Maria Helena Ornellas

Age-related changes in natural killer cell receptors from childhood through old age

Most studies on natural killer (NK) cells and aging have focused on overall cell numbers and global cytotoxic activity. NK cell functions are controlled by surface receptors belonging to three major families: killer cell immunoglobulin-like receptors (KIRs), natural cytotoxicity receptors (NCRs), and C-type lectins. The expression of these receptors was investigated from childhood through old age in T, NKT- and NK cells and also in the CD56(dim) (cytotoxic) and CD56(bright) (responsible for cytokine production) NK cell subsets. A decrease in the expression of activating receptors (NKp30 and NKp46) was observed in NK cells in elderly individuals. KIR expression was increased only in the CD56(bright) subset. Children presented similar results regarding expression of NKp30 and KIR, but not NKp46. NKG2D expression was decreased in T cells of elderly subjects. Analysis of KIR genotype revealed that KIR2DL5 and KIR2DS3 were significantly associated with old age. Cytotoxic activity was preserved from childhood through old age, suggesting that the increase of the absolute number of CD56(dim), observed in elderly, may represent a compensatory mechanism for the receptor expression alterations. This initial study provides the framework for more focused studies of this subject, which are necessary to determine whether the changing balance of NK receptor expression may influence susceptibility to infectious, inflammatory, and neoplastic diseases.

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.

Translocation 11;14 in three children with acute lymphoblastic leukemia of T-cell origin

Clinical, karyotypic, immunophenotypic, and molecular profiles of three TALL cases carrying a t(11;14) are discussed and compared with data in the literature. As previously reported, t(11;14)(p13;q11) was associated in one patient with a TALL profile of intermediate stage of maturation (CD7+, CD4+, CD8+). However, the same translocation was found to be present in another patient with a more immature, pro-TALL profile (CD7+, CD4-, CD8-). Both patients showed molecular rearrangements of the TCR beta chain gene. A third patient, with a very immature pro-TALL profile (CD34+, CD7+, CD4-, CD8-), carrying a t(11;14)(p15;q11), showed molecular rearrangements of the TCR beta and gamma chain genes, while the IgH chain genes were in germline configuration. Our data indicate that t(11;14) can also be present in TALLs of more immature stages of intrathymic development; the significant factor determining the clinical behavior of TALLs is apparently related more to cell differentiation than to the presence of this chromosome rearrangement.

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.

CDKN2A (p14ARF/p16INK4a) and ATM promoter methylation in patients with impalpable breast lesions

Early detection of breast cancer increases the chances of cure, but the reliable identification of impalpable lesions is still a challenge. In spite of the advances in breast cancer detection, the molecular basis of impalpable lesions and the corresponding circulating biomarkers are not well understood. Impalpable lesions, classified by radiologists according to the Breast Imaging Reporting and Data System in the categories 3 and 4, can be either benign or malignant (slow growing or aggressive). In this article, we report the DNA methylation pattern in CDKN2A (p14(ARF)/p16(INK4a)) and in ATM gene promoters from 62 impalpable lesions, 39 peripheral blood samples, and 39 saliva samples, assessed by methylation-specific polymerase chain reaction method. ATM showed the greatest percentage of methylation in DNA from lesions (benign and malignant), blood (even with p16(INK4a)), and saliva, followed by p16(INK4a) and p14(ARF). Among the malignant cases, ATM promoter was the most hypermethylated in lesion DNA and in blood and saliva DNAs, and p14(ARF), the least. The highest percentage of p16(INK4a) methylation was found in the blood. Finally, our data are relevant because they were obtained using impalpable breast lesions from patients who were carefully recruited in 2 public hospitals of Rio de Janeiro.

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

Benzene poisoning, clinical and blood abnormalities in two Brazilian female gas station attendants: two case reports

BackgroundBrazilian gas station workers are chronically exposed to benzene, toluene, xylene (BTX) during their working time. Describe below two cases of latin female gas station workers with benzene poisoning symptoms and miscarriage history.Case presentationIn both cases were identified complex chromosomal rearrangements (CCR) with fluorescence in situ hybridization, applied to whole chromosome paints by chromosomes 1, 2 and 4. The lower natural killer cell (NK) cells have also been observed in cases correspondents, especially the rare type of NK (NKbright) in their peripheral blood cells.ConclusionsIt is known that acquired chromosomal aberrations are positively correlated with cancer and reproductive risk. In concordance, lower NK cytotoxicity increases the risk for cancer, as well. Thus, this is the first study providing hints on a possible causative relation of lower NK cytotoxicity and increase rates of chromosomal rearrangements including CCRs.

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.

Experimental validation of the complement protein C3a down expression in the plasma of patients with squamous cell carcinoma of the penis

OBJECTIVES We have previously shown the importance of the complement system in differentiating between patients with squamous cell carcinoma of the penis (SCCP) and controls. These patients had low expression of C3a and C4 fragments. Therefore, in this study, we investigated the complement protein C3a as a potential circulating biomarker in these patients by a commercially available enzyme-linked immunosorbent assay (ELISA) test. PATIENTS AND METHODS Plasma samples from 39 patients with SCCP, 15 patients with prostate cancer, and 50 healthy male subjects were evaluated using the ELISA-Bioscience OptEIA Kit human anti-C3a (BD). The nonparametric Mann-Whitney test was used for comparison of means among the groups. RESULTS The complement protein C3a was found down expressed in patients with SCCP (P<0.05) in comparison to either subjects with good health or subjects with prostate cancer. CONCLUSION Experimental validation of the down expression of C3a was well succeeded using a commercial ELISA kit. Complement system fragment C3a is down expressed in patients with SCCP. Besides, C3a is also low expressed in the plasma of patients with initial prostate cancer when compared to healthy subjects. These results suggest that the innate immune response might be suppressed in patients with these malignancies.