BRCA1/2 mutation screening and LOH analysis of lung adenocarcinoma tissue in a multiple-cancer patient with a strong family history of breast cancer

Abstract

BACKGROUND: Germline mutations in BRCA1/2 greatly elevate risks of breast and ovarian cancers, but the role of these genes in tumourigenesis of other cancer types is still being investigated. OBJECTIVE: We report on an investigation of BRCA1/2 mutations and their loss of heterozygosity (LOH) in a patient with a strong family history of breast cancer who was diagnosed with consecutive primary cervical, ovarian and lung carcinomas. METHODS AND RESULTS: BRCA1/2 mutation screening of the proband revealed a common familial breast- and ovarian cancer-associated germline BRCA2 mutation (3034del4bp). We then performed LOH analysis for BRCA2 in lung adenocarcinoma tissue of the patient. Using the laser-capture microdissection (LCM) technique, we obtained pure populations of neoplastic cells from which DNA could be extracted. Mutation analysis by denaturing high-performance liquid chromatography (DHPLC) and direct sequencing revealed loss of the mutant allele in the adenocarcinoma tumour tissue. CONCLUSION: To our knowledge, this is the first report of investigation for LOH for BRCA2 in primary lung adenocarcinoma tissue of a patient with multiple primary tumours related to a familial germline BRCA2 mutation. Interestingly, it was the mutant, not the wild-type, allele which was lost in the lung adenocarcinoma tissue.

Figure 1

Pedigree of the patient's family showing strong family history of breast cancer (BC). Filled symbols (black) represent cancer cases. Ages of disease onset are shown in years. The index patient is indicated with an arrow.

Figure 2

DHPLC elution profiles of 463 base pair PCR fragments of BRCA2 exon 11. A: Heteroduplex profile with two peaks from control DNA that was found to be heterozygous for the common exon 11 BRCA2 mutation 3034del4bp. B: Homoduplex profile (DHPLC) in amplified wild-type DNA. C: DHPLC analysis of amplified genomic DNA of our patient showing a heteroduplex profile similar to the profile of the mutant control DNA sample. D: DHPLC elution profile of amplified lung tumour DNA showing only one peak indicating LOH, either of the wild-type or the mutant allele.

Figure 3

Results of direct sequencing-amplified genomic (A) and lung tumour DNA (B) of our patient. A: Direct sequencing reveals heterozygosity beginning from position 3034 of the BRCA2 gene (C/T, arrow), that was shown to be a deletion of 4 base pairs (AAAC) leading to frame shift and premature termination of translation in position 958 of BRCA2 protein. B: "Homozygosity" for the wild-type allele around position 3034 of BRCA2, most likely as a result of chromosomal deletion of the mutant allele, was found by sequencing analysis (arrow).