Teresa de Souza Fernandez

Chromosomal alterations associated with evolution from myelodysplastic syndrome to acute myeloid leukemia

Several studies have demonstrated the prognostic value of cytogenetic analysis in MDS both for survival and progression to AML. However it is unknown which are the numerical or structural abnormalities required for leukemic transformation. In this report we studied clinically and cytogenetically 127 patients: 125 with primary MDS and two with AML with a previous history of MDS. Thirty-one patients (24%) showed evolution of the disease during the follow-up study. Chromosomal abnormalities found at diagnosis in patients that progressed toward AML included: del(5)(q15), +6, del(6)(q21), t(5;8)(q32;q22),-7, del(7)(q22), der(7)t(1;7)(q10;p10), t(7;11)(p15;p15), +8, del(11)(q23), del(12p), del(3)(q21), del(20)(q12) and complex karyotypes. Eight of these patients were studied cytogenetically during transformation and showed acquisition of chromosomal alterations involving dup(1q), +8, del(11)(q23), and translocations between chromosomes 1 and 8 or 7 and 17. In addition we also observed gain of ploidy and monosomy 21. These results suggest that chromosomal alterations during evolution of the disease include special chromosome gains or abnormalities of chromosomes 1, 7, 8, 11 and 17 with involvement of ETV-1, Hox-A9, Pax 4, MLL genes besides a putative gene mapped at 17q25. We also applied the International Prognostic Scoring System (IPSS) to 114 patients, excluding those submitted to allogeneic bone marrow transplant. Our patients were classified into four distinct risk groups. The analysis of risk groups presented by 27 patients who showed evolution of the disease revealed 18 at the high risk group and four at the intermediate-2 group. From the intermediate-1 risk group only five patients showed evolution of the disease. Three of these patients evolved from RA to RAEB with gain of a del(11)(q23) or an expansion of a del(12)(p12) clone. Our results suggest that some chromosomal alterations are responsible for each step in the evolution of the disease. As the pathway of evolution is not unique it has been very difficult to define what genetic alteration comes first. However from several results in the literature and our own, it seems that some chromosomal alterations may predict the evolution of the disease and are correlated with short survival, as for example the trisomy of chromosome 8, and might be incorporated in the high risk group in the IPSS. This score system has been proved to be useful for predicting survival and evolution from MDS to AML.

Correlation of N-ras point mutations with specific chromosomal abnormalities in primary myelodysplastic syndrome

A cytogenetic and N-ras point mutation study was done in patients with primary myelodysplastic syndrome (MDS) from Rio de Janeiro, Brazil, in order to evaluate the progression of preleukemic states to overt leukemia. Cytogenetic analysis was performed in 50 patients with MDS and clonal chromosomal abnormalities were detected in 19 (38%) of them. Patients with refractory anemia (RA) or with ringed sideroblasts (RARS) presented normal karyotypes or single abnormalities as del(5q) or -Y, while patients in more advanced states as RA with excess of blasts (RAEB), RAEB in transformation (RAEB-t) and chronic myelomonocytic leukemia (CMML) showed complex karyotypes and single abnormalities involving chromosomes 7 or 8, which were related to poor prognosis and elevated risk of transformation to acute myeloid leukemia (AML). The frequency of ras activation was studied in these 50 patients with MDS. Samples of bone marrow were screened for oncogenic point mutations by DNA amplification followed by oligonucleotide hybridization analysis (PCR-ASO) at codon 12 of N-ras proto-oncogene. We detected N-ras point mutations in 21 patients (42%). Progression from MDS to AML was observed in 9 patients (18%). The correlation analysis between N-ras point mutations and specific chromosomal abnormalities indicated that although mutated N-ras was found in cells with del(5q) and monosomy 7, cells with those abnormalities and normal N-ras were also identified. Otherwise trisomy of chromosome 8 showed a correlation with N-ras point mutations and in all cases, patients showed progression of MDS to AML during the follow-up study. MDS comprises a heterogeneous group of hematopoietic disorders and probably several steps are implicated in the evolution to AML. In this work we suggest that one possible pathway of leukemogenesis in MDS includes N-ras point mutations in association with trisomy of chromosome 8.

