Abnormal Metaphases Research Articles

Pomalidomide Therapy in Myelofibrosis: 2-Year Follow-up of a Randomized Phase 2 Study.

Abstract 1904Poster Board I-927 BACKGROUND:Pomalidomide is an IMiDsó immunomodulatory drug that was created by chemical modification of thalidomide with the intent to reduce toxicity and enhance therapeutic activity. In a phase-2 randomized study of patients with myelofibrosis (MF), treatment with pomalidomide alone or in combination with a short course of prednisone was shown to be well tolerated and active in alleviating anemia ( JCO 2009). Herein, we report observations during the longer-term follow-up of those patients who had responded to a pomalidomide-containing treatment regimen. METHODS:Both primary and post polycythemia vera/essential thrombocythemia MF were included in the current study. Protocol eligibility criteria included absolute neutrophil count of × 109/L, platelet count of 50 × 109/L and hemoglobin level of < 10 g/dL. Patients with prior thalidomide or lenalidomide therapy were excluded from study participation. Response was assessed by the International Working Group for Myelofibrosis Research and Treatment criteria (IWG-MRT) (Blood. 2006). Pomalidomide was administered daily and continued indefinitely in responding patients. Prednisone was given for the first 3 months only, in a tapering dose schedule, starting at 30 mg/day. RESULTS:A total of 84 patients were enrolled from April 2007 through May 2008 and received treatment with pomalidomide alone 2 mg/day (n=22), pomalidomide 2 mg/day + prednisone (n=19), pomalidomide 0.5 mg/day + prednisone (n=22) or prednisone alone (n=21). Short-term toxicity details have previously been published (Tefferi et al. JCO 2009) and were remarkable for the lack of neuropathy and low prevalence of grade 3 or 4 myelosuppression (5–16%).Based on an intent-to-treat analysis, 16 (25%) of the 63 patients receiving pomalidomide alone or with a 3-month course of prednisone responded at a median of 1.8 months (range 0.9–6.9) from treatment initiation; all responses represented improvement in anemia with little or no effect on splenomegaly. Specifically, 12 of the 16 treatment responders became transfusion independent and the remaining 4 had a hemoglobin increase of > 2 g/dL (median best hemoglobin response = 11.9 g/dL; range 8.5–15.9). Responding patients usually displayed increases in platelet count but there was no consistent change in serum lactate dehydrogenase level.As of August 2009, ten (63%) of the 16 treatment responders were still in anemia remission and receiving pomalidomide therapy; median time on treatment was 19 months (range, 14–26) and median response duration was 17 months (range, 13-24). No new side effects emerged during this period. Six patients discontinued treatment because of either loss of response (n=5) or progressive splenomegaly/leukocytosis (n=1); 3 patients with continued anemia remission have had substantial increases in their spleen size, which qualifies as spleen progression per IWG-MRT. Among 9 patients with available cytogenetic information, all 3 with unfavorable karyotype relapsed while none of the 6 with favorable karyotype did.A small number of patients (n=7) had follow-up bone marrow examination at 1 to 2 years, at the discretion of their treating physician; none of these patients displayed any change in bone marrow fibrosis or JAK2V617F mutational status. Suppression of a monosomy 7 clone was documented in two patients. In one, the percentage of abnormal metaphases decreased from 90% to 15% after 11 months of treatment with pomalidomide but increased back to 100% four months later, when the patient relapsed while still on treatment. In the second patient, the percentage of abnormal metaphases was 65% at baseline, remained at 75% after 10 months of pomalidomide therapy, but decreased to 7% after one month of treatment discontinuation for relapse; a repeat examination in this patient after another 3 months (still off therapy) showed monosomy 7 and new additional abnormalities (including 4q21q25 deletion) in 65% of metaphases evaluated. CONCLUSION:Longer-term follow-up of pomalidomide-treated patients with myelofibrosis confirm the drug's favorable side effect profile. Also, the majority of treatment responders maintain their remission, which is exclusively anemia remission, for at least one year and several remain in remission after 2 years of therapy. The drug has limited effect on splenomegaly and progressive splenomegaly might occur in some patients despite continued remission in anemia. The presence of unfavorable cytogenetic abnormalities predicts relapse. Disclosures:Gale:Celgene: Employment.

Aberrations of 6q13 mapped to the COL12A1 locus in chondromyxoid fibroma

Chondromyxoid fibroma, a rare benign bone tumor, may be mistaken for chondrosarcoma. Although cytogenetic studies of chondromyxoid fibroma are few, rearrangements of the long arm of chromosome 6, frequently expressed as an inv(6)(p25q13), are prominent. In this study, conventional cytogenetic analysis of 16 chondromyxoid fibroma samples from 14 patients revealed rearrangements of chromosome 6 in 10 of 11 clonally abnormal specimens. In addition to 6q13 rearrangements, recurrent 6p25 and 6q25 anomalies were detected. Notably, an identical t(6;9)(q25;q22) translocation was identified in two cases, suggesting that it represents a distinct translocation of chondromyxoid fibroma. In an effort to further define the aberrant 6q13 breakpoint and identify the molecular consequences, a fluorescence in situ hybridization (FISH)-based positional cloning strategy on chondromyxoid fibroma abnormal metaphase and interphase cells using a series of bacterial and plasmid artificial chromosome (BAC/PAC) probe combinations spanning a 6.1 Mb region was employed. The breakpoint on 6q13 was located within the COL12A1 gene, a collagen gene purportedly involved in another benign bone tumor, subungual exostosis. The findings of this study expand our knowledge of chromosomal alterations in chondromyxoid fibroma, identify COL12A1 as the likely gene candidate within the recurrent 6q13 breakpoint, and provide an alternative approach for detecting 6q13 anomalies in nondividing cells of chondromyxoid fibroma. The latter could potentially be utilized as an adjunct in diagnostically challenging cases.

