Membrane Fc-IgG and C3b receptors on myeloid leukaemia cells: a comparison with cytoplasmic acid naphthyl acetate esterase cytochemistry

Membrane receptors for IgG and C3b were examined on blast cells from 57 cases of acute myeloid leukaemia. These acute leukaemias were classified as myeloblastic, myelomonocytic or monocytic following morphological, cytochemical, and immunological investigations. The membrane receptors of leukaemic blast cells appear to be directly related to the degree of monocytic differentiation with the lowest receptor activities found in acute myeloblastic leukaemia. A comparison was also made between receptor and cytoplasmic acid naphthyl acetate esterase (ANAE) activities in 29 morphologically and immunologically-defined myelomonocytic and monocytic leukaemias. This study revealed that the receptor-positive "monocytic component" in a significant proportion of cases showed unexpectedly weak or negative ANAE reactions suggesting a more cautious approach to the interpretation of ANAE cytochemistry in acute leukaemias. The normal development of cytoplasmic ANAE and membrane receptors is also discussed and compared with their abnormal patterns of expression associated with leukaemic transformation.     

Stimulating capacity of blast cells from patients with chronic myelocytic leukaemia, in blastic crisis in ''one-way'' mixed lymphoycte reaction: lack of evidence for T lymphoblastic conversion.

It has long been suggested that the blastic transformation in some patients with Ph1-positive chronic myelocytic leukaemia (CML) may be lymphoid in nature. It has recently been postulated that some patients with CML may undergo a T lymphoblastic crisis because the leukaemic blasts from these patients have high terminal deoxynucleotidyl transferase (TdT) activity and that some patients may undergo a non-T/non-B lymphoblastic crisis since leukaemic blasts from a majority of morphologically lymphoid type CML-BC cases react with antiserum specific for non-T/non-B acute lymphoblastic leukaemia (ALL). The present study shows that leukaemic blasts from each of six patients with Ph1-positive chronic myelocytic leukaemia-blastic crisis (CML-BC) exerted a strong stimulation on allogeneic lymphocytes in 'one-way' mixed lymphocyte reaction. There was no apparent difference in stimulating capacity between morphologically myeloid type (four cases) and lymphoid type (two cases). The stimulating capacity of leukaemic blasts from patients with CML-BC was quite similar to that of blasts from all patients with acute myeloblastic leukaemia (AML) and from some patients with non-T/non-B type ALL. Leukaemic blasts from a patient with T-cell type ALL and cultured leukaemic T lymphoblastoid cells (2 lines) consistently failed to stimulate while cultured leukaemic null-cells (4 lines) consistently exerted a strong stimulation in 'one-way' mixed lymphocyte reaction. These observations suggest that leukaemic cells from patients with CML-BC, morphologically lymphoblastic type, are not T lymphoblasts although the possibility that these cells are non-T/non-B lymphoblasts cannot be ruled out entirely.     

Lysine metabolism in acute leukemia]

The metabolism of 14C-lysine by leukaemic cells in acute myeloblastic, myelomonocytic, lymphoblastic and chronic myeloid leukaemia with blast crisis was studied. The investigations included lysine metabolism to CO2, lipids, organic acids and nucleotides and its incorporation into cellular proteins. The obtained results were compared with determinations carried out in granulocytes and lymphocytes of healthy subjects. Cells in acute leukaemias metabolized 14C-lysine in a similar range. In relation to normal cells the range of lysine metabolism to lipids in the leukaemic cells was significantly higher (p less than 0.01), while that of organic acids was significantly lower (p less than 0.05). The activity of 14C-lysine metabolism depended on the number of blast cells in the sample and the type of acute leukaemia. Neoplastic cells in blast crisis and in acute myeloblastic leukaemia incorporated more actively 14C-lysine into proteins than cells in acute myelomonocytic and acute lymphoblastic leukaemia (p less than 0.05). Similar differences in lysine metabolism were observed between myelomonocytes and blast cells from acute lymphoblastic leukaemia (p less than 0.05).     

