Detection Of Chromosomal Aberrations Research Articles

Detection of chromosomal aberrations in multiple myeloma with fluorescence in situ hybridization

To detect of chromosomal abnormalities in multiple myeloma (MM) patients with fluorescence in situ hybridization (FISH). FISH was performed in 20 MM patients using 5 specific DNA probes. The difference in chromosomal abnormalities was compared by FISH and other routine cytogenetic tests. Eighteen of the 20 patients showed chromosomal abnormalities (90%). The positive rates of t(14q32), del(13q14), dup(1q21), and p53 gene were 65% (13 in 20), 55% (11 in 20), 25% (5 in 20), and 15%(3 in 20), respectively. The abnormal rate of the conventional chromosome examination was 15% only. FISH is more sensitive than traditional chromosomal tests and can be used as an index in prognostic evaluation for MM. Del(13q14) and t(14q32) are the most common chromosomal abnormalities in MM patients.

Evaluation of SureFISHtm DNA-FISH Probes for Validation of Array-CGH Aberrations

Evaluation of SureFISHtm DNA-FISH Probes for Validation of Array-CGH Aberrations

Authentication of primordial characteristics of the CLBL-1 cell line prove the integrity of a canine B-cell lymphoma in a murine in vivo model.

Cell lines are key tools in cancer research allowing the generation of neoplasias in animal models resembling the initial tumours able to mimic the original neoplasias closely in vivo. Canine lymphoma is the major hematopoietic malignancy in dogs and considered as a valuable spontaneous large animal model for human Non-Hodgkin's Lymphoma (NHL). Herein we describe the establishment and characterisation of an in vivo model using the canine B-cell lymphoma cell line CLBL-1 analysing the stability of the induced tumours and the ability to resemble the original material. CLBL-1 was injected into Rag2−/−γc −/− mice. The generated tumor material was analysed by immunophenotyping and histopathology and used to establish the cell line CLBL-1M. Both cell lines were karyotyped for detection of chromosomal aberrations. Additionally, CLBL-1 was stimulated with IL-2 and DSP30 as described for primary canine B-cell lymphomas and NHL to examine the stimulatory effect on cell proliferation. CLBL-1 in vivo application resulted in lymphoma-like disease and tumor formation. Immunophenotypic analysis of tumorous material showed expression of CD45+, MHCII+, CD11a+ and CD79αcy+. PARR analysis showed positivity for IgH indicating a monoclonal character. These cytogenetic, molecular, immunophenotypical and histological characterisations of the in vivo model reveal that the induced tumours and thereof generated cell line resemble closely the original material. After DSP30 and IL-2 stimulation, CLBL-1 showed to respond in the same way as primary material. The herein described CLBL-1 in vivo model provides a highly stable tool for B-cell lymphoma research in veterinary and human medicine allowing various further in vivo studies.

Health hazards among workers in plastic industry

Styrene is a basic building block for manufacturing thousands of products throughout the world. The present study aimed to (1) detect the presence of styrene and/or its metabolites in the workers in one of the Egyptian plastic factories; (2) demonstrate some common health effects of styrene exposure among the same group by some laboratory investigations and compare them with the unexposed healthy individuals; and (3) correlate the duration of styrene exposure and its level in the blood with the severity of the demonstrated health effects. This study was conducted in one of Egyptian plastic factories. The exposed group was 40 male workers, ranging in age from 18 to 33 years (23.20 ± 4.09), working 12 h/day with 1 day off, and working without any protective equipment. A control group of 50 unexposed healthy males matched with the exposed group for age (21-35 yrs (23.40 ± 4.05)), sex, socioeconomic status, and smoking habit is selected. Written individual consent is obtained from all participants followed by (a) a full medical and occupational history and full clinical examination; (b) ventilatory function tests: forced vital capacity (FVC), slow vital capacity, forced expiratory volume in the 1st second (FEV₁)%, FEV₁/FVC%, peak expiratory flow, and mid-expiratory flow 25-75%; (c) analyses of β₂ microglobulin; blood styrene level; and urinary mandelic acid; and (d) cytogenetic study. The study results showed a statistically significant difference between the exposed and the control groups as regard the blood styrene level, urinary mandelic acid level, β₂ microgloblin in urine, and chromosomal study. The study also showed a statistically significant correlation between the duration of styrene exposure and ventilatory function parameters, also between the duration of styrene exposure and some detectable chromosomal aberrations. Our study recommends the implementation of preemployment and periodic medical examinations and health education programs using personal protective equipments and following the recommended allowable concentrations of styrene exposure.