Detection of chromosomal abnormalities associated with chronic lymphocytic leukemia: what is the best method?

B-cell chronic lymphocytic leukemia (CLL) follows a heterogeneous clinical course, for which several biological markers may predict clinical outcome. Cytogenetic aberrations are considered major prognostic indicators for predicting the survival of CLL patients. Given the difficulties in obtaining abnormal metaphases in CLL, fluorescent in situ hybridization (FISH) with specific probes is generally used to detect the most frequent abnormalities. To determine the best strategy for identifying cytogenetic abnormalities, we compared results obtained by FISH analysis on peripheral blood mononuclear cells with those obtained by FISH after culture with mitogens. We studied 46 CLL patients selected from two different institutions. The most frequent structural aberrations leading to loss of genetic material were loss of the 13q14 region, in 32 cases (70%), and loss of TP53 in 11 cases (24%). Of the 46 cases patients, 10 patients (21.7%) had deletion of the ATM locus at 11q22 and 8 patients (17%) had trisomy 12. Results could be interpreted as discordant in four cases in which the abnormal clone was small and both values were around the threshold. FISH performed on stimulated cells detected the same frequency of abnormalities as FISH performed without culture. This equivalence between the two techniques allows performing both conventional cytogenetic and FISH analyses on the same sample.

Preclinical Study of Proproten-100 for Mutagenicity

Experiments on CBA/CaLac mice showed that single and course administration of proproten-100 did not increase the percent of abnormal metaphases in red bone marrow cells and produced no genotoxic effects in Drosophila melanogaster wing cells in the test of somatic mosaicism.

The usefulness of interphase fluorescence in situ hybridization at diagnosis of myeloma in addition to metaphase cytogenetics

e19558 Background: Multiple myeloma is characterized by the accumulation of malignant plasma cells within the bone marrow and regarded as incurable, but remissions may be induced with steroids, chemotherapy, thalidomide and stem cell transplants. The clinical heterogeneity of myeloma is dictated by the cytogenetic aberrations present in the clonal plasma cells. Fluorescence in situ hybridization (FISH) overcomes the limitations of standard cytogenetics and allows for the detection of numerical and structural chromosomal abnormalities in both metaphase spreads and interphase nuclei. Methods: We evaluated the chromosome abnormalities in 34 MM patients using conventional cytogenetics and interphase FISH with 6 probes such as IGH/CCND1, IGH/FGFR3, IGH/MAF, DS13S319/LAMP1, IGH/BAP, and p53/CEP17. Results: Cytogenetic abnormalities were found in 24 (70.6%) of the 28 MM patients. 10 (35.7%) patients had abnormal metaphases by conventional cytogenetics. Interphase FISH results were abnormal in 21 (61.8%) patients and 11 (52.3%) patients had abnormal interphase FISH but normal metaphases. The evidence of the loss of D13S319 with or without loss of LAMP1 was found in 6 (21.4%) patients, and loss of p53±CEP17 for 2 patients, IGH-BAP for 9 (26.5%) patients, IGH/FGFR3 for 2 patients, and IGH/CCND1 for 7 (20.6%) patients, respectively. However, there were none positive for IGH/MAF. Chromosome 13 abnormalities and IGH rearrangement is correlated with poor clinical outcome. Conclusions: Interphase FISH can provide useful information to evaluate the presence of prognostic chromosome abnormalities in addition to metaphase cytogenetics. And it should be used in the routine evaluation of multiple myeloma. No significant financial relationships to disclose.

Mechanisms of the Formation of Multinuclear Malignant Plasma Cells in the Novel AL/MM Human Cell Lines, ALMC-1 and ALMC-2: Implications for Tumor Cell Growth Control.

Malignant plasma cell (PC) disorders are clonal diseases based on their immunoglobulin variable region sequence signature. However, intratumor phenotypic, genetic, and morphologic heterogeneity is often observed, fueling the notion that a subpopulation of the malignant PC clone may have extensive self-renewal properties whereas others may not. Little information exists regarding the relationship between these putative tumor subpopulations that is instrumental to our understanding of the growth, progression and therapeutic targeting of PC disorders. We recently described establishment of two novel cell lines from one patient initially diagnosed with primary amyloidosis (ALMC-1), who eventually progressed to a combination of AL and symptomatic multiple myeloma (MM; ALMC-2). Although both cell lines are clonally related, they exhibit a number of intriguing differences, including a marked variability in their makeup of mononuclear (MoN) and multinuclear cells (2 − >10 nuclei; MuN). The ALMC-1 cell line consistently displays a much larger fraction of MuN cells (~50%) than the ALMC-2 cell line (~20%). By contrast, the growth rate of the ALMC-2 cells is significantly higher than ALMC-1 cells suggesting an inverse relationship between growth rate and fraction of MuN cells. It has been demonstrated that MoN plasmablastic morphology is a predictor of poor survival in MM, however the clinical significance of MuN PCs, which are frequently observed in bone marrow biopsies, has yet to be determined. We therefore used the ALMC-1 and ALMC-2 cell lines to begin to test the hypothesis that the MoN fraction in both cell lines contains cells with extensive self-renewal properties whereas the MuN cells are no longer capable of cell division. The central questions become by what mechanism are MuN PCs generated and how are MoN PCs maintained? Two hypotheses that can explain the dual persistence of both types of cells are:random cell fusion;regulated or sporadic cell division of MoN cells into 2 daughter cells, one of which divides normally and the other one fails to undergo cytokinesis.To address the first possibility, each cell line was transfected with GFP or YFP. Single positive (GFP or YFP) cells were then mixed and cocultured for assessing the frequency of dual positive cells by flow cytometry at various time points. However, studies to date suggest this mechanism does not occur. To begin to address the second possibility and determine whether extrinsic factors, e.g., cytokines, may regulate the rate of generation of MuN PCs, we cultured both cell lines +/− IL-6 and IGF-I and assessed numbers of MoN and MuN PCs. Initial analyses suggest that IL-6, but not IGF-I, increases the proportion of MoN PCs in both cell lines. To address the hypothesis that MuN PCs are no longer capable of growth, cells were labeled with bromodeoxyuridine (BrDU) to assess the capability of MuN cells to synthesize DNA. We observed that MuN PCs incorporating BrDU did so uniformly in each nucleus present in the cell. We then assessed the ability of cell division of MuN cells by quantitating normal vs abnormal metaphases in both cell lines and observed that >90% of metaphases were present in MoN PCs, suggesting that MoN PCs largely give rise to MuN PCs and the latter are likely compromised in cytokinesis. As centrosomes play a critical role in regulating cell division and centrosome abnormalities have been noted in MM by others, we next used gamma-tubulin immunofluorescence to detect centrosomes. MoN PCs exhibited uniformly normal centrosome numbers and positioning while MuN PCs showed clear centrosomal abnormalities including accumulation of centrosomes, centrosome clustering, and multiple directional centrosome polarization, suggesting a mechanism by which the MuN cells may be generated. In summary, our data suggest that MuN PCs lack growth potential and that intrinsic and/or extrinsic factors may drive accumulation of these cells. These observations support the possibility that patients with a larger proportion of MuN PCs may have differences in clinical outcome from those patients with largely MoN PCs. To test this, we have begun hematopathological analysis of a large series of patients diagnosed with various plasma cell disorders. Knowledge of the signals that regulate the balance between MoN and MuN cells could lend itself toward identification of therapeutic modalities that may maximize differentiation of tumor cells into end-stage cells.

