Loss of lenalidomide-induced megakaryocytic differentiation leads to therapy resistance in del(5q) myelodysplastic syndrome.

Interstitial deletion of the long arm of chromosome 5 (del(5q)) is the most common structural genomic variant in myelodysplastic syndromes (MDS)1. Lenalidomide (LEN) is the treatment of choice for patients with del(5q) MDS, but half of the responding patients become resistant2 within 2 years. TP53 mutations are detected in ~20% of LEN-resistant patients3. Here we show that patients who become resistant to LEN harbour recurrent variants of TP53 or RUNX1. LEN upregulated RUNX1 protein and function in a CRBN- and TP53-dependent manner in del(5q) cells, and mutation or downregulation of RUNX1 rendered cells resistant to LEN. LEN induced megakaryocytic differentiation of del(5q) cells followed by cell death that was dependent on calpain activation and CSNK1A1 degradation4,5. We also identified GATA2 as a LEN-responsive gene that is required for LEN-induced megakaryocyte differentiation. Megakaryocytic gene-promoter analyses suggested that LEN-induced degradation of IKZF1 enables a RUNX1-GATA2 complex to drive megakaryocytic differentiation. Overexpression of GATA2 restored LEN sensitivity in the context of RUNX1 or TP53 mutations by enhancing LEN-induced megakaryocytic differentiation. Screening for mutations that block LEN-induced megakaryocytic differentiation should identify patients who are resistant to LEN.

Occurrence of acute myeloid leukemia in hydroxyurea-treated sickle cell disease patient.

Hydroxyurea (HU) has been widely used in sickle cell disease. Its potential long-term risk for carcinogenesis or leukemogenic risk remains undefined. Here, we report a 26 y old African-American female with Sickle Cell Disease (SCD) who developed refractory/relapsed acute myeloid leukemia (AML) 6 months after 26 months of HU use. That patient's cytogenetics and molecular genetics analyses demonstrated a complex mutation profile with 5q deletion, trisomy 8, and P53 deletion (deletion of 17p13.1). P53 gene sequence studies revealed a multitude of somatic mutations that most suggest a treatment-related etiology. The above-mentioned data indicates that the patient may have developed acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) as a direct result of HU exposure.

Chromosomal effects of non-alkylating drug exposure in oncology personnel.

Therapy-related leukemia has been a recognized sequela of cancer treatment for decades with "signature" abnormalities of chromosomes 5, 7, and 11 observed in treated patients. Risk to oncology personnel handling anti-cancer agents has also been documented by non-specific measures of genotoxicity in blood and urine. Using chromosomal markers applied in clinical practice, we previously demonstrated in oncology workers, a dose-related increase in abnormalities of chromosomes 5 and 7, known to be targets of alkylating agent exposure. In the analysis presented here, we extended that work to also assess damage resulting from non-alkylating drug exposure. Peripheral blood lymphocytes from oncology personnel (N = 63) and non-exposed controls (N = 46) was collected and examined using the fluorescent in situ hybridization technique with probes for targets on chromosomes 5, 7, and 11. Participants recorded drug handling events over a 6 week period. Important co-variates were considered. Examining chromosomal outcomes as a function of drug handling frequency, we employed Poisson Regression to obtain incident rate ratios (IRRs) for selected drug handling frequencies. We found a dose-related increase in the IRR for aberrations in all three chromosomes 5, 7, and 11, reaching statistical significance for chromosome 5, as a function of non-alkylating drug handling. This suggests that the targeting of chromosome 5 is not limited to alkylating agent exposure, as some recent evidence in treated patients has also shown. Thus, the pattern of insult observed in treated patients appears to extend to oncology personnel exposed in the workplace.

Therapy-related myelodysplastic syndrome and acute myeloid leukemia in patients with chronic lymphocytic leukemia treated with fludarabine and cyclophosphamide.

We retrospectively studied four cases of t-MDS/AML among 210 (1.9%) consecutive patients with CLL treated at a single center with fludarabine and cyclophosphamide (FC) either as the first- or second-line therapy. The median follow-up of the whole cohort of patients was 46months (range: 7-60). Two of these patients (2/130, 1.7%) had been treated with FC only, and two more (2/80, 2.3%) with CHOP and CHOP+FND, respectively, prior to FC. The median age was 61.5years (range: 49-71); three were male. They developed t-MDS/AML after a median latency period of 41months (range: 7-56) from the FC completion. Chromosomal aberrations with an adverse prognostic impact were present in the karyotype of all four patients, including abnormalities of chromosome 5 in three of them, and a rare chromosomal translocation in one patient. Median survival after t-MDS/AML diagnosis was 4months (range: 2-8). Although the agents administered prior to FC make it difficult to assess the risk of t-MDS/AML attributable to FC, this report might be a valuable addition to the literature.

