Alwin Krämer

Centrosome replication, genomic instability and cancer.

Karyotypic alterations, including whole chromosome loss or gain, ploidy changes, and a variety of chromosome aberrations are common in cancer cells. If proliferating cells fail to coordinate centrosome duplication with DNA replication, this will inevitably lead to a change in ploidy, and the formation of monopolar or multipolar spindles will generally provoke abnormal segregation of chromosomes. Indeed, it has long been recognized that errors in the centrosome duplication cycle may be an important cause of aneuploidy and thus contribute to cancer formation. This view has recently received fresh impetus with the description of supernumerary centrosomes in almost all solid human tumors. As the primary microtubule organizing center of most eukaryotic cells, the centrosome assures symmetry and bipolarity of the cell division process, a function that is essential for accurate chromosome segregation. In addition, a growing body of evidence indicates that centrosomes might be imortant for initiating S phase and completing cytokinesis. Centrosomes undergo duplication precisely once before cell division. Recent reports have revealed that this process is linked to the cell division cycle via cyclin-dependent kinase (cdk) 2 activity that couples centriole duplication to the onset of DNA replication at the G1/S phase transition. Alterations in G1/S phase regulating proteins like the retinoblastoma protein, cyclins D and E, cdk4 and 6, cdk inhibitors p16INK4A and p15INK4B, and p53 are among the most frequent aberrations observed in human malignancies. These alterations might not only lead to unrestrained proliferation, but also cause karyotypic instability by uncontrolled centrosome replication. Since several excellent reports on cell cycle regulation and cancer have been published, this review will focus on the role of centrosomes in cell cycle progression, as well as causes and consequences of aberrant centrosome replication in human neoplasias.

Addition of sorafenib versus placebo to standard therapy in patients aged 60 years or younger with newly diagnosed acute myeloid leukaemia (SORAML): a multicentre, phase 2, randomised controlled trial

BACKGROUNDnPreclinical data and results from non-randomised trials suggest that the multikinase inhibitor sorafenib might be an effective drug for the treatment of acute myeloid leukaemia. We investigated the efficacy and tolerability of sorafenib versus placebo in addition to standard chemotherapy in patients with acute myeloid leukaemia aged 60 years or younger.nnnMETHODSnThis randomised, double-blind, placebo-controlled, phase 2 trial was done at 25 sites in Germany. We enrolled patients aged 18-60 years with newly diagnosed, previously untreated acute myeloid leukaemia who had a WHO clinical performance score 0-2, adequate renal and liver function, no cardiac comorbidities, and no recent trauma or operation. Patients were randomly assigned (1:1) to receive two cycles of induction therapy with daunorubicin (60 mg/m(2) on days 3-5) plus cytarabine (100 mg/m(2) on days 1-7), followed by three cycles of high-dose cytarabine consolidation therapy (3 g/m(2) twice daily on days 1, 3, and 5) plus either sorafenib (400 mg twice daily) or placebo on days 10-19 of induction cycles 1 and 2, from day 8 of each consolidation, and as maintenance for 12 months. Allogeneic stem-cell transplantation was scheduled for all intermediate-risk patients with a sibling donor and for all high-risk patients with a matched donor in first remission. Computer-generated randomisation was done in blocks. The primary endpoint was event-free survival, with an event defined as either primary treatment failure or relapse or death, assessed in all randomised patients who received at least one dose of study treatment. We report the final analysis. This trial is registered with ClinicalTrials.gov, number NCT00893373, and the EU Clinical Trials Register (2008-004968-40).nnnFINDINGSnBetween March 27, 2009, and Nov 28, 2011, 276 patients were enrolled and randomised, of whom nine did not receive study medication. 267 patients were included in the primary analysis (placebo, n=133; sorafenib, n=134). With a median follow-up of 36 months (IQR 35·5-38·1), median event-free survival was 9 months (95% CI 4-15) in the placebo group versus 21 months (9-32) in the sorafenib group, corresponding to a 3-year event-free survival of 22% (95% CI 13-32) in the placebo group versus 40% (29-51) in the sorafenib group (hazard ratio [HR] 0·64, 95% CI; 0·45-0·91; p=0·013). The most common grade 3-4 adverse events in both groups were fever (71 [53%] in the placebo group vs 73 [54%] in the sorafenib group), infections (55 [41%] vs 46 [34%]), pneumonia (21 [16%] vs 20 [14%]), and pain (13 [10%] vs 15 [11%]). Grade 3 or worse adverse events that were significantly more common in the sorafenib group than the placebo group were fever (relative risk [RR] 1·54, 95% CI 1·04-2·28), diarrhoea (RR 7·89, 2·94-25·2), bleeding (RR 3·75, 1·5-10·0), cardiac events (RR 3·46, 1·15-11·8), hand-foot-skin reaction (only in sorafenib group), and rash (RR 4·06, 1·25-15·7).nnnINTERPRETATIONnIn patients with acute myeloid leukaemia aged 60 years or younger, the addition of sorafenib to standard chemotherapy has antileukaemic efficacy but also increased toxicity. Our findings suggest that kinase inhibitors could be a useful addition to curative treatment for acute myeloid leukaemia. Overall survival after long-term follow-up and strategies to reduce toxicity are needed to determine the future role of sorafenib in treatment of this disease.nnnFUNDINGnBayer HealthCare.