Cytogenetic Analysis of Normal Human B Cells Following CpG Stimulation: Implications for Interpretation of CpG Induced CLL Metaphase Analysis

BACKGROUND: Karyotype analysis of metaphase cells for discovering genome-wide cytogenetic abnormalities has played an important role in prognosis and counseling of patients with chronic lymphocytic leukemia (CLL). Due to the low proliferative rate of CLL B cells, karyotype analysis has been of limited clinical utility in this disease. Thus, fluorescence in situ hybridization (FISH) on interphase cells has replaced karyotype analysis for the assessment of recurrent genetic changes in CLL. Recently, immunostimulatory CpG-oligodeoxynucleotides (CpG) have been shown to induce primary CLL B cell proliferation via engagement of toll-like receptor 9 (TLR9), thereby rendering karyotype analysis feasible and providing for a useful and reliable method to discover new cytogenetic abnormalities in CLL B cells. However, we and others have shown that CpG stimulation of CLL and normal B cells also induces robust expression of activation induced cytidine deaminase (AID), a mutagenic enzyme that is linked to the development of cytogenetic abnormalities. It is therefore plausible that cytogenetic abnormalities observed following CpG stimulation of CLL B cells may not reflect de novo abnormalities but rather reflect CpG induced-AID mediated cytogenetic alterations. We began to address this possibility by assessing the effects of CpG stimulation on the karyotypes of normal blood B cells.HYPOTHESIS: Since CpG stimulation induces AID expression in CLL and normal B cells, we hypothesize that:robust B cell activation by CpG could result in cytogenetic abnormalities thereby contributing to lymphomagenesis; andif CpG induces cytogenetic abnormalities that are linked to disease initiation and/or progression, such information would be of great value in the interpretation of cytogenetic data obtained from CpG-stimulated CLL B cells.METHODS: We performed interphase FISH analysis on freshly isolated blood B cells, and metaphase karyotype analysis on CpG stimulated B cells from 17 healthy blood donors. Pure (>98%) B cells obtained by negative selection, were hybridized at isolation with a probe set to identify deletion 6q, 11q, 13q, and 17p, trisomy 12, and IGH gene rearrangements, and cells were also stimulated for 5 days with 10 μg/ml CpG (CpG 2006, synthesized in-house) plus IL-2 and IL-15 for metaphase analysis. At least 20 post-CpG metaphase spreads were analyzed for each sample and abnormalities were considered clonal using the accepted convention (gains or translocations are present in two or more cells and loss present in at least 3 cells). The expression of AID in the B cells pre- and post-CpG stimulation was assessed by RT-PCR.RESULTS: FISH analysis of freshly isolated B cells showed that all 17 subjects were normal. After CpG stimulation, all samples examined exhibited strong AID expression as revealed by RT-PCR. By karyotype analysis, none of the 17 CpG stimulated samples showed evidence of clonal abnormalities. However, one or more nonclonal chromosomal abnormalities were observed in 7 out of 17 (41.2%) samples. Among those 7 samples, 4 samples had 1 abnormal metaphase out of a minimum of 20 examined while the other 3 samples exhibited two or more cells, each with distinct abnormalities. Some of those nonclonal abnormalities have also been observed in CLL (i.e., trisomy 3, trisomy 12 and 14q32 rearrangements). This is a much higher incidence of nonclonal abnormalities than has been seen with peripheral blood cells cultured by other methods.CONCLUSION: Our data have significance for the biology of CLL and for clinical practice. First, our data suggest that signaling through TLR9, and perhaps other receptors that likewise induce robust B cell AID expression, may represent a natural mechanism by which B lineage cells acquire chromosomal abnormalities during normal immune responses that may predispose toward neoplastic transformation. Second, given that none of the CpG-induced metaphases in normal B cells resulted in clonal abnormalities in the 5 day timeframe, our data also validate the clinical utility of CpG as a valuable tool for the cytogenetic analysis of CLL B cells since only clonal abnormalities are currently scored. However, it remains possible that non-clonal abnormalities seen in CpG-stimulated CLL B cells may be relevant to the disease process if future work shows that CpG stimulation uncovers recurring non-clonal defects of chromosomes/genes associated with tumorigenic events.

