The activity of nucleolar organizer regions of human bone marrow cells studied with silver staining. I. Chronic myelocytic leukemia

The activity of nucleolar organizer regions (NORs) in chromosomes and interphase nuclei of bone marrow (BM) cells from 21 patients with chronic myelocytic leukemia (CML), including seven patients at the time of blastic crisis (BC), has been studied with silver nitrate staining. The average numbers of Ag-NOR per metaphase in PHA-stimulated peripheral blood lymphocytes from patients with CML and normal individuals were 7.1 +/- 0.3 and 7.4 +/- 0.1, respectively, indicating no statistical difference between them. Those in BM cells from patients with CML, as in the normal donors, were more heterogeneous compared to PHA-stimulated lymphocytes, and most of the metaphases (up to 67%) did not contain silver-stained NORs. The average number of Ag-positive NORs in BM mitoses from untreated patients in the chronic phases of CML and from those in the BC were similar (4.9 +/- 0.3 and 4.8 +/- 0.4, respectively). As for NORs of the Ph chromosome, they were Ag-positive in the majority of patients, including 9 of 14 in the chronic phase and 3 of 7 in the BC. This article contains some data in support of the authors' previous assumption regarding the correlation between BM Ag-NOR patterns and the degree of maturity of the cells tested in mitosis.     

Natural cytotoxicity in adult acute leukemia

Natural cytotoxic activity was studied in 32 adult patients with acute non-lymphoid leukemia (ANLL) and 27 adult patients with acute lymphoblastic leukemia (ALL). Thirteen patients with active ANLL had a significantly reduced natural cytotoxicity in peripheral blood compared with that of healthy controls, 5.0 +/- 3.4 versus 27.1 +/- 11.5 (p less than 0.0005). Patients with ANLL in remission had a normal natural cytotoxicity (p greater than 0.05). Patients with active ANLL had a significantly reduced number of Leu-7-positive cells (p less than 0.001), while patients in remission had normal proportions of these cells (p greater than 0.05). Peripheral blood from patients with ALL showed reduced natural killer (NK) cell activity, both in active disease and in remission (p less than 0.0005). The percentage of Leu-11b (CD 16)-positive cells was increased in patients in remission from ALL (p less than 0.05). Bone marrow cells from patients with ALL in remission had a reduced natural cytotoxicity, 2.8 +/- 4.0 versus 16.3 +/- 9.0 in bone marrow controls (p = 0.01). In contrast, patients with ANLL in remission showed a normal bone marrow cytotoxicity (p greater than 0.05) while patients with active ANLL had a reduced NK cell activity (p less than 0.03). The low NK activity observed in leukemic patients may be of importance for the pathogenesis of the diseases. Acute non-lymphoid leukemia (ANLL) is a malignant disease of the multipotential hemapoietic stem cell. The stem cell is capable of differentiating into various cell lineages, i.e. erythroid, granulocytic, monocytic and megacariocytic cell lineages.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chromosome studies in patients with acute nonlymphocytic or acute lymphocytic leukemia submitted to bone marrow transplantation--results of a European cooperative study

The chromosomal data of 58 acute nonlymphocytic leukemia (ANLL) patients and of 32 acute lymphocytic leukemia (ALL) patients submitted to bone marrow transplantation and collected from nine institutions are reported. Chromosomal studies were available at diagnosis in 19 cases with ANLL: seven had a partially or completely abnormal pattern. Forty-one patients had a chromosome study before bone marrow transplantation and all had a normal pattern. Thirteen patients with ALL were studied at diagnosis: five had a partially or completely abnormal karyotype. Of 20 cases analyzed before bone marrow transplantation, only one has maintained the abnormal pattern of diagnosis in part of the cells. Karyotypes were available in nine ANLL patients relapsed after bone marrow transplantations. Two showed the same clonal abnormalities seen at diagnosis; in three other cases the leukemic clone of relapse carried an additional chromosome abnormality with respect to the pattern at diagnosis and four more cases presented at relapse complex abnormalities; two of them had a cytogenetically normal pattern at diagnosis. In four of ten relapsed ALL cases chromosomes analyses were available. A relapse in donor cells and a hyperdiploid pattern were observed in two cases, respectively, while a normal, recipient pattern was documented in the other two cases. Serial chromosome studies performed in acute leukemia patients after bone marrow transplantation may allow the detection of different chromosomal patterns of relapse. In those cases who relapsed with a cell clone cytogenetically different from the pattern at diagnosis, a direct role played by the conditioning treatment in the pathogenesis of the relapsing disease may be hypothesized.     

