Detection Of Chromosomal Aberrations Research Articles

Translocation (2;11)(q37;q23) in therapy-related myelodysplastic syndrome after treatment for acute promyelocytic leukemia

Treatment of acute promyelocytic leukemia (APL) with a combination of anthracycline-based chemotherapy and all- trans retinoic acid (ATRA) leads to very high rates of complete remission and survival. There are only a limited number of publications on the development of therapy-related myelodysplastic syndrome (MDS) or acute myeloid leukemia during follow-up of APL. Although drugs targeting at DNA-topoisomerase II characteristically induce translocations involving 11q23, this was seldom seen in patients treated for APL. We report on a patient initially diagnosed with APL. Response to therapy was monitored by fluorescence in situ hybridization (FISH) and reverse-transcriptase polymerase chain reaction for the PML-RARα rearrangement. Consecutive samples showed a swift and complete reduction of PML-RARα rearranged cells. Twenty months after diagnosis, however, conventional cytogenetics revealed a complex karyotype with a translocation involving 11q23 and loss of chromosomes 7q and Xq. FISH analysis with the MLL probe identified 2q37 (harboring the SEPT2 gene) as the translocation partner of chromosome 11. We consider the rather unique t(2;11)(q37;q23) as the primary event causing therapy-related MDS in our patient. This case stresses the importance of conventional karyotyping to be performed on a regular basis in all treated APL patients for the early detection of chromosomal aberrations that indicate the development of therapy-related MDS or acute myeloid leukemia.

C021 SNP-array karyotyping complements routine metaphase cytogenetics in the detection of chromosomal aberrations including uniparental disomy in MDS

C021 SNP-array karyotyping complements routine metaphase cytogenetics in the detection of chromosomal aberrations including uniparental disomy in MDS

Comparison of Nicolaides’ risk evaluation for down’s syndrome with a novel software: an analysis of 1,463 cases

The individual risk assessment of fetal Down's syndrome based on measurement of nuchal translucency (NT) according to Nicolaides, optionally complemented by the determination of PAPP-A and free beta HCG has progressively supplanted other search strategies for fetal aneuploidies. It could be shown that this diagnostic strategy equally detects other numeric aneuploidies at a comparable rate. A positive test result is also predictive for the presence of a fetal malformation. In this field, several computer programs are available for clinical use. The objective of our study was to re-evaluate the first consecutive 1463 NT-risk calculations determined by Nicolaides' method and to compare the risk calculation to the JOY software (NT-risk calculation module, JOY Patient Database) introduced in 2002. At the Department of Obstetrics and Gynecology, Hannover Medical School, 1463 consecutive complete data sets comprising first trimester screening performed between May 2, 2000 and June 26, 2003 and corresponding fetal outcome were analysed using risk assessment based on the Nicolaides method (PIA Fetal Database NT-Module) and compared with the risk evaluation as determined by the JOY software (JOY Patient Database NT module). A risk exceeding 1:300 was considered to indicate the need for further invasive testing. In a first step, only cytogenetically detectable chromosomal aberrations were analysed. Then, a second evaluation including fetal malformations was performed. Among the 1463 cases, 1445 (98.77%) fetuses revealed to be cytogenetically healthy. Both softwares showed identical detection rates at the genetic and somatic level:13 cases of Down-Syndrome (0.89%), 2 cases of trisomy 18 (0.14%), one case of triploidy, one Turner-Syndrome, one Klinefelter-Syndrome (0.07% each) were detected. A positive test result was found in 15 cases ending in a spontaneous abortion, intrauterine death or peripartum death (1.03%) and in 22 cases of fetal malformation (1.50%). At the level of genetic detection the test positive rate dropped from 92 (PIA) to 71 (JOY) (-22.8%). At the level of combined adverse outcome the test positive rate was reduced from 100 (PIA) to 76 (JOY) (-22.0%), thus yielding in a marked improvement of the characteristic test performance parameters. The novel, recently developed JOY software package allowed reliable evaluation of the risk for aneuploidy with increased specificity whereas sensitivity was unchanged. Our data suggest an improvement of the screening for aneuploidy when using this novel software: With an identical detection rate, the number of unnecessary invasive measures may be reduced.