Stimulation of B-Cell Lymphoproliferations with CpG-Oligonucleotide DSP30 Plus IL-2 Is More Effective than with TPA to Detect Clonal Abnormalities.

Conventional cytogenetics (CC) of B-cell lymphoproliferations remains difficult because of low mitotic in vitro activity of the leukemic cells. Therefore, mitogen stimulation of B-cells is required to analyze an adequate number of metaphases. Chromosome abnormalities using CC with TPA can be detected in up to 50% of chronic lymphocytic leukemia (CLL), but the development of FISH techniques has allowed the detection of selected chromosome abnormalities in more than 80% of CLL. However, FISH is restricted, since information is only available for the genes/loci for which probes are used. So, for a comprehensive genetic analysis, CC is essential because it provides an overview of all microscopically visible chromosome abnormalities important as prognostic factors. The use of the immunostimulatory CpG-oligonucleotide DSP30 to effectively induce cell cycle progression of CLL cells in vitro has been reported. This proliferation is markedly enhanced upon addition of Interleukine-2 to cultures. To our knowledge and to date, there has been no direct comparison of classical TPA versus DSP30+IL-2. DSP30+IL-2 stimulation has been successfully tested in CLL but no data are available for other lymphoproliferations. We cultured 132 B-cell lymphoproliferations (80 CLL and 52 other B-cell lymphoid neoplasms (BCLN)) in parallel, in presence of TPA or DSP30+IL-2. The objective of this study was to evaluate the suitability of DSP30+IL-2 as a routine B-cell mitogen for metaphase cytogenetics. CC successfully analyzed 94.9% of CLL and 98.1% of BCNL with more than 80% abnormal karyotypes. For CLL, failures of karyotypes were more frequent in cultures with DSP30+IL-2 (14%) than in those with TPA (4%). The rate of failures were similar for BCLN (6% versus 4%). For CLL, there were significantly fewer metaphases in DSP30+IL-2 than in TPA spreads (mean of 50 versus 72 metaphases per slide respectively, p=0.0007), as well as for BCLN (mean of 50 versus 71 metaphases per slide respectively, p=0.009). However, the proportion of abnormal metaphases was significantly higher in DSP30+IL-2 (mean of 59%) compared to TPA cultures (mean of 26%, p=0.00265) for CLL and for BCLN (mean of 57% versus 33%, p=0.0065). Stimulation with DSP30+IL-2 allowed to detect more abnormalities, more abnormal subclones and more complex karyotypes in CLL and in the majority of BCLN. Though FISH exploration using a large probe panel has yielded valuable results in lymphoproliferative diseases, it underestimates the heterogeneity of chromosomal aberrations. Complexity of chromosomal changes, recently associated to unfavorable outcomes, can only be assessed with CC. In conclusion, our results in both CLL and BCLN indicate that the immunostimulatory oligonucleotide DSP30 in combination with IL-2 is an easy and efficient stimulus in metaphase generation for routine chromosomal banding.

Using Combined High Density SNP/CNV Arrays as a Clinical Karyotyping Tool in Myeloid Malignancies.

We have evaluated various types of single nucleotide polymorphism arrays (SNP-A) as a karyotyping platform in over 600 cases of bone marrow failure and various myeloid disorders including MDS, MDS/MPD, and AML and in over 360 controls. We have shown that SNP-A not only reliably confirms chromosome gains and losses identified by metaphase cytogenetics (MC) but also allows for detection of previously cryptic chromosomal lesions. Moreover, through the ability of combining copy number measurement with genotyping, SNP-A also enables detection of copy-neutral loss of heterozygosity (LOH), a lesion that cannot be recognized using traditional MC. We have previously shown that this type of lesion occurs frequently in myeloid disorders. Recently, ultra-high density SNP-A were introduced (Affymetrix 6.0 array platform) with modified chemistry and a combination of SNP (>906,600) and copy number variant probes (CNV >946,000) to allow for precise delineation of the location and size of submicroscopic chromosomal defects, copy-neutral LOH and germline CNVs. This technology will likely supplant previous SNP-A platforms in performance and opens a new era in clinical cytogenetic diagnostics. In this pilot trial, we applied the Affymetrix SNP 6.0 array to samples obtained at diagnosis from 193 patients (59 AML, 115 MDS, 19 MDS/MPD). For 95 samples, both Affymetrix 250K and 6.0 arrays were applied; 6.0 SNP-A allowed for more identification of 33 regions of copy number aberrations and 11 copy-neutral LOH, suggesting a higher resolution of this technology over 250K arrays. By MC, 43% (71/167) of patients had chromosomal aberrations including a complex karyotype; 48% (81/167) had a normal karyotype and 9% (15/167) showed no growth. By 6.0 SNP-A, aberrations were detected in 78% (150/193) of patients. Significantly, among the samples with either a normal karyotype or non-informative results, aberrations were found by SNP-A in 57% and 73%, respectively. In an illustrative case with a balanced translocation by MC, copy number changes were found at the junctions, indicating that the translocation breakpoints contained small deletions. Comparison of 6.0 SNP-A results with MC showed concordance for detection of MC-defined lesions. However, 9% (15/167) of defects were only detected by MC and could not be detected by SNP 6.0 array in patients with a low proportion of abnormal metaphases. Gains and losses detected by SNP-A 6.0 required a sensitivity threshold of 20% clonal cells. We identified recurrent lesions not previously reported as being CNV. These comprised 238 regions of loss, 257 regions of gain, and 97 regions of copy-neutral LOH. When feasible (or if indicated to resolve a discordance with MC or to exclude CNV), DNA from sorted CD3+ cells was used as a reference. Recurrent losses were located in 37%(71/193) patients at 5q, 7p, 7q, 13q, 17p, 20q, 1p, 12p, 3q and 4q. Recurrent gains in 28%(55/193) patients included 13q, 11p, 11q, 21q, 3p, 8p, 8q, 9q, 19p and 5p. Somatic copy-neutral LOH was identified in 20%(39/193) patients at 13q, 1p, 5q, 6p, 7q, 11q, 17q, 3p, 4q, and 12q. Homozygous deletion was observed in one patient with a 6 Mb loss at 5q34 (not visible on MC) within a heterozygous 5q11.2–5q35.3 deletion Copy neutral LOH was found in 29% (56/193) patients overall. AML patients appeared to have more frequent copy neutral LOH(34%) than MDS (24%), but not significant (P=0.2). The 5%(9/193) of patients had more than 2 chromosomes with copy-neutral LOH. Among the samples showing a normal karyotype, 20% (16/81) of patients showed copy -neutral LOH as the sole aberration without additional copy number aberrations. To our knowledge, this is the first systematic application of SNP-A 6.0 in a large cohort of patients with myeloid malignancies. Our results demonstrate that this technology complements, but does not replace conventional MC for identification of abnormalities in myeloid neoplasia.

