Characterization of a monoclonal antibody having selective reactivity with normal and neoplastic plasma cells

A myeloma cell-reactive monoclonal antibody (MoAb), MM4, was generated from BALB/c mice immunized with alternate injections of cells from two human multiple myeloma (MM) cell lines. Screening by the enzyme-linked immunosorbent assay (ELISA) technique showed that MM4 reacted with human MM cell lines (7 of 7 positive), as well as bone marrow aspirates from MM patients (4 of 4 cases positive). MM4 did not react with marrow aspirates from control patients (3 cases), or with peripheral blood mononuclear (PBM) cells from normal subjects, lymphocytic (12 cases) and myelogenous (8 cases) leukemia patients. In addition, MM4 was negative with polymorphonuclear leukocytes and RBCs from normal donors. By means of the immunoperoxidase technique, the MM4-reactive antigen was detected in paraffin-embedded, Zenker formalin-fixed bone marrow biopsies of MM (12 of 12 cases positive), Waldenstrom's macroglobulinemia (2 of 2 cases positive), asymptomatic plasma cell dyscrasia (4 of 4 cases positive), and certain lymphomas (2 of 5 cases positive). Marrow biopsies from lymphocytic (5 cases) and myelogenous (5 cases) leukemias were uniformly negative. The MM4-reactive antigen also was expressed on plasma cells generated from pokeweed mitogen (PWM)-stimulated normal PBM cultures. The pattern of reactivity of MM4 with lymphocytes of B origin was similar to that of the plasma cell MoAb PCA-1. Competitive binding studies showed, however, that these two MoAbs recognized distinct antigenic determinants. These observations suggest that MM4 may be useful for the study of human plasma cell dyscrasias.     

Cytogenetic studies on 53 childhood acute nonlymphocytic leukemia

Cytogenetic study in 53 children (aged less than 15 years) with acute non-lymphocytic leukemia (ANLL) were studied. The cytogenetic findings were compared with those of ANLL patients (136 aged less than 19 years and 747 aged over 20 years) in the Fourth International Workshop on Chromosomes in Leukemia (IV IWCL) and also with those of childhood acute lymphoblastic leukemia (ALL) cases (previously reported as our 124 ALL case). Of the ANLL patients, 77.4% had acquired chromosomal clonal abnormalities. As abnormalities, t(15;17), all cases which were seen in M3 or M3V cases, t(8;21), which was seen in M1 or M2, and rearrangements of 11q23, which were seen in M5, were more frequently seen than was reported at the IV IWCL (20.8%, 17.0% and 7.5% vs 6.3%, 6.3% and 3.2% respectively). 5q-, monosomy 7, t(6;9) and t(9;22), which have been noted previously in this disease, were not seen. Besides structural abnormalities, some cytogenetic differences in numerical abnormality between ALL and ANLL were observed as follows: 1) Hyperdiploidy of greater than 51 chromosomes noted in ALL was not found in ANLL. 2) Isolated trisomy 8 was frequently found in ANLL, but not in ALL. 3) Loss of a sex chromosome was frequently found in ANLL, but not in ALL. Our study revealed a different frequency of non-random chromosome abnormality in children with ANLL as compared with that of adults, and clarified the differences in numerical abnormalities, as well as structural abnormalities, between ALL and ANLL.     

Effects of homo-aza-steroids on acute non-lymphocytic leukaemia cell proliferation in vitro