Temporal variation in nucleolar organizer region expression in bone marrow cells of individuals with leukemia

Silver staining was used to study nucleolar organizer region (NOR) expression in bone marrow cells obtained at two or, in one case, three time points from each of six leukemia patients. Using three measures of silver positivity, we observed that NOR expression was influenced by both metaphase stage and time. Silver positivity decreased significantly from one metaphase stage to the next, from prometaphase through late metaphase. When this variable was controlled for, significant changes in NOR activity were documented in comparisons between disease stages in the patients examined. However, patterns of NOR expression were not consistently associated with disease stage. These results indicate that in previous reports both the metaphase stage effect and the temporally changing nature of NOR activity have, as unrecognized variables, influenced observations of heterogeneity in NOR expression.     

Lack of correlation between telomere length and telomerase activity and expression in leukemic cells

The expression of three components of telomerase complex (hTR, hTERT, TP1) along with telomerase activity and telomere length in leukemic cells was investigated. Cells were isolated from peripheral blood and/or bone marrow of children with acute lymphoblastic (ALL) and non-lymphoblastic (ANLL) leukemia. Expression of three components of telomerase as well as telomerase activity was found in all leukemic cells. Chemiluminescent detection of terminal restriction fragments (TRF) from DNA isolated from ALL cells showed variable patterns expressing considerable heterogeneity of telomere length. The ALL cells appeared to have both long and short telomere lengths, in contrast to normal peripheral lymphocytes, which produced limited pattern of TRF. The ANLL cells produced predominantly short telomere pattern despite high telomerase activity and expression. It can be concluded that high telomerase activity and expression in leukemic cells is not always correlated with long telomeres (TRF pattern).     

Sister chromatid exchanges in leukemic patients

Sister chromatid exchange (SCE) was studied in PHA-stimulated peripheral blood lymphocytes from 36 newly diagnosed and untreated leukemic patients: 16 with acute lymphoblastic leukemia (ALL), 10 with acute nonlymphocytic leukemia (ANLL), and 10 with chronic myelocytic leukemia (CML). The metaphases analyzed show no chromosomal abnormalities. The mean SCE frequency (mean +/- SE) for each group of patients was: 6.8 +/- 0.4, 6.6 +/- 0.3, and 7.0 +/- 0.6 per mitosis, respectively, which was significantly lower than the mean SCE score for 30 controls (8.7 +/- 0.2). No differences in SCE score among ALL, ANLL, and CML and a similar SCE frequency by chromosome number and group allowed consolidation of all the cases into a single group of 36 leukemic patients (6.8 +/- 0.3). When the frequency of SCE was compared by chromosome number and group between the leukemic patients with the control group, a significant decrease in SCE frequency was observed due to a low SCE score in almost all the complements, except chromosome #1. It is suggested that the low SCE rate is related to the leukemic process itself.     

Cytogenetic study in a mentally retarded child with bloom syndrome and acute lymphoblastic leukemia

Bloom syndrome (BS) was diagnosed in a 7-year-old boy during hospitalization for acute lymphoblastic leukemia (ALL). The patient had most of the signs of BS along with some atypical manifestations: absence of telangiectases, obesity, and moderate mental retardation. Results of the cytogenetic studies were fully consistent with the diagnosis of BS: the occurrence of quadriradial figures and a very high incidence of sister-chromatid exchanges (SCE). This child's ALL was of non-B, non-T type with the presence, at the time of diagnosis, of a marrow clone including two markers. A Yq– chromosome was detected in about 10% of PHA-stimulated lymphocytes but neither in bone marrow cells nor in skin fibroblasts. This case is the fifth instance of ALL out of 104 registered cases of BS.     