Ber-MAC3: new monoclonal antibody that defines human monocyte/macrophage differentiation antigen.

A new monoclonal antibody Ber-MAC3 is reported. It recognises a formol sensitive epitope of a not yet clustered monocyte/macrophage specific 140 kilodalton glycoprotein that is expressed on the cell surface and in the cytoplasm. In 30 cases of acute and chronic leukaemia, Ber-MAC3 staining was restricted to 15 myeloid leukaemias of M4 and M5 types. The tumour cells of two cases of true histiocytic malignancies were Ber-MAC3 positive, whereas those of all 280 malignancies of lymphocytic origin were negative. The latter included 52 cases of Hodgkin's disease and 41 cases of Ki-1 positive anaplastic large cell lymphomas which had previously been classified as true histiocytic lymphomas. Ber-MAC3 therefore seems to be of considerable value for selective identification of monocytes and macrophages at a certain stage of differentiation and seems to be suitable for diagnosing myelomonocytic or monocytic leukaemia and neoplasms of true histiocytic origin.     

The value of immunohistochemistry for CD14, CD123, CD33, myeloperoxidase and CD68R in the diagnosis of acute and chronic myelomonocytic leukaemias

Rollins‐Raval M A & Roth C G
(2012) Histopathology  60, 933–942 The value of immunohistochemistry for CD14, CD123, CD33, myeloperoxidase and CD68R in the diagnosis of acute and chronic myelomonocytic leukaemias Aims: In the absence of adequate aspirate films and touch imprints, distinction of chronic myelomonocytic leukaemia (CMML) from acute myeloid leukaemia with monocytic differentiation (Mo‐AML) may be difficult solely on the basis of bone marrow biopsy morphological features. The aim of this study was to evaluate the diagnostic utility of a novel immunohistochemical panel for the diagnosis of acute and chronic myelomonocytic leukaemias in bone marrow biopsies. Methods and results: Immunohistochemical labelling for CD14, CD123, CD33, myeloperoxidase (MPO) and CD68R was assessed in 49 myeloid neoplasms with monocytic differentiation (24 CMMLs and 25 Mo‐AMLs) and compared with that of 15 non‐monocytic acute myeloid leukaemias (NM‐AMLs) and 17 non‐neoplastic controls. More than 20% CD14 immunohistochemistry (IHC)+ cells were seen only in Mo‐AMLs and CMMLs, although Mo‐AMLs showed wide variability and overlapped with other categories. More than 20% CD68R IHC+ cells had the highest sensitivity and specificity for Mo‐AML. Discrepant MPO–/CD33+ expression was specific for Mo‐AML but insensitive. A subset of blasts in Mo‐AMLs and NM‐AMLs were weakly CD123+. Conclusions: A significantly increased number of CD14+ cells raises the possibility of a myelomonocytic neoplasm but does not distinguish between CMML and Mo‐AML. Significantly increased numbers of CD68R IHC+ cells and a discrepant MPO–/CD33+ staining pattern are specific for Mo‐AML but are best utilized in a comprehensive panel.     

Bone marrow evaluation of monocytosis

An absolute peripheral blood (PB) monocytosis is defined as greater than 1000 monocytes/μl. The differential diagnosis of an absolute peripheral monocytosis includes a reactive monocytosis and a neoplastic process that may be associated with various haematological neoplasms, that is chronic myelomonocytic leukaemia, myeloid and lymphoid neoplasms with eosinophilia and rearrangement of PDGFRB, juvenile myelomonocytic leukaemia, chronic myeloid leukaemia, as well as acute myeloid leukaemias with a prominent monocytic component. The diagnostic approach to a PB monocytosis, including appropriate evaluation of cytomorphological features and flow cytometric analysis of the PB, associated bone marrow findings, as well as the cytogenetic and molecular findings, will be discussed.     