Chromosome 5 and 7 abnormalities in oncology personnel handling anticancer drugs.

OBJECTIVE: To determine the frequency of "signature" chromosomal abnormalities in oncology workers handling anticancer drugs. METHODS: Peripheral blood from health care personnel (N = 109) was examined with probes for targets on chromosomes 5, 7, and 11. The effect of drug-handling frequency on chromosome abnormalities was assessed. RESULTS: An excess of structural (0.18 vs 0.02; P = 0.04) and total abnormalities (0.29 vs 0.04; P = 0.01) of chromosome 5 was observed in the high-exposure group compared with the unexposed. Increased incidence rate ratios (IRRs) for abnormalities of chromosome 5 (IRR = 1.24; P = 0.01) and for either chromosome 5 or 7 (IRR = 1.20; P = 0.01) were obtained at 100 handling events. Effect sizes were augmented 2- to 4-fold when alkylating agent handling alone was considered. CONCLUSIONS: Biologically important exposure to genotoxic drugs is apparently occurring in oncology work settings despite reported use of safety practices.

An erythroid differentiation signature predicts response to lenalidomide in myelodysplastic syndrome.

BACKGROUND: Lenalidomide is an effective new agent for the treatment of patients with myelodysplastic syndrome (MDS), an acquired hematopoietic disorder characterized by ineffective blood cell production and a predisposition to the development of leukemia. Patients with an interstitial deletion of Chromosome 5q have a high rate of response to lenalidomide, but most MDS patients lack this deletion. Approximately 25% of patients without 5q deletions also benefit from lenalidomide therapy, but response in these patients cannot be predicted by any currently available diagnostic assays. The aim of this study was to develop a method to predict lenalidomide response in order to avoid unnecessary toxicity in patients unlikely to benefit from treatment. METHODS AND FINDINGS: Using gene expression profiling, we identified a molecular signature that predicts lenalidomide response. The signature was defined in a set of 16 pretreatment bone marrow aspirates from MDS patients without 5q deletions, and validated in an independent set of 26 samples. The response signature consisted of a cohesive set of erythroid-specific genes with decreased expression in responders, suggesting that a defect in erythroid differentiation underlies lenalidomide response. Consistent with this observation, treatment with lenalidomide promoted erythroid differentiation of primary hematopoietic progenitor cells grown in vitro. CONCLUSIONS: These studies indicate that lenalidomide-responsive patients have a defect in erythroid differentiation, and suggest a strategy for a clinical test to predict patients most likely to respond to the drug. The experiments further suggest that the efficacy of lenalidomide, whose mechanism of action in MDS is unknown, may be due to its ability to induce erythroid differentiation.

Leukaemia-specific chromosome damage detected by comet with fluorescence in situ hybridization (comet-FISH).

Acute myeloid leukaemia (AML) is associated with exposure to benzene and treatment with chemotherapeutic agents. It is thought to arise from damage to specific regions of DNA, resulting in chromosome rearrangements or loss. For instance, a deletion on the long arm of chromosome 5 [e.g. del(5q31)] is common in AML patients previously treated with alkylating agents, such as melphalan, or exposed to benzene. Translocations of the MLL gene at 11q23 are frequently observed in AML arising from treatment with topoisomerase II inhibitors, such as etoposide. Our goal was to determine whether or not breakage at 5q31 and 11q23 is selectively induced by these chemical agents. To address this question, the comet assay combined with fluorescence in situ hybridization (comet-FISH) was used to detect DNA breakage in the specific chromosomal regions in an in vitro model. TK6 lymphoblastoid cells were exposed to melphalan, etoposide or the benzene metabolite, hydroquinone (HQ), at various concentrations. HQ, melphalan and etoposide induced DNA breaks at both 5q31 and 11q23 chromosome regions in a dose-dependant manner. However, HQ produced significantly more DNA damage at 5q31 than at 11q23. Etoposide produce slightly more DNA damage at 11q23 and melphalan had a somewhat greater effect at 5q31, but not significantly so. Thus, HQ and melphalan act similarly, perhaps explaining some similarities between benzene- and alkylating agent-induced AML. Comet-FISH also appears to be a useful approach for detecting and comparing damage to specific chromosome regions of significance in leukaemogenesis.

