Spectrin isoforms: differential expression in normal hematopoiesis and alterations in neoplastic bone marrow disorders

Spectrins are large, rod-like, multifunctional molecules that participate in maintaining cell structure, signal transmission, and DNA repair. Because little is known about the role of spectrins in normal hematopoiesis and leukemogenesis, we immunohistochemically stained bone marrow biopsy specimens from 81 patients for αI, αII, βI, and βII spectrin isoforms in normal reactive marrow (NRM), myelodysplastic syndrome, myeloproliferative neoplasm, acute myeloid leukemia (AML) with well-characterized cytogenetic abnormalities, acute erythroid leukemia (EryL), and acute megakaryoblastic leukemia (MegL). In NRM, spectrin isoforms were differentially expressed according to cell lineage: αI and βI in erythroid precursors; αII and βII in granulocytes; and βI and βII in megakaryocytes. In contrast, 18 (44%) of 41 AMLs lacked αII spectrin and/or aberrantly expressed βI spectrin (P = .0398; Fisher exact test) and 5 (100%) of 5 EryLs expressed βII spectrin but lacked βI spectrin. The frequent loss and/or gain of spectrin isoforms in AMLs suggests a possible role for spectrin in leukemogenesis.     

Immunoreactivity of MIC2 (CD99) in acute myelogenous leukemia and related diseases.

MIC2 is characteristically expressed in lymphoblastic lesions and Ewing's/primitive neuroectodermal tumor sarcomas. Although MIC2 has recently been reported in chloroma and rare terminal deoxynucleotidyl transferase-positive acute myelogenous leukemia (AML), the incidence and the significance of MIC2 (CD99) immunoreactivity in myeloid lesions is not clear. In this study, we evaluated MIC2 positivity in a variety of myeloid diseases and normal marrow to determine its incidence and distribution in myeloid diseases; its correlation with flow cytometric and cytogenetic data in AML; and its association with leukemic transformation, relapse, and chloroma formation. Paraffin sections of 11 chloromas and 94 bone marrow core biopsies from 66 patients were stained with CD99 monoclonal antibody 12E7. Of 94 bone marrow core biopsies, there were 30 AML (fragment antigen binding M0 to M6), 23 remissions, 5 relapses, 12 myeloproliferative disorders, 13 myelodysplastic syndromes, and 11 normal marrows from patients who did not have leukemia. CD99 immunoreactivity was evaluated with light microscopy. MIC2 expression was seen in leukemic blasts in 6 of 11 chloromas (55%) and 13 of 30 AML (43%) but rarely in myeloproliferative disorders, myelodysplastic syndromes, remission, and normal marrow. CD99 tended to be positive in M1-, M3-, and HLA-Dr-negative AML and negative in AML with relapse. MIC2 expression did not correlate with the karyotype independent of French-American-British Cooperative Group classification and the disease remission or occurrence of chloroma in AML. We concluded that MIC2 is commonly expressed in leukemic blasts of AML and is not predictive of leukemic transformation from myeloproliferative disorders and myelodysplastic syndromes or chloroma formation. Caution should be taken when using MIC2 as a marker for Ewing's sarcoma/ primitive neuroectodermal tumor or lymphoblastic lymphoma on paraffin sections of either soft tissue or bone marrow specimens.     

Flow cytometric detection of intracellular myeloperoxidase, CD3 and CD79a. Interaction between monoclonal antibody clones, fluorochromes and sample preparation protocols

Detection of intracellular myeloperoxidase (MPO), CD79a and CD3 has become the most specific tool for the assignment of myeloid, B- and T-lymphoid lineages in acute leukemias. In order to establish the best combination of monoclonal antibody reagent and sample preparation technique for the intracellular detection of these three markers, we compared six different cell fixation-permeabilization kits (Cytofix/Cytoperm, Fix and Perm, Intraprep, Intrastain, Permeacyte and Permeafix) using 12 fluorochrome conjugates derived from seven monoclonal antibody (mAb) clones. A total of 21 samples corresponding to normal peripheral blood (n=4), normal bone marrow (n=3), acute myeloblastic leukemia (AML, n=6), precursor B-acute lymphoblastic leukemia (ALL, n=6) and T-ALL (n=2) cases, were analysed in two centers. All fixation/permeabilization methods resulted in decreased side scatter and mostly increased forward scatter as compared to erythrocyte-lyse-washed and 1% paraformaldehyde fixed samples. The autofluorescence levels of the leukocyte populations was only significantly increased with use of the Cytofix/Cytoperm kit and mildly with the other techniques. In addition, non-specific staining increased significantly for combinations of any anti-MPO mAb with the Cytofix/Cytoperm kit and for the CD3 clone S4.1 combined with any intracellular method. Anti-MPO antibodies gave a stronger fluorescence signal when conjugated to PE than when coupled to FITC. In conclusion, MPO-7-PE, UCHT-1-PE (CD3) and any HM57-PE conjugate (CD79a) in combination with Fix and Perm, Intraprep, Intrastain or Permeafix, provided specific staining of the respective markers in sufficient intensities. Thus, combined selection of fixation/permeabilization kits and monoclonal antibody reagents against CD3, CD79a and MPO is required for obtaining optimal cytoplasmic detection of these antigens.     

