Treatment discontinuation following low-dose TKIs in 248 chronic myeloid leukemia patients: Updated results from a campus CML real-life study.

TKIs long-term treatment in CML may lead to persistent adverse events (AEs) that can promote relevant morbidity and mortality. Consequently, TKIs dose reduction is often used to prevent AEs. However, data on its impact on successful treatment-free remission (TFR) are quite scarce. We conducted a retrospective study on the outcome of CML subjects who discontinued low-dose TKIs from 54 Italian hematology centers participating in the Campus CML network. Overall, 1.785 of 5.108 (35.0%) regularly followed CML patients were treated with low-dose TKIs, more frequently due to relevant comorbidities or AEs (1.288, 72.2%). TFR was attempted in 248 (13.9%) subjects, all but three while in deep molecular response (DMR). After a median follow-up of 24.9 months, 172 (69.4%) patients were still in TFR. TFR outcome was not influenced by gender, Sokal/ELTS risk scores, prior interferon, number and last type of TKI used prior to treatment cessation, DMR degree, reason for dose reduction or median TKIs duration. Conversely, TFR probability was significantly better in the absence of resistance to any prior TKI. In addition, patients with a longer DMR duration before TKI discontinuation (i.e., >6.8 years) and those with an e14a2 BCR::ABL1 transcript type showed a trend towards prolonged TFR. It should also be emphasized that only 30.6% of our cases suffered from molecular relapse, less than reported during full-dose TKI treatment. The use of low-dose TKIs does not appear to affect the likelihood of achieving a DMR and thus trying a treatment withdrawal, but might even promote the TFR rate.

Efficacy and safety of dasatinib in imatinib-resistant or -intolerant patients with chronic myeloid leukemia in blast phase.

Dasatinib is an inhibitor of BCR-ABL and SRC-family kinases for patients with imatinib-resistant or -intolerant chronic myelogenous leukemia (CML). In this international phase II trial, dasatinib was administered orally (70 mg twice daily) to patients with myeloid blast phase (MBP, n=109) or lymphoid blast phase (LBP, n=48) CML. After a minimum follow-up of 12 months (range 0.03-20.7 months), major hematologic responses were induced in 34% (MBP-CML) and 35% (LBP-CML) of patients. Major cytogenetic responses were attained in 33% (MBP-CML) and 52% (LBP-CML) of patients and complete cytogenetic responses were attained in 26 and 46%, respectively. Median progression-free survival was 6.7 (MBP-CML) and 3.0 (LBP-CML) months. Median overall survival was 11.8 (MBP-CML) and 5.3 (LBP-CML) months. Overall, dasatinib had acceptable tolerability. Fluid retention events were more frequent in the MBP-CML than the LBP-CML cohort: pleural effusion occurred in 36 and 13% (all grades) and 15 and 6% (grades 3/4), respectively. Other non-hematologic side effects were primarily grade 1/2; grade 3/4 events were recorded in

NF-kB inhibition as a strategy to enhance etoposide-induced apoptosis in K562 cell line.

NF-kB is a transcription factor that mediates antiapoptotic signals in several cancer cell lines. Here we have demonstrated that the cytotoxic drug, Etoposide, activates NF-kB in K562, a chronic myeloid leukemia blast crisis cell line. Treatment with the NF-kB inhibitors MG-132, Bay11-7082, and Resveratrol impedes Etoposide-induced NF-kB activation, rendering K562 sensitive to Etoposide-induced apoptosis. Stable expression of mutant form of IkB-alpha, which retains NF-kB inactive in the cytoplasm of cells, confirmed the data obtained with molecular inhibitors. Both inhibitors and stable expression of SR-IkB are associated with down-modulation of the antiapoptotic protein Bcl-xL, suggesting that the survival pathway activated by Etoposide involves NF-kB-mediated Bcl-xL expression.

Partial deletions of long arm of chromosome 6: biologic and clinical implications in adult acute lymphoblastic leukemia.

