Luize Otero

Cytogenetic analysis of 100 consecutive newly diagnosed cases of acute lymphoblastic leukemia in Rio de Janeiro.

We report the cytogenetic analysis of newly diagnosed Brazilian children with acute lymphocytic leukemia (ALL). We investigated 100 ALL cases from four different institutions in Rio de Janeiro. The frequency of chromosomal abnormalities was 92.3%. The karyotype profile and recurrent abnormalities found in this study do not differ essentially from those described by other groups. Although the Brazilian population is usually the product of different ethnic groups, our results show that the frequency of each recurrent abnormality is similar to that found in populations without our degree of diverse ethnic composition. Hence, our results suggest that childhood ALL in Brazil has the same biological features as that in developed countries, supporting the use of similar treatment protocols. We can therefore expect to reach the same survival rates in the coming years, depending possibly on the efficacy of the support therapy and extent of social assistance.

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.

Additional t(1;11)(q21;q23) with mixed lineage leukemia rearrangement in T-blastic crisis of a Ph-positive chronic myeloid leukemia.

To the Editor: Chronic myeloid leukemia (CML) is characterized by the proliferation and the accumulation of myeloid cells and theirs progenitors. During the initial indolent chronic phase, the Philadelphia chromosome (Ph) is usually the sole cytogenetic anomaly, but as the disease progresses into the accelerated phase, and eventually into aggressive blast crisis (BC), secondary chromosomal aberrations, such as +8, i(17q) and +Ph become frequent (1). These additional chromosomal abnormalities are associated with shorter survival and lower remission rates (2). In addition, molecular abnormalities in fundamental genes that control cell cycle and proliferation program can arise as p53, RB1, RAS, CMYC, p16 and AML-EVI-1 (3). We describe a case of a patient with T-cell blastic crisis of CML presenting the Ph chromosome and t(1;11)(q21;q23) with positive mixed lineage leukemia (MLL) gene rearrangement shown by FISH analysis as well as its correlation with the clinical course of the disease. A 15-yr-old female was diagnosed in October 1999 exhibiting a typical myeloproliferative scenario shown both by peripheral blood and bone marrow with very low leukocyte alkaline phosphatase score. Haematologic parameters were as follows: Hb 9.0 g ⁄dL, platelets 595 · 10 ⁄L, Ht 27.2% and white blood cell count 402 x 10 ⁄L. No cytogenetic and FISH studies were performed at this time. Initially, the patient have responded to the cytoreduction with hydroxyurea. However, 30 d after therapy, despite an important reduction in leukocyte count, she developed a massive ganglionar bulky disease in cervical and mediastinal regions. Lymph node biopsy exhibited a diffuse lymphoid blastic infiltration. The immunophenotype of peripheral blood and bone marrow cells showed a T-lineage profile with CD45, CD7 and cCD3 without any positive myeloid marker (Becton & Dickinson, San Jose, CA, USA). Cytogenetic analysis of bone marrow cells after GTG banding showed the karyotype 46,XX,t(9;22)(q34;q11)[16] ⁄ 46,XX,t(1;11)(q21;q23),t(9;22)(q34;q11)[4] according to the International System of Human Cytogenetic Nomenclature (4) (Fig. 1A and B). The molecular analysis of bcr-abl rearrangement was done by RT-PCR as previously described (5). It was positive for b2a2. FISH analysis of bone marrow cells using the dual color MLL probe (LSI MLL Dual Color Break Apart Rearrangement Probe – Vysis) showed one fusion signal and two split signals were found in 70% of the cells corresponding to the MLL rearrangement (Fig. 1C). The patient had a poor clinical outcome with primary refractoriness to the chemotherapeutic approaches. Although she has been indicated for allogeneic bone marrow transplantation, but 7 months after the diagnosis, the patient died due to resistant disease. The clinical distinction between BC of CML and de novo Ph in acute leukemia is not always clear. The features used to distinguish the Ph ALL from the CML in BC include the presence of chromosomally normal cells accompanying the Ph clone at diagnosis or later in the disease, as well as the achievement of true haematological and, in some cases, cytogenetic remission (6). Blastic phase of CML shows a slightly different karyotypic pattern of evolution, depending on the myeloid vs. lymphoid nature of blast cells (7). The case reported here is uncommon as the CML in T cell BC is very rare (8). Cytogenetic, imunophenotyping and clinical studies also confirmed the diagnosis. Cytogenetic analysis showed the Ph chromosome in 100% of bone marrow cells and in 40% of these cells an additional chromosomal aberration involving the 11q23 region was observed. FISH analysis revealed the MLL gene rearrangement. Rearrangements involving 11q23 are well documented in haematopoietic malignancies (9). In about 50–70% of cases, the molecular

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.