Micronucleus frequency in human lymphocytes after exposure to diphenylamine in vitro

Diphenylamine (DPA) is an antioxidant compound that occurs naturally in several vegetables. It is widely applied in agriculture for preservation of the quality of apples and pears, and used for controlling superficial scald, a disorder that renders fruits of a number of apple cultivars unfit for the market. Because of its anti-oxidative properties, DPA also has several industrial applications. The potential genotoxic effect of DPA on human lymphocytes has previously been investigated in only two studies, which focused on detection of chromosome aberrations and sister chromatid exchange, respectively. In the present analysis, we evaluated micronucleus (MN) formation in freshly isolated human peripheral lymphocytes exposed to different concentrations (0.625, 1.25, 2.50, 5.0 and 10.0μg/ml) of DPA. Peripheral venous blood was collected from ten healthy subjects, and a total of 10,000 bi-nucleated cells were analyzed. Results indicated that DPA significantly increased the micronucleus frequency at concentrations of 1.25μg/ml and higher. Significant differences in the MN frequency were also found between the lower dose (0.625μg/ml) and all other doses tested, with the exception of 1.25μg/ml. Our results indicate a potential cytogenetic effect of DPA on human cells in vitro and require further in vivo studies to clarify the actual genotoxicity of this compound and the consequent risks for human health.

Developmental and genetic aspects of congenital diaphragmatic hernia

Congenital diaphragmatic hernia (CDH) is a frequent occurring cause of neonatal respiratory distress and occurs 1 in every 3,000 liveborns. Ventilatory support and pharmaceutical treatment of the co-occurring lung hypoplasia and pulmonary hypertension are insufficient in, respectively, 20% of isolated cases and 60% of complex ones leading to early perinatal death. The exact cause of CDH remains to be identified in the majority of human CDH patients and prognostic factors predicting treatment refraction are largely unknown. Their identification is hampered by the multifactorial and heterogenic nature of this congenital anomaly. However, application of high-resolution molecular cytogenetic techniques to patients' DNA now enables detection of chromosomal aberrations in 30% of the patients. Furthermore, recent insights in rodent embryogenesis pointed to a specific disruption of the early mesenchymal structures in the primordial diaphragm of CDH-induced offspring. Together, these data allowed for the introduction of new hypotheses on CDH pathogenesis, although many issues remain to be resolved. In this review, we have combined these new insights and remaining questions on diaphragm pathogenesis with a concise overview of the clinical, embryological, and genetic data available.

SNP-based arrays complement classic cytogenetics in the detection of chromosomal aberrations in Wilms’ tumor

Wilms' tumors have characteristic chromosomal abnormalities, such as the 11p13 deletion, in a subset of cases. This is one of the very few reports comparing single nucleotide polymorphism (SNP) array analysis with conventional karyotyping of Wilms' tumors. A total of 43 frozen tumor samples were analyzed using the Affymetrix Cytogenetics Whole-Genome 2.7M array. The findings from the SNP array analysis were then compared with those from conventional karyotyping. A comparison between SNP array and conventional karyotype findings was possible in 38 of 43 specimens (88.4%). The SNP array and classic cytogenetic results were concordant in 33 of 38 specimens (87%). SNP array analysis was able to support the findings of classic cytogenetics. The SNP array detected regions of loss of heterozygosity (LOH) in 41 of 43 (95%) specimens. However, it did not detect balanced translocations and inversions that were observed by conventional cytogenetics. Our results show that the data generated from these platforms are complementary. The SNP array also detected additional gains and losses as well as regions of LOH with associated disomy, which are likely to represent segmental uniparental disomy. The observed discrepancies can be explained by the inherent limitations of each technique.