Genome-wide analysis of DNA copy number changes and LOH in CLL using high-density SNP arrays

AbstractRecurrent genomic aberrations are important prognostic parameters in chronic lymphocytic leukemia (CLL). High-resolution 10k and 50k Affymetrix SNP arrays were evaluated as a diagnostic tool for CLL and revealed chromosomal imbalances in 65.6% and 81.5% of 70 consecutive cases, respectively. Among the prognostically important aberrations, the del13q14 was present in 36 (51.4%), trisomy 12 in 9 (12.8%), del11q22 in 9 (12.8%), and del17p13 in 4 cases (5.7%). A prominent clustering of breakpoints on both sides of the MIRN15A/MIRN16-1 genes indicated the presence of recombination hot spots in the 13q14 region. Patients with a monoallelic del13q14 had slower lymphocyte growth kinetics (P = .002) than patients with biallelic deletions. In 4 CLL cases with unmutated VH genes, a common minimal 3.5-Mb gain of 2p16 spanning the REL and BCL11A oncogenes was identified, implicating these genes in the pathogenesis of CLL. Twenty-four large (> 10 Mb) copy-neutral regions with loss of heterozygosity were identified in 14 cases. These regions with loss of heterozygosity are not detectable by alternative methods and may harbor novel imprinted genes or loss-of-function alleles that may be important for the pathogenesis of CLL. Genomic profiling with SNP arrays is a convenient and efficient screening method for simultaneous genome-wide detection of chromosomal aberrations.

Chromosomal Aberrations in Early Embryos of Weatherfish (Misgurnus fossilis L.) Exposed to Crude Cyanobacterial Extract and Semipurified Compound of Microcystins - a Pilot Study

The genotoxicity of semipurified compound of microcystins and crude extract of cyanobacteria was analyzed and assessed using detection of chromosomal aberrations in early life stages of weatherfish. For 96 hours, weatherfish eggs at an eye spot stage were exposed to semipurified compound of microcystins (MC-LR: ΣMC= 1: 2.6) and crude cyanobacterial extract (MC-LR: ΣMC= 1: 2.1) both at doses of 130, 50 and 13 μg l-1. Hatching time in individual groups and aberrations in the behaviour of hatched specimens were recorded. Cumulative mortality and the proportion of deformed specimens were determined; materials for histological and cytogenetic screening were simultaneously sampled. Cumulative mortality (28 and 26%, respectively) increased after the application of higher doses (130 and 50 μg l-1) of crude cyanobacterial extract (p ⪬ 0.05). In these groups, hatched embryos showed reduced agility and stayed in clusters and in the group with the highest dose of crude cyanobacterial extract white spots on yolk sacks appeared. Cytogenetic screening revealed both chromatid (gaps) and chromosomal aberrations (rings, dicentrics), percentage of which increased with the increased concentration of microcystins and the higher doses of crude cyanobacterial extract. The highest number of aberrant metaphases was recorded after the application of the highest concentrations of cyanobacterial extract and microcystin (p ⪬ 0.05). Embryo mortality was affected especially by crude cyanobacterial extract, therefore other toxic elements than microcystins present in the extract were probably involved.

Radiological Emergency Response: The National Biological Dosimetry Response Plan

This presentation will discuss new developments in emergency biological dosimetry and the CRTI (CBRN Research and Technology Initiative) National Biological Dosimetry Response Plan (NBDRP). Biological dosimetry is a technique used to estimate the biological consequences of a radiation exposure and is largely based on chromosome aberration detection. The NBDRP will establish a national network of laboratories to respond to a nuclear event for the purposes of rapid radiation dose estimation for crisis management and for long-term health risk assessment. In the event of a large-scale radiation accident or deliberate act of terrorism this will help guide the actions of emergency officials, emergency responders and health care personnel by providing timely biological dose estimates. The presentation will outline the research initiatives to develop modern techniques used for biological dosimetry. The overall purpose of this presentation is to provide the audience with a brief introduction to radiobiology, radiation-induced DNA damage, the health risks associated with radiation exposure, and the latest cytogenetic techniques used to estimate the risk.