Centrosome aberrations as a possible mechanism for chromosomal instability in non-Hodgkin's lymphoma

Recently, centrosome aberrations have been described as a possible cause of aneuploidy in many solid tumors. To investigate whether centrosome aberrations occur in non-Hodgkins lymphoma (NHL) and correlate with histologic subtype, karyotype, and other biological disease features, we examined 24 follicular lymphomas (FL), 18 diffuse large-B-cell lymphomas (DLCL), 33 mantle cell lymphomas (MCL), and 17 extranodal marginal zone B-cell lymphomas (MZBCL), using antibodies to centrosomal proteins. All 92 NHL displayed numerical and structural centrosome aberrations as compared to nonmalignant lymphoid tissue. Centrosome abnormalities were detectable in 32.3% of the cells in NHL, but in only 5.5% of lymphoid cells from 30 control individuals (P<0.0001). Indolent FL and MZBCL contained only 25.8 and 28.8% cells with abnormal centrosomes. In contrast, aggressive DLCL and MCL harbored centrosome aberrations in 41.8 and 35.0% of the cells, respectively (P<0.0001). Centrosomal aberrations correlated to lymphoma grade, mitotic, and proliferation indices, but not to the p53 labeling index. Importantly, diploid MCL contained 31.2% cells with abnormal centrosomes, while tetraploid samples harbored centrosome aberrations in 55.6% of the cells (P<0.0001). These results indicate that centrosome defects are common in NHL and suggest that they may contribute to the acquisition of chromosomal instability typically seen in NHL.

JAK2-V617F mutation in a patient with Philadelphia-chromosome-positive chronic myeloid leukaemia

In July, 2000, a 50-year-old man presented with leukocytosis and splenomegaly (21 cm). Leucocyte concentration was 93×109/L, haemoglobin 150 g/L, and platelets 345×109/L (figure 1). A differential blood count showed 54% neutrophils, 2% lymphocytes, 13% myelocytes, 7% metamyelocytes, 2% promyelocytes, 1% blasts, and 7% basophils. Lactate dehydrogenase (LDH) concentration was increased at 484 U/L. 17 months previously, the blood count had been in the normal range. Bone-marrow aspiration was dry and bone-marrow biopsy showed marked myeloid hyperplasia, increased megakaryopoiesis, and the beginning of fibrosis. Cytogenetic analysis revealed a chromosome translocation (t[9;22][q34;q11]) in all ten metaphases examined. Expression of B3A2 BCR-ABL mRNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR) in peripheral-blood leucocytes.

Blastic Plasmacytoid Dendritic Cell Neoplasia (BPDC) in Elderly Patients: Results of a Treatment Algorithm Employing Allogeneic Stem Cell Transplantation with Moderately Reduced Conditioning Intensity

