Multilineage Involvement Research Articles

Multilineage involvement of Philadelphia chromosome positive acute lymphoblastic leukemia.

Acute lymphocytic leukemia (ALL) is considered a clonal disease restricted to the lymphoid compartment. The Philadelphia chromosome (Ph) is found in a subset of ALL with poor prognosis. Here we present the largest series of Ph+ ALL analyzed for involvement of the myeloid compartment. For the first time at a single cell level the presence of Ph in lineages other than lymphoid is demonstrated. Granulocytes from nine patients diagnosed with BCR-ABL + ALL (eight Ph+, one Ph-) were purified using two layer density gradient separation. They were further identified by the morphology of DAPI-stained nuclei and studied for the presence of the Ph by fluorescence in situ hybridization (FISH) using a BCR-ABL dual-color probe. Ph was demonstrated in 30 to 93% of granulocytes in all patients. FISH identified major and minor BCR gene breakpoints (M-bcr and m-bcr). In one patient, with CD19+/34+/33-/2-/3-/7-/10- lymphoblasts, involvement of B cells (CD19+), T cells (CD3+), myeloid (CD13+), erythroid (glycophorin A+) cells was found by FISH following fluorescence-activated cell sorting (FACS). The diagnosis of ALL as opposed to lymphoblastic transformation of CML was established based on clinical and laboratory data including Western blot results demonstrating the presence of p190/m-bcr in five of the nine cases studied. Results suggest that Ph+ ALL originates from a pluripotent stem cell.

Acute myelogenous leukaemia

Age represents one of the most important adverse prognostic factors in acute myeloid leukemia (AML). The therapeutic results for patients older than 60 years accrued into clinical trials of intensive chemotherapy are largely unsatisfactory (complete remission rates rarely superior to 50–60%; median relapse-free survival usually less than 12 months). Because only 30–40% of elderly patients are actually entered into these trials, the overall failure of current treatments appear even more disappointing when considered in the context of the whole population of older individuals with AML. This appears primarily due to intrinsic differences in the biology of leukemia itself and to host-related factors (i.e. reduced tolerance to chemotherapy and comorbidity). AMLs of older subjects display several biological overlaps with secondary AMLs including multilineage involvement, phenotype, unfavorable cytogenetics and elevated activity of multidrug resistance genes. The clinical application of biologically-based prognostic factors may enable to separate patients who may actually benefit from aggressive chemotherapy from those who should be offered attenuated/palliative treatments or enrolled upfront into experimental trials of new drugs or biologic/immunologic treatments. This may hopefully result in a ‘risk-adapted’ strategy aimed at improving disease free survival and/or quality of life for patients with differing risk profiles.

Fluorescence in Situ Hybridization Analysis of t(3; 12)(q26;p13): A Recurring Chromosomal Abnormality Involving the TEL Gene (ETV6) in Myelodysplastic Syndromes

We have identified a new recurrent reciprocal translocation between chromosome 3 and 12 with breakpoints at bands 3q26 and 12p13, t(3;12)(q26;p13) in the malignant cells from five patients with acute transformation of myelodysplastic syndrome or blast crisis of chronic myelogenous leukemia. t(3;12)(q26;p13) appears as a rare but nonrandom event present in various myeloid leukemia subtypes, which is frequently associated with dysplasia of megakaryocytes, multilineage involvement, short duration of any blastic phase, and a very poor prognosis. Here, we report the molecular cytogenetic analysis of the t(3;12). Fluorescence in situ hybridization results indicate that the 3q26 breakpoints are quite heterogeneous and occur 5' of MDS1, 3' of EVI1, or between MDS1 and EVI1. Our results are very similar to those observed in other 3q26 rearrangements in which breakpoints were shown to occur over considerable distances 5' and 3' of EVI1. Fluorescence in situ hybridization investigations proved that, in three myelodysplastic syndrome cases with t(3;12)(q26;p13), the 12p 13 breakpoint occurred within the TEL gene.