Effects of copper ions released from metallic copper on CHO-K1 cells

The aim of this study was to investigate the cytotoxic and genotoxic effect of copper extracts obtained from metallic copper in Chinese hamster ovary (CHO-K1) cell line using neutral red (NR), sister chromatid exchange (SCE), chromosomal aberrations (CA) and cell-cycle kinetics tests. Cells were cultured in Ham-F10 with different copper-containing extracts obtained after the immersion of copper disks for 1, 2, 3, 9, 12, 24, 48 and 72 h in culture medium. Results from cytotoxicity assay showed an inverted U-shape response evidenced in changes in lysosomal activity and mitotic index. The analysis of CA revealed an increase of abnormal metaphases for copper concentration (cCu) in the 5.67–7.42 mg/L dose-range ( p < 0.001). In addition, SCE frequencies were higher for treated cells when compared with controls in the 1.56–7.42 mg/L concentration range ( p < 0.001). The absence of metaphases indicated cytotoxicity for cCu ≥ 10.85 mg/L. Results show that cells close to copper-containing materials releasing copper ions are susceptible to cytotoxic and genotoxic effects.

Genotoxic studies in hypertensive and normotensive rats treated with amiodarone

Amiodarone, a benzofuran derivative, is a very effective antiarrhythmic medication, but has potential to cause side effects. Although its cytotoxicity potential is very well-known, there are few reports about its genotoxicity effects. Since amiodarone has not been investigated in genotoxicity studies, and the spontaneously hypertensive rat (SHR) is a well-characterized model for hypertension, the aim of the present study was to perform cytogenetic analysis on chromosome aberrations in bone marrow cells of SHRs and normotensive Wistar-Kyoto rats (WKYs) that received oral amiodarone treatment for 4 weeks. Amiodarone activity was also monitored using electrocardiograms. The presence of bradycardia in amiodarone-treated rats confirmed that this drug was really active. Metaphase analysis on bone marrow cells showed that there were significant differences in total chromosomal damage and percentage abnormal metaphase between WKY and SHR negative controls. In the SHR negative control, the frequencies of basal chromosomal aberrations and abnormal metaphases were significantly higher ( p < 0.05). There were high numbers of chromosomal aberrations in all amiodarone-treated groups, compared with negative controls. In amiodarone-treated groups, the most frequent chromosomal aberration was chromatid breaks. More chromosomal aberrations were found in WKYs that received amiodarone, with a statistically significant difference in comparison with negative controls ( p < 0.05). However, in SHR rats there was no significant difference between the amiodarone and negative groups regarding chromosomal damage induction. These results showed that treatment with amiodarone was genotoxic in WKYs, but not in SHRs. Further studies are needed to confirm whether amiodarone is genotoxic or efficient and harmless, among humans undergoing therapy.

The effect of low temperature on the microtubules in root meristem cells of spring and winter cultivars of wheat Triticum aestivum L.

We have studied the response of the interphase and mitotis microtubule arrays in root meristem cells of spring and winter cultivars of wheat Triticum aestivum L. (Moskovskaya 35 and Moskovskaya 39) during cold stress (1 h at 0 degrees C) and acclimation to cold (3-48 h at 0 degrees C). Our data show that interphase microtubules are more resistant to cold than mitotic arrays in both cultivars. During cold stress the density of endoplasmic microtubules increases in interphase cells of winter plants, yet no changes are detected in cells of spring plants. In mitotic cells of both wheat cultivars the density of microtubules within the kinetochore fibers decreases, yet this effect is more evident in the cells of spring plants. During acclimation to cold of both cultivars, we have observed the disorganization of the interphase cortical arrays and the enhanced growth of endoplasmic microtubule arrays, composed of microtubule converging centers. However, the reaction of mitotic microtubule arrays differs in the cells of winter and spring plants. In winter plants, during prophase diffuse tubulin "halo" accumulates first at perinuclear area, followed by the appearance of the microtubule converging centers. In spring plants, we have observed the formation of the prophase spindle, yet later the prophase spindle is not detected. Metaphase cells of both cultivars show similar aberrations of the mitotic spindle, accumulation of abnormal metaphases and the excessive formation of microtubule converging centers. In telophase cells of both cultivars, acclimation induces similar reaction, resulting in the disorganization of the phragmoplast and the formation of multiple microtubule converging centers. The latter are detected in the perinuclear areas of the daughter cells in winter plants and in the cortical cytoplasm of cells in spring plants. Our data point to the common pathways of microtubule response to cold treatment (0 degrees C). The excessive formation of the microtubule converging centers indicates the activation of microtubule assembly during prolonged cold treatment.