The proportion of abnormal karyotypes in acute leukemia samples related to method of preparation

Bone marrow and peripheral blood were studied from 200 patients with acute leukemia [109 with acute myeloid leukemia (AML), 91 with acute lymphoblastic leukemia (ALL)] who had samples cultured for varying times and who had a mixture of chromosomally abnormal and normal cells. The mean percentage of abnormal metaphase cells increased with culture time. The peak was reached at 48 hours and declined slightly after 72 hours in culture for ALL patients. The mean percentage of abnormal cells increased up to 72 hours in culture for AML patients. In 68 patients (31 AML and 37 ALL), cytogenetic data were available from samples processed with both direct preparations and culture methods. The percentage of abnormal cells increased after culture in 49 patients (23 AML and 26 ALL), while it decreased or remained at the same level in 19 patients. For AML patients, the mean percentage of abnormal cells was significantly different between direct (38%) and cultured preparations (63%), (p less than 0.001). Seven of 9 patients with AML who showed a greater than 50% increase in abnormal cells after culture had either a t(8;21), t(15;17), or abnormalities involving 11q23. The two patients who showed a significant decrease in abnormal cells both had a translocation involving 11q13. Compared with ALL, more AML patients showed greater than 80% abnormal bone marrow metaphase cells at diagnosis or at relapse.     

Application of nucleolar organizer region staining technique to air-dried blood smears]

Silver staining of nucleolar organizer regions (NOR) was applied to air-dried peripheral and bone marrow smears of normal subjects and leukemic patients. Specimens were fixed in buffered acetone formalin. Even in smears kept for 2 years at room temperature, the stainability of Ag-NOR was well preserved. By dipping Giemsa-stained smears in 5% trichloracetic acid and then placing them in methanol for 5 minutes, the stain was leached out. After the dye had been removed, the smears were clearly stained by a Ag-NOR staining technique. The mean number of Ag-NOR per nucleus of mature granulocytes and mononuclear cells in normal peripheral bloods was 0.59 and 1.43 respectively. The mean number of Ag-NOR per nucleus of peripheral and bone marrow leukemic cells from patients with acute leukemia and chronic myelocytic leukemia in blastic crisis was 2.32 and 2.66 respectively. On the other hand, the mean number of Ag-NOR per nucleus of peripheral leukemic cells from patients with chronic lymphocytic leukemia was 1.48. These results suggest that acute leukemia cells possess a more active proliferating potential. The Ag-NOR staining technique is very simple and might be useful for investigation of hematologic cells.     

Variation in nucleolar organizer activity in lymphocytes of females with adenocarcinoma

With the use of Ag staining and conventional light microscopy, a difference is visualized in the frequency of nucleolar organizer regions between PHA-stimulated lymphocytes from normal females and females with various types of adenocarcinoma. This difference in NOR activity is more pronounced in G chromosomes than D chromosomes. It is feasible that rDNA gene activity in PHA-stimulated lymphocytes is augmented in patients with adenocarcinoma due to the presence of plasma mediators arising from cellular malignancies. The NOR regions located on G chromosomes are considered to be more sensitive to these mediators than those on D chromosomes.     

High resolution chromosomes of the t(9;22) positive leukemias

A combined bromodeoxyuridine (BrdU) and actinomycin D (AMD) treatment of methotrexate (MTX) synchronized bone marrow cells from four patients with chronic myelogenous leukemia (CML), and two each with acute lymphocytic (ALL) and acute nonlymphocytic (ANLL) leukemia were used to define the breakpoints involved in the t(9;22) found in subgroups of these three disorders. An additional 20 patients with CML were studied with the MTX technique alone. All cases showed the same breakpoints at subbands 9q34.1 and 22q11.2. In CML, it was possible to further define the breakpoint of chromosome 22 to subband q11.21.     

Chromosome abnormalities in acute lymphoblastic leukemia

Less information is available on the cytogenetic abnormalities in marrow cells of patients with acute lymphoblastic leukemia (ALL) than on abnormalities in acute nonlymphocytic leukemia (ANLL); nonetheless, some patterns of karyotypic change in ALL are evident. Even with banding, about 50% of patients appear to have a normal karyotype. The modal chromosome number tends to be higher in ALL than in ANLL. Every patient with B-cell ALL has had an abnormality of one chromosome No. 14 that involved the translocation of material to the end of the long arm. Among seven reported cases, the translocation was from 8q in three patients and 11q in one. Cells with a haploid or near-haploid (24–35) chromosome number have been reported in five patients with ALL and in four patients in a lymphoid blast crisis of chronic myelogenous leukemia. The karyotype in the four ALL patients whose cells were analyzed with banding was remarkably consistent. All patients had the haploid number, usually with both sex chromosomes, plus an additional No. 10, 18, and 21. Evolution of the karyotype, which occurs in the leukemic cells of about 50% of patients, involves cells of patients who had an initially normal or an initially abnormal karyotype. The evidence regarding a correlation between the presence of an abnormal clone prior to treatment and response to treatment is contradictory at present. Some chromosome abnormalities, such as the presence of a Philadelphia (Ph¹) chromosome, a 14q+ chromosome, or a haploid clone, are associated with a relatively short survival.     

