Hidden aberrations diagnosed by interphase fluorescence in situ hybridisation and spectral karyotyping in childhood acute lymphoblastic leukaemia

Acute lymphoblastic leukaemia (ALL) is the most common oncologic disease in childhood, accounting for approximately 25% of all paediatric malignancies. Based on clinical risk criteria and modern laboratory investigations including immunophenotyping, cytogenetics and molecular genetics, patients can be divided into prognostic groups and assigned to risk-adjusted treatment protocols. The karyotype is an independent prognostic indicator and has for some aberrations that are associated with a poor outcome a direct impact on the choice of treatment. Cytogenetic analysis in ALL is often hampered by poor chromosome morphology, few malignant metaphases, undetectable chromosomal rearrangements due to regions of a similar size and banding pattern and sometimes only normal metaphases derived from normal cells are found after cell culture. Structural as well as numerical aberrations may therefore remain undetected using conventional G-banding. The application of modern molecular cytogenetic techniques including a broad set of fluorescence in situ hybridisation (FISH) methods and recent developments in comparative genomic hybridisation to DNA microarrays, together with molecular methods such as Southern blotting and RT-PCR has greatly improved the detection rate of genetic changes in ALL. This review emphasises the value of increasing the resolving power of the cytogenetic investigation by spectral karyotyping (SKY) and interphase FISH in identifying prognostically important and novel chromosomal rearrangements as a complement to conventional banding analysis. The results of investigations performed on cases with ALL have shown that interphase FISH is valuable and in many cases even mandatory for the detection of prognostically important genetic abnormalities and should therefore consistently be employed in the routine cytogenetic investigations in ALL. Likewise, SKY is a valuable tool for the cytogenetic analysis. Thus, the results of several different investigations described in this review revealed that SKY yielded additional information in 97/157 (62%) cases with chromosomal aberrations detected by G-banding, and in 10/66 (15%) cases with normal G-banding.     

Cytogenetics and oncogenes in leukemia.

Cytogenetic and molecular studies have assumed an increasing role in the evaluation and management of patients with leukemia. Many cytogenetic findings have become well established as important independent indicators of prognosis in the acute leukemias. There has been a recent explosion of knowledge about the genes involved in leukemogenesis and the manner in which their structure or expression is altered by chromosomal translocations or point mutations. Application of newer techniques such as the polymerase chain reaction and in situ hybridization has begun to allow quantitation of residual leukemia cells after therapy. Cytogenetic and molecular findings should allow us to use more individualized therapy in treating leukemia, as well as therapy targeting leukemia-specific abnormal gene products.     

Applying Cytogenetic and Molecular Information in the Clinic: Implications for the Treatment of Chronic Myeloid Leukemia

The tyrosine kinase inhibitor (TKI) imatinib mesylate has changed dramatically the outcome of patients with chronic myeloid leukemia (CML). Most patients achieve a complete cytogenetic response (CCyR), and many also achieve profound molecular responses. These excellent clinical responses translate into very favorable long-term outcomes. The prognostic impact of achieving cytogenetic and molecular responses at specific time points on response duration and survival has made it even more important to appropriately monitor TKI response in patients with CML. To this end, sophisticated molecular techniques are increasingly available to clinical laboratories. The clinical implications of TKI response monitoring in CML are herein reviewed.     

Usefulness of cytogenetics in leukemias

Present study consists of cytogenetic evaluation in 141 cases referred to our centre for various leukemias. This includes 110 cases of CML, 10 of ALL, 16 of AML (M3), 2 of AML(M2), 2 of MDS and 1 of CMML. The conventional cytogenetic study was carried out in all the cases using G Banding technique. Of the 141 patients studied, 17 patients showed secondary chromosomal alterations along with primary chromosomal alterations. In two patients of CML with secondary chromosomal alteration t(4:9:22), molecular cytogenetic technique (FISH) has been carried out which has confirmed the primary observations revealed by the conventional cytogenetic technique. Other secondary alterations were numerous and would have been missed if only FISH or PCR technique would have been used for diagnosis. We observed from our study that advanced molecular techniques like FISH and PCR cannot replace the conventional cytogenetic study but are useful as supportive and confirmative diagnostic tools.     