Screening microarrays of novel monoclonal antibodies for binding to T-, B- and myeloid leukaemia cells

We have developed a microarray (DotScan) that enables rapid immunophenotyping and classification of leukaemias and lymphomas by measuring the capture of cells by immobilized dots of 82 CD antibodies [Belov, L., de la Vega, O., dos Remedios, C.G., Mulligan, S.P., 2001. Immunophenotyping of leukemia using a cluster of differentiation antibody microarray. Cancer Res. 61, 4483; Belov, L., Huang, P., Barber, N., Mulligan, S.P., Christopherson, R.I., 2003. Identification of repertoires of surface antigens on leukemias using an antibody microarray. Proteomics 3, 2147]. The DotScan technology has been used to investigate the properties of 498 new antibodies submitted to the HLDA8 Workshop. These antibodies have been applied as 10 nl dots to a film of nitrocellulose on a microscope slide to make an HLDA8 microarray. After blocking the remaining nitrocellulose surface, individual arrays were incubated with each of 7 cell types from a human leukaemia cell panel consisting of three cell lines, CCRF-CEM (a T-cell acute lymphocytic leukaemia), MEC-1 (derived from B-cell chronic lymphocytic leukaemia) and HL-60 (a promyelocytic leukaemia), and four leukaemias from patients: a T-cell prolymphocytic leukaemia, a B-cell chronic lymphocytic leukaemia, and two acute myeloid leukaemias. Leukaemia cells were captured by those immobilized antibodies for which they expressed the corresponding surface molecule. Unbound cells were gently washed off, bound cells were fixed to the arrays and dot patterns were recorded using a DotScan array reader and quantified using DotScan data analysis software. The data obtained show the unique expression profiles of the 7 cell types in the leukaemia cell panel obtained with the DotScan microarray, and the differential capture patterns for these 7 cell types screened against the 498 antibodies in the HLDA8 microarray constructed for this study.     

Nucleolar organizer regions in acute and chronic leukaemias.

A silver staining technique for nucleolar organizer regions (NORs) has been applied to bone marrow biopsies of various types of acute and chronic leukaemias. This method could be easily evaluated on resin-embedded bone marrow obtained from acute lymphocytic leukaemia (n = 12), acute myelogenous (n = 16), chronic lymphocytic (n = 16) and chronic granulocytic (n = 20) leukaemia. A significant difference (p < or = 0.1) was only found between the AgNOR numbers in nuclei of lymphocytes from acute and chronic leukaemia (mean of 1.23 to 1.40 and 1.58) and those of cells from acute and chronic myelogenous leukaemia (from a mean of 5.00 to 9.17 per nucleus). However, no significant difference was observed among cells of various types of acute and chronic myelogenous leukaemias, despite of their markedly higher staining intensity and proliferative activity. The greatest mean of AgNOR numbers was counted in monoblasts of acute myelomonocytic leukaemia. It is suggested, that higher AgNOR counts in nuclei of more malignant leukaemic cells are in parallel with their mitotic activity and could be related to their elevated cell turn-over.     

Leu-M1 antigen expression in acute leukaemias

Leu-M1 is a differentiation antigen present in human myelomonocytic cells. Seventy-seven acute leukaemias were retrospectively stained with anti-Leu-M1 using the immunoperoxidase technique on Bouin-fixed paraffin-embedded sections. The subjects were 44 acute lymphoblastic leukaemias (ALL) and 33 acute myeloid leukaemias (AML) previously characterized by cytochemical and immunologic (cell suspension) methods. Leu-M1 was positive in all the AML and in half of the ALL cases. These results suggest that Leu-M1 does not allow differentiation between AML and ALL. For the ALL cases Leu-M1 was positive in 15/28 B-cell types, 4/12 T-cell type and 3/4 'null'-cell type cases. Thus, this antibody is of no assistance in defining types B, T, or 'null' in ALL. Leu-M1 was also studied on paraffin sections of 34 high grade malignant lymphomas. The antibody was negative in all 13 B-cell lymphomas (lymphoblastic: 6; immunoblastic: 7) and in all 4 'null' cell lymphomas. It was positive in 4/9 peripheral T-cell type, the other T-cell lymphomas (lymphoblastic: 5; immunoblastic: 3) remaining negative. Thus, Leu-M1 may be positive in T-cell lymphomas but it is negative in B-cell lymphomas and is always negative in B or T lymphoblastic types. It seems that lymphoblasts are Leu-M1 negative in non-Hodgkin's lymphoma and may be Leu-M1 positive in leukaemias.     