Summary. Homo-aza-steroids (modified steroid molecules) in their esterified forms have been used extensively as carrier molecules of alkylating agents against several neoplastic malignancies in vivo and in vitro. We studied the effects of two homo-aza-steroid carrier molecules alone, namely 3β-hydroxy-13α-amino-13,17-seco-5α-androstan-17-oic-13, 17-lactam (compound 1) and 13α-amino-13,17-seco-1,3,5-estratrien-17-oic-13,17-lactam (compound 2), on human acute non-lymphocytic leukaemia cell proliferation in vitro.
We used peripheral blood samples from 27 untreated ANLL patients (eight M1, four M2, two M3, six M4, three M5a, two M5b and two M6, according to FAB criteria). Proliferative activity was estimated by using thymidine uptake and the percentage of cells in metaphase in 24, 48 and 72 h of culture. Exposure of human leukaemic blasts with either of the two compounds resulted in enhanced cell proliferation in M1, M2, M4, M6 and M5a (only by compound 2) cases, whereas there was no significant effect in the M3 and M5b cases.
Our results indicate that the two compounds tested exhibit stimulatory effect on cell proliferation, particularly in blast cells possessing a relatively smaller degree of differentiation (Ml and M6 cases exhibiting CD34 and CD7). Further research is needed to study the cell growth effect and the therapeutic potential of these steroid molecules in human blood malignancies in vitro and in vivo.     

Distribution of complement receptors on human normal and malignant mononuclear cells

Mononuclear cells from normal human subjects and patients with chronic lymphocytic leukemia (CLL), chronic lymphosarcoma cell leukemia (LCL), and hairy cell leukemia (HCL) were labeled with fluoresceinated, purified human C3b (FI-C3b) and analyzed using the fluorescence- activated cell sorter (FACS). FI-C3b labeled 17.6% +/- 6.0% of peripheral blood mononuclear cells (PBM) from 20 normal subjects, which, when separated by the FACS, consisted of B lymphocytes and approximately 5% monocytes. Analyses in which either monocytes or B lymphcoytes were excluded from consideration demonstrated that both these cell types were labeled by the FI-C3b with a heterogeneous distribution of fluorescence intensity, indicating either heterogeneity of CR density or variable avidity of individual CR for the FI-C3b. FACS profiles of PBM ( < 5% monocytes) from 14 of 15 patients with CLL showed a homogeneous distribution of very low fluorescence intensity, with > 60% of the cells being slightly more fluorescent than unlabeled controls. This low, homogeneous distribution of fluorescence is strikingly similar to profiles of CLL cells labeled with anti-Ig reagents and suggests homogeneity of low CR density and/or avidity. Similarly, CR+ mononuclear cells from five patients with HCL and three patients with LCL displayed more homogeneous FI-C3b labeling than normal CR+ PBM. Homogeneity of FI-C3b binding to CLL, LCL, and HCL cells further supports the concept for a clonal origin for these disorders.     

Rearrangements of chromosome 3 involving bands 3q21 and 3q26 are associated with normal or elevated platelet counts in acute nonlymphocytic leukemia

Fourteen patients with acute nonlymphocytic leukemia (ANLL) or dysmyelopoietic syndromes were found to have abnormalities involving the long arm of chromosome 3. In eight patients, the structural rearrangements involved both bands 3q21 and 3q26 and included t(3;3) (four patients), inv(3) (three patients), and ins(5;3) (one patient). Before treatment, seven of these eight patients had platelet counts above 100,000 per microliter, five had normal or elevated platelet counts, and four had significantly elevated platelet counts (600,000 to 1,731,000 per microliter). In each of the eight cases, normal or elevated platelet counts were associated with marked abnormalities of megakaryocytopoiesis, including increased numbers of megakaryocytes and numerous micromegakaryocytes. Classification within the French-American-British system was difficult in most of these cases; however, the leukemia in five of the eight patients with abnormalities of chromosome 3 that involved both bands 3q21 and 3q26 was classified as M4. The remaining six of the 14 patients had translocations between chromosome 3 and another chromosome. None involved both bands 3q21 and 3q26, and a break in either q21 or q26 was noted in only two patients. One of the six, who had ANLL (M4) with a normal platelet count, had a 3;5 translocation which involved band 3q25. These data suggest that in patients with ANLL, abnormalities of chromosome 3 which simultaneously involve bands 3q21 and 3q26 are associated with unusually high platelet counts.     

