Marion Kiechle

A comparison of BRCA1 mutation analysis by direct sequencing, SSCP and DHPLC

The most sensitive screening technique for genes that predispose patients for particular cancers is direct sequencing. However, sequencing of complex genes is technically demanding, costly and time-consuming. We have tested alternate screening techniques to find a fast sensitive method for detecting alterations of DNA in the large BRCA1 gene prior to sequencing. Sequencing of this gene is particularly arduous because it lacks clearly defined mutation sites. The single-strand conformation polymorphism (SSCP) technique is one of the most frequently used pre-screening methods but its sensitivity and efficiency is not completely satisfying. We have compared the SSCP assay with a newly developed technique called denaturing high performance liquid chromatography (DHPLC) to screen the BRCA1gene. We studied 23 patients at high risk for early onset breast and ovarian cancer and four controls. In these patients, a total of 113 fragments with sequence variations in the BRCA1 gene could be identified. The DHPLC technique resolved 100% of the DNA alterations that were observed in cycle sequencing. In contrast, mutation analysis by SSCP accounted for 94% of the detected variations. In addition, DHPLC screening allowed us to discriminate between different alterations in a single fragment, because of the characteristic elution profiles of the DNA molecules. Polymorphisms that were present in our samples could be predicted by means of DHPLC testing independently of sequence analysis. We conclude that DHPLC is a highly potent screening method for genetic analyses. It is highly sensitive, efficient and economical and can be automated.

A highly sensitive, fast, and economical technique for mutation analysis in hereditary breast and ovarian cancers

Mutation analysis of complex genes without hotspots for sequence variations, such as BRCA1, is time‐consuming and expensive. Of all currently available methods, direct sequencing has the highest sensitivity, but also the highest costs. Other techniques, such as SSCP, DGGE, and PTT, are more economical but, depending on the experience of the investigator, have at best a sensitivity of 90%. We investigated in a prospective study the feasibility and accuracy of the DHPLC technique. We present the application of the DHPLC protocol for BRCA1 mutation detection on a HPLC device from Bio‐Tek Kontron Instruments (Neufahrn, Germany). DNA from 46 women with hereditary breast and ovarian cancer undergoing genetic testing for BRCA1 mutations were tested. Of 1,518 amplicons analyzed by DHPLC, corresponding to 33 fragments spanning the entire BRCA1 gene, 626 were also directly sequenced. The comparison demonstrated that DHPLC detected all alterations found by direct sequencing. No false‐positive signals were seen in cases of homozygous sequences. Further, no false‐negative results were ever obtained in women with mutations or polymorphisms, or both. In cases of known genetic variations, the nature of the alterations could be predicted by DHPLC. We also compared different separation matrices. Up to about 500 injections, no significant differences in sensitivity could be observed between poly(styrene divinylbenzene) and end‐capped silica based columns. However, after more than 500 injections, the resolution of hetero‐ from homoduplex deteriorated rapidly on silica columns. Hum Mutat 14:333–339, 1999.

Mutation analysis of p53 in ovarian tumors by DHPLC

Up to now, ovarian carcinomas represent a major health problem among female cancers because they are the leading cause of death from gynecological malignancy. A high proportion of these tumors selects for mutations in the p53 gene. There is evidence that inactivation of the p53 protein could indicate poor prognosis and chemoresistance of patients. To set up a fast and sensitive test for p53 defects in tumor tissues, we analyzed ovarian cancer cells by denaturing high-performance liquid chromatography (DHPLC). A primer set spanning the whole coding region of p53 with seven fragments was designed and appropriate heteroduplex detection in DHPLC analysis was elaborated. The analysis of 45 ovarian tumor specimens yielded 17 genetic alterations (38%) occurring exclusively in the malignant tissue of the patients. In addition, frequent polymorphisms present in normal compared to tumor tissue could serve as a tool for the rapid identification of loss of heterozygosity (LOH) in the tumor. We observed that LOH in intron 2 or 3 correlated well with a lack of one allele in mutated fragments. In conclusion, DHPLC screening appears to be a sensitive and effective test for genetic alterations in tumors with p53 involvement. Since p53 mutations point to a poor prognosis state in several cancers, a fast screening of tumor material for genetic variations may have implications for further individual treatment of patients.

Genetic Imbalances in Precursor Lesions of Endometrial Cancer Detected by Comparative Genomic Hybridization

