Samples and control DNA for the HRM optimisation and targeted mutation detection were obtained from the Diagnostic Molecular Pathology laboratory at the Peter MacCallum Cancer Centre and from the Kathleen Cunningham Foundation Consortium for Research into Familial Breast Cancer (kConFab).
The highest mutation detection ratio was obtained in breast ovarian cancer families fulfilling the criteria for hereditary disease (75%=21/28), decreasing to 23.1% (9/39) in families with familial breast and ovarian cancer (P<0.0001).
As expected, a higher mutation detection rate was obtained in breast and ovarian (HBOC) cancer families fulfilling criteria I and II (96/160, or 60%) as compared to breast cancer only (HBC) families fulfilling the same criteria (114/412, or 27.7%).
A significantly higher mutation detection ratio was obtained within the group of TNBC patients (7/30; 23.3 %; 95 % CI = 9.9 42.3 %) compared to the premenopausal breast cancer group without TNBC (6/78; 7.7 %; 95 % CI = 2.9 16.0 %) (p = 0.0432).
Not surprisingly, the highest mutation detection ratio was obtained within the subgroup of TNBC patients diagnosed before the age of 50 (5/14; 35.7 %; 95 % CI = 12.7 64.9 %).> The BRCA2 c.7934delG Afrikaner founder mutation was identified in 2 (white) patients, one with TNBC and one diagnosed with premenopausal breast cancer.
The presence of an ovarian cancer case turned out to be a good indicator for the presence of a causative BRCA mutation: the highest mutation detection rates were obtained in apparently sporadic patients diagnosed with both breast and ovarian cancer (73,7%) and in breast-ovarian cancer families (about 60%).
Considering the percentage represented by this variant in patients (26%) and the obtained mutation detection rate (60.5%), this change would represent the 15.7% of all the potential arSTGD associated alleles.
In light of this rather extensive coverage, it is likely that a similar sensitivity for mutation detection could to be obtained when utilizing a reduced coverage, thus opening up the possibility of barcoding of two or more samples on one SMRT cell.
In familial cases, we obtained a mutation detection rate of 36.7% (210/572) in sporadic bilateral breast, bilateral ovarian or breast, and ovarian cancer patients 43.6% (24/55) but only 10.7% (13/121) in early onset unilateral breast cancer patients.
As depicted in Table 5, all methods evaluated in this study, including classic dideoxy-sequencing, performed optimally with favorable FFPE-DNA; for unfavorable FFPE-DNA however, translating into 1 in 4 5 patients, genotyping results could mostly be obtained by targeted mutation detection with Q-PCR only.