Blood-group antigen expression during pancreatic cancer induction in hamsters

The expression of blood group-related and tumor-associated antigens was examined in pancreatic adenocarcinomas and in the normal pancreas of hamsters to determine if this expression correlated with the host blood group and/or stage of carcinogenicity, respectively. Pancreatic tumors were induced by 4 weekly treatments of hamsters with N-nitrosobis(2-oxopropyl)amine (BOP) and analyzed immunohistochemically during different stages of tumor progression with polyclonal antibodies (PoAbs) and monoclonal antibodies (MoAbs) against A, B, O and Lewis (Le) isoantigens, including X, Y and CA 19-9 monosialoganglioside (gastrointestinal cancer antigen, GICA), as well as with PoAbs detecting human carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP) and the beta-subunit of human chronic gonadotropin (beta-HCG). The red blood cells of both control and tumor-bearing hamsters expressed AB and Le(a+b+)-like blood group types, as detected by polyvalent antisera. However, none of the MoAbs reacted with the hamster red blood cells. In the pancreas, all PoAbs against blood group antigens reacted with hyperplastic ducts and ductules at very early stages of carcinogenesis, as well as with neoplastic lesions, but not with normal pancreatic cells, except for the acinar cells, which were stained with PoAb-B, PoAb-Lea and PoAb-Leb. None of the MoAbs showed any affinity for the normal pancreatic cells; however, they reacted to various degrees with induced hyperplastic and neoplastic tissue. Reactivities of several MoAbs with malignant cells were greater than those with hyperplastic lesions: MoAb-B was highly reactive with all induced lesions, MoAb-A less reactive, and MoAb-H and MoAb-Ley (which has 6 sugar chains) detected only some cancer cells. Neither of the two MoAb-Lex (with 5 carbohydrate chains) reacted with carcinoma cells, although they did bind to a few hyperplastic cells. Neither MoAb-Lea and MoAb CA 19-9, nor PoAbs against CEA, AFP and beta-HCG, reacted with any normal, hyperplastic or malignant cells. These results demonstrate the differential reactivity of these PoAbs and MoAbs in normal and malignant pancreatic tissue and show that blood group antigens, especially the B isoantigens, are specific markers for induced pancreatic duct tumors in hamsters.     

Expression of SSEA-I antigen (3-fucosyl-N-acetyl-lactosamine) on normal and leukaemic human haemopoietic cells: modulation by neuraminidase treatment

Several mouse monoclonal antibodies (MoAbs) considered specific for the myeloid lineage recognize the same carbohydrate structure (3-fucosyl-N-acetyl-lactosamine) which is similar to the murine antigen SSEA-I. We have investigated the expression of this antigen with six different well-characterized murine IgM MoAbs on normal, leukaemic, and cultured cells by immunofluorescence and immunoelectron microscope cytochemistry. The cells were studied before and after neuraminidase treatment since epitopes recognized by these MoAbs may be masked by sialic acid. Among the recognizable normal marrow or blood cells, all these MoAbs specifically labelled the granulocytic lineage from the promyelocyte to the polymorph. After neuraminidase treatment, monocytes became labelled. All the other lineages remained unstained. Several cell lines were studied. Six of eight lymphoblastoid cell lines were stained by these MoABs; reactivity was increased by neuraminidase. One Burkitt cell line and two T cell lines were also found to be positive. These antibodies were tested on leukaemic cells. In acute non-lymphocytic leukaemia they usually labelled promyelocytes, more mature granulocytic and monocytic precursors but did not label myeloblasts; after neuraminidase treatment, these myeloblasts became stained. No labelling was observed on leukaemic proerythroblasts and promegakaryoblast before and after neuraminidase treatment except in one case of promegakaryoblastic leukaemia in which the SSEA-I antigen and platelet peroxidase were expressed in the same cell. In addition, six cases of common acute lymphoblastic leukaemia were studied; the blasts became positive after desialylation. Two examples of T cell acute leukaemia were essentially negative. We conclude, therefore, that the reactivity of haemopoietic cells with these MoAbs alone does not represent a criterion sufficient to sustain their myeloid origin since the SSEA-I antigen may be expressed at the surface of all cell lineages in the early phases of haemopoietic differentiation.     

Proliferation and maturation effects of in-vivo granulocyte-macrophage colony stimulating factor in acute non-lymphocytic leukaemia.