Translocation (3;3) in a patient with thrombocytopenia and erythroid dysplasia

We describe a case with a translocation between the two chromosomes 3 with breakpoints q21 and q26, associated with a unique constellation of hematologic abnormalities. Megakaryocytic dysplasia and peripheral thrombocytosis are the most common abnormalities associated with this chromosome abnormality. Our patient had acute myeloid leukemia (AML), thrombocytopenia, dyserythropoiesis, and an increased myeloid/erythroid ratio but no megakaryocytic dysplasia. Although multilineage involvement has been reported, erythrocyte dysplasia associated with thrombocytopenia and without megakaryocyte dysplasia appears to be extremely rare. Our case supports the speculation that #3 abnormalities with breakpoints q21 and q26 affect a pluripotent stem cell and suggests that megakaryocytic involvement may not be pathognomonic in hematologic abnormalities with t(3;3) (q21;26).

Morphologic characteristics of erythroleukemia (Acute Myeloid Leukemia; FAB‐M6): A CALGB study

We have reviewed the clinical, morphologic, immunophenotypic, and cytogenetic features of 52 patients with erythroleukemia (FAB Cooperative Group; AML-M6) studied by the Cancer and Leukemia Group B (CALGB). The purpose of this study was to correlate morphology with the clinical features, immunophenotypes, and karyotypes of neoplastic cells, and with the response to therapy of patients with AML-M6. Thirty-three patients (63%) were male, median age 59 (range 16-81) years, 47 patients (90%) were white, and 42 patients (81%) had a performance status of < 2. Myelodysplastic changes were observed in at least 1 cell lineage in all cases, and in 2 cell lineages in 45 of 52 (86%) cases. Fifty percent or more of cases studied were positive for CD11b, CD13, CD15, CD33, glycophorin-A, and HLA-DR markers. Fourteen of 27 cases (52%) in whom karyotypic analyses were conducted had cytogenetic abnormalities. Five (19%) were simple (< 3 karyotypic abnormalities), while 9 (33%) were complex (> or = 3 abnormalities). We observed either a complete or partial loss of chromosomes 5, 7, or 12p, or the presence of trisomy 8, in 11 of 27 (41%) patients. Cases of AML-M6 were divided into group 1 (14 patients with bone marrow proerythroblasts and basophilic erythroblasts > 25% of all erythroblasts) and group 2 (38 patients with proerythroblasts and basophilic erythroblasts < or = 25% of all erythroblasts). We observed no significant differences between groups 1 and 2 in regard to sex, age, race, performance status, percentage of blood erythroblasts or myeloblasts, percentage of bone marrow erythroblasts, and periodic acid-Schiff (PAS) or myelodysplasia scores. Six of 6 (100%) patients of group 1, and 7 of 21 (33%) patients of group 2, had normal karyotypes (P = .006). Nine of 13 (69%) patients of group 1 and 15 of 33 (45%) patients of group 2 had a complete remission (CR) (P = .2). Eight of 11 (73%) cytogenetically normal patients achieved CR: 5 of 6 (83%) in group 1, and 3 of 5 (60%) in group 2. Five of 12 (42%) cytogenetically abnormal patients achieved CR. No difference in duration of survival (group 1, median = 4.6 months vs. group 2, median = 10.2 months; P = .93) was observed between the 2 groups. We conclude that AML-M6 is typified by multilineage involvement of hematopoietic cells. The morphology of erythroblasts in patients with AML-M6 may correlate with cytogenetic abnormalities and rate of CR.

Abnormal platelet von willebrand factor (vWF) as a marker of abnormal function in megakaryocytic dysplasia

The myelodysplastic syndromes (MDS) are neoplastic disorders of the hemopoietic system; multilineage involvement is also evidenced by specific cellular dysfunctions. The von Willebrand factor (vWF), synthesized and processed in the megakaryocytes (MK), is stored in the alpha granules of the platelets. The platelet vWF multimeric pattern was studied in 18 patients with MDS, and in 4 with pernicious anemia (PA), to investigate whether the processing of vWF is abnormal in the megakaryocytic dysplasia. An abnormal multimeric pattern was observed in 10/18 MDS and 4/4 PA patients. The abnormality of this specific protein is the discrete expression of the basic disorder, and is reversible when hemopoiesis is normalized. Although the data do not allow any conclusion, abnormal synthesis is the likely explantation of the abnormality.

Translocation t(8;13)(p11;q11-12) in stem cell leukemia/lymphoma of T-cell and myeloid lineages.

