Conventional Chromosome Banding Analysis Research Articles

Application of fluorescence in situ hybridization technique to verify the clonalities of non-clonal cytogenetic abnormalities identified in Myelodysplastic syndrome

To assess the value of fluorescence in situ hybridization (FISH) technique for the verification of the clonalities of non-clonal cytogenetic abnormalities (n-CCA) identified by conventional chromosome banding analysis (CBA) in patients with Myelodysplastic syndrome (MDS). Clinical data and results of karyotyping and FISH assays for 91 patients of MDS with n-CCA identified by CBA were retrospectively analyzed. In total 94 non-clonal +8, 5q-, -7/7q- or 20q- were detected by CBA, among which 43 (45.7%) were verified to be clonal abnormalities by FISH. The detection rates for +8, 5q-, -7/7q- and 20q- by FISH were 47.6% (30/63), 25% (2/8), 41.7% (5/12), 40% (2/5) and 66.7% (4/6), respectively, with the positive cells accounting for 4% to 90% of all counted cells, with a median value of 7%. The 91 patients were divided into three groups including ≥ 20, 10 ~< 20 and < 10 based on the numbers of metaphase cells in CBA, and the detection rates by FISH for the three groups were 43.7% (31/71), 33.3% (3/9) and 63.6% (7/11), respectively, which showed no statistically difference (P > 0.05). Continuous CBA and FISH surveys were conducted for 26 patients who received supportive treatment, and the results revealed that 91.7% (11/12) of FISH-verified positive abnormalities had persisted, whereas 92.9% (13/14) of the n-CCA verified as negative by FISH was transient. Nearly half of the CBA identified n-CCA have been verified as clonal aberrations by FISH, and the FISH detection rate showed no correlation with the number of metaphase cells. FISH test is strongly recommended for verifying the clonalities of n-CCA detected by CBA, and continuous cytogenetic survey of the patients with MDS is necessary.

CpG-ODN Stimulation in Population Screening Monoclonal B-Cell Lymphocytosis

Synthetic CpG-oligodeoxynucleotides (CpG-ODN) induce proliferation in normal and malignant lymphocytes B (LyB). This effect is widely exploited in CLL conventional chromosome banding analysis (CBA), which has become reliable only after the cultivation of CLL LyB using CpG-ODN stimulation in combination with IL-2. Monoclonal B-cell lymphocytosis (MBL) differs from CLL mainly in clone size. Only cytogenetic data on recurrent chromosomal aberrations analyzed by fluorescence in situ hybridization (FISH) are available for population screening MBL (sMBL). In sMBL the clone of malignant LyB is typically below 10/µL. We compared CpGODN stimulation in healthy donors and in individuals with sMBL. LyB and MBL LyB count were determined by flow cytometry in 15 samples from healthy subjects and 12 MBL cases. Mitotic indices were determined and CBA was done after cultivation of samples by CpG-ODN + IL2. In MBL samples, FISH analysis was performed on isolated LyB. MBL LyB clones in sMBL cases presented less than 1% of WBC and up to 33% of LyB. The MBL group was therefore compared to the group of healthy donors. Although normal and MBL group did not differ in WBC, overall LyB, and normal LyB count, a significantly higher mitotic index was observed in MBL samples (p = 0.0139). We were able to accomplish CBA in all samples which revealed a normal karyotype in all but one case. In this particular sMBL case FISH performed on isolated LyB showed 5% trisomy 12 which was later confirmed by CBA on CpG stimulated blood sample in 15% of metaphases. Our study, which was done on MBL cases obtained by population screening, confirmed that CpGODN preferentially induced proliferation in MBL LyB over normal LyB. Therefore, CBA can also be successfully accomplished in sMBL and can be used to additionally confirm clonality as well as to improve sensitivity of FISH analysis. Due to coexistence of comparable size of normal and malignant LyB, MBL can serve as a model for exvivo studying of LyB stimulation by CpG-ODN.