BACKGROUND: Granulocyte-macrophage colony stimulating factor (GM-CSF) can affect the treatment of acute non-lymphocytic leukaemia (ANLL) by supporting normal haemopoiesis and by enhancing the proliferation and the maturation of leukaemic cells. METHODS: A human recombinant E. Coli synthesized GM-CSF was administered to 7 patients with ANLL at a dose of 5 or 10 micrograms/kg/day for 7 days, prior to antileukaemic treatment. Peripheral blood neutrophil and blast cell count was monitored daily. Changes in marrow blast cell morphologic features, mitotic index, and the reactivity to a panel of monoclonal antibodies were assessed after 3 and 7 days of treatment. RESULTS: Peripheral blood neutrophil and blast cell count increased in 6/7 cases. The mitotic index of marrow cells increased in 6/7 cases. A significant maturation occurred along the granulocytic line (2 cases) and the monocytic line (1 case). Mild to moderate eosinophilia developed in 3/7 cases. Early stem cell markers (CD 34 and HLA-DR) were not lost, or actually increased. Myelomonocytic markers (CD 33, CD 13, and CD 14) rose and fell. Expression of the multidrug resistance-associated 170 kd glycoprotein remained stable. CONCLUSIONS: These data showed that in vivo GM-CSF more consistently enhanced the proliferation than the maturation of leukaemic cells.     

P—糖蛋白表达在急性非淋巴细胞白血病中的意义

为探讨成人急性非淋巴细胞白血病（ＡＮＬＬ）Ｐ－糖蛋白（Ｐ－ｇｐ）表达及其与临床、生物学特征的关系，用单克隆抗体ＵＩＣ２及流式细胞术间接免疫荧光法对１６９例ＡＮＬＬ（初治１５２例，难治７例，缓解１０例）患者Ｐ－ｇｐ进行检测。结果显示：初治者Ｐ－ｇｐ表达率为２９％，明显低于难治者的７１％，而缓解者无Ｐ－ｇｐ表达。ＡＮＬＬ亚型中以杂合型急性白血病及急性单核细胞白血病表达较高，阳性率分别为６７％及４７％。Ｐ－ｇｐ表达与ＣＤ３４、ＣＤ７、ＣＤ１４或ＣＤ４２ｂ／ＣＤ６１高度相关。细胞遗传学研究显示：Ｐ－ｇｐ在染色体预后差组表达最高（５４％），而在预后好组表达低（７％）。Ｐ－ｇｐ＋ＡＮＬＬ的完全缓解率为２３％，明显低于Ｐ－ｇｐ－者的７６％。结果提示：Ｐ－ｇｐ表达是成人初治ＡＮＬＬ化疗耐药的指标，综合免疫分型及核型分析可以更准确地判断预后及指导治疗。     

Blood-group antigen expression during pancreatic cancer induction in hamsters

Summary The expression of blood group-related and tumor-associated antigens was examined in pancreatic adenocarcinomas and in the normal pancreas of hamsters to determine if this expression correlated with the host blood group and/or stage of carcinogenicity, respectively. Pancreatic tumors were induced by 4 weekly treatments of hamsters withN-nitrosobis(2-oxopropyl)amine (BOP) and analyzed immunohistochemically during different stages of tumor progression with polyclonal antibodies (PoAbs) and monoclonal antibodies (MoAbs) against A, B, O and Lewis (Le) isoantigens, including X, Y and CA 19-9 monosialoganglioside (gastrointestinal cancer antigen, GICA), as well as with PoAbs detecting human carcinoembryonic antigen (CEA), α-fetoprotein (AFP) and the Β-subunit of human chorionic gonadotropin (Β-HCG). The red blood cells of both control and tumor-bearing hamsters expressed AB and Le(^a+b+)-like blood group types, as detected by polyvalent antisera. However, none of the MoAbs reacted with the hamster red blood cells. In the pancreas, all PoAbs against blood group antigens reacted with hyperplastic ducts and ductules at very early stages of carcinogenesis, as well as with neoplastic lesions, but not with normal pancreatic cells, except for the acinar cells, which were stained with PoAb-B, PoAb-Le^a and PoAb-Le^b. None of the MoAbs showed any affinity for the normal pancreatic cells; however, they reacted to various degrees with induced hyperplastic and neoplastic tissue. Reactivities of several MoAbs with malignant cells were greater than those with hyperplastic lesions: MoAb-B was highly reactive with all induced lesions, MoAb-A less reactive, and MoAb-H and MoAb-Le^y (which has 6 sugar chains) detected only some cancer cells. Neither of the two MoAb-Le^x (with 5 carbohydrate chains) reacted with carcinoma cells, although they did bind to a few hyperplastic cells. Neither MoAb-Le^a and MoAb CA 19-9, nor PoAbs against CEA, AFP and Β-HCG, reacted with any normal, hyperplastic or malignant cells. These results demonstrate the differential reactivity of these PoAbs and MoAbs in normal and malignant pancreatic tissue and show that blood group antigens, especially the B isoantigens, are specific markers for induced pancreatic duct tumors in hamsters.     

