic Alterations ◽

Fish Analysis ◽

Hematopoietic Stem ◽

Who 2008

Abstract Abstract 3420 Background: Numerical karyotypic abnormalities such as −7/del(7q) and del(13q) are occasionally seen in patients with bone marrow (BM) failure who do not have typical signs of myelodysplasia. The WHO 2008 defined this subset of BM failure as MDS-U because of its likely association with a risk of evolving into leukemia, while the presence of isolated abnormalities including +8, del(20q), and -Y was not considered to be presumptive evidence of MDS. Previous studies showed that BM failure patients with del(13q) responded to immunosuppressive therapy (IST) and had a favorable prognosis (Ishiyama K et al, Br J Haematol; 117: 747. 2002; Sloand, JCO 2010). However, the clinical features of del(13q) BM failure remain unclear due to its low incidence as well as the frequently associated karyotypic abnormalities. Objectives/Methods: To characterize the clinicopathological features of patients with BM failure with del(13q), this study reviewed the clinical data of 1705 BM failure patients (733 with AA, 286 with MDS-RCUD, 149 with RCMD, 60 with MDS-U) whose blood was examined for the presence of glycosylphosphatidyl-inositol anchored protein (GPI-AP)-deficient granulocytes and erythrocytes from May 1999 through July 2010. Genomic DNA was isolated from the peripheral blood cells of 7 patients with 13q- and was subjected to SNP array-based genome-wide analysis for genetic alterations using GeneChip® 250K arrays to identify the gene locus that is commonly deleted as a result of 13q-. Results: The 13q- clone was found in 25 (1.5%) of the 1705 patients. All the 13q- patients were classified as MDS-U, due to the absence of significant dysplasia to fulfill the criteria for MDS defined by the WHO 2008 classification. BM was hypocellular in 17 patients and normocellular in 6. Seventeen patients had a clone with 13q- alone, while the remaining 8 patients had a clone with 13q- and other numerical abnormalities including –Y, +mar in 2, and −20, del(7q), +8, der(1;7) in 1. A significant increase in the percentage of GPI-AP- granulocytes was detected in 366 (50%) of 733 patients with AA and 115 (23%) of 495 patients with MDS. GPI-AP- cells were detected in all (100%) of the 17 patients with 13q- alone. On the other hand, the prevalence of increased GPI-AP− cells in patients with 13q- plus other abnormalities and in those with the normal karyotype was 38% (3/8) and 43% (405/937), respectively. Fifteen patients with 13q- alone were treated with IST (ATG + cyclosporine in 6 and cyclosporine ± anabolic steroid in 9) and all of them achieved either a PR or a CR, while in the patients with 13q- plus other abnormalities, the response rate to IST was 40%. A total of 106 patients with the normal karyotype were treated with ATG+CsA (48) or CsA±AS (58) and the response rates were 73% and 85%, respectively. None of the 17 13q- patients progressed to advanced MDS or AML during the follow-up period of 3 to 108 months (median: 52 months) while 2 of 8 patients with 13q- plus other abnormalities developed AML. The 5-year overall survival rates of the patients with 13q-, those with 13q- plus other abnormalities, and patients with a normal karyotype were 84%, 45%, and 91%, respectively. The percentage of 13q- clones increased in 5 patients, and decreased in 3 patients after successful IST. When GPI-AP- and GPI-AP+ granulocytes were subjected to a FISH analysis using a 13q probe (13q14.3), the 13q- clones were detected only in of GPI-AP+ granulocytes, suggesting that 13q- cells are derived from non-PIG-A mutant HSCs. SNP arrays identified 13q13.3 to 13q14.3 regions in all cases. Conclusions: MDS-U with 13q- is a benign BM failure syndrome characterized by a good response to IST and a markedly high prevalence of GPI-AP cells. Patients with this type of BM failure may be inappropriately treated with hypomethylating agents or hematopoietic stem cell transplantation from unrelated donors, which is associated with high therapy-related mortality. Therefore, del(13q) should be eliminated from the intermediate prognosis group defined by the IPSS, and BM failure with del(13q) should be managed as idiopathic AA. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Array CGH As a Complementary Tool in the Diagnosis of Myelodysplastic Syndromes

Blood ◽

10.1182/blood.v120.21.3827.3827 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3827-3827

Author(s):

María Abáigar ◽

Eva Lumbreras ◽

Irene Rodríguez ◽

Javier Sánchez-del-Real ◽

María Díez-Campelo ◽

...

