Detailed analysis of therapy-driven clonal evolution of TP53 mutations in chronic lymphocytic leukemia.

In chronic lymphocytic leukemia (CLL), the worst prognosis is associated with TP53 defects with the affected patients being potentially directed to alternative treatment. Therapy administration was shown to drive the selection of new TP53 mutations in CLL. Using ultra-deep next-generation sequencing (NGS), we performed a detailed analysis of TP53 mutations' clonal evolution. We retrospectively analyzed samples that were assessed as TP53-wild-type (wt) by FASAY from 20 patients with a new TP53 mutation detected in relapse and 40 patients remaining TP53-wt in relapse. Minor TP53-mutated subclones were disclosed in 18/20 patients experiencing later mutation selection, while only one minor-clone mutation was observed in those patients remaining TP53-wt (n=40). We documented that (i) minor TP53 mutations may be present before therapy and may occur in any relapse; (ii) the majority of TP53-mutated minor clones expand to dominant clone under the selective pressure of chemotherapy, while persistence of minor-clone mutations is rare; (iii) multiple minor-clone TP53 mutations are common and may simultaneously expand. In conclusion, patients with minor-clone TP53 mutations carry a high risk of mutation selection by therapy. Deep sequencing can shift TP53 mutation identification to a period before therapy administration, which might be of particular importance for clinical trials.

Frequent chromatin rearrangements in myelodysplastic syndromes--what stands behind?

Myelodysplastic syndromes (MDS) represent a clinically and genetically heterogeneous group of clonal haematopoietic diseases characterized by a short survival and high rate of transformation to acute myeloid leukaemia (AML). In spite of this variability, MDS is associated with typical recurrent non-random cytogenetic defects. Chromosomal abnormalities are detected in the malignant bone-marrow cells of approximately 40-80 % of patients with primary or secondary MDS. The most frequent chromosomal rearrangements involve chromosomes 5, 7 and 8. MDS often shows presence of unbalanced chromosomal changes, especially large deletions [del(5), del(7q), del(12p), del(18q), del(20q)] or losses of whole chromosomes (7 and Y). The most typical cytogenetic abnormality is a partial or complete deletion of 5q- that occurs in roughly 30 % of all MDS cases either as the sole abnormality or in combination with other aberrations as a part of frequently complex karyotypes. The mechanisms responsible for the formation of MDS-associated recurrent translocations and complex karyotypes are unknown. Since some of the mentioned aberrations are characteristic for several haematological malignancies, more general cellular conditions could be expected to play a role. In this article, we introduce the most common rearrangements linked to MDS and discuss the potential role of the non-random higher-order chromatin structure in their formation. A contribution of the chromothripsis - a catastrophic event discovered only recently - is considered to explain how complex karyotypes may occur (during a single event).

[Chromosome banding analysis of peripheral blood lymphocytes stimulated with IL2 and CpG oligonucleotide DSP30 in patients with chronic lymphocytic leukemia]. / Cytogenetické vysetrení lymfocytu periferní krve po stimulaci IL2 a CpG oligonukleotidem DSP30 u pacientu s chronickou lymfocytární leukemií

BACKGROUND: Chromosomal aberrations play an important role as prognostic factors in chronic lymphocytic leukemia (CLL). These aberrations are mostly detected by fluorescent in situ hybridization (FISH), as chromosomal banding analysis has been scarce due to low proliferative activity of malignant B-lymphocytes in vitro. In 2006, a new method using stimulation with IL-2 and CpG oligonucleotide DSP30 for metaphase generation in CLL was published [1]. The objective of our study was to verify the efficacy of stimulation and to evaluate if the method is suitable for routine diagnostics. PATIENTS AND METHODS: In total, peripheral blood samples of 369 CLL patients were analyzed in parallel by chromosomal banding analysis and by FISH probes for 13q14, 11q22-23, CEP12 and 17p13. RESULTS: Out of 369 patients, 307 (83%) were successfully stimulated for metaphase generation. Chromosomal aberrations were detected in 243 (79%) out of 307 patients evaluated by chromosomal banding analysis. Other aberrations that are not included into standard FISH panel were detected in patients karyotypes, e.g. del(6q), del(14q), t(14;18)(q32;q21), t(11;14)(q13;q32) and t(18;22)(q21;q11). One hundred and three (42%) patients showed complex aberrant karyotype not detected by FISH analysis. CONCLUSION: Stimulation with IL-2 and oligonucleotide DSP30 is an efficient method how to induce proliferation of malignant B-lymphocytes and allows detection of a substantial number of chromosomal aberrations in addition to those detected by standard FISH panel. Using this method in routine diagnostics is helpful particularly in identification of patients with complex aberrant karyotype.