Epigenetic alterations of p15INK4B and p16INK4A genes in pediatric primary myelodysplastic syndrome

We studied the methylation status of the p15INK4B and p16INK4A genes in 47 pediatric patients with primary MDS, its correlation with subtype, and the role of p15INK4B and p16INK4A in the evolution of MDS toward AML. Aberrant methylation of the p15INK4B gene was detected in 15 of 47 patients (32%), whereas only four patients demonstrated methylation of the p16INK4A gene (8%). The frequency of p15INK4B methylation was significantly higher in RAEB and RAEB-t subtypes (p < 0.003). Aberrant methylation of the p16INK4A gene was also more frequent in the subtypes that characterize advanced stages of the disease (p < 0.05). Evolution of disease was verified in 17 (36%) of the 47 patients. The association of p15INK4B and p16INK4A methylation status with evolution of disease was clearly significant (p < 0.008 and p < 0.05, respectively). These results suggest that methylation of the p15INK4B and p16INK4A genes is an epigenetic biomarker of pediatric disease evolution.

TET2 expression level and 5-hydroxymethylcytosine are decreased in refractory cytopenia of childhood.

Myelodysplastic syndromes (MDS) are myeloid malignancies characterized by ineffective hematopoiesis, dysplasia, peripheral cytopenia and increased risk of progression to acute myeloid leukemia. Refractory cytopenia of childhood (RCC) is the most common subtype of pediatric MDS and has overlapping clinical features with viral infections and autoimmune disorders. Mutations in TET2 gene are found in about 20-25% of adult MDS and are associated with a decrease in 5-hydroxymethylcytosine (5-hmC) content. TET2 deregulation and its malignant potential were reported in adult but not in pediatric MDS. We evaluated the gene expression and the presence of mutations in TET2 gene in 19 patients with RCC. TET2 expression level was correlated with 5-hmC amount in DNA and possible regulatory epigenetic mechanisms. One out of 19 pediatric patients with RCC was a carrier of a TET2 mutation. TET2 expression and 5-hmC levels were decreased in patients when compared to a disease-free group. Lower expression was not associated to the presence of mutation or with the status of promoter methylation, but a significant correlation with microRNA-22 expression was found. These findings suggested that TET2 downregulation and low levels of 5-hmC are inversely related to miR-22 expression. The existence of a regulatory loop between microRNA-22 and TET2 may play a role in MDS pathogenesis.

Cytogenetic and immunophenotypic evidence of independent clonal origins of concomitant chronic lymphocytic leukaemia and acute myeloid leukaemia