Keyword(s):

Risk Stratification ◽

Myelodysplastic Syndromes ◽

Copy Number ◽

Normal Karyotype ◽

Chromosome 8 ◽

Comparative Genomic ◽

Whole Genome ◽

Acgh Analysis ◽

Conventional Cytogenetics ◽

Copy Number Changes

Abstract Abstract 3827 Background: Myelodysplastic syndromes (MDS) are a heterogeneous group of hematological disorders in which diagnosis, risk stratification, and treatment selection are based on morphological and cytogenetic studies in bone marrow (BM) samples. MDS are characterized by several recurrent chromosomal abnormalities, most of them unbalanced, with a widely variable prognosis. The assessment of these genomic defects is essential for a correct risk stratification of these patients. However, conventional cytogenetic (CC) techniques are not sufficient for the study of all MDS patients, because of the high proportion of normal karyotypes (40–50%) and unsuccessful cytogenetics (10%) (defined as the absence of mitosis). Array-based comparative genomic hybridization (aCGH) technology allows the screening of copy number changes among the whole genome in one single experiment and offers a higher resolution than conventional cytogenetics. Aims: To assess the potential application of aCGH in the clinical diagnosis of MDS as complementary tool to conventional cytogenetics. Patients and Methods: The study cohort comprises a total of 263 patients: MDS (203) and MDS/MPN (60) patients that have been previously studied by CC and FISH. Among the whole series, 33 (12.5%) patients had no successful cytogenetic results due to the absence of mitosis. In the remaining 230 patients with evaluable metaphases, 42 (16%) had an aberrant, while 188 (71.5%) presented a normal karyotype. Within this last group, 141 had ≥20 good-quality metaphases evaluated, 37 had 10–20 metaphases studied, and 10 patients had ≤10 successful metaphases. Copy number changes were analysed in all patients included in the study using NimbleGen Human CGH 12×135K Whole-Genome Tiling Array (Roche NimbleGen). Sex-matched human commercial DNA samples were used as reference. Data were analysed using the segMNT algorithm in NimbleScanv2.6 Software. Subsequently all genomic abnormalities found by aCGH analysis were confirmed by FISH. Results: Using aCGH methodology, copy number changes (greater than 600 bp) were detected in 54 patients of the global series: 4.3% of the normal karyotype patients, 88.1% of cases with abnormal cytogenetics, and 27.3% of patients with unsuccessful cytogenetics. Overall a high correlation (94.3%) between the cytogenetic changes observed by CC and CGH arrays was observed. Thus aCGH analysis revealed the same genomic abnormalities showed by CC in 88.1% of patients. In the remaining 11.9% genomic results were discordant between aCGH and CC, because of the presence of balanced translocations, not assessable by aCGH, and clonal cell populations below 30%. Furthermore, additional genomic abnormalities (n=36) not detected by CC were found by aCGH. The most frequent aberrations were losses affecting chromosomes 5 (33%), 7/7q (17%), 20q (14%), and Y (14%), as well as gains involving chromosome 8 (14%). Interestingly, other abnormalities, mainly losses, were found in chromosomes 4, 12, and 17. Focusing on the 188 patients with normal karyotype by CC, the aCGH profiling results were concordant with cytogenetics in 98% of those patients with ≥20 metaphases studied and in 92% of those with 10–20 metaphases. However, only 80% of those patients with ≤10 successful metaphases and no changes by CC displayed no copy number changes by aCGH. The most frequent abnormality found by aCGH among these normal karyotype cases was the presence of 5q deletion (2%), while other chromosomes affected were 7, 8, 11, 12 and 20. All these abnormalities were confirmed by FISH. Regarding the patients with unsuccessful cytogenetics, 72.7% of cases displayed a normal aCGH profile, while 27.3% showed at least one genomic imbalance The most frequent genomic aberrations were losses in 4q (6%), 5q (12%) and 7q (9%), and gain of chromosome 8 (6%). In addition, three of these cases showed a complex karyotype, showing more than 5 abnormalities. Conclusion: The use of aCGH karyotyping in the diagnosis of MDS could be used as a complementary technique to conventional karyotyping in the evaluation of MDS patients. Mainly in patients with unsuccessful cytogenetics and those with normal karyotype and <20 good-quality metaphases evaluated. Disclosures: Hernández: Celgene: Research Funding.