Innovations in image cytometry

WHEN it comes to Cytometry, the quantitative biology of single cells, it should not be forgotten that historically, microscopy and imaging are the mothers of cytometry and flow cytometry is just another way of imaging individual cells. Both image and flow cytometry have made great advances in the last few decades. Recent instruments can measure substantially more colors at a time, are faster and more and more automated and are easily standardized. Flow cytometry has been a ‘‘mature’’ technology for some time whereas image cytometry still surprises with innovations in hardware and software thereby pushing technology and therewith cellular analysis and the understanding of their nature forward to new frontiers. Fluorescence microscopy has been for a long time a standard technology in all fields of medicine and biology. However, with respect to excitation of light sources, many of these instruments are still using arc lamps for optical inspection of the samples by the eye as well as for digital imaging and image cytometry. Unfortunately, arc lamps have several disadvantages including short lifetime, some health risks (particularly with mercury arc lamps), high energy consumption, uneven field of emission, fluctuation in the light intensity, and so on. Particularly, light intensity fluctuations render problems to (quantitative) image cytometry and various technological efforts have been undertaken by the instrument manufacturers to compensate for this obstacle. The recent advances in the development of light emitting diodes (LEDs) render them as new, alternate light sources providing a high spectral range for conventional and advanced fluorescence microscopy. Wessels and colleagues (this issue, page 188) from G€ ottingen, Germany review the cutting-edge developments of LEDs for microscopy, which will presumably replace the arc lamp over the next few years. These advances will allow improved and simplified imaging and enable for image cytometry. One of the commonly used applications of image cytometry is the counting of subcellular objects. In the not so far away past, which one may term the premolecular biology times, it was common sense to believe that chromosomes and genes are located at random within the nuclear membrane. However, after the ingenious development of chromosome site specific fluorescence in-situ hybridization (FISH) probes and innovations in microscopy spearheaded by the Cremer brothers (1) it became evident by sophisticated nuclear distance measurements of FISH spots that ‘‘God doesn’t play dice.’’ The nuclear DNA is extremely well organized, displays a delicate 4D spatiotemporal ballet within the cells’ nucleus and chromosomes anchor at definite sites of the nuclear membrane. Interestingly, the latest amazing discovery is that it is not carved in stone that interphase chromosomes have to be located at the nuclear membrane: in animals with night vision they are located in the center of the rods of the retina forming a biological optical waveguide (2). Furthermore, FISH spot counting and measurement are of high relevance also in tumor diagnosis and detection of chromosomal aberrations. Several automated and semi-automated methods to analyze such images have been developed (3). But when it comes to 3D, accurate manual or automated analysis are still a challenge. Now, Ram and colleagues (this issue, page 198) from the University of Arizona in Tucson, USA present a new approach to 3D for automated FISH-spot counting from 3D confocal images. Their approach consists of a two step model by which first the images are segmented and spots detected followed by the classification of these spots. This new technique is substantially more accurate then previously described methods.

Molecular diagnostics in lymphoma: why, when and how to apply

Current strategies for diagnosing lymphomas include molecular methodologies next to histomorphological assessment supplemented by immunohistochemistry. Especially PCR-based clonality analysis and detection of chromosome aberrations by FISH can support the diagnosis and classification in difficult cases. We have to ensure that these existing molecular methodologies are used to their full potential. Yet, such analyses are not required in every situation. Ongoing optimization, dissemination of (new) protocols, standardized interpretation, and education are essential elements to ensure best practice for patient care. Here we discuss why, when, and how to apply these molecular diagnostic methods in the field of lymphoma.

Genetic analysis of a supernumerary derivative chromosome 15

To detect and analyze a supernumerary derivative chromosome 15 with combined cytogenetic and molecular techniques, and to discuss the correlation between genomic copy number variations (CNVs) and clinical phenotypes. G-banded chromosome analysis and multiplex ligation-dependent probe amplification (MLPA) were carried out. The whole genome of the patient was also analyzed with array-comparative genome hybridization(array-CGH). G-banding analysis indicated that the patient has a karyotype of 47, XY, + mar, with the supernumerary chromsome derived from 15q11-13 region spanning 9.8 Mb from locus 20477397 to 30298155. CNVs of 15q11-13 are associated with mental retardation, language development delay and autistic disorder. Conventional cytogenetic analysis with array-CGH may provide a platform for accurate detection of chromosomal aberrations, which can faciliate the study of genome rearrangement underlying various diseases.

MicroRNA Profiling in Patients with CLL B Cells Expressing the Unmutated IGHV1-69 Gene