Within 285 adult acute lymphoblastic leukemias (ALL) included in the multicenter GIMEMA 0496 trial and prospectively studied by conventional cytogenetics, 18 cases (6%) with long arm deletion of chromosome 6 (6q) were identified. These cases were divided into: (i) del(6q) only (n = 6); (ii) del(6q) plus other numerical and/or structural abnormalities (n = 8); (iii) del(6q) and other 'specific' translocations (n = 4). The biologic and clinical features of the patients carrying this anomaly, as well as their outcome, were compared with those of 267 patients without del(6q). A T cell phenotype was more frequently associated with del(6q) cases in general (P = 0.001) and particularly with cases presenting del(6q) as the isolated abnormality (P = 0.0027). No significant difference with respect to multidrug resistance (MDR)/P glycoprotein expression was observed between the two groups of patients (21% vs 28% of MDR-positive cases, respectively). A BCR-ABL fusion transcript was less frequently detected in cases with del(6q) (11%) compared with those without the anomaly (29%). p15 and p16 deletions were identified by Southern blot analysis in 21% of cases with del(6q) and in 26% of cases without del(6q). In this latter group, a T cell phenotype was less frequently associated with p15 and/or p16 deletion than in the group carrying del(6q) (36% vs 100% of cases, P = 0.011). Overall, patients with ALL and del(6q) had a high complete remission (CR) rate (83%); however, they had a lower 18 month event-free survival (31% vs 41%) and a higher relapse rate (70% vs 37%, P = 0.02) compared with patients without del(6q). To date, this is the largest series of adult ALL cases reported with del(6q) homogeneously treated, which have also been prospectively studied for MDR expression and for the detection of known fusion genes. This anomaly, as an isolated change, identifies a subset of cases with hyperleukocytosis (median WBC count 52 x 10(9)/l) and a strict correlation with a T cell phenotype. Overall, del(6q) seems to be associated with an unfavorable clinical outcome, although this finding will need to be confirmed by extended FISH analysis.

Quantitative assessment of WT1 expression by real time quantitative PCR may be a useful tool for monitoring minimal residual disease in acute leukemia patients.

In order to verify if quantitative assessment of the WT1 transcript amount by the real time quantitative PCR (RQ-PCR) can be used as a marker for minimal residual disease detection, the WT1 transcript amount was determined in BM and PB samples of patients with myeloid and lymphoid acute leukemia, in normal controls, in regenerating bone marrow samples and in purified CD34-positive cells from normal subjects. In 10 patients bearing a fusion gene transcript suitable for minimal residual disease quantitative assessment, we performed a simultaneous analysis of the WT1 and of the fusion-gene transcript at sequential time intervals during follow-up. Sequential WT1 analysis was also performed in five AML patients lacking additional molecular markers. The data obtained show that normal and regenerating BM samples and purified CD34-positive cells consistently express minimal amounts of WT1 transcript and that this is extremely low and frequently undetectable in normal PB. By contrast, high levels of WT1 expression are present in the BM and PB samples of all acute leukemia (AL) cases at diagnosis. The WT1 levels during follow-up were found to follow the pattern of the other molecular markers (fusion gene transcripts) used for MRD monitoring and increased WT1expression in the BM and/or PB during follow-up of AL patients was always found to be predictive of an impending hematological relapse.

Incidence and significance of cryptic chromosome aberrations detected by fluorescence in situ hybridization in acute myeloid leukemia with normal karyotype.