Molecular cytogenetic monitoring from CD34+ peripheral blood cells in myelodysplastic syndromes: First results from a prospective multicenter German diagnostic study

The gold standard of cytogenetic analysis in myelodysplastic syndromes (MDS) is conventional chromosome banding (CCB) analysis of bone marrow (BM) metaphases. Most aberrations can also be detected by fluorescence-in situ-hybridization (FISH). For this prospective multicenter German diagnostic study (www.clinicaltrials.gov: #NCT01355913) 360 patients, as yet, were followed up to 3 years by sequential FISH analyses of immunomagnetically enriched CD34+ peripheral blood (PB) cells using comprehensive FISH probe panels, resulting in a total number of 19,516 FISH analyses. We demonstrate that CD34+ PB FISH correlates significantly with CCB analysis and represents a feasible method for a reliable non-invasive cytogenetic monitoring from PB.

Equivalence of BCR-ABL Transcript Levels

AbstractAbstract 2495 Introduction:Response to therapy in patients with chronic myeloid leukemia (CML) is monitored by both cytogenetic assessment of bone marrow metaphases and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) of peripheral blood samples. Although standardization of BCR-ABL quantification according to the international scale (IS) provides results comparable between different laboratories, current treatment recommendations are still predominantly based on cytogenetic analyses. The aim of this work was to determine a BCR-ABL expression level according to the IS (BCR-ABLIS) which corresponds to complete cytogenetic remission (CCyR). Patients:1,329 paired cytogenetic and molecular assessments of 557 chronic phase CML patients on imatinib-based therapies from the German CML study IV, all during follow-up, have been considered. Median number of samples per individual was 3 (range 1–24). Only molecular evaluations derived from peripheral blood and from patients expressing b2a2 and/or b3a2 BCR-ABL transcripts were considered. Participating laboratories provided results standardized according to the international scale. For cytogenetic analyses at least 20 metaphases, evaluated by conventional chromosome banding analysis, were required. Paired molecular and cytogenetic analyses were performed within an interval of <3 days. Methods:Data was split randomly at the rate of 2:1 into learning and validation sample. 10,000 subsamples that contained no more than one observation per patient were repeatedly drawn randomly from the original learning sample, according to the bootstrap method Using these subsamples, the BCR-ABLIS expression level providing the best discrimination between CCyR and no CCyR was determined choosing the smallest p-value of the exact Fisher test. Only cut-off levels with p-values which were significant after adjustment for multiple testing were considered.To confirm these results, again 10,000 subsamples were randomly drawn from the original validation sample. For the cut-off points discovered in the learning sample, sensitivity and specificity for predicting a CCyR from the molecular data and the p-values from the Fisher test were calculated within each of these subsamples. Results:1329 cytogenetic analyses using a median of 25 metaphases (range, 20–30) of which 75% were in CCyR were compared with 1329 molecular data with a median BCR-ABLIS expression level of 0.055% (range, 0–237%). In the learning sample the most frequently found BCR-ABLIS value was 0.35%, but all values between 0.2% and 1.1% were similarly well-suited for separating between patients in CCyR and not in CCyR. Based on this range, two potential cut-off levels were chosen for validation, 0.35% and 1%. While the first one represents a value, where one would rather expect to underestimate the number of CCyRs, the latter represents a value, where one would rather expect to overestimate the number of CCyRs. For the level of 0.35% the p-value was below 0.05 for 67.2% of the 10,000 subsamples, the median p-value was 0.017. With a median value of 88.4%, CCyR cases were correctly identified while cases that were not in CCyR were correctly assessed with a median rate of 71.4%. For the 1% BCR-ABLIS level, the p-value was below 0.05 for 72.0% of the 10,000 subsamples with a median p-value of 0.011. At a median value of 96.0%, CCyR cases were correctly identified while cases that were not in CCyR were correctly assessed with a median rate of 60.0%. When restricting on BCR-ABLIS values between 0.1 and 10.0%, the median concordance rate was 76%. Conclusions:Compared to molecular analyses, cytogenetic assessment is imprecise and limited with regard to sensitivity. Thus, it is not surprising to find a range of molecular data equivalent to CCyR but not a one-to-one cut-off point. However, for practical reasons it seems desirable to have such a cut-off point. Since lack of CCyR is considered a failure criterion according to the ELN treatment recommendations, it is suggested to keep the proportion of patients small that are considered erroneously not to be in CCyR. Additionally, a considerable amount of patients in major molecular remission or even better is present in the sample, so there might be a slight bias towards lower cut-off values. Thus, BCR-ABLIS expression of <1% is considered to be equivalent to CCyR in chronic phase CML patients treated with imatinib with a median concordance rate of 89%. Disclosures:Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Hochhaus:Novartis, BMS, MSD, Ariad, Pfizer: Consultancy Other, Honoraria, Research Funding. Müller:Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding.

