P53 gene mutations in acute myelogenous leukaemia

A polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) assay was used to identify the exons which contained point mutations in the conserved regions (exons 4-8) of the p53 gene in 49 acute myelogenous leukaemia (AML) patients. SSCP analysis in our study was consistent with the results of subsequent direct DNA sequencing in detecting point mutational change in exons 5 and 8 of one AML patient and in exons 7 and 8 of two additional AML patients. The mutations were located at codons 245 and 273, which have been found in many other tumours, and codons 178 and 290, which have not been reported previously. All of the p53 proteins in which we detected point mutations were immunoprecipitated by the p53 monoclonal antibody PAb 240, which has been shown to recognize a mutant conformation of p53 protein. Thus, our results indicate that functional inactivation of the p53 gene by point mutational change might be one of the mechanisms underlying disease progression of AML.     

Biological characteristics of CD7 positive acute myelogenous leukaemia

We studied the biological characteristics of CD7+ acute myelogenous leukaemia (AML). We diagnosed nine out of 88 consecutive AML cases as CD7+ AML based on myeloperoxidase positivity and surface antigen expression. In eight of these nine cases more than 20% of leukaemic blasts were found to coexpress both CD7 and a myeloid-associated antigen, CD33, by a two-colour flow-cytometric assay, while in the remaining case more than 90% of blasts were positive for CD7 and myeloperoxidase. CD7+ AML was most frequently observed in M1 among AML subtypes according to the FAB classification. An early stage-specific antigen, CD34 was also expressed on leukaemic blasts from eight of these nine cases. Neither the T-cell receptor (TcR)-beta nor the TcR-gamma gene was clonally rearranged in any of the cases. We then studied the proliferative responses to stimulation by various growth factors. Among interleukin-3 (IL-3), granulocyte/macrophage colony-stimulating factor (GM-CSF), and granulocyte-CSF (G-CSF), IL-3 showed the strongest stimulatory effect on DNA synthesis and leukaemic blast colony formation in 8/9 and 6/8 CD7+ AML cases examined, respectively. On the other hand, the strongest stimulatory effect exerted by IL-3 on blast colony formation was observed in only six out of the 33 CD7- AML cases examined. Furthermore, CD7+ AML blasts could proliferate in response to stem cell factor (SCF); SCF alone showed stimulatory effects on blast colony formation (7/8 cases), and in 5/7 SCF-responding cases, stimulatory effects of SCF were more potent than those of IL-3. In addition, SCF enhanced blast colony formation synergistically with IL-3 in four of these seven cases. These data suggest that progenitor cells of CD7+ AML may possess the biological properties characteristic of immature haematopoietic stem cells.     

Cytochemical and immunological characteristics of acute monocytic leukaemia

Immunological and cytochemical findings are presented from 12 cases of morphologically unequivocal acute monocytic leukaemia (AMoL). The results indicate considerable heterogeneity and three main non-morphological subgroups were identified. The blast cells from half the patients were positive for the presence of both cytoplasmic alpha naphthyl acetate esterase (ANAE) and monocyte-associated membrane determinants whereas the cells from three cases lacked detectable monocytic antigens despite the presence of strong cytochemical ANAE activity. A further three cases expressed monocytic antigens but were cytochemically unreactive for ANAE. These cytochemical results, which were extended by electrophoretic studies of ANAE isoenzymes, suggest that the absence of significant cytoplasmic ANAE activity does not preclude the diagnosis of AMoL and that serum lysozyme estimations may be of value in the recognition of immunocytochemically-atypical cases.     

Detection of donor cell derived acute myelogenous leukaemia in a patient transplanted for chronic myelogenous leukaemia using fluorescence in situ hybridization

The recurrence of leukaemia following allogeneic bone marrow transplantation appears to develop rarely in donor cells. However, the standard method for assigning the origin of recurrence, metaphase analysis, can be unreliable. We have applied the technique of fluorescence in situ hybridization (FISH) directly on archival Wright stained bone marrow slides obtained from a patient who developed acute myelogenous leukaemia (AML) following allogeneic bone marrow transplantation (BMT) for chronic myelogenous leukaemia (CML). Using a chromosome-specific DNA probe we linked a chromosomal aberration, previously detected by conventional metaphase analysis, directly to morphologically identifiable blast cells. In this way we were able to assess cell-lineage involvement of the secondary leukaemia and assign a donor origin.     