To the Editor: Although chronic B-cell leukaemia (B-CLL) is the most common leukaemia in the Western world, the association of CLL and acute leukaemia (AL) is a rare event. The presentation of acute myeloid leukaemia (AML) concomitantly with CLL is unusual and, so far, only 16 cases have been described (1). In the most of these cases the characterization of the diseases has been made by morphology and immunophenotyping studies (1–4), and only in two cases was a cytogenetic study carried out (5, 6). We report a case with the simultaneous occurrence of CLL and AML without previous exposure to a cytotoxic agent or irradiation. Immunophenotyping and cytogenetic studies were performed, and the clonal origins of the diseases are discussed. A 70-yr-old Portuguese woman was hospitalized with a one-month history of daily fever (38–39 uC), gum bleeding, an episode of epistaxis and weight loss. Physical examination was significant for cervical and inguinal lymphadenopathy, gum hypertrophy, and an enlarged liver and spleen (6 and 3 cm, respectively). The patient also had high arterial blood pressure and congestive heart failure. The leukocyte count was 61,600 cells/mm with 31% blast cells and 59% lymphocytes. Five months before this admission her haemogram had been normal. A bone marrow (BM) aspirate revealed approximately 27% blast cells and 49% apparently mature lymphocytes. Cytochemical analysis revealed the blastic cells to be positive Sudan black. The great majority of cells were negative to PAS, and few cells were weakly positive; a-naphtyl butyrate was also negative. LDH measured 5261 UI. Immunophenotyping studies showed the presence of two cell populations with different light-scatter in BM. Large cells which corresponded to blast cells had an immature myeloid immunophenotype, (HLA-Dr, CD13, CD33, CD34), and small cells, corresponding to a mature B cell population, displayed a typical B-cell phenotype (CD19, CD5, HLA-DR, weak SMIg and K). ANLL-M2 was diagnosed. Hydroxyurea 1.5 g/d was started. The number of blast cells was reduced in subsequent days, although the total leukocyte count had increased due to an increase in the absolute number of lymphocytes (93,000 cells/mm in 5 d with 93% lymphocytes and 5% blasts). The patient died on the tenth day of hospitalization due to complications of both metabolic functions and infection. A pathologic leg fracture was also report-ed. The karyotype of bone marrow cells was obtained after cultures in RPMI 1640 with 20% foetal calf serum (Gibco) and pokeweed mitogen at 37 uC for 72 h. Cell cultures were pulsed with colcemid (0.06 mg/ml) in the last hour of incubation. Cells were subsequently harvested by standard procedures (hypotonic shock with 0.075 M KCl) and fixed in methanol–acetic acid (3:1). GTG banding was per-formed as described by Seabright (7), and chromosomes were identified and arranged according to the International System for Cytogenetic Nomenclature (8). The cytogenetic analysis showed the presence of two abnormal clones: 47,XX,+12 [10]/46,XX,del(5)(q31),t(8;13) (q22;q21) [4]/46,XX [6]. Although other cases of CLL associated with AML have been reported (1–6), the present study showed some peculiarities. There were no data suggesting the presence of AML after the diagnosis of CLL, since the patient had received no prior chemotherapy or radiotherapy and the haemogram obtained 5 months before had been normal. The cytogenetic study showed two abnormal clones, and as yet only two cases reporting concomitant CLL and AML included cytogenetic studies (5, 6). The patient described by Lima et al. (5) had chromosome aberrations commonly associated with CLL and AML in the same metaphases, suggesting that both diseases might be derived from a single cell clone. The cytogenetic analysis for the case reported by Mateu Eur J Haematol 2001: 66: 281–283 Printed in UK. All rights reserved Copyright # Munksgaard 2001

Acute myeloid leukemia of donor origin after allogeneic stem cell transplantation from a sibling who harbors germline XPD and XRCC3 homozygous polymorphisms

A 54-year-old woman was diagnosed with infiltrative ductal breast carcinoma. Two years after treatment, the patient developed an acute myeloid leukemia (AML) which harbored del(11q23) in 8% of the blast cells. The patient was submitted for allogeneic stem cell transplantation (aSCT) from her HLA-compatible sister. Ten months after transplantation, she relapsed with an AML with basophilic maturation characterized by CD45low CD33high, CD117+, CD13-/+, HLA Drhigh, CD123high, and CD203c+ blast cells lacking expression of CD7, CD10, CD34, CD15, CD14, CD56, CD36, CD64, and cytoplasmic tryptase. Karyotype analysis showed the emergence of a new clone with t(2;14) and FISH analysis indicated the presence of MLL gene rearrangement consistent with del(11q23). Interestingly, AML blast cell DNA tested with microsatellite markers showed the same pattern as the donors, suggesting that this AML emerged from donor cells. Additionally, polymorphisms of the XPA, XPD, XRCC1, XRCC3 and RAD51 DNA repair genes revealed three unfavorable alleles with low DNA repair capacity.In summary, we report the first case of AML involving XPD and XRCC3 polymorphisms from donor origin following allogeneic stem cell transplantation and highlight the potential need for careful analysis of DNA repair gene polymorphisms in selecting candidate donors prior to allogeneic stem cell transplantation.

Translocation (11;11)(p13- p15;q23) in a child with therapy-related acute myeloid leukemia following chemotherapy with DNA-topoisomerase II inhibitors for Langerhans cell histiocytosis.

