Cytogenetic and myelodysplastic alterations after autologous hemopoietic stem cell transplantation.

Secondary myelodysplastic syndrome/acute myelogenous leukemia (MDS/AML) are today considered a primary complication of autologous hematopoietic stem cell transplantation. In our Center, 83 autografted patients underwent bone marrow (BM) biopsy and cytogenetic analysis at fixed intervals. Twelve patients developed non-clonal cytogenetic abnormalities and 10 patients clonal abnormalities, five of whom (three - 7, one - 5 and one t(9;11)) developed secondary MDS/AML. MDS was also diagnosed in two patients with a normal karyotype. In brief, seven patients (three males, four females; median age 36 years) developed MDS/AML 12-48 months (median 14) after autografting. The FAB diagnosis was AML-M2 in one, chronic myelomonocytic leukemia in two and refractory anemia with excess of blasts in transformation in four cases. Two patients presented a BM biopsy picture of MDS with fibrosis; none of them experienced leukemic transformation. Four MDS patients died, three of leukemic transformation and one of BM insufficiency; the two remaining patients are still living and untransformed. Our data underline the leukemogenic role of previous treatments, even if it is not possible to exclude that underlying disease and/or conditioning therapy may be involved.

Haematopoietic abnormalities after autologous stem cell transplantation in lymphoma patients.

Haematopoietic reconstitution after autologous stem cell transplantation (ASCT) was evaluated at different times in 26 lymphoma patients. All of the patients showed a significant decrease in the number of both committed (CFU-C) and more primitive progenitor cells (LTC-IC). The expansion of bone marrow progenitor cells in a 'stroma-free' long-term liquid culture system supplemented with SCF, IL-3, IL-6 and GM-CSF from 19 transplanted patients was significantly reduced compared to normal controls. The stromal cell compartment, evaluated by means of a CFU-F assay, was also greatly reduced. The number of haematopoietic and stromal cell progenitors was, nevertheless, very similar to their pre-transplant values. Bone marrow histology, which was evaluated at different times after transplant, showed an increase in reticulin fibres, the dilatation of parenchymal sinusoids and some morphological evidence of trilineage dysplasia in 11 patients; however, the same abnormalities were seen in the majority of pre-transplant samples. No cytogenetic abnormalities were observed in 15 patients before transplant, but four subsequently developed persistent clonal karyotypic alterations and five showed non-clonal abnormalities that generally disappeared over time. Our data suggest that both the stromal and the haematopoietic compartments are somehow damaged after ASCT for lymphoma; however, these defects generally pre-exist the transplant conditioning regimen and seem to become less pronounced over time.

Response of myelodysplastic syndrome bone marrow cells to multiple cytokine stimulation in liquid cultures: an in situ hybridization study.

BACKGROUND AND OBJECTIVE: Myelodysplastic syndrome progenitor cells can be grown and expanded in long term bone marrow liquid cultures in the presence of multiple cytokines. In this study we investigated the pattern of differentiation and response to growth factors in six cases of myelodysplastic syndrome (MDS) with well-defined cytogenetic abnormalities by means of conventional cytogenetics and fluorescence in situ hybridization (FISH). METHODS: Bone marrow cells were grown in stroma-free liquid cultures in the presence of SCF, IL-3, IL-6 and GM-CSF. RESULTS: IN three cases a CFU-GM expansion comparable to normal controls was observed, together with a decrease or increase of cells with abnormal karyotype. Two cases showed no response to growth factor stimulation, morphological signs of terminal myeloid differentiation and increase (one case) or decrease (one case) in the percentage of abnormal FISH signals along the cultures. In one additional case, while CFU-C expansion was present, clearcut leukemic transformation was observed in the culture, together with a sharp decrease in the percentage of abnormal FISH signals, indicating a leukemic transformation of MDS progenitor cells with a normal karyotype. INTERPRETATION AND CONCLUSIONS: Our data indicate that FISH analysis is generally a poor indicator of clonality in MDS; nevertheless, determining the kinetics of cytogenetically abnormal clones in liquid bone marrow cultures may provide insight as to the growth abnormalities of MDS progenitor cells and may be useful prior to in vivo growth factor administration.

Unrelated mismatched cord blood transplantation in an adult with secondary AML.