Genome Wide SNP Analysis Reveals Frequent Cryptic Clonal Chromosomal Aberrations Including Uniparental Disomy (UPD) in Waldenstrom's Macroglobulinemia.

Abstract 3932Poster Board III-868 BackgroundWaldenstrom's macroglobulinemia (WM) is a rare lymphoproliferative disorder characterized by bone marrow (BM) infiltration of lymphoplasmacytic cells that secrete monoclonal IgM antibody. Approximately 50% of patients (pts) with WM exhibit a normal karyotype using either conventional chromosome banding analysis (CBA) or FISH approach. However, CBA is a low resolution method and FISH only target previously described abnormalities. Comparative genomic hybridization (CGH) array delineated the minimal deleted region on 6q deletion, the most frequent aberration in WM, and pointed out the role of NFKB pathway key regulators genes. However, tumour suppressor gene or oncogene involved in WM physiopathology is not known, to date. Partial uniparental disomy (UPD) induced by copy neutral loss of heterozygosity (LOH) are important mechanisms for tumour suppressor gene inactivation or oncogene activation in cancer. Recently, Single Nucleotide Polymorphism (SNP) based arrays had been described as a powerful high resolution method allowing both the detection of LOH and copy number alteration (CNA) analysis in the same experiment, a major advantage over CGH array. Our aim was to identify new CNA and LOH involved in WM pathogenesis using SNP arrays analysis. Material and Methods. BM samples of 12 pts with WM (7 males, mean age: 67 years, 7 symptomatic pts) were analysed. DNA was extracted following CD19 B cells selection. Genome-Wide Human SNP Array 6.0 (Affymetrix chips) was used to detect both LOH and CNA. In 6 pts, paired samples (tumor / normal T lymphocytes) were used as an intra-individual reference to identify germline polymorphisms. In the 6 left pts, CN polymorphisms were excluded using copy-number variants database (http://projects.tcag.ca/variation) and the reference genotyping data from the HapMap project. Size, position and location of genes were identified with UCSC Genome Browser HG18 assembly, LOH and CNA using genotyping console 3.02 software (Affymetrix) and Partek genomic suite. FISH analysis was performed to detect deletion 6q; 13q14, 11q22, TP53, trisomy 4 and 12 chromosomal aberrations using Vysis probes. ResultsSNP array detected 35 CNA (23 gains, 12 losses) in 9/12 pts, with a mean of 2.9 abnormalities per patient (range 0 to 10). 91% of CNA were observed in symptomatic pts. 50% of CNA were < 5 mb in size, the lower limit of detection by CBA. 15 cryptic aberrations were identified among the somatic CNA spread over 7 pts. These 11 gains and 4 losses are identified by *. Deletions were observed on chromosomal segments 1p36*, 3p21.*, 6q16-q27 (2pts), 8p21*, 8p23-q24, 9p21*, 11q22 (2pts), 16p13*, 19p13*. One cryptic homozygous deletion in 13q14 was identified. Gains were observed on chromosomal segments 3p26, 4p16-q35 (3pts), 4q24*, 5p15 (2pts)*, 5p14, 7p22, 12p13-q24, 16p13*, 17q11*, 18p11-q23, 19p13-q13, Xq21* (5pts), Xq22*, Xq25*, Xp22-q28, Xq27-q28*(2pts).We also identified 42 LOH in 9/12 pts (mean of 3.5 per genome, range 0 to 7) dispersed on 17/23 chromosomes. The LOH observed in the absence of CNA loss are consistent with UPD in 35 cases (83%). 59% of LOH were observed in symptomatic pts. The interstitial or telomeric UPD regions varied in size, from 0.4 to 154mb. Two recurrent regions were identified on chromosome 4 (1.2mb involving genes DCLK2 and LRBA) and 13 (0.8mb involving genes KPNA3, ARL11 and SETDB2), probably subsequent to mitotic recombination. No UPD was observed on chromosome 6q.SNP arrays detected all FISH and CBA findings, except in one pt with subclonal deletion of TP53. Of the 5 pts with normal CBA or FISH, SNP array detected a CNA and 4 UPD in one patient and 7 UPD in an other patient. The remaining 3 cases (25%) had no detected CNA or LOH.Overall, SNP array detected a total of 77 genetic aberrations (CNA + LOH) (6.4 per genome, range 0 -16) in this cohort allowing the selection of 976 relevant genes (159 implicated in cellular growth and proliferation, 165 in cancer, 69 in cell cycle). ConclusionNew cryptic clonal chromosomal lesions were detected using high resolution SNP array in this study. Several abnormalities were recurrent. We described a high frequency of UPD in WM, that might contribute to the inactivation of tumour suppressor genes by mutations or epigenetic alterations and subsequently to the regulation of tumor progression in WM. Further confirmation of the role of these candidate genes is underway. Disclosures:Leleu:Celgene: Research Funding; Janssen Cilag: Research Funding; Chugai: Research Funding.