We report a new case of therapy-related acute myeloid leukemia in a child with Langerhans cell histiocytosis. This patient was previously treated with a protocol of multidrug chemotherapy, containing a relatively low dose of etoposide (total dose of 900/m(2)). Twenty-six months after the end of the therapy, the patient returned to the hospital with fever and anemia. The white blood cell count was 53 x 10(9)/L. The bone marrow examination showed massive infiltration with French-American-British acute myeloid leukemia classification M4 blast cells. The patient did not respond to an intensive treatment with high dose ARA-C and idarubicin. He died 6 months later. The cytogenetic abnormality of the blast cells was a t(11;11)(p13 -15;q23), that has not been described before in a secondary leukemia case.

Epigenetics in Cancer: The Myelodysplastic Syndrome as a Model to Study Epigenetic Alterations as Diagnostic and Prognostic Biomarkers

Epigenetics is characterized as hereditary changes in gene activity and expression that occur without alteration in DNA genomic sequence. It is known that epigenetics corresponds basically by two majority modifications: DNA methylation and histone modifications. Epigenetics events are reversible without primary DNA base sequence changes, resulting in possible modulation of the gene expression. The accurate DNA modifications and chromatin changes are important to normal embryonic development, to correct tissue cells differentiation, to precise cell cycle progression and cell death control. However, since epigenetics is also crucial to regulate gene expression, uncontrolled and/or incorrect modifications can unbalance the genetic expression profile and result in cellular transformation from normal to malignant cells.

Karyotype abnormalities and their clinical significance in a group of chronic myeloid leukemia patients treated with hematopoietic stem cell transplantation

CONTEXT AND OBJECTIVE Following hematopoietic stem cell transplantation (HSCT), karyotyping is a valuable tool for monitoring engraftment and disease status. Few studies have examined the prognostic significance of karyotypes in patients who underwent HSCT for chronic myeloid leukemia (CML). The objective of this study was to evaluate the significance of pretransplantation cytogenetic status in relation to outcomes following HSCT in CML patients. DESIGN AND SETTING Case series study at Instituto Nacional do Câncer (INCA), Rio de Janeiro, Brazil. METHODS Cytogenetic analysis was performed by G banding on 39 patients treated with HSCT. RESULTS Thirty-one patients were in the chronic phase and eight were in the accelerated phase. Prior to HSCT, additional chromosomal abnormalities on the Philadelphia (Ph) chromosome were found in 11 patients. The most frequent additional abnormality was a double Ph, which was observed in four cases. Following HSCT, full chimeras were observed in 31 patients (79.5%). Among these, 23 (82.3%) had presented Ph as the sole abnormality. Mixed chimeras were observed in seven patients, of which three had additional abnormalities. Only one case did not present any cytogenetic response. Five patients presented cytogenetic relapse associated with clinical relapse following HSCT. Twenty-seven patients are still alive and present complete hematological and cytogenetic remission. CONCLUSION In our study, the presence of additional abnormalities was not associated with worse outcome and relapse risk. Also, no differences in survival rates were observed. Our study supports the view that classical cytogenetic analysis remains an important tool regarding HSCT outcome.

Complex karyotype and N-RAS point mutation in a case of acute megakaryoblastic leukemia (M7) following a myelodysplastic syndrome.

The development of acute megakaryoblastic leukemia (ANLL-M7) following myelodysplastic syndrome (MDS) has been described only in a few reports, and the mutations necessary for this transformation are still unknown. In this study, we describe a case of ANLL-M7 with a previous history of MDS presenting a complex karyotype 46,XX, t(4;11)(q21;q23),del(5)(q13q33),t(12;13)(p13;q21) and N-RAS point mutation. During MDS, the patient showed a hypercellular myelogram with dysplasia of the three myeloid lineages and the clinical symptoms characteristic of the 5q- syndrome. During the follow-up, we observed the appearance of two additional subclones, one with an isochromosome 17q and another with polyploidy. The presence of an isochromosome 17q in one subclone and polyploidy in another is probably due to the genetic instability generated by the malignant transformation.