How to address second and therapy-related acute myelogenous leukaemia

Secondary acute myelogenous leukaemia (AML), as compared to de novo AML, occurs in the more elderly population, is independently more resistant to cytotoxic chemotherapy, has a higher relapse rate, and a worse prognosis. Secondary AML (sAML) is a heterogeneous disease, both biologically and clinically, even within the World Health Organization subgroups of sAML. Outcomes are the poorest in subgroups with sAML arising from an antecedent haematologic disorder which has been previously treated (ts-AML), and sAML in patients <55 years of age. This review describes the suboptimal outcomes of contemporary therapy, to support the notion of an unmet need for innovative treatment strategies in sAML. Despite the recent approval of CPX-351, long-term outcomes for this high-risk disease remain dismal. Resistance mechanisms to intensive chemotherapy contribute to relapse. Targeted immune therapy may avoid multidrug resistance mechanisms, but are unlikely to provide long-term remission due to a complex and rapidly evolving clonal disease profile. Advances for sAML will likely be accomplished by CAR T cell therapy or bispecific antibodies providing a bridge to allogeneic stem cell transplantation. Therefore, focus should be placed on novel strategies that can augment the untargeted effector function of allogeneic grafts.     

Aberrant expression of HLA-G antigen in interferon gamma-stimulated acute myelogenous leukaemia

We have analysed the expression of HLA-G in 40 leukaemia samples of various subtypes [seven cases of acute lymphoblastic leukaemia (ALL), 28 cases of acute myelogenous leukaemia (AML), three cases of chronic myelogenous leukaemia (CML) and two cases of chronic lymphocytic leukaemia (CLL)] by flow cytometry using HLA-G-specific monoclonal antibody. No leukaemia samples expressed HLA-G without incubation with interferon (IFN)-gamma. However, six out of 28 (21%) AML samples expressed HLA-G upon incubation with IFN-gamma. These six samples derived from one out of seven M2, two out of eight M4 and three out of five M5. The results indicated that AML cells, especially myelomonocytic leukaemia samples, are capable of expressing the HLA-G molecule.     

Chronic myelogenous leukaemia

In the last decade, improvements in both non-transplant and transplant therapy have extended the lives of patients with CML, particularly those in chronic phase. The future will probably bring a greater understanding of molecular leukaemogenesis and options for treating CML. Non-transplant therapies in development include novel agents and combination therapy. Transplant strategies seek to decrease regimen-related toxicity and directly manipulate the immune system to eradicate disease. Clinical and laboratory science seems poised to add novel therapies to the armamentarium against CML. It is exciting to contemplate what reviews of CML written 10 years from now will discuss.     

Advances in the immunological monitoring of childhood acute lymphoblastic leukaemia

In children with acute lymphoblastic leukaemia (ALL), measurements of minimal residual disease (MRD) during therapy provide crucial information about the response to treatment and the risk of relapse. Flow cytometry is a practical and widely applicable tool for monitoring MRD in patients with ALL. This approach is based on the identification of immunophenotypes expressed by leukaemic cells but not by normal lympho-haematopoietic cells in bone marrow and peripheral blood. These phenotypes can identify one leukaemic cell among 10 000 normal cells and are currently applicable to at least 90% of patients with ALL. A strong correlation between flow cytometric measurements of MRD during clinical remission and treatment outcome has been demonstrated, suggesting that these assays should be incorporated into treatment protocols.     

Acute fungal thyroiditis in a patient with acute myelogenous leukaemia.

Acute suppurative thyroiditis of any origin is uncommon, but fungal infections of the gland are particularly rare. Haematogenous spread is the usual route of infection. We here present the case of a recently encountered patient with neutropenic fever and Candida thyroiditis. Fine-needle aspiration biopsy greatly aided the diagnosis. In immunocompromised patients, the specimens should be treated with special stains to detect the presence of opportunistic organisms; if any are found, appropriate therapy should be initiated.     

