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.     

Cytogenetic analysis of hematologic malignancies in Hong Kong. A study of 98 cases.

The karyotypes of 98 patients between the ages of 8 and 81 years with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), and chronic myeloid leukemia (CML) are presented. Although the well-described cytogenetic abnormalities associated with particular FAB subtypes in the West were observed, certain important local differences were noted. In ALL, hyperdiploidy was rarely observed, whereas the Philadelphia chromosome was observed in 50% of abnormal karyotypes. In AML, the t(8;21) was infrequently observed in M2 case, whereas trisomy 4 and 6, rarely reported elsewhere, formed 12% of the abnormal cases. In MDS, the incidence of -5/5q- and/or -7/7q- was 83% of cases with aberrant cytogenetic findings. Neither i(17q) nor an extra Ph was seen in 26 cases of CML including 9 cases of accelerated phase/blast crisis. In addition, previously unreported cytogenetic abnormalities occurring as single cases are presented. These findings are discussed in the context of geographical heterogeneity of chromosomal abnormalities in leukemia and emphasize the importance of continued epidemiologic studies of cytogenetics in hematologic malignancies.     

Contribution of multiparameter genetic analysis to the detection of genetic alterations in hematologic neoplasia. An evaluation of combining G-band analysis, spectral karyotyping, and multiplex reverse-transcription polymerase chain reaction (multiplex RT-PCR)

We investigated 150 acute myeloid leukemia (AML) patients and 48 acute lymphoblastic leukemia (ALL) patients by multiplex RT-PCR to 7evaluate the adjuvant diagnostic effect, vis-à-vis G-banding and spectral karyotyping (SKY), and the potentials of this method for providing means for monitoring residual disease by real-time quantitative RT-PCR. An abnormal G-banded karyotype was found in 57% of AML and 68% of ALL cases. Ninety-six patients were investigated by SKY in parallel which extended or confirmed the G-banding finding in 94/96 cases. In patients with an abnormal G-banded karyotype, classification of chromosomes involved in structural aberrations by SKY was possible in 98% of the cases and SKY extended the G-banded karyotype in 34% of cases. In 32 cases, an mRNA hybrid was detected by PCR. These cases constitute 16% of the cases investigated at diagnosis (AML: 11% and ALL: 31%). In 13 of these cases, we detected an mRNA hybrid the equivalent of which was not found by G-banding or SKY (AML: 4% and ALL: 13%). By including multiplex RT-PCR, we were able to detect abnormalities in 62% of the investigated patients as opposed to 59% by G-banding. Genetic techniques complement each other and selection of relevant and targeted primer kits for the multiplex RT-PCR assay is recommended.     

Combination assay of IAP and ADA in hematologic malignancies

We measured the levels of adenosine deaminase (ADA) and immunosuppressive acid protein (IAP) in 10 patients with acute myeloid leukemia (AML), 5 with acute lymphoblastic leukemia (ALL), 8 with chronic myeloid leukemia (CML), 7 with myelodysplastic syndrome (MDS), 5 with malignant lymphoma (ML), 3 with multiple myeloma (MM) and one with adult T cell leukemia. On admission, the level of IAP was abnormally high in all cases of AML and ALL 50% of CML cases, 71.4% of MDS cases, 60% of ML cases and none of MM cases. ADA was elevated in all cases of ALL, 77.8% of AML and CML cases, 57.1% of MDS cases, 60% of ML cases and 33.3% of MM cases. In 7 patients with AML, the level of IAP returned to normal when they achieved complete remission. On the other hand, the level of ADA had already returned to normal even during induction therapy. ADA showed a positive correlation with the absolute number of peripheral blasts and lactic dehydrogenase both in AML and ALL. These results suggest that ADA indicates the activity of leukemia and IAP indicates the immunocompetence of the host.     

