Atypical D-FISH patterns of BCR/ABL gene rearrangements in 169 chronic myeloid leukemia patients

The Philadelphia (Ph) chromosome, a hallmark chromosomal anomaly observed in 95 percent of chronic myeloid leukemia (CML) cases, is known to involve the Abelson (ABL) proto-oncogene on chromosome 9 and the breakpoint cluster region (BCR) gene on chromosome 22, producing BCR/ABL mRNA encoding an abnormal tyrosine kinase protein. In the process of generating BCR-ABL fusion, the deletion of residual BCR or ABL occurs in 15-30 percent of CML patients. In addition, some rearrangements are complex, and do not yield the ABL/BCR fusion due to the involvement of a third chromosome in the rearrangement. The possible role of these deletions and complex rearrangements in disease outcome is an ongoing topic of research. We report our results of cytogenetic analysis with GTG banding and fluorescence in situ hybridization using dual color dual fusion probe (D-FISH) from Vysis Inc, USA in 169 (109 male and 60 female) CML patients registered at The Gujarat Cancer and Research Institute (GC and RI) from April 2004 to December 2005. GTG banding was carried out in 123 cases having analyzable metaphases. Of these 123 cases, D-FISH revealed atypical signal patterns in 57 patients (46%), and 12 cases revealed additional complex translocations indicative of disease progression. Out of 57 cases with atypical FISH patterns, 22 included metaphase FISH results, and the rest had only interphase FISH performed. In addition to the hallmark Philadelphia chromosome, other chromosomal aberrations in CML revealed heterogeneity of molecular events. Pooling of more data may lead to identification of new CML sub-groups and hence help in the analysis of clinical trials. Patients enrolled in our prospective study of prognostic significance will be followed up for disease free and overall survival in correlation with ABL-BCR deletion status.     

Targeting the kinase activity of the BCR-ABL fusion protein in patients with chronic myeloid leukemia

Imatinib mesylate is a major advance in the therapy of patients with chronic myelogenous leukemia (CML). Imatinib mesylate binds to the inactive conformation of BCR-ABL tyrosine kinase suppressing the Philadelphia chromosome positive clone in CML. Clinical studies have yielded impressive results in all phases of CML. With higher rates of complete cytogenetic response with imatinib, molecular monitoring of disease is now advisable in assessing response and determining prognosis. Emergence of resistance to imatinib may be manifest at the hematologic, cytogenetic, or molecular levels in patients who remain in chronic phase, or may be evidenced by the development of more advanced CML phases. Resistance and eventual clinical failure of imatinib occurs in most patients with blastic phase disease. Resistance may occur at the level of Bcr-Abl, with reduction or loss of imatinib effectiveness as a kinase inhibitor, or, despite retention of its inhibitory ability, with changes in the ability to deliver an effective dose at the cellular level, and/or, the leukemia becoming less dependent on Bcr-Abl. The various mechanisms underlying these differing, non-mutually exclusive, mechanisms of resistance must be understood to develop corresponding therapeutic remedies. We review the current data on imatinib in CML, the criteria for diagnosis of imatinib resistance, and the mechanisms that underlie such resistance in CML.     

Genetic Characteristics of the Human Hepatic Stellate Cell Line LX-2

The human hepatic cell line LX-2 has been described as tool to study mechanisms of hepatic fibrogenesis and the testing of antifibrotic compounds. It was originally generated by immortalisation with the Simian Vacuolating Virus 40 (SV40) transforming (T) antigen and subsequent propagation in low serum conditions. Although this immortalized line is used in an increasing number of studies, detailed genetic characterisation has been lacking. We here have performed genetic characterisation of the LX-2 cell line and established a single-locus short tandem repeat (STR) profile for the cell line and characterized the LX-2 karyotype by several cytogenetic and molecular cytogenetic techniques. Spectral karyotyping (SKY) revealed a complex karyotype with a set of aberrations consistently present in the metaphases analyses which might serve as cytogenetic markers. In addition, various subclonal and single cell aberrations were detected. Our study provides criteria for genetic authentication of LX-2 and offers insights into the genotype changes which might underlie part of its phenotypic features.     

