Relationship between chromosomal changes complexity and disease aggressiveness in myeloid and lymphoid disorders

In this paper are presented four cases, with unusual chromosomal abnormalities, identified at the first presentation, among over 100 patients with myeloid and lymphoid acute and chronic leukemias cytogenetically investigated. The complexity and nature of cytogenetic abnormalities was in direct relationship with the disease evolution. The first case, a 22 years old man with acute lymphoblastic leukemia type L3, exhibited many structural changes in bone marrow cells with diploid number of chromosomes: del(3)(q26); del (5)(p13); t(8;14) (q24;q32); del(9)(p11q11);inv(15)(p12qter). The second case, a 62 years old woman, diagnosed as poorly differentiated acute leukemia, refractory to treatment, showed hiperdiploidy (48–54 chromosomes) and 3–4 markers derived from chromosomes 5 and 12. The third case, a young man of 27 years old, diagnosed as acute myeloid leukemia, apart of Philadelphia chromosome, presented trisomy 16, both in diploid and aneuploid cells. None of these three patients did respond to any medical therapy. Their rapid death was a powerful proof of the correlation between the complexity of genome changes and disease aggressiveness. In the fourth case, a constitutional translocation t(3;5)(q26.3;q21) identified in a 72 years old woman with essential thrombocythemia, appeared not to be involved in the etiology of the disease. In this case, the treatment with hydroxyurea was successful and the disease evolution was favourable. In conclusion, we appreciate that in the three cases of myeloid and lymphoid leukemias it was a direct relationship between the complexity of genomic changes and the aggressiveness of the disease.     

Correlations between cytogenetics and morphology in myelodysplastic syndromes

In order to detect possible relationships between cytogenetic abnormalities and morphologic features in myelodysplastic syndromes (MDS), 48 patients with MDS were investigated. Clonal cytogenetic abnormalities were present in bone marrow cells from 27 patients (56%). The most frequent single anomaly was del (5 q) (10 cases), followed by monosomy 7 (3 cases), trisomy 8 (3 cases) and del (20 q) (2 cases). Complex anomalies were present in 6 patients. Morphologically, according to the French-American-British (FAB) classification: 17 cases were considered as refractory anemia (RA), 17 as RA with excess of blasts (RAEB), 2 as RAEB in transformation, 2 as acquired idiopathic sideroblastic anemia and 10 as chronic myelomonocytic leukemia. With regard to the FAB classification, del (5 q) was often associated with RA and complex cytogenetic anomalies with RAEB. When myelodysplasia was studied in individual myeloid lineages, del (5 q) was associated with hypolobulated megakaryocytes, monosomy 7 with micromegakaryocytes and complex chromosomal anomalies with the association of two or more features of dysmegakaryocytopoiesis. Del (11 q) was associated with increased iron storage and del (20 q) with marked dyserythropoiesis. No correlation was observed between cytogenetic anomalies and features of dysgranulocytopoiesis.     

Prognostic significance of additional chromosomal abnormalities in Ph positive bone marrow cells in chronic myeloid leukemia

We studied the prognostic significance of combination of different chromosomal abnormalities and genomic mutations in Ph+ chronic myeloid leukemia patients. In general 49 cytogenetic analyses of bone marrow aspirate from 35 patients (11 of these observed in dynamics) have been carried out. The additional chromosome changes were found in 25.07% cases and approximately 80% of anomalies appeared in the blast phase. Among the secondary chromosomal abnormalities extra Ph-chromosome appeared mostly in different combinations with trisomy of chromosomes 8 and 19. Appearance of clonal differences of cells, high rate of genome mutations and of microchromosomes had negative prognostic significance on disease proceeding.     

