NSE/αNAE positivity in B-lineage acute lymphoblastic leukemia: revisiting a potential cytochemical diagnostic pitfall

Cytochemical staining for leukemia typing is declining in hematology laboratories, but the use of flow cytometry may not be possible in some settings. Aberrant cytochemical nonspecific esterase/α-naphthyl acetate esterase (NSE/αNAE) positive B-lymphoblasts can cause confusion with monoblasts, a potentially dangerous pitfall. This unusual cytochemical NSE/αNAE positivity had been associated with relatively poorer outcome of acute lymphoblastic leukemia (ALL) in the era prior to the advent of routine multicolor flow cytometric immunophenotyping. We reviewed morphological, cytochemical and flow-cytometric data from five cases of B-lineage ALL that showed NSE/αNAE positivity and were diagnosed definitively using multi-parametric flow cytometric immunophenotypic analysis. Diffuse or dot-like (localized) strong cytochemical NSE/αNAE activity was detected in all cases and all showed one or more features of high risk disease. The number of NSE/αNAE positive blasts in the marrow varied from 10 to 75%. The morphological differential diagnoses included T-lymphoid lineage ALL and acute monoblastic leukemia (AML-M5). Flow cytometric data revealed B-lineage antigens and the absence of monocytic or other myeloid markers resolved the diagnosis. These cases underscore the importance of immunophenotyping in all cases of suspected ALL regardless of the cytochemical findings. Although the numbers are small, the association with high risk disease observed in all five of our cases may corroborate the previously reported poor prognostic value of such aberrant cytochemical staining.     

Associations between the two serum proteins haptoglobin and transferrin and leukaemia

Haptoglobin and transferrin (TF) types were determined for 134 patients with leukaemia of the four most common types: acute lymphocytic (ALL), chronic lymphocytic (CLL), acute myelocytic (AML) and chronic myelocytic leukaemia (CML). The phenotype HP1 was found to have an increased incidence in the total patient group due to an increased incidence in those with AML, ALL and CML compared with controls, but not in those with CLL. Although tests of association applied to each of the samples of the four common types of leukaemia produced no significant chi 2 values, they did indicate that the relative incidence (RI) was just under 2 for the groupings of the acute forms ALL and AML, the myelocytic forms AML and CML and for the combination of ALL, AML and CML, respectively. All these associations were statistically significant (p less than 0.05). Analysis of TF subtypes and leukaemia indicated a significantly increased frequency of TF C1C1 among leukaemia patients compared with controls (p less than 0.005). Analysis of the samples of each of the four common types suggested that while the RI was raised in all but ALL patients, the association was significant only in AML patients (p less than 0.05). However, when the two myelocytic types were combined the RI was 2.3 and the association was highly significant (p less than 0.005). No such association could be detected in the lymphocytic forms.     

Immunophenotyping of acute myeloid leukaemia: relevance of analysing different lineage-associated markers

The immunophenotype of 135 previously untreated patients with FAB defined acute myeloid leukaemia (AML) was studied at diagnosis. The panel of reagents included monoclonal antibodies (MoAb) recognising myeloid-associated determinants (CD11, CD13, CD14, CD33 and others) as well as MoAb directed towards lymphoid antigens (CD7, CD10, CD19) and TdT. The results indicate that CD13 and/or CD33 are consistently expressed in AML and only rarely in ALL blasts (131/135 + ve cases, versus 4/130 in ALL). Lymphoid antigen expression was rarely detected when CD10 and CD19 were investigated in AML (0.9% and 2% + ve cases, respectively), whereas significant positivities were found for TdT and CD7 (20% and 10% respectively). Concerning FAB subtypes, two new MoAb (LAM3 and LAM7) proved very useful in the specific recognition of AML with monocytic features. The phenotype CD13+ and/or CD33+, CD9+, HLA-DR- was found to be almost exclusive for M3 AML. The response to induction chemotherapy was analysed in CD7+ and in TdT+ patients. In the latter group a statistically significant lower response rate was found with respect to TdT-ve-AML patients.     

Cytogenetic analysis and clinical significance of chromosome 7 aberrations in acute leukaemia

The analysis was performed on bone marrow cells derived from 96 patients with acute leukaemia (AL): 76 with acute myelogenous leukaemia (AML) and 20 with acute lymphoblastic leukaemia (ALL). Aberrations of chromosome 7 were revealed in 20 (21%) of 96 analysed cases: in 14 (18%) with AML and in six (30%) with ALL. Structural aberrations, present in 13 patients (eight with AML and five with ALL), were unbalanced and led to partial monosomy (12 cases) or trisomy (four cases) of chromosome 7. Twelve (86%) out of 14 AML and all the ALL patients with chromosome 7 aberrations had complex karyotypes in their bone marrow cells. Monosomy 7 and 7q losses were frequently observed in the AML group, whereas, in the ALL group, gains in 7q and losses in the short arms constituted most chromosome 7 aberrations. The occurrence of monosomy, or of losses in 7q, results in a worse response to induction therapy in AML patients. The complete remission (CR) rate was significantly lower in this group in comparison to the group of AML patients with a normal karyotype (p = 0.01) in bone marrow cells.     