Philadelphia-negative chromosomal evolution during treatment for chronic myeloid leukemia

Chromosome evolution is one of the major mechanisms of disease progression and resistance in chronic myeloid leukemia (CML) patients. However, the clinical significance of chromosomal evolution in the Philadelphia (Ph)-negative clone during therapy is not fully understood. We evaluated 94 CML patients in the chronic phase of CML during treatment of the disease. Six of them had Ph-negative chromosome abnormalities during treatment. Four patients with a single abnormality and a good molecular response showed no obvious complications from the chromosomal changes, while two other patients who had complex abnormalities and previous treatment had poor outcomes. Our results highlight the need for close monitoring of this kind of patient, not only on a molecular level but also at the cytogenetic level.     

Cytogenetic and FISH findings are complementary in childhood ALL

Primary genetic abnormalities of leukemia cells have important prognostic significance in childhood acute leukemia. In the last two years 30 newly diagnosed or recurrent childhood ALL bone marrow samples were analyzed for chromosomal abnormalities with conventional G-banding and interphase-fluorescence in situ hybridization (I-FISH) using probes to detect BCR/ABL fusions, cryptic TEL/AML1 and MLL rearrangements and p16(9p21) tumor suppressor gene deletions. G-banded karyotype analysis found clonal chromosomal aberrations in 50% of cases. With the use of complementary I-FISH techniques, ALL-specific structural and numerical changes could be identified in 70% of the patients. Nine cases (30%) had subtle chromosomal aberrations with prognostic importance that had not been detected in G-banded analysis. Conventional G-banding yielded additional information (rare and complex structural aberrations) in 19% of patients. The most common aberration (30%) was AML1 copy number increase present in G-banded hyperdiploid karyotype as a chromosome 21 tetrasomy in the majority of cases; one case displayed 5-6 copies and in another case amplification of AML1 gene on der(21) was combined with TEL/AML1 fusion of the homologue AML1 gene and deletion of the remaining TEL allele. High hiperdiploidy was detected in 6 cases, in one patient with normal G-banding karyotype. TEL/AML1 fusion signals were identified in four patients. Deletion of p16 locus was found in eight cases (23%), of which only two had cytogenetically visible rearrangements. G-banding in combination with I-FISH has produced major improvements in the sensitivity and accuracy of cytogenetic analysis of ALL patients and this method helps to achieve a more precise identification of different risk categories in order to choose the optimal treatment.     

Identification of frequent cytogenetic aberrations in hepatocellular carcinoma using gene-expression microarray data

Background  

Hepatocellular carcinoma (HCC) is a leading cause of death worldwide. Frequent cytogenetic abnormalities that occur in HCC suggest that tumor-modifying genes (oncogenes or tumor suppressors) may be driving selection for amplification or deletion of these particular genetic regions. In many cases, however, the gene(s) that drive the selection are unknown. Although techniques such as comparative genomic hybridization (CGH) have traditionally been used to identify cytogenetic aberrations, it might also be possible to identify them indirectly from gene-expression studies. A technique we have called comparative genomic microarray analysis (CGMA) predicts regions of cytogenetic change by searching for regional gene-expression biases. CGMA was applied to HCC gene-expression profiles to identify regions of frequent cytogenetic change and to identify genes whose expression is misregulated within these regions.     

Molecular cytogenetic insights into the ageing syndrome Hutchinson-Gilford Progeria (HGPS)

Hutchinson-Gilford Progeria Syndrome (HGPS) is an extremely rare genetic disorder characterized by premature ageing in childhood and serves as a valuable model for the human ageing process in general. Most recently, point mutations in the lamin A (LMNA) gene on chromosome 1q have been associated with the disease, however how these mutations relate to the complex phenotype of HGPS remains to be established. It has been shown that fibroblasts from HGPS patients are frequently resistant to immortalization with telomerase (hTERT), consistent with the idea that the loss of a dominant acting HGPS gene is a pre-requisite for immortalization. In this study we report the first detailed cytogenetic analysis of hTERT-immortalised HGPS cell lines from three patients and one corresponding primary fibroblast culture. Our results provide evidence for a cytogenetic mosaicism in HGPS with a distinctive pattern of chromosome aberrations in all the HGP clones. Chromosome 11 alterations were observed at a high frequency in each immortalised HGPS cell line but were also present at a lower frequency in the corresponding primary cells. Moreover, we were able to identify the 11q13-->q23 region as a potential site of breakage. Our results are therefore consistent with a role of chromosome 11 alterations in the escape from senescence observed in HGPS cells. In addition to this defined rearrangement, we consistently observed complex chromosomal rearrangements, suggesting that HGPS displays features of chromosomal instability.     