Pathologic diagnosis of acute lymphocytic leukemia

With present knowledge, the optimal management of individual patients with acute leukemia requires that every case be studied by morphology, cytochemistry, cytogenetic, immunologic and molecular techniques. An algorithm for diagnostic evaluation and classification of ALL is provided in Fig. 11. Other techniques, such as DNA or cDNA [figure: see text] microarray, are at present important research tools but have not yet had a major effect on patient care. More detailed studies of individual patients need to be conducted at specialized cancer centers, where preservation of cells, DNA, RNA, or protein is possible. Such investigations will yield important information on the clinical importance of the expression of various markers, the prevalence and relevance of bilineage and biphenotypic leukemias, and above all will reveal the mechanisms of leukemogenesis and of disease evolution. Such insights will further aid clinicians in treating ALL and in preventing refractory disease.     

Monitoring bcr-abl by polymerase chain reaction in the treatment of chronic myeloid leukemia

The elucidation of the molecular biology of chronic myeloid leukemia (CML) has provided a paradigm for understanding leukemogenesis, targeted drug development, and disease monitoring at the molecular level. Minimal residual disease (MRD) monitoring by fluorescence in situ hybridization and polymerase chain reaction (PCR) has become an important tool in predicting relapse after allogeneic transplant, allowing for early intervention strategies such as donor lymphocyte infusion. MRD monitoring is important for assessment of disease status in patients who obtain a complete cytogenetic remission, and this approach is likely to play an important role in following patients to determine who will relapse on imatinib mesylate therapy. This review focuses primarily on MRD monitoring by PCR.     

The clinical role of molecular genetics in soft tissue tumor pathology

Cytogenetic and molecular analysis of soft tissue tumors has yielded a wealth of information over the past decade. Some of the genetic aberrations that have been identified appear to be fairly specific for individual tumor types. It is because of this specificity that these findings harbor the promise to become useful as diagnostic and/or prognostic markers. Technical advances that allow the application of cytogenetic and molecular techniques to archival material have been crucial in this respect.Molecular genetics has already become an integral part of the work-up of some tumors, e.g., small cell sarcomas of childhood, which demonstrate fairly characteristic translocations, often involving the Ewing's sarcoma gene. Some genetic abnormalities have become established as prognostic marker, such as the deletion of the short arm of chromosome 1 for neuroblastomas.Soft tissue tumor pathology has also benefitted from major advances in identifying genes that are critical in mesenchymal differentiation or cell cycle control. MyoD is a good example of a such a gene, that has become useful as a diagnostic tool in rhabdomyosarcomas.Beyond potential practical applications of cytogenetic and molecular analyses in the diagnosis of these tumors, we also review their impact on several philosophical concepts concerning soft tissue neoplasia.     

Nuances of Morphology in Myelodysplastic Diseases in the Age of Molecular Diagnostics

Morphologic dysplasia is an important factor in diagnosis of myelodysplastic syndrome (MDS). However, the role of dysplasia is changing as new molecular genetic and genomic technologies take a more prominent place in diagnosis. This review discusses the role of morphology in the diagnosis of MDS and its interactions with cytogenetic and molecular testing. Recent changes in diagnostic criteria have attempted to standardize approaches to morphologic diagnosis of MDS, recognizing significant inter-observer variability in assessment of dysplasia. Definitive correlates between cytogenetic/molecular and morphologic findings have been described in only a small set of cases. However, these genetic and morphologic tools do play a complementary role in the diagnosis of both MDS and other myeloid neoplasms. Diagnosis of MDS requires a multi-factorial approach, utilizing both traditional morphologic as well as newer molecular genetic techniques. Understanding these tools, and the interplay between them, is crucial in the modern diagnosis of myeloid neoplasms.     

Study of Cytogenetic Alterations and Association With Prognostic Factors in Indian Children With B-Lineage Acute Lymphoblastic Leukemia

BackgroundAcute lymphoblastic leukemia (ALL) is a heterogeneous disorder with coexistence of multiple clones. The mortality rate in children with ALL has reduced to 15% to 20% in developed countries. However, it continues to be high in India (25%-35%), which may be attributed to ethnic variation and differences in disease biology. The treatment and outcome in ALL are dependent on risk stratification, which is derived from prognostic factors like leukocyte count, age at diagnosis, immunophenotypic subtypes and, most importantly, cytogenetic alterations. Chromosomal rearrangements are important initiating events in leukemogenesis.Approximately, 75% of children with ALL harbor a recurring chromosomal alteration detectable by karyotyping, fluorescence in situ hybridization, or other molecular techniques. The present study was planned to compare the prevalence of various cytogenetic alterations with western.literature and to see the association of these cytogenetic alterations with other prognostic factors as well as survival outcome.MethodsWe enrolled, 117 children of 1 to 14 years of age with newly diagnosed B-cell ALL from August 2014 to Mar 2016 prospectively. Patients were monitored for response to prednisolone, postinduction complete morphological remission minimal residual disease assessment, and were followed for a minimum 2 years.Results and DiscussionWe observed that poor-risk cytogenetic alterations were more prevalent, whereas good-risk cytogenetic alterations were less frequent in our patient cohort as compared with western studies.ConclusionsWe observed that event-free survival is significantly less in those with poor-risk cytogenetics. We have also highlighted nonrecurrent alterations observed in our study group.     