Three new monoclonal antibodies that define a unique antigen associated with prolymphocytic leukemia/non-Hodgkin's lymphoma and are effectively internalized after binding to the cell surface antigen

Prolymphocytic leukemia (PLL) is closely related to chronic lymphocytic leukemia (CLL), but present with distinctive clinical/laboratory features and associated with much worse prognosis. In this study, we generated three new IgG1-kappa monoclonal antibodies (MoAbs), termed SN8, SN8a and SN8b, by use of an unconventional approach, ie, by using an isolated B PLL antigen preparation to immunize mice. These MoAbs, particularly SN8, showed a highly selective reactivity to B PLL and B non-Hodgkin's lymphoma (NHL) among various human leukemia-lymphoma specimens tested; eg, SN8 was capable of effectively distinguishing B PLL from B CLL as well as from hairy cell leukemia (HCL) cell specimens. The cell surface antigen defined by the three MoAbs was determined to be a covalently linked heterodimeric glycoprotein complex (gp49/40) consisting of a 49,000 dalton (alpha-chain) and a 40,000- dalton component (beta-chain). Epitope comparison showed that the epitope defined by SN8 (SN8 epitope) is in close proximity to SN8a epitope but in a distant position from SN8b epitope. Western blot analysis showed that both SN8 and SN8a epitopes are on the beta-chain, but SN8b epitope was not detected on either the alpha- or the beta- chain of the reduced antigen in the same analysis. Binding of either SN8 or SN8b to the cell surface gp49/40 did not cause significant downregulation of the antigen expression whereas binding of SN8a to the antigen caused small (approximately 20%) decrease in the antigen expression. Among the various normal peripheral blood cells, only a subpopulation (6.0% to 24.2% among different specimens derived from different donors) of B cells reacted with the SN8 series MoAbs; these MoAbs showed no significant reactivity against T cells, granulocytes, monocytes, erythrocytes, and platelets. Minimal or no significant reactivity (0 to 2.6% among different specimens) was detected against normal bone marrow cells. Ricin A-chain conjugates of the three MoAbs are all strongly effective for specific killing of SN8 antigen- expressing leukemia cells in the absence of any potentiators; furthermore, the addition of 10 mmol/L NH4Cl, a potentiator, enhanced strongly the cytotoxic activities of the SN8, SN8a, and SN8b conjugates. Thus, each of the three MoAbs was effectively internalized after binding to the cell surface antigen.     

Anti-M monoclonal antibodies cross-reacting with variant Mg antigen: an example of modulation of antigenic properties of peptide by its glycosylation