Epigenetic inactivation of the miR-124-1 in haematological malignancies

miR-124-1 is a tumour suppressor microRNA (miR). Epigenetic deregulation of miRs is implicated in carcinogenesis. Promoter DNA methylation and histone modification of miR-124-1 was studied in 5 normal marrow controls, 4 lymphoma, 8 multiple myeloma (MM) cell lines, 230 diagnostic primary samples of acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), chronic myeloid leukaemia (CML), chronic lymphocytic leukaemia (CLL), MM, and non-Hodgkin's lymphoma (NHL), and 53 MM samples at stable disease or relapse. Promoter of miR-124-1 was unmethylated in normal controls but homozygously methylated in 4 of 4 lymphoma and 4 of 8 myeloma cell lines. Treatment of 5-Aza-2'-deoxycytidine led to miR-124-1 demethylation and re-expression of mature miR-124, which also associated with emergence of euchromatic trimethyl H3K4 and consequent downregulation of CDK6 in myeloma cells harboring homozygous miR-124-1 methylation. In primary samples at diagnosis, miR-124-1 methylation was absent in CML but detected in 2% each of MM at diagnosis and relapse/progression, 5% ALL, 15% AML, 14% CLL and 58.1% of NHL (p<0.001). Amongst lymphoid malignancies, miR-124-1 was preferentially methylated in NHL than MM, CLL or ALL. In primary lymphoma samples, miR-124-1 was preferentially hypermethylated in B- or NK/T-cell lymphomas and associated with reduced miR-124 expression. In conclusion, miR-124-1 was hypermethylated in a tumour-specific manner, with a heterochromatic histone configuration. Hypomethylation led to partial restoration of euchromatic histone code and miR re-expression. Infrequent miR-124-1 methylation detected in diagnostic and relapse MM samples showed an unimportant role in MM pathogenesis, despite frequent methylation found in cell lines. Amongst haematological cancers, miR-124-1 was more frequently hypermethylated in NHL, and hence warrants further study.     

Human leukaemia-associated antigens expressed by acute lymphocytic leukaemias and their detection with heterologous antisera to T, B-, and non-T-non-B subtype AL blasts.

Antisera from rabbits and goats against subtypes of acute lymphocytic leukaemia (ALL with T-cell markers, ALL with B-cell markers, Non-T-non-B ALL) were tested for their specificity in complement-dependent in-vitro cytotoxicity testing. After absorption of the fivefold diluted antisera with erythrocytes and spleen cells of allogenous donors they reacted with ALL cells, but not with leukaemias of other types (AML, CLL, CML), lymphocytes of healthy donors, enriched B-lymphocytes, enriched T-lymphocytes, PHA-stimulated lymphocytes, cord lymphocytes and bone marrow lymphocytes of patients in remission. In the reactions of the antisera against ALL cells the subtype of ALL is of major importance: Six rabbit antisera and one goat antiserum against T-subtype ALL reacted in all 19 tests with the leukaemia cells of 5 patients with T-cell ALL and in all 9 tests with thymocytes of 3 donors, but only in 14 out of 41 tests with the leukaemia cells of 14 Non-T-non-B ALL patients. One antiserum against a B-subtype ALL lysed B-cell ALL (1/1), but not T-cell ALL (0/3), Non-T-non-B-cell ALL (1/5) and thymocytes (0/2). Four antisera against Non-T-non-B-subtype ALL reacted in 22 out of 46 tests with the Non-T-non-B cells of 17 ALL patients, but did not react with the leukaemia cells of 4 children with T-cell ALL (0/16), one child with B-cell ALL (0/1) thymocytes of 2 donors (0/4). The reactions of the anti-ALL sera with fetal liver cells, complete absorbability of the antileukaemic activity of the antisera with fetal tissue and the reactions of an anti-fetal serum with ALL cells point to the existence of fetal antigen components as leukaemia-associated antigens.     

Bacterial glutaminase in treatment of acute leukaemia.