Detection of Chromosomal Aberrations in CLL and Correlation with Clinical Staging.

Chromosome abnormalities detected in CLL are important markers for determination of treatment approaches. This study was aimed to evaluate usefulness of FISH panels for testing chromosome aberrations in CLL and to correlate the aberrations with clinical staging of disease and responses to chlorombucil therapy. Cytogenetics and FISH analyses from bone marrow samples of 67 CLL patients were performed according to standard unstimulated short term culture methods. CLL specific FISH panel included probes for 11q22.3 (LSIATM), CEP12, 13q14 (LSID13S19 and LSID13S25), 17p13.1 (LSIp53). FISH analysis was performed in all samples. The most commonly seen abnormality was 13q14 deletion followed by deletions of ATM, p53 and trisomy12. Of 19 cases with 13q14 deletion, 11 had only this abnormality whereas the others had trisomy12, p53 and ATM loci deletions (multiple abnormalities). Of 11 cases with single abnormality, 8 were at stage A, 3 was at stage C and all patients achieved benefits from chlorombucil therapy except one patient. Failed therapy response was seen in 9 patients with multiple abnormalities and the disease was at stage C (4 cases) and B (5 cases). Patients showing p53 and ATM deletions related with poor prognosis had progressive disease and no benefit could be achieved from chlorombucil therapy. Of 6 cases with trisomy 12, 4 were at early stage and response to the therapy was good but in two cases at later stage, therapy benefit could be achieved in one while no benefit was seen in the other one. The results showed that FISH analysis is more informative than karyotype analysis in CLL. Detection of p53 and ATM loci deletions is an important marker for administration of second level therapy.

Outpatient-Based Therapy with Oral Fludarabine and Alemtuzumab for Asian Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia (CLL).

Chronic lymphoproliferative disorders are considered rare in Asian countries. A recent 3-year study of 342 consecutive leukemia patients from 2 regional hospitals in Hong Kong, however, showed that CLL was diagnosed in 19% of Chinese patients with leukemia. Intravenous (IV) alemtuzumab (Campath-1H) in combination with fludarabine has been shown to be an effective chemoimmunotherapy regimen for treatment of CLL, yielding responses even in patients with relapsed/refractory disease who did not respond to therapy with either agent alone. Because alemtuzumab and fludarabine appear to be effective as monotherapy when given subcutaneously and orally, respectively, we performed a phase 2 single-arm clinical study to assess the safety and efficacy of subcutaneous (SC) alemtuzumab in combination with oral fludarabine for the treatment of patients with CLL. Patients with relapsed/refractory CLL who had failed ≥1 prior therapy were eligible to self-administer, on an outpatient basis, SC alemtuzumab 30 mg, 3 times per week on days 1, 3, and 5 and oral fludarabine 40 mg/m2/day on days 1–5, of a 28-day cycle. Depending upon response, patients received 2 to 6 cycles: if hematological CR was attained without molecular remission (assessed by clonal IgH rearrangement), SC alemtuzumab was given only on day 1 with fludarabine for subsequent cycles. Therapy was discontinued if molecular CR was attained. Patients were all males with ages ranging from 60 to 81 (median 72 years). The CLL FISH panel showed that 2 patients had 11q23 (consistent with ATM gene deletion) and trisomy 12 abnormalities, 1 had 17p13.1 deletion (consistent with p53 deletion), 1 had 13q14 deletion, whereas another had no detectable chromosomal aberrations. The baseline mean lymphocyte counts of all patients decreased from 63,610 to 42,610 cells/mL after the first cycle of treatment, and further decreased to a significantly lower mean of 1,500 cells/mL after completion of 2 to 6 cycles of therapy. Both the platelet counts (mean 114,000 cells/mL prior to and 111,400 cells/mL after therapy) and hemoglobin levels (11.7 g/dL prior to and 11.66 g/dL after therapy) remained unaffected. No cytomegalovirus reactivation was documented, but 2 patients developed a rapidly reversible episode of neutropenic sepsis treated with empiric antibiotics and transient administration of G-CSF. 3 patients attained a complete haematological response, while two had a good partial response, as defined by the NCI-WG response criteria. Based on bone marrow assessment and 4-color flow cytometry, 2 patients also attained complete molecular remission. These responses are comparable to those observed with patients from Western countries, who are treated with alemtuzumab and fludarabine combinations. In light of the promising results, the combination of oral fludarabine and SC alemtuzumab should be extended to larger multicenter clinical trials.