Umbilical cord blood (CB) has been widely used for related and unrelated transplants in pediatric patients. We present the case of an adult with secondary AML who received an unrelated, one-antigen mismatched CB transplant due to the lack of a matched donor. The patient was a 26-year-old female (35 kg/bw) who had received an autologous bone marrow transplant for Hodgkin's disease in April 1994 and, 6 months later, developed secondary MDS (RAEB, 46, XX, -7, +mar), which slowly evolved into acute myelogenous leukemia. In May 1995, she was transplanted with a 165 ml CB unit containing a total of 1.6 x 10(9) nucleated cells, 11 x 10(6) CD34+ cells and 7.2 x 10(5) CFU-GM. GVHD prophylaxis consisted of standard CsA and methotrexate. Myeloid engraftment occurred on day +28 (PMN > 500) and full donor chimerism was confirmed twice (on days +33 and +56) by means of cytogenetics and DNA microsatellite analysis. Erythroid and megakaryocytic engraftment was documented by immunohistochemical analysis of a bone marrow biopsy on day +40, showing the presence of erythroblastic islands and isolated CD61+ immature cells. The patient did not develop GVHD but died on day +56 from idiopathic interstitial pneumonia and multiorgan failure. To our knowledge, this is one of the first case reports of unrelated mismatched CB transplantation in an adult.

Response of myelodysplastic syndrome marrow progenitor cells to stimualtion with cytokine combinations in a stroma-free long-term culture system.

The effect of an ex vivo expansion culture system using multiple cytokine combinations was evaluated in 38 cases of myelodysplastic syndrome (MDS) with the aim of overcoming the defective in vitro growth of haemopoietic progenitor cells. A combination of four growth factors (GF) including SCF, IL-3, IL-6 and GM-CSF was identified as the optimal combination for expanding clonogenic progenitor cells in MDS bone marrow liquid cultures. The cultures of 50% of the patients (19/38) responded to GF stimulation (mean CFU-GM fold increase 15.65+/-48 at week 4) and showed morphological features of normal and/or dysplastic myeloid differentiation. In 12/38 cases (31%), complete unresponsiveness to multiple cytokine stimulation was observed; a small number of patients (7/38) showed progressive leukaemic growth along the cultures with the presence of 100% immature blasts at week 4. GM-CSF and c-kit receptors, analysed by immuno-histochemistry in 10 patients, were over-expressed in responding patients and either lacking or down-regulated in non-responders. Fluorescence in situ hybridization (FISH) analysis of cultured interphase cells of nine patients (trisomy 8 in eight patients) showed a clear-cut increase in the percentage of cells with three signals in the two responding patients, thus indicating the expansion of a MDS clone. Multiple cytokine liquid cultures seem to be able to override the refractoriness of MDS progenitor cells to GF stimulation in many cases, revealing a heterogeneity which may have prognostic implications and should be considered in ex-vivo and in vivo clinical trials with cytokine combinations.

5q- in a case of chronic myelogenous leukemia relapsed after allogeneic bone marrow transplantation.

Clonal chromosome and/or hematological abnormalities typically observed in myelodysplastic syndromes (MDS) have been described with increased frequency after autologous bone marrow transplantation (BMT) for lymphoma. We report the case of a woman with chronic myelogenous leukemia (CML) allografted with her HLA-identical sibling who, 5 years after the transplant and under immunosuppressive treatment for chronic graft host disease (GVHD), suffered a cytogenetic relapse associated with a 5q- deletion in the host metaphases. These findings suggest that myelodysplastic changes, possibly related to the chemo-radiotherapy conditioning regimen, may also present after allogeneic BMT.

Frequent p53 gene involvement in splenic B-cell leukemia/lymphomas of possible marginal zone origin.

A phenotypic and molecular evaluation was made of 15 patients with mature B-cell leukemia/lymphoma showing exclusive spleen and bone marrow involvement. According to French-American-British criteria, these cases could not be classified as classical B-cell chronic lymphocytic leukemia, hairy cell leukemia and its variant forms, splenic lymphoma with villous lymphocytes, or leukemic phase non-Hodgkin's lymphoma (NHL; follicular or intermediate type). The immunophenotype pattern (high surface Ig and CD25 expression, and little or no reactivity with CD5, CD23, and CD11c) and cytomorphologic features of these neoplasms suggested an origin in the marginal zone of the spleen. Molecular analysis did not show any involvement of the dominantly acting oncogenes generally associated with lymphoid malignancies (c-myc, bcl-2, bcl-1, Ras), but mutations of the p53 tumor suppressor gene involving exons 5, 6, and 8 were found in 6 cases (6 of 15, 40%). In 4 cases, the p53 alterations consisted of a point mutation leading to amino acid substitution. In the remaining 2 cases, an insertion or deletion resulting in a frame-shift of the protein was observed. In all but 1 of the cases, the wild-type sequence at the mutation site was barely visible, implying the loss of the normal p53 allele in leukemic cells. All of the cases showed a clinical course compatible with that of low-grade NHL, regardless of the p53 loss/mutation. Overall, our data suggest the existence of a form of splenic B-cell leukemia/lymphoma of possible marginal zone origin in which p53 inactivation may play an important pathogenetic role.