Acute leukemia cytogenetics: an evaluation of combining G-band karyotyping with multi-color spectral karyotyping

We have, during a 12-month period, evaluated the adjuvant effect of combining G-band karyotyping and multi-color spectral karyotyping (SKY) in acute leukemia patients. Forty-four cases were evaluated; fewer cases than those routinely analyzed by G-band cytogenetics had mitoses left for SKY analysis. Of the 44 patients, 35 were acute myeloid leukemia (AML) and 9 acute lymphatic leukemia (ALL) cases. Twenty-seven of 35 AML and 7 of 9 ALL patients had an abnormal G-band karyotype. Thirteen of these 34 abnormal cases had a simple clonal chromosome aberration, and the remaining 21 cases had a complex karyotype. The SKY confirmed the simple karyotype in 11 and in 7 with a complex karyotype. In 13 of the cases with a complex karyotype, ambiguous structural aberrations were classified, in 6 of these, SKY disclosed cryptic translocations. Thus, SKY either extended or confirmed G-band karyotypes in 31 of 34 analyzed abnormal cases. Cases where SKY did not reveal the abnormal clone showed only few abnormal mitoses by G-banding (2/23, 2/25, and 4/27). Additional or confirmatory information was therefore obtained in 91% of analyzed cases, and SKY proved to be a valuable additional tool for hematologic cytogenetics.     

Impact of cellular CD71 (transferrin receptor 1) expression in Egyptian acute leukemia: correlation with clinicopathologic features

Cellular transferrin receptor 1 (CD71) has been identified as a proliferation marker. Inferior outcome with higher expression was observed in many solid tumors. This study objected to assess the expression of CD71 in patients with acute leukemia and to address its prognostic significance and relations to clinicopathologic features. The study included 34 acute myeloid leukemia (AML) and 64 acute lymphoblastic leukemia (ALL) newly diagnosed cases from Mansoura Oncology Center. CD71 was analyzed on blast cells by flow cytometry. CD71 expression was significantly elevated in both AML and ALL. Antigen expression apparently increased in T-ALL, while in AML there was a trend toward a gradual increase of antigen expression in relation to maturation evidence of myeloid subtypes. CD71 expression correlated positively with total leukocyte count in ALL cases and negatively with platelet count in AML cases. In ALL, higher CD71 expression was associated with higher relapse rate and was an independent prognostic factor of overall survival (HR 1.8; 95 % CI 1.2–4.1). In conclusion, CD71 is overexpressed in acute leukemia; it predicts adverse clinical outcome in ALL. In addition, CD71 antagonism could be a possible therapeutic target in acute leukemia.     

Automated hematology analysers and spurious counts Part 2. Leukocyte count and differential

Using hematology analysers, white blood cell (WBC) counts and differentials (either three or five parameters) may be ascertained after Red Blood Cell (RBC) lysis and analysis using either impedance and/or optical (laser) technology. Cells or particles not destroyed by lytic agents are enumerated as WBC: abnormal particles may be observed on WBC differential scattergrams, if performed, appearing as a variable number of dots, which location may help to ascertain the nature of the abnormality. Spuriously low WBC counts are rare, mainly related to agglutination in the presence of ethylenediamine tetra-acetic acid. Cryoglobulins, lipids, insufficiently lysed RBC, erythroblasts and platelet aggregates are common situations increasing WBC counts. So far, many current high performance analysers clearly identify and enumerate erythroblasts now. In normal patients and in reactive disorders automated differential provides true and accurate results. However, failure to enumerate accurately basophilic granulocytes and monocytes is not uncommon. Using myeloperoxidase cytochemistry to ascertain differential may lead to slide review if the enzyme expression is low or absent. Low number of abnormal cells (blasts, lymphoma cells, dysplastic granulocytes) may be missed, more frequently if leukopenia is present. In many but not all instances flagging and/or an abnormal WBC differential scattergram will alert the operator. Although these flags are sensitive enough to allow the identification of several spurious counts, only the most sophisticated analysers have optimal flagging, whereas more simple ones, especially those without a WBC differential scattergram, do not demonstrate the same sensitivity for the detection of abnormal results.     