The use of monoclonal antibody Y1/82A in the identification of acute myeloblastic and monocytic leukemias

A newly developed monoclonal antibody (Y1/82A), which binds a cytoplasmic antigen in peripheral blood monocytes and tissue macrophages, was tested for its ability to detect monocytic differentiation in acute myeloid leukemia, i.e., to distinguish French-American-British (FAB) groups M4 and M5 from FAB groups M1, M2, M3, M6 and M7. Staining was performed by the alkaline phosphatase-antialkaline phosphatase (APAAP) immunocytochemical technic on bone marrow smears from 29 cases of acute myeloid leukemia, on 17 normal peripheral blood and/or bone marrow smears, on bone marrows from 10 cases of lymphoid leukemia, and on lymph nodes of 13 patients with lymphoma. Neoplastic cells from 11 of 11 patients with either M4 or M5 leukemia had positive results, whereas only 2 out of 18 cases of M1, M2, M3, M6, and M7 leukemia had positive results. In normal samples, only peripheral blood monocytes, bone marrow macrophages, and megakaryocytes stained. Lymphoid neoplasms were unreactive. These results suggest that monoclonal antibody Y1/82A may be a useful reagent in detecting cases of M4 and M5 acute myeloid leukemia and that it offers a valuable alternative to nonspecific esterase cytochemistry.     

Dysmegakaryopoiesis resembling acute megakaryoblastic leukemia in treated acute myeloid leukemia

Acute myeloid leukemia (AML) is characterized by trilineage dysplasia, including atypical megakaryocytes. Acute megakaryoblastic leukemia (FAB M7) is particularly associated with atypical megakaryocytic hyperplasia (AMH). Fifteen patients with nonmegakaryoblastic AML developed AMH after therapy, comprising 12.6% of cases of AML diagnosed from 1986 to 1989. Platelet counts were normal in nine patients and decreased in six. Blasts comprised less than 5% of cells in 40% of the biopsies, ranged from 5-15% in 53%, and comprised more than 30% of cells in 7%. Numerous small hypo- and hyperlobated megakaryocytes were seen in all specimens, often occurring in clusters, and were more easily seen in sections than in smears. Subsequent biopsies in 13 patients showed a remission marrow in seven, increased blasts in three, and AML in three; none showed AMH. AMH resembling acute megakaryoblastic leukemia may be seen transiently after treatment of AML.     

CD79 alpha expression in acute myeloid leukemia. High frequency of expression in acute promyelocytic leukemia.

CD79 alpha is a subunit of an intracytoplasmic protein reported to be specific for B lymphocytes, including immature B lineage cells. To evaluate expression of the CD79 alpha antigen in acute myeloid leukemia (AML), we studied forty-eight cases of AML by paraffin section immunohistochemistry. The cases included four MO, nine M1, nine M2, ten M3, ten M4, and six M5 AMLs using criteria of the French-American-British cooperative group. Eleven cases demonstrated cytoplasmic staining for the CD79 alpha antigen, including one M1, nine M3, and one M5 AML. These CD79 alpha-positive cases represented 5% of all non-promyelocytic AMLs and 90% of all acute promyelocytic leukemias studied. All acute promyelocytic leukemias had the characteristic t(15;17)(q24;q21), including two cases of the microgranular variant (M3v). No other B-lineage-associated antigens were found in the CD79 alpha-positive cases, with the exception of a subpopulation of CD19-positive leukemic cells in one patient. The two non-promyelocytic leukemias that expressed CD79 alpha had no evidence of t(15;17) and did not express any additional B-lineage-associated antigens that might suggest a mixed lineage proliferation. This study demonstrates that CD79 alpha expression in acute leukemia is not restricted to B-lineage acute lymphoblastic leukemias and that CD79 alpha expression is frequently associated with t(15;17) acute myeloid leukemia.     