Abstract 2846The immunoglobulin heavy chain variable (IGHV) 1-69 gene, is rearranged in chronic lymphocytic leukemia (CLL) B cells in approximately 20% of the patients, is typically found without somatic mutations, and its expression is often associated with an aggressive clinical course. Unique microRNA (miRs) signatures have been found to be associated with various CLL prognostic markers such as: IGHV mutation status, ZAP-70 expression, and chromosomal aberrations. A karyotype specific signature of some of these miRs has emerged that can differentially segregate patients with adverse clinical behavior and can predict the time from diagnosis to initial treatment.We had interphase FISH available on 281 of the 452 IGHV1-69 cases analyzed in our companion study. We investigated the miRs signature of 53 of these cases that had CLL cells with a sole cytogenetic lesion. All of these cases expressed unmutated (UM) IGHV1-69. These samples included: 18 that had isolated del 11q, 14 that had trisomy 12, 10 that had del 17p and 11 had no detectable chromosomal abnormalities. We did not include any cases that had del 13q. Expression data of the miRs were analyzed by the multiclass comparison within BRB Array Tools to identify a specific set of miRs differentially expressed among the various cytogenetic classes (p=0.05). The data were normalized using the quantiles method. We compared the miRs profiles of CLL cells having defined cytogenetic aberrations with those described in a previously published cohort of 61 CLL patients not selected for IGHV use (Visone et al. Blood 2009).In our cohort of 53 IGHV1-69 cases, we observed significant differences in the expression level of 33 miRs between CLL cells of patients that had no detectable chromosomal aberrations and those of patients with cytogenetic lesions. Of these 2 miRs (miR-543 and miR-769-3p) were published in the cohort of 61 CLL patients. We identified significant differences in the miR expression level of 91 miRs between CLL cells with trisomy 12 and those with other chromosomal aberrations or not. Of these 6 (miR-125b-2*, miR-103, miR-155, miR-107, miR-340 and miR-222) had been previously described in the 61 CLL cohort. We found significant differences in the miR expression level of 26 miRs between CLL cells with the 11q deletion and those with other chromosomal aberrations or not, none of these was previously identified in the 61 CLL cohort. We observed significant differences in the miR expression level of 20 miRs between CLL cells with the 17p deletion and those with other chromosomal aberrations or not, of these only 1 (miR-618) had already been described in the 61 CLL cohort.Since in our companion study on 452 IGHV1-69 cases we found that ZAP-70 expression was a strong predictor of time to first treatment (TFS). We investigated whether CLL expression of ZAP-70 was associated with the peculiar expression of particular miRs in this cohort. Of these 53 patients, 33 (62%) cases had CLL cells that were ZAP-70 positive and had a median TFS of 2.4 years, whereas the 20/53 patients with CLL cells lacking ZAP-70 had a median TFS of 4.0 years.We found that miR-618 and miR-639 were commonly overexpressed in patients with CLL B cells carrying the 11q or 17p deletions and lacking ZAP-70 whereas miR-22 was down-regulated in CLL cells from patients carrying the trisomy 12 or no chromosomal aberrations and lacking ZAP-70. There was a significant difference in miR-618 expression between patients with CLL cells carrying the 11q or 17p chromosomal aberrations with or without ZAP-70 (9.6 vs 10.8, respectively p<0.0001). In this initial investigation we conclude that in patients with CLL cells expressing the UM IGHV1-69 gene mir-618 appears to be associated with ZAP-70 expression, most apparent in cases with CLL cells carrying 11q or 17p deletions. Mir-618 is known to target the histone deacetylase 4 (HDAC4) which may be involved in the regulation of ZAP-70 expression. Further work needs to be performed to establish the miR-618 prognostic value in CLL. Disclosures:No relevant conflicts of interest to declare.

Chromosome Aberrations by Conventional Karyotyping in Chronic Lymphocytic Leukemia Carrying No Aberration by Fluorescence in Situ Hybridization: Correlation with Prognostic Parameters and Clinical Features

