Preleukemic State Research Articles

Prognostic Importance of Mutations in the ras Proto-Oncogenes in De Novo Acute Myeloid Leukemia

AbstractMutations of the N- and K-ras genes are the most frequent genetic aberrations in acute myeloid leukemia (AML) and their detection in preleukemic conditions such as the my-elodysplastic syndrome (MDS) suggests a role in the earliest phases of leukemogenesis. Despite these observations, little is known about the clinical importance of ras mutations in AML. We studied the clinical impact of ras mutations in 99 patients with de novo AML. All patients were treated in two prospective multicenter trials. The polymerase chain reaction was used to amplify areas surrounding the codons 12, 13, and 61 of the three ras genes N-, K-, and H-ras from DNA from bone marrow cells, ras mutations were detected by an algorithm based on allele-specific oligonucleotide hybridization. Eighteen of 99 (18%) patients harbored mutations in either N- or K-ras. All of the observed mutations occurred in N-ras (N = 10) and K-ras (N = 5) or concurrently in both N-and K-ras (N = 3). There were no significant differences between ras-negative and ras-positive patients according to age, sex, blood counts, cytogenetic abnormalities, or French-American-British classification. However, univariate analysis suggested a longer survival in ras-positive patients (P = .11). When adjusted for age, which was the most important factor affecting outcome, the presence of a ras mutation emerged as a significant predictor for improved survival (P = .03) along with lower bone marrow blast counts (P = .02) and better cytogenetic category (P = .01). However, the presence of an aberrant ras allele was strongly correlated with lower bone marrow blast counts (P = .007). Thus, whether a mutation in the N-ras or K-ras proto-oncogenes directly affects treatment outcome or indirectly through an association with lower leukemic burden remains to be determined. Nevertheless, these findings counter the prevailing bias that oncogene mutations lead to more aggressive behavior in human malignancies.

Prognostic importance of mutations in the ras proto-oncogenes in de novo acute myeloid leukemia

Mutations of the N- and K-ras genes are the most frequent genetic aberrations in acute myeloid leukemia (AML) and their detection in preleukemic conditions such as the myelodysplastic syndrome (MDS) suggests a role in the earliest phases of leukemogenesis. Despite these observations, little is known about the clinical importance of ras mutations in AML. We studied the clinical impact of ras mutations in 99 patients with de novo AML. All patients were treated in two prospective multicenter trials. The polymerase chain reaction was used to amplify areas surrounding the codons 12, 13, and 61 of the three ras genes N-, K-, and H-ras from DNA from bone marrow cells, ras mutations were detected by an algorithm based on allele-specific oligonucleotide hybridization. Eighteen of 99 (18%) patients harbored mutations in either N- or K-ras. All of the observed mutations occurred in N-ras (N = 10) and K-ras (N = 5) or concurrently in both N- and K-ras (N = 3). There were no significant differences between ras-negative and ras- positive patients according to age, sex, blood counts, cytogenetic abnormalities, or French-American-British classification. However, univariate analysis suggested a longer survival in ras-positive patients (P = .11). When adjusted for age, which was the most important factor affecting outcome, the presence of a ras mutation emerged as a significant predictor for improved survival (P = .03) and along with lower bone marrow blast counts (P = .02) and better cytogenetic category (P = .01). However, the presence of an aberrant ras allele was strongly correlated with lower bone marrow blast counts (P = .007). Thus, whether a mutation in the N-ras or K-ras proto-oncogenes directly affects treatment outcome or indirectly through an association with lower leukemic burden remains to be determined. Nevertheless, these findings counter the prevailing bias that oncogene mutations lead to more aggressive behavior in human malignancies.

