Incidence and prognostic significance of karyotype abnormalities in de novo primary myelodysplastic syndromes: a study on 331 patients from a single institution.

The impact of clinical parameters, International Prognostic Scoring System (IPSS) scores/cytogenetic categories, and some single cytogenetic defects on overall survival (OS) and time to myelodysplastic syndromes (MDS)/AML progression (progression-free interval (PFI)) was evaluated in 331 MDS patients. Statistical analysis demonstrated that OS and PFI were significantly affected by all these parameters. Since single 7q- showed a better survival than the poor IPSS cytogenetic category (P=0.009), it was considered as a new prognostic entity ('modified IPSS categories'). In multivariate analysis OS was significantly influenced by age, marrow blast cell percentage, number of cytopenias and either modified or standard IPSS cytogenetic categories; hazard ratios for MDS/AML progression were influenced by all the former, except for age and cytopenias. Multivariate analysis of del(7)(q31q35) confirmed the results of univariate analysis, but the Akaike Information Criterion showed no difference in evaluating OS and PFI between the modified and standard IPSS cytogenetic grouping. In conclusion, (i) chromosome defects as grouped by IPSS and blast cell percentage are the most relevant parameters for predicting OS and PFI; (ii) the prognostic power of the IPSS cytogenetic grouping is not ameliorated by the introduction of del(7)(q31q35) as a new entity; (iii) complex karyotypes have a prognostic value independent of blast cell percentage.

Is FISH a relevant prognostic tool in myelodysplastic syndromes with a normal chromosome pattern on conventional cytogenetics? A study on 57 patients.

Conventional cytogenetics (CC) at clinical diagnosis shows a normal karyotype in 40-60% of de novo myelodysplastic syndromes (MDSs). Fluorescence in situ hybridization (FISH) might detect occult aberrations in these patients. Therefore, we have used FISH to check 57 MDS patients who were karyo-typically normal on CC. At clinical diagnosis, FISH revealed a clonal abnormality in 18-28% interphase cells from nine patients, five of whom also presented the same defect on metaphase FISH. In five out of nine patients, the occult defect effected a change in the international prognostic scoring system (IPSS). An abnormal FISH pattern was significantly correlated with marrow blast cell percentage (P<10(-3)) and IPSS (P<10(-3)). Patients with an occult abnormality showed an overall survival and event-free survival significantly inferior in comparison to those of patients with normal FISH (P<10(-3), P<10(-3)). Death and AML progression were 15- and eight-fold more frequent in FISH abnormal patients. In conclusion, occult defects (1) are revealed in about 15% of CC normal MDS patients, (2) are overlooked by CC either because of the poor quality of metaphases or their submicroscopic nature, (3) are clinically relevant as they may cause a change in the IPSS category and may identify a fraction of CC normal patients with an unfavorable clinical outcome.

Acute myeloid leukemia (AML) having evolved from essential thrombocythemia (ET): distinctive chromosome abnormalities in patients treated with pipobroman or hydroxyurea.

ET is a chronic myeloproliferative disorder rarely evolving into AML, sometimes preceded by a myelodysplastic syndrome (MDS). Such transformations mostly occur in patients treated with radiophosphorous ((32)P) or alkylating agents, especially busulfan. Recently, concern has also arisen about the long-term safety of hydroxyurea (HU). Pipobroman (PI), a well tolerated and simple to use drug, constitutes a valid alternative to those cytoreductive treatments. The present study reports on 155 ET patients treated at our institution from 1985 to 1995, and monitored until December 2000. A good control of thrombocytosis was achieved with PI as the only treatment in 106 patients and with HU in 23 patients. Twenty-six patients received no treatment. After a median follow-up of 104 months, seven patients (four treated with HU, and three with PI) developed AML whereas one patient treated with PI developed MDS. A significant difference in progression-free survival was observed between HU- and PI-treated patients (P = 0.004). A short-arm deletion of chromosome 17 was most frequently detected in HU-treated patients, while a long-arm trisomy of chromosome 1 and a monosomy 7q were seen in PI-treated patients. No TP53 mutation was discovered in the six patients studied (two HU-treated and four PI-treated). We conclude that these cytogenetic abnormalities are not linked to the natural history of the disease, but rather that they might be induced by the cytoreductive treatment.