A glutaminase-asparaginase enzyme from Achromobacter sp has antitumour activity in vitro and in animals. Glutaminase was administered in doses of 3500-20 000 IU/m2 body surface area/day to six patients with acute lymphoblastic leukaemia (ALL) and three patients with acute myeloid leukaemia (AML). The enzyme had a blood half life of 80 minutes but depletion of blood glutamine persisted for 12 hours after single doses. Seven patients, including four (two with AML and two with ALL) resistant to asparaginase, received repeated doses of glutaminase. Antileukaemic effects were observed in all seven; one elderly patient developed metabolic acidosis. Study of this new antileukaemic agent in patients with acute leukaemia at an earlier stage of their disease is now justified.     

Acute lymphatic leukemia with pre-B-cell characteristics

54 patients affected with acute lymphatic leukaemia (ALL) in the period from 1978-1984 were examined concerning the frequency of pre-B-ALL in comparison with other immunological subtypes of ALL. For this purpose the following markers were tested: 1. Cytoplasmatic IgM 2. Rosette formation with sheep erythrocytes 3. T-cell specific antigen 4. C-ALL antigen 5. Surface immunoglobulin By means of these markers the patients were divided into 5 immunological subtypes of ALL, viz. 1. Pre-B-ALL (16.6%) 2. O-ALL (37.1%) 3. C-ALL (27.9%) 4. T-ALL (16.6%) 5. B-ALL (1.8%) The immunological subtypes of ALL were compared by means of clinical parameters (age, initial leukocyte number, mediastinal tumor, initial CNS involvement). No difference could be detected between O-ALL, pre-B-ALL and C-ALL, whereas T-ALL showed a distinctly worse prognosis.     

Diagnostic application of monoclonal antibody KB90 (CD11c) in acute myeloid leukaemia

The expression of membrane CD11c by leukaemic blast cells was examined (indirect immunorosetting) in 75 cases of acute leukaemia (myeloid, n = 60; lymphoid, n = 15) and evaluated as a potential marker for the diagnostic discrimination between monocytic (AMML-M4 and AMoL-M5) and non-monocytic (M1, M2 and M3) AML subtypes. Preliminary studies of normal bone marrow cells indicated that CD11c expression was not restricted to cells of monocytic lineage but was also present, with apparent lower density, on significant proportions of mature and immature granulocytes. Examination of acute myeloid leukaemia (AML) subtypes revealed that the non-monocytic leukaemias (n = 33) were CD11c-, defined as less than 30% positive cells, whereas all but one of the AMML-M4 (n = 13) and AMoL-M5 (n = 14) cases were CD11c+. All 15 cases of lymphoblastic leukaemia (ALL) showed less than 5% CD11c+ blasts. Membrane CD11c expression was also compared to the more widely used markers of monocytic differentiation; cytoplasmic alpha-naphthyl acetate esterase (ANAE) and membrane CD14 expression. This analysis showed that all 13 AMML-M4 leukaemias studied, including seven cases that were CD14- and eight that were ANAE-, were CD11c+. In addition, the AMoL-M5 cases (all of which were ANAE+) could be phenotypically subdivided into CD11c+ CD14+ (n = 9), CD11c+ CD14- (n = 4) and CD11c- CD14- (n = 1) subgroups. The study also confirmed that the discriminitive ability and sensitivity of the immunorosetting procedure for the detection of membrane CD11c compared favourably to immunofluorescent staining intensities as measured by flow cytometry.     

Monoclonal antibodies for diagnosis of immunodeficiencies

Progress in immunophenotyping is characterized by the availability of monoclonal antibodies and an increased number of clusters of differentiation consisting of reagents with known specificity and defined reactivity patterns. Technical improvements have lead to standardization of immunofluorescence staining procedures and broad application of flow cytometry. These developments have contributed to better diagnosis of immunodeficiencies characterized by the lack of certain lymphocyte subsets or more broadly expressed, functionally important cell-surface molecules. Antibodies valuable for routine immunophenotyping of immunodeficiencies as well as examples of the different antibody groups desirable for immunofluorescence studies are presented. When used in concert with clinical and other laboratory tests, immunophenotyping provides a valuable instrument for differential diagnosis of defects in the immune system. As a consequence, detection of new defects of cell surface antigens and respective cell subpopulations is facilitated and a basis is provided for further study of the genetic and molecular regulatory aspects of immunologic disorders.     

Cell markers in the recognition of acute myeloblastic leukaemia subtypes

The diagnosis of acute myeloblastic leukaemia (AML) is based on cell morphology, cytogenetic and molecular changes, cell markers and clinical data. Our aim was to establish whether morphology and cell markers are comparable in the evaluation of AML. Bone marrow smears were analysed, and flow cytometry and monoclonal antibodies were used to determine cell type and maturity. Morphology and cell markers correlated differently in different AML subtypes.     