Prenatal diagnosis of metabolic diseases on chorionic villi obtained before the ninth week of pregnancy.

Nine pregnancies at risk for various metabolic disorders were monitored by prenatal diagnosis on chorionic villi obtained between the sixth and ninth weeks of pregnancy. A diagnosis of an affected fetus was made in five cases (Sandhoff, Tay-Sachs (2), Pompe's, GM1), while metachromatic leukodystrophy, GM1 (2), and Pompe's were excluded in four cases. It is concluded that chorionic villi are a reliable tissue for prenatal diagnosis of metabolic disorders also when obtained before the ninth week.

Microgranular variant of acute promyelocytic leukemia in children.

PURPOSE: The microgranular variant (M3v) of acute promyelocytic leukemia (APL) rarely has been reported in a pediatric series of acute nonlymphoblastic leukemia (AnLL). We reviewed the clinical and biologic features of childhood M3v cases in our AnLL series. PATIENTS AND METHODS: From January 1970 to January 1991, 11 children with M3v were admitted and treated at our center. A diagnosis was made according to French-American-British (FAB) criteria. Morphologic examination, cytochemical analysis, and immunophenotyping were performed by a single pathologist. From January 1984, the diagnosis was confirmed by cytogenetic and, subsequently, by molecular analysis on frozen material. RESULTS: In our series, the overall incidence of children with APL was unusually high, 31.2% of the AnLL and M3v constituted one case in every four cases of APL. Even restriction of the analysis to the time when either cytogenetic and DNA studies confirmed the diagnosis, the incidence did not change. The immunophenotype of M3v cases was identical to that described for the hypergranular type, but an unexpected association of CD2 with M3v was shown. The onset was characterized by marked hyperleukocytosis (median WBC count, 87 x 10(9)/L) unlike classic APL. Disseminated intravascular coagulation (DIC) was always present and severe. Hyperleukocytosis and DIC were responsible for the high incidence of deaths for hemorrhagic events in the first days after onset (eight of 11 patients). CONCLUSIONS: In our experience, for unknown reasons, M3v may occur in childhood more than generally was considered. The clinical course and prognosis seem worse in M3v than in typical APL cases.

Seven cases of trisomy 3 mosaicism in chorionic villi.

This paper describes seven cases of confined chorionic mosaicism with trisomy 3. The chromosomally abnormal cell line in chorionic villi was revealed in three cases at diagnostic CVS and in four cases at the evacuation of the uterine cavity after a missed abortion had been diagnosed by ultrasound. In two of these cases, the abortion occurred after apparently normal development of the fetus during the second trimester of pregnancy. An evaluation of the effect of confined chorionic mosaicism with trisomy 3 on the viability of the conceptus has been attempted.

Relevance of ultrastructural immunocytochemistry in the characterization of unclassifiable leukemias: correlation with phenotypic and genic studies.

Nine cases of acute leukemia presenting unusual phenotype were studied by light microscopy (LM) cytochemistry and transmission electron microscopy (TEM) immunocytochemistry with the immunogold staining (IGS) method; in addition, cytogenetic and molecular analyses were performed. The presence of myeloperoxidase (MPO) was studied at TEM in combination with immunophenotype to identify minor populations not characterizable at LM. Four of nine cases had no TEM/MPO reactivity, whereas the remaining five showed variable percentages of positive cells. Of the MPO negative cases, one was a megakaryoblastic leukemia with a positive platelet peroxidase (PPO) reaction, and three were lymphoid. Among the peroxidase positive cases, the percentage of MPO reactive cells was higher at TEM than at LM examination. In case 5 TEM analysis indicated that cells with some MPO reactivity at LM were non neoplastic myeloid cells. With this combined technique in cases 1 and 2 we excluded the presence of the MPO enzyme in CD15 positive lymphoid cells and, in another case, we documented the existence of CD19/MPO positive cells. The value of cytochemistry and immunology at the ultrastructural level for the characterization of blast cells and for the precise diagnosis of leukemia with "unusual" phenotype is illustrated.

Myelodysplastic syndromes in childhood. Report of 21 patients from Italy and West Germany.