Blastic plasmacytoid dendritic cell neoplasm (BPDC), formerly known as blastic NK cell lymphoma, is a rare hematopoietic malignancy preferentially involving skin, bone marrow, and lymph nodes. The overall prognosis of BPDC is dismal, with a median overall survival (OS) of only 12 to 14 months despite aggressive chemotherapy. Anecdotal reports suggest that younger patients might benefit from myeloablative therapy with autologous or allogeneic stem cell transplantation (alloSCT). However, with a median age at diagnosis beyond 60 years, BPDC primarily affects elderly patients. Here, we present for the first time evidence that also in elderly patients, alloSCT for BPDC is feasible and may result in sustained remission if conditioning with moderately reduced intensity is used. Between 2006 and 2009, 6 patients were treated at our institution who fulfilled the diagnostic criteria for BPDC. Median age was 67 (range: 55-80) years. All responded to acute leukemia-type induction therapy. Whereas 2 patients who were ineligible for alloSCT rapidly died of disease recurrence, 4 patients underwent alloSCT from unrelated donors as part of first-line (n = 1) or salvage treatment (n = 3). Two patients allografted in remission live disease free 57 and 16 months post-alloSCT, whereas 2 patients transplanted with active disease achieved complete remission but relapsed 6 and 18 months after transplantation, respectively. In conclusion, reduced-intensity conditioning (RIC) alloSCT from unrelated donors is feasible and seems to be effective in elderly patients with BPDC, suggesting that alloSCT should be pursued aggressively in patients with this otherwise fatal disease up to 70 years of age.

Gene expression patterns in acute myeloid leukemia correlate with centrosome aberrations and numerical chromosome changes.

Centrosomes, which mediate accurate chromosome segregation during mitosis, undergo duplication precisely once per cell division at the G1/S boundary. Recently, we described centrosome aberrations as a possible cause of aneuploidy in acute myeloid leukemia (AML) and found a correlation of the percentage of cells carrying abnormal centrosomes to their cytogenetic risk profile. To elucidate the molecular events responsible for the development of centrosome aberrations in AML, tumor RNA of 29 AML samples was hybridized to cDNA microarrays. The microarrays comprised some 2800 different genes with relevance to hematopoiesis, tumorigenesis and mitosis and included a set of 359 centrosome-associated genes. We identified two gene expression signatures, which allowed an accurate classification according to the extent of centrosome aberrations and the ploidy status in 28 of 29 patients each. Specifically, 18 genes were present in both signatures, including genes that code for cell cycle regulatory proteins (cyclin A2, cyclin D3, cyclin H, CDK6, p18INK4c, p21Cip1, PAK1) and centrosome-associated proteins (pericentrin, α2-tubulin, NUMA1, TUBGCP2, PRKAR2A). In conclusion, the high expression of centrosome-associated genes matches the description of centrosome aberrations in several tumor types. Moreover, in AML the identification of G1/S-phase stimulatory genes suggests that one mechanism of aneuploidy induction might be the deregulation of centrosome replication at the G1/S boundary.

Chromosomal instability correlates with poor outcome in patients with myelodysplastic syndromes irrespectively of the cytogenetic risk group

Chromosomal instability (CIN), defined by an elevated frequency of the occurrence of novel chromosomal aberrations, is strongly implicated in the generation of aneuploidy, one of the hallmarks of human cancers. As for aneuploidy itself, the role of CIN in the evolution and progression of malignancy is a matter still open to debate. We investigated numerical as well as structural CIN in primary CD34‐positive cells by determining the cell‐to‐cell variability of the chromosome content using fluorescence‐in situ‐hybridization (FISH). Thereby, CIN was measured in 65 patients with myelodysplastic syndromes (MDS), acute myeloid leukaemia (AML) and control subjects. Among MDS patients, a subgroup with elevated levels of CIN was identified. At a median follow‐up of 17.2 months, all patients within this ‘high CIN’ subgroup had died or progressed to AML, while 80% of MDS patients with normal CIN levels had stable disease (P < 0.001). Notably, there was no statistically significant difference between ‘normal CIN’ and ‘high CIN’ MDS patients regarding established risk factors. Hence, elevated CIN levels were associated with poor outcome, and our method provided additional prognostic information beyond conventional cytogenetics. Furthermore, in all three MDS patients for whom serial measurements were available, development of AML was preceded by increasing CIN levels. In conclusion, elevated CIN levels may be valuable as an early indicator of poor prognosis in MDS, hence corroborating the concept of CIN as a driving force in tumour progression.