Download Full-text

Lenalidomide in Myelodysplastic Syndromes with 5q Deletion. Results From the Italian National Cancer Registry

Blood ◽

10.1182/blood.v120.21.3850.3850 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3850-3850

Author(s):

Cristina Mecucci ◽

Andrea Roncadori ◽

Roberta La Starza ◽

Francesco Arcioni ◽

Alessandro Levis ◽

...

Keyword(s):

Median Time ◽

Myelodysplastic Syndromes ◽

Normal Karyotype ◽

Hematological Toxicity ◽

Laboratory Findings ◽

Transfusion Independence ◽

Drug Agency ◽

Conventional Cytogenetics ◽

Grade 3 ◽

5Q Deletion

Abstract Abstract 3850 Background: Lenalidomide (LEN) is available in Italy for patients with intermediate-1- and low-risk myelodysplastic syndromes (MDS) associated with 5q deletion (5q-) since October 2008, based on a local disposition of the Italian Drug Agency (AIFA) issued according to a national law (Law 648/96). LEN is an oncological drug subject to intensive monitoring and, when it is prescribed in MDS, physicians are requested to enter the patient into a registry, which is entirely web-based. Methods: This observational, retrospective, multicenter study (registered at www.clinicaltrials.govasNCT01347944) has enrolled patients at 40 centres of 43 authorized in Italy. The study was approved by the ethic committee of each participating institution. The purpose was to analyse data available in the Registry and to integrate them with additional clinical and laboratory findings by filling up new e-forms in all cases with MDS and 5q- receiving LEN from October 31st, 2008 until the present. Diagnosis, treatment, follow-up and re-evaluation of patients were considered. Results: Study population included patients pre-treated and not pre-treated, with or without the integration form at the end of treatment. Pre-treated were defined as patients who were given LEN (from 1 to 12 cycles, median 3) before entering the AIFA registry. 158 patients (42 pre-treated and 116 not pre-treated) were eligible for this preliminary analysis. Median age was 75 (range, 38–89 years); 6 patients were younger than 50. There were 108 females (68.35%) and 50 males (31.6%) (table). Hematological findings evaluated at diagnosis were: hemoglobin (<8.5g/dL, 43% and >8.5g/dL, 57%), macrocytosis (MCV >98 fL, 54.4%), platelet count (<140×109/L, 24.7%; between 140 and 400×109/L, 56.3%; >400×109/L, 19%). Median white blood cell count was 4× 103/L with 54% neutrophils. Bone marrow blasts ≤5% were found in 75.9% of cases. The 5q- was detected by conventional cytogenetics in 131/158 cases (82.9%): it was isolated in 110 cases, associated with one additional in 15, and in complex karyotypes in 6. In 2 cases with normal karyotype and in 25 cases with failed cytogenetics the 5q- was demonstrated by fluorescence in situ hybridization (FISH). These cases could not be grouped as isolated or non-isolated. Patients received LEN at a daily dose of 10mg or 5 mg for 21 of 28 days as the starting dose. The dose and schedule was adjusted mainly based on blood count and hematological toxicity. Median time on treatment was 15.5 months (range, 1–45). Major and minor criteria for hematological and cytogenetic response are under evaluation. Transfusion independence at 6 months was reached in 77/103 cases (74.75%). Hematological toxicity could be evaluated in 148 cases. A total of 555 neutropenia events (36% grade 3 and 12.9% grade 4) and 422 thrombocytopenia events (10.9% grade 3 and 6.16% grade 4) were seen. Among 69 evaluable cases, 11 (15.9%) developed acute myeloid leukemia. In this group the median age was 72 (range, 62–85) and there were 4 males and 7 females. The median number of LEN cycles was 6 (range, 2–31). The median time from diagnosis of MDS was 34 months (range, 4–68). Chromosome 5q- at diagnosis was demonstrated by conventional cytogenetics in 8 cases (6 isolated and 2 with one additional change) and by FISH in 3. Comments: The Italian Drug Agency (AIFA) Registry provided us with a large series of MDS cases with 5q- to investigate appropriatness of LEN prescription and management. As expected LEN was successful to obtain transfusion independence in this patient population. Hematological toxicity was manageable. The number of cases with leukemic evolution is in line with data reported in the literature. These cases will be further investigated. Disclosures: Mecucci: CELGENE: Research Funding.