Detection of chromosomal aberrations by comparative genomic hybridization during transformation of human breast epithelial cells in vitro

Breast cancer is the most frequent malignancy in women. It is well recognized that tumorigenesis is a multistep process resulting from the accumulation of sequential genetic alterations. In breast cancers LOH has been described on one or both arms of multiple chromosomes. Comparative genomic hybridization (CGH) analysis was performed to identify chromosomal imbalances in the breast epithelial cells (HBEC). We have used a human in vitro-in vivo system in which the environmental carcinogen benz(a)pyrene (BP) and the c-Ha-ras oncogene were utilized for inducing in vitro transformation of HBEC. Immortal MCF-10F cells were treated with BP which resulted in the transformed cell line BP-1 that was further enhanced by transfection with the c-Ha-ras to generate the cell line BP-1-Tras. This cell line is tumorigenic when injected in severe combined immunodeficient (SCID) mice, generating the tumor cell line BP-1-Tras T J#4. Our comparative genomic hybridization analysis indicates that the most overrepresented segment after cell transformation and in the BP-1, BP-1-Tras and in the tumor cell line were 1p (80%), 5q21-ter (80%), 8q24.1 (90%) and Xq27-28 (60%). DNA sequence amplification at 10p14-15 was observed in BP-1-Tras T J#4 cells. Allelic losses of chromosome 4, 8p11-21 and 15q11-12, occur after cell transformation and are maintained consistently during tumorigenesis.

COmbined Binary RAtio fluorescence in situ hybridiziation (COBRA-FISH): development and applications

The ability to probe for the location of DNA sequences in morphologically preserved chromosomes and nuclei by fluorescence in situ hybridization (FISH) provided for cytogenetics a quantum leap forward in resolution and ease of detection of chromosomal aberrations. COBRA-FISH, an acronym for COmbined Binary RAtio-FISH is a multicolor FISH methodology, which enables recognition of all human chromosome arms on the basis of color, thus greatly facilitating cytogenetic analysis. It also permits gene and viral integration site mapping in the context of chromosome arm painting. Here we review the principle, practice and applications of COBRA-FISH.

Molecular cytogenetic study of 126 unselected T-ALL cases reveals high incidence of TCRβ locus rearrangements and putative new T-cell oncogenes

Chromosomal aberrations of T-cell receptor (TCR) gene loci often involve the TCRalphadelta (14q11) locus and affect various known T-cell oncogenes. A systematic fluorescent in situ hybridization (FISH) screening for the detection of chromosomal aberrations involving the TCR loci, TCRalphadelta (14q11), TCRbeta (7q34) and TCRgamma (7p14), has not been conducted so far. Therefore, we initiated a screening of 126 T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma cases and 19 T-ALL cell lines using FISH break-apart assays for the different TCR loci. Genomic rearrangements of the TCRbeta locus were detected in 24/126 cases (19%), most of which (58.3%) were not detected upon banding analysis. Breakpoints in the TCRalphadelta locus were detected in 22/126 cases (17.4%), whereas standard cytogenetics only detected 14 of these 22 cases. Cryptic TCRalphadelta/TCRbeta chromosome aberrations were thus observed in 22 of 126 cases (17.4%). Some of these chromosome aberrations target new putative T-cell oncogenes at chromosome 11q24, 20p12 and 6q22. Five patients and one cell line carried chromosomal rearrangements affecting both TCRbeta and TCRalphadelta loci. In conclusion, this study presents the first inventory of chromosomal rearrangements of TCR loci in T-ALL, revealing an unexpected high number of cryptic chromosomal rearrangements of the TCRbeta locus and further broadening the spectrum of genes putatively implicated in T-cell oncogenesis.