Alu family variations in neoplasia.

Human chromosomes contain about one million copies of dispersed repeats of the Alu family which are distributed non-randomly. In this study we have compared the pattern of hybridization of tritiated Alu-probes on chromosomes of PHA-stimulated lymphocytes of normal donors and of non-stimulated bone marrow cells of acute leukemia patients, and found regular differences in this pattern over some chromosome bands (3q26, 8p11-p12, 14q24, 15q21, 6q22) between normal individuals and leukemia patients. These data were interpreted as indicative of somatic variation of the Alu family in acute leukemia. Possible mechanisms of the variation and the role of the Alu family in chromosome rearrangements in neoplasia are discussed.     

Terminal deoxynucleotidyl transferase (TdT) in acute nonlymphocytic leukemia. A clinical, morphologic, cytochemical, immunologic, and ultrastructural study

The classification of acute leukemia is essential for proper therapy and may be based on morphologic, cytochemical, immunologic, or even ultrastructural studies. Terminal deoxynucleotidyl transferase (TdT) is expressed in most patients with acute lymphocytic leukemia (ALL) and a minority of patients with acute nonlymphocytic leukemia (ANLL). Thirteen patients with ANLL and greater than 30% blasts positive for TdT were studied to establish the clinical, light microscopic, cytochemical, immunologic, and ultrastructural correlates of this phenomenon. Most patients demonstrated some morphologic and cytochemical features of monocytic differentiation. On cytochemical stains, nine had greater than 3% Sudan black-positive blasts. Diffuse alpha naphthyl acetate esterase (ANAE) staining of leukemic cells was present in nine cases, though extremely weak in seven. Blasts in ten patients did not express any other markers of lymphoid differentiation except TdT. However, two patient's immature cells bore CD10 common ALL antigen (CALLA) and CD19 (B4). Ultrastructural studies confirmed nonlymphoid differentiation in all ten patients studied, with a prominent monocytic component present in nine. In no case was a second population of lymphoblasts identified to account for TdT positivity. These patients responded poorly to conventional therapy for ANLL, with complete remissions in 3 of 13 (23%). With conventional therapy for ALL, complete remission was achieved in only two of nine (22%) patients. However, four of seven (57%) patients had a complete remission with high-dose cytosine arabinoside regimens. The authors' studies suggest that patients with TdT-positive ANLL represent a distinct subset that usually displays ultrastructural evidence for monocytic differentiation and is clinically significant in that these patients respond poorly to conventional therapy for both ALL and ANLL. Recognition of the monocytic lineage of these cases by light microscopic examination is difficult because they are often poorly differentiated morphologically and express only weak nonspecific esterase positivity.     

Childhood biphenotypic leukemia: detection of mixed lymphoid and myeloid populations in bone marrow specimens

In a retrospective study, consecutive bone marrow biopsies and smears from 104 children with leukemia were analyzed for expression of lymphoid and myeloid lineage-associated features. Eighty-six cases were diagnosed as acute lymphoblastic leukemia (ALL), 14 cases as acute non-lymphocytic leukemia (ANLL), and one case as chronic myelogenous leukemia (CML). Finally, three children were classified as biphenotypic leukemia demonstrating mixed populations of lymphoid and myeloid blast cells from the onset of the disease. Thus, leukemia with a dual phenotype was assessed in 2.9% of all cases examined. The recognition of bilineage origin even by conventional methods such as morphology, cytochemistry and marker studies may be important for the selection of an effective treatment.     

