The biological mechanisms responsible for the association of specific karyotypes with prognosis in acute myeloid leukaemia (AML) remain largely unclear. A prospective study was performed to evaluate how far cytogenetically defined prognostic subgroups of AML differ in their proliferative activity as a potential mechanism for differential sensitivities to S-phase-specific induction chemotherapy comprising cytosine arabinoside (AraC). One hundred and eighty-seven patients with de novo AML were included in the study; 25 patients with a favourable [inv(16), t(8;21), t(15;17)] karyotype, 99 with a normal karyotype, 29 with an unfavourable karyotype (-5, 5q-, -7, 7q-, complex aberrations) and 34 with cytogenetic aberrations of unknown prognostic significance (all others). The favourable group demonstrated the highest ex vivo proliferative activity (PA) (3.41 pmol/10(5) cells), significantly (P = 0.02) exceeding the unfavourable group with the lowest PA (0.72) and the group with a normal karyotype (1.06) or with karyotype of unknown significance (1.05) that both demonstrated an intermediate PA. Samples with a high PA (> median of the whole group) were more likely to produce interleukin 3, granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte CSF (G-CSF) (56%, 43% and 50%) than cells with a low PA (33%, 36% and 36%; n.s.), The effect of priming by exogenous GM-CSF or G-CSF was significantly more pronounced in samples with a low PA than in rapidly proliferating samples (P < 0.01). For the whole group, a high PA was closely associated with an increased incorporation of AraC triphosphate (AraCTP) into the DNA (P < 0.0001). Clinically, a high PA was associated with a better complete remission (CR) rate in the normal (95% versus 62%) and the unfavourable group (75% versus 33%). The significant differences in proliferative activity between cytogenetic subgroups of AML are associated with increased cytosine arabinoside pharmacodynamics and constitute one potential mechanism for the different response of cytogenetic subgroups to AraC-based induction therapy.
Continuous leukemia–lymphoma cell lines are important research tools, in particular as starting material for the cloning of recurrent translocations. In 1998, we established the continuous leukemia cell line MUTZ-5 and its two simultaneous sister cell lines MUTZ-6 and MUTZ-7. The primary specimen was obtained from the peripheral blood of a 26-year-old man with B cell precursor acute lymphoblastic leukemia at relapse carrying a t(12;13). The immunoprofile of MUTZ-5 corresponds to that of a precursor B cell. The immunoglobulin heavy chain gene was found to be rearranged. Despite receptor expression, none of the cytokines examined enhanced proliferation; several cytokines had significant inhibitory effects. Giemsa-banding cytogenetics showed the following karyotype which was identical in all three sister cell lines: 45<2n>X, −Y, t(12;13)(p12;q13–14). The karyotype and DNA fingerprinting confirmed the malignant nature and the authenticity of the cell line, excluding cross-contamination with other cells. MUTZ-5 represents a new unique leukemia B cell line; its scientific significance lies in the t(12;13).
Philadelphia chromosome-positive (Ph+) acute leukaemia usually shows lymphoblastic morphology and a B-precursor phenotype. The bone marrow aspirate of a 9-year-old boy showed a L3 blast cell morphology in 90% of cells; immunophenotyping revealed a mature B-blast population. The translocation t(9;22) (q34;q11) was seen in 45 out of 50 metaphases, and expression of the corresponding bcr1/abl fusion transcripts, but no IgH/myc co-localization or splitting of c-myc, was demonstrated. Chemotherapy according to the Berlin-Frankfurt-Munster non-Hodgkin's lymphoma (NHL-BFM 95) protocol with maintenance according to the BFM acute lymphoblastic leukaemia (ALL-BFM 90) protocol resulted in continuing complete remission of 54 months. The occurrence of Ph+ Burkitt's leukaemia might reflect multiple-step cancer development.