Clonal remissions in acute myeloid leukaemia are commonly associated with features of trilineage myelodysplasia during remission

Clonal haemopoiesis has previously been demonstrated in some 30% of patients in remission of acute myeloid leukaemia (AML). Whilst a 'clonal remission' in many such patients may represent a skewed X-chromosome inactivation pattern in haemopoietic cells, its relationship to an underlying preleukaemic state remains uncertain. We therefore analysed the clonal status of 48 female patients in remission of AML using X-chromosome linked restriction fragment length polymorphisms (RFLPs) within the X-linked PGK and HPRT genes and the DXS255 (M27 beta) locus, and carried out in conjunction a detailed study of the morphological and karyotypic features of the patients' bone marrows. During remission, 35 patients (73%) with AML demonstrated nonclonal haemopoiesis, and their bone marrows were morphologically normal. Remission haemopoietic tissue in nine cases (19%) showed a skewed X-chromosome inactivation pattern and remission bone marrows in these patients had features of trilineage myelodysplasia (TMDS), with seven having similar features at presentation. Analysis of constitutional DNA showed a non-clonal pattern in seven of these patients, but was unsuccessful in two cases. These nine patients with post-chemotherapy TMDS were considered to have true clonal haemopoiesis. Four patients (8%) with a skewed X-chromosome inactivation pattern had normal remission bone marrows. Analysis of constitutional DNA showed a skewed pattern in two of these patients, but was unsuccessful in two cases. Cytogenetic investigation during remission in the nine patients with TMDS showed a normal karyotype in four cases and the acquisition of new karyotypic abnormalities in three cases. In contrast, 10 female patients in remission of de novo acute lymphoblastic leukaemia (ALL) were shown to have non-clonal haemopoiesis. We conclude that the majority of patients with AML who achieve remission after cytoreductive chemotherapy have non-clonal haemopoiesis, and when clonal remissions are observed these are commonly associated with the development of trilineage myelodysplasia in the bone marrow, with or without karyotypic abnormalities. True clonal remission in association with morphologically normal haemopoiesis is a rare entity, the significance and frequency of which remain uncertain.

Detection of p53 mutations using nonradioactive SSCP analysis: p53 is not frequently mutated in myelodysplastic syndromes (MDS).

p53 is one of the most frequently mutated genes in human cancers. Since p53 has been implicated in lymphatic and some myeloid leukemias, such as the blastic phase of chronic myelogenous leukemia, we sought to address the role of p53 gene mutations within exons 4-9 in myelodysplastic syndromes (MDS), a myeloid preleukemic condition. In order to avoid the potential hazard of using radioactive single-strand conformation analysis (SSCP), we used a nonradioactive SSCP method based on the silver stain of small minigels. In cell lines with known point mutations of the p53 gene, aberrant migrating bands were found. Serial dilutions indicated a sensitivity comparable to radioactive methods. Furthermore, a common polymorphism within the 4th exon of the p53 gene was easily detected. However, of 17 primary samples from patients with MDS, none harbored a p53 gene mutation. We conclude that this nonradioactive method can easily be used to screen for p53-gene mutations, and that p53-gene mutations do not play a major role in the pathogenesis of MDS.

Frequency of clonal remission in acute myeloid leukaemia

Analysis of X-chromosome inactivation patterns in females has been used to assess clonality of various tumours and for prenatal diagnosis of X-linked disorders. Studies with these methods in acute myeloid leukaemia suggest that a significant proportion of cases have clonal remissions (ie, persistence of the malignant clone), which may represent return to a preleukaemic state. We therefore analysed X-chromosome inactivation patterns with differential methylation patterns of heterozygotes for three DNA probes, HPRT, PGK, and M27β, in leukaemic patients and normal controls. As expected, blast cells from 67 of 68 analysable samples (99%) were monoclonal or had a skewed X-inactivation pattern. A skewed pattern in remission was also found in 26 of 77 patients (34%), proportion only slightly greater than control (16/75, 21%). In 7 of 10 patients with a skewed pattern in myeloid cells there was similar skewing in the T cells, which is compatible with the concept of a constitutively skewed X-chromosome inactivation pattern of haemopoietic cells in these patients. Our study illustrates the difficulty of interpreting clonality in individual tumour samples and emphasises the importance of comparisons with non-malignant tissue of the same cell type from that individual and from normal control populations.

