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.     

The use of fluorodeoxyuridine synchronization for cytogenetic investigation of acute lymphoblastic leukemia.

The role of fluorodeoxyuridine (FUdR) synchronization in cytogenetic analysis of acute lymphoblastic leukemia (ALL) was investigated using samples of bone marrow (BM) (10 patients) and peripheral blood (PB) (2 patients), prepared for chromosome analysis using both 24-hour unstimulated cultures (24-hr) and cultures synchronized with FUdR. The mitotic index (MI) in FUdR was lower than in 24-hr in 8 of 10 BM and 2 of 2 PB cultures. The quality of the metaphases was the same in both cultures. The FUdR had a lower percentage of abnormal cells than the 24-hr in the 7 BM samples with a normal/abnormal population and sufficient analyzable cells in each culture for comparison (p less than 0.05). PB FUdR cultures yielded only normal cells. We conclude that FUdR cultures are inferior to 24-hr cultures for chromosome analysis in ALL.     

Cytogenetic study of circulating blasts in leukemias

A special cultivation technique of separated blasts of peripheral blood in suspension culture has been used for cytogenetic diagnosis of patients suffering from acute nonlymphocytic leukemia, chronic myeloid leukemia, and refractory anemia with excess of blasts. Twenty-three patients were examined; in ten cases isolation of blasts was performed on Ficoll-Verografin and in the remaining 13 patients further separation of T-lymphocyte precursors by means of sheep erythrocytes was performed. Remarkably, a 100% success rate was attained in all cultivations. The optimum harvesting time was 72-96 hours; the rate of cell division per cultivation was determined by means of bromodeoxyuridine incorporation; second mitoses were revealed only after 96-hour cultivation. In all patients, except one, abnormal karyotypic changes were ascertained in separated blasts of peripheral blood cultivations. In most cases the morphology of chromosomes obtained from separated blasts of peripheral blood cultivations was of excellent quality.     

CDCP1 identifies a broad spectrum of normal and malignant stem/progenitor cell subsets of hematopoietic and nonhematopoietic origin

CUB-domain-containing protein 1 (CDCP1) is a novel transmembrane molecule that is expressed in metastatic colon and breast tumors as well as on the surface of hematopoietic stem cells. In this study, we used multiparameter flow cytometry and antibodies against CDCP1 to analyze the expression of CDCP1 on defined hematopoietic cell subsets of different sources. In addition, CDCP1 expression on leukemic blasts and on cells with nonhematopoietic stem/progenitor cell phenotypes was determined. Here we demonstrate that a subset of bone marrow (BM), cord blood (CB), and mobilized peripheral blood (PB) CD34+ cells expressed this marker and that CDCP1 was detected on CD34(+)CD38- BM stem/progenitor cells but not on mature PB cells. Analysis of leukemic blasts from patients with acute lymphoblastic leukemia, acute myeloid leukemia, and chronic myeloid leukemia in blast crisis revealed that CDCP1 is predominantly expressed on CD34(+)CD133+ myeloid leukemic blasts. However, CDCP1 was not strictly correlated with CD34 and/or CD133 expression, suggesting that CDCP1 is a novel marker for leukemia diagnosis. Stimulation of CD34+ BM cells with CDCP1-reactive monoclonal antibody CUB1 resulted in an increased (approximately twofold) formation of erythroid colony-forming units, indicating that CDCP1 plays an important role in early hematopoiesis. Finally, we show that CDCP1 is also expressed on cells phenotypically identical to mesenchymal stem/progenitor cells (MSCs) and neural progenitor cells (NPCs). In conclusion, CDCP1 is not only a novel marker for immature hematopoietic progenitor cell subsets but also unique in its property to recognize cells with phenotypes reminiscent of MSC and NPC.     

Fluorescence in situ hybridization (FISH) on peripheral blood smears for monitoring Philadelphia chromosome-positive chronic myeloid leukemia (CML) during interferon treatment: A new strategy for remission assessment