[Azathioprine-associated myelodysplastic syndrome with cytogenetic aberrations]. / Azathioprin-assoziiertes myelodysplastisches Syndrom mit zytogenetischen Aberrationen.

HISTORY AND ADMISSION FINDINGS: A 49-years-old female with a history of multiple sclerosis and on medication with azathioprine over 5 years amounting to a cumulative dosage of 45 g presented with fatigue and sinus tachycardia. INVESTIGATIONS: Haematological analysis revealed a pancytopenia with a sustained normochromic anemia (hemoglobin 6,2 g/dl), a mild leukopenia (leucocytes 3500/ microl) and thrombocytopenia (platelets 22000/ microl) requiring platelet substitution. Bone marrow aspirate revealed a dysplasia of all three lineages with reduced thrombopoiesis and ineffective erythropoiesis. Cytogenetic analysis detected a complex aberrant clone including loss of the critically deleted regions in 5q31 and 7q31, as well as structural changes of 12p. The diagnosis was refractory cytopenia with multilineage dysplasia according to the WHO classification of myelodysplastic syndromes. TREATMENT AND COURSE: While allogeneic sibling transplantation was planned the patient developed spontaneous recurrent subdural haematoma and died due to persistent bleeding refractory to platelet substitution. CONCLUSION: Treatment with azathioprine may - depending on its cumulative dosage - lead to pancytopenia and subsequent development of myelodysplasia or secondary leukaemia, respectively. Complex genetic alterations involving chromosome 7 are characteristic. Long-lasting treatment with azathioprine needs critical assessment.

Therapy-related erythroleukemia caused by the administration of UFT and mitomycin C in a patient with colon cancer.

A 54-year-old man with colon cancer underwent hemicolectomy. He received postoperative adjuvant chemotherapy with UFT (tegafur/uracil at a 1 : 4 molar ratio) and mitomycin C (MMC) for 3 years. Three years and 4 months after the start of chemotherapy, pancytopenia was noted. Bone marrow aspiration smear demonstrated an increased number of immature erythroblasts, including megaloblasts and myeloblasts. Chromosomal analysis demonstrated structural and numerical abnormalities of 5, 7, 15, and 17. Therapy-related erythroleukemia, acute myeloid leukemia (AML), M6, was diagnosed. The disease progressed after 5 months, and the patient was received chemotherapy with cytosine arabinoside, aclacinomycin, and granulocyte colony-stimulating factor (CAG), and showed a partial hematological response. Careful monitoring for the generation of therapy-related leukemia is needed when UFT and MMC are used for postoperative adjuvant chemotherapy for colorectal cancer.

Genetic susceptibility to oral cancer and the expression of common fragile sites. a study of 100 patients.

The expression of bleomycin-induced fragile sites (FS) in the blood lymphocytes of 150 individuals (100 oral cancer patients and 50 age and sex matched controls) is described. FS expression frequencies in oral cancer patients were significantly higher when compared with controls. FS expression was site specific in oral cancer patients. Chromosome 5 was the most affected, with four of its FS expressing in high frequencies. Enhanced expression of FS at the centromeric region was observed in the patient group. This study emphasizes the role of FS in the genetic susceptibility to oral cancer.

Karyotype analysis of tumorigenic human bronchial epithelial cells transformed by chrysolite asbestos using chemically induced premature chromosome condensation technique.

We examined karyotypic changes of tumorigenic human bronchial epithelial cell lines transformed by asbestos fibers. Using Calyculin A mediated premature chromosome condensation (PCC) assay and Giemsa-trypsin banding, we showed that the common changes of all tumorigenic cell lines were the loss of one or two copies of chromosome 5, the monosomy of chromosome 19 and the increased trisomy of chromosome 8. The results indicate that the karyotypic change of chromosome 5, 8 and 19 could play an important role in asbestos-induced tumorigenic conversion of human bronchial epithelial cells from an immortalized to tumorigenic state.

The benzene metabolites hydroquinone and catechol act in synergy to induce dose-dependent hypoploidy and -5q31 in a human cell line.