Abstract 1459Background. Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease. Adverse prognostic parameters include stage, CD38 and/or ZAP70 expression, the unmutated configuration of the variable region of the Ig heavy chain gene (IGHV) and the presence of specific chromosome aberrations. Using fluorescence in situ hybridization (FISH) with probes detecting trisomy 12 and deletions at 13q14, 11q22–23/ATM and 17p13/p53, approximately 60–80% of the cases carry an abnormality. Patients without FISH aberrations (“normal” FISH) have a relatively favourable outcome. Improved mitotic stimulation using oligonucleotide (odn) and interleukin-2 (IL-2) can detect karyotype aberrations in chromosome regions not covered by the classical 4-probe panel. This study was designed to assess whether the presence of karyotypic aberrations in CLL patients with “normal” FISH may have a correlation with established clinical and prognostic parameters.Methods. Eighty-four patients with “normal” FISH results referred to our laboratory between 2007 and June 2011 were included in the present report. These patients represent consecutive CLL cases diagnosed and treated, according to the NCI criteria, at 4 GIMEMA CLL group centres between 2006 and 2011. Indications for treatment included advanced stage (Binet C) or disease progression. The Matutes immunophenotypic score was calculated giving 1 point to CD5+, CD23+, CD22weak+, sIg weak+ and FMC7- and only patients with a score ≥3 (i.e. typical CLL) were included. The coexpression of the CD38 and CD19 antigens and ZAP-70 was tested on fresh PB cells with a 20% cut-off for positivity. IGVH genes were amplified from genomic DNA and sequenced according to standard methods and the cut-off of 98% homology to the germline sequence was chosen to discriminate between mutated (<98% homology) and unmutated (≥98% homology) cases, as reported previously. FISH studies were performed using probes for the following regions 13q14, 12q13; 11q22/ATM; 17p13/TP53. Conventional cytogenetic analysis using immunostimulatory CpG-oligonucleotide DSP30 plus IL2 (2μmol/L TibMolBiol / IL2 100U/mL Stem Cell Technologies Inc) was performed on the same samples that had been used for FISH studies. At least 20 metaphases were karyotyped. Stage, time to first treatment (TTFT) were obtained by review of clinical records at each centre.Results. An abnormal karyotype was found in 30/84 cases (35,7%) with “normal” FISH. The chromosome aberrations affected regions not covered by the 4-probe panel used by FISH. Recurring aberrations were: interstitial deletions of 14q and of 7q in 5 and 3 cases, respectively; 14q32 translocations in 2 cases, balanced and unbalanced structural changes in 1 case each. A complex karyotype with 3 or more aberrations was found in 12 patients. The correlations between karyotype and clinicobiological parameters are shown in tables 1 and 2.Table 1:Correlation between karyotype and salient clinicobiologic parametersParameterNormal karyotype (54 cases)Abnormal karyotype (30 cases)p (Fischer's exact test)Binet Stage A/B/C45/9/020/7/30.04Matutes score3/4/54/24/257/6/120.035CD38pos/neg11/4112/180.079ZAP-70 pos/neg16/3115/110.083IGHV Unmutated/ Mutated4/208/80.037Table 2:Factors affectingTTFT in univariate snalysisNumber of casesMedian TTFT months (se)pKaryotype Normal Abnormal50 34Not reached at 54 months 18 months (6.7)<0.0001Binet stage AB-C65 19Not reached at 54 months 12 months (2.1)0.0006IGHV Unmutated Mutated12 2815 months (2.5) Not reached at 54 months0.0007At multivariate analysis, the following factors were independently predictive of shorter TTFT: advanced disease Binet stage (p=0.049, Hazard Ratio 2.39 [se: 1.06]), and abnormal karyotype (p<0.001, Hazard ratio 6.8 [se: 3.28]). IGHV mutations were not included in the TTFT analysis, because the data were not available for all patients.Conclusions. Conventional karyotyping using odn + IL-2 as mitogens is an effective method for the detection of chromosome aberrations in approximately 1/3 of patients with “normal” FISH on a conventional 4-probe panel. Recurrent aberrations include interstitial deletions at 14q and 7q. The current data show that, in CLL patients with “normal” FISH, conventional cytogenetic analysis using odn + IL-2 identifies a subset of cases with adverse clinical and prognostic features. Disclosures:Rizzotto:CELGENE CORPORATION: Research Funding. Cuneo:Roche: Consultancy, Speakers Bureau.

Relative Prognostic Significance of ZAP-70 and IGHV1-69 Expression in Chronic Lymphocytic Leukemia,