Double Philadelphia‐chromosome: a resistance factor on the imatinib mesylate therapy for chronic myeloid leukemia

Sir, chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder of a pluripotent hematopoietic stem cell. It is characterized by a reciprocal translocation t(9;22)(q34;q11), leading to the formation of the Philadelphia (Ph) chromosome. The molecular product is the creation of the bcr-abl chimeric gene, which encodes a protein with elevated kinase activity (Jacob et al., 2002). Progression of CML to blast crisis is preceded or accompanied by additional chromosomal abnormalities as +8, +Ph, i(17)(q10), loss of Y chromosome and less frequently +19 (Heim & Mitelman, 1995). Since 1999, imatinib mesylate (IM), which competes for the ATP binding of the BCR-ABL kinase, has been used in the treatment of CML. However, several types of IM resistance mechanisms have been identified, including genomic amplification of bcr-abl, overexpression of the multi drug resistance P glycoprotein, point mutation of the abl tyrosine kinase domain and the occurrence of additional chromosomal abnormalities (Hochhaus & Hughes, 2004). We report five cases that presented double Ph chromosome before IM therapy and did not reached cytogenetic and hematologic responses after the therapy. Between January 2001 and December 2004, 48 patients with CML were treated with IM after interferon-alpha resistance at the Hematology Unit of the National Institute of Cancer – Brazil. In five patients (10.4%), the cytogenetic analysis of bone marrow cells after GTG banding showed the presence of double Ph chromosome. All the patients presented in accelerated phase. FISH analysis of bone marrow cells using the LSI bcr/abl Dual Fusion Probe (Vysis) confirmed the amplification of the bcr-abl gene in the five cases (Figure 1). The patients with double Ph did not show cytogenetic response to IM and one of them evolved with clonal cytogenetic evolution as showed in Table 1. Four patients died due disease evolution. One patient was indicated to hematopoetic stem cell transplantation (HSCT) and actually he is in complete hematological remission and with full donor chimerism revealed by the cytogenetics and the RT-PCR analysis. The clinical, cytogenetic and molecular data of these patients are described in the Table 1. Amplification of bcr-abl gene has been described as mechanism of IM resistance. We described five patients who presented the double Ph chromosome before IM therapy. All these patients had not reached hematological and cytogenetic response to therapy. FISH analysis confirmed the amplification of the bcr-abl gene. Similar report was described by Ossard-Receveur et al. (2005) in a group of five patients who presented an amplification of bcr-abl due the duplication of the Ph-chromosome and IM resistance. Their results were similar to ours in some aspects, for example: clonal cytogenetic evolution was observed after the therapy. In three cases, complete remission was reached only after HSCT. Previous reports of patients with CML showed that IM prior to HSCT did not result in increased transplant-related morbidity or worse outcomes following HSCT (Oehler et al., 2007). So, the early detection of double Ph may, therefore, be important in treatment modification to overcome IM resistance. According to our results and a review of literature, we suggest that patients with double Ph should LETTER TO THE EDITOR INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY

Transient chromosomal rearrangements in three children with acute lymphoblastic leukemia after unrelated cord blood transplantation

Transient chromosomal rearrangements in three children with acute lymphoblastic leukemia after unrelated cord blood transplantation

Patients with chronic myeloid leukemia treated with imatinib who showed the appearance of clonal cytogenetic abnormalities in Philadelphia chromosome-negative cells.

Patients with chronic myeloid leukemia treated with imatinib who showed the appearance of clonal cytogenetic abnormalities in Philadelphia chromosome-negative cells

Dicentric t(8;13)(q10;q10) as an additional chromosomal abnormality in a case of acute promyelocytic leukemia with very poor outcome.