Dasatinib in imatinib-resistant or -intolerant CML patients: data from the clinical practice of 6 hematological centers in the Czech Republic.

Dasatinib is effective second line treatment for patients with chronic myeloid leukemia (CML) resistant or intolerant to imatinib. We report here the first experiences with dasatinib therapy in 71 CML patients resistant or intolerant to imatinib from the real clinical practice of 6 hematological centers in the Czech Republic. Dose 100 mg daily and 70 mg twice daily was administered to patients with chronic phase (CP) and advanced phases (AP) CML. In chronic phase (n=46), complete hematological reponse (CHR) was achieved in 97%, major cytogenetic reponse (MCgR) in 77% and complete cytogenetic response (CCgR) in 67%. Major molecular reponse (MMR) was achieved in 19/31 patients in median of 10 months. In advanced phase (n=25), CHR was attained in 77%, MCgR in 39%, CCgR in 33% and MMR in 2/18 patients. Eleven different baseline mutations were followed up in 15 patients. Dasatinib eliminated mutations in most of the patients, but 3 patients acquired a new one. Novel mutations were detected under dasatinib therapy in 2 patients. Dasatinib was well tolerated, cytopenias were common and was managed by dose modification. The estimated progression free survival (PFS) at 12 months was 97+/-3% in CP and 62+/-21% in AP. The median time to treatment failure was 605 days in AP while it was not reached in CP patients. Our clinical experiences, described here, confirmed that dasatinib is associated with high response rates especially in imatinib resistant or intolerant CML patients in chronic phase.

Negative prognostic significance of two or more cytogenetic abnormalities in multiple myeloma patients treated with autologous stem cell transplantation.

Malignant plasma cells in multiple myeloma (MM) are frequently characterized by complex karyotypes and chromosome instability. These cytogenetic changes are considered important prognostic indicators in MM patients. We have studied samples from 68 patients with newly diagnosed MM who were treated with high-dose chemotherapy and autologous stem cell transplantation. G-banding revealed abnormal karyotypes in 14 of 55 patients (25%) who had informative conventional cytogenetics. The combination of cytoplasmic immunoglobulin light chain labeling and interphase fluorescent in situ hybridization (cIg-FISH) revealed the presence of genetic aberrations in 53 of 68 patients (78%). Chromosome 13 abnormalities were found in 33 patients (50%) and IgH rearrangements in 36 patients (56.25%). In IgH positive patients we performed subsequent examinations of IgH affecting translocations t(4;14) and t(11;14) and we found translocation t(11;14) in 8 patients (12.5%) and t(4;14) in 10 patients (15.5%). The occurrences of others chromosomal abnormalities with known prognostic impact in MM were as follows: del(17)(p13) was present in 5 patients (9.8%) and gain 1q21 in 14 patients (36%). Analysis of survival of patients with different cytogenetic abnormalities revealed shorter overall survival (OS) in patients with IgH rearrangements (p=0.020) and trend to shorter OS in patients with gain 1q21 (p=0.064), respectively. Remarkably, patients with two or more aberrations had significantly shorter overall survival (p=0.001), time to progression (p=0.036) and progression free survival (p=0.008). Our results show a high incidence of chromosomal abnormalities in MM patients and confirm the prognostic impact of selected chromosomal aberrations as well as cumulative effect of multiple cytogenetic changes occurring simultaneously.