Fluorescent in situ hybridization (FISH) in bone marrow and peripheral blood of leukemia patients: implications for occupational surveillance

Although there has been a rapid rise in the application of fluorescent in situ hybridization (FISH) analysis of bone marrow tissue for the staging and prognosis determination of hematopoietic malignacies such as the chronic and acute leukemias, it's application as a surveillance tool for leukemogen exposed high risk occupational cohorts is understandably limited by the invasiveness of sample collection. While some small occupational studies have been performed using FISH in peripheral blood with promising results, some of the basic assumptions made in utilizing the FISH technique have not been fully explored. These include selection of the correct hematopoietic cell to assay (myeloid or lymphoid); selection of appropriate chromosomal markers and the sensitivity of peripheral blood FISH in detecting unbalanced genomic abnormalities. In this study, we performed a pilot 'validation' exercise utilizing the FISH technique and standard metaphase cytogenetics, comparing results in tandem pairs of peripheral blood with bone marrow cells, where clonal abnormalities arise. Samples were taken from patients with known chromosomal lesions associated with active leukemia. We carefully chose markers most frequently associated with leukemogen-inducing DNA damage and probes that have been utilized successfully in clinical practice. Ten de novo or therapy-related acute myeloid leukemia (t-AML) patients underwent bone marrow cell karyotyping and fluorescent in situ hybridization (FISH) analysis. Parallel peripheral blood samples were concommitently drawn and evaluated with FISH using the same probes. In six of eight paired samples treated with a 3-day phytohemagglutinin (PHA) stimulation, typically used to assay lymphocytes and their progenitors, we detected abnormal clones. In one of the two remaining cases, we identified an abnormal clone in both bone marrow and PHA-stimulated peripheral blood, although at a level in the peripheral blood sample that would typically be reported as "non-diagnostic" for clinical purposes. These results suggest that use of FISH in PHA stimulated peripheral blood samples with probes commonly employed in t-AML evaluations (chromosomes 5q, 7q, 8, 11q) to detect cytogenetic abnormalities in peripheral blood represents a potentially promising though as yet, under-utilized approach for the occupational surveillance of workers exposed to leukemogens, especially if it could be linked to automated high-throughput assays for increased sensitivity.     

Preleukemic syndromes.

Acute nonlymphocytic leukemia (ANLL) is preceded by a hematologic illness representing the "preclinical" stages of the disease in many patients. This "preclinical stage" or preleukemic stage is difficult to recognize by conventional hematologic morphologic techniques. A prospective study was carried out to determine whether cytogenetic studies would be helpful in the recognition of preleukemic states and whether the presence of cytogenetic abnormalities would have prognostic significance. A study of 284 patients with suspected preleukemia has yielded 62 patients with progression to overt ANLL. Cytogenetic abnormalities were found in 30% of suspected preleukemic patients, whereas 53% of the patients progressing to acute leukemia had cytogenetic abnormalities. These studies show that the presence of cytogenetic abnormalities aid in the recognition of preleukemia but are not specific for early leukemia. Patients with cytogenetic abnormalities are more likely to develop overt ANLL. Banded chromosome studies demonstrated cytogenetic abnormalities in the preleukemic phase in 13 of 26 patients. A variety of clonal chromosomal abnormalities were observed.     

Cytogenetic and molecular cytogenetic investigations in relapse of B-cell chronic lymphocytic leukemia

Сhromosomal abnormalities have been analyzed in bone marrow cells of 61 patients with relapse of B-cell chronic lymphocytic leukemia. The cytogenetic results have allowed the structural stratification of the obtained karyotypes into ten groups of clones: normal, normal/near tetraploid, abnormal/normal, abnormal/ near tetraploid/normal, evolution of clonal chromosome abnormalities; evolution of clonal chromosome abnormalities/normal, evolution of clonal chromosome abnormalities/near tetraploid/normal, independent clones, independent/normal clones; and independent/near tetraploid/normal clones. The identified structural rearrangements included translocations, deletions, insertions, and duplications; however, deletions with the involvement of bands 17p12, 13q12–q14, 11q14, and 11q23 dominated (63.8%). The application of i-FISH helped to show the presence of one to four abnormalities per karyotype. The identified cytogenetic and molecular cytogenetic rearrangements may signify a multilevel nature of the process underlying the development of resistant karyotypes. The results obtained under both methods have revealed the presence of a heterogenic cell population with possibly different levels of chemotherapy resistance.     

