Assessment of genomic instability in breast cancer and uveal melanoma by random amplified polymorphic DNA analysis

Abstract

Some types of cancer have been associated with abnormal DNA fingerprinting. We used random amplified polymorphic DNA (RAPD) to generate fingerprints that detect genomic alterations in human breast cancer. Primers were designed by choosing sequences involved in the development of DNA mutations. Seventeen primers in 44 different combinations were used to screen a total of 6 breast cancer DNA/normal DNA pairs and 6 uveal melanoma DNA/normal DNA pairs. Forty-five percent of these combinations reliably detected quantitative differences in the breast cancer pairs, while only 18% of these combinations detected differences in the uveal melanoma pairs. Fourteen (32%) and 12 (27%) primers generated a smear or did not produce any band patterns in the first and second cases, respectively. Taking into account the ability of RAPD to screen the whole genome, our results suggest that the genomic damage in breast cancer is significantly higher than in uveal melanoma. Our study confirms other reports that the molecular karyotypes produced with random priming, called amplotypes, are very useful for assessing genomic damage in cancer. Genomic instability is a hallmark of neoplastic transformation and a herald of genomic damage. The existence of an additional type of genomic instability has been described, the microsatellite mutator phenotype pathway. Progress in molecular cancer genetics has facilitated the detection of these mutations. In the last decade, representation differential analysis and comparative genomic hybridization (CGH) were added to methods like flow cytometry, restriction fragment length polymorphisms of polymorphic minisatellite loci and allelotyping, a PCR amplification of informative microsatellite loci.Another promising approach has been introduced, arbitrarily primed PCR 1 combined with random amplified polymorphic DNA (RAPD). 2 Both PCR-based techniques, called amplotyping, use fingerprinting to quantitatively and qualitatively evaluate band alterations. 3 This random priming method allows comparison of normal and tumor tissues at a minimum of 20 -40 genome sites in a single PCR, though evidence suggests that the number of compared loci runs into the hundreds if we take into account that a gel band consists of many comigrating fragments. 4,5 It also includes internal controls and detects moderate gains of chromosomal sequences (trisomy/tetrasomy) that would otherwise escape detection.Previously, we addressed the problems of reproducibility and contamination in random priming. In the present work, we used RAPD fingerprinting to assess the genomic damage present in breast cancer and in uveal melanoma cells. For this task, we designed primers based on sequences that are supposed to play an important role in the mechanisms leading to DNA alterations. Human gene mutations appear to be caused by multiple mechanisms whose relative importance is probably governed by local primary and secondary DNA structure. 7 We have searched for consensus sequences located in the immediate vicinity of gene mutations and for "hot s...

Integrative Genomic Analysis of Aneuploidy in Uveal Melanoma

“…Consistent with 8p loss being a later event in tumor progression, we recently identified a metastasis modifier locus on 8p that is deleted in tumors with more rapid onset of metastasis. 7 We studied the temporal sequence in which chromosomal alterations accumulated in individual tumors, based on the assumption that aneuploidy accrues over time, with more advanced tumors having more aneuploidy than less advanced tumors (2). A similar analysis was done in UM (26), but this previous study relied on karyotype analysis of cultured tumor cells, which could be associated with cell culture artifacts and does not provide the same resolution as microarray-based genome-wide techniques.…”

“…Uveal melanoma (UM) is an attractive solid tumor for studying aneuploidy because it is remarkably uniform in its clinical, histopathologic, and genetic features, and it metastasizes in up to 50% of patients (4,5). UM displays recurrent chromosomal rearrangements but less generalized chromosomal instability than many other solid tumors (6,7).…”

p53 codon 72 polymorphisms and random amplified polymorphic DNA analysis of non-melanoma skin cancer through archival formalin-fixed paraffin-embedded tissue

“…On the other hand, various studies have applied RAPD-PCR for the analysis of genetic alterations in human tumors and revealed frequent occurrence of genetic polymorphisms in various types of tumors, for instance, lung cancer (20), squamous cell carcinoma of the head and neck (21), brain tumor (22), ovarian cancer (23), breast cancer (24,25), hepatocellular carcinoma (26,27), lymphoma (28) and in leukemia (29). Histological processing of FFPE tissue.…”

Identification of Genomic Markers by RAPD-PCR Primer in Leukemia Patients

“…It has been used as a mean for identifying ability of primer OPA-09 for genetic polymorphism the genetic alterations in human tumors and revealed analysis and detection of DNA markers in genomic DNA frequent occurrence of genetic polymorphisms in various of AML, CML and CLL. types of tumors, for example, lung cancer (Ong et al, 1998), squamous cell carcinoma of the head and neck MATERIALS AND METHODS (Maeda et al, 1999), brain tumor (Misra et al, 1998), ovarian cancer (Sood andBuller, 1996), breast cancer Blood samples collection and DNA extraction: Blood (Singh and Roy, 2001;Papadopoulos et al, 2002), samples were collected from leukemia patients attending hepatocellular carcinoma (Zhang et al, 2004; Al-Bairuni University Hospital and Al-Assad University 2005), lymphoma (Scarra et al, 2001) …”