Research on the molecular basis of neoplasms: malignant melanoma as an example

Knowledge about genetic alterations occurring in human tumors has dramatically increased following the development of cytogenetic and molecular techniques. Various alterations have been characterized: chromosomal damages, oncogene activations, loss of genetic material. Some of those alterations, such as c-abl rearrangements in certain leukemias, are characteristic of a certain type of malignancy. However, in most tumors, no such correlation has been demonstrated. We review here the genetic alterations discovered in human malignant melanomas.     

Cytogenetic and molecular aspects of Philadelphia negative chronic myeloproliferative disorders: clinical implications

Chronic myeloproliferative disorders (CMPD) are clonal disorders of the hematopoietic stem cell. The myeloid lineage shows increased proliferation with effective maturation, while peripheral leukocytosis, thrombocytosis or elevated red blood cell mass are found. In Philadelphia negative CMPD recurrent cytogenetic abnormalities occur, but no specific abnormality has been defined to date. The spectrum of cytogenetic aberrations is heterogeneous ranging from numerical gains and losses to structural changes including unbalanced translocations. The most common chromosomal abnormalities are 20q-, 13q-, 12p-, +8, +9, partial duplication of 1q, balanced translocations involving 8p11 and gains in 9p. Cytogenetic analysis of CMPD by conventional or molecular techniques has an important role in establishing the diagnosis of a malignant disease, adding also more information for disease outcome. Molecular studies may detect the possible role of candidate genes implicated in the neoplastic process, addressing new molecular target therapies. FIP1L1/PDGFRalpha rearrangements, as well as alterations of PDGFRbeta or FGFR1 gene have been found to be associated with specific types of CMPD. Recently, a novel somatic mutation, JAK2V617F, has been reported in most of the polycthemia vera (PV) patients, as well as in a lower percentage in essential thrombocythemia (ET) or idiopathic myelofibrosis (IMF) patients. This finding represents the most important advance in understanding of the molecular mechanisms underlined the pathogenesis of CMPD, contributing to the classification and management of patients.     

Chromosomal imbalances in human lung cancer

A wealth of cytogenetic data has demonstrated that numerous somatic genetic changes are involved in the pathogenesis of human lung cancer. Despite the complexity of the genomic changes observed in these neoplasms, recurrent chromosomal patterns have emerged. In this review, we summarize chromosomal alterations identified in small cell and non-small cell lung cancer, using classical and molecular cytogenetic techniques. These analyses have uncovered a set of chromosome regions implicated in lung cancer development and progression. However, many of the target genes remain unknown. Newer technology, such as array-CGH, when combined with cDNA microarrays and tissue microarrays, will facilitate the integration of genomic and gene expression data and pave the way toward a molecular classification of lung carcinomas. The molecular implications of consistent chromosome imbalances found in lung cancer to date are also discussed.     

Cytogenetics and fluorescence in-situ hybridization in detection of hematological malignancies

BACKGROUND: The technique of Fluorescence In-Situ Hybridization (FISH), a hybrid of cytogenetics and molecular biology has increased the resolution and application of cytogenetics in various neoplastic processes. In various types of leukemias, primary investigation by conventional cytogenetic [CC] technique followed by FISH has increased our understanding of the abnormal clonal formation involving different gene region. AIMS: Present study is aimed to use different kinds of in-house FISH probes in various hematological malignancies and its correlation with conventional cytogenetic finding. MATERIAL AND METHODS: Cytogenetic study was carried out in 360 patients either from peripheral blood or from bone marrow cells suspected for various types of leukemias. Four of 360 cases were further selected for FISH study by using different types of in-house probes, such as BAC [Bacterial Artificial Chromosome], PAC [Phague Artificial Chromosome], alphoid, PCP [Partial Chromosome Paint] and WCP [Whole Chromosome paint]. RESULTS: The results confirmed breakpoints of inversion 16 and del 16 in case 2 and 3 respectively. Whereas, case 1 did not confirm the cytogenetic findings of t(15;17) by PML/RARa fusion signals as multiple cell lines were involved in the patients. PCP and WCP were helpful in the identification of the marker chromosome in case 1. Telomeric and centromeric probes confirmed the cytogenetic findings of t(5;7) in case 4. CONCLUSION: We observe from this study that, in addition to the conventional cytogenetic study, FISH study provide further confirmation of chromosomal rearrangements. This facilitates our understanding of the neoplastic process more precisely for the better prognostication of the patient.     