Interferon-α (IFN-α) alone or in combination with cytostatic drugs can induce major and durable cytogenetic responses in about 20 to 25% of chronic myeloid leukemia (CML) patients. Since these patients have a significant survival benefit, more frequent follow-up investigations have become clinically important but require bone marrow (BM) aspirates. The aim of our study was to evaluate interphase fluorescence in situ hybridization (IPF) on peripheral blood (PB) smears as a rapid and reliable method to quantify Ph-positive myeloid cells. IPF analysis was performed on 49 PB samples from 36 patients in the chronic phase of CML and at different stages of cytogenetic remission. IPF results of 30 PB samples were compared with those from BM aspirates simultaneously obtained from the same patients to evaluate the correlation of Ph-positive cells. Further, the hypermetaphase FISH (HMF) technique was performed on cultured BM preparations of 31 patients for comparison with IPF results on PB. An excellent correlation was observed between the IPF results obtained on PB and BM samples (r = 0.98, y = x − 0.6, p < 0.0001). The mean difference between HMF from BM, on the one hand, and IPF from PB, on the other hand, was 3.2% (SD = ± 8.4%). Seventy percent of samples were identically classified in one of the four subgroups of cytogenetic response. Thirty percent were classified in neighbouring response groups. We conclude that FISH performed on PB is a rapid and reliable method for assessing the cytogenetic response of CML patients on IFN-α based therapies, allowing more frequent and less invasive follow-up investigations although it is not able entirely to replace routine analysis of BM. Genes Chromosomes Cancer 21:90–100, 1998. © 1998 Wiley-Liss, Inc.     

Usefulness of anti-CD117 in the flow cytometric analysis of acute leukemia

We assessed the diagnostic usefulness of adding anti-CD117 to our existing flow cytometric profile in the analysis of 150 consecutive cases of acute leukemia (de novo or relapsed acute myelogenous leukemia [AML], AML arising in myelodysplastic syndrome, blast crisis of chronic myelogenous leukemia [CML], acute lymphoblastic leukemia, acute unclassifiable leukemia, and biphenotypic leukemia). CD117 was expressed on more than 10% of blasts in 64% of de novo AMLs (42/66), 95% of relapsed AMLs (19/20), 75% of AMLs arising from a myelodysplastic syndrome (6/8), and 25% of myeloid blast crisis in CMLs (1/4). CD117 was not expressed in acute lymphoblastic, acute biphenotypic, or unclassified leukemia or lymphoid blast crisis of CML. The specificity, positive predictive value, sensitivity, and negative predictive value of CD117 for AML were 100%, 100%, 69%, and 62%, respectively. CD117 is a specific marker for myeloblastic leukemias. Sensitivity is greatest in French-American-British M2 and relapsed AML. Intensity of CD117 expression is dim. Despite the high specificity and positive predictive value, the addition of anti-CD117 to our panel did not prove essential for the assignment of blast lineage.     

Correlation between chromosomal abnormalities and blast phenotype in the blast crisis of Ph-positive CGL

We carried out cytogenetic analysis in 23 patients with Ph-positive chronic granulocytic leukemia in blast crisis. In all cases the type of blast cell was characterized by cytochemistry, immunologic markers, and ultrastructural studies. Twelve cases were classified as myeloid transformation, six as lymphoid, two as mixed (lymphoid and myeloid), and two were unclassifiable. Duplication of Ph was the most frequent abnormality in the whole series. Trisomy 8, i(17q) and trisomy 19 were seen only in patients with myeloid blast crisis (53%, 30%, and 23%, respectively). Our findings suggest that the nature of additional chromosome abnormalities arising in blasts with features of myeloid differentiation are different from those in blasts showing lymphoid differentiation.     

The use of monoclonal antibodies against primary myeloid granules in normal and leukemic cells.

Three monoclonal antibodies, K101, D46, and H36/71 (CD15), reactive with membrane components of primary granules of human promyelocytes, were studied to assess their binding to normal and leukemic cells. Using the alkaline phosphatase antialkaline phosphatase technique, these antibodies were applied to sections of normal organs and to peripheral blood and bone marrow films from hematologically normal individuals and patients with hematologic malignancies. In control experiments, antibodies showed reactivity with cytoplasmic constituents of granulocytes from the promyelocytic to the neutrophilic stage. In acute myeloid leukemia, antibody K101 was positive (more than 20% of blasts) in 13 of 21 (62%) cases, while antibody D46 was positive in 11 of 17 (65%) cases. Antibody H36/71 was positive in only 4 of 24 (17%) cases of acute myeloid leukemia. At least one marker was present in 6 of 8 (75%) cases of acute lymphoblastic leukemia with myeloid antigen-positive blasts and was negative in 20 cases of acute lymphoblastic leukemia with myeloid antigen-negative blasts. These results support the view that abnormal granules (with defective expression of the D46, K101, and H36/71 antigens) form in blastic and leukemic cells of patients with acute myeloid leukemia. Data also suggest that membrane components of myeloid granules are made in the cytoplasm of cells from some acute lymphoblastic leukemia patients with myeloid antigen-positive blasts.     