Evaluation of microsatellite amplifications at chromosomal locus 3q26 as surrogate marker for premalignant changes in mucosa surrounding head and neck squamous cell carcinoma

We analyzed tumor and surrounding mucosal samples of head and neck squamous cell carcinoma by fragment analysis for gain of genomic material as a potential indicator for oncogenic transformation. Our aim was to evaluate the potential value of this fast and sensitive polymerase chain reaction (PCR)-based method for intra-operative detection of chromosomal aberrations as a surrogate marker for incomplete tumor resection. Biopsies of the primary tumors and adjacent macroscopically nonmalignant mucosa 1 and 2 cm away from the tumor margins were collected from 20 patients. DNA were isolated, and 11 microsatellite markers at loci 3q25.31∼3q28 were amplified by PCR. Allelic losses or gains were analyzed by capillary electrophoresis. Imbalanced alleles were common in the samples evaluated. In the median gains of the informative loci were detected in 67% of the primary tumors, 22% of the samples taken at 1 cm and 15% of the samples taken at 2 cm distance. We observed gains of 3q at least in one microsatellite in all primary tumors, in 15 (1 cm) and 12 (2 cm) nonmalignant mucosa samples. Gain of genetic material is frequent in tumor-surrounding mucosa. Detection of small chromosomal aberrations is possible using the PCR-based, highly sensitive, and fast-to-perform fragment analysis technique. The value for the diagnosis and prognosis will have to be proven in follow-up studies.

Detection of Chromosome Aberrations in the Second Trimester Using Genetic Amniocentesis: Experience During 1995-2004

To retrospectively investigate the 10-year experience of prenatal diagnosis of fetal chromosome aberrations by second-trimester amniocentesis. Data were collected at Taichung Veterans General Hospital between 1995 and 2004 from cytogenetic analyses of cultured amniocytes from second-trimester amniocentesis. The main indications for amniocentesis included advanced maternal age, abnormal maternal serum screening results, and abnormal ultrasound findings. Chromosome aberrations included autosomal aneuploidies, sex chromosome aneuploidies, polyploidies, and rearrangements. Variant chromosomes were considered to be normal and excluded. A total of 7,028 amniocenteses were performed and analyzed for chromosome aberrations. Among these, 4,026 (57.29%) were for advanced maternal age, 1,500 (21.34%) for abnormal maternal serum screening results, 553 (7.87%) for abnormal ultrasound findings, and 949 (13.50%) for other reasons. The highest detection rate of chromosome aberrations was in cases undergoing amniocentesis for abnormal ultrasound findings (8.86%), followed by other reasons (2.74%), abnormal maternal serum screening results (2.60%), and advanced maternal age (2.31%). Chromosome aberrations were detected in 207 cases (2.90%), including fetuses of 93 older mothers, 39 mothers with abnormal serum screening results, 49 mothers with abnormal ultrasound findings, and 26 mothers with other reasons for amniocentesis. Of fetuses with chromosome aberrations, 144 (69.56%) had trisomy 13, trisomy 18, trisomy 21, or sex chromosome disorder. The other 63 cases (30.44%) included balanced translocation, unbalanced abnormality, inversion, and marker chromosome. For daily practice, our data could offer a database for proper genetic counseling, such as termination issues and future pregnancies.

Affymetrix GeneChip® system: moving from research to the clinic

Affymetrix Inc.’s GeneChip® technology has become the industry standard in microarray-based research. Due to the high density of content per array that can be achieved today (an industry leading 6.5 million features) the GeneChips can be used for high-throughput mutation detection, single nucleotide polymorphism genotyping, expression profiling and detection of chromosomal aberrations. This in turn opens the way for clinical applications in genetics, cytogenetics, pharmacogenetics, oncology and pathogen recognition. Establishing standards is a central issue in improving data quality and, in combination with automatic, easy-to-interpret reports, will form the basis of the clinical applicability. Future pricing policies and the resolving of ethical considerations will also dictate the technology’s full translation from research into clinical laboratories.