Refractory or advanced myeloid leukemias have a poor prognosis. Allogeneic stem cell transplantation (alloSCT), the only therapeutic option which has the potential of inducing long term remissions, is associated with a 30–40% transplant related mortality (TRM), if conventional, myeloablative conditioning regimens are used. Achieving engraftment with fludarabine-based reduced intensity conditioning regimens may decrease toxicity and improve results. We therefore developed a tandem strategy combining a cytoreductive intensive chemotherapy with upfront reduced intensity conditioning for alloSCT. Thirty-three patients received fludarabine 4 x 30 mg/m2, AraC 4 x 2 g/m2, and Amsacrine 4 x 100 mg/m2. After a three day pause they received 4Gy TBI followed by Cytoxan 2 x 40 mg/kg or 2 x 60 mg/kg (urd) and ATG 3 x 10 mg/kg or 3 x 20 mg/kg (urd). Six patients had secondary AML after MDS, 5 of them with unfavorable karyotypes, one patient had RAEB-t, fifteen patients had primarily or relapsed refractory disease, one patient was in 2. CR after an autotransplant, three patients had de novo AML with unfavorable karyotypes in 1. CR, 2 patients had CML in second chronic phase and five patients had CML in blast crisis. Twelve patients received grafts from HLA-identical siblings and 21 from matched unrelated donors. Median patient age was 49 with a range from 19–61 years. Overall survival was 43% at a median follow-up time of 164 days (range 30 to 472 days). All patients surviving more than 30 days engrafted and all but one patient with CML blast crisis achieved a CR. Compared to a historical control, TRM could be reduced from 38% to 15% at day + 100. Five patients relapsed at d +48, d +57, d +66, d +70 and d +106 respectively. One of these patients refused further treatment and died from leukemia. Another patient was treated with intensive chemotherapy followed by GM-CSF and a second peripheral stem cell transplantation without prior conditioning and achieved again a CR. However, later she developed an extramedullary CNS relapse from which she finally died. The third relapsed patient had an extramedullary relapse in the skin. She was treated identical and developed acute GVHD with the skin nodules regressing. The remaining two patients were still on immunosuppression, which then was discontinued. One of these two went into CR after discontinuation of immunosuppression while the other progressed and is now receiving low-dose Ara-C. Causes of death were recurrent or persistent leukemia in 3 patients, acute GVHD in one patient and infection in 4 patients. One patient died from pulmonary bleeding as a complication of a diagnostic procedure. In summary, treatment with HD-AraC, fludarabine and amsacrine followed by alloSCT with reduced intensity conditioning is safe and has the potential of inducing sustained remissions in high risk myeloid leukemias.
Abnormalities in chromosomes of malignant cells comprise one of the independent prognostic factors in acute lymphoblastic leukemia in children. Depending on the type of the abnormality, the patients are assigned to the different risk groups [1,3,13]. Studies on isoenzymes and immunological markers provide valuable information on aetiology of ALL; however, the mystery of leukemic transformation or persistent malignancy are not yet cleared. Classification of ALL according to cytogenetic features, both modal and structural, turned out to be more promising approach [9]. The presence of specific cytogenetic abnormalities within leukemic cells prompted molecular studies on the breaking points within the genes and their protein products [15].
Allogeneic stem cell transplantation involves infusion of hematopoietic stem cells from an HLA-matched donor together with T lymphocytes from the same donor. These donor T lymphocytes facilitate engraftment and may remove residual leukemic cells, which have survived the conditioning treatment [1]. This Graft-versus-leukemia (GvL) effect is often accompanied by graft-versus-host disease (GvHD) which may be life-threatening. Several approaches are being pursued in order to minimize GvHD while at the same time preserving or enhancing the GvL effect. Suicide gene therapy constitutes one of these approaches [2–8]. Prior to infusion into the patient, donor lymphocytes are genetically modified to express the Herpes Simplex Virus Thymidine Kinase (HSV-TK) gene. This allows specific elimination of those cells in vivo with ganciclovir (GCV), when GvHD symptoms become too severe.
The main purpose of treatment of acute leukaemia is a selection of patients who are potentially curable by appropriate therapy. Relapsed acute leukaemia still remains often an incurable disease. However there is a late relapse group within AML patients (pts) whose prognosis is not so bad. Idarubicin, antileukaemic drug, was investigated in late 1970′s with phase 1 clinical trial in 1980 (Casazza et al. 1980). Later its activity in relapsed and refractory acute leukaemia was found (Kaplan et al. 1982). We report herein the results of the treatment of primary resistant and early and late relapsed AML with salvage regimen containing Idarubicin.