Centrosome aberrations in hematological malignancies

As the primary microtubule organizing center of most eukaryotic cells, centrosomes play a fundamental role in proper formation of the mitotic spindle and subsequent chromosome separation. Normally, the single centrosome of a G1 cell duplicates precisely once prior to mitosis in a process that is intimately linked to the cell division cycle via cyclin‐dependent kinase (cdk) 2 activity that couples centrosome duplication to the onset of DNA replication at the G1/S transition. Accurate control of centrosome duplication is critical for symmetric mitotic spindle formation and thereby contributes to the maintenance of genome integrity. Numerical and structural centrosome abnormalities are hallmarks of almost all solid tumors and have been implicated in the generation of multipolar mitoses and chromosomal instability. In addition to solid neoplasias, centrosome aberrations have recently been described in several different hematological malignancies like acute myeloid leukemias, myelodysplastic syndromes, Hodgkins as well as non‐Hodgkins lymphomas, chronic lymphocytic leukemias and multiple myelomas. In analogy to many solid tumors a correlation between centrosome abnormalities on the one hand and karyotype aberrations as well as clinical aggressiveness on the other hand seems to exist in myeloid malignancies, chronic lymphocytic leukemias and at least some types of non‐Hodgkins lymphomas. Molecular mechanisms responsible for the development of centrosome aberrations are just beginning to be unraveled. In general, two models with distinct functional consequences can be envisioned. First, centrosome aberrations can arise as a consequence of abortive mitotic events and impaired cytokinesis. Second, evidence has been provided that centrosome amplification can also precede genomic instability and arise in normal, diploid cells. Accordingly, this review will focus on recent advances in the understanding of both, causes and consequences of centrosome aberrations in hematological malignancies.

Clonal Heterogeneity As Detected by Metaphase Karyotyping Is an Indicator of Poor Prognosis in Acute Myeloid Leukemia

PURPOSEnIn acute myeloid leukemia (AML), studies based on whole-genome sequencing have shown genomic diversity within leukemic clones. The aim of this study was to address clonal heterogeneity in AML based on metaphase cytogenetics.nnnPATIENTS AND METHODSnThis analysis included all patients enrolled onto two consecutive, prospective, randomized multicenter trials of the Study Alliance Leukemia. Patients were newly diagnosed with non-M3 AML and were fit for intensive chemotherapy.nnnRESULTSnCytogenetic subclones were detected in 418 (15.8%) of 2,639 patients from the whole study population and in 418 (32.8%) of 1,274 patients with aberrant karyotypes. Among those, 252 karyotypes (60.3%) displayed a defined number of distinct subclones, and 166 (39.7%) were classified as composite karyotypes. Subclone formation was particularly frequent in the cytogenetically adverse group, with subclone formation in 69.0%, 67.1%, and 64.8% of patients with complex aberrant, monosomal, and abnl(17p) karyotypes (P < .001 each). Two-subclone patterns typically followed a mother-daughter evolution, whereas for ≥ three subclones, a branched pattern prevailed. In non-core binding factor AML, subclone formation was associated with inferior event-free and overall survival and was confirmed as an independent predictor of poor prognosis in multivariate analysis. Subgroup analysis showed that subclone formation adds prognostic information particularly in the cytogenetic adverse-risk group. Allogeneic stem-cell transplantation improved the prognosis of patients with subclone karyotypes as shown in landmark analyses.nnnCONCLUSIONnCytogenetic subclones are frequent in AML and permit tracing of clonal evolution and architecture. They bear prognostic significance with clonal heterogeneity as an independent adverse prognostic marker in cytogenetically adverse-risk AML.

Enzymatic assay for quantitative analysis of ( d )-2-hydroxyglutarate

Levels of (d)-2-hydroxyglutarate [D2HG, (R)-2-hydroxyglutarate] are increased in some metabolic diseases and in neoplasms with mutations in the isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) genes. Determination of D2HG is of relevance to diagnosis and monitoring of disease. Standard detection methods of D2HG levels are liquid-chromatography–mass spectrometry or gas-chromatography–mass spectrometry. Here we present a rapid, inexpensive and sensitive enzymatic assay for the detection of D2HG levels. The assay is based on the conversion of D2HG to α-ketoglutarate (αKG) in the presence of the enzyme (d)-2-hydroxyglutarate dehydrogenase (HGDH) and nicotinamide adenine dinucleotide (NAD+). Determination of D2HG concentration is based on the detection of stoichiometrically generated NADH. The quantification limit of the enzymatic assay for D2HG in tumor tissue is 0.44xa0μM and in serum 2.77xa0μM. These limits enable detection of basal D2HG levels in human tumor tissues and serum without IDH mutations. Levels of D2HG in frozen and paraffin-embedded tumor tissues containing IDH mutations or in serum from acute myeloid leukemia patients with IDH mutations are significantly higher and can be easily identified with this assay. In conclusion, the assay presented is useful for differentiating basal from elevated D2HG levels in tumor tissue, serum, urine, cultured cells and culture supernatants.