Plasma soluble interleukin-2 receptor (sIL-2R) levels in patients with acute leukemia

The plasma soluble interleukin-2 receptor (sIL-2R) level was higher in 137 patients with acute leukemia (1,489 +/- 1,798 U/ml, including 98 cases of acute myeloid leukemia (AML), 1,063 +/- 1,414 U/ml, and 39 cases of acute lymphoblastic leukemia (ALL), 2,561 +/- 2,194 U/ml), compared to 49 normal control subjects, 421 +/- 151 U/ml). The ALL patients showed elevated plasma sIL-2R levels more frequently than the AML patients (92.3% vs 44.9%). No patient with either hypoplastic AML or AML with multilineage dysplasia and only 1 of 13 patients with acute promyelocytic leukemia (APL) had an elevated plasma sIL-2R level. All the My+ ALL patients (15 cases) showed elevated plasma sIL-2R levels. Plasma sIL-2R levels were significantly lower after chemotherapy in the ALL patients, but were not significantly lower in the AML patients. IL-2R was expressed on the leukemic cells in 36 (53.7%) of 67 AML and in 9 (21.4%) of 42 ALL cases. None of the AML M3, M4, M5, M6, or M7 subgroups showed IL-2R expression. The My+ ALL patients (42.9%, 6/14) showed IL-2R expression more frequently than the other ALL subgroups (10.7%, 3/28) (p = 0.025). The plasma sIL-2R level was correlated with the proportion of leukemic cells expressing IL-2R in acute leukemia. However, there were many cases, particularly ALL cases, who had elevated plasma sIL-2R levels without IL-2R expression on their leukemic cells. These results suggest that the plasma sIL-2R level is a valuable marker for monitoring ALL after chemotherapy, particularly in My+ ALL cases, and that the T cell immune reaction to leukemia appears to be much higher in ALL patients than in AML patients.     

Leukemic blasts detected by the Technicon H-1 blood cell counter

The authors studied the usefulness of the flagging system of the Technicon H-1 Cell Counter using samples from 45 patients with acute leukemia, 24 with acute lymphoblastic leukemia (ALL), and 21 with acute nonlymphoblastic leukemia (ANLL). Results by the automated method were compared with those by the eye-count method. Four out of 24 specimens were falsely negative; no false positive results were found. For ANLL specimens, there were no false positives or negatives. When 1% or more of the cells were blasts by the eye-count method, the automated system almost always flagged the sample. The results suggested that the flagging system programmed in the H-1 is useful for the diagnosis and follow-up of leukemia.     

Use of the Technicon H-1 in the characterization of leukemias

The Technicon H-1 is a hematology analyzer that performs a complete blood cell count and white blood cell differential using both cytochemistry and flow technology. Two white blood cell cytograms are produced based on peroxidase activity and nuclear characteristics of the cells. Ninety cases of leukemia were studied. The 25 cases of acute lymphocytic leukemia (ALL) could be distinguished from the 39 cases of acute nonlymphocytic leukemia as the lymphocyte percentage was greater than 50% in the ALL cases and the mean peroxidase index value was 0 in 80% of the cases. The ALL cases and the chronic lymphocytic leukemia cases also had different cytogram patterns. Subtypes of acute nonlymphocytic leukemia could not be absolutely distinguished, although promyelocytic leukemias (M3) had characteristic cytograms and a monocyte percentage above 15% suggested a monocytic component (M4 or M5). Chronic myelogenous leukemia likewise seemed to have a recognizable pattern. Since a sample takes only 60 s to process, information is readily available. The unique data available from this instrument should provide a significant advancement in the automated hematology field.     