AMLl is the most frequent target for chromosomal translocations in acute leukemia. AML-1 binds the canonical DNA sequence TGT/cGGT in concert with core binding factor ß to activate or repress transcription. The chromosomal translocation fusion proteins that disrupt this heterodimeric transcription factor create constitutive repressors. Our work has focused on defining the molecular mechanism of transcriptional regulation by these cancer-associated fusion proteins. We have found that the t(8;21) fusion protein makes multiple contacts with the mSin3A and N- CoR co-repressors. In addition, the fusion protein also contacts histone deacetylases 1, 2, and 3 independent of the co-repressors. The t(12;21) fusion protein also interacts with mSin3A and N-CoR, but appears to interact only with histone deaceylase 3. By contrast, the inv(16) fusion protein appears to act as an AML-1 co-repressor and makes indirect associations with co-repressors and histone deacetylases. Therefore, the use of histone deacetylases may be a common feature of these translocations fusion proteins and may provide an opportunity for therapeutic intervention.
In this study we examined the expression pattern of monocytic acute myeloid leukemia (AML) blasts in comparison to monocytes from healthy donors using a cDNA array technology to identify differentially expressed genes. CD 14 positive cells were sorted from the peripheral blood of patients with a monocytic AML (FAB M5) using MACS microbeads. Buffy coats obtained from healthy blood donors served as controls. The purity of enriched CD 14 positive cells was assessed by flow cytometry and was constantly above 97%. RNA was isolated, radiolabeled cDNA was generated and subsequently hybridyzed to an Atlas™ Human Cancer cDNA Expression Array. This expression profile consists of 588 selected genes thought to play important roles in tumoroge- nesis. Comparing the expression profiles of monocytic AML cells and normal monocytes, we identified a number of differentially expressed genes, including Stat 1, interferon regulatory factor 1, and AXL (tyrosine-pro- tein kinase receptor) which were found to be overexpressed in malignant blasts. Other genes such as cyklin-dependent kinase inhibitor l(p21) and integrin beta 8 were markedly downregulated compared to normal monocytes. Our study demonstrates the power of sophisticated cell separation techniques in combination with cDNA expression arrays as a feasible screening technique to generate detailed expression profiles of well defined cell populations and to identify differentially expressed genes.
The present development, replacing the term “cytogenetics (and genomics)” with “cytogenomics” is reviewed and the necessity to do this is discussed. Also, the word “cytogenomics” is defined as including, besides methods such as banding cytogenetics or genomics, also many more fields like the DNA, its structure and content (including hetero and euchromatin) with epigenetic changes, but also repetitive elements and topologically associating domain, interphase architecture, and many more. Certainly, technical approaches to studying genomes are part of the cytogenomics field. Overall, the term cytogenomics is intended to be integrative and includes the vision to create novel concepts in biology and medicine.
The prognosis for patients with acute myelogenous leukemia (AML) has not improved substantially in more than a decade. Regimens based on an anthracycline (e.g. daunorubicin) and cytarabine, induce complete remissions in 60-80% of patients under the age of 55 years with a projected long-term disease free survival of 25-50%; the figures for older patients are 25-30% and <15%, respectively. Higher doses with stem cell support have not been associated with major benefit [9]. Clearly new approaches are needed to improve the outlook for these patients
Chronic myelogenous leukemia (CML) is characterized by a reciprocal translocation between long arms of chromosomes 9 and 22 that produces the Philadelphia chromosome. The resulting chimeric BCR-ABL fusion gene is thought to be the molecular cause of the disease. Typical symptom is the marked expansion of myeloid cells that mature normally. The natural history of CML is progression from chronic phase to a rapidly fatal blast crisis within three to five years. Blast crisis is often preceded by an accelerated phase in which increasing doses of initial therapy are required. Treatment options have become more complex in recent years, allogeneic stem cell transplantation is still the only curative option. Recent data show that interferonalpha prolongs life in CML patients, as compared to chemotherapy. New therapies based on the causative molecular abnormality of CML are showing great promise. Imatinib (formerly STI571; Glivec) is an orally administered selective inhibitor of the BCR-ABL tyrosine kinase. Preliminary results from three international phase 11 studies are promising with good hematological and cytogenetical response rates and manageable side effects. Combination with cytostatic drugs may help to overcome the problem of secondary resistance in advanced disease. Longterm observation is required to demonstrate whether imatinib will prolong life in comparison with other methods of treatment.
The combination of chemotherapy with all-trans retinoic acid (ATRA) improved prognosis of acute promyelocytic leukemia (APL) when compared to treatment concepts with chemotherapy alone before the availability of ATRA. Using ATRA as single agent complete hematological remission (CR) rates over 90% were achieved. However, cure was only possible, if ATRA was combined with a potent chemotherapy. These observations suggest that the antileukemic effectiveness of the chemotherapy is of critical importance for cure. Therefore, it was the concept of the present study of the AMLCG for patients with newly diagnosed APL to integrate the positive effects of the differentiating substance, ATRA, into a chemotherapy strategy of proven high curative potential.