Antigen shared by human hemopoietic precursor cells and T and B lineage cells

In this study, we investigated antigens present at the surface of acute myeloblastic leukemia (AML) cells by using the monoclonal antibody (MAb) approach. The MAb AGF43 reacted with acute myeloid and lymphoid leukemia cells and chronic lymphocytic leukemia cells and was unreactive against chronic myeloid leukemia cells. A large proportion of AML blasts showing minimal or no differentiation (AML-M1) were intensely labeled by AGF43 in contrast to a smaller percentage of blasts showing partial differentiation (AML-M2 and acute myelomonocytic leukemia). The AGF43 antigen is expressed by bone marrow lymphoid (TdT+) and myeloid (CFU-GM) progenitor cells, 95% of B cells and 65% of T cells in the blood and absent from monocytes. Only 17% of normal myeloblasts were weakly stained by AGF43. Sections of tonsil and spleen were used to confirm that, unlike antibodies to MHC class II antigens, AGF43 stained a majority of T cells and macrophages were unreactive. In conclusion, the MAb AGF43 identifies a new precursor cell antigen. The distribution of this antigen during normal myelopoiesis and on AML cells support the suggestion that acute myeloid leukemias originate in pluripotent or closely related myeloid stem cells.     

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.     

Immunohistochemical detection of VEGF in the bone marrow of patients with acute myeloid leukemia. Correlation between VEGF expression and the FAB category

We studied vascular endothelial growth factor (VEGF) expression in bone marrow sections obtained from 3 healthy donors and 41 patients with acute myeloid leukemia (AML) of various French-American-British (FAB) subtypes by immunohistochemical analysis using an anti-VEGF antibody. In normal bone marrow, the anti-VEGF antibody reacted with myeloid progenitor cells and megakaryocytes but not with erythroid cells or mature granulocytic cells. High levels of VEGF were found in the bone marrow in patients with AML-M1, -M2, -M3, -M4, -M4Eo, and -M5. In these leukemias, the vast majority of myeloblasts (> 90%) expressed VEGF. By contrast, in AML-M0, the percentage of VEGF-positive blasts was lower in most cases (median, 42%), and if at all detectable, these blast cells contained only trace amounts of VEGF. In AML-M3 and -M4Eo, maturing granulocytes failed to express VEGF similar to granulocytes in normal bone marrow. In AML-M6, myeloblasts exhibited VEGF, whereas erythroid cells did not. In AML-M7, blast cells and megakaryocytes were identified as major sources of VEGF. In summary, VEGF expression in the bone marrow is restricted to certain stages of differentiation and maturation of myeloid cells and correlates with the FAB category.     

Histological and cytogenetic characterization of bone marrow in relation to prognosis and diagnosis of myelodysplastic syndromes

Bone marrow (BM) histology of 102 myelodysplastic syndromes (MDS) patients was analyzed retrospectively. All the cases were reclassified according to the World Health Organization (WHO) classification. Karyotype study was conducted for all except one. Fifteen of the MDS cases were hypoplastic. The cellularity in bone marrow histology is sometimes ineffective in the differential diagnosis of MDS and aplastic anemia (AA). Nonetheless, a marked decrease in the number of megakaryocytes (average, 0.3/mm(2); range, 0-2/mm(2)) even in the hyperplastic foci of the marrow of AA was the most important histological feature differentiating AA from MDS, whereas the number of megakaryocytes increased in most MDS cases (44/mm(2); range, 1-240/mm(2)) and also in hypoplastic MDS (14/mm(2); range, 8-26/mm(2)). Hyperplastic marrow had a significantly high frequency of progress to acute myeloid leukemia (AML) and hypoplastic MDS had a lower rate of progress to AML. Severe myelofibrosis had a significantly poor prognosis. An increase in CD34-positive cells in MDS indicated a high rate of progress to AML. As for the patients with refractory cytopenia with multilineage dysplasia (RCMD; the new category under the WHO classification), the increased number of megakaryocytes was correlated with poor prognosis.     

Incidence, sensitivity, and specificity of leukemia-associated phenotypes in acute myeloid leukemia using specific five-color multiparameter flow cytometry

We assessed the usefulness of 5-color multiparameter flow cytometry to detect leukemia-associated phenotypes (LAPs) in the bone marrow of patients with newly diagnosed acute myeloid leukemia (AML) and determined its usefulness for detection of minimal residual disease (MRD). Overall, 94% of patients (51/54) with AML had LAPs at diagnosis. The frequency of leukemic bone marrow/median frequency of LAPs in normal or regenerating bone marrow samples using maximum log difference statistics revealed that CD2, CD56, CD11b, CD7, and CD19 expression on AML blasts represented the most sensitive and reliable markers for detection of MRD. Serial dilutional experiments showed that the sensitivity level of immunophenotyping was between 10-4 and 10-5 and that the approach was highly reproducible.Immunophenotypic analysis using a CD45 gating strategy, 5-color staining, and an extensive panel of monoclonal antibodies allowed the identification of LAPs in 94% of AML cases, and these immunophenotypes can be used for MRD monitoring with a sensitivity limit of 10-4 to 10-5.     