Acute promyelocytic leukemia (APL) is a specific type of acute myeloid leukemia (AML) cytogenetically characterized by a reciprocal translocation between chromosomes 15 and 17, t(15;17) (q22;q21), which results in a PML/RARA and RARA/PML. Chromosomal rearrangements, in addition to t(15;17), have been reported in 25–40% of APL patients [1]. Trisomy of chromosome 8 is the most frequent secondary anomaly and other abnormalities involving chromosomes 9, 17, 7, 21, 16, 6 and 12 have been described with less frequency [2]. The prognostic value of chromosomal abnormalities besides to t(15;17) remained uncertain in previous studies. We described a case of APL with very poor outcome and cytogenetically characterized with dic(8;13)(q10;q10) in addition to t(15;17). In June 2008, a 21-year-old woman was referred to the hospital with epistaxis and menorrhagia. Physical examination revealed pallor, tachycardia and discrete hepatomegaly. Peripheral blood examination showed hemoglobin level of 6.0 g/dL, platelets count 266 10/L and white blood cells count 226 10/L (92% of which consisted of promyelocytes). Coagulation assays revealed prolongation in prothrombin time (25.8 s and INR 2.6) and normal activated partial thromboplastin time (32.8 s). The bone marrow aspirate revealed hypercellular marrow with 95% of atypical hypergranular promyelocytes (many of them with abundant Auer rods) [Figure 1(A)]. Immunophenotypic analysis of bone marrow cells was positive for CD13 (94%), CD33 (94%), CD117 (94%), CD64 (94%), but negative for HLA-DR and other lymphoid cell antigens. To detect the PML– RARA fusion gene, FISH analysis was performed using a PML–RARA two colour direct labelled probe (Aquarius probes, Cytocell, Oxforshire, UK). This analysis detected a fusion signal in 85% of the bone marrow cells [Figure 1(B)]. The chromosomal analysis revealed 46,XX,t(15;17)(q22;q21)[11]/ 45,XX,dic(8;13)(q10;q10),t(15;17)(q22;q21)[5]/46, XX[4] [Figure 1(C)]. A diagnosis of APL, FAB M3, was made according to the above data. The remission induction therapy consisted of all-trans retinoic acid (ATRA) and anthracycline-based chemotherapy (given on day 1 after the first dose of ATRA, because of the high presenting white cells count). However, despite the fresh frozen plasma and platelet replacement to keep coagulation time and platelet count above 506 10/L, the patient died due to fatal central nervous system hemorrhage on day 3 of induction therapy. According to the Southwest Oncology Group (SWOG), the t(15;17) with or without secondary chromosomal abnormalities has been associated with favourable prognosis [3]. However, the prognostic significance of the additional abnormalities remains controversial. Several studies have shown that secondary chromosomal abnormalities do not influence therapeutic response

Hyperdiploid karyotype in a child with hypocellular primary myelodysplastic syndrome.

To the Editor: Myelodysplastic syndrome (MDS) is unusual in childhood (1). Monosomy 7 is the most common acquired chromosomal abnormality in children with MDS (2). In this report, we describe a rare case of hyperdiploid karyotype in a child with hypocellular primary MDS, classified as refractory cytopenia (RC) (2) and submitted to bone marrow transplant (BMT). Cytogenetic and immunophenotyping studies were performed and their values in diagnosis and prognosis are discussed. A 16-year-old girl was referred in August 1999 to the Bone Marrow Unit, CEMO-INCA (Rio de Janeiro) with the suspicion of MDS-RC. She presented pancytopenia and hypocellularity of bone marrow showing megaloblastoid maturation. She was treated with vitamin B12 and folate with no response. In January 2000, a cytogenetic study revealed a normal karyotype. Granulocyte colonystimulating factor (G-CSF) was administered without success. In March 2001, the patient was indicated for human leucocyte antigen (HLA)identical sibling BMT. In order to choose the conditioning regimen, the diagnosis was discussed between hypoplastic MDS and aplastic anaemia (AA). Other laboratory tests were performed. Bone marrow analysis showed hypoplasia with the mielogram revealing some dysplastic features. Bone marrow biopsy revealed hypocellularity, with a decrease of erythroid cells with megaloblastoid changes, a decrease of granulocytic cells and the presence of dysmorphic megakaryocytes. The abnormal localisation of immature progenitor cells (ALIP) was not present. Cytogenetic analysis of bone marrow cells after GTG banding showed 41 normal cells (93%) and three (7%) with a hyperdiploid karyotype 51,XX,+4,+6,+8,+14,+20, according to the International System of Human Cytogenetic Nomenclature (3). Immunophenotyping was performed. A panel of the following directly conjugated antibodies was used: CD45, CD4, CD8, CD2, CD3, CD19, CD10, CD33, CD34, CD61, CD7, anti-HLA-Dr (Becton & Dickinson, San Jose, CA, USA). The immunophenotypic abnormalities observed in this patient were: hypogranular neutrophils demonstrated by CD45 vs. side light scatter, CD10 granulocytes and myeloid lineage expressing non-myeloid antigens such as CD2. The number of megakaryocytes detected by CD61 cells was 16.59%. This value is considered increased according to Stetler-Stevenson et al. (4) and are compatible with MDS patients. The percentage of CD34 cells was 1.72%. Based on the morphological, immunophenotypic and cytogenetic studies the final diagnosis was MDSRC. Her sister’s bone marrow was infused after conditioning with busulfan (16 mg/kg) and cyclophosphamide (120 mg/kg). Graft-vs.-host disease (GVHD) prophylaxis consisted of cyclosporin A (CSA) (3 mg/kg from day 1) and methotrexate (15 mg/m on day +1 and 10 mg/m days +3 and +6) post-transplant. Neutrophil and platelet engraftment were achieved on days 17 and 20, respectively. She developed acute grade II GVHD (skin and liver) at day 42. Chronic progressive GVHD was diagnosed at her 11th month posttransplant along with liver dysfunction. Prednisone, CSA and mycophenolate mophetil were used. She had normalisation of hepatic enzymes. The patient is now 27 months post-transplant and remains in cytogenetic remission and complete donor chimaerism. The present case has proved to be peculiar in different aspects. In childhood MDS, Acar et al. described the first case of hyperdiploid karyotype in a patient (6-month-old boy) who had congenital anomalies, hypercellular bone marrow and classified as refractory anaemia with excess of blasts (RAEB) (5). Hyperdiploid karyotype in MDS was also described in a young woman (27-year old) with hypercellular bone marrow (6). According to literature, the present case represents the first case of paediatric MDS without congenital anomalies showing a hyperdiploid karyotype in a hypocellular Eur J Haematol 2003: 71: 399–401 Printed in UK. All rights reserved Copyright Blackwell Munksgaard 2003