Chromosomes and causation of human cancer and leukemia. LIV. Near-tetraploidy in acute leukemia

Near-tetraploid cell populations were observed in a case of T-cell acute lymphoblastic leukemia (T-ALL) and in one of acute myeloblastic leukemia (AML). In the ALL case, hyperdiploid chromosomal changes, characterized by an isochromosome 17q [i(17q)], as well as other changes, were seen at the onset of the disease. At the first relapse, hypertetraploid cells appeared in about 10% of the mitoses in the bone marrow (BM), and by the second and third relapses, the hypertetraploidy was present in more than 90% of the mitoses in the BM. Even though karyotypic instability was evident, all abnormal karyotypes contained one or two i(17q) at every sampling. In spite of karyotypic instability at each relapse, karyotypic evolution was observed whenever relapse occurred. A normal female karyotype was confirmed in the BM of each period. Immunologic examinations performed at each sampling revealed no recognizable changes before and after the appearance of tetraploidy. In the AML case, which was classified as FAB M2, cytogenetic examination was performed at diagnosis and relapse. In both, hypotetraploid cells were observed in over 60% of the BM cells; the modal chromosome number was 90. Banding analysis was successful at relapse, and a pseudodiploid clone characterized by t(8;21) and a hypotetraploid clone with two t(8;21) and a loss of two Y chromosomes were observed in the same BM sample. A normal male karyotype was also observed in BM cells. In both cases, giant and bizarre blasts were seen in the BM. A close correlation between near-tetraploid mitoses and giant and bizarre blast cells in BM smears of the same samples was observed. Previously published tetraploid acute leukemia cases analyzed with banding methods were accumulated and compared with our two cases.     

Covert preleukemia driven by MLL gene fusion

Acute leukemia is considered to be a two- or multiple-step process. Although there is a considerable knowledge regarding the character of the "first hit," the nature of the "second hit" remains unanswered in most of the cases including leukemias with MLL gene rearrangement. We demonstrate here a striking sequence of events, which include a covert, protracted preleukemic phase characterized by a dominant MLL/FOXO3A clone with intact myeloid differentiation and the subsequent acquisition of a secondary genetic abnormality, leading to overt lymphoblastic leukemia. Backtracking of the secondary acute lymphoblastic leukemia (sALL) with the MLL rearrangement showed no blasts in the bone marrow (BM) during the protracted preleukemic phase. However, at the same time (more than 1 year before the sALL diagnosis) the MLL/FOXO3A was present in up to 90% of BM cells including myeloid lineage, suggesting that the fusion arose in a multipotent progenitor. To identify potential "second hit" precipitating sALL we compared DNA in preleukemic versus fully leukemic samples. The analysis revealed a 10 Mb gain on 19q13.32 in the sALL, absent in the preleukemic specimen. These data provide insight into the dynamics of leukemogenesis in secondary leukemia with MLL rearrangement.     

Electron microscopic and cytochemical studies of peroxidase-negative acute nonlymphoblastic leukemia

Cytochemical and ultrastructural studies of 42 patients with acute nonlymphoblastic leukemia (ANLL) lacking myeloperoxidase as detected by light microscopy were performed. These ANLL patients were classified into minimally differentiated acute myeloid leukemia (AML-M0) (26 cases), mixed-lineage leukemia (9 cases), and acute undifferentiated leukemia (AUL) (7 cases). Ultrastructural morphology revealed blasts from AML-M0 as very immature myeloid cells, blasts from mixed-lineage leukemia as having partially lymphoid cell features, and blasts from AUL as undifferentiated blasts with myeloid nature to some extent. Ultrastructurally, myeloperoxidase activity was positive in 81% of AML-M0 patients and less positive in the mixed-lineage leukemia and AUL patients. The DNA in the nuclei of the AML-M0 blasts was dispersed, as was observed in the immature myeloid cells. Some of the blasts from the mixed-lineage leukemia patients showed condensation of DNA, resembling blasts from acute lymphoblastic leukemia (ALL). The distribution of periodate-reactive glycoconjugates revealed that glycogen particles were absent or, if present, very scanty in AML-M0. Most of the blasts from the AUL patients lacked glycogen particles, and the blasts from those with mixed-lineage leukemia contained clustered glycogen particles, as was observed in the blasts from all patients. These findings disclosed that three subtypes of immature ANLL showed different cytochemical and ultrastructural features in accordance with their immunophenotypical classification.     