Download Full-text

Interest of the XN-10® Analyzer to Screen for Myelodysplastic Syndromes on Complete Blood Count (CBC) Especially with Hemoglobin Levels and Neut-WX

Blood ◽

10.1182/blood.v128.22.5518.5518 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5518-5518

Author(s):

Robin Boutault ◽

Sebastien Tremblais ◽

Mathilde De Oliveira Lopes ◽

Pierre Peterlin ◽

Yannick Le Bris ◽

...

Keyword(s):

Myelodysplastic Syndromes ◽

Blood Count ◽

Complete Blood Count ◽

Conflicts Of Interest ◽

Normal Karyotype ◽

University Hospital ◽

Multilineage Dysplasia ◽

End Point ◽

Group 2 ◽

A Prospective Study

Abstract A prospective study was performed over one year at Nantes University Hospital in France, in order to investigate whether suspected myelodysplastic syndromes (MDS) could be detected on a complete blood count (CBC), the most rapid laboratory investigation. Indeed, the recently developed XN-10® (Sysmex, Kobe, Japan), provides novel CBC parameters witch could be useful to discriminate such patients from normal samples or from cytopenia of other etiology. Seventy-nine patients were enrolled in the study, for whom a diagnosis of MDS was concluded based on CBC, bone marrow smears examination and karyotype. All patients were free of treatment, including transfusions, at inclusion. They were 40 men and 39 women with a median age of 77,9 years (range 36,4-92,4). CBC were performed on a Sysmex analyzer XN-10®, including investigation of reticulocytes and fluorimetric analysis of platelets. For comparison with normal values, results from 776 healthy samples, for which CBC were performed on the same analyzer and generated no flag, were used. All had parameters within the normal range according to age. The classical parameters of hemoglobin level, Mean Corpuscular Volume (MCV), reticulocytes, platelets and neutrophil counts were recorded. In addition, the extra-parameters, immature reticulocytes fraction (IRF%), platelets by fluorescence (PLT-F) and immature platelets fraction (IPF%), were taken into account. The neutrophils median position on the three axes as well as their dispersion (Neut-WX) were also measured by the analyser. The primary end-point was to discriminate between MDS and healthy patients and the secondary end-point was to distinguish MDS with excess blasts, MDS with multilineage dysplasia and MDS with single lineage dysplasia within the MDS group and by comparison with controls. According to the WHO 2016 classification, 27 patients in the cohort had MDS with excess blasts, 26 MDS with multilineage dysplasia (among whom 7 had ring sideroblasts [RS], group 2), 16 MDS-RS and single lineage dysplasia, 7 MDS with single lineage dysplasia and 3 MDS with isolated del(5q). Forty-four patients had a normal karyotype and 28 displayed anomalies classically reported in MDS, including 5 complex karyotypes. Among the latter, 4 were associated with MDS with excess blasts. Both classical and extra parameters indeed showed significant differences between the subgroups tested. Among the whole group of MDS patients, a number of parameters of all lineages were statistically different from the healthy cohort. The median level of hemoglobin was 9,8 g/dL (range 4,7-14,9), (p<0,0001), the median MCV 104,3 fL (range 75,4-123,9; p<0,0001), reticulocyte counts 44,3x109/L (range 8-165,9; p=0,041) and IRF% 16,7% (range 2,4-50,9; p<0,0001). An hemoglobin value below 11,5 g/dL was strongly suggestive of MDS with a sensitivity of 81% and specificity of 100%. The median platelet count was 164x109/L (range 8-505; p<0,0001) and median IPF% 8,8% (1,2-42; p<0,0001). Among leukocyte parameters, the MDS median neutrophil count was significantly lower at 2,15x109/L (range 0,17-13,67; p<0,001) and the Neut-WX value increased above 350. The latter, by itself, allowed to make a diagnosis of MDS with a sensitivity of 73,1% and a specificity of 96,9%. When considering the three MDS subgroups of MDS with excess blasts, multilineage or single lineage dysplasia, although each of them was significantly different from controls for hemoglobin levels, MCV, IRF% and neutrophil counts (p<0,0001), they could not be discriminated by these parameters. In the subgroup of MDS with single lineage dysplasia, platelet counts were similar to those of controls, yet significantly higher than for MDS with excess blast or with multilineage dysplasia (p=0,004 and p=0,029 respectively). Taken together, this study demonstrates that a simple CBC allows to screen for MDS using thresholds of 11,5 g/dL for hemoglobin and of 350 for Neut-WX. Blood smear examination should be performed in this situation even if the XN-10® analyzer does not raise an alarm, especially in unknown older patients. Disclosures No relevant conflicts of interest to declare.

Download Full-text