Some monoclonal antibodies (MoAbs) directed against blood group M- related epitope of glycophorin A (GPA) were found to agglutinate rare variant erythrocytes carrying GPA of Mg type. In contradistinction to normal GPA-M or -N, the N-terminal portion of GPA-Mg is not glycosylated. Therefore, the multipin peptide synthesis was used for testing the specificity of the cross-reacting MoAbs. Among several anti- M and anti-N MoAbs tested, only three anti-M (E3, E6, 425/2B) agglutinated Mg erythrocytes and showed binding to the synthetic octapeptides corresponding to N-terminal sequences of GPA-M (SSTTGVAM), GPA-N (LSTTEVAM), and GPA-Mg (LSTNEVAM). Testing multiple peptide analogs (window and replacement analysis) showed that these MoAbs were specific for peptidic epitope in which Met8 and Val6 were the most essential amino acid residues. The amino acid replacements Ser<-->Leu1 or Gly<-->Glu5 (M v N) and Thr4<-->Asn4 (M and N v Mg) had no or negligible effect on the reaction of synthetic peptides with the MoAbs. However, when Ser2, Thr3, and Thr4 carry O-linked sialooligosaccharides (normal GPA-M or -N), the MoAbs recognize Gly5- and sialic acid- dependent blood group M-related epitope. An interesting finding concerning anti-M/Mg MoAbs described here is the fact that glycosylation of amino acid residues adjacent to the most important part of peptidic epitope not only differentially modulates the proper exposure of peptidic epitope, but also alters the requirement for some amino acid residues present within the epitope. Pathologic conditions, including hematologic disorders, are often accompanied by alterations in protein glycosylation, resulting not only from differences in the structure of antigen polypeptide chain, but also from changes in specificity or expression of enzymes involved in glycosylation. Our present findings draw attention to possibility of the bidirectional modulation of protein antigenicity by glycosylation and may be helpful in interpretation of some results obtained with MoAb used for diagnostic or other purposes.     

Mast cell typing: demonstration of a distinct hematopoietic cell type and evidence for immunophenotypic relationship to mononuclear phagocytes

The immunologic surface marker profile of human mast cells (MCs) was established using a combined toluidine/immunofluorescence staining procedure [49 monoclonal antibodies (MoAbs) tested]. Ascites (n = 9) MCs as well as enzymatically dispersed MCs from all organs tested (lung n = 11, skin n = 7, intestinum n = 10) exhibited an identical marker profile. MCs were recognized by MoAbs clustered as CD9 (anti-gp24), CD33 (anti-gp67), and CD45 (anti-gp220) as well as by MoAbs directed against membrane-bound IgE. MoAB YB5B8 (anti-gp145) selectively recognized MCs. Most significantly, however, MCs were stained by MoAbs MAX1 (anti-gp65), MAX3 (anti-gp68), MAX11 (anti-gp65), and MAX24 (anti-gp65). These antibodies bind to surface membrane antigens associated with a late stage of monocyte/macrophage differentiation. Thus, our results provide definite evidence that MCs share surface membrane markers with mononuclear phagocytes. In contrast, MCs are stained neither by lymphatic markers (CD1-8, 10, 19-24) nor by myelomonocytic markers (CD11-17). MCs also lack the interleukin-2 (IL-2) receptor (CD25), the T10 antigen (CD38), and most of the myelocytic markers expressed on peripheral blood (PB) basophils. Thus, MCs displayed a unique phenotype within the hematopoietic system. This new approach enabled us to enrich human lung MCs to a purity greater than 95% by means of negative selection with complement-mediated cell lysis. Purified MCs were subsequently stained with MoAbs and analyzed by flow cytometry, which confirmed the results obtained from the double-staining experiments. We next examined cultured metachromatic cells derived from bone marrow (BM) and peripheral blood colony-forming units (CFU). These metachromatic cells previously could not be classified by morphologic criteria alone and have therefore been termed basophil-like/MC-like cells. In this study, toluidine blue-positive cells obtained from either pooled multipotential colonies (day 14-CFU-GEM) or pooled myelocytic colonies (day 16/17-CFU-GM/G/M) were recognized by MoAbs MY7 (CD13), VIM12 (CD11b), and VIM2, as well as by an anti-IgE MoAb, after preincubation with IgE. In contrast, CFU-derived metachromatic cells were not stained by MoAb YB5B8. This marker profile corresponds to the immunologic phenotype of blood basophils and excluded a detectable formation of mature MCs in colonies derived from cultured hematopoietic stem cells.     

A continuous spectrum of hypercoagulability exists in acute nonlymphoblastic leukemia.