Bone marrow necrosis intravitally recognized in four cases of blastic leukaemia.

Bone marrow necrosis (BMN) is a rare intravitally recognized finding in acute leukaemia with an uncertain clinical significance. The clinical events in 4 patients with AML, ALL, AMoL and blastic transformation of CGL in whom bone marrow cytology and histology revealed BMN are reviewed. One patient with BMN at clinical presentation of AML entered complete, long lasting remission with marrow restoration after the standard DAT therapy. In the three remaining patients survival after BMN diagnosis was 6, 11, and 14 weeks. Clinical, haematological, histological and marrow scanning findings and their significance for early diagnosis and means to asses the extent and evaluation of BMN will be discussed. In contrast to the most earlier reports, BMN does not appear to confer a poor prognosis in all patients with blastic leukaemia.     

Risk of primary haematologic cancers following incident non-metastatic breast cancer: A Danish population-based cohort study

BackgroundBreast cancer survivors may have increased risk of subsequent haematologic cancer. We compared their risk of haematologic cancers with the general population during 38 years of follow-up.MethodsUsing population-based Danish medical registries, we assembled a nationwide cohort of women diagnosed with incident non-metastatic breast cancer during 1980–2017, with follow-up through 2018. We compared breast cancer survivors with the general population by computing standardised incidence ratios (SIR) and 95% confidence intervals (CI).ResultsAmong 101,117 breast cancer survivors, we observed 815 incident haematologic cancers (median follow-up: 7.9 years). We observed excess risk of acute myeloid leukaemia (AML) (SIR: 1.65, 95%CI: 1.33–2.01), particularly in women who received chemotherapy (SIR: 3.33, 95%CI: 2.24–4.75) and premenopausal women (SIR: 3.23, 95%CI: 2.41–4.25). The risk of acute lymphoid leukaemia (ALL) was increased (SIR: 2.25, 95%CI: 1.29–3.66), whereas the risk of chronic lymphoid leukaemia (CLL) was decreased (SIR: 0.66, 95%CI: 0.53–0.82). An additional analysis showed elevated risk of CLL 0–6 months after breast cancer diagnosis (SIR: 3.00 95%CI: 1.75–4.80).ConclusionCompared to the general population, breast cancer survivors had elevated risk of AML, particularly when treated with chemotherapy. The risk of ALL was elevated, whereas the risk of CLL was lower. The higher risk of CLL in the first six months after diagnosis likely reflects surveillance bias—due to intensified diagnostic efforts at breast cancer diagnosis and treatment—prompting earlier detection. This has likely reduced the long-term risk of CLL in breast cancer survivors.     

Binding of lectins to human leukaemic cells

The interactions of five different lectins: peanut (PNA), lentil (LEN), wheat germ (WGA), soybean (SBA), Asparagus pea (FBP) with leukaemic cells obtained from 31 children: 25 with acute lymphoblastic leukaemia (ALL) and 6 with acute myeloblastic leukaemia (AML) were examined in this study. The relationship of lectin-binding ability to cells cytomorphological, cytochemical and immunological features and its potential clinical application were investigated. It has been shown that PNA and LEN receptors were found in the majority of blast cells. The SBA reacting cells were found only in few patients and FBP binding was not found in studied ALL and AML cells. There was a clear difference in the WGA binding capacity in ALL cells with L1 and L2 characteristics respectively. No differences were found in PNA. WGA and LEN reactivity between PAS negative and PAS positive leukaemic cells. Only PNA of all studied lectins seemed to differentiate T- from B-ALL blast cells. Only WGA binding of ALL cells showed the positive correlation to the risk index value.     

Decreasedin vitro chemosensitivity of tumour cells in patients suffering from malignant diseases with a poor prognosis

The aim of the study was to assess the predictive value of MTTin vitro assay for evaluation of tumour cell resistance/sensitivity to cytotoxic drugs. We analyzed 105 samples of malignant cells of different origin. The study included patients with a diagnosis of acute and chronic lymphatic leukaemia, acute and chronic myeloid leukaemia, non-Hodgkin lymphoma, carcinoma of the lung, stomach and liver, rhabdomyosarcoma and breast carcinoma. The results demonstrate outstanding chemosensitivity in the majority of childhood acute lymphoblastic leukaemias, medium chemosensitivity of adult haematopoietic malignant diseases and chemoresistance of solid tumour cells. Our preliminary data suggest a good correlation betweenin vitro MTT assay and clinical curability of individual malignant diseases.Abbreviations ALL acute lymphoid leukaemia - AML acute myeloid leukaemia - CML chronic myeloid leukaemia - LCS₅₀ 50% leukaemic cell survival     