Clinical significance of cytogenetic aberrations in bone marrow of patients with diffuse large B-cell lymphoma: prognostic significance and relevance to histologic involvement

Background

Although knowledge of the genetics of diffuse large B-cell lymphoma (DLBCL) has been increasing, little is known about the characteristics and prognostic significance of cytogenetic abnormalities and the clinical utility of cytogenetic studies performed on bone marrow (BM) specimens. To investigate the significance of isolated cytogenetic aberrations in the absence of histologic BM involvement, we assessed the implication of cytogenetic staging and prognostic stratification by a retrospective multicenter analysis of newly diagnosed DLBCL patients.

Methods

We analyzed cytogenetic and clinical data from 1585 DLBCL patients whose BM aspirates had been subjected to conventional karyotyping for staging. If available, interphase fluorescence in situ hybridization (FISH) data were also collected from patients.

Results

Histologic BM involvement were found in 259/1585 (16.3%) patients and chromosomal abnormalities were detected in 192 (12.1%) patients (54 patients with single abnormalities and 138 patients with 2 or more abnormalities). Isolated cytogenetic aberrations (2 or more abnormalities) without histologic involvement were found in 21 patients (1.3%). Two or more cytogenetic abnormalities were associated with inferior overall survival (OS) compared with a normal karyotype or single abnormality in both patients with histologic BM involvement (5-year OS, 22.0% vs. 52.7%; P < 0.001) and those without BM involvement (31.8% vs. 66.5%; P < 0.001). This result demonstrated that BM cytogenetic results have a significant prognostic impact that is independent of BM histology. The following abnormalities were most frequently observed: rearrangements involving 14q32, 19q13, 19p13, 1p, 3q27, and 8q24; del(6q); dup(1q); and trisomy 18. In univariate analysis, several specific abnormalities including abnormalities at 16q22-q24, 6p21-p25, 12q22-q24, and -17 were associated with poor prognosis. Multivariate analyses performed for patients who had either chromosomal abnormalities or histologic BM involvement, revealed IPI high risk, ≥ 2 cytogenetic abnormalities, and several specific chromosomal abnormalities, including abnormalities at 19p13, 12q22-q24, 8q24, and 19q13 were significantly associated with a worse prognosis.

Conclusions

We suggest that isolated cytogenetic aberrations can be regarded as BM involvement and cytogenetic evaluation of BM improves staging accuracy along with prognostic information for DLBCL patients.

     

Radiation damage in patients treated by total-body irradiation, bone marrow grafting, and cyclosporin

The bone marrow (BM) and peripheral blood (PB) from 63 patients were assessed for the presence of chromosomal aberrations after bone marrow transplantation (BMT) following total body irradiation (TBI) for leukemia. Forty-one patients showed no abnormalities in either BM or PB, and 22 had aberrations in either BM or PB or both. Only stable aberrations were found in the BM, but both stable and unstable abnormalities were present in the PB, the majority showing only unstable aberrations. Among the 25 patients who had a leukemic relapse, clonal chromosomal abnormalities were found in the BM of 12 out of the 16 cases for whom marrow was studied at the time of the relapse. A statistically significant negative correlation between leukemic relapse and graft versus host disease (GvHD) was found, but the relationships between chromosome damage and leukemic relapse, GvHD, and the pretransplant radiation dose and between the radiation dose and both leukemic relapse and GvHD were not significant.     

Oral Ezatiostat HCl (TLK199) and Myelodysplastic syndrome: A case report of sustained hematologic response following an abbreviated exposure

Treatment options for patients with lower risk non-del(5q) myelodysplastic syndromes (MDS) who fail erythroid stimulating agents are restricted to one of the hypomethylating drugs with an expected response rate of ~50%. Ezatiostat HCl, an agent with the potential for producing multi-lineage responses in this population is currently in clinical investigation phase. This case report describes a 77 year old male who received less than two cycles of therapy with ezatiostat HCl which had to be aborted due to intolerable side effects, but which produced a sustained normalization of all three blood counts. This trilineage response has now lasted for more than a year. Interestingly, the patient began with a del(5q) abnormality and responded briefly to lenalidomide. Upon relapse of the anemia, a bone marrow showed the disappearance of the del(5q) but the appearance of a new clonal abnormality t(2;3). Given that the patient had a complete cytogenetic response to a truncated exposure to lenalidomide followed by a trilineage response to an even briefer course of ezatiostat HCl suggests a potential role for ezatiostat HCl in del(5q) patients who relapse following lenalidomide.     

