Cancer begins with multiple cumulative epigenetic and genetic alterations that sequencially transform a cell, or a group of cells in a particular organ. The early genetic events might lead to clonal expansion of pre-neoplastic daughter cells in a particular tumor field. Subsequent genomic changes in some of these cells drive them towards the malignant phenotype. These transformed cells are diagnosed histopathologically as cancers owing to changes in cell morphology. Conceivably, a population of daughter cells with early genetic changes (without histopathology) remain in the organ, demonstrating the concept of field cancerization. With present technological advancement, including laser capture microdisection and high-throughput genomic technologies, carefully designed studies using appropriate control tissue will enable identification of important molecular signatures in these genetically transformed but histologically normal cells. Such tumorspecific biomarkers should have excellent clinical utility. This review examines the concept of field cancerization in several cancers and its possible utility in four areas of oncology; risk assessment, early cancer detection, monitoring of tumor progression and definition of tumor margins.

Genomic rearrangements are a frequent source of instability, but the mechanisms involved are poorly understood. A 2.5-kbp poly(purine⅐pyrimidine) sequence from the human PKD1 gene, known to form non-B DNA structures, induced long deletions and other instabilities in plasmids that were mediated by mismatch repair and, in some cases, transcription. The breakpoints occurred at predicted non-B DNA structures. Distance measurements also indicated a significant proximity of alternating purine-pyrimidine and oligo(purine⅐pyrimidine) tracts to breakpoint junctions in 222 gross deletions and translocations, respectively, involved in human diseases. In 11 deletions analyzed, breakpoints were explicable by non-B DNA structure formation. We conclude that alternative DNA conformations trigger genomic rearrangements through recombination-repair activities. G ross chromosomal rearrangements are a common source of genetic instability (1). Thus, characterization of the underlying molecular mechanisms of mutagenesis is fundamental for our understanding of human disease. A hallmark of gross deletions is the presence of short homologous tracts (typically 2-8 bp) at the breakpoints (2), a finding that has prompted speculation as to the two distinct mechanisms postulated to be responsible for their formation. The slipped mispairing model (3) envisages that during lagging strand DNA synthesis, distantly located repeats are brought into close proximity by the looping out of the single-stranded region, thereby enabling the replication complex to ''jump'' from the proximal to the distal repeat and hence bypass the looped structure. Alternative models propose that various types of repetitive sequence elements may serve as substrates for intra-or intermolecular recombination (2, 4). Neither model is satisfactory; slipped mispairing is inconsistent with deletions greater than Ϸ500 bp and deletions manifesting Ͻ4-bp homologies (5-9), whereas the recombination model does not provide a rationale for the initiation of the process.Specific sequence motifs such as direct and inverted repeats, (RY⅐RY) n and (R⅐Y) n , in which R represents purine and Y represents pyrimidine, and four closely spaced G-rich direct repeats [i.e., (G⅐C) 3 ] undergo structural transitions from the orthodox right-handed B-helical duplex to higher energy state non-B structures (slipped hairpin͞loops, cruciforms, left-handed Z-helices, triplexes, and tetraplexes, respectively) (10-12) under torsional stress (negative supercoiling) in vivo.Early articles in bacteria and hamster cells reported isolated cases in which deletions could occur by a recombination-repair reaction mediated by cruciform structures forming at each breakpoint (13,14). Recently, the breakpoint junctions of the human constitutional translocations t(1;22), t(4;22), t(11;22), and t(17;22), which involve a common locus on chromosome 22q11.2, were found to coincide with large (Ͼ95 bp) cruciform structures (15-18), suggesting that this conformation may predispose specific loci to genomic rearrangements.The po...

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The expansion of triplet repeat sequences (TRS) associated with hereditary neurological diseases is believed from prior studies to be due to DNA replication. This report demonstrates that the expansion of (CTG⅐CAG) n in vivo also occurs by homologous recombination as shown by biochemical and genetic studies. A two-plasmid recombination system was established in Escherichia coli with derivatives of pUC19 (harboring the ampicillin resistance gene) and pACYC184 (harboring the tetracycline resistance gene). The derivatives contained various triplet repeat inserts ((CTG⅐CAG), (CGG⅐CCG), (GAA⅐TTC), (GTC⅐GAC), and (GTG⅐CAC)) of different lengths, orientations, and extents of interruptions and a control non-repetitive sequence. The availability of the two drug resistance genes and of several unique restriction sites on the plasmids enabled rigorous genetic and biochemical analyses. The requirements for recombination at the TRS include repeat lengths >30, the presence of CTG⅐CAG on both plasmids, and recA and recBC. Sequence analyses on a number of DNA products isolated from individual colonies directly demonstrated the crossing-over and expansion of the homologous CTG⅐CAG regions. Furthermore, inversion products of the type [(CTG) 13 (CAG) 67 ]⅐[(CTG) 67 (CAG) 13 ] were isolated as the apparent result of "illegitimate" recombination events on intrahelical pseudoknots. This work establishes the relationships between CTG⅐CAG sequences, multiple fold expansions, genetic recombination, formation of new recombinant DNA products, and the presence of both drug resistance genes. Thus, if these reactions occur in humans, unequal crossing-over or gene conversion may also contribute to the expansions responsible for anticipation associated with several hereditary neurological syndromes.Genetic instabilities (expansions and deletions) of triplet repeat sequences (TRS) 1 ((CTG⅐CAG), (CGG⅐CCG), or (GAA⅐TTC)) are a hallmark of certain hereditary neurological diseases (1, 2). Numerous workers in human genetics have proposed DNA replication, gene conversion, recombination, and related processes as the mechanism(s) responsible for these alterations in repeat sequence lengths. Subsequent in vivo studies in genetically tractable systems (1, 3) and in vitro investigations (4) have demonstrated expansions and deletions during DNA replication, probably by slipped strand misalignment due to preferential hairpin formation of TRS. Similar molecular studies in vivo on gene conversion and recombination are lacking.Several human genetic studies on patient materials reported haplotype analyses, especially related to myotonic dystrophy (DM) and the fragile X syndrome, which implicated gene conversion and/or unequal crossing-over (types of recombination) to genetic instabilities. In the first report, Korneluk and coworkers (5, 6) proposed unequal crossing-over (5) and gene conversion (6) as the mechanisms responsible for the expansions and deletions observed in the (CTG⅐CAG) mutation during DM transmission. This conclusion was derived from haplotype a...