In vitro effects of 2‐methoxyestradiol on MCF‐12A and MCF‐7 cell growth, morphology and mitotic spindle formation

The influence of 2-methoxyestradiol (2ME) was investigated on cell growth, morphology and spindle formation in a tumorigenic (MCF-7) and non-tumorigenic (MCF-12A) epithelial breast cell line. Inhibition of cell growth was more pronounced in the MCF-7 cells compared to the MCF-12A cells following 2ME treatment. Dose-dependent studies (10(-5)-10(-9) M) revealed that 10(-6) M 2ME inhibited cell growth by 44% in MCF-12A cells and by 84% in MCF-7 cells (p-value < 0.05). 2ME-treated MCF-7 cells showed abnormal metaphase cells, membrane blebbing, apoptotic cells and disrupted spindle formation. These observations were either absent or less prominent in MCF-12A cells. 2ME had no effect on the length of the cell cycle between S-phase and the time a mitotic peak was reached in either cell line but MCF-7 cells were blocked in mitosis with no statistically significant alterations in the phosphorylation status of Cdc25C. Nevertheless, Cdc2 activity was significantly increased in MCF-7 cells compared to MCF-12A cells (p-value < 0.05). The results indicate that 2ME disrupts mitotic spindle formation and enhances Cdc2 kinase activity, leading to persistence of the spindle checkpoint and thus prolonged metaphase arrest that may result in the induction of apoptosis. The tumorigenic MCF-7 cells were especially sensitive to 2ME treatment compared to the normal MCF-12A cells. Therefore, differential mechanism(s) of growth inhibition are evident between the normal and tumorigenic cells.

The tubulin-depolymerising agent combretastatin-4 induces ectopic aster assembly and mitotic catastrophe in lung cancer cells H460

The relationship between microtubular dynamics, dismantling of pericentriolar components and induction of apoptosis was analysed after exposure of H460 non-small lung cancer cells to anti-mitotic drugs. The microtubule destabilising agent, combretastatin-A4 (CA-4) led to microtubular array disorganization, arrest in mitosis and abnormal metaphases, accompanied by the presence of numerous centrosome-independent "star-like" structures containing tubulin and aggregates of pericentrosomal matrix components like gamma-tubulin, pericentrin and ninein, whereas the structural integrity of centrioles was not affected by treatment. On the contrary, in condition of prolonged exposure or high concentrations of CA-4 such aggregates never formed. Treatment with 7.5 nM CA-4, which produced a high frequency "star-like" aggregates, was accompanied by mitotic catastrophe commitment characterized by translocation of the proapoptotic Bim protein to mitochondria activation of caspases-3/9 and DNA fragmentation as a result of either prolonged metaphase arrest or attempt of cells to divide. Drug concentrations which fail to block cells at mitosis were also unable to activate apotosis. A detailed time-course analysis of cell cycle arrest and apoptosis indicated that after CA-4 washout the number of metaphases with "star-like" structures decreased as a function of time and arrested cells proceeded in anaphase. After 4 h, the multiple alpha- and gamma-tubulin aggregates coalesced into two well-defined spindles in a bipolar mitotic spindle organization. Overall, our findings suggest that the maintenance of microtubular integrity plays a relevant role in stabilising the pericentriolar matrix, whose dismantling can be associated with apoptosis after exposure to microtubule depolymerising agents.

Total fertilization failure and molecular abnormalities in metaphase II oocytes

Total fertilization failures (TFF) are rare events of IVF by intracytoplasmic sperm injection (ICSI). When male factor is excluded, the lack of identifiable aetiological criteria raises the question of the reliable clinical management. The goal of this study was to identify molecular abnormalities in metaphase II (MII) oocytes yielding TFF. The nuclear mature MII oocytes mRNA expression profiles were compared between a 30-year-old patient who had experienced three successive TFF (egg number = 39) and control patients with fertile cohorts diagnosed with tubal or male infertility. The mRNA abundance for the 30,000 genes of the genome was evaluated by microarray and, for selected genes, by quantitative-polymerase chain reaction analysis. Transcriptional analysis of unfertilized MII oocytes revealed an altered gene expression profile associated with TFF. Meiosis, cell growth and apoptosis controlling genes were mis-expressed with important fold changes. The results reveal that, despite passing the pre-IVF morphological examination, high-grade oocytes may carry substantial molecular abnormalities at the gene expression level associated with failure of MII oocyte activation. In the absence of an identifiable defect causing TFF, this microarray approach allows improvement of clinical therapeutic management: informed counselling about alternate therapeutic solutions could be proposed.

A rule-based computer scheme for centromere identification and polarity assignment of metaphase chromosomes

Automatic centromere identification and polarity assignment are two key factors in the automatic karyotyping of human chromosomes. A multi-stage rule-based computer scheme has been investigated to automatically detect centomeres and determine polarities for both abnormal and normal metaphase chromosomes. The scheme first implements a modified thinning algorithm to identify the medial axis of a chromosome and extracts three feature profiles. Based on a set of pre-optimized classification rules, the scheme adaptively identifies the centromere and then assigns corresponding polarity. An image dataset of 2287 chromosomes acquired from 24 abnormal and 26 normal Giemsa metaphase cells is utilized to optimize and test the scheme. The overall accuracy is 91.4% for centromere identification and 97.4% for polarity assignment. The experimental results demonstrate that our scheme can be successfully applied to diverse chromosomes, which include those severely bent and abnormal chromosomes extracted from cancer cells.

Deletion 13q in B-CLL: Interphase FISH Reveals More 13q- Than Does CpG Stimulation.