Lactoferrin-deficient neutrophil polymorphonuclear leucocytes in leukaemias: a semiquantitative and ultrastructural cytochemical study

Semiquantitative analysis of lactoferrin deficiency in neutrophil polymorphonuclear leucocytes in various haematological and non-haematological disease was carried out by scoring polymorphonuclear leucocytes stained for lactoferrin by the immunoperoxidase method. The staining patterns for lactoferrin were classified into four types (0-III) based on the intensity of reaction, and the sum of the ratings of 100 polymorphonuclear leucocytes was considered as "lactoferrin score" with a possible range of 0-300. As a result, significantly low lactoferrin-scores were frequently observed in acute leukaemias and the acute phase of chronic leukaemias. Of 35 cases with leukaemias, lactoferrin-negative polymorphonuclear leucocytes (type 0) were observed in the following cases: eight cases of acute myelogenous leukaemia (8/14), a case of chronic myelogenous leukaemia (1/10) in blast crisis, one of acute promyelocytic leukaemia (1/1), one of acute monocytic leukaemia (1/2), and a case of chronic myelomonocytic leukaemia (1/2) in a transitional phase to an acute myelomonocytic leukaemia. In two cases of acute myelogenous leukaemia, in which the majority of polymorphonuclear leucocytes were negative for lactoferrin, ultrastructural cytochemical study revealed total lack of specific granules in these polymorphonuclear leucocytes. This suggests that lactoferrin is localised in the specific granules of neutrophils as has been postulated previously by others.     

Cytochemical profile of B and T leukaemic lymphocytes with special reference to acute lymphoblastic leukaemia

The PAS and acid phosphatase reactions showed a different pattern of positivity in the cells of lymphoproliferative disorders according to their B or T cell nature. In B-cell leukaemias (chronic lymphocytic and prolymphocytic) a low proportion of lymphocytes gave a positive result with the acid phosphatase reaction, while the majority were PAS positive in granular form. In contrast, in the T-prolymphocytic and T-lymphoblastic leukaemias the acid phosphatase reaction was positive in the majority of cells, while the PAS reaction was only positive in a minority. The significance of these findings, particularly for the recognition of a distinct T-cell variant of acute lymphoblastic leukaemia, is discussed.     

Antibody-dependent haemolytic activity of human leukaemic cells.

Antibody-dependent cellular cytotoxicity (ADCC) of human leukaemic blood cells against human RBC treated with IgG isoantibody was studied by the 51Cr-release method. ADCC in this particular system is a property of normal phagocytic cells of the monocytic and myeloid series while lymphocytes are inactive. Well differentiated leukaemic monocytes from patients with acute monocytic leukaemia were highly cytotoxic and engulfed opsonized RBC. Promyelocytic leukaemic cells from two patients with acute promyelocytic leukaemia were cytotoxic and phagocytic. Seven patients with low differentiated acute myeloblastic leukaemia had no cytotoxic or phagocytic blood cells. Leukaemic B cells from patients with chronic lymphocytic leukaemia or prolymphocytic leukaemia lacked cytotoxic and phagocytic properties. It is concluded that ADCC against isoantibody-treated human RBC may be a tool to distinguish between well and poorly differentiated leukaemic cells of the monocytic or myeloid series.     