Detection of chromosomal aberrations by fluorescence in situ hybridization in cervicovaginal biopsies from women exposed to diethylstilbestrol in utero

Epidemiologic studies have associated estrogens with human neoplasms such as those in the endometrium, cervix, vagina, breast, and liver. Perinatal exposure to natural (17beta-estradiol [17beta-E(2)]) and synthetic (diethylstilbestrol [DES]) estrogens induces neoplastic changes in humans and rodents. Previous studies demonstrated that neonatal 17beta-E(2) treatment of mice results in increased nuclear DNA content of cervicovaginal epithelium that precedes histologically evident neoplasia. In order to determine whether this effect was associated with chromosomal changes in humans, the frequencies of trisomy of chromosomes 1, 7, 11, and 17 were evaluated by the fluorescence in situ hybridization (FISH) technique in cervicovaginal tissue from 19 DES-exposed and 19 control women. The trisomic frequencies were significantly elevated in 4 of the 19 (21%) DES-exposed patients. One patient presented with trisomy of chromosomes 1, 7, and 11, while trisomy of chromosome 7 was observed in one patient. There were two patients with trisomy of chromosome 1. Trisomy of chromosomes 1, 7, 11, and 17 was not observed in the cervicovaginal tissue taken from control patients. These data suggest that DES-induced chromosomal trisomy may be an early event in the development of cervicovaginal neoplasia in humans.

Threshold of Nuchal Translucency for the Detection of Chromosomal Aberration: Comparison of Different Cut-offs

This study evaluated the sensitivities and false positive rates of the screening test using ultrasonographic measurement of thickness of nuchal translucency (NT) with different cut-offs for chromosomal aberration in a Korean population. We included 2,570 singleton pregnancies undergoing ultrasound between 11 weeks and 14 weeks of gestation in this study. We analyzed the sensitivities of NT alone for screening chromosomal aberration using three cut-offs -2.5 mm, 3.0 mm, and 95th percentile for each crown rump length (CRL). There were 31 chromosomal aberrations (1.2%) including 12 cases of trisomy 21. The numbers of chromosomal aberrations that were detected by NT with different cut-offs of 2.5 mm, 3.0 mm and the 95th percentile CRL were 22, 18 and 23, respectively. At a threshold of 2.5 mm, the sensitivity and the false positive rate for total chromosomal aberrations were 67.7% and 6.3%, respectively. At 3.0 mm, those were 54.8% and 3.5%, respectively. At the 95th percentile CRL, those were 70.9% and 5.8%, respectively. The use of CRL-dependent cut-offs for nuchal translucency improves the detection of chromosomal aberrations when compared to fixed cut-offs in a Korean population.

Detection of chromosomal aberrations by fluorescencein situhybridization in cervicovaginal biopsies from women exposed to diethylstilbestrolin utero

Detection of chromosomal aberrations by fluorescence<i>in situ</i>hybridization in cervicovaginal biopsies from women exposed to diethylstilbestrol<i>in utero</i>

Chromosomal aberrations in radiation workers of radiology departments in Northern Iran-Babol

We determined the frequency of chromosomal aberrations using the metaphase analysis method in radiation workers of radiology departments in Babol. Blood samples were prepared from 62 radiology workers and 29 individuals as controls, and lymphocytes were cultured in a culture medium. Results showed that the mean frequencies of the total chromosomal aberration in radiation workers are significantly higher than those in controls. It is also shown that there is no significant correlation between the frequency of chromosomal aberration and age, sex and smoking habit of all individuals in both radiation workers and control groups. Also no significant correlation was seen between the frequency of chromosomal aberrations in radiation workers and the years of work as well as any history of over dose. The higher frequency of chromosomal aberration in lymphocytes of radiation workers compared with non-exposed controls could be due to the accumulative effect of radiation, finally leading to detectable chromosomal aberrations.