Prognostic impact of acute myeloid leukemia classification. Importance of detection of recurring cytogenetic abnormalities and multilineage dysplasia on survival

To evaluate the prognostic impact of acute myeloid leukemia (AML) classifications, specimens from 300 patients with 20% or more bone marrow myeloblast cells were studied. Specimens were classified according to the French-American-British Cooperative Group (FAB), the World Health Organization (WHO), the Realistic Pathologic Classification, and a cytogenetic risk group scheme. Cases with fewer than 30% blast cells did not have a 5-year survival significantly different from cases with 30% or more blast cells, and survival was similar for the low blast cell count group and cases with multilineage dysplasia and 30% or more blasts. Categories of AML with recurrent cytogenetic abnormalities of t(15;17), t(8;21), inv(16)/t(16;16), and 11q23 showed significant differences in 5-year survival. No significant difference was identified between AMLs arising from myelodysplasia and de novo AMLs with multilineage dysplasia, but all cases with multilineage dysplasia had a worse survival than all other AMLs and other AMLs without favorable cytogenetics. FAB types M0, M3, and M4Eo showed differences in survival compared with all other FAB types, with M0 showing a significant association with high-risk cytogenetics and 11q23 abnormalities. Other FAB groups and WHO AML, not otherwise categorized subgroups did not show survival differences. These findings suggest that the detection of recurring cytogenetic abnormalities and multilineage dysplasia are the most significant features of current AML classification.     

The specificity of immunophenotypic alterations in blasts in nonacute myeloid disorders

Data regarding flow cytometry (FC) in nonacute myeloid disorders is confounded by variable gating strategies and controls limited to normal bone marrow (BM) samples. Blasts in diagnostic BM samples of myelodysplastic syndromes (MDSs), myeloproliferative neoplasms (MPNs), and chronic myelomonocytic leukemias (CMMLs) were compared with 20 nonneoplastic cytopenias/cytoses (CCs) and negative staging BM samples using 4-color FC. Blasts in 10 of 20 CCs showed immunophenotypic differences vs control samples. Immunophenotypic alterations were identified in 18 of 21 MDSs, 11 of 14 MPNs, and 7 of 7 CMMLs vs control samples and 13 (62%) of 21 MDSs, 7 (50%) of 14 MPNs, and 3 (43%) of 7 CMMLs vs CCs. Neoplastic-specific blast immunophenotypic changes included expression of CD7, CD11b, CD15, CD36, and CD56; CD34 overexpression; HLA-DR variability; lack of CD13 and CD33; underexpression of CD13, CD33, CD45, and HLA-DR; and partial loss of CD13, CD33, CD38, and CD117. In all cases, blasts were CD34+. Several blast immunophenotypic alterations are shared in neoplastic and nonneoplastic BM samples. Approximately 40% to 60% of neoplastic BM samples exhibited aberrancies not seen in reactive BM samples.     

Cytogenetic follow-up of patients with nonlymphocytic leukemia. II. Acute nonlymphocytic leukemia

Bone marrow (BM) karyotypes of 86 patients with acute nonlymphocytic leukemia (ANLL) were studied at the time of diagnosis; 39 of them (45%) were normally diploid and 47 (55%) showed acquired abnormalities. The median survival was no longer in the diploid group than in the aneuploid one. Nonrandom aberrations were often found: trisomy 8 (15 times), monosomy 7 (7 times), and t(8;21) (7 times). Two patients with acute promyelocytic leukemia presented with the t(15;17) in BM cells. Serial cytogenetic studies performed in 17 cases showed that karyotypic evolution closely followed the clinical evolution. Complete remission, obtained in 10 cases, was characterized by BM metaphases with a normal karyotype. Relapse after a period of complete remission was documented four times; the BM metaphases then showed the original abnormal karyotype with additional changes that, in three cases, were limited to a new structural aberration.     

Cytogenetic analysis of cryopreserved bone marrow cells

We studied the results of culturing and karyotyping of fresh BM and defrosted BM cells from 11 patients with cytogenetic abnormalities with the diagnosis of acute myeloid leukemia (AML). In 8 of 11 patients the cytogenetic results from the fresh and defrosted BM were comparable with respect to the amount and quality of abnormal metaphases. In only one patient one abnormal cell-line (out of two) re-appeared in the frozen BM, probably due to selection, and in three patients we found a distinct decrease of amount and quality. In conclusion, cytogenetic analysis of cryopreserved BM cells can be a reliable alternative to analysis of fresh cells.     

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.     

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.