Cell death by apoptosis in acute leukaemia

We have previously demonstrated that when freshly isolated childhood T-cell acute lymphoblastic leukaemia cells are incubated in growth medium after isolation from blood, chromatin is rapidly cleaved into nucleosomal sized fragments that are multiples of 200 bp. The fragmentation is similar to that observed in other types of cells undergoing apoptosis or programmed cell death. In this study we describe a more comprehensive approach to the study of DNA fragmentation in leukaemia. Fragmentation was observed in freshly isolated cells from patients with T-cell acute lymphoblastic leukaemia and in one with common acute lymphoblastic leukaemia. Frozen samples of T-cell acute lymphoblastic leukaemia, common acute lymphoblastic leukaemia, and acute myeloid leukaemia cells also showed fragmentation of DNA. However, no fragmentation was evident in normal leukocytes treated under the same conditions. Ultrastructural studies on the isolated leukaemia cells demonstrate that the chromatin cleavage observed biochemically is associated with morphological changes characteristic of apoptosis.     

The immunological classification of leukaemia based on a rapid microcytotoxicity test.

We describe a rapid, accurate, and reproducible cytotoxic antibody test for the immunological classification of leukaemia. Well characterized heteroantisera and monoclonal antibodies were distributed in a microcytotoxicity tray referred to as a leukaemia screening tray (LST). Leukaemia cells were tested for the presence of Ia-like, ''blast'', thymocyte, common acute lymphoblastic leukaemia (cALL), and acute myeloblastic leukaemia (AML) antigens. Utilizing this technique, T ALL could be distinguished from non-T ALL, ALL and AML could be differentiated, the myeloid blast crisis of chronic myelogenous leukaemia (CML) could be distinguished from the lymphoid blast crisis, and the T lymphocyte and B lymphocyte lymphoid leukaemias were readily identified. It has been shown that subclassification of leukaemia according to surface markers, as described here, offers considerable improvement in the diagnosis and treatment of leukaemia over the classical morphological methodologies. Because the test can be completed in 2 hr and unlimited amounts of monoclonal antibodies are available, the LST or similar tests should become universally available in the future to supplement morphological data and replace other lengthy enzyme and rosetting tests.     

Human lymphocyte markers defined by antibodies derived from somatic cell hybrids. II. A hybridoma secreting antibody against an antigen expressed by human B and null lymphocytes.

A hybridoma (FMC4) has been derived which secretes antibody showing selective reaction with human B lymphocytes, monocytes and some null lymphocytes. Few, if any, T lymphocytes in normal blood are stained, although stimulation of lymphocytes with PHA leads to an increase in the proportion of cells reacting with the hybridoma antibody. The antibody reacts with B and null lymphoblastoid cell lines but not with T cell lines. B chronic lymphocytic leukaemia (CLL) cells but not T-CLLs are stained and null-type acute lymphoblastic leukaemia (ALL) cells but not T-type ALL also react. Normal blood myeloid cells do not react with FMC4 supernatant whilst some myeloid leukaemias do. The expression of the antigen reacting with FMC4 supernatant suggests that FMC4 may secrete an antibody against the human equivalent of the Ia antigen.     

Diagnosis of acute myeloid leukaemia with the use of monoclonal anti-neutrophil elastase (NP57) reactive with routinely processed biopsy samples

The reactivity of a monoclonal anti-neutrophil elastase antibody (NP57) with routinely processed biopsy samples from various acute leukaemias has been examined and compared with that of chloroacetate esterase and CD15 (hapten X), two other myeloid cell-associated markers detectable in paraffin sections. No staining was seen with these markers in 14 cases of acute lymphoblastic leukaemia. In contrast the neoplastic cells in 27 of 37 acute myeloid leukaemias were NP57 positive. Twenty of these were also positive for chloroacetate esterase, whereas CD15 was expressed in only six cases. These results indicate that detection of elastase with monoclonal NP57 forms a useful supplement to traditional methods for the histopathological diagnosis of acute myeloid leukaemias.     

Detection of terminal transferase in paraffin sections with the immunoperoxidase technique.