Translocation (8;16) in a patient with acute myelomonocytic leukemia, occurring after treatment with fludarabine for a low-grade non-Hodgkin's lymphoma.

We describe a 65-year old woman who developed a t(8;16)(p11;p13) positive acute myeloid leukemia (AML)-M4 without a prior myelodysplasia thirty-six months after a low-grade non-Hodgkin's lymphoma treated with alkylating agents (chlorambucil and cyclophosphamide) and fludarabine, a purine analog with a significant activity in lymphoproliferative disorders. The t(8;16)(p11;p13) is present in 0.4% of AML of M4-M5 cytotype. In the present case it was identified by conventional cytogenetics; involvement of the MOZ and CBP genes was demonstrated by fluorescence in situ hybridization, but not by reverse transcription polymerase chain reaction. The patient died of sepsis after the first course of induction chemotherapy. This is the first t(8;16) AML-M4 arising after fludarabine treatment of which the leukemogenic role in our case is very difficult to ascertain. Most t(8;16) t-AML cases had received anthracyclines with or without cyclophosphamide; none was ever administered chlorambucil. Our patient was never given anthracyclines and the cumulative doses of chlorambucil and cyclophosphamide employed were low.

Trisomy 11 and a complex t(11;11;22) in a patient with acute myelomonocytic leukemia (AML-M4) following myelodysplasia (MDS): a cytogenetic study of a mechanism of leukemogenesis.

We describe a 73-year-old man diagnosed with acute myelomonocytic leukemia (AML-M4) following myelodysplasia with trisomy 11 and with a t(11;11;22). This is the first case with both abnormalities present in the same cells and with the t(11;11;22) involving a chromosome 11 already duplicated at 11q23. This band contains the MLL gene that undergoes partial tandem duplication in patients with +11, which is "promiscuous," being translocated with a large number of genetic partners. Our patient had a complex karyotype that was completely defined by in situ hybridization. This technique demonstrated that the t(11;11;22) derivative with a duplication of band 11q23 carried from three to four copies of MLL. Two copies of the gene were close to each other and centromeric to the break-point region. Therefore, a partial tandem duplication of the MLL gene might have happened before the occurrence of t(11;11;22). Considering the associated chromosome defects, the monosomy for the long arm of chromosome 7, due to an unbalanced translocation t(7;17), further underlines the possibility that a partial tandem duplication of the MLL gene might have taken place.

A complex translocation (5;7) in a patient with acute nonlymphocytic leukemia evolved from a myelodysplastic syndrome.

Complete or partial monosomy for the long arms of chromosomes 5 or 7 or both is frequently observed in therapy-related myelodysplastic syndromes and acute nonlymphocytic leukemia. Sporadic cases have been reported in which partial monosomy is due to unbalanced translocations. The patient described herein carries one such rearrangement. 46,XY,t(1;2) (q32;p23),del(5)(q13),der(7)(5qter-->5q22::7p15-->7 q21:),del(12)(p12), resulting in partial monosomy for the long arms of chromosomes 5 and 7 and in partial monosomy for the short arm of chromosome 7.

Detection and monitoring of trisomy 8 by fluorescence in situ hybridization in acute myeloid leukemia: a multicentric study.