Abstract 3891The leukemia B cells of patients (pts) with chronic lymphocytic leukemia (CLL) express a restricted repertoire of immunoglobulin heavy chain variable region (IGHV) genes. Moreover, certain IGHV genes appear over-represented in this repertoire. Among these, IGHV1-69 was first identified as most frequently expressed, being used by the CLL cells of nearly 20% of all pts (PNAS, 86:5913–7, 1989). CLL cells most frequently express IGHV1-69 without somatic mutations and often with restricted D and JH segment use, providing for stereotypic motifs in the heavy chain third complementarity determining region (HCDR3). We addressed whether there were peculiar biologic or clinical features of pts with CLL that used IGHV1-69. For this, we studied 452 pts identified in a cohort of 2,866 followed by the CLL Research Consortium (CRC) found to have CLL cells that express IGHV1-69. This accounted for 16% of all pts. We found that 420 of 452 CLL samples (93%) express IGHV1-69 without somatic mutation (≥98% sequence homology with germline IGHV1-69), which is in significant contrast to the frequency use of UM IGHV among CLL samples that do not use IGHV1-69. As noted for CLL pts in general, there is a strong association between mutation status and clinical behavior. Among pts that use IGHV1-69, the 32 of 452 pts that use MU IGHV1-69 had a highly indolent clinical course, with a median time from diagnosis to initial treatment (TFS) of 17.3 years. This was significantly longer than the median TFS of 2.8 yrs for the 452 pts that used UM IGHV1-69 (p<0.0001). Among the pts that used UM IGHV1-69 we identified a stereotypic HDCR3 motif shared by more than one patient in 249/452 (55%) of the cases. Multivariate analysis failed to discriminate any significant differences in the median TFS of pts that used IGHV1-69 that had a stereotypic CDR3 motif versus pts who had an idiosyncratic HCDR3 (2.9 yrs vs 3.0 yrs, respectively p=0.14). Interphase FISH for common cytogenetic aberrations in CLL were available for 281 of the 452 cases. Among these, 58% of the cases had deletions at 17p (18%), 11q (23%), or trisomy 12 (17%). The remaining cases had no detectable chromosomal abnormalities (24%) or isolated deletion of 13q (18%).The CLL cells of all 452 pts were examined for ZAP-70. There was a strong association between the expression of ZAP-70 and use of UM IGHV1-69. However, the association between ZAP-70 expression and use of UM IGHV1-69 was not absolute. Only 70% with UM IGHV1-69 were ZAP-70 positive, as were 12.5% that used M IGHV1-69. Of all 452 pts that expressed IGHV1-69, 300 (66%) had ZAP-70 positive CLL cells. These pts had a median TFS of 2.3 yrs, which was significantly shorter than that of the remaining 152 pts with ZAP-70-negative CLL, who had a median TFS of 4.3 yrs (p<0.0001). Moreover, of the 420 pts that used UM IGHV1-69, 296 (70%) had CLL cells that expressed ZAP-70; these pts had a median TFS of 2.3 yrs. This was significantly shorter than the median TFS of pts with CLL cells that express UM IGHV1-69, but were ZAP-70 negative 4.1 yrs (p<0.0001).A Cox regression model revealed that although the presence of detectable chromosomal aberrations was associated to a shorter median TFS, ZAP-70 was a stronger predictor of short TFS (HR for 13q =1.1 p =0.03, HR for trisomy 12 =1.2 p =0.03, HR for 11q =1.6 p =0.03, HR for 17p =1.8, p =0.03) (HR for ZAP-70 positive = 1.8, p=0.0004).The Cox regression model was used to assess the associations of ZAP-70, and the use or not of the UM IGHV1-69 gene with TFS (p-value <0.05 were considered as significant). We investigated these associations using a previously published cohort of characterized 705 CLL pts (Blood.2008;112:1923). The HR associated with the expression of ZAP-70 (HR=3.2) (p<0.0001) was significantly higher than if either the UM IGHV1-69 or the cases with UM IGHV other than IGHV1-69 were incorporated into the model (HR=1.9 and HR=1.6 respectively, p=0.001).We conclude that cases that use IGHV1-69 are peculiar in that they more frequently use UM IGHV and appear to have a higher frequency of adverse cytogenetic features than CLL cases at large. In addition, we found that CLL-cell expression of ZAP-70 can segregate pts that use UM IGHV1-69 into subgroups with disparate clinical behavior, despite the fact that all patients use the same IGHV gene. Moreover, multivariable analyses revealed that ZAP-70 was strongest predictor of short TFS among all other considered prognostic parameters in this distinctive cohort of pts. Disclosures:Kipps:Igenica: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Abbott Industries: Research Funding; Genentech: Research Funding; GSK: Research Funding; Gilead Sciences: Consultancy, Research Funding; Amgen: Research Funding.

The detection of chromosome anomalies by QF‐PCR and residual risks as compared to G‐banded analysis

To determine the detection rate of clinically significant chromosome abnormalities using quantitative fluorescent polymerase chain reaction (QF-PCR) of fetal DNA in comparison with G-banded analysis of cultured amniotic fluid cells and determine the residual risk if QF-PCR were performed alone for low-risk cases. Amniotic fluid samples were prospectively categorized based on the likelihood of the fetus having a chromosome anomaly. QF-PCR results were compared with the G-banded findings. The distribution of patients and the rates of clinically significant anomalies in each risk category were determined. A total of 4176 amniotic fluid samples were studied. Among these, 331 cases with abnormalities were detected by both methods and an additional 19 abnormal cases were detected by G-banding only. Five of those undetected by QF-PCR were considered clinically significant, four of which were referred due to an elevated a priori risk (>4%). If QF-PCR is performed in all cases and G-banding performed only in higher risk cases, the residual risk for a clinically significant chromosome abnormality will be as low as 0.083%. This study suggests that QF-PCR alone is appropriate for patients with uncomplicated pregnancies, who are referred solely for an increased risk of a common trisomy.