Fluorescence in situ hybridization: molecular probes for diagnosis of pediatric neoplastic diseases

Fluorescence in situ hybridization (FISH) has become an important tool for diagnosing neoplasia in children. With probes designed to identify specific chromosomes and chromosomal regions, FISH is commonly used to detect the specific chromosomal abnormalities associated with hematologic diseases and solid tumors. Variations of FISH currently being investigated, such as comparative genomic hybridization, multicolor FISH, and microchip arrays, will probably result in additional uses of FISH in both research and clinical cytogenetic laboratories. Although FISH has disadvantages when compared with conventional cytogenetics and molecular methods, FISH will continue to be important in analyzing chromosomal abnormalities of tumors in children.     

Cytogenetics and cancer

Techniques based on fluorescence in situ hybridization (FISH) have bridged the gap between molecular genetics and conventional cytogenetics. Since its introduction in the late 1980s, advanced FISH-based methods have greatly enhanced the cytogenetic analysis of hematopoietic and solid tumors and are rapidly gaining ground in clinical cytogenetic diagnostics. As interest in FISH technologies has grown, it has inspired an era of new FISH-based technologies such as multiplex FISH, spectral karyotyping, and comparative genomic hybridization. In this review, the focus is on the impact of these technologies in the field of cancer genetics.     

Cancer cytogenetics and molecular-genetics - clinical implications (review)

Cancer genetics has become a burgeoning area of both cytogenetic and molecular genetic research and practical clinical application of the findings of such research in human cancer and leukemia. This review summarizes the specific types of chromosomal and associated molecular genetic alterations in leukemia and cancer. The primary objective is to acquaint physicians of various disciplines with the application of cytogenetics and molecular genetics as diagnostic, prognostic and therapeutic indices, as approaches to the evaluation of minimal residual disease and as guides for differentiation therapy and the molecular localization of oncogenes and tumor suppressor genes related to gene therapy in leukemias and cancers.     

High-resolution copy number array in the molecular cytogenetic diagnostics of pediatric malignant hematological disorders

The presence of genetic alterations was investigated by SNP array in combination with conventional and spectral karyotyping and fluorescence in?situ hybridization analysis of 75 consecutive pediatric bone marrow samples. The samples were collected at diagnosis from all children diagnosed with malignant hematological disease between 2006 and 2010 at a single diagnostic center in Gothenburg, Sweden. Conventional cytogenetic and molecular genetics techniques are up to this date essential for the clinical laboratories but there is a need for higher resolution techniques in order to identify new genetic markers that eventually can improve the management of these disorders. Here, we conclude that the addition of SNP array-based karyotyping combined with conventional cytogenetics increase the diagnostic accuracy of pediatric hematological malignancies. The two techniques enhance the cytogenetic image and should not be contrasted as they meet distinct important roles. Since balanced translocations cannot be detected with the SNP arrays of today and tumor-specific translocations are very important diagnostic and prognostic indicators we suggest that SNP-based array is a valuable adjuvant tool in the cytogenetic diagnostics of pediatric leukemias but cannot replace currently used techniques, i.e. G-banding, SKY and FISH analysis.     

Molecular features of B-cell lymphoma

Malignant transformation of B cells can occur at various steps of lymphocyte development, starting from early B-cell progenitors up to mature B cells, which reflects the heterogeneity of B-cell malignancies with regard to their biologic and clinical behavior. The genetic characterization of B-cell neoplasms during the past two decades has elucidated the mechanisms underlying B-cell lymphomagenesis and led to a more precise definition of lymphoma subgroups. This progress is reflected in the upcoming World Health Organization classification for hematologic neoplasms, which stresses the diagnostic importance of recurrent genetic alterations in leukemias and lymphomas. In the recent past, several genes deregulated by such recurrent chromosomal aberrations have been identified. In addition, the recent introduction of microarray technology has now allowed a more global assessment of gene dysregulation in B-cell oncogenesis and provided a new means for more exactly defining the molecular hallmarks of distinct lymphoma subtypes. This review will focus on recently described molecular features of B-cell lymphomas discovered by the application of new molecular cytogenetic techniques, advanced breakpoint cloning strategies, and microarray approaches.