Terminal deoxynucleotidyl transferase (TdT) is an early T-cell differentiation marker in a number of species, including man. We have demonstrated TdT in the nuclei of cortical thymocytes in paraffin-embedded sections of calf, rat, and human thymus by indirect immunoperoxidase techniques. In addition, these techniques have been used to verify and extend enzyme assay results by detecting TdT in blast cells from 10 patients with convoluted T-cell lymphoma/leukemia, 1 patient with acute granulocytic leukemia, 1 patient with chronic granulocytic leukemia in blast crisis, and 1 patient with non-T non-B acute lymphocytic leukemia.     

Correlation if circulating immune complexes and disease status in patients with leukaemia.

The occurrence of soluble immune complexes (IC) was investigated in 177 serum samples from 92 patients with various leukaemias using the Raji cell immunoassay. In general, patients with myeloproliferative diseases had a higher incidence and higher quantities of IC than did patients with lymphoproliferative disorders. Elevated levels of IC were found in the sera of patients as follows: 17% with chronic lymphocytic leukaemia (mean value of 13.1 microgram/ml), 67% with acute lymphocytic leukaemia (54.1 microgram/ml), 65% with chronic myelocytic leukaemia (86.7 microgram/ml), 70% with acute myelocytic leukaemia (202.5 microgram/ml) and 56% with acute myelomonocytic leukaemia (41.9 microgram/ml). Patients in terminal blastic crisis of chronic myelocytic leukaemia had the highest levels, with a mean level of 1,364.1 microgram/ml. Serial samples were obtained, as available, from individual patients during the course of the disease in an attempt to relate severity with the incidence and quantity of IC. No significant correlation could be made between the occurrence or levels of IC and the presence of absence of systemic symptoms. Similarly, no correlations could be made between levels of IC and haematological parameters, infection, or therapy. However, the data does indicate a positive relationship between the levels of IC and the progressive state of the leukaemia, especially, the myelocytic leukaemias.     

Monoclonal antibody (Y1/82A) with specificity towards peripheral blood monocytes and tissue macrophages.

A new monoclonal antibody, Y1/82A, was raised against phytohaemagglutinin activated peripheral blood mononuclear cells. Using an immunohistochemical technique it was shown that Y1/82A reacts against peripheral blood and bone marrow monocytes and resident macrophages from essentially all human tissues. Y1/82A bound to determinants present in leukaemic cells from patients with acute myelomonocytic leukaemia and acute monocytic leukaemia, but not to neoplastic cells from patients with malignant lymphoproliferative disorders or malignant epithelial tumours. Y1/82A failed to react with other cell types, with the exception of osteoclasts and megakaryocytes. Analysis by Western blotting showed that the antigen detected by antibody Y1/82A was associated with intracellular granules in macrophages. Monoclonal antibody Y1/82A may be useful in the diagnosis of monocytic leukaemias and histiocytic neoplasms and in the identification of macrophages in tissues from various inflammatory and neoplastic conditions.     

Serum and leukocyte lactate dehydrogenase activity in leukaemias

Lactate dehydrogenase (LHD) content of serum and leukocytes was examined in 42 haematologically normal healthy volunteers and in 34 patients suffering from various types of leukaemia. All patients were studied at the time of presentation and before any therapeutic intervention. Serum LDH was elevated in all types of leukaemia. In acute myeloid leukaemia (AML) a significant elevation of leukocyte LDH activity (p less than 0.005) was noted. In acute lymphoblastic leukaemia leukocyte (ALL), LDH was significantly elevated when compared to normal lymphocyte LDH (p less than 0.01) levels, but not when compared to total normal leukocyte LDH levels. In chronic leukaemias, leukocyte LDH levels were not significantly different from the normal. Comparison of LDH isoenzyme pattern in peripheral blood cells with that of serum, both in normal and in leukaemia cases showed more "M" type enzyme in the cells than in the serum. However, the "M" type enzyme was significantly elevated only in AML cases (p less than 0.005). Serum LDH and peripheral blood leukocyte count compared in normal subjects and in leukaemia cases showed no correlation.