Two patient groups were used in this study: a control group of 10 histologically normal
tissues from patients (CT1-10) with no breast tumors and a group of 44 breast tumor
patients with different entities of primary breast cancer (HER2?+?1–15, TN1-16 and
ERPR+?1–13) of which both tumor and normal tissues were available.
SNP selection
Technical validation of the selected SNP assays was performed using the 10 histologically
normal breast tissues from the control group. All SNP assays had a sufficient sensitivity,
yielding cycle-threshold values of 35 (threshold 0.007), a cut-off above which results
are routinely considered less reliable in our laboratory. In addition, little or no
cross-reaction of the SNP assays’ allele 1 corresponding probes (VIC reporter) with
allele 2, and of the allele 2 corresponding probes (FAM) with allele 1 was observed.
This resulted in clear clustering of homozygous allele 1, homozygous allele 2 and
heterozygous patients. The panel of 11 SNP markers (Table 1) was therefore used to analyze the clinical specimens (Additional file 1: Table S1 and Additional file 2: Table S2).
SNP analysis of patient materials
To minimize the influence of DNA concentration on VIC/FAM ratio and therefore data
interpretation, a correction on all delta Rn FAM data was performed as described in
the Materials and Methods section. Fig. 2 shows the corrected scatterplot of representative SNP 8 for histologically normal
tissues from the control group and paired histologically normal and tumor samples
of all patients in the breast tumor group. Ratios of each sample pair (histologically
normal and tumor tissues) were connected.
Fig. 2. Visualization of VIC/FAM ratios that were generated using representative SNP 8 after
FAM correction. Scatterplot of SNP 8 analyses of histologically normal tissues from
the control group and paired histologically normal and tumor tissues from the group
of breast tumor patients. Connected symbols indicate normal and tumor tissue of a
single patient. X-axis: delta Rn VIC (T?=?Thymine), Y-axis: corrected delta Rn FAM
(C?=?Cytosine)
Subsequently, the delta Rn VIC/FAM ratios of all informative SNPs of the histologically
normal tissues from both groups (10 tissues from the control groups and 44 tissues
from the breast tumor group) were used to gain insight in normal data distribution.
Combining the data of the histologically normal tissues from these groups was justified
due to the mutual similarity of delta Rn VIC/FAM ratio distribution amongst both groups.
To determine the HER2/TOP2A allelic (im)balance, fluorescent ratios of the panel of 11 informative SNP’s were
analyzed using the cut-off values based on the interquartile range of data distribution
amongst the histologically normal samples.
Histologically normal tissues
As expected, all informative SNP results (total of 54 SNPs of 10 patients) of the
breast tissues from the control group fell within the normal IQR range (?1.5 to 1.5
IQR for SNP 1, 2 and 4–11 and ?1 to 1 IQR for SNP 3). Overall results are depicted
in Additional file 1: Table S1, Fig. 2 represents the corrected scatterplot of representative SNP 8.
The 44 histologically normal samples of the breast tumor patient group generated SNP
results that fell within the normal IQR range and are therefore in agreement with
the normal range observed in the control group. Only 1 out of the total of 208 informative
SNPs yielded an equivocal result (patient TN2, SNP 11, triplicate analysis yielded
2 equivocal results and 1 result was found to indicate allelic instability).
Breast tumor tissues
Results of IHC and SISH are depicted in Additional file 2: Table S2. The SNP assays were used to investigate in more detail the genomic composition
of the HER2/TOP2A locus. The results are summarized in Additional file 2: Table S2.
HER2+ group
Within the group of patients with tumors overexpressing HER2, the SNP data of patients
HER2+ 2–5 and 7–15 were consistently indicative of an instable HER2/TOP2A locus. IHC scores were 3+, SISH ratios were 2.0–15.2 and TC% ranged from 40 to 80 %.
SNP 1 in patient HER2+ 8 generated an equivocal result. SNP results from patient HER2+
1 and 6 showed inconsistent results between informative SNPs. All informative SNPs
were therefore subsequently repeated in duplicate for these patients yielding identical
results. SNP results from patient HER2+ 1 showed allelic balance of SNPs 8 and 9,
whereas results from SNPs 1 and 10 were found to indicate allelic instability. IHC
yielded a 3+ score, whilst the SISH ratio was 1.1. The sample had a TC% of 30Â %. SNP
results from patient HER2+ 6 were also inconsistent. SNPs 1, 2 and 8 were indicative
of allelic stability, while results from SNPs 4, 7 and 11 showed allelic instability.
IHC, SISH ratio and TC% in this patient were 3+, 4.1 and 80Â %, respectively.
Triple negative group
In the triple negative group, patients TN1-4, 6, 9, 10 and 12–16 showed SNP results
consistent with instability of the HER2/TOP2A locus. IHC scores, SISH ratios and TC% varied from 0–2+, 0.7–1.9 and 25–90 %, respectively.
Several SNPs generated equivocal results in patients TN1 (SNP 3 3x equivocal), 3 (SNP
4 3x equivocal), 8 (SNP 1 2x equivocal, 1x normal and SNP 2 3x equivocal), 10 (SNP
3 2x equivocal, 1x allelic instability) and 13 (SNP 1 and 3 both 3x equivocal). Patients
TN5, 7 and 11 revealed no signs of HER2/TOP2A allelic imbalance, IHC scores were 0–1+, SISH yielded ratios of 1.1–1.6 and the samples
contained 30–95 % tumor cells. In patient TN8, the 2 SNPs that were informative (SNPs
1 and 2) yielded equivocal results. IHC score, SISH ratio and TC% for this patient
were 1+, 0.9 and 70Â %, respectively.
Estrogen and progesterone positive group
From the ERPR+ group, patients ERPR+?1–3, 6 and 7 yielded SNP results indicative of
HER2/TOP2A locus instability. IHC scores, SISH ratios and TC% varied from 0–1+, 0.5–1.1 and
30–70 %, respectively. Equivocal results were generated in patients ERPR+?3 (SNP 3
3x equivocal) and 7 (SNP 3 1x equivocal, 1x allelic instability and 1x normal). The
ratio of SNP 3 was found to be equivocal (equivocal, allelic instability and normal)
in patient ERPR+?7. The breast tumor tissues from patients ERPR+?4, 5 and 8–13 showed
a stable HER2/TOP2A locus, IHC scores were 0–1+, SISH ratios ranged from 1.0–1.5 and a TC% was 50–70 %.
Patient ERPR+?8 and 11 generated an equivocal result utilizing SNP 8 (2x equivocal,
1x allelic instability) and 4 (3x equivocal), respectively.