To better define the incidence and significance of cryptic chromosome lesions in acute myeloid leukemia (AML), fluorescence in situ hybridization (FISH) studies were performed in interphase cells and, when appropriate, in metaphase cells and in morphologically intact BM smears. Fifty-five adult de novo AML (group A) and 27 elderly AML or AML after myelodysplastic syndrome (AML-MDS) (group B) were tested using probes detecting the following anomalies: -5, -7, +8, deletions of 5q31, 7q31, 12p13/ETV6, 17p13/p53, 20q11. All the patients had a normal karyotype in more than 20 cells and tested negative for the common AML-associated fusion genes. No patient in group A was found to carry occult chromosome anomalies, whereas 8/27 patients in group B (P < 0.0001) showed 5q31 or 7q31 deletion (three cases each), a 17p13/p53deletion or trisomy 8 (one case each) in 33-60% interphase cells. Metaphase cells showed only one hybridization signal at 5q31 (three cases) and 7q31 (one case), whereas two normal signals at 7q31 and chromosome 8 centromeres were seen in two patients with 7q deletion and trisomy 8 in interphase cells. The majority of blast cells (76-94%) carried the chromosome anomaly in all cases; erythroid involvement in a minority of cells was seen in three patients. In group B, the presence of occult chromosome anomalies was associated with exposure to myelotoxic agents in the workplace (5/8 cases vs 3/19, P = 0.026) and with a lower complete remission rate (0/6 patients vs 7/12, P = 0.024). We arrived at the following conclusions: (1) cryptic chromosome deletions in the order of a few hundred kb magnitude may be found in a fraction of elderly AML or MDS-related AML and not in de novo adult AML with normal karyotype; (2) these chromosome lesions are usually represented by submicroscopic rearrangements; (3) they display a specific pattern of cell-lineage involvement arguing in favor of their role in the outgrowth of the leukemic blast cells; (4) they are associated with a history of exposure to myelotoxic agents in the workplace and, possibly, with resistance to induction treatment.

MEC1 and MEC2: two new cell lines derived from B-chronic lymphocytic leukaemia in prolymphocytoid transformation.

We report the establishment and characterization of two cell lines, MEC1 and MEC2, that grew spontaneously on two subsequent occasions from the peripheral blood (PB) of a patient with B-chronic lymphocytic leukemia (B-CLL) in prolymphocytoid transformation. The patient was EBV-seropositive, his leukemic cells were EBNA negative, but the spontaneously grown cell lines are EBNA-2 positive. In liquid culture MEC1 cells grow adherent to the vessel wall and as tiny clumps; MEC2 cells do not adhere and form large clumps. The doubling time of MEC1 is 40h and of MEC2 is 31h. Both cell lines express the same light (kappa) and heavy chains (mu, delta) as the fresh parental B-CLL cells at the same high intensity, share the expression of mature B cell markers (CD19, CD20, CD21, CD22), differ in the expression of CD23 and FMC7, are CD11a+, CD18+, CD44+, CD49d+, CD54+ and express at high levels both CD80 and CD86. CD5 is negative on MEC1 cells (as on the vast majority of parental cells) and it has been lost by MEC2 cells after several months of culture. The cells have a complex karyotype. The tumour origin of MEC1 and MEC2 has been demonstrated by Southern blot analysis of the IgH loci and by Ig gene DNA sequencing. They use the VH4 Ig family and have not undergone somatic mutations (94.8% homology with germline Ig gene 4-59). Cytofluorographic analysis and RT-PCR reveal that MEC1 and MEC2 overexpress Bcl-2 together with Bax, express large amounts of Bcl-xL and trace amounts of Bcl-xS.

Isochromosome 6p and deletion of 6q characterize two related cytogenetic clones in a patient with immunoblastic lymphoma.

In a case of immunoblastic lymphoma we observed the presence of either a deletion of the long arm of chromosome 6 or of an isochromosome, i(6p), which occurred alternatively in metaphase cells. This suggests a selective pressure for loss of heterozygosity of genes located on 6q and is in accordance with the hypothesis that one or more tumor suppressor genes might be located on the long arm chromosome 6.

Analysis of the p53 gene in myeloid malignancies associated with chromosomal abnormalities involving the short arm of chromosome 17.

p53 protein is encoded by a tumor-suppressor gene located on the short arm of chromosome 17. We looked for mutations or rearrangements of the p53 gene in five patients with acute transformation of a chronic myeloproliferative disorder and cytogenetic anomalies involving the short arm of chromosome 17. Two patients had a isochromosome i(17q); three more patients showed the presence of unbalanced translocations involving chromosome 17. One of these patients had a single base pair deletion, causing a frameshift mutation, in the exon 5 of the p53 gene. The karyotype of this patient showed a translocation t(5;17)(q11;p11), with loss of a normal homologue of both chromosomes 5 and 17. In all other cases the configuration of the p53 gene, as tested by PCR-SSCP analysis of exons 5 to 9 and Southern blot, was normal. Our data suggest that mutations of the p53 gene occur in a minority of hemopoietic malignancies characterized by monosomy for the short arm of chromosome 17. However, the unbalanced translocation t(5q;17p) could be a chromosomal abnormality specifically associated with loss of function of the p53 gene.