The t(15;17) translocation is the cytogenetic hallmark of acute promyelocytic leukemia (APL). This reciprocal translocation results in two chimeric fusion genes PML/RARα and RARα/PML. In order to assess the minimal residual disease, RT-PCR methods have been developed to detect mRNA transcripts of the PML/RARα fusion gene. The sensitivity of these methods is about 10-4. Patients who test PCR positive by this method after treatment are likely to relapse. However, a fraction of patients are at risk to relapse, in spite of PCR negativity. In order to identify these patients we developed a RT-PCR method for the detection of the RARα/PML fusion gene with a sensitivity of 10-6. This transcript can be detected in over 70% of APL patients. We compared the results of this more sensitive RT-PCR with the results of the previously established RT-PCR for the PML/RARα fusion gene and the outcome of the patients. 254 bone marrow and/or peripheral blood samples of 54 patients younger than 60 years with newly diagnosed APL and under follow-up were investigated. The follow-up time of the individual patients was 1 to 47 months (median 13 months). In all patients PML/ RARa transcripts could be detected at diagnosis, whereas the RARα/PML transcripts were detected in 48 patients (89%). Three out of the 48 patients relapsed hematologically (6.3%). In all three patients, the RARα/PML transcripts persisted or could be detected earlier than the PML/RARα transcripts indicating the clinical value of double monitoring. The longer persistance of RARα/PML in five patients after induction therapy demonstrates individual differences in the depletion of the malignant clone which may have prognostic significance. We conclude that RT-PCR of the reciprocal transcript RARα/PML can contribute to earlier identification of patients at high risk of relapse.
Previous preclinical and clinical work with 8chloro-cAMP (8-Cl-cAMP) has raised questions whether it works as a cAMP analog or as a nucleoside analog, 8-Cl-Adenosine (Ado). Although degradation of 8-Cl-cAMP to 8-ClAdo in culture medium or plasma has been shown, cellular pharmacology is missing. Additionally, conversion of 8-Cl-Ado to 8-ClcAMP in the cell and its effects via a Protein Kinase A (PKA) dependent pathway is possible. However, there was no measurable increase in PKA activity by 8-Cl-Ado. The phorbol ester PMA, which blocks both glucocorticoid and cAMP mediated cell death, was not able to block 8-Cl-Ado mediated apoptosis. These data suggested that the mechanism of action of 8-Cl-Ado is via a separate pathway than either glucocorticoid or PKA signaling. In multiple myeloma (MM) and leukemia cells, both 8-Cl-cAMP and 8-Cl-Ado incubation resulted in the accumulation of 8Cl-Ado mono-, di-, and tri-phosphate (8-ClATP). Accumulation of 8-Cl-ATP was dependent on both the exogenous concentration of 8-Cl-Ado and incubation time. At the 10 μM level of 8-Cl-Ado, more than 400 pM 8-ClATP accumulated in MM cells after 12 hours. Similar incubation with 8-Cl-cAMP also resulted in accumulation of 8-Cl-ATP, albeit at a lower level. The formation of 8-Cl-ATP from 8-Cl-cAMP was inhibited by more that 80% in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine in the medium, suggesting extracellular conversion of 8-Cl-cAMP to 8-Cl-Ado. Parallel to accumulation of 8-Cl-ATP, there was a decline in the endogenous level of cellular ATP pool. This was associated with inhibition of RNA synthesis but did not affect DNA synthesis. Among the RNA species, there was maximum inhibition of mRNA, which was consistent with highest incorporation of 8-Cl-Ado residues. Taken together, these data demonstrate that 8-Cl-cAMP serves as a prodrug for 8-ClAdo. Because 8-Cl-Ado induces apoptosis in MM and leukemia cells resistant to traditional chemotherapeutic drugs, this analog may be a potential new agent for these diseases.