The detection of chromosome anomalies by QF-PCR and residual risks as compared to G-banded analysis

The detection of chromosome anomalies by QF-PCR and residual risks as compared to G-banded analysis

Single-cell copy number variation detection

Detection of chromosomal aberrations from a single cell by array comparative genomic hybridization (single-cell array CGH), instead of from a population of cells, is an emerging technique. However, such detection is challenging because of the genome artifacts and the DNA amplification process inherent to the single cell approach. Current normalization algorithms result in inaccurate aberration detection for single-cell data. We propose a normalization method based on channel, genome composition and recurrent genome artifact corrections. We demonstrate that the proposed channel clone normalization significantly improves the copy number variation detection in both simulated and real single-cell array CGH data.

STUDY OF MOTOR VEHICLE EXHAUST EXPOSURE,AS A RISK FOR GENOTOXICITY AND NEPHROTOXICITY AMONG PROFESSIONALDRIVERS IN ZAGAZIG CITY, EGYPT.

Objectives: vehicle exhaust consists of many toxic components and professional drivers are occupationally exposed to vehicle exhaust during their routine daily work. Hence, the aim of the present study is to evaluate genotoxic and nephrotoxic risk of vehicle exhaust in professional drivers in Zagazig city, Al-Sharkia Governorate, Egypt.Subjects and Methods: A comparative cross sectional study was carried out on 33 professional drivers and 34 control office workers as non-exposed control group. A structured questionnaire was offered for all subjects through personal interview.Peripheral blood samples were collected from all participants for detection of chromosomal aberrations and measurement of blood lead level. Urine samples also were obtained from all participants for laboratory analysis to detect levels of B2 –microglobulin, microalbumin and trans,trans- muconic acid. Results: A significant increase was observed in the ercentage of chromosomal aberrations in non smoker and smoker professional drivers when compared to their respective control groups. Also a significant increase in the mean values of B2 microglobulin and microalbumin was observed in studied professional drivers when compared to their control. The results showed that professional drivers have significanthigher blood lead level and urinary trans,trans- muconic acid level as compared to control group, with positive correlation to the duration of exposure. Conclusion: Thus the present study suggests that the induction of cytogenetic changesand renal damage might be due to the cumulative effect of smoking, lead toxicity, benzene toxicity, leaded gasoline and prolonged duration of exposure to vehicle exhaust.