Molecular mechanisms of tumor progression in chronic myeloproliferative disorders.

We have investigated the involvement of tumor suppressor genes (p53 and RB1) and dominantly acting oncogenes (Ras family genes) in BCR/ABL positive and negative chronic myeloproliferative disorders (CMPD) at different stages of the disease, including 26 cases of BCR/ABL+ chronic myeloid leukemia (CML) blast crisis, 9 myelosclerosis with myeloid metaplasia, 4 polycythemia vera, 10 essential thrombocythemia, 1 juvenile CML, and 8 BCR/ABL- CML. The presence of mutations in p53 exons 5 through 9, as well as in RB1 exons 10-27 and in N-, K-, H-Ras exons 1 and 2 was tested by the PCR-Single Strand Conformation Polymorphism technique and by PCR-Direct Sequencing. In addition, Southern blot analysis was used to investigate the occurrence of gross rearrangements in the p53 gene as well as loss of heterozygosity at 17p13, the site of p53. Acute phase BCR/ABL-CMPD cases displayed a high frequency of p53 (2/7) and Ras (3/7) lesions, whereas BCR/ABL- CMPD in chronic phase displayed only germline p53 and Ras sequences. Conversely, p53 inactivation was restricted to only 1/26 cases of BCR/ABL+ CML blast crisis. No alterations in the RB1 gene were detected in any of the cases analyzed. These data indicate that p53 inactivation and/or Ras activation might play a role in acute transformation of BCR/ABL- CMPD and that the molecular mechanisms of tumor progression may be different in BCR/ABL+ versus BCR/ABL-CMPD.

Genetic analysis of p53 and RB1 tumor-suppressor genes in blast crisis of chronic myeloid leukemia.

We have investigated the involvement of the p53 and RB1 tumor-suppressor genes in 26 cases of chronic myeloid leukemia (CML) blast crisis, including 17 myeloid, eight lymphoid, and one megakaryoblastic crisis. The presence of p53 mutations in exons 5 through 9 was tested by the PCR-single-strand conformation polymorphism (SSCP) assay, followed by PCR-direct sequencing; in addition, loss of heterozygosity (LOH) at 17p13, the site of the p53 gene, was assayed by Southern blot. Given the variability of the mechanisms of inactivation of the RB1 gene in human tumors, a combination of Southern blot and mutational analysis by PCR-SSCP was used. p53 mutations were restricted to one case of myeloid blast crisis, showing a CGC-->TGC (Arg-->Cys) mutation at codon 283; two additional cases displayed LOH at 17p13 in the absence of p53 mutations. No molecular lesions of the RB1 gene were detected in any of the cases analyzed. These data indicate that inactivation of p53 and RB1 is a rare event in the molecular pathogenesis of CML acute transformation.

Cytogenetics and occupational exposures in acute nonlymphocytic leukemia and myelodysplastic syndrome. Working Group on the Epidemiology of Hematolymphopoietic Malignancies in Italy.

Studies on the association between cytogenetic aberrations and environmental or occupational exposures of patients with acute nonlymphocytic leukemia and myelodysplastic syndrome were selected from the literature and reviewed. Chromosome abnormalities as a whole and specific aberrations involving chromosomes 5, 7, 8, and 21 were considered. Occupational exposures were also considered as a whole or, whenever possible, as specific substances. An association between all occupations and any chromosome aberration has been observed in earlier studies but was not confirmed in later studies. Similar results have been observed when specific aberrations, or specific substances (ie, solvents) have been considered. Methodological issues and the comparability of the reviewed studies are discussed, and suggestions are made for refining epidemiologic investigations.

Myeloid leukemias and myelodysplastic syndromes: chemical exposure, histologic subtype and cytogenetics in a case-control study.

