Introduction
From the 25,000 genes in the human genome, approximately 350 genes have been causally linked to the development of cancer. Variant or aberrant function of these so-called cancer genes may result from changes in genome copy number (through amplification, deletion, chromosome loss, or duplication), changes in gene and chromosome structure (through chromosomal translocation, inversion, or other rearrangements that lead to chimeric transcripts or deregulated gene expression) and point mutations (including base substitutions, deletions, or insertions in coding regions and splice sites). The vast majority (90%) of cancer genes are mutated or altered through chromosomal aberrations in somatic tissue, about 10% are altered in the germ line, thereby transmitting heritable cancer susceptibility through successive generations. In addition to high resolution chromosome banding and advanced chromosomal imaging technologies, chromosome aberrations in cancer cells can be analyzed with an increasing number of large-scale, comprehensive genomic and molecular genetic technologies – including fluorescence in situ hybridization (FISH).
Chromosomal translocation (t) is the process by which a break in at least two different chromosomes occurs,
with exchange of genetic material between the chromosomes.
Translocation, Dual-Fusion Assay
Dual-fusion, dual-color FISH assays for translocation utilizes large probes that span 2 breakpoints or flanking regions on the different chromosomes. Dual-fusion, dual-color FISH is optimal for detection of low levels of nuclei possessing a simple balanced translocation, as it greatly reduces the number of normal background nuclei with an abnormal signal pattern.
Translocation, Dual-Fusion Assay
Expected signal pattern:
In normal intact cells, two separate red and two separate green individual signals will be visible, whereas a reciprocal translocation will generate two fused red/green signals (often appearing as single yellow signals), accompanied by one red and one green signal (representing the normal chromosomes).
Translocation, Break-Apart or Split Assay
FISH using dual-color, break-apart probes is very useful in the evaluation of genes known to have multiple translocation partners; the differently colored probes hybridize to targets on opposite sides of the breakpoint of the affected gene.
Translocation, Break or Split Array
Expected signal pattern:
In normal cells two sets of red/green-fused signals (representing the two alleles) will be visible. In an abnormal diploid cell, in which one allele has been split by a translocation, a separated red and green signal will be visible in addition to the normal fused signal.
Chronic Myeloproliferative Disorders (CMPD)
Chromosomol translocations in chronic myeloproliferative diseases (CMPD) almost invariably result in epxression of constitutively activated fusion tyrosine kinases. The hallmark of these diseases is CML, where the BCR/ABL activated tyrosine kinase results from the balanced reciprocal Philadelphia chromosome translocation t(9;22).
Leukemia and Lymphomas
Chronic Myelogenous Leukemia (CML)
CML is a malignant chronic myeloproliferative disorder (MPD) of the hematopoietic stem cell. All CML have a t(9;22) causing fusion of the 3’ ABL region at 9q34 with the 5’ BCR region at 22q11. This chimeric BCR/ABL gene encodes a constitutively activated protein tyrosine kinase with profound effects on cell cycle, adhesion, and apoptosis. Understanding this process has led to the development of the drug imatinib mesylate (Gleevec™), the first in a new class of genetically targeted agents, this is a major advance in cancer treatment. Several different approaches are used to analyze the BCR/ABL t(9;22)(q34;q11) by FISH each providing different details about this translocation.
BCR/ABL Product Family
The Philadelphia chromosome is an abnormally short chromosome 22 that is one of the two chromosomes involved in a translocation with chromosome 9. This translocation t(9;22)(q34;q11) takes place in a single bone marrow cell and, through the process of clonal expansion, gives rise to the leukemia. ABL and BCR are normal genes on chromosomes 9 and 22, respectively. The ABL gene encodes a tyrosine kinase enzyme whose activity is tightly controlled. In the formation of the Ph translocation, two fusion genes are generated: BCR-ABL on the Ph chromosome and ABL-BCR on the chromosome 9 participating in the translocation. The BCR-ABL gene encodes a protein with deregulated tyrosine kinase activity. The presence of this protein in the CML cells is strong evidence of its pathogenetic role. The efficacy in CML of a drug that inhibits the BCRABL tyrosine kinase has provided the final proof that the BCR-ABL oncoprotein is the unique cause of CML. The Poseidon portfolio contains now 4 different probes for BCR/ABL to suit all needs for the detection of t(9;22) by FISH:
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