Cytogenetic and comparative genomic hybridization findings in four cases of breast cancer after neoadjuvant chemotherapy

To assess a potential common pattern of genetic alterations in chemotherapy-resistant tumors we analyzed four tumors from breast cancer patients (patients 1–4) after neoadjuvant chemotherapy, by comparative genome hybridization (CGH) and conventional chromosome banding analysis. All patients showed structural aberrations involving chromosomes 1, 5, 11, 16, and 17. In CGH analysis, the patients showed typical imbalances for ductal breast cancer: gains of 1q (3 patients), 5q (2 patients), 8q (3 patients), and X (4 patients) and losses of 1p33∼p36 (3 patients), 16q (3 patients), 17p (3 patients), 19 (4 patients), and 22q (4 patients). Other recurrent imbalances of atypical pattern for ductal breast cancer were gain of 4q21∼q32 (2 patients), 20q21∼q22 (2 patients), and 21 (2 patients) and loss of 20p (3 patients). Three patients showed involvement of several regions bearing genes of drug resistance ( MDR1 [HUGO symbol: ABCB1], BCRP [HUGO symbol: ABCG2], MRP1 [HUGO symbol: ABCC1], RFC1); the fourth patient displayed an amplification in the region of MYC (alias c-myc), thus providing—at the level of the light microscope—an explanatory background for the ability of their tumors to survive anthracycline-, taxane- and cyclophosphamide-based chemotherapy. Conventional cytogenetic analysis and CGH displayed highly coincidental findings in the tumors of four patients after neoadjuvant chemotherapy for breast cancer.

Modern molecular cytogenetic techniques in genetic diagnostics

During the past decade, fluorescence in situ hybridization (FISH) has become an important complementing application in genetic diagnostics. The use of variable FISH techniques enhances the thorough interpretation of numerical and complex chromosome aberrations, bridging the gap between conventional chromosome banding analysis and molecular genetic DNA studies. This review gives a brief overview of the different molecular cytogenetic FISH techniques and applications currently used in routine genetic diagnostics and focus on their advantages and limitations.

Multicolor fluorescence in situ hybridization in clinical cytogenetic diagnostics.

Multicolor fluorescence in situ hybridization is a technology that has vastly expanded the diagnostic repertoire of the clinical cytogenetics laboratory. The limitations of conventional chromosome banding analysis can often be overcome by the high sensitivity and specificity of multicolor fluorescence in situ hybridization tests. This article reviews the latest multicolor fluorescence in situ hybridization tests (including multiplex fluorescence in situ hybridization, spectral karyotyping, cross-species color banding, and comparative genomic hybridization) that are currently limited to a few select clinical cytogenetic laboratories, but may soon have more dominant roles in clinical cytogenetic practice.