An unusual hematologic neoplasia has been described recently in which the predominant clinical features include T-cell lymphoma, myeloid hyperplasia, and eosinophilia. The multilineage involvement in this disorder suggests transformation of a primitive stem cell. Abnormal karyotypes have been described in three such cases, including one case with t(8;13)(p11.2;q12) and a second case with t(8;13)(p23;q14). We report translocation of chromosomes 8 and 13 in lymph node karyotypes from two patients with this syndrome. Fluorescence in situ hybridization confirmed an identical translocation, t(8;13)(p11;q11-12), in lymphoma cells from each patient. The translocation breakpoints are of particular interest because the FLT3 receptor tyrosine kinase gene has been mapped 13q12. FLT3 is expressed highly in hematopoietic progenitor cells and in myeloid and lymphoid acute leukemias.

Immunophenotype of mitotic cells with clonal chromosome abnormalities demonstrating multilineage involvement in acute myeloid leukemia

Eleven patients with acute myeloid leukemia (AML) were studied with a technique that simultaneously identifies cytogenetic abnormality and immunophenotype of the same mitotic cell. To determine the cell lineages with abnormal karyotypes, monoclonal antibodies in the alkaline phosphataseantialkaline phosphatase (APAAP) detection procedure were used. The granulocytic/monocytic lineage was involved in the leukemic process in all 11 patients. In nine patients, we also detected abnormal karyotypes in the erythrocytic and/or megakaryocytic lineages. All four patients with secondary AML showed involvement of the granulocytic/monocytic, erythrocytic, and megakaryocytic lineages into the leukemic process, as compared with five of seven patients with de novo AML. One patient with trisomy 8 showed erythrocytic participation in the leukemic process, but in another the erythrocytic lineage had only normal karyotypes. Thus, in AML, the chromosome abnormalities apparently usually originate at the multipotent progenitor cell stage, since in addition to granulocytic/monocytic lineages, erythrocytic and/or megakaryocytic lineages were also involved. Some patients show involvement of granulocytic/monocytic lineages only, however, suggesting that the target cell belongs to a more mature committed progenitor cell stage.

Therapy associated leukaemia

A dose related risk of acute leukaemia, myelodysplasia and other cancers is seen in patients treated with certain drugs, particularly alkylating agents, and radiotherapy either alone or in combination. Treament associated acute myeloid leukaemia (tAML) and myelodysplasia have biological and clinical features in common and are distinct from the coresponding de-novo disorders. tAML generally occurs between 2–11 years from administration of chemo/radiotherapy with few cases thereafter. Patients may present with myelodysplasia and severe cytopenia with abnormalities in all cell lines or as an acute leukaemia which may be difficult to classify because of multi-lineage involvement. Clonal cytogenetic abnormalities usually including either loss or interstitial deletion of the long arm of chromosomes 5 or 7 are frequently identified. Critical regions deleted in all patients with these lesions have been localised at 5q23-32 and 7q22-31; regions carrying the genes for several haemopoietic growth factors, receptors and oncogenes. The prognosis of patients with tAML is poor with low remission rates and a median survival of 6 months. Complex karyotypes or lesions of chromosomes 5 or 7 seen in the majority of cases are associated with a particularly poor outlook. The risks of this most serious complication of therapy should be weighed carefully against possible benefits.

Morphologic, immunologic and cytogenetic studies in erythroleukaemia: evidence for multilineage involvement and identification of two distinct cytogenetic‐clihicopathological types