The optimal post remission treatment of acute myeloid leukemia(AML) in patients over 60years remains controversial and is regularly disappointing This is explained by a higher incidence of adverse prognostic factors but also host related factors which limit high dose chemotherapy delivery We report here the feasibility of peripheral blood stem cell( PBSC) collection and tolerance of Melphalan 140 mg/m2 and PBSC support in AML first complete remission (CR1) patients. Thirty one patients received classical Daunorubicin(DNR)/Arac induction:66 years(61–80) 17 de novo AML, 14 secondary AML. Complete remission, toxic death and resistance rate were 58%, 16%, and 26% respectively. Twelve/18 CR1(66%) underwent apheresis after 2 consolidations( DNR/Arac then Cyclophosphamide(CY)/VP16) which collected a median of 7,7(3–23) CD34/kg with a median of 2(2–5) cytapheresis These 12 patients received Melphalan followed by PBPC infusion at Day 0 and GCSF at Day 6 with excellent tolerance. Median neutrophil reconstitution> 1.109 /l and platelet> 50.109/l were observed at Day 11(10–14) and Day 15 respectively,median Day of hospital discharge was Day 14(0–24). The median 1 year overall survival (OS), disease free survival(DFS) is 42%(26–60) and 47%(26–69) respectively. This preliminary report suggest that high dose Melphalan can be safely incorporated in the therapeutic strategy of elderly AML patients. Given the 50% relapses already observed, a second intensification also supported by PBSC should be considered.
Cytogenetic findings in pretreatment bone marrow are increasingly being used for treatment stratification in adult de novo acute myeloid leukemia (AML). Herein we discuss the role of prognostic factors in cytogenetically defined groups of adults with AML, such as dose of post-induction cytarabine for AML with t(15;17)(q22;q12) and an internal tandem duplication (ITD) of the FLT3 gene in AML with normal cytogenetics. For AML with t(15;17)(q22;q12) or PML/RARα current management is based on the use of all-trans-retinoic acid (ATRA) combined with anthracycline therapy. Post-induction therapy often includes cytarabine, although the benefit of cytarabine is unclear. We present data from Cancer and Leukemia Group B (CALGB) suggesting that higher-dose cytarabine as post-induction therapy has activity, at least in the absence of ATRA therapy as part of induction. For AML with normal cytogenetics high-dose cytarabine as post-induction therapy has been shown to improve outcome. However, only 40% of patients who achieve complete remission are long-term disease-free survivors when treated with chemotherapy alone. An important current goal is to identify molecular abnormalities in AML patients with normal cytogenetics that have prognostic significance and could potentially serve as therapeutic targets. We describe recently published data from CALGB that show that patients with an ITD of the FLT3 gene, especially those who lack a FLT3 wild-type allele, have a very poor prognosis, despite treatment with current dose-intensive regimens. Such patients may benefit from therapy with a FLT3 tyrosine kinase inhibitor.
Cancer patients are confronted with many challenges, which can potentially affect their quality of life. Sexual dysfunction may seem a secondary issue when faced with the diagnosis of Leukemia and the ensuing therapy, but it is a major contributor to the quality of life of these patients. It is also one of the most sensitive issues, including body image, self-confidence, interest and desire, arousal, infertility, functional ability, orgasm, gender role identity, frequency of sexual activity and satisfaction.
Today most strategies for immune therapy and gene therapy of cancer are based on the utilization of adaptive immune responses. Thus, MHC-restricted CD4 and CD8 T cells capable of recognizing tumors are induced through vaccination procedures in vivo or such T cells are selected and expanded ex vivo for subsequent adoptive transfer into patients who may be unable to elicit adequate responses in vivo. In melanoma patients it has been seen that the prevalence of MHC-restricted T cells is associated with tumor regression (Jäger et al. 1996, Kawakami et al. 2000, Nestle et al. 1998, Rosenberg et al. 1998, Rosenberg 1999). However, many clinical responses are short-lived and it has been found that tumor variants emerge that escape immune detection due to altered expression of the MHC-peptide ligands that serve to activate MHC-restricted effector T cells (Garrido et al. 1997, Jäger et al. 1996, Lehmann et al. 1995, Maeurer et al. 1996, Riker et al. 1999).
Refractory disease still remains the main cause of treatment failure in acute myeloid leukemia (AML). Life expectancy in patients with CR achieved after second line therapy for primary drug resistance, relapse or poor pretreatment is really short (~6 months). Any attempt to prolong remission duration in this high risk group patients should be of certain value. It’s a well established fact that INF-alfa and ATRA combination leads to a synergy in antiproliferative, antiangiogenic, antiviral and differentiating effects (Moore DM, 1994).