Detection of terminal deoxynucleotidyl transferase in acute leukemias using monoclonal antibodies directed against native and denatured sites

Monoclonal antibodies (MoAb) directed against human terminal deoxynucleotidyl transferase (TdT) have been developed recently. The authors evaluated the reactivity of two TdT MoAb, one directed against a native site and the other against a denatured site, in bone marrow samples from patients with acute lymphoblastic leukemia (ALL) and acute myelogenous leukemia (AML). Results were correlated with the immunophenotype and compared with those obtained with an anti-TdT polyclonal antibody (PoAb). The authors found that 39 of the 45 children (87%) with ALL were positive with the anti-TdT PoAb, while only 25 of 45 (56%) were positive using MoAb. In the 41 of 45 cases of ALL for which marker studies were available, there was no relationship between immunophenotype and reactivity with the PoAb or either MoAb. Five cases of AML were studied and two were positive using the PoAb, but none showed staining with the MoAb. The authors' findings demonstrate that, although MoAb may be used to detect TdT in acute leukemia, the two MoAb used do not correlate with immunophenotype and are less sensitive than the PoAb. However, the MoAb appears to demonstrate more specificity for ALL than the PoAb, since it was not reactive in PoAb+ AML.     

Megakaryocytes,erythropoietic and granulopoietic cells express CAL2 antibody in myeloproliferative neoplasms carrying CALR gene mutations

Testing for the CALR mutation is included in the updated WHO criteria for essential thrombocythaemia (ET) and primary myelofibrosis (PMF). We report on the application of the CAL2 monoclonal antibody, raised against the mutated CALR gene to myeloid cases. The immunostain was used on 116 acute myeloid leukaemias (AML) and 66 myeloproliferative neoplasms (MPN) or myelodysplastic syndromes/myeloproliferative neoplasms (MDS/MPN). None of AML cases was stained by the CAL2 antibody, while 20/66 MPNs and MDS/MPNs appeared positive. Fourteen of the latter cases were studied by molecular techniques, and all showed aberrations of the CALR gene. In addition, CAL2 positivity was found in some small‐sized elements besides megakaryocytes. By double staining, these elements corresponded to small megakaryocytes as well as both erythroid and myeloid precursors. This finding suggests possible occurrence of CALR gene abnormalities in a stem cell.     

Chronic myelomonocytic leukemia: The role of bone marrow biopsy immunohistology.

The World Health Organization criteria for diagnosing chronic myelomonocytic leukemia (CMML) are largely based on findings observed in the peripheral blood and bone marrow aspirate. A specific diagnostic role for the bone marrow biopsy has not been adequately explored. We examined whether bone marrow biopsy supplemented by immunohistochemistry may be helpful in distinguishing CMML from cases of chronic myelogenous leukemia and atypical chronic myeloid leukemia (aCML). We immunostained 25 cases of CMML with paraffin reactive antibodies which included CD68 (KP1), CD68R (PG-M1), and CD163, and compared the results with those observed in six cases of chronic myelogenous leukemia and in three cases of atypical CML. In addition, we examined whether CD34 immunohistochemistry could be useful in separating cases of CMML with less than 10% blasts (type-1) from cases of CMML with blasts accounting for 10-19% (type-2), and cases of CMML in acute transformation to acute myeloid leukemia (blasts > or = 20%). The presence of nodules of plasmacytoid monocytes was investigated by CD123 staining. CD42b was used to highlight abnormal megakaryocytes. Our results demonstrate significant differences between the groups. CD34 analysis allowed separating CMML type-1 from type-2 and the former from CMML in acute transformation. CD123-positive plasmacytoid monocyte nodules were found only in CMML and not in the other two disease groups. Overlap between CMML and the other two groups were observed with CD68 immunostaining. CD68R was more restricted to bone marrow macrophages and monocytes than CD68, but the differences between CMML and chronic myelogenous leukemia or atypical CML were still not significant. Although CD42b immunostaining facilitated the detection of dwarf megakaryocytes often present in CMML, the distinction between those and the small forms seen in chronic myelogenous leukemia was still problematic.     

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.     