The predictive value of initial cytogenetic studies in 148 adults with acute nonlymphocytic leukemia: a 12-year study (1970-1982)

When leukemic blood or marrow specimens from 148 adults with newly diagnosed acute nonlymphocytic leukemia (ANLL) were studied with chromosome banding techniques, 79 were found to have clonal abnormalities. Among 130 treated patients, the 53 with initially normal karyotypes had a significantly longer survival rate than the 16 in whom no normal metaphases were observed (p = 0.02). The 55 patients with both normal and abnormal metaphase cells had an intermediate survival. Once a complete remission had been attained, however, there was no significant difference in median survival between those patients with entirely normal karyotypes and those with abnormal karyotypes. Among the various FAB morphologic subsets of ANLL, the differences in complete remission rate and overall survival between the various cytogenetic subsets were greatest in acute myelogenous leukemia (AML, M1 + M2). The presence of an abnormal clone was a more important predictor of clinical outcome (p = 0.02) than the presence of normal stem cell clones. Aneuploidy alone (hyperdiploidy or hypodiploidy) was not of predictive value, indicating that the use of banding techniques to identify structural rearrangements in pseudodiploid cells was essential. Clonal chromosomal abnormalities were nonrandom and acquired, and specific abnormalities were closely associated with specific clinical-pathologic subsets of ANLL. All 13 patients with acute promyelocytic leukemia and adequate cytogenetic specimens had t(15;17); this translocation was not found in any other subset of ANLL. Six patients with AML (M2) had t(8;21) or a variant of this rearrangement. Seven patients had inv(16)(p13q22) associated with acute myelomonocytic leukemia (AMMoL, M4) and abnormal marrow eosinophils. Two patients had ins(3;3) and thrombocytosis. Four patients had a translocation involving 11q, but none of these had acute monocytic leukemia (AMoL, M5); no patient had del(11q).     

Secondary acute non-lymphocytic leukemia with monosomy 7 arising 9 years after acute lymphoblastic leukemia in childhood

We report a case of pediatric acute non-lymphocytic leukemia (ANLL) with monosomy 7 occurring in a child successfully treated for acute lymphoblastic leukemia (ALL) nine years earlier. Acquired monosomy 7 is currently recognized as a distinct therapy-related cytogenetic abnormality which nonrandomly occurs as a late complication of cytotoxic therapy used in the treatment of both malignant and nonmalignant disease. Most commonly, this occurs as a disorder of bone marrow morphology and function characterized as a myelodysplastic syndrome (MDS) or ANLL. This case report emphasizes the need for continued evaluation of long-term survivors of childhood cancer to identify and minimize therapy-related side effects without compromising successful management.     

Geographic heterogeneity of neoplasia-associated chromosome aberrations

Using a database comprising 13,266 cytogenetically abnormal neoplasms, the geographic heterogeneity of neoplasia-associated chromosomal abnormalities was investigated by comparing the frequencies of characteristic aberrations in consecutive series of patients with the same diagnosis. Significant frequency differences between geographic areas were found for the aberrations +8, i(17q), +19, and an additional Ph1 chromosome in chronic myeloid leukemia (CML); -5, 5q-, and +8 in acute nonlymphocytic leukemia (ANLL); t(8;21) in ANLL-M2; t(15;17) in ANLL-M3; 5q- and -7 in myelodysplastic syndromes (MDS); t(9;22) and +21 in acute lymphocytic leukemia (ALL); t(14;18) in follicular lymphoma; -8 and -22/22q- in meningioma; and structural abnormalities of 12q in pleomorphic adenoma of the salivary glands (PAS). No geographic incidence variation was detected for -7 and +21 in ANLL; +8 in MDS; 6q- and +8 in ALL; +12 in chronic lymphocytic leukemia; 6q- in non-Hodgkin's lymphoma (NHL); t(8;14) in Burkitt's lymphoma; t(11;22) in Ewing's sarcoma; i(12p) in germ cell tumors; 1p- in neuroblastoma; structural abnormalities of 3q, 8q, and 9p in PAS; or 3p- in renal cell carcinoma. Intraregional frequency similarities between cytogenetically identical abnormalities in related tumor types were also analyzed. No significant correlations were found regarding the incidence of 5q- in ANLL and MDS, 6q- in ALL and NHL, -7 in ANLL and MDS, +8 in ANLL and CML, +8 in ANLL and MDS, +8 in ALL and ANLL, or +21 in ALL and ANLL. The findings indicate that some geographic heterogeneity of tumor-associated aberrations exists both in hematologic neoplasms and in solid tumors.(ABSTRACT TRUNCATED AT 250 WORDS)