Immunophenotyping of acute lymphoblastic leukemia using immunohistochemistry in bone marrow biopsy specimens

Flow cytometry is the preferred method of diagnosing and immunophenotyping acute lymphoblastic leukemia (ALL). However, there are situations in which immunohistochemical staining (IH) of bone marrow trephine biopsy specimens can be used to provide immunophenotypic information. To evaluate the use of IH and to confirm its value in diagnosing and typing of ALL, we studied 50 cases of denovo ALL that were previously classified into pre B, T and B by morphologic, cytochemical and FC methods. Paraffin embedded bone marrow trephine biopsies sections were stained using a panel of antibodies,namely, myeloperoxidase (MPO), terminal deoxynucleotidyl transferase (TdT), CD10, CD20, CD79a, CD3. The cases included 37 pre BALL, 10 T ALL and 3 mature BALL. TdT was the most commonly expressed antibody and was positive in 41 of 50 cases of ALL (82%) and in 95% of pre B ALL cases. CD79a and CD10 were positive in 68% and 65% of pre B ALL cases, respectively. CD79a showed similar positivity in B ALL cases (66%). CD 20 was positive in 66% of mature B ALL cases but less positive in pre B ALL (22%). CD3 was positive in 70% of T ALL cases and negative in other ALL subtypes. All of the cases were negative for MPO. Diagnosis and immunophenotyping of acute lymphoblastic leukemia is possible using immunohistochemical staining of bone marrow trephine biopsies.     

Hybrid acute leukemia: therapeutical implications of immunological phenotyping.

We phenotyped blood and bone marrow cells from a patient with acute Ph1+ acute leukemia longitudinally during the four months he received intensive chemotherapy. At presentation this case of biphenotypic acute leukemia had two immunologically different types of blast cells, one expressed CD10 (CALLA), CD13 (MY7) and CD33 (MY9) but lacked CD20 (B1), the other type expressed no CD10 or CD33. The phenotype, during AML induction therapy, changed to a more CD10+, CD20+ ALL one. ALL therapy based on these findings induced improvement in bone marrow function but the patient died of septicemia at day 134. The use of concomitant immunophenotyping (IP) and cell cycle analysis had shown proliferation advantage of the more lymphoid malignant cells. These results suggest that it is possible to induce lineage-associated changes in the phenotype of hybrid malignant cells and that these leukemias might be treated best according to longitudinal immunophenotyping of the blast cells.     

MLL-rearranged infant leukaemia: A ‘thorn in the side’ of a remarkable success story

Advances in treatment of childhood leukaemia has led to vastly improved survival rates, however some subtypes such as those characterised by MLL gene rearrangement (MLL-r), especially in infants, continue to have high relapse rates and poor survival. Natural history and molecular studies indicate that infant acute lymphoblastic leukaemia (ALL) originates in utero, is distinct from childhood ALL, and most cases are caused by MLL-r resulting in an oncogenic MLL fusion protein. Unlike childhood ALL, only a very small number of additional mutations are present in infant ALL, indicating that MLL-r alone may be sufficient to give rise to this rapid onset, aggressive leukaemia in an appropriate fetal cell context. Despite modifications in treatment approaches, the outcome of MLL-r infant ALL has remained dismal and a clear understanding of the underlying biology of the disease is required in order to develop appropriate disease models and more effective therapeutic strategies.     

Specificities of heterologous antisera against human leukaemia cells. 1. Reactions against leukaemia cells.

Rabbit or goat antisera directed to ALL, CLL, AML and CML cells were investigated in cytotoxicity tests with different leukaemia and normal cells as targets. After absorptions with erythrocytes and spleen cells from allogeneic donors the antisera killed only leukaemia cells. There was no reaction with remission leukocytes or blood leukocytes from normal donors. Anti-ALL-Sera reacted in 35 out of 49 tests with ALL cells from 13 patients. Apparently the ALL antisera which were directed to the T cell subtype of ALL preferentially affected ALL cells of this subtype. Cross reactions with cells from CLL, AML and CML were not found. Anti-CLL-sera reacted in 10 out of 12 tests with CLL cells from 4 donors, and in 4 out of 20 tests with ALL cells from 7 donors and also with the cells of a CML patient. AML cells from two patients were not killed. Antisera against AML and CML showed extensive cross reactions with cells of myelocytic and lymphocytic leukaemias. Absorption tests demonstrated the presence of two antibody specificities in AML antisera, one of which being directed to a common antigen of AML and ALL cells and another against an antigen of myelocytic leukaemia cells.