Clinical aspects and genetics of Williams-Beuren syndrome. Clinical and molecular genetic study of 44 patients with suspected Williams-Beuren syndrome

The suspected diagnosis of Williams-Beuren syndrome (WBS), which is a retardation syndrome with great clinical variability, was cause for comparison of molecular genetic, molecular cytogenetic analysis to clinical symptoms. The results of the genetical analysis of a microdeletion of the elastin gene region on chromosome 7 were compared to the clinical symptoms. Are there any differences between symptoms in case of deletion or non-deletion? How informative are the molecular genetic, molecular cytogenetic analysis? 44 patients with suspected diagnosis of WBS were examined using molecular genetic and molecular cytogenetic methods. The clinical symptoms as general symptoms, heart anomaly, dysmorphic signs and unusual neurobehavioural features were reported during clinical investigation in standardized questionnaires. The genomic DNA of the patients and their parents was analyzed using microsatellite markers. In some cases (e.g. uninformative microsatellite studies) we also used fluorescence in situ hybridization (FISH) with an elastin gene probe and performed a conventional chromosome banding analysis. 15 patients had a microdeletion. 4 patients had a deletion of the paternal allel and 7 patients showed the deletion of the maternal allel. The polymorphisms were of limited informativeness. In 2 cases microsatellite analysis was not able to determine whether the paternal or the maternal allel had been lost. In 2 cases the microsatellite analysis was uninformative so that FISH analysis was performed. All FISH analysis performed had an informative result. 80% of the children with a microdeletion of chromosome 7q11.23 showed the typical dysmorphic signs, 70% exhibited the typical WBS behaviour pattern, 50% had a specific heart anomaly. In contrast, in the group of children without a chromosomal microdeletion only 30-40% showed typical dysmorphic signs, only 10% had a typical heart anomaly and none of them showed specific behavioural changes. We found no indication to association of specific symptoms with paternal versus maternal origin of the deletion. The FISH analysis combined with a conventional chromosome banding analysis is very informative for diagnostic values. The results are compared to data of literature. Children with developmental retardation and WBS dysmorphic signs and an unusual behaviour should be examined by a molecular cytogenetic FISH analysis. If a microdeletion of band 7q11.23 is found a special cardiologic examination should be offered.

Variant intra Philadelphia translocation with rearrangement of BCR-ABL and ABL-BCR within the same chromosome in a patient with cALL

A unique variant Philadelphia translocation accompanied by the loss of the short arm of chromosome 9 in a 32-year-old female with common acute lymphoblastic leukemia (cALL) is described. Furthermore, supernumerary chromosome 8 material was found as an insertion into the long arm of chromosome 2 and/or as ring chromosomes in addition to two normal chromosomes 8. The chromosomal abnormalities were identified by combined conventional chromosome banding analysis and fluorescence in situ hybridization (FISH). The BCR-ABL rearrangement was confirmed by FISH and reverse transcriptase-polymerase chain reaction (RT-PCR) studies. Possible mechanisms leading to this variant intra Philadelphia translocation are discussed. The aberrations found have prognostic implications, because 9p anomalies confer an adverse effect to the already poor prognosis of Philadelphia-positive ALL.

Chromosome aberrations in B-cell chronic lymphocytic leukemia: reassessment based on molecular cytogenetic analysis.