Karyotypic Evolution in a Patient with a Preleukemic State of Adult T-cell Leukemia (Pre-ATL) progressing to Overt ATL

Karyotypic evolution in a 56-year-old male with a preleukemic state of adult T-cell leukemia (pre-ATL) that progressed to overt ATL is described. The patient was born in the Kagoshima district, an endemic ATL area in Japan. A clone with inv(14)(q11q32) which was not detected in a pre-ATL state appeared during the early phase of overt ATL. These findings suggest that inv(14)(q11q32) may have played an important role in leukemogenesis in the present case. Based on our cytogenetic data obtained from both the pre-ATL and overt ATL phases, a model of the leukemogenic process of ATL is proposed.

Myelodysplastic syndrome associated with bone marrow fibrosis.

Bone marrow biopsy (BMB) in myelodysplastic syndrome (MDS) frequently reveals a slight alteration in the reticulin stroma which does not have any clinical significance. However, in a minority of cases, full-blown bone marrow fibrosis (BMF) can be found. Primary MDS patients with BMF show distinct clinico-pathological features and an unfavourable prognosis mainly attributable to complications deriving from pancytopenia and continuous transfusions, while leukemic transformation occurs only rarely. Since BMF may characterize other hematological disorders, primary MDS with BMF should be included in the differential diagnosis particularly with malignant myelofibrosis (MM) and idiopathic myelofibrosis (IMF). Secondary MDS with BMF represent a variety of preleukemic conditions in subjects treated for previous neoplasias. Unlike the primary forms, they do not form a clearcut clinico-pathological entity.

Bone marrow depletion by 89Sr complements a preleukemic defect in a long terminal repeat variant of Moloney murine leukemia virus.

We previously described a preleukemic state induced by Moloney murine leukemia virus (Mo-MuLV) characterized by hematopoietic hyperplasia in the spleen. Further experiments suggested that splenic hyperplasia results from inhibitory effects in the bone marrow, leading to compensatory extramedullary hematopoiesis. An enhancer variant of Mo-MuLV, Mo + PyF101 Mo-MuLV, fails to induce preleukemic hyperplasia and has greatly reduced leukemogenicity, indicating the importance of this state to efficient leukemogenesis. An alternative method for induction of preleukemic hyperplasia was sought. Treatment of mice with 89Sr causes specific ablation of bone marrow hematopoiesis and compensatory extramedullary hematopoiesis in spleen and nodes. NIH Swiss mice were inoculated neonatally with Mo + PyF101 Mo-MuLV and treated with 89Sr at 6 weeks of age. Approximately 85% developed lymphoid leukemia with a time course resembling that caused by wild-type Mo-MuLV. In contrast, very few animals treated with Mo + PyF101 Mo-MuLV or 89Sr alone developed disease. In approximately one-third of cases, the Mo + PyF101 Mo-MuLV proviruses were found at common sites for wild-type Mo-MuLV-induced tumors (c-myc, pvt-1, and pim-1), indicating that this virus is capable of performing insertional activation in T-lymphoid cells. These results support the proposal that splenic hyperplasia results from inhibitory effects in the bone marrow. They also indicate that Mo + PyF101 Mo-MuLV is blocked in early and not late events in leukemogenesis.

Concepts of remission, curability and lineage involvement in relationship to the problems of minimal residual leukaemia