Analysis of CD38 and ZAP70 mRNA expression among cytogenetic subgroups of Iranian chronic-lymphocytic-leukemia patients

Chromosomal abnormalities and ZAP70 expression profile are two major independent prognostic markers in B-cell chronic lymphocytic leukemia. We investigated a possible correlation between these two markers. ZAP70 expression using real-time RT-PCR was examined in 20 B-cell chronic lymphocytic leukemia patients with del13q14, 13 patients with del11q22, 15 patients with trisomy 12, and 16 patients with no detected chromosomal abnormalities. Molecular analysis revealed that ZAP70 expression in the del13q subgroup was the same as in the control group, while it increased 2.78-fold in the del11q subgroup and 2.95-fold in the trisomy 12 subgroup, compared to the 15 cases in the control group. Comparison of the mean and standard deviation of the ZAP70 expression profile within the subgroups showed it to be highly variable among the individuals of the del11q and trisomy 12 subgroups, versus tight clustering for the del13q subgroup. Therefore, there is a correlation between del13q aberration, which has good prognosis with normal levels of ZAP70 expression. Due to a high degree of variation, no conformity is seen for del11q and trisomy 12 subgroups, making this grouping poor for prognostic discrimination. As a result, neither of these markers can serve as sole discriminators to determine the course of the disease; the use of both markers improves prognostic assessment.     

Philadelphia-negative chromosomal evolution during treatment for chronic myeloid leukemia

Chromosome evolution is one of the major mechanisms of disease progression and resistance in chronic myeloid leukemia (CML) patients. However, the clinical significance of chromosomal evolution in the Philadelphia (Ph)-negative clone during therapy is not fully understood. We evaluated 94 CML patients in the chronic phase of CML during treatment of the disease. Six of them had Ph-negative chromosome abnormalities during treatment. Four patients with a single abnormality and a good molecular response showed no obvious complications from the chromosomal changes, while two other patients who had complex abnormalities and previous treatment had poor outcomes. Our results highlight the need for close monitoring of this kind of patient, not only on a molecular level but also at the cytogenetic level.     

A nine-year cytogenetic follow-up of a patient injected with thorotrast

Summary Chromosomal abnormalities in the peripheral lymphocytes of a Thorotrast case were followed up for almost nine years during which four direct bone marrow observations were made. Chromosomal abnormalities, both Cu type (dicentrics, rings, and acentric fragments) and Cs type (reciprocal translocations, inversions, deletions, duplications, and others), were observed with an average frequency of about 7.5% and 8%, respectively. The frequency of chromosomal abnormalities showed no significant changes during the nine years. Formation of clones of cells with identical chromosomal abnormalities was observed both in bone marrow and peripheral lymphocytes. There were clones common to myeloid and lymphoid cell series, which may be regarded as evidence for the presence of pluripotent stem cells in man. One such common clone had a translocation involving chromosomes 2 and 22. Banding analysis revealed a break in chromosome 22 at the q12 or q12/13 band interface. The frequency of the clone remained fairly constant within 5% for several years and the patient showed no indication of leukemia or any other blood disease. The finding seems to suggest a genetic factor relating to the development of chronic myelocytic leukemia (CML), which generally shows the Ph¹ chromosome resulting from a translocation of chromosome 22 with the break at the q11 or q11/12 band interface. The increase in the number of cells lacking the Y chromosome was observed in our final bone marrow sample, but the phenomenon may be attributed to the age of the patient rather than to the direct effect of radiation. Results of the cytogenetic follow-up study seem to indicate the importance of studies in this direction for a better understanding of radiation effects on human beings.     