Chronic exposure to high concentrations of benzene is associated with an increased incidence of myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). Studies of patients occupationally exposed to benzene show a pattern of cytogenetic aberrations involving loss of all or part of chromosomes 5 and/or 7 as well as trisomy 8 and we have previously reported that hydroquinone (HQ) induces deletions of 5, 7 and 8. Benzene metabolism is a requirement for bone marrow toxicity and the phenolic metabolites, HQ and catechol (CAT), have been implicated in benzene hematotoxicity. A research project was designed to determine whether CAT by itself and in conjunction with HQ could directly induce loss of chromosome 5 and/or 7 and gain of chromosome 8. Using fluorescence in situ hybridization with chromosome-specific 5, 7, and 8 probes we demonstrate that 5 to 150 uM CAT does not produce chromosomal aberrations, however CAT and 25 uM HQ can act in synergy to induce dose dependent loss of these chromosomes. In addition HQ/CAT selectively induces -5q which is not observed for HQ only. These results demonstrate for the first time that CAT/HQ act in synergy to induce specific chromosome loss found in secondary MDS/AML.

Survival of cells with bleomycin-induced chromosomal lesions in the cultured lymphocytes of lung cancer patients.

In a previous study of lung cancer, we showed that bleomycin, a radiomimetic agent, induced breaks preferentially on chromosomes 4 and 5. The molecular cytogenetic study reported here, using chromosome painting and G banding, was designed to assess whether the chromatid breaks induced by bleomycin could survive as chromosome-type aberrations after treated lymphocyte populations were allowed to recover in a drug-free medium for one or two cell generations and whether the survival rates of lesions on chromosomes 4 and 5 differed in cases with lung cancer and controls. The findings from 16 cases and 14 controls showed that in samples allowed to recover for 48 h, most aberrations were of the chromosome type. The proportion of chromosome 5 abnormalities surviving as chromosome-type aberrations was significantly higher in the cells of lung cancer cases (13.4%) than in controls (4.6%; P < 0.0001). However, no significant differences in survival of chromosome 4 abnormalities were detected between cases and controls. The proportions of chromosome 5q13-q22 abnormalities were 5.3% in the cases and 0.6% in the controls (P < 0.0001). 5q13-q22 regions encompassed 38.4% of all abnormalities on chromosome 5 in the cases but only 14.5% in the controls. Therefore, the survival rate of chromosome 5 lesions (especially those at 5q13-q22) in lymphocytes might be used as a biomarker to identify populations at high risk for lung cancer.

Chromosome aberrations induced by etoposide (VP-16) are not random.

The clastogenic effect of etoposide, an anti-cancer chemotherapeutic drug, was investigated in vitro on lymphocytes of 5 healthy donors. The analysis of the first division metaphases arising after mutagenesis in G1 phase shows that chromosome-type aberrations are much more frequent than chromatid-type lesions. The distribution in relation to chromosome lengths of the 439 breakpoints that were accurately identified is not random: chromosomes 1, 11 and 17 are most frequently involved, while chromosomes 4, 5 and X are seldom affected. This non-random distribution may be related to chromosome structure, since R-band-rich chromosomes are significantly more affected than G-band-rich chromosomes.

Acute leukemia and cytogenetic abnormalities complicating melphalan treatment of primary systemic amyloidosis.

We followed up 153 patients with biopsy-proven primary systemic amyloidosis to determine their risk for acute nonlymphocytic leukemia or a dysmyelopoietic syndrome. In 10 patients cytogenetic abnormalities developed consistent with alkylator-induced damage to hematopoietic cells. In this group, the total melphalan dose ranged from 476 to 2450 mg (median, 1764 mg) administered over 21 to 92 months (median, 38 months). Eight of the 10 patients died as a direct result of pancytopenia, 1 died of progressive renal amyloid, and 1 remains alive with persistent complex cytogenetic abnormalities. Four patients had acute nonlymphocytic leukemia; 5 had a dysmyelopoietic syndrome; and 1 had a nondiagnostic bone marrow examination. Although only 6.5% of the entire group had leukemia or a dysmyelopoietic syndrome, the actuarial risk in patients surviving 3.5 years was 21%. Median survival from onset of dysmyelopoietic syndrome or acute leukemia was 8.1 months.

[Characteristics of chromosomal and mitotic disorders in patients with acute non-lymphoblastic leukemia exposed to chemical factors]. / Kharakter khromosomnykh i mitoticheskikh narushenii u bol'nykh ostrymi nelimfoblastnymi leikozami, podvergavshikhsia vozdeistviiu khimicheskikh faktorov.

Cytogenetic and mitotic disorders were studied in 151 patients with acute non-lymphoblastic leukemia. A significant rise in the incidence of clonal disorders with predominant affection of chromosomes 5 and 7, and an increase in the percentage of cases with the presence of pathologic mitoses in hemopoietic cells have been recorded in patients who, due to their occupation, have been exposed to potentially mutagenic chemical substances.