Minimal residual leukaemia (MRL) is due to chemotherapeutic failure. Chemoincurability in chronic myeloid leukaemia and in myelodysplastic syndrome is the norm and is the result of tumour defect arising within the marrow stem cell compartment. We propose that this is indeed the state of affairs in the majority of adult acute leukaemias and such tumours derived from stem cells are chemoincurable. The proportion of acute leukaemias which belong to this category can only be cured by allogeneic bone marrow transplantation though conventional chemotherapy and autotransplant may result in prolonged periods of remission and a return to a preleukaemic state in some patients. A proportion of the acute leukaemias occurring predominantly in children are presently curable by chemotherapy. It is hypothesized that these chemocurable leukaemias derive from the compartment of haemopoietic progenitors already irreversibly committed to a single lineage. Some recent studies using markers of the leukaemic clone to determine the origin of in vitro myeloid colony forming cells support this concept. Intrinsic and/or acquired genetic chemoresistance represents a supplementary restriction to the chemocurability of acute leukaemias. New methods of detecting MRL are sensitive enough to detect up to one leukaemic cell in 10(6) bone marrow mononuclear cells. However, it is possible that even such sensitive techniques will not be sufficient to determine whether patients in complete remission continue to harbour leukaemic cells in the stem cell compartment when the marker of interest might not be expressed.(ABSTRACT TRUNCATED AT 250 WORDS)

Exposure histories in acute nonlymphocytic leukemia patients with a prior preleukemic condition

A subset of patients with acute nonlymphocytic leukemia (ANLL) have evidence of a myelodysplastic syndrome (MDS), low infiltrate leukemia, or other preleukemic condition that may be present for several months before onset of disease. The hypothesis that these conditions act as markers for environmentally induced cancer was examined in 270 ANLL patients, 46 with a preleukemic phase and 224 with an acute onset. Although the effects of previously identified risk factors (male sex, age older than 50 years, prior cytotoxic therapy) were demonstrated, no associations with common environmental conditions (cigarette smoking, alcohol use, occupations with exposure to chemicals or radiation) were present with the exception of hobbies involving potential chemical exposure, odds ratio (OR) and 95% confidence intervals = 4.2 (1.4 to 12.3) and self-reported exposure to pesticides, OR = 10.2 (1.8 to 63.1). These may be chance associations although a previous case-control study of MDS reported similar findings.

Decreased ABH blood group antigen expression associated with preleukemic conditions and acute leukemia: loss of detectable B, then A antigens in a group AB patient progressing from a myelodysplastic syndrome to leukemia

Abstract Decreased or absent expression of blood group antigens is well known to occur in acute leukemia. In some carcinomas, the malignant solid tumor cells have also been shown to lose normal blood group antigen expression. In both carcinoma and hematologic malignancies, these findings have been associated with a more aggressive behavior of the neoplasm. A 34-year-old, group AB, Rh positive woman was diagnosed with a preleukemic condition, myelodysplastic syndrome, in December 1988. In April 1989 B antigen expression was markedly decreased, then absent; in November 1989 the patient had progressed to acute leukemia. In April 1990 neither A nor B antigens could be detected, there was disease progression, and the patient died 19 days after A and B antigens were undetectable. Decreased expression of normal blood group antigens may prove useful in differentiating benign from malignant conditions and may indicate a more grave prognosis. Immunohematology 1991;7:89–93.

Cytogenetic implication in adult T-cell leukemia

The close association between adult T-cell leukemia (ATL) and human T-cell leukemia virus type I (HTLV-I) has been established. Nevertheless, the mechanism of progression of ATL by HTLV-I infection is still uncertain, because the virus contains no typical oncogene and no significant expression of the viral RNA has been generally found. I propose a model of leukemogenic process in ATL based on our cytogenetic data and molecular results in the literature. It seems that the rearrangement of some proto-oncogene and alpha-chain gene of the T-cell antigen receptor (TCR-alpha) is necessary for the development to overt ATL. A deficiency in the rearrangement of proto-oncogene to TCR-alpha may result in only a minor proliferation of abnormal lymphocytes and remain in the preleukemic state of ATL or in the HTLV-I carrier state.