Identification of 3q21q26 syndrome by "multipoint" interphase FISH analyses in childhood myeloid leukemia

Cytogenetic syndrome involving bands 3q21 and 3q26, known as "3q21q26 syndrome" has been observed in adult patients with acute myelogenous leukemia (0.5-2%), chronic myelogenous leukemia in blast crisis (20%), myelodysplastic syndromes and myeloproliferative disorders. In the present study bone marrow samples from two boys (12 and 16 years), diagnosed with CML and AML respectively, were investigated using conventional cytogenetic methods, interphase "multipoint" fluorescence in situ hybridization (FISH), dual color-FISH and multiplex FISH. The "multipoint" FISH analysis identified in de novo childhood AML case an inv(3)(q21q26) and a complex 3q rearrangement including inversion and duplication in the CML case. The "3q21q26 syndrome" is associated with normal or elevated platelet counts with marked abnormalities of megakaryocytopoiesis, involvement of multiple hematopoietic lineages. The affected patients were resistant to conventional chemotherapy and had a short survival. This syndrome is very rare in de novo childhood AML, and simultaneous presence of 3q inversion and duplication, to our knowledge, has not yet been identified in hematological malignancies. The results of our study emphasize the importance of classical and modern cytogenetic analysis in the diagnosis of hematologic malignancies, because in the majority of cases they can provide additional diagnostic information for the clinicians in deciding the best therapeutic approach, precise classification and prognosis of the disease.     

Hematopoietic stem cell expansion and distinct myeloid developmental abnormalities in a murine model of the AML1-ETO translocation

The t(8;21)(q22;q22) translocation, which fuses the ETO gene on human chromosome 8 with the AML1 gene on chromosome 21 (AML1-ETO), is one of the most frequent cytogenetic abnormalities associated with acute myelogenous leukemia (AML). It is seen in approximately 12 to 15% of AML cases and is present in about 40% of AML cases with a French-American-British classified M2 phenotype. We have generated a murine model of the t(8;21) translocation by retroviral expression of AML1-ETO in purified hematopoietic stem cells (HSC). Animals reconstituted with AML1-ETO-expressing cells recapitulate the hematopoietic developmental abnormalities seen in the bone marrow of human patients with the t(8;21) translocation. Primitive myeloblasts were increased to approximately 10% of bone marrow by 10 months posttransplant. Consistent with this observation was a 50-fold increase in myeloid colony-forming cells in vitro. Accumulation of late-stage metamyelocytes was also observed in bone marrow along with an increase in immature eosinophilic myelocytes that showed abnormal basophilic granulation. HSC numbers in the bone marrow of 10-month-posttransplant animals were 29-fold greater than in transplant-matched control mice, suggesting that AML1-ETO expression overrides the normal genetic control of HSC pool size. In summary, AMLI-ETO-expressing animals recapitulate many (and perhaps all) of the developmental abnormalities seen in human patients with the t(8;21) translocation, although the animals do not develop leukemia or disseminated disease in peripheral tissues like the liver or spleen. This suggests that the principal contribution of AML1-ETO to acute myeloid leukemia is the inhibition of multiple developmental pathways.     

Cytogenetic studies in children with Acute Lymphocitic Leukemia-B in Costa Rica

Chromosome analyses were performed on bone marrow of 177 pediatric patients with Acute Lymphocitic Leukemia at the "Hospital Nacional de Nifios". The standard cytogenetic techniques now belongs to the panel of mandatory analyses performed at diagnosis of our acute leukemia patients and represent a major advantage to be effective and independent prognostic factors, essential for therapeutic choices. Cytogenetic results were achieved in 83% of the bone marrow samples: normal karyotypes represented 29% and abnormal karyotypes 71% with the follow distribution: t(9;22) 3%; t(l;19) 5%; t(4;11) 3%, Hyperdiploidy 39%; other chromosomal abnormalities 21%. Systematic cytogenetic analyses are essencial to define morpho-immunologic sub-types of leukemia and to detect new translocations that allows to understand hematopoiesis and leukemogenesis.     