Abnormalities of leukocyte histograms resulting from microorganisms

The authors report a series of 13 patients seen in their laboratory during October 1985 to August 1988 in which the presence of bacterial, fungal, or malarial parasites visible on peripheral smear was correlated with an abnormal leukocyte histogram. Samples submitted for complete blood count and differential counts were analyzed with Coulter S-Plus VI (seven specimens) or S-Plus STKR (six specimens) instrumentation. Organisms visualized on the Wright-stained peripheral smears included Histoplasma capsulatum (two), Candida sp. (four), Plasmodium sp. (three), and Staphylococcus sp. (four). Two patients had a diagnosis of acquired immune deficiency syndrome (AIDS); intravascular catheters were present in five other patients. In all cases the leukocyte histograms were abnormal. The instrument flagged abnormalities of the R1 region in four patients and multiple regions in nine patients. Similar flags were produced by the in vitro addition of bacteria or fungi to whole blood. These studies document that the presence of microorganisms in the peripheral blood can result in spurious white blood cell (WBC) counts or electronic differentials. The authors' findings indicate that the possibility of circulating organisms should be considered when abnormal WBC flags are detected with Coulter instrumentation.     

Podocalyxin: a marker of blasts in acute leukemia

Podocalyxin is a CD34 family member expressed by podocytes, vascular endothelium, mesothelium, and a subset of hematopoietic progenitors. Podocalyxin expression was not observed in the hematopoietic cells of normal adult bone marrow samples. However, podocalyxin was expressed by blasts in 30 (77%) of 39 cases of acute myeloid leukemia (AML), 22 (81%) of 27 cases of acute lymphoblastic leukemia (ALL), and 13 (87%) of 15 cases of cutaneous myeloid sarcoma. No correlation with CD34 expression by immunohistochemical analysis was seen. Wilms tumor 1 (WT1) expression was detected in blasts in 17 AML cases (44%) and 21 ALL cases (78%). There was no correlation between WT1 and podocalyxin expression. We conclude that podocalyxin is expressed commonly by blasts in ALL and AML. Analysis of the expression of CD34 and podocalyxin increases sensitivity for the immunophenotypic detection of leukemic blasts compared with the analysis of CD34 alone. Therefore, podocalyxin seems to complement CD34 as a useful hematopoietic blast marker. The physiologic role of podocalyxin in leukemic blasts remains unknown.     

Different characteristics identified by single nucleotide polymorphism array analysis in leukemia suggest the need for different application strategies depending on disease category

The purpose of this study was to evaluate the detection rate of chromosomal rearrangements in leukemia using single nucleotide polymorphism array (SNP‐A) in combination with metaphase cytogenetics (MC), with the aim of proposing a practical approach for clinical karyotyping applications of SNP‐A. The Genome‐Wide Human SNP Array 6.0 (Affymetrix, Santa Clara, CA) was applied in 469 patients with a variety of hematologic malignancies. Combined use of SNP‐A with MC improved the detection rate in comparison with MC alone: acute myeloid leukemia (AML) with normal karyotype (NK), 32% versus 0%; core binding factor (CBF)‐AML 40% versus 29%; myelodysplastic syndrome (MDS), 54% versus 39%; chronic myeloid leukemia (CML), 24% versus 3%; and acute lymphoblastic leukemia (ALL), 88% versus 63%. Different patterns of abnormalities (especially the type, size, and location) were noted in the leukemia subtypes. Copy neutral loss of heterozygosity lesions was detected in 23% of AML‐NK, 3% of CBF‐AML, 25% of MDS, 2% of CML, and 20% of ALL. SNP‐A also provided information on cryptic deletions and a variety of aneuploidies in ALL, while the benefit was minimal in CML. In conclusion, different patterns of abnormal lesions were presented according to the disease category, thus requiring a different approach of adopting SNP‐A‐based karyotyping among different leukemia subtypes. © 2012 Wiley Periodicals, Inc.     

Low incidence of TEL/AML1 fusion and TEL deletion in Korean childhood acute leukemia by extra-signal fluorescence in situ hybridization