An uncommon case of a child with del(5q) and hypocellular myelodysplastic syndrome.

To the Editor: Myelodysplastic syndrome (MDS) in children is an uncommon disorder [1]. We report a child with hypocellular MDS and del(5q) after an ovarian germ tumor only treated surgically. An 11-year-old female with anaplastic dysgerminoma treated surgically returned after 26 months with pancytopenia. The bone marrow morphology showed 3% of blasts, megakaryocytic hypoplasia, marrow cellularity of 10%. Cytogenetic analysis of bone marrow cells showed: 46,XX,del(5)(q15q35)[15]/46,XX[28]. FISH analysis using probes for EGR1 gene at 5q31 region and CSF1R gene at 5q33-q34 region showed loss of these regions. She was diagnosed with hypocellular MDS, refractory cytopenia (RC). Cytogenetic analysis from peripheral blood stimulated with phytohemagglutinin showed normal constitutional karyotype. FISH analysis in paraffin-embedded ovarian tumor, using the same probes utilized for bone marrow cells did not show del(5q). The patient received a matched marrow transplant from her HLA-identical brother. Forty months after transplant, she was in good clinical condition and without symptoms of GVHD. Complete remission and 100% donor chimerism were observed by bone marrow analysis using GTG banding, FISH, and VNTR. Compared to adult MDS, del(5q) is a rare chromosomal abnormality in childhood [2]. In adult MDS, most of cases with del(5q) as sole chromosomal abnormality represents a distinct clinical entity, 5q− syndrome and is characterized by erythroid hypoplasia, normal or elevated platelet count, medullary blast count <5%, and a relatively benign clinical course [3]. This case is the second child reported with RC and del(5q) as a sole chromosomal abnormality. However, our patient and the child described previously had thrombocytopenia, a not common characteristic in 5q− syndrome in adults [2]. The del(5q) can vary considerably with the involvement of all bands between 5q11 and 5q33-q34. Some studies suggest that haploinsufficiency of individual genes such as EGR1, CSF1R, and RPS14 may contribute to malignant transformation [3,4]. The present case is the second del(5q) in a child with MDS who received allogeneic HSCT. The first case was described by Pitman et al. [2]. Both cases showed a favorable clinical response. Because HSCT has emerged as the therapy of choice for pediatric MDS, more cases with del(5q) in pediatric MDS must be related to establish the clinical characteristics and compare with adult MDS. Childhood MDS following ovarian germ tumor is a rare event. Mascarello et al. [5] described a link between these two neoplasms using cytogenetic and FISH analysis. However, we did not find an association using these methods. MDS occasionally occurs in cancer survivors treated only surgically, suggesting a possible predisposition [6]. We must consider a possible association between the two neoplasms as well as further analyses like the investigation of specific polymorphisms, a gene expression, or an array CGH that would help in this context.