New cytogenetic aberrations found in a case of aggressive retinoblastoma

We describe a case of retinoblastoma with an atypical presentation and previously unreported cytogenetic aberrations. A 19-month-old girl with left intraocular retinoblastoma was treated with enucleation and chemotherapy. The disease showed aggressive evolution within a short period between diagnosis and relapse. Eight months after diagnosis, a new large tumor was present in the orbit of the right eye, with diffuse bone pain, pancytopenia and diffuse infiltration into the bone marrow and the central nervous system. The child did not respond to treatment and died. Cytogenetic studies made with G-banding, FISH and SKY analysis showed chromosomal aberrations commonly associated with retinoblastoma, including del(13q), i(6p), +1, and monosomy 16, along with others that had not been reported previously, including dup(5q), dic(15;22) and add(14q). The new chromosomal aberrations may be related to the aggressiveness of the disease in this case.     

Flow cytometric DNA content in myelodysplastic syndromes

DNA flow cytometric analysis of unfixed bone marrow cells stained with propidium iodide was carried out in 33 patients with untreated primary myelodysplastic syndromes. Patients with stable clinical course for up to 3 years had higher fractions of cells in S and G2 phases (22.7 +/- 12.4% and 12 +/- 3.6%) than those who developed acute leukemia and/or died early in the course of disease (14.4 +/- 8.5% and 6.6 +/- 4%). Median survival was more than 36 mo in patients with S + G2 cell fraction higher than 24%, and 14 mo in the remaining 16 patients with lower values (P less than 0.01). Analyses repeated after 3-24 mo showed no major changes in cell proliferation pattern in ten out of 11 patients. The remaining patient had sharp decrease in S and G2 cell fraction 3 mo before the transition into acute leukemia. The DNA index (DI) of bone marrow cells was calculated to assess ploidy. However, comparative evaluation of cytologic, cytogenetic, and flow cytometric data suggest that, under our experimental conditions, the DI may be influenced by factors such as the degree of chromatin compactness.     

Prognostic significance of some clinical, morphological and cytogenetic findings in refractory anaemia (RA) and RA with sideroblasts

In 27 patients initially diagnosed as refractory anaemia (RA) or RA with sideroblasts (RA-S) according to the FAB-classification a number of clinical, morphological and cytogenetic parameters were correlated for prognostic significance. From these correlations it emerged that severe cytopenia is centrally positioned with regard to clinical course in RA and RA-S. Positive correlations were found to initial diagnosis, clonal cytogenetic abnormalities, progression to RA with an excess of blasts (RAEB) or acute myeloid leukaemia (AML), the percentage of bone marrow blast cells and prolonged half life for radioactively labeled iron. The degree of peripheral blood granulocytopenia, alone, was correlated to bone marrow hypoplasia. Moreover, the frequency of abnormal karyotypes was inversely correlated to bone marrow cellularity and proportional to the frequency of bone marrow blast cells. From these relationships it may be proposed that chromosome abnormalities are associated with prolonged blast cell generation times and inhibition of blast cell maturation resulting in reduced marrow cellularity and blast cell accumulation, and, in the peripheral blood, falling percentages of neutrophil granulocytes. With the blast cell accumulation the bone marrow cellularity again becomes hyperplastic and the preleukaemic condition is transformed into RAEB or AML.     

Cytogenetics and molecular genetics of acute lymphoblastic leukemia

An important factor in the diagnosis of acute lymphoblastic leukemia (ALL) is that karyotype is an independent prognostic indicator, with an impact on the choice of treatment. Outcome is related to the number of chromosomes. For example, high hyperdiploidy (51-65 chromosomes) is associated with a good prognosis, whereas patients with near haploidy (23-29 chromosomes) have a poor outcome. The discovery of recurring chromosomal abnormalities in the leukemic blasts of patients with ALL has identified a large number of genes involved in leukemogenesis. Certain specific genetic changes are related to prognosis. The ETV6/AML1 fusion arising from the translocation (t12;21) (p13;q22) has been associated with a good outcome; the BCR/ABL fusion of (t9;22)(q34;q11), rearrangements of the MLL gene, and abnormalities of the short arm of chromosomes 9 involving the tumor suppressor genes p16INK4A have a poor prognosis. Unfortunately, the classification of patients into prognostic groups based on cytogenetics is not always as predicted. Even when other clinically based risk factors are taken into account, some patients with good-risk cytogenetic features will relapse. In the search for new measures of prognosis, it has recently emerged that the level of minimal residual disease following induction therapy can be a reliable predictor of outcome in ALL.