As part of a national effort to identify biomarkers for the early detection of cancer, we developed a rapid and high-throughput sequencing protocol for the detection of sequence variants in mitochondrial DNA. Here, we describe the development and implementation of this protocol for clinical samples. Heteroplasmic and homoplasmic sequence variants occur in the mitochondrial genome in patient tumors. We identified these changes by sequencing mitochondrial DNA obtained from tumors and blood from the same individual. We confirmed previously identified primary lung tumor changes and extended these findings in a small patient cohort. Eight sequence variants were identified in stage I to stage IV tumor samples. Two of the sequence variants identified (22%) were found in the D-loop region, which accounts for 6.8% of the mitochondrial genome. The other sequence variants were distributed throughout the coding region. In the forensic community, the sequence variations used for identification are localized to the D-loop region because this region appears to have a higher rate of mutation. However, in lung tumors the majority of sequence variation occurred in the coding region. Hence, incomplete mitochondrial genome sequencing, designed to scan discrete portions of the genome, misses potentially important sequence variants associated with cancer or other diseases.

Homo(purine•pyrimidine) sequences (R•Y tracts) with mirror repeat symmetries form stable triplexes that block replication and transcription and promote genetic rearrangements. A systematic search was conducted to map the location of the longest R•Y tracts in the human genome in order to assess their potential function(s). The 814 R•Y tracts with ≥250 uninterrupted base pairs were preferentially clustered in the pseudoautosomal region of the sex chromosomes and located in the introns of 228 annotated genes whose protein products were associated with functions at the cell membrane. These genes were highly expressed in the brain and particularly in genes associated with susceptibility to mental disorders, such as schizophrenia. The set of 1957 genes harboring the 2886 R•Y tracts with ≥100 uninterrupted base pairs was additionally enriched in proteins associated with phosphorylation, signal transduction, development and morphogenesis. Comparisons of the ≥250 bp R•Y tracts in the mouse and chimpanzee genomes indicated that these sequences have mutated faster than the surrounding regions and are longer in humans than in chimpanzees. These results support a role for long R•Y tracts in promoting recombination and genome diversity during evolution through destabilization of chromosomal DNA, thereby inducing repair and mutation.

Genetic recombination is a robust mechanism for expanding CTG⅐CAG triplet repeats involved in the etiology of hereditary neurological diseases (Jakupciak, J. P., and Wells, R. D. (1999) J. Biol. Chem. 274, 23468 -23479). This two-plasmid recombination system in Escherichia coli with derivatives of pUC19 and pACYC184 was used to investigate the effect of triplet repeat orientation on recombination and extent of expansions; tracts of 36, 50, 80, and 36, 100, and 175 repeats in length, respectively, in all possible permutations of length and in both orientations (relative to the unidirectional replication origins) revealed little or no effect of orientation of expansions. The extent of expansions was generally severalfold the length of the progenitor tract and frequently exceeded the combined length of the two tracts in the cotransformed plasmids. Expansions were much more frequent than deletions. Repeat tracts bearing two G-to-A interruptions (polymorphisms) within either 171-or 219-base pair tracts substantially reduced the expansions compared with uninterrupted repeat tracts of similar lengths. Gene conversion, rather than crossing over, was the recombination mechanism. Prior studies showed that DNA replication, repair, and tandem duplication also mediated genetic instabilities of the triplet repeat sequence. However, gene conversion (recombinational repair) is by far the most powerful expansion mechanism. Thus, we propose that gene conversion is the likely expansion mechanism for myotonic dystrophy, spinocerebellar ataxia type 8, and fragile X syndrome.Several hereditary neurological diseases including myotonic dystrophy, fragile X syndrome, spinocerebellar ataxia type 8, and Friedreich's ataxia result from expanded TRS 1 CTG⅐CAG, CGG⅐CCG, and GAA⅐TTC within or near their genes (reviewed in Ref. 1). For these diseases, the TRS expansions occur by hundreds of repeats and can appear rapidly within a pedigree. However, for other diseases (Huntington's disease, spinocerebellar ataxia type 1, and Kennedy's disease), the CAG⅐CTG repeats expand to a smaller extent (tens of repeats) and occur in exons and, hence, lengthen the oligoglutamine tracts in the relevant proteins. The earlier age of onset and the increased severity of most of these neurological diseases in successive generations (clinically referred to as anticipation) are correlated to the lengths of the TRS. Long tracts of TRS are unstable and show repeat size polymorphisms in successive generations and in different tissues. In addition to these observations in humans, the molecular mechanisms of TRS instabilities have been investigated in Escherichia coli (2-14), yeast (15-21), and transgenic mice (22-26).The molecular mechanisms of genetic instability of TRS have been intensively studied because of their pivotal role in the disease pathogenesis. A number of investigations have revealed the involvement of misalignment-mediated DNA synthesis (1-16). These expansions and deletions are thought to be due to the formation of unusual DNA secondary structures and ...

Background: Nuclear mitochondrial pseudogenes (numts) are a potential source of contamination during mitochondrial DNA PCR amplification. This possibility warrants careful experimental design and cautious interpretation of heteroplasmic results.