Analysis of BRCA2 loss of heterozygosity in tumor tissue using droplet digital polymerase chain reaction.
Loss-of-heterozygosity (LOH) analysis of archival tumor tissue can aid in determining the clinical significance of BRCA variants. Here we describe an approach for assessing LOH in formalin-fixed, paraffin-embedded (FFPE) tissues using variant-specific probes and droplet digital polymerase chain reaction (ddPCR). We evaluated LOH in 2 related breast cancer patients harboring a rare missense BRCA2 variant of unknown clinical significance (c.6966G>T; M2322I). Conventional PCR followed by Sanger sequencing suggested a change in allelic abundance in the FFPE specimens. However, we found no evidence of LOH as determined by the allelic ratio (wild type-variant) for BRCA2 in both patients' archival tumor specimens and adjacent normal control tissues using ddPCR. In summary, these experiments demonstrate the utility of ddPCR to quickly and accurately assess LOH in archival FFPE tumor tissue.
The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University School of Medicine, Baltimore, MD 21231.
Rory L
RL
Cochran
The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University School of Medicine, Baltimore, MD 21231.
Karen
K
Cravero
The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University School of Medicine, Baltimore, MD 21231.
David
D
Chu
The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University School of Medicine, Baltimore, MD 21231.
Bracha
B
Erlanger
The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University School of Medicine, Baltimore, MD 21231.
Patricia Valda
PV
Toro
The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University School of Medicine, Baltimore, MD 21231.
Julia A
JA
Beaver
The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University School of Medicine, Baltimore, MD 21231.
Daniel J
DJ
Zabransky
The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University School of Medicine, Baltimore, MD 21231.
Hong Yuen
HY
Wong
The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University School of Medicine, Baltimore, MD 21231.
Justin
J
Cidado
The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University School of Medicine, Baltimore, MD 21231.
Sarah
S
Croessmann
The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University School of Medicine, Baltimore, MD 21231.
Heather
H
Parsons
The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University School of Medicine, Baltimore, MD 21231.
Minsoo
M
Kim
The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University School of Medicine, Baltimore, MD 21231.
Sarah J
SJ
Wheelan
The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University School of Medicine, Baltimore, MD 21231.
Pedram
P
Argani
The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University School of Medicine, Baltimore, MD 21231.
Ben
B
Ho Park
D000483:Alleles; D024682:BRCA2 Protein / Q000235:genetics*; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D018270:Carcinoma, Ductal, Breast / Q000235:genetics* / Q000473:pathology; D005260:Female; D005838:Genotype; D006801:Humans; D019656:Loss of Heterozygosity; D008875:Middle Aged; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*
D024682:BRCA2 Protein
BRCA2; Droplet digital PCR; Familial breast cancer; LOH; VUS