Advances in molecular genetic diagnosis of leukemia]

Recently, Molecular genetics has remarkably advanced and it is introduced in medicine. The use of recombinant DNA methods for the diagnosis of leukemias is reported with special reference to the contribution of cytogenetic findings, such as specific chromosome aberrations previously obtained. Therefore, cytogenetic studies on Ph1 chromosome and other specific aberrations found in leukemias are historically reviewed. Using Southern blotting, PFGE, PCR, and in situ chromosome mapping techniques we have analyzed many cases with CML and cases with ALL. We found M-bcr rearrangements not only in standard Ph1, but also in complex types and in Ph1 (-) ve CML. Chromosomal in situ hybridization was very informative identifying transposition of bcr and abl genes between chromosomes 22 and 9. In this connection, FISH (fluorescence in situ hybridization) technique was developed by us, which is expected to have an exceptional power of analysis. ALL had either M-bcr or m-bcr rearrangements, the latter being identified by PFGE. Next, application of PCR technique that enables to obtain more than 10(5) copies of target sequences could monitor minimal residual diseases in CML. Recently, the relevant gene were cloned respectively in FAB-M2 and APL (FAB-M3), so that detection of minimal residual diseases will be successfully performed in these types of leukemia. Finally, targeting chemotherapy using antisense sequences is prospectively described.     

Cytogenetic genotype-phenotype studies: improving genotyping, phenotyping and data storage

High-resolution molecular cytogenetic techniques such as genomic array CGH and MLPA detect submicroscopic chromosome aberrations in patients with unexplained mental retardation. These techniques rapidly change the practice of cytogenetic testing. Additionally, these techniques may improve genotype-phenotype studies of patients with microscopically visible chromosome aberrations, such as Wolf-Hirschhorn syndrome, 18q deletion syndrome and 1p36 deletion syndrome. In order to make the most of high-resolution karyotyping, a similar accuracy of phenotyping is needed to allow researchers and clinicians to make optimal use of the recent advances. International agreements on phenotype nomenclature and the use of computerized 3D face surface models are examples of such improvements in the practice of phenotyping patients with chromosomal anomalies. The combination of high-resolution cytogenetic techniques, a comprehensive, systematic system for phenotyping and optimal data storage will facilitate advances in genotype-phenotype studies and a further deconstruction of chromosomal syndromes. As a result, critical regions or single genes can be determined to be responsible for specific features and malformations.     

The effect of the spontaneous level of the chromosome aberration rate on its alterations after a single local irradiation

With X-ray examinations of the stomach and duodenum, changes in chromosomal aberration frequencies in peripheral blood lymphocytes were shown to depend on the spontaneous level of these aberrations in patients exposed to a single local irradiation by 25 R. The cytogenetic efficiency of radiation exposure was found to be local in patients with the lower frequency of spontaneous chromosomal aberrations. A genetic genesis of the revealed dependence is supposed. The effect of irradiation on the correlation between radiosensitivity and radioresistance of the cytogenetic process is regarded as a possible mechanism of the appearance of the observed dependence.     

Genetic features of B-cell chronic lymphocytic leukemia

The genetic features of B-cell chronic lymphocytic leukemia (CLL) are currently being reassessed by molecular cytogenetic techniques such as fluorescence in situ hybridization (FISH). Conventional cytogenetic studies by chromosome banding are difficult in CLL mainly because of the low in vitro mitotic activity of the tumor cells, which leads to poor quantity and quality of metaphase spreads. Molecular genetic analyses are limited because candidate genes are known for only a few chromosomal aberrations that are observed in CLL. FISH was found to be a powerful tool for the genetic analysis of CLL as it overcomes both the low mitotic activity of the CLL cells and the lack of suitable candidate genes for analysis. Using FISH, the detection of chromosomal aberrations can be performed at the single cell level in both dividing and non-dividing cells, thus circumventing the need of metaphase preparations from tumor cells. Probes for the detection of trisomies, deletions and translocation breakpoints can be applied to the regions of interest with the growing number of clones available from genome-wide libraries. Using the interphase cytogenetic FISH approach with a disease specific set of probes, chromosome aberrations can be found in more than 80% of CLL cases. The most frequently observed abnormalities are losses of chromosomal material, with deletions in band 13q14 being the most common, followed by deletions in 11q22-q23, deletions in 17p13 and deletions in 6q21. The most common gains of chromosomal material are trisomies 12q, 8q and 3q. Translocation breakpoints, in particular involving the immunoglobulin heavy chain locus at 14q32, which are frequently observed in other types of non-Hodgkin's lymphoma, are rare events in CLL. Genes affected by common chromosome aberrations in CLL appear to be p53 in cases with 17p deletion and ataxia telangiectasia mutated (ATM), which is mutated in a subset of cases with 11q22-q23 aberrations. However, for the other frequently affected genomic regions, the search for candidate genes is ongoing. In parallel, the accurate evaluation of the incidence of chromosome aberrations in CLL by FISH allows the correlation of genetic abnormalities with clinical disease manifestations and outcome. In particular, 17p abnormalities and deletions in 11q22-q23 have already been shown to be among the most important independent prognostic factors identifying subgroups of patients with rapid disease progression and short survival. In addition, deletion 17p has been associated with resistance to treatment with purine analogs. Therefore, genetic abnormalities may allow a risk assessment for individual patients at the time of diagnosis, thus giving the opportunity for a risk-adapted management.     