The most common recognized cytogenetic change in B-CLL is a deletion involving band 13q14.3. Dewald found a heterozygous or homozygous 13q– in 92% of CLL patient samples using the FISH probe D13S319 that hybridizes to 13q14.3 (BJH 121:287, 2003). This contrasts with the usually normal conventional chromosome analysis (CCA) results partly because CLL cells divide infrequently but also because the 13q– is often visualized only by FISH (Stockero, Ca Genet Cytogenet 166:152, 2006). Mayr recently showed that CpG stimulation can reveal a chromosomally abnormal CLL clone in metaphase cells (Blood 107:742, 2006). The purpose of the present study was to compare the incidence of microdeletion and visible 13q deletion in CLL using interphase FISH and CCA after CpG culture on the same peripheral blood specimens.METHOD: We compared the chromosome 13 results by interphase FISH (fresh uncultured cells) and by a 20 metaphase CCA from 5–day CpG cultures. Our unselected CLL cohort (n=40) represented a typical and relatively high risk population: median age 61 (range 36–75), 48% CD38–positive, 62% ZAP–70 positive, and 48% IgVH unmutated. In addition 70% were previously treated for progressive disease, with all Rai stages represented.CpG RESULTS: By CCA of 40 patients, CpG stimulation revealed a clonal (multiple abnormal cells) or nonclonal (one abnormal cell) abnormal karyotype in 32 (80%) and a normal karyotype in 8 of the 40 patients (20%). Among the 32 abnormal cases, each chromosome 13 pair appeared normal in 17 and was abnormal in 15 (one was monosomy 13, 8 were 13q–, and 6 had a 13q translocation). CpG did not reveal a homozygous 13q abnormality in any patient. In total, CpG revealed a 13q abnormality in 15 of 40 (38%) patients (8 had multiple abnormal metaphases and 7 had only one abnormal metaphase).FISH RESULTS: By interphase FISH, 29 of 40 (73%) patients had a 13q–. The deletion was heterozygous in 18 patients, homozygous in 7, and mixed homo– and heterozygous in 4. Of the 18 with a heterozygous 13q loss by FISH, CpG revealed an abnormal 13 in only 8. Of the 11 patients with homozygous or mixed homo– and heterozygous 13q– by FISH, CpG revealed a heterozygous 13q abnormality in only 6. Of the 8 patients with a normal CpG karyotype, by FISH 5 had a 13q– in one or both 13s. Of the 9 patients with heterozygous 13q loss by CpG, the FISH result was 13q– in 4 and homozygous or mixed homo– and heterozygous 13q– in 5. Of the 6 with a chromosome 13 translocation by CpG, FISH revealed heterozygous 13q– in 4, mixed homo– and heterozygous 13q– in one, and in one case the 13q FISH result was within normal limits. In this latter case, CpG stimulation followed by metaphase FISH analysis confirmed that the apparently balanced translocation harbored a microdeletion 13q. Interphase FISH analysis confirmed the presence of a clonal deletion 13q in all 7 of the patients with a CpG metaphase result of a non-clonal (one metaphase cell) 13q abnormality.CONCLUSIONS:1.Even though CpG induces CLL metaphases in cell culture and reveals many chromosome abnormalities that cannot be identified by interphase FISH, cytogenetic evaluation of CLL by CpG alone significantly underestimates the incidence of 13q deletions.2.When a 20-cell analysis after CpG stimulation reveals a 13q abnromality in only one (apparently nonclonal) metaphase cell, concurrent interphase FISH analysis often confirms clonality for the 13q defect.

Tracking the MDS Clone in Peripheral Blood (PB) Cells from Patients (pts) with Myelodysplastic Syndrome (MDS) Undergoing Azacitidine (AZAC)-Based Therapy.

The genetic hallmark of MDS is a gain or loss of chromosomal loci identified in bone marrow (BM) cells in ∼50% of pts at diagnosis. We previously demonstrated in longitudinal chromosome study a modulating effect of chronic AZA C therapy on the MDS clone which subdivided patients into five distinct cytogenetic groups with statistically significant differences in survival (P=0.0003) (Najfeld et al, ASH 2004). Our goal was, therefore, to investigate whether PB cells can be used in substitute of BM cells for detection of genomic defects to monitor the clone during AzaC-based therapy. Chromosomal and interphase (I-) FISH studies were performed using BM and PB cells at baseline and following AzaC-based therapy. I-FISH studies were evaluated with a panel of six probes [EGR1 (5q31), D7S522 (7q31), D8Z2 (8p11.1-q11.1), MLL (11q23), Rb1 (13q14), D20S108 (20q12)]. Of the 47 pts studied, 25 (53%) had a normal karyotype and disomy for the MDS panel of six probes. The other 22 pts (47%) were cytogenetically abnormal, showing concordant results in 16 of 22 pts (73%) for cytogenetic and I-FISH genomic defect in BM cells (showing ≤20% frequency difference). The remaining 6 pts had a mean of 77% (range 37.5–100%) of abnormal metaphase cells and a mean of 42% (range 1.8–73.3%) of abnormal BM interphase nuclei. The 35% difference in frequency seen between metaphase and interphase BM cells is attributed to the proliferative advantage of metaphase cells with a complex karyotype. The frequency of the abnormal MDS-marked clone in BM and PB cells was concordant in 13 of 20 pts (65%). Hematological response of these pts was PR=1, hematological improvement=3, stable disease=2, too early for evaluation=6, and 1 patient had no hematological response after 10 months of AZA C treatment. The remaining 7 pts had a mean of 59% (range 48–68%) of abnormal BM metaphase cells and a mean of 21% (range 7–41%) of abnormal PB interphase nuclei. The MDS abnormal clone was detectable in more than two-fold higher frequency in BM compared to PB prior to treatment in 35% of pts. Preliminary sequential studies in discordant pts revealed a transition between the BM and PB cells to concordant frequencies within 4–5 months after AzaC-based therapy. These early observations suggest that monitoring AzaC-based therapy can be achieved using peripheral blood cells in 80% of pts. Remarkably, one pt with monosomy 7, a notoriously poor IPSS indicator, demonstrated a hematological improvement, full cytogenetic and 97% FISH remission, both in the BM and PB after 4 months of AzaC-based therapy. To examine AzaC's response on cell lineage involvement in MDS, purified BM and PB cells were subjected to FISH analysis before and during treatment from patients who were cytogenetically abnormal at baseline. Similar frequencies of genomic imbalances were seen in purified BM and PB derived CD34+ (97% vs. 96%) and CD15+ cells (94% vs. 98%), indicating that these cell populations had a similar response to AZAC therapy, as monitored in PB or BM, during the initial treatment period (4–5 months). Purified BM and PB-derived T-lymphocytes (CD3+/4+/8+) had normal disomic patterns before and during AzaC-based therapy, indicating that T-cells were not involved in the MDS clone in these pts. In contrast, BM and PB derived B-cells (CD19+) had slightly discordant results, showing a mean of 83% vs. 56% respectively of MDS-marked clone, indicating a trend towards greater response in PB- derived B-cells when compared to BM-derived CD19+ cells. In summary, our results demonstrates that although the MDS abnormal clone may be detected in both the BM and PB at the start of therapy, due to the proliferative advantage of the abnormal clone the optimal tissue should be bone marrow. This is the first study demonstrating that tracking the MDS-marked clone during the AZA-C therapy is feasible in peripheral blood cells.