Chromosome Aberrations, Gene Mutations and Expression Changes, and Prognosis in Adult Acute Myeloid Leukemia

Pretreatment clinical features and prognosis of patients with acute myeloid leukemia (AML) are strongly influenced by acquired genetic alterations in leukemic cells, which include microscopically detectable chromosome aberrations and, increasingly, submicroscopic gene mutations and changes in gene expression. Cytogenetic findings separate AML patients into three broad prognostic categories: favorable, intermediate and adverse. The cytogenetic-risk classifications differ somewhat for younger adult patients and those aged 60 years or older. In many instances, patients with specific cytogenetic findings, e.g., those with a normal karyotype or those with either t(8;21)(q22;q22) or inv(16)(p13q22)/t(16;16)(p13;q22) [collectively referred to as core-binding factor (CBF) AML] can be further subdivided into prognostic categories based on the presence or absence of particular gene mutations or changes in gene expression. Importantly, many of these molecular genetic alterations constitute potential targets for risk-adapted therapies. In this article, we briefly review major cytogenetic prognostic categories and discuss molecular genetic findings of prognostic significance in two of the largest cytogenetic groups of patients with AML, namely AML with a normal karyotype and CBF AML.

Immunostimulatory CpG-Oligonucleotide Activated Metaphase Cytogenetics Is Feasible in Routine Diagnostics of Chronic Lymphocytic Leukaemia and Reveals More Abnormalities Than Interphase FISH.

Genetic characterization of chronic lymphocytic leukemia (CLL) cells by fluorescence in situ hybridization (FISH) has identified genetic aberrations in 80% of CLL patients. In contrast, conventional metaphase cytogenetics detected chromosomal aberrations in only 40–50% of all cases. Immunostimulatory CpG-oligonucleotides (CpG-ODN) in combination with interleukin 2 (IL-2) have recently been reported to induce proliferation in CLL B cells. Therefore, we evaluated this stimulus for its efficacy in metaphase generation for the detection of chromosomal aberrations by cytogenetic banding techniques. In addition these results were compared with data obtained by interphase FISH. For metaphase induction 107 cells were cultured in growth medium with the CpG-ODN DSP30 and IL-2. After 2 days colchicine was added for another 24h before preparation. Of the 133 samples that were included in this study, all cases could be successfully analyzed by interphase FISH with a rate of 79% aberrations like deletions of chromosomes 13q (57%, in 38% as sole abnormality), 11q (17%), trisomy 12 (13%), 6q (7%), 17p (6%) and translocations involving 14q32 (4%). By FISH 55% of all samples showed a single aberration, 22% two aberrations and 2% 3 aberrations. In comparison, 126 cases (95%) could be successfully analyzed by cytogenetic banding techniques. 102 (81%) of the 126 samples showed chromosomal aberrations, which involved all different chromosomes. 9 cases with a normal karyotype in conventional cytogenetics revealed genetic aberrations by FISH. In all but 1 of these cases the aberrant clone represented < 30% of the cell population. In addition to the aberrations that were detected by the FISH probes, a substantial amount of other aberrations was revealed by chromosome banding analysis. Interestingly, 10 cases of a total of 27 cases without abnormalities detected by FISH displayed aberrations in chromosome analysis indicating that the true amount of CLL patients with aberrations exceeds 79%. Overall, 47 samples (37%) showed chromosomal aberrations in chromosome banding analysis in addition to FISH analysis. Compared to FISH analysis, which detected 2 cases with 3 aberrations, metaphase cytogenetics detected 22 cases with 3 or more unbalanced aberrations, which can be considered as a complex aberrant karyotype. Loss of p53 referred to as del(17p13) in FISH has attracted considerable attention, because of the poor clinical outcome of affected patients. In our study, all del(17p13) cases (n=7) displayed either a loss of the short arm of chromosome 17 (n=6) or a complete loss of chromosome 17 (n=1) indicating that chromosomal regions in addition to the p53 locus might be relevant for this phenotype. Furthermore, numerous recurrent aberrations have been identified in this study beyond the aberrations that are detected by FISH. Of note are gains of 2p and 3q, losses in 11q13 and gains in 11q23–25, gains in 13q31–34, gains of 17q21–25 and cases with trisomy 18 and 19, which occurred in parallel to trisomy 12. The results of the present study show that immunostimulatory CpG-oligonucleotides plus interleukin 2 are a simple and cheap stimulus for efficient metaphase induction in CLL. The rate of aberrations detected by conventional banding techniques was even slighty increased compared to FISH analysis, however, the complexity of cytogenetic aberrations is underestimated by FISH analysis in a large portion of CLL cases (37%).