A consumption coagulopathy syndrome has frequently been reported in association with some cases of acute nonlymphoblastic leukemia (ANLL) and mainly in acute promyelocytic leukemia (M3). Eighteen cases of ANLL have been studied on admission, before chemotherapy was started. Levels of antithrombin III (AT-III), protein C (PC), protein S (PS), thrombin-antithrombin complex (T-AT-III), tissue plasminogen activator, plasminogen (Pg), alpha-2-antiplasmin (alpha-2-AP), D-dimer (DD) and fibrinogen (Fg) were determined. The results showed normal levels of AT-III and PS, decreased levels of PC, alpha-2-AP, Pg and Fg in some cases, and an elevation of DD and T-AT III complex in almost all patients. There was a continuous evolution of data from M1 cases in which only slight alterations were seen up to M3 cases where all those pathologic data were observed.     

Immunoglobulin heavy chain and T-cell receptor beta chain gene rearrangements in acute non lymphoid leukemia

The occurrence of immunoglobulin heavy chain (IgH) and/or T-cell receptor (TcR) gene rearrangements has been reported in some cases of acute non lymphoid leukemia (ANLL), and variously interpreted as reflecting "aberrant gene expression" or "lineage promiscuity" of the leukemic cell. In an attempt to verify the incidence, FAB distribution and immunophenotypic correlates of gene rearrangements in ANLL, we analyzed the configuration of IgH and TcR beta chain genes in 70 patients with ANLL. In all cases myeloid (CD13, CD33, CD14, CD15) and lymphoid (CD7, CD2, CD10, CD19, TdT) antigenic determinants were analyzed in conjunction with conventional morpho-cytochemical characterization. Clonal rearrangements of the IgH gene were identified in 6/70 ANLL patients (8.6%), whereas in only 2/48 (4.2%) were T beta rearrangements documented. Concerning FAB subtypes, IgH or T beta rearrangements were detected in the less differentiated forms MO and M1 (3 cases), as well as in 2 M4 and 1 M5a cases. With the exception of a higher incidence of gene rearrangements in TdT+ ANLL, no significant correlation was found with other immunophenotypic markers.     

Immunological definition of acute promyelocytic leukemia (FAB M3): a study of 39 cases

Acute promyelocytic leukemia (FAB-M3) is a distinct entity among acute non-lymphoid leukemias (ANLL) with peculiar morphological, biological, clinical and prognostic features. An atypical form of M3 (M3v) could be confused with other FAB ANLL and therefore the diagnosis of this variant requires ultrastructural analysis and/or cytogenetic study and/or selective gene rearrangement studies. The immunological phenotype of blast cells in 39 APL patients was studied at diagnosis. The diagnosis of M3 FAB type was ascertained in 32 and the diagnosis of M3v in 7 cases. Using a large series of monoclonal antibodies (mAb), the APL blast cells were B and T cell antigens-negative, HLA-DR constantly negative, CD13- and/or CD33-positive, CD9-positive. Among ANLL this phenotype seems to be closely related to APL both in M3 type and M3v subtype. Because the diagnosis of APL (M3 or M3v) is important in order to establish the specific therapeutic approach, the discriminant capacity of the immunological typing to identify M3 and mainly M3v (hypogranular) could be determinant for a "quick" diagnosis.     

Characteristics of red cell pyruvate kinase (PK) and pyrimidine 5''nucleotidase (P5N) abnormalities in acute leukaemia and chronic lymphoid diseases with leukaemic expression

Red cell pyruvate kinase (PK), pyrimidine 5'nucleotidase (P5N) and reduced glutathione content (GSH) were studied in 126 untreated patients with acute leukaemia (AL, 80 cases), chronic lymphocytic leukaemia (B-CLL, 38 cases) and B-cell lymphoma with leukaemic expression (LSCL, eight cases). Acute leukaemias were classified into lymphoblastic (ALL) and non-lymphoblastic (ANLL), the latter have been further sub-divided into four different variants according to FAB morphological criteria (1976). A significant decrease of PK activity was observed only in the ANLL group, leading to a clear-cut difference with the ALL group where a normal value was obtained. The decrease of P5N activity was similar in all the morphological variants of ANLL and no abnormalities in the low PEP assay system or after fructose 1,6-bisphosphate (Fru 1,6-P2) activation were observed. P5N activity was found to be significantly decreased in all groups of patients except in B-CLL, where it was normal. In regards to the different morphological groups of ANLL, a striking decrease of P5N activity was observed in the M3 variant. Although red cell GSH content was significantly increased in all groups of patients, no correlation was demonstrated between the raised GSH levels and the decreased P5N activities.     