Syndecan-1/CD138 expression in normal myeloid,acute lymphoblastic and myeloblastic leukemia cells

Stabilization of cell surface antigens and preservation of ultrastructural integrity are important aspects of immunoelectron microscopical studies. In the present study, 4 anti-syndecan-1/CD138 (B-B2, B-B4, MI15, 1D4) monoclonal antibodies (mAbs) were applied in combination with periodatelysine-paraformaldehyde (PLP) fixation and indirect pre-embedding peroxidase electron microscopical immunocytochemistry to analyse the localization and function of these molecules in normal myeloid cells, acute lymphoblastic leukemia (ALL) cells and acute myeloblastic leukemia (AML) cells. One case of normal human bone marrow, 3 cases of untreated AML and 2 cases of untreated ALL were studied. Samples were immediately fixed for 4 h in freshly-prepared PLP fixative in 0.037 mol/L phosphate buffer, pH 7.4, containing 10 mmol/L sodium metaperiodate, 75 mmol/L lysine, and 2% paraformaldehyde. Expression of syndecan-1 was found at the plasma membrane of all cell types. Staining intensity at the membrane of AML cells was stronger than that on the membrane of normal myeloid and ALL cells. We conclude that anti-syndecan-1/CD138 mAbs in combination with the method described here are a suitable tool for detection of cell surface syndecan molecules in cells originating from progenitor cells that can differentiate in both myeloid and lymphoid cells.     

Discordant immunophenotypic profiles of adhesion molecules and cytokines in acute myeloid leukemia involving bone marrow and skin

We investigated the role of adhesion molecules in skin involvement by acute myeloid leukemia (AML) using immunohistochemical analysis. Ten paired cases of skin and bone marrow biopsy specimens from patients with myeloid leukemia cutis (MLC) and 15 bone marrow biopsy specimens from patients without MLC were studied with antibodies directed against CD29, CD34, CD54, CD62-L, CD183, and cutaneous lymphocyte antigen (CLA). CLA was expressed in all cases of leukemia whereas CD54 was negative within blasts. CD62-L was expressed in 4 of 10 specimens of marrow infiltrates with MLC and 6 of 10 specimens of matching skin infiltrates; in marrows without MLC, only 2 of 15 were positive. CD29 was expressed in 1 of 10 marrow infiltrate specimens with MLC and 4 of 10 matching skin infiltrate specimens; in marrows without MLC, only 1 of 15 were positive. CD183 was expressed in 1 of 10 marrow infiltrate specimens with MLC and 4 of 10 matching skin infiltrate specimens; in marrows without MLC, CD183 was negative. The gain of CD62-L, CD29, and CD183 expression in bone marrow and skin infiltrates in leukemia cutis, relative to bone marrow infiltrates of cases without MLC, suggests a role for these markers in AML homing to skin.     

伴11号染色体异常的急性髓系白血病的临床和细胞遗传学研究

目的 研究11号染色体异常在急性髓系白血病中的发生率及与临床和预后的关系.方法 采用R带常规显带技术进行染色体检查,对356例急性髓系白血病患者的核型进行分析.结果 356例急性髓系白血病患者中检出11号染色体异常患者34例,占9.55%;其中20例(58.8%)涉及11q23,7例11p15易位(20.6%),5例-11(14.7%),其他少见的核型改变有:+11,t(11;14).11q23中,M4、M5占70%;且有10例同时合并有其他染色体异常.30例进行正规化疗的患者,13例缓解,缓解率低于同期急性髓系白血病的总缓解率(43.3% vs64.0%);伴11q23的急性髓系白血病的缓解率低于染色体正常的急性髓系白血病患者(45% vs67%);11q23伴其他染色体异常的缓解率低于伴单纯11q23者(30% vs60%).7例涉及11p15易位患者3例缓解,2例早期复发.5例-11患者缓解2例.结论 11q23是11号染色体异常中最为常见的核型改变,且多见于急性髓系白血病的M5型,并可能与急性单核细胞白血病的发病有关;伴11号染色体异常的急性髓系白血病患者预后较差.     

急性髓系白血病中CD64表达的敏感性及特异性研究

研究急性髓系白血病(AML)免疫分型中CD64的敏感性和特异性.用系列抗原对132例AML患者的骨髓细胞进行直接标记,并用流式细胞术进行分析.结果表明:在AML中,CD64对急性粒-单细胞白血病(M4)和急性单核细胞白血病(M5)中敏感性最高(分别为96.4%和100%),在其他AML(M0、M1、M2、M3,M6、M7)中表达均较低.CD64对M4和M5中的特异性为56.5%.因此,CD64有助于AML中M4、M5的鉴别诊断.