Endometrial hyperplasia is regarded as a precursor lesion of endometrioid adenocarcinomas of the endometrium. The genetic events involved in the multistep process from normal endometrial glandular tissue to invasive endometrial carcinomas are primarily unknown. We chose endometrial hyperplasia as a model for identifying chromosomal aberrations occurring during carcinogenesis. Comparative genomic hybridization (CGH) was performed on 47 formalin-fixed, paraffin-embedded specimens of endometrial hyperplasia using the microdissection technique to increase the number of tumor cells in the samples and reduce contamination from normal cells. CGH analysis revealed that 24 out of 47 (51%) samples had detectable chromosomal imbalances, whereas 23 (49%) were in a genetically balanced state. The incidence of aberrant CGH profiles tended to parallel dysplasia grade, ranging from 22% aberrant profiles in simple hyperplasia to 67% in complex hyperplasia with atypia. The most frequent imbalances were 1p, 16p, and 20q underrepresentations and 4q overrepresentations. Copy number changes in 1p were more frequent in atypical complex hyperplasia than in complex lesions without atypical cells or simple lesions (42% versus 20% and 0%). Our results show that endometrial hyperplasia reveals recurrent chromosomal imbalances which tend to increase with the presence of atypical cells. The most frequent aberrations in endometrial cancer, 1q and 8q overrepresentations, are not present or are rare in its precursor lesions. This analysis provides evidence that tumorigenesis proceeds through the accumulation of a series of genetic alterations and suggests a stepwise mode of tumorigenesis.

An easy and reliable procedure of microdissection technique for the analysis of chromosomal breakpoints and marker chromosomes.

Microdissection in combination with reverse painting fluorescence in-situ hybridization (FISH) is a very effective method to identify breakpoints and rearrangements of derived chromosomes and reveal the chromosomal origin of marker chromosomes. We describe an innovation that allows a convenient, fast and safe isolation of microdissected fragments as currently available protocols. The microdissected chromosomes are harvested in a collection drop located in a movable micropipette adjusted to a second micromanipulator under microscopic observation. We used this technique to analyze several cytogenetic aberrations. In order to evaluate the efficiency of our microdissection procedure, we compared the results obtained with microdissection probes made from only one fragment with those obtained with more than six microdissected fragments. In all cases, the single- fragment microdissections were sufficient to provide probes.

FISH-microdissection (FISH-MD) analysis of complex chromosome rearrangements

We combined the techniques of fluorescence in situ hybridization (FISH) and chromosomal microdissection in one experiment (FISH-MD). This novel method permits rapid identification of the composition, origin, and breakpoints of rearranged chromosomes. Rearranged chromosomes are first identified by multicolor-FISH, then the fluorophore-labeled derivative chromosomes are directly isolated by microdissection and reverse painted to identify the breakpoints.

Comparison of comparative genomic hybridization and interphase fluorescence in situ hybridization in ovarian carcinomas: possibilities and limitations of both techniques

Comparative genomic hybridization (CGH) is a valuable technique for cytogenetic analysis of solid tumors. To evaluate the reliability of CGH, we examined DNA of 10 ovarian carcinomas after CGH analysis with single- and double-locus fluorescence in situ hybridization (FISH). The FISH experiments, involving 5 chromosomes (chromosomes 3, 6, 8, 12, and 18) with different FISH probes, confirmed the CGH results in 66.2% of cases (92 of 139 investigated loci). In 4 patients, inconsistent results (41 loci) were related to polyploidy, because CGH cannot detect polyploid karyotypes. The remaining 6 discordant loci can be referred to limitations in both techniques. Re-evaluation of FISH and CGH results by one other is therefore recommended to overcome these technical artifacts. Nevertheless, CGH is of potential value in characterizing chromosomal alterations and might help in generating tumor-specific sets of FISH probes to obtain genetic information of prognostic value within a few days.

Highly comprehensive karyotype analysis by a combination of spectral karyotyping (SKY), microdissection, and reverse painting (SKY-MD).

A technique disclosing most information about chromosome modifications is the technique of choice for the analysis of chromosome alterations. The newly developed method for microdissection of fluorescence-labeled chromosomes (FISH-MD) can improve upon this expectation in combination with 24-color spectral karyotyping (SKY). The highly efficient way to detect chromosome modifications by SKY and the detailed specification of aberrant chromosomes by FISH-MD prompted us to use both techniquesin a combined approach called SKY-MD. First, an overview of chromosomal aberrations is obtained by spectral karyotyping and subsequently the derivative chromosomes recognized are characterized in a highly specific manner by microdissection and reverse painting. A small quantity of isolated material dissected directly from a 24-color metaphase is sufficient to obtain very detailed information about the chromosome regions and the breakpoints involved in the derivative chromosomes. Therefore, the combination of spectral karyotyping and microdissection in one procedure, and reverse painting can characterize chromosomal aberrations with a degree of specificity hitherto unknown from individual karyotyping experiments. In this article we compare the efficiency of both the SKY technique and that of classical microdissection with the efficiency obtained by SKY-MD.

Ten novel BRCA1 and BRCA2 mutations in breast and/or ovarian cancer families from northern Germany.

Germline mutations in the BRCA1 and BRCA2 gene account for the majority of high‐risk breast/ovarian cancer families. We have screened such families from Northern Germany by using DHPLC analysis and subsequent direct sequencing techniques. In ten families we identified six novel BRCA1 and 4 novel BRCA2 mutations comprising four frame shift mutations, one nonsense and one splice site mutation in the BRCA1 gene as well as three frameshift mutations and one nonsense mutation in the BRCA2 gene. Our analysis contributes to the further characterisation of the mutational spectrum of BRCA1 and BRCA2. Hum Mutat 16:529–530, 2000.