TEL/AML1 fusion in acute leukemia results from cryptic translocation of chromosome 12 and 21, the presence of which suggests a favorable prognosis. The incidence of TEL/AML1 fusion in B-lineage ALL is approximately 25%, but the incidence in Korea has not yet been reported. To investigate the incidence of TEL/AML1 fusion and TEL deletion, bone marrow specimens from 77 Korean children with newly diagnosed acute leukemia were analyzed by FISH. We applied extra-signal FISH to discriminate a true TEL/AML1 fusion from a false-positive fusion signal. To determine the cut-off value of the TEL/AML1 fusion signal, 20 normal bone marrow specimens and 28 normal peripheral blood specimens were also analyzed. The frequency of patients with TEL/AML1 fusion was 13.3% (4 cases) among 30 B-lineage ALL and 9.5% among 42 ALL. One TEL/AML1 fusion-positive patient was also found among 4 acute biphenotypic leukemias. TEL/AML1 fusion was not found in any samples from patients with T-lineage ALL or AML. The incidence of TEL deletion was 6.7% (2 cases) among 30 B-lineage ALL and 4.8% among 42 ALL. The incidences of TEL/AML1 fusion and TEL deletion in Korean children with acute leukemia appear to be lower than those in other countries, suggesting a racial difference.     

Immunoalkaline phosphatase labeling of terminal transferase in hematologic samples

Early studies of terminal transferase (TdT) expression in acute leukemia indicated that this enzyme is only found in acute leukemia of lymphoblastic type (ALL). More recently, however, several reports have suggested that TdT-positive blast cells may be found in a substantial number of cases of acute myeloid leukemia (AML). In this study, a sensitive immunoalkaline phosphatase procedure (the APAAP technic) has been used to stain normal and neoplastic blood and bone marrow samples for TdT with the use of both polyclonal and monoclonal anti-TdT antibodies. As expected, most cases of ALL studied (63 of 65) were TdT-positive. In addition, however, blast cells in 22 out of 59 (37%) cases of AML were stained by anti-TdT antibodies. Both nuclear and cytoplasmic localization were seen in each type of acute leukemia. These findings, together with previous immunocytochemical and biochemical studies, suggest that a substantial number of cases of AML express TdT (usually in a minority of blast cells) and that the frequency with which these cases are detected is directly related to the sensitivity of the staining technic used.     

Electrophoretic mobility of leukocytes in leukemia patients

Lymphocytes isolated from the blood of healthy individuals (analyzed on Parmoquant-2 apparatus) showed mean electrophoretic mobility (EM) of 1.00 +/- 0.05 um sec-1 V-1 cm and the histogram appeared to be an asymmetric curve - sum of B and T lymphocytes subpopulation. In 11 patients with chronic lymphatic leukemia (CLL) and in 4 with acute lymphatic leukemia (ALL) the histogram showed a domination of one of the population. On the other hand, in patients with chronic myeloid leukemia (CML) the histograms seem to be a sum of various populations of different mobility which was illustrated by curves of a long base and several peaks.     

CD117在急性白血病中的表达及临床意义

目的探讨CD117在急性白血病中的表达及其临床意义。方法应用CD45/SSC设门,直接荧光标记法,经流式细胞术对73例急性髓系白血病（AML）和47例急性淋巴细胞白血病（ALL）进行CD117的检测。结果对照组、ALL及AML3组CD117的表达阳性率差异有统计学意义（χ2=41.681,P﹤0.01）。AML组CD117的表达阳性率（58.9%）明显高于对照组（0）及ALL组（8.5%）。CD117/CD34的共表达率ALL组明显低于AML组（4.3%vs45.2%,P﹤0.05）。AML组CD117＋患者的CR率为60.5%,CD117-患者为76.7%,两者比较差异无统计学意义（P〉0.05）;而CD117＋/CD34＋患者的CR率为54.5%,明显低于CD117-/CD34-患者的CR率（88.2%,P﹤0.05）。结论 CD117可作为辅助诊断AML的髓系标志抗原,CD34＋/CD117＋可作为进一步排除ALL的指标。CD117＋/CD34＋可能是AML中一类预后不良的特殊亚型,可以作为AML预后判断的指标之一。     

Coexistence of inversion 16 and the Philadelphia chromosome in acute and chronic myeloid leukemias : report of six cases and review of literature

We report 5 cases of chronic myelogenous leukemia (CML) and 1 case of acute myeloid leukemia (AML) with the dual presence of t(9;22) and inv(16). The 6 patients were 5 men and 1 woman with a median age of 42.5 years. All cases were BCR-ABL+ with p210 products detected in all CML cases and a p190 product detected in the AML case. An increase in bone marrow eosinophils was detected in 3 of 5 cases, and abnormal eosinophils were identified in these 3 cases. The CBFbeta-MYH11 fusion gene was confirmed in all 3 CML cases and the 1 AML case tested, and this correlated with the presence of abnormal eosinophils with coarse basophilic granules. Of 5 patients with CML, 4 had a rapid transformation to myeloid accelerated phase of blast crisis. The coexistence of t(9;22) and inv(16) in CML seems to correlate with more rapid transformation.     