In B-cell chronic lymphocytic leukemia (B-CLL) clonal chromosome aberrations are detected in approximately 40-50% of tumors when using conventional chromosome banding analysis. Most studies find trisomy 12 to be the most frequent chromosome aberration, followed by structural aberrations of the long arm of chromosomes 13 and 14. Trisomy 12 and the "14q+" marker are associated with shorter survival times, while the patients with 13q abnormalities have a favorable outcome, similar to those with a normal karyotype. The development of molecular cytogenetic techniques has greatly improved our ability to detect chromosome aberrations in tumor cells. Using fluorescence in situ hybridization, chromosome aberrations can be detected not only in dividing cells but also in interphase nuclei, an approach referred to as interphase cytogenetics. The prevalence of specific aberrations in B-CLL is currently being reassessed by interphase cytogenetics. By far the most frequent abnormality are deletions involving chromosome band 13q14, followed by deletions of the genomic region 11q22.3-q23.1, trisomy 12, deletions of 6q21-q23, and deletions/mutations of the TP53 tumor suppressor gene at 17p13. The evaluation of the true incidence of these aberrations now provides the basis for more accurate correlations with clinical characteristics and outcome. Deletions/mutations of the TP53 gene have been shown to be associated with resistance to treatment and to be an independent marker for poor survival. 11q deletions have been associated with extensive nodal involvement, rapid disease progression, and short survival times. Whether trisomy 12, 13q14, and 6q deletions have a prognostic impact awaits further study. The application of these molecular cytogenetic techniques will also contribute to the identification of the pathogenetically relevant genes that are affected by the chromosome aberrations in B-CLL.

Design and Validation of DNA Probe Sets for a Comprehensive Interphase Cytogenetic Analysis of Acute Myeloid Leukemia

The objective of this study was to design DNA probe sets that enable the detection of chromosome aberrations in acute myeloid leukemia (AML) by interphase cytogenetics using fluorescence in situ hybridization (FISH) and to compare the results of interphase cytogenetics with those of conventional chromosome banding analysis. One hundred five consecutive patients with adult AML entered on a multicenter treatment trial were studied with a comprehensive set of DNA probes recognizing the most relevant AML-associated structural and numerical chromosome aberrations: translocations t(8;21), t(15;17), and t(11q23); inversion inv(16);chromosomal deletions (5q-, 7q-, 9q-, 12p-, 13q-, 17p-, and 20q-); and chromosomal aneuploidies. Interphase cytogenetics was particularly sensitive for detecting the AML-specific gene fusions: 3 additional cases of inv(16) and 1 additional case of t(8;21) were identified by FISH that were missed by banding analysis, whereas equal numbers of t(11q23) and t(15;17) were detected. Five additional cases of trisomy 8q, 3 more cases of trisomy 11q, and 2 more cases of trisomies 21q and 22q were shown by FISH. These aberrations were either masked in complex karyo-types or identified in cases in which conventional banding analysis failed. On the other hand, the DNA probes selected were not informative to detect 1 case of 5q-, 9q-, and 20q-. In 5 cases, clonal aberrations were detected on banding analysis for which no FISH probes were selected. In conclusion, interphase cytogenetics proved to be more sensitive for detecting AML-specific chimeric gene fusions and some partial trisomies. Interphase cytogenetics provides a powerful technique complementary and, with further development of diagnostic DNA probes, even an alternative to chromosome banding studies for the cytogenetic analysis of AML.

The use of FISH with chromosome-specific repetitive DNA probes for the follow-up of leukemia patients: Correlations and discrepancies with bone marrow cytology

The use of fluorescence in situ hybridization (FISH) for the purpose of repeated follow-up examination of bone marrow samples from 38 leukemia patients was investigated. On the basis of conventional cytogenetic analysis, patients with acute leukemia whose leukemic cells carried numerical chromosomal aberrations were selected and followed with repetitive DNA probes that specifically hybridize to one chromosome type. Repeated cytogenetic metaphase analyses would have been laborious and not sensitive or quantitative enough to follow declining numbers of aberrant cells. FISH, as an interphase cytogenetic technique, provides a rapid and simple alternative with high sensitivity. Although FISH data before and after chemotherapy were in agreement with bone marrow cytology in 30 of 38 patients, discrepancies were noticed in specific cases. These could be explained by the presence of cytogenetically distinct subclones that behave differently during treatment, the presence of differentiated leukemic cells, changes in the chromosomal constitution caused by clonal relapse, or the fact that a numerical aberration is found by conventional chromosome banding analysis while the target region to which the probe is directed is still present in the nucleus as a diploid set.