Lymphoma and Chromosomal Instability in Artemis Deficient Mice - A Challenge for Long Term Malignancy Free Survival and Choice of Optimal Conditioning Regimen in Artemis Gene Therapy Trials.

Correction of Artemis deficiency, which causes a T−/−B−/− negative and radiosensitive type of SCID, is subject of murine gene therapy trials as an alternative treatment protocol for Hematopoietic Stem Cell Transplantation (HSCT). A conditioning regimen consisting of irradiation or busulfan in mice is indispensable for a succesfull engraftment. Gene therapy related toxicity on the basis of vector integration and conditioning regimen has been observed previously. Herein we provide evidence that Artemis deficiency harbours an additional risk factor for the development of lymphoproliferative disease. We provide in vivo evidence, that non gene corrected Artemis mice are able to develop lymphoma facilitated by irradiation and/or aging. Artemis−/− mice were irradiated at 3 Gy. Lymphoma were detected in 2 out of 10 Artemis−/− mice at the age of 4 to 6 months. Lymphoproliferative disease were exclusively of T-cell origin and arose from the thymus carrying a highly CD4+ or CD4+/CD8+ phenotype. Additionally, increased chromosomal rearrangements were detected in bone marrow cells of irradiated Artemis−/− mice compared to non irradiated control mice. Karyograms of bone marrow cells of 3 Gy-irradiated, 4 months old mice were stained with the Spectral Karyotyping (SKY) method. Analysis of 5 out of 20 metaphases revealed chromosomal translocations whereas in two out of 5 abnormal metaphases chromosome 15 was involved in the translocations. On contrary in karyograms of 4 to 6 weeks old Artemis−/− Sca1+ cells, which were used for viral transduction twenty out of twenty metaphases showed no obvious rearrangement. Even stimulation with cytokines in the course of viral transduction (SCF, FLT3L, TPO, IL6 and IL11) for five days did not increase chromosomal instability. Here we provide evidence, that Artemis−/− mice have an additional increased risk of developing hematopoietic malignancies, due to chromosomal instability which is independent of gene therapy. Human cells might not harbour the same riskfactor. However chromosomal instability in human Artemis−/− cells needs to be analysed in more detail for the choice of an optimal conditioning regimen and long term malignancy free survival in clinical trials.

Natural history of patients with myelodysplastic syndromes (MDS) with interstitial deletions of chromosome 5q detected on G-banded karyotyping

7077 Background: MDS includes a heterogenous group of hematopoietic stem cell malignancies characterized by ineffective hematopoiesis and recurring clonal cytogenetic abnormalities with poorly understood functional consequences. Interstitial deletion of chromosome 5 including band q31 is of special interest currently because of the striking effectiveness of lenalidomide therapy in patients with this lesion. Methods: We reviewed the clinical and pathological features of 175 consecutive patients with 5q31 deletions whose final diagnosis was MDS from 1996 to 2006. Patients not meeting the definition of a clonal chromosomal anomaly (i.e., only 1 abnormal metaphase) and those with &gt;20% marrow blasts (now defined as AML) were excluded. Results: The median age at diagnosis was 73 years old (range 8–91). The 5 most common MDS subtypes (WHO classification) were RAEB2 (38 cases), 5q- syndrome (33 cases), MDS NOS (26 cases), RCMD (22 cases), and RAEB1 (20 cases). Chromosomal breakpoints encountered most frequently were q13q33 (86 cases), q15q33 (38 cases), and q22q33 (14 cases). The 5q deletion was present as a sole cytogenetic anomaly in 31%, was present along with one other anomaly in 10%, and was part of complex karyotype in 59%. Median time to death from confirmation of diagnosis at our institution was only 7.2 months (range 1 month –1.8 years). Survival was greatest in those diagnosed with 5q- syndrome, but the median survival time of 1.8 years (p=0.001) was lower than reported in other series. Survival was also superior in women (7.9 months, versus 6.6 months for men, p=0.05) and those with non-complex cytogenetics (1.44 versus 0.41 years, p&lt;0.001). Poorer survival was noted in those with prior chemotherapy (median 6.6 months versus 7.6 months, p=0.01). Differences in breakpoint (p=0.55) and number of metaphases involved with the 5q abnormality (p=0.08) did not have a statistically significant impact on survival. Conclusion: Classic 5q- syndrome as defined by the WHO represents only a minority of MDS patients with interstitial 5q deletions. Although the short survival in our series compared with previous reports may be due in part to referral bias, it is also possible that deletions of chromosome 5q are less benign than previously suspected. No significant financial relationships to disclose.