Chromosomal Aberration Network In Myeloproliferative Neoplasms

AbstractAbstract 318The classical myeloproliferative neoplasms (MPNs) comprise of three entities: polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Despite distinct phenotypic features of MPN entities they share characteristics like clonal hematopoiesis, risk for thrombosis and bleeding and tendency to transform to secondary acute myeloid leukemia (post-MPN AML). In order to investigate the genetic lesions associated with MPN a large single-center cohort of 311 MPN patients was analyzed for chromosomal aberrations using high resolution Affymetrix SNP 6.0 arrays. The cohort included 150 patients with PV, 90 with ET, 68 with PMF and 3 with post-MPN AML. Of the 311 patients, 144 (46%) had a normal karyotype and 167 (54%) harbored 1 to 8 detectable chromosomal aberrations. We found 51 gains, 102 deletions and 143 uniparental disomies (UPDs). A total of 13 recurrent chromosomal defects (more than three events) were detected. We investigated if either the number of chromosomal aberrations in a patient or specific types of lesions associate with a certain patient group defined by clinical criteria. Chromosomal aberrations were equally distributed among the three MPN entities and only 9pUPD showed significant clustering with PV. We did not detect an association between the number of chromosomal aberrations and disease duration. Patients positive or negative for JAK2 mutations did not differ significantly in the frequency of chromosomal aberrations (except of the association of 9pUPD with JAK2 positive MPN). Patients with complex karyotype were significantly older than patients with normal karyotype (P<0.001). Transformation to post-MPN AML is an important complication in MPN. To investigate associations between chromosomal changes and transformation, we included additional 19 post-MPN AML patients from another center into the study (total N=22). Patients in the post-MPN AML group harbored significantly more chromosomal lesions (P<0.001). Recurrent aberrations of chromosomes 1q, 7p, 7q, 5q, and 3q strongly associated with post-MPN AML. When we reviewed the clinical data of patients in chronic phase MPN harboring the leukemia-associated aberrations, they showed features of disease progression, and some transformed to AML at a later follow-up. We were able to map a common deleted region (CDR) on chromosome 4 to the tet oncogene family member 2 (TET2), a gene frequently deleted in myeloid disorders. On chromosome 7p we mapped a CDR to the Ikaros transcription factor (IKZF1) and a 7q CDR mapped to a novel putative tumor suppressor, the cut-like homeobox 1 gene (CUX1). Interestingly, in one patient who carried a UPD of chromosome 7q we did not detect a mutation in the CUX1 gene but an R288Q mutation was found in the EZH2 gene. Chromosome 7 aberrations in our cohort were strongly linked to post-MPN AML. Our results show that at least three chromosome 7 genes (IKZF1, CUX1, and EZH2) are relevant in leukemic transformation. In addition to chromosome 7, we found gains of chromosome 1q equally relevant in post-MPN AML. We mapped the common 1q amplification to a 3.5 Mbp region that contained the MDM4 gene. Mdm4 is a known negative regulator of p53 and was frequently shown amplified in various cancers. This result prompted us to investigate the relevance of the p53 pathway in post-MPN AML and we sequenced TP53 in all 22 leukemic patients and found mutations in 6 cases (27.3%). Interestingly, none of the patients with TP53 mutation carried an MDM4 amplification. Taken together, 10 out of 22 post-MPN AML cases (45.5%) had evidence of a p53-related defect. To gain deeper insight into the pathways involved in transformation to post-MPN AML we sequenced genes commonly affected in de novo AML, and found two patients with mutations in FLT3, two patients with RUNX1 mutations, two patients with either IDH1 or IDH2 mutations. We conclude that lesions known to play an important role in de novo AML are present only in a fraction of post-MPN AML patients. In this study we show that aberrations of the p53 pathway together with the chromosome 7 lesions affecting IKZF1 and CUX1 are present in 64% of all post-MPN AML patients. Our data give insight into the genetic complexity and heterogeneity of MPN patients in chronic phase as well as in post-MPN AML. The marked genetic heterogeneity of MPN patients will render targeted therapies challenging and underlines the requirement of personalized treatments. Disclosures:No relevant conflicts of interest to declare.

Combined simultaneous analysis of morphology and fluorescence in situ hybridization as a novel sensitive method for detecting small populations of malignant cells in patients with a high risk of cancer, premalignant lesions, and minimal residual disease

Fluorescent in situ hybridization (FISH) is a valuable tool for detection of chromosome aberrations in clinical practice of malignancies. However, the relatively high false positive and false negative rates of this method complicate its application to analysis of small populations of pathological cells. Combined simultaneous analysis of morphology and FISH using a Duet™ multiparametric cell scanning system (Bio-View, Rehovot, Israel) has been developed and introduced into clinical practice. The novel method has been demonstrated to increase the specificity of identification of pathological cells in patients with various hematological malignancies and, hence, estimate the efficiency of treatment. Studies on different premalignant disorders of the oral cavity mucosa have demonstrated that the combined method allows the identification of individual pathological cells and, hence, early detection of malignant and premalignant cells that may be overlooked by traditional histomorphological methods.

Genotoxic effect of occupational exposure to cadmium

Many studies proved the genotoxic effect of cadmium (Cd) exposure and highlighted the importance of the cytogenetic studies as a sensitive and effective means for early detection of Cd-induced mutagenicity. The relationship between occupational exposure to Cd and increased risk of cancer, particularly lung cancer, has been explored in number of epidemiological studies. The aim of this study is to assess the role of chromosomal abnormalities and sister chromatid exchange as sensitive indicators for the genotoxicity of occupational exposure to Cd. Cytogenetic studies was done for 40 workers (27 smokers and 13 non-smokers) exposed to Cd dust and fumes with 40 control subjects (28 smokers and 12 non-smokers) not exposed to Cd before. Both exposed and control groups were similar in age and other sociodemographic factors. Clinical examination, laboratory investigation including urinary and blood Cd, cytogenic analysis for detection of chromosomal aberrations and sister chromatid exchange and environmental study of the work places were done. Statistical analysis of cytogenetic studies revealed the presence of significant elevation of chromosomal aberrations and sister chromatid exchanges of the exposed group. Urinary and blood Cd of the exposed group were significantly higher than that of the control group. These abnormalities were not significantly affected with age, duration of exposure, smoking habits, blood and urinary Cd. In conclusion, the study adds more proof that Cd exposure has a genotoxic effect and highlighted the importance of using cytogenetic studies as a sensitive and effective means for early detection of Cd-induced mutagenicity.