Anti-CD10 immunoperoxidase staining of paraffin-embedded acute leukemias: comparison with flow cytometric immunophenotyping

CD10 is common in B-precursor acute lymphoblastic leukemia (ALL) but is rare in acute myeloid leukemia (AML). However, until recently, analysis for CD10 has generally required fresh or frozen tissue. 56C6 is a monoclonal antibody that is now commercially available for the detection of CD10 in routinely processed paraffin-embedded tissue. Immunoperoxidase stains for CD10 on paraffin-embedded bone marrow core biopsy specimens (B5-fixed, decalcified) and marrow aspirate clots (formalin-fixed) were compared with flow cytometric immunophenotyping for CD10 on fresh cell suspensions in 20 cases of AML and in 30 cases of ALL. CD10 detection by immunohistochemistry agreed with CD10 by flow cytometry in 98% (49 of 50) of acute leukemias. The results matched in 100% (20 of 20) of AML. Five percent (1 of 20) of AMLs expressed CD10. Two of the AMLs with monocytoid differentiation were interpreted as negative for CD10 by flow cytometry, although these had nonspecific dim immunofluorescence for multiple markers, including CD10, and these cases were negative by immunohistochemistry. CD10 detection by immunohistochemistry agreed with CD10 by flow cytometry in 97% (29 of 30) of ALL. Eighty-four percent (21 of 25) of B-precursor ALL and 40% (2/5) of T-lineage ALL expressed CD10 by immunohistochemistry. In 1 case of B-precursor ALL, CD10 was dimly positive in 24% of the blasts by flow cytometry but negative by immunohistochemistry. We conclude that immunohistochemical staining of paraffin-embedded tissue, either B5- or formalin-fixed, is an effective method for the detection of CD10 in acute leukemia. This technique is useful in distinguishing AML from ALL.     

Acute lymphoblastic leukemia. Survey of immunophenotype, French-American-British classification, frequency of myeloid antigen expression, and karyotypic abnormalities in 210 pediatric and adult cases

Immunophenotypic studies are essential to distinguish acute lymphoblastic leukemia (ALL) from minimally differentiated acute myeloid leukemia (AMLM0) and to classify ALL into immunologic subtypes. Frequently, immunophenotyping identifies myeloid antigen expression in ALL, causing a potential diagnostic problem. To evaluate the immunophenotype of ALL, we studied 210 cases of pediatric and adult ALL by flow cytometry and compared the results with the French-American-British (FAB) Cooperative Group classification and the karyotypic findings. Myeloid-associated antigens were expressed in 78 (45.6%) of precursor B-cell ALL cases. Pediatric precursor B ALLs had a higher frequency of myeloid antigen expression than did adult cases. All mature B-cell ALL cases were negative for TdT and myeloid antigens. Myeloid antigen expression was less frequent in T-cell ALL cases compared with precursor B-cell ALL cases. Of the 192 cases submitted for cytogenetic analysis, 147 were abnormal. The most common chromosomal translocation was the Philadelphia chromosome, which was more likely to have L2 blast morphology and a precursor B immunophenotype. Myeloid antigen expression was present in 70.8% of Ph-positive cases (P = .008). Chromosome rearrangements involving 11q23 also showed an increased frequency of myeloid antigen expression. Chromosome translocations involving regions of T-cell receptor genes were present in 24% of T-cell ALL cases. A high percentage of ALL cases, however, had various other cytogenetic abnormalities, many of which involved less well-studied chromosomal regions.