Cytogenetic studies of human T-cell leukemia virus type I carriers: A family study

Recently, the chromosome 14q11 anomaly has been reported to be specific to adult T-cell leukemia (ATL), and this anomaly has also been confirmed in the preleukemic state of adult T-cell leukemia (pre-ATL) patients. Because the cytogenetic abnormality at the stage of human T-cell leukemia virus type I (HTLV-I) carrier remains uncertain, we performed cytogenetic studies of lymphocytes stimulated with phytohemagglutinin in three HTLV-I carriers and three non-HTLV-I carriers in an ATL family. As a result, in three HTLV-I carriers, four of 311 cells examined (1.3%) had chromosome 14q11 anomaly. However, in three non-HTLV-I carriers, none of 260 cells examined had chromosome 14q11 anomaly. These results suggest that chromosome 14q11 anomaly is already present at the stage of HTLV-I carrier and seems to be an important cytogenetic clue to the pathogenesis of ATL.

Preleukemic hematopoietic hyperplasia induced by Moloney murine leukemia virus is an indirect consequence of viral infection

We previously showed that neonatal mice inoculated with Moloney murine leukemia virus (M-MuLV) exhibit a preleukemic state characterized by splenomegaly and increased numbers of hematopoietic progenitors. An M-MuLV variant with greatly reduced leukemogenic potential, Mo+PyF101 M-MuLV, does not generally induce this preleukemic state. In order to investigate the mechanism involved in M-MuLV induction of preleukemic hyperplasia, we tested the CFU-mixed myeloid and erythroid (CFUmix) from M-MuLV- and Mo+PyF101 M-MuLV-inoculated mice for the presence of virus by antibody staining and for the release of infectious virus. The majority of CFUmix colonies from both M-MuLV- and Mo+PyF101 M-MuLV-inoculated mice contained infectious virus even though M-MuLV-inoculated mice showed elevated levels of CFUmix while the Mo+PyF101 M-MuLV-inoculated mice did not. This indicates that direct infection of hematopoietic progenitors was not sufficient to induce hyperplasia. Rather, hematopoietic hyperplasia may result indirectly from infection of some other cell type.

Combined infection by Moloney murine leukemia virus and a mink cell focus-forming virus recombinant induces cytopathic effects in fibroblasts or in long-term bone marrow cultures from preleukemic mice

We described previously a preleukemic state in mice inoculated with Moloney murine leukemia virus (M-MuLV) characterized by generalized hematopoietic hyperplasia in the spleen. To investigate this further, long-term bone marrow cultures (LTBMC) from preleukemic mice were established. Surprisingly, LTBMC from M-MuLV-inoculated preleukemic mice showed less hematopoiesis than LTBMC from control mice. This resulted from a quantitative defect in establishment of bone marrow stromal cells in the LTBMC. This phenomenon could also be observed in LTBMC from normal mice infected in vitro with a stock of M-MuLV containing a mink cell focus-forming virus (MCF) derivative (M-MCF), but not in LTBMC infected with M-MuLV alone. This implicated MCF derivatives in the reduction in bone marrow stromal cells. The phenomenon could also be detected in infected NIH 3T3 cells. Combined infection of M-MuLV plus M-MCF resulted in fewer cells, in comparison to uninfected cells or cells infected with either virus alone. Further studies indicated that this was predominantly due to an inhibition in cell growth rather than to cell lysis. The cytopathic effect did not appear to result from overreplication of viral DNA, as measured by Southern blots. Thus, combined infection with M-MuLV and an MCF derivative had cytostatic effects on cell growth. This phenomenon might also contribute to the leukemogenic process in vivo.

口腔内に初発症状の発現をみたＲｅｆｒａｃｔｏｒｙ ａｎｅｍｉａ ｗｉｔｈ ｅｘｃｅｓｓ ｏｆ ｂｌａｓｔｓ（ＲＡＥＢ）の１例

According to the FAB classification, refractory anemia with excess of blasts (RAEB) is a subtype of myelodysplastic syndrome (MDS) and a preleukemic condition that is often transformed into acute myelogenous leukemia (AML).We encountered a patient with RAEB who complained of oral symptoms. The patient, a 52-year-old male, visited our hospital due to swelling of the lower jaw on the right side. After treatment such as tooth extraction and incision, poor healing of the extraction cavity was observed. Based on the results of close examination, a diagnosis of RAEB was made. The condition progressed to AML after 9 months.It seems to be important that oral symptoms were the initial symptoms that led to the diagnosis of RAEB in this patient.