We conducted a case control study of 50 acute myeloid leukemias (AML), 17 chronic myeloid leukemias (CML), 19 myelodysplastic syndromes (MDS), and 246 controls. The cases were classified according to the French-American-British (FAB) classification, and chromosome aberrations were recorded according to the International System for Human Cytogenetic Nomenclature. Exposure to suspected leukemogenic agents was assessed blindly by an industrial hygienist. Increased risks were noted for mechanics, welders, electricians, and drivers among men and among farmers and textile workers among women. Increased SMRs for leukemias in a census-based cohort study conducted in the same area (Torino) were previously reported for electricians and drivers among men and for textile workers among women. We detected nonstatistically significant increased relative risks for exposure to benzene (odds ratio, OR = 1.7), petrol refining products (1.9), polycyclic aromatic hydrocarbons (1.7), and electromagnetic fields (1.6) in men; in women, a statistically significant association with exposure to pesticides was detected [OR = 4.4; 95% confidence interval (CI) 1.7-11.5]. Although exposure to pesticides was confined to AML, MDS cases included a high proportion of subjects exposed to benzene and electromagnetic fields. No particular histologic subtype of AML was associated with chemical exposures except for that of pesticides with the M4 category. Chromosome aberrations were not associated with chemical exposures (OR = 1.0), but a nonstatistically significant excess was noted in association with electromagnetic fields (OR = 2.1).

Molecular defects associated with the acute phase CML.

Parts of the Bcr/Abl hybrid transcript supposed to be important for its transforming ability were sequenced in a series of CML blast crises, in order to evaluate the possible presence of alterations responsible for the disease transition from the chronic to the acute phase. In addition, the N- and Ki-ras as well as the p53 involvement was investigated by exploring their structure and expression in the same patients. We used traditional types of molecular analysis including Southern and Northern blot, together with methods that allow a rapid detection of point mutations and microdeletions, such as SSCP, single strand conformation polymorphism and direct sequencing. The results obtained may be summarized as follows: no alterations were found in the parts of the Bcr/Abl transcripts investigated in the present study (SH2, SH3 and the region surrounding codon 832); p53 alterations were observed in 5% and N- and Ki-RAS mutations in 5% of the cases examined. These molecular defects are therefore responsible for the clinical progression of the Ph1-positive CML only in a minority of cases.

Karyotypic analysis of gastric carcinoma cell lines carrying an amplified c-met oncogene.

MKN 45 is a poorly differentiated gastric carcinoma cell line from which the subclone GTL 16 was obtained. Both lines carry an amplification unit derived from chromosome 7 sequences and containing an activated c-met oncogene. Karyotypic analysis showed that GTL 16 derived from a subclone of MKN 45 after endoreduplication. Several clonal abnormalities are evident in both lines; some are frequently observed in gastrointestinal tumors (loss of 17p and monosomy 18). Other consistent anomalies include 6q-, t(8;10) and t(5;8), and inv(16). A marker chromosome (M1), which was previously shown to contain the c-met amplification unit, is constantly duplicated in all GTL 16 metaphases; in contrast, most unidentified markers are retained in only a single copy in GTL 16 cells. These data are in agreement with the hypothesis that the c-met oncogene activation in these gastric cancer cell lines might be related to a gene dosage effect.

Transient cytogenetic relapse in a Ph1-positive chronic myelogenous leukemia patient previously treated with alpha-interferon.

Therapy with alpha-interferon (IFN alpha) can suppress the Ph1-positive hemopoiesis in a percentage of patients with chronic myelogenous leukemia (CML). We used IFN alpha to treat a 30-year-old CML patient, characterized by favourable prognostic signs (such as low leukocytosis, absence of splenomegaly and no increase in bone marrow blasts) at diagnosis, and obtained a complete remission, as evaluated by Southern blot and cytogenetic analysis, after one year of treatment. However, the polymerase chain reaction (PCR) revealed the persistence of a minimal residual disease. The IFN alpha therapy was stopped and the hematological status remained stable until eighteen months later, when a cytogenetic analysis revealed the appearance of a clone characterized by t(9;22) and trisomy 8, accounting for 30% of bone marrow metaphases. This cell population spontaneously regressed in the following months, before any cytotoxic treatment. However, as leukemic cells, detected by PCR, were still present, the patient received a high dose chemotherapy, which induced the complete eradication of the Ph1-positive clone, as demonstrated by the absence of bcr-abl transcript at the PCR reaction. Molecular and cytogenetic remission persist one year later, without any further therapy.