[Radiotherapy induced glioblastoma in a child previously treated for cerebellar medulloblastoma (case report and review of the literature)]. / Radioterapií indukovaný glioblastom u dítete léceného pro meduloblastom mozecku (kasuistika a prehled literatury).

Malignant gliomas represent the most frequent radiotherapy induced ("secondary") solid tumor. Their prognosis remains extremely poor despite of aggressive multimodal treatment. We present a case report of a 16 years old boy who developed cerebellar glioblastoma six years following the combined treatment for medulloblastoma. Clinical history, pathological and cytogenetic findings of the case are discussed along with treatment possibilities.

Efficacy of high-resolution comparative genomic hybridization (HR-CGH) in detection of chromosomal abnormalities in children with acute leukaemia.

The efficient detection of chromosomal aberrations in childhood acute leukaemias presents a significant component in the diagnostics of this frequent malignant disease. We used comparative genomic hybridization (CGH) and high-resolution comparative genomic hybridization (HR-CGH) to determine the frequency of chromosomal changes in 33 children with acute leukaemia (AL). The yields of chromosomal abnormalities were compared with the results obtained using conventional cytogenetics (G-banding) and fluorescence in situ hybridization (FISH). Conventional cytogenetics revealed chromosomal changes in 17 (52 %) of studied patients. The employment of FISH together with G-banding analysis identified chromosomal changes in 27 (82 %) of the AL patients investigated. CGH detected changes in DNA copy numbers in 24 (73 %) patients, 40 losses and 67 gains were found in total. HR-CGH disclosed 98 losses and 97 gains in 26 (79 %) patients. In comparison with CGH, HR-CGH analyses unveiled 88 new chromosomal aberrations: 58 losses and 30 gains. The most commonly gained chromosomes were 21 (22.5 %), X (15 %), 18 (12,5 %) and 17 (10 %). The most common losses involved sub-regions or arms of chromosomes 7 (15 %), 9 (12.5 %), 16, 19 and 1 (10 % each). Cytogenetic and molecular cytogenetic analyses of 33 childhood acute leukaemias revealed chromosomal changes in total 31 (94 %) patients. The evaluation of HR-CGH sensitivity proved that the minimal cell population of malignant cells in which a certain chromosomal change could be found was close to the 20 - 30 % level. Our results confirm the benefits of HR-CGH in detecting chromosomal changes in childhood AL. Supplementing G-banding and FISH with the HR-CGH diagnostic method increases the detection of unbalanced structural chromosomal rearrangements and can reveal small cell clones with gains and losses of whole chromosomes in hyperdiploid AL.

The optimization of sample treatment for spectral karyotyping with applications for human tumour cells.

Spectral karyotyping (SKY) represents an important tool for the investigation of the complex chromosomal rearrangements (CCRs) in many human malignancies which may be difficult to characterize by conventional banding techniques. The main goal of our work was to optimize the most important steps in the preparation of molecular cytogenetic slides for a SKY protocol. This approach consisted of optimization of both the aging procedure and protease pretreatment of the slides, with special regard given to the preservation of chromosome structure and shape, as well as to the intensity of hybridization signals. The best results were obtained with a chemical aging procedure using SSC or ethanol in combination with trypsin pretreatment applied at a higher concentration for a shorter period of pretreatment. A resulting protocol for SKY also applicable to human solid tumour cells was subsequently proposed. The practical potential of the SKY technique was demonstrated on examples of two types of human embryonal tumours--neuroblastoma and Wilms' tumour, in which some kinds of chromosomal aberrations were not detectable by means of classic cytogenetic methods.

[The significance and current diagnostic options of cytogenetic changes in multiple myeloma]. / Význam a soucasné moznosti diagnostiky cytogenetických zmen u mnohocetného myelomu.

Multiple myeloma is a malignant disease caused by malign transformation of B-cells, their clonal proliferation and the accumulation of myeloma cells in the bone marrow. These cells are set apart by a pronounced genetic instability. Chromosomal abnormalities are probably the most important prognostic factors in myeloma which influence the division of the patients into individual sub-groups each with a different developmental process of the disease and thus a different approach during treatment. The study summarizes the methodological approaches and current options in classical and molecular cytogenetics during the examination of cytogenetic changes in multiple myeloma. It presents the most common types of numerical and structural chromosomal changes found in the karyotype of multiple myeloma patients, and their prognostic importance.