Patterns of BCR/ABL Gene Rearrangements in Chronic Myeloid Leukemia with Complex t(9;22) Using Fluorescence In Situ Hybridization (FISH)

Chronic myeloid leukemia (CML) is characterized by the reciprocal translocation t(9;22)(q34;q11.2) which fuses the ABL1 oncogene on chromosome 9 with the BCR gene on chromosome 22. It is the BCR/ABL protein that drives the neoplasm and the ABL/BCR is not necessary for the disease. In the majority of CML cases, the BCR/ABL fusion gene is cytogenetically recognizable as a small derivative chromosome 22(der 22), which is known as the Philadelphia (Ph) chromosome. However, approximately 2-10% of patients with CML involve cryptic or complex variant translocations with deletions on the der(9) and/or der(22) occuring in roughly 10-15% of CML cases. Fluorescence in situ hybridization (FISH) analysis can help identify deletions and complex or cryptic rearrangements. Various BCR/ABL FISH probes are available, which include dual color single fusion, dual color extra signal (ES), dual color dual fusion and tri color dual fusion probes. To test the utility of these probes, six patients diagnosed with CML carrying different complex variant Ph translocations were studied by G-banding and FISH analysis using the BCR/ABL ES, BCR/ABL dual color dual fusion, and BCR/ABL tricolor probes. There are differences among the probes in their ability to detect variant rearrangements, with or without accompanying chromoso me 9 and/or 22 deletions, and low level disease.     

Molecular cytogenetics: Diagnosis and prognostic assessment

This review describes molecular cytogenetic techniques for detection and characterization of genetic aberrations associated with human disease. The techniques of fluorescence in situ hybridization, primed in situ labeling and comparative genome hybridization are described, as are probes for repeated sequences, whole chromosomes and specific loci. Also reviewed are applications of these technologies to pre- and neonatal diagnosis and to the characterization of human malignancies.     

Label-free MS(E) proteomic analysis of chronic myeloid leukemia bone marrow plasma: disclosing new insights from therapy resistance

Chronic myeloid leukemia (CML) is a pluripotent hematopoietic disorder that is currently considered incurable. The tyrosine kinase product of the Philadelphia chromosome, P210 BCR-ABL, provided a pathogenetic explanation for the initiation of the CML chronic phase and is the molecular therapeutic target for the disease. Imatinib mesylate, an orally available BCR-ABL kinase inhibitor, can induce haematologic and cytogenetic remission of CML. However, imatinib resistance occurs frequently, resulting in relapse. New treatment strategies are focusing on resistant CML stem cells and the bone marrow stroma. The identification of novel pathways and mechanisms in the bone marrow microenvironment could significantly contribute to the development of such strategies. In this work, we used a high-resolution label-free MS(E) proteomic approach to identify differential protein expression in the CML bone marrow plasma of responsive and resistant patients. Oxidative lipid metabolism and regulation of the switch from canonical to noncanonical WNT signaling may contribute to CML resistance in the bone marrow compartment.     