Nine Italian and 12 German children fulfilled the criteria of myelodysplastic syndromes (MDS), according to the French-American-British (FAB) classification. All patients belonged to the more aggressive subtypes of myelodysplastic syndromes. Four presented with refractory anemia with excess of blasts (RAEB), 16 presented with refractory anemia with excess of blasts in transformation (RAEB-T), and 1 had chronic myelomonocytic leukemia (CMML). Dyserythropoiesis and dysgranulopoiesis were seen in all patients, and dysmegakaryopoiesis was seen in most patients. Cytogenetic studies in 13 of the 21 children showed karyotype abnormalities in 8; 5 had monosomy 7. Eleven patients were treated with intensive chemotherapy soon after diagnosis; 6 achieved complete remission (CR), and 2 of them are alive and still in complete remission after 48 and 69 months. Low-dose cytosine arabinoside (Ara-C) was given in six children without improvement. Bone marrow transplantation after progression of the disease has produced complete remission lasting 28 + months now in one of two patients. Four patients received only symptomatic treatment. The rate of 5-year survival for the total group was 20% (SD 9%). We conclude that children with MDS may benefit from more aggressive treatment, but that in general the survival rate is poor and similar to that observed in adults with the same subtypes of this disease.

Chromosome changes in human monocytic cell lines with in vitro spontaneous malignant transformation.

Serial chromosome studies were performed on four monocytic cell lines established from bone marrow samples of patients suffering from hematopoietic disorders other than leukemia. A spontaneous in vitro transformation towards a malignant phenotype has been found to be related to the karyotype evolution. The correlation between the chromosome changes of these cell lines and those described in human cancer and leukemia is discussed.

Biphenotypic Philadelphia positive, monosomy seven acute leukaemia: ultrastructural immunocytochemical study.

A case of Ph1 positive acute leukaemia is presented in which an additional chromosome change, monosomy 7 was found. There was no clinical evidence of a pre-existing chronic myeloid leukaemia. Cytochemistry and immunology showed a predominant HLA-DR+, TdT+, cALL- phenotype, with a small percentage of HLA-DR+, Leu-Ml+ and cALL- cells. The true biphenotypic nature of this case was clearly shown by transmission electron microscopy using the immunogold method combined with myeloperoxidase (MPO). Two distinct phenotypes, lymphoid (cALL+, MPO-) and myeloid (LeuMl+, MPO+) were identified with this technique. An immuno-scanning electron microscope technique was also used to study this case, which demonstrated the presence of two different surface morphologies.

First trimester fetal karyotyping: one thousand diagnoses.

Cytogenetic investigations for diagnostic purposes were performed on 1000 first trimester samples of chorionic villi (CVS) in two laboratories using similar techniques. Fetal karyotyping was the primary indication for CVS in 912 and maternal age was the major indication in 758 of them. The risk category "previous child/fetus with chromosome abnormality" included 74 diagnoses, while the category "chromosome abnormality in one of the parents" included 38 diagnoses. Sex determination was the primary indication for CVS in 53 pregnancies. The overall incidence of chromosomal abnormalities was 70, of which 47 were balanced and 23 unbalanced. The results are detailed for each of the risk categories and the incidence of abnormal karyotypes is given for each year of maternal age. In the maternal age of 35-37 years the incidence of unbalanced karyotypes was 2.9% and in the years 38 onwards it was 6.6%. The incidence of unbalanced karyotypes was about 4% when the sampling was made in the weeks 9 to 12 but six abnormal karyotypes were found among 39 CVS performed at the eight week of gestation. The 11 trisomies of the type not found at birth were clustered between the 8th and the 10th week of pregnancy. The technical problems encountered in this experience and the preliminary estimates of fetal loss are discussed.

Reproductive failure and parental chromosome abnormalities.

The effect of ascertaining on estimates of the frequency of parental chromosome abnormalities in couples with a previous history of pregnancy wastage was investigated by comparing three samples which differ in the ascertainment modality but not in the cytogenetical approach. The incidence of chromosome abnormalities was higher in the sample of 441 couples selected essentially on clinical criteria (6%) than in the two samples (659 and 479 couples) selected retrospectively from the files of two cytogenetic laboratories (4.6 and 3.2%). The comparison of these results with similar data, based on large samples reported in the literature, indicated that sample size may be relevant in producing the wide ranges of variation of the frequency of chromosomal abnormalities. Using our data and those from three other samples of greater than 300 couples a reasonable estimate of the overall incidence of chromosomal abnormalities in couples with a previous history of fetal wastage is approximately 5%.