[Comparison of standard prognostic factors with the deletion of 13q14 detected by interphase fluorescence in situ hybridization on separated and unseparated bone marrow cells in multiple myeloma]. / Porovnání standardních prognostických faktoru u pacientu s mnohocetným myelomem s delecí 13q14 stanovenou metodou interfázní fluorescencní in situ hybridizace na separovaných a neseparovaných bunkách kostní direne.

BACKGROUND: Cytogenetic abnormalities of chromosome 13 are emerging as important prognostic factors in multiple myeloma and have been associated with poor prognosis. METHODS AND RESULTS: The occurrence of 13q14 deletion and other standard laboratory parameters were determined in 40 patients with multiple myeloma. We found that interphase fluorescence in situ hybridization using a locus specific probe for RB1 gene on immunomagnetically selected myeloma cells was more sensitive than non selected cells. The 13q14 deletion was found in 10 of 40 (25.0%) of bone marrow samples without cell selection and in 25 of 40 (62.5%) of samples with CD138+ enriched myeloma cells. Negative correlation was found between albumin and the 13q14 deletion in separated (p = 0.003) as well as in cells without selection (p = 0.010). No significant correlation was found in overall survival of separated and unseparated cells (p = 0.830; p = 0.260) and a similar result was obtained for treatment response after transplantation of separated cells (p = 0.520) or non-separated cells (0.190). CONCLUSIONS: Our results confirm that immunomagnetic selection of CD138+ cells increases the probability of detection of the 13q14 deletion in bone marrow samples. The correlation was found between albumin and the 13q14 deletion in both of type of cells.

Location of the BCR/ABL fusion genes on both chromosomes 9q34 in Ph negative chronic myeloid leukemia.

We present two patients with Ph-negative chronic myeloid leukemia (CML) and fusion signal BCR/ABL on both chromosomes 9, located in region 9q34. The first case was a 27 years old man with CML. Molecular studies (RT-PCR) revealed the rearrangement in the major-BCR region and expression of chimeric BCR/ABL mRNA of b3a2 configuration. By classical cytogenetic studies (G-banding) karyotype 46,XY was found in short-term cultivated bone marrow cells and phytohemagglutinin (PHA) stimulated peripheral lymphocytes. FISH studies revealed the BCR/ABL fusion signals on both chromosomes 9 and green BCR signals on both chromosomes 22 in all mitoses studied. Detection of the alleles of ABL1 intragenic STR locus by fluorescence PCR followed by fragmentation analysis in the patient and his parents provided no information about transmission of the ABL gene. Quantitative assessment of BCR/ABL transcript level by RT-PCR showed 60 and 70% BCR/ABL positivity in two peripheral blood samples at 6,5 and 10,5 months after diagnosis, respectively, which does not correspond to the expression from two identical BCR/ABL hybrid genes. Therefore, the possible mechanism of the origin of two BCR/ABL fusion signals present on both chromosomes 9 could not be resolved and remains speculative. The second case was a 53 years old male with diagnosis of chronic phase of CML, with first sign of acceleration one month after diagnosis and death because of sepsis in blastic phase within four months. The cytogenetic findings were identical to those in case No. 1., i.e. karyotype 46, XY by G-banding, two BCR/ABL fusion signals on both chromosomes 9 and RT-PCR molecular studies proved b3a2 breakpoints. It is generally accepted that prognosis of the patients with fused BCR/ABL gene located on chromosome 9 is poor. The presence of two fused genes could be anticipated as two Ph chromosomes in accelerated and blastic phases of the disease. However, in our study, quantitative findings of BCR/ABL transcripts did not corresponded to the expression of two BCR/ABL genes originating from duplication. If this assumption is correct then the expression of both fused genes BCR/ABL was in case No. 1 equally suppressed and total expression reached about the level of one BCR/ABL gene.