BACKGROUND AND OBJECTIVE: The role of fluorescence in situ hybridization (FISH) in the detection and monitoring of trisomy 8 (+8) in acute myelogenous leukemia (AML) has not been defined exactly. This multicentric study was performed in order to: i) analyze the sensitivity of interphase FISH with respect to conventional chromosome analysis (CCA) in detecting +8; ii) compare the results of FISH and CCA in the quantitation of the frequency of +8-positive cells; iii) analyze the possible role of FISH in the cytogenetic follow-up of patients with +8. DESIGN AND METHODS: One hundred and ninety-eight nonconsecutive patients with a diagnosis of AML seen at five centers over a 3-year period were studied by CCA and FISH with a chromosome 8-specific centromeric probe. Two hundred interphase cells were scored in each test and the cut-off for the recognition of +8 was set at 3%. An irrelevant pericentromeric probe was used as negative control in those cases with an apparently normal karyotype and trisomy 8 in interphase cells. FISH studies were conducted at diagnosis and, in 14 cases with +8, on 1.5 occasions during follow-up. RESULTS: Karyotype aberrations were seen in 121 cases (61.1%), with +8 being present in 38 of them (16 as the sole aberration). Interphase FISH detected +8 in 37/38 cases; in a patient with 1/10 metaphases with +8, 2.3% interphase cells with 3 signals were seen. Fourteen additional cases with occult +8 were detected by FISH, which showed 4-22% interphase cells with three signals; 6 patients had an abnormal karyotype without +8, 3 had a normal karyotype, 5 had no analyzable mitoses. In 24 cases with > 15 analyzable metaphases, percent variations between CCA and FISH in the estimation of the size of the trisomic clone ranged between 0.4% and 51%, median value 22%. Underestimation of the percent of trisomy 8 by FISH occurred in all 10 cases with > 90% +8 metaphases. In 7/14 cases investigated sequentially, FISH detected 5-35% trisomic cells in the BM after induction therapy (4 CR, 3 PR); 4 cases relapsed with +8 at 8-15 months. The absence of +8 in remission marrows was documented in the remaining 7 cases, 4 of which relapsed at 20-32 months. INTERPRETATION AND CONCLUSIONS: It is concluded that FISH was a valuable method in this multicentric study since it showed greater sensitivity than CCA in detecting minor clones with +8, in patients with both normal and abnormal karyotypes. The role of FISH in the cytogenetic follow-up of trisomies in AML patients may be promising.

Intensive chemotherapy followed by donor PBSC in ANLL relapsed after allogeneic BMT.

We describe a 42-year-old man with ANLL-M4 in relapse after allogeneic BMT, in whom a new CR was obtained by conventional chemotherapy followed by the infusion of his female donor PBSC. At the time of BMT he was in CR. Six months later a full hematological relapse occurred an a three drug 5-day regimen was started. Two days after the end of chemotherapy he received donor PBSC collected by two leukaphereses after mobilization with G-CSF, given subcutaneously at 5 micrograms/kg/day for 7 days. The mononuclear PBSC were 4.2 x 10(8)/kg; the CD34 positive cells were 8.2 x 10(6)/kg and the CFU-GM were 14 x 10(4)/kg. Two days after PBSC infusion the patient received G-CSF at a dose of 5 micrograms/kg/day. Hemopoietic recovery occurred promptly on day + 13 and Y-FISH revealed 14% of Y-spot positive cells in the marrow. On day +20 hematological and cytogenetic remission was documented. The percentage of recipient cells decreased from day +36 onwards following the occurrence of a grade II GVHD, from which the patient recovered 1 week later with oral cyclosporin A and intravenous high-dose steroids. At present (day +200 from relapse) the patient is still in CR with 3% of Y-spot positive cells.

The CD11/CD18 granulocyte adhesion molecules in myelodysplastic syndromes.

We have evaluated the function of granulocytes in 14 patients suffering from myelodysplastic syndrome (MDS). We also evaluated the functional and immunochemical activities of five monoclonal antibodies (MoAbs) reactive with the CD11/CD18 leucocyte adhesion molecules of granulocytes. Granulocytes showed a decrease in chemotaxis (P < 0.001) and in aggregation (P < 0.01) using various agents as a stimulus. Cytofluorimetric and immunoenzymatic assays with alkaline phosphatase (APAAP) analysis showed decreased expression of the CD11b/CD18 receptor detected by OKM1 (P < 0.001). Despite LFA-1 and-CD11a/CD18 was expressed in normal amounts. The studies of upregulation of granulocytes CD11b/CD18 and image analysis of immunochemical preparation (APAAP) demonstrated decreased expression of CD11b/CD18 in granulocytes from MDS compared to controls (P < 0.001). We conclude that granulocyte dysfunction in MDS may be correlated with decreased expression of surface CD11b/CD18 leucocyte adhesion molecules or their structural modification.