Sequential combination of karyotyping and RNA-sequencing in the search for cancer-specific fusion genes

Cancer-specific fusion genes are often caused by cytogenetically visible chromosomal rearrangements such as translocations, inversions, deletions or insertions, they can be the targets of molecular therapy, they play a key role in the accurate diagnosis and classification of neoplasms, and they are of prognostic impact. The identification of novel fusion genes in various neoplasms therefore not only has obvious research importance, but is also potentially of major clinical significance. The “traditional” methodology to detect them began with cytogenetic analysis to find the chromosomal rearrangement, followed by utilization of fluorescence in situ hybridization techniques to find the probe which spans the chromosomal breakpoint, and finally molecular cloning to localize the breakpoint more precisely and identify the genes fused by the chromosomal rearrangement. Although laborious, the above-mentioned sequential approach is robust and reliable and a number of fusion genes have been cloned by such means. Next generation sequencing (NGS), mainly RNA sequencing (RNA-Seq), has opened up new possibilities to detect fusion genes even when cytogenetic aberrations are cryptic or information about them is unknown. However, NGS suffers from the shortcoming of identifying as “fusion genes” also many technical, biological and, perhaps in particular, clinical “false positives,” thus making the assessment of which fusions are important and which are noise extremely difficult. The best way to overcome this risk of information overflow is, whenever reliable cytogenetic information is at hand, to compare karyotyping and sequencing data and concentrate exclusively on those suggested fusion genes that are found in chromosomal breakpoints.This article is part of a Directed Issue entitled: Rare Cancers.     

FISH panels for hematologic malignancies

Cytogenetic analysis of hematological malignancies has played a crucial role in the diagnosis and clinical management of patients, as well as in providing fundamental insights into the genetic basis of the pathogenesis of these diseases. Leukemias and lymphomas have lent themselves readily to karyotypic analysis and undoubtedly represent the greatest successes of cytogenetics in human cancer. Several cytogenetic changes have been shown to have considerable prognostic significance also and are being used as measurable targets for response to therapy. Molecular characterization of the recurrent cytogenetic abnormalities has identified genes involved in leukemogenesis and formed a basis for specific treatment strategies. Fluorescence in situ hybridization (FISH) analysis, since its introduction, has revolutionized the field and enabled a more precise determination of the presence and frequency of genetic abnormalities. It is particularly indispensable where metaphase cytogenetics may be difficult in the largely quiescent cells of some hematological malignancies, particularly the lymphoid disorders. FISH probes have been used extensively to detect nonrandom abnormalities in interphase nuclei and the true incidence of chromosome abnormalities has been proven to be much higher than that detected by conventional chromosomal analysis. The avail- ability of a comprehensive line of commercial probes for rapid identification of critical genetic aberrations has contributed to the widespread use of this technique. It has also led to the current practice in most laboratories to test for genetic aberrations by using FISH panels that have been designed to detect genetic changes important not only in the diagnosis of leukemias and lymphomas, but also because of their association with prognosis, to identify high-risk populations in specific hematological cancers, so they can be targeted for aggressive therapy.     

Balanced complex chromosomal rearrangements (BCCR) with at least three chromosomes and three or more breakpoints: report of three new cases

Balanced complex chromosomal rearrangements (BCCR) encompass a heterogeneous group of rare chromosomal aberrations. In this paper, we report three cases of BCCRs. In two the probands were referred for either genetic counseling or prenatal management. One case was ascertained after chromosome analysis performed because of psychiatric manifestations; this was an isolated finding. We also outline the molecular cytogenetic techniques, which were essential in confirming and precisely delineating the BCCRs identified in these patients. In addition the various aspects of genetic counseling for this type of chromosomal rearrangement, highlighting the details particular to each individual case are discussed. We discuss the classification for this type of chromosomal mutation.     

Protocols for cytogenetic studies of human embryonic stem cells

All cultured cells develop chromosome changes over time, including cultures of human embryonic stem cells (hESC), but only those cells with adaptive chromosomes changes survive. The most frequent chromosome changes in hESC cultures are trisomy 12 and trisomy 17. Cells with these trisomies are indistinguishable from normal cells by appearance and also demonstrate typical markers of pluripotency, making them difficult to identify without cytogenetic analysis. Early detection of these cells is essential since cells with trisomy 12 and 17 can replace the normal cell population in 5-10 passages. Cytogenetic analysis using G-banding is considered to be the gold standard for detecting chromosome abnormalities and, when used in combination with interphase FISH, provides a sensitive method for early detection of cytogenetic aberrations, such as full and partial trisomies of chromosomes 12 and 17. The following discussion describes the cytogenetic methods used in our laboratory to study cultured hESCs, along with recommendations for integrating these methods into a plan for routine cell line quality control.     

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.