Clinical features, as well as morphology, immunophenotype and cytogenetics were retrospectively studied in 20 patients with an original diagnosis of erythroleukaemia (EL) reclassified according to the FAB criteria. Fifteen patients had de novo EL, five patients had therapy-related EL. Myelodysplasia preceded the onset of EL in eight cases and myelodysplastic features involving multiple haemopoietic lineages were observed at leukaemia presentation in all cases. Immunologic findings confirmed multilineage involvement, showing sub-population of cells expressing platelet-associated markers in more than 50% of cases tested and the presence of a myelomonocytic component, besides glycophorin A-positive cells. Cytogenetically, major karyotype aberrations (MAKA), defined by the presence of three or more aberrant events in the same clone, were observed in 14 cases, minor karyotype aberrations (MIKA) were observed in four cases and normal karyotype in two cases. No differences in the cytological-cytogenetic picture of our patients with de novo EL, and with therapy-related EL were found suggesting that aetiological factors and/or pathogenetic mechanisms common to EL and secondary leukaemia may exist. All patients with MAKA had leftward shift of erythropoiesis with proerythroblasts and basophilic erythroblasts usually representing more than 50% of all erythroid cells. In patients with MIKA or normal karyotype, maturatio of erythroid cells, though morphologically abnormal, was quantitatively preserved and early erythroblasts never exceeded 25% of erythroid cells. Clinically, the haemoglobin level at presentation, as well as in the proportion of patients achieving complete remission after chemotherapy, appeared to be lower in the maturation arrest-MAKA group as compared to the preserved maturation-MIKA/normal karyotype group. Median survival was shorter in the former group (3.5 months) than in the latter (median 13 months). Morphologic-immunologic-cytogenetic studies thus allow for the identification of two distinct cytogenetic-clinicopathological types of EL.

Immunophenotype of Abnormal Metaphases Demonstrating Multilineage Involvement in Myelodysplastic Syndromes.

Eight patients with myelodysplastic syndrome (MDS) and chromosome abnormalities were studied by a combined method which allows simultaneous analysis of the karyotype and immunophenotype of the same mitotic cell. To determine the cell lineages with abnormal karyotype, monoclonal antibodies in the alkaline phosphatase-antialkaline phosphatase (APAAP) staining methods were used All patients with MDS showed metaphases with abnormal karyotype in cells belonging to granulocytic/monocytic lineage. In 7 patients studied the leukaemic process also involved erythrocytic and/or megakaryocytic lineages. In 4 patients with 5q- chromosome it was evident that at least the granulocytic/monocytic and erythrocytic cell lineages participated in the clonal proliferation. One patient with 5q- chromosome at initial diagnosis showed a shift from megakaryocytic to a granulocytic/monocytic cell population at the time of acute leukaemia These findings indicate multi-lineage involvement in patients with MDS who have karyo-typic abnormalities.

Karyotype evolution and multilineage involvement of Philadelphia chromosome-positive clones in blastic transformation of two patients with chronic myelocytic leukemia

Simultaneous analysis of the cell morphology and karyotypes on single colonies was carried out in two patients with Philadelphia chromosome (Ph1)-positive chronic myelocytic leukemia in blastic transformation in order to clarify the origin of leukemic cells involved. Patient no. 1 was in a typical myeloblastic transformation and patient no. 2 in “basophilic transformation.” Both patients exhibited karyotype evolution in blastic phase (BP), so that we could differentiate BP clones with additional chromosomal abnormalities from chronic phase (CP) clones with only Ph1 among single colonies. The number of single colonies yielding two or more analyzable metaphases was 18 in patient no. 1, and 19 in patient no. 2. Among these colonies, only three in patient no. 1 and none in patient no. 2 were from CP clones and 15 in patient no. 1 and 19 in patient no. 2 were from BP clones. Morphological examination revealed that not only blasts but also mature neutrophils, eosinophils, basophils, macrophages, and erythroblasts were derived from BP clones. These results suggested that (1) BP clones developed at the pluripotent stem cell level, (2) additional chromosomal abnormalities were not restricted to occur in a specific cell line representative in BP; and (3) BP clones, if not all, may retain capacity for maturation and differentiation.

A fluorescent dye which recognizes mature peripheral erythrocytes of myeloproliferative disorders

Based on the previous finding that erythrocytes from patients with chronic myelogenous leukemia stain with the fluorescent dye merocyanine 540, erythrocytes from patients with other myeloproliferative disorders were examined for their ability to bind the membrane probe. As assessed by both fluorescence staining and a quantitative dye removal assay, all samples of erythrocytes from patients with chronic myelogenous leukemia, polycythemia vera, myelofibrosis with myeloid metaplasia and essential thrombocythemia bound more dye than did erythrocytes from normal, healthy individuals. Erythrocytes from three of six patients with acute myelogenous leukemia also showed increased affinity for the dye. In contrast, erythrocytes from three patients with acute lymphocytic leukemia and one with unclassifiable leukemia bound only normal amounts of dye. The procedures described may be useful as a supplemental aid to diagnosis of myeloproliferative disorders or for investigation of hematological diseases where multilineage involvement is suspected.