Extra translocation +der(1q9p) is a prognostic indicator in myeloproliferative disorders.

An identical extra derivative chromosome resulting from a translocation between the long arm of chromosome 1 and the short arm of chromosome 9, +der(1q9p), has been observed in three patients with a myeloproliferative disorder. Two patients had polycythemia vera in transformation (erythroleukemia in one patient and refractory anemia in the second), whereas the third patient had myelofibrosis which later evolved into acute myelomonocytic leukemia. The two patients who developed overt leukemia did not receive any previous cytotoxic treatment. Non-isotopic in situ hybridization was performed in two patients, allowing for the localization of the breakpoints in 1q12 and 9q12. A similar rearrangement has been previously described in patients with polycythemia vera, either at diagnosis or in advanced stages of the disease. These data suggest that this chromosome abnormality may be consistently associated with myeloproliferative disorders showing a high propensity to transformation, which is not treatment related, and the finding of the +der(1q9p) may represent a poor prognostic sign when observed in the chronic phase.

Effect of recombinant human IL-3 on the mitotic index and karyotype of hemopoietic cells.

The proliferative induction by hemopoietic growth factors may provide a useful tool to improve the mitotic yield of hemopoietic cells, allowing a more accurate cytogenetic analysis in hematologic malignancies. For such a purpose, we studied the effects of the recombinant human IL-3 (rhIL-3) on the mitotic index and the karyotype of bone marrow cells from 14 patients with myelodysplastic (MDS) and myeloproliferative syndromes (MPS). The mitotic response to IL-3 of normal bone marrow samples was also evaluated. Total bone marrow cells were cultured for 24 to 72 hours either in presence or absence of rhIL-3. In most cases, IL-3--stimulated samples showed a considerably higher (4-70 times) mitotic index than unstimulated controls. Although a great patient-to-patient variability was observed, a common pattern of mitogenic response to IL-3 emerged among MPS, MDS, and normal cases. At 48 hours of incubation, the mean mitotic index from MPS and MDS cases stimulated with IL-3 was significantly higher (p less than 0.01) than unstimulated controls, whereas the mean mitotic increase from normal samples did not reach statistical significance (p greater than 0.1). Even though not statistically evaluable, a similar trend of response was observed at 24 and 72 hours of culture. Chromosome studies of MPS and MDS cases showed the same karyotype either in stimulated and unstimulated samples.

Different suppression of Ph1 positive hemopoiesis induced by intensive chemotherapy in lymphoid and myeloid blast crisis of CML.

BACKGROUND: The suppression of Ph1-positive hemopoiesis is a major goal in the treatment of CML; in this context the CML patients in blast crisis obtaining a complete clinical remission represent a useful model to investigate the behavior of the Ph1-positive and negative clones during bone marrow repopulation after ablative therapy. METHODS: Seven CML patients in blast crisis (four lymphoid and three myeloid) who obtained a complete clinical remission after an intensive polychemotherapy treatment were evaluated by cytogenetic and molecular analysis both in blast and remission phases. Standard cytogenetic and Southern techniques were employed; in addition, minimal residual disease status (MRD) was ascertained by amplification (PCR) of the specific bcr-abl chimeric transcripts. RESULTS: After a single cycle of induction, all lymphoid cases displayed a complete restoration of Ph1-negative hemopoiesis; by contrast, one myeloid blast crisis showed a partial suppression of Ph1-positive hemopoiesis only after two cycles of chemotherapy, and in the remaining two cases the hematological remission was indeed a reversion to the chronic phase. CONCLUSIONS: Ph1-positive chronic clones present in lymphoid blast crisis showed a higher degree of sensitivity to intensive chemotherapy than those present in the myeloid cases. This observation further suggests that the growth properties of the Ph1-positive clones are highly variable from case to case and probably tend to progress during the time-course of the disease.