High la (HLA-DR) and low CD11b (Mo1) expression may predict early conversion to leukemia in myelodysplastic syndromes

The FAB classification of myelodysplastic syndromes (MDS) has been useful in predicting prognosis; however, additional methods are required to detect patients at high risk for early conversion to acute nonlymphoblastic leukemia (ANLL). Using a panel of monoclonal antibodies to myelomonocytic surface antigens (MMSA) and flow cytometry, we studied bone marrow cells from 26 patients with MDS of all five FAB subtypes. The MMSA studied included la (HLA-DR), CD11b (Mo1), CD14 (Mo2, My4), CD13 (My7), and CD33 (My9). Marrows were considered 'positive' for a given MMSA if the percentage of reactive cells exceeded the upper limit of the normal range. Twenty-four of twenty-six patients (92.3%) were CD13 (My7)+, suggesting that CD13 may serve as a diagnostic marker for MDS. Ten of twelve patients who developed ANLL during a median follow-up of 44 weeks were la(HLA-DR)+. The Kaplan-Meier estimated median time to leukemia (TTL) was 16 weeks for la+ patients and 88 weeks for la– patients (P = 0.004). All six patients who developed ANLL before 16 weeks from diagnosis were la+, while none of the la– patients converted to ANLL before 24 weeks. Nine of thirteen patients with low CD11b (Mo1) expression (<53% reactive cells) developed ANLL, compared with only two of 11 patients with high CD11b expression (>53% reactive cells). Kaplan-Meier estimated TTL was 29 weeks for patients with low CD11b, compared to 160 weeks for patients with high CD11b (P < 0.05). Patients who met both criteria, la+ and low CD11b, represented the poorest prognostic subgroup, with median TTL of 13 weeks compared with 88 weeks for the others (P = 0.017), la and CD11b patterns were not specific for MDS subtype, and their expression did not correlate with blast count. These data suggest that MDS patients whose bone marrow cells demonstrate high la (HLA-DR) and low CD11b (Mo1) expression represent a poor prognostic subgroup with short TTL. These patients may be candidates for early aggressive or investigational treatment.     

Structural polymorphism of glycophorins demonstrated by immunoblotting techniques

We have explored the polymorphism of the glycophorin system in the human erythrocyte membrane using the immunoblotting techniques and examining 52 individuals selected without prior bias as to their serologic state and ten documented serologic variants of M, N, S, s blood group system. Polyclonal antisera to alpha glycophorin and to alpha glycophorin CNBr carboxyl terminal fragment C (residues 82-131) and M and N specific monoclonal antibodies (MoAbs) were used. The first two reagents detect specific regions of the alpha glycophorin molecule and all electrophoretically resolved species of glycophorins immunologically related to alpha and delta glycophorins (delta glycophorin, [alpha-delta] hybrids and other glycophorins with an alteration in the carboxyl terminal segment); the M and N MoAbs identified the glycophorin species containing or lacking the M or N determinant in the amino terminal octapeptide structures. We find that immunoblotting confirmed in all cases the serologically determined phenotype; we also find that polymorphic forms of the glycophorin system are relatively infrequent; immunoblotting, independent from serologic testing, was capable of detecting five mutants, two most likely S-s-U-phenotypes; a new glycophorin species was detected in normal red cells with both antiglycophorin and antipeptide C sera, which is not evident with MoAbs; immunoblots of known glycophorin variants (En(a-), U-, Mg, Mi I, II, III, V, and Sta) confirmed but also extended our knowledge of the abnormal glycophorins involved; and the He+ and Wrb(-) cells showed normal patterns.