Impaired platelet haemostasis in patients with myelodysplastic syndrome

One third of patients with myelodysplastic syndrome die before the development of leukemia from infectious and hemorrhagic complications; after the onset of acute myeloleukemia this figure reaches 59%. Therefore, the relevance of studying hemostatic disorders in preleukemic conditions is dictated primarily by the need to preserve the lives of patients. Literature data indicate the absence of significant changes in the plasma-coagulation component of hemostasis and fibrinolytic processes. At the same time, hematopoietic dysfunction in myelodysplastic syndrome necessarily includes megakaryocytic bone marrow, which cannot but affect the functional state of the platelet apparatus, the assessment of which was the main task of this work.

Detection of fos oncogene products by monoclonal antibody FO-120 in lymphoproliferative disorders

We investigated the expression of fos oncogene proteins in lymphoproliferative disorders, using a monoclonal antibody (FO-120) that was prepared against a synthetic oligopeptide of fos protein (amino acid sequence from 127 to 152). Although peripheral blood leukocytes were rarely positive for FO-120, they were transiently stained after lectin (PHA) stimulation. After culture with IL-2 for 1 or 2 weeks, less than 40% of the lymphocytes weakly reacted with FO-120, whereas strongly positive cells were detected in more than 70% of cells in half the T-cell lines established from preleukemic state of adult T-cell leukemia (pre-ATL) and all of ATL derived T-cell lines. All in vivo specimens of non-Hodgkin's malignant lymphomas, except for one case of T-cell lymphoma were also strongly positive. In addition, the extent of the antibody reactivity correlated with the histopathological grade of malignancy in B-cell lymphoma. The reactivity to most of AILD-IBL lesions overlapped with that to T-lymphomas, and could be distinguished from that to reactive lesions. FO-120 appears to be a useful tool for detecting early neoplastic changes in lymphoproliferative disorders.

A hypothesis regarding the development of acute myeloid leukemia from preleukemic disorders: The role of protooncogenes

Acute nonlymphocytic leukemia is often preceded by a preleukemic phase that can be characterized by a reduction in hematopoiesis, by ineffective hematopoiesis, or by a myeloproliferative/myelodysplastic state. The time between onset of the preleukemic state and the appearance of leukemia is quite variable, and at times such evolution does not occur. These clinical observations are compatible with the multistep hypothesis of tumor development [1]. In studies underway in our laboratory, chronic myelogenous leukemia (CML) is being used as a model system to study the steps in and possible mechanisms underlying the development of preleukemia and its evolution to acute leukemia. Chronic myelogenous leukemia is particularly suited for this role because the chronic phase of the disease is an easily identifiable myeloproliferative state that invariably evolves into acute leukemia. In the discussion that follows, this clinical entity is used to develop a general model for the preleukemias and their evolution to acute leukemia.

The oncogenicity of chlorambucil in rheumatoid arthritis.

Chlorambucil is useful in patients with rheumatoid arthritis (RA) refractory to other agents but there is concern about the risk of haematological malignancy with this agent. A retrospective survey was performed to assess the incidence of all types of malignancy in 39 patients treated with chlorambucil (mean daily dose 4.25 mg, mean duration of treatment 25 months). These patients were compared with 30 patients with RA who received contemporaneously, the purine analogues azathioprine or 6-mercaptopurine (mean dose 100 mg, mean duration of treatment 24 months). Eight patients treated with chlorambucil and one patient receiving purine analogues developed cutaneous malignancy (p = 0.03). In the chlorambucil-treated patients these were mostly multiple and recurrent. Three patients treated with chlorambucil developed myeloid leukaemia or a preleukaemic state, whilst no patient treated with purine analogues developed this complication. The use of chlorambucil in RA is associated with an increased risk of cutaneous as well as haematological oncogenesis.