Telomerase activity in acute myelogenous leukaemia: clinical and biological implications

We examined telomerase activity in myeloid leukaemic cell lines, normal haemopoietic cells, and leukaemic blasts from acute myelogenous leukaemia (AML) patients. Normal bone marrow mononuclear (BMNC) cells expressed low telomerase activity. Higher telomerase activity was detected in 10 myeloid leukaemic cell lines compared to normal BMNC cells. Treatment with 1,25(OH)₂D₃, and vitamin D₃ analogues, EB1089 and KH1060, reduced telomerase activity in vitamin D₃-sensitive HL-60 cells, whereas vitamin D₃ insensitive K562 cells did not change its activity. This down-regulation of telomerase activity by EB1089 was associated with induction of p21 protein. The rank order of telomerase activity was leukaemic CD34⁻ cells > leukaemic CD34⁺ cells > normal CD34⁻ cells > normal CD34⁺ cells. Telomerase activity was positive in all of the AML patients tested; however, heterogeneity of telomerase activity was found amongst this group. Therefore we compared telomerase activity with clinical response. Unexpectedly, we found that a higher rate of complete remission was noted in AML patients with higher telomerase activity. No association between telomerase activity and biological parameters including percentage of S-phase, cytotoxicity to cytosine arabinoside and percentage of CD34⁺ cells in AML blasts was found. These results suggest that telomerase activity in AML patients is detected with high frequency, but is heterogenous. Expression level of telomerase activity may have a clinical implication in AML patients regarding clinical response.     

Variable differentiation patterns of acute myelogenous leukaemia blasts in liquid suspension cultures

To study the ability of acute myelogenous leukaemia blasts to spontaneously differentiate in vitro, bone marrow and/or blood mononuclear cells from 63 patients with acute myelogenous leukaemia were incubated in liquid suspension cultures containing human serum, without addition of chemical inducers of differentiation. Cultures were examined weekly for disappearance or persistence of blasts, and for appearance of morphologically recognizable granulocytes and macrophages. Culture outcomes were extremely variable, ranging from lack of appearance of differentiated cells to complete disappearance of blasts with replacement by mature cells. In 50 cases an increase (25-185%) in the absolute number of differentiated cells in culture was noted during the culture period. Full differentiation was seen exclusively in cultures from 13/48 (27.1%) patients studied at diagnosis, as compared to 0/20 patients studied at relapse (P less than 0.01). The ability to fully or partially differentiate in culture was lost to a significant degree at relapse (13/20 patients) as compared to diagnosis (48/48 patients, P = 0.0001). At diagnosis full differentiation in culture was associated with a significantly higher remission rate than partial differentiation (89% versus 40%, P less than 0.02). Origination of mature cells from leukaemic rather than normal precursors was suggested by the appearance of Auer rods in mature cells in seven cases, by the correlation of types of differentiated cells seen in culture with the FAB class of leukaemia and by cytogenetic data in one case.     

Hypercalcaemia complicating acute myelogenous leukaemia: a syndrome of multiple aetiologies

Hypercalcaemia complicating acute myelogenous leukaemia is a rare but well-recognized phenomenon. In most cases the pathogenetic mechanism causing the hypercalcaemia remains poorly understood. We recently studied in detail two patients with acute myelogenous leukaemia who developed hypercalcaemia during the course of their illness. The results of these studies conclusively excluded primary hyperparathyroidism or ectopic parathyroid hormone production as causes of the patients' hypercalcaemia. In vitro studies carried out on short-term suspension cultures of one patient's peripheral blood blast cells demonstrated production of a factor with potent bone resorbing activity, distinct from parathyroid hormone (iPTH) and prostaglandin E2 (PGE2). Further characterization of the bone resorbing factor suggested that it bore some similarity to osteoclast activating factor (OAF). Hypercalcaemia in the other case appeared to be due to a combination of skeletal invasion by malignant cells, and to ectopic secretion of an unidentified humoral factor with bone resorbing activity. These two cases demonstrate that the hypercalcaemia complicating acute myelogenous leukaemia may be due to a variety of mechanisms distinct from parathyroid hormone production.     

A possible role of rifampicin in prolonging remission duration in acute myelogenous leukaemia

The remission duration (RD) of 22 adult acute myelogenous leukaemia patients who received maintenance rifampicin treatment during 24 episodes was significantly longer than RD in 29 complete remission patients who received conventional maintenance chemotherapy (25 versus 9 months, p less than 0.003). Although this study was not randomized, the observed RD in the rifampicin group suggests a suppressive activity upon regrowth of residual disease in acute myelogenous leukaemia during apparent complete remission.