Assay of phagocytic cell functions.

The fundamental role of the immune system is recognition of the self from the non-self; in this way the principal functions of the immune system can be summarized as: resistance against the cells and foreign substances which are potentially damaging the tissues; identification of neoplastic cells to be destroyed. The cells which have this role are essentially lymphocyte, neutrophils and macrophages: extracellular and cellular humoral factors also play their role into the inflammatory process. In fact, we define the normal responses of phagocyte as the capacity of the specific phagocytic cell to respond to various stimuli and to migrate to the location of the damage. This complex cellular defense mechanism comprises several steps that can be summarized as following: opsonization of particles to be ingested, adhesion and aggregation of phagocytes to vascular endothelium, migration of phagocytes through the vessel walls, chemotaxis of phagocytes towards pathogenic agents, recognition of the particles/antigens by the phagocytes which subsequently adhere to their surface, ingestion of the particles with formation of a phagosome. This process is completed with the fusion of the phagosome with cellular granules (lysosomes) and formation of phagolysosomes, degranulation and release of the enzyme laden granules into the phagolysosome, lysis and killing of ingested particles and bacteria. It is clear from this schematic summary, that the response to pathogens can be very complex and each of the processes involved in the above described steps could be deranged leading to various pathological changes. We analyze the most classical and new methods to study the physiopathology of granulocytes, which are important for clinical diagnosis of phagocyte diseases or for phagocytic dysfunction in various syndromes and in neoplastic patients.

Phagocyte activation in coronary artery disease.

Recent studies suggest that granulocytes (PMNs) play a role in the pathogenesis of acute and chronic myocardial ischemia and extension of myocardial injury. Granulocytes can release a variety of molecules mediating tissue injury which act synergistically with other molecules and cells. The aim of our investigation was to evaluate the granulocyte function in patients affected by coronary artery disease (CAD) and during coronary angioplasty (PTCA). We studied 20 patients suffering from CAD. The PMN's aggregating activity was greater in the coronary sinus than in the aorta (P < 0.01). The increase in aggregating activity was evident in patients who were smokers: their cells release significantly lower quantities of leukotriene C4 (P < 0.025). In the 20 patients who underwent coronary angioplasty we analyzed superoxide release after stimulation with phorbol-myristate-acetate (PMA). The results showed a greater decrease of PMN's superoxide production in the coronary sinus than in the aorta (P < 0.05). In all patients affected by CAD we evaluated the PMN's expression of CD11b/CD18 membrane integrins. In these patients the increase in expression of CD11b/CD18 was statistically significant in comparison with the controls (P < 0.01). This increase in expression correlates with a higher aggregation (r = 0.87, P < 0.001). The potential role of leukocytes, oxygen radicals, leukotrienes and granulocyte enzymes in the pathophysiology of myocardial injury due to regional ischemia and reperfusion is an area of intense investigation. This paper presents studies carried out in vivo which have been instrumental in demonstrating the role of granulocytes as mediators of myocardial ischemia.

Correlation between CD11b/CD18 and increase of aggregability of granulocytes in coronary artery disease.

Recent studies suggest that granulocytes (PMNs) play a role in the pathogenesis of acute and chronic myocardial ischemia and extension of myocardial injury. Rabbit-derived antiserum-dependent reduction of circulating PMNs in the dog or using monoclonal antibody anti-CD11b/CD18 of PMNs resulted in smaller myocardial infarcts. Experience in humans shows the modification of PMN function in angina and during myocardial ischemia. In our studies, patients affected by coronary artery disease presented an increase in granulocyte aggregability in coronary sinus and showed a related higher expression of CD11b/CD18 in coronary sinus with respect to aorta leukocytes. The potential role of this modification of PMNs was analyzed.