Comparable cancer‐relevant mutation profiles in synchronous ductal carcinoma in situ and invasive breast cancer

Abstract Background Ductal carcinoma in situ (DCIS) comprises a diverse group of preinvasive lesions in the breast and poses a considerable clinical challenge due to lack of markers of progression. Genomic alterations are to a large extent similar in DCIS and invasive carcinomas, although differences in copy number aberrations, gene expression patterns, and mutations exist. In mixed tumors with synchronous invasive breast cancer (IBC) and DCIS, it is still unclear to what extent invasive tumor cells are directly derived from the DCIS cells. Aim Our aim was to compare cancer‐relevant mutation profiles of different cellular compartments in mixed DCIS/IBC and pure DCIS tumors. Methods and results We performed targeted sequencing of 50 oncogenes in microdissected tissue from three different epithelial cell compartments (in situ, invasive, and normal adjacent epithelium) from 26 mixed breast carcinomas. In total, 44 tissue samples (19 invasive, 16 in situ, 9 normal) were subjected to sequencing using the Ion Torrent platform and the AmpliSeq Cancer Hotspot Panel v2. For comparison, 10 additional, pure DCIS lesions were sequenced. Across all mixed samples, we detected 23 variants previously described in cancer. The most commonly affected genes were TP53, PIK3CA, and ERBB2. The PIK3CA:p.H1047R variant was found in nine samples from six patients. Most variants detected in invasive compartments were also found in the corresponding in situ cell compartment indicating a clonal relationship between the tumor stages. A lower frequency of variants were observed in pure DCIS lesions. Conclusion Similar mutation profiles between in situ and invasive cell compartments indicate a similar origin of the two tumor stages in mixed breast tumors. The lower number of potential driver variants found in pure DCIS compared with the in situ cell compartments of mixed tumors may imply that pure DCIS is captured earlier in the path of progression to invasive disease.


| Tumor tissue samples
Fresh frozen tissue from patients with mixed tumors (invasive ductal carcinoma with synchronous in situ lesion) or pure DCIS was collected at the Fresh Tissue Biobank, Department of Pathology, Uppsala University Hospital, Sweden. Histopathological evaluation of all cases was performed by a pathologist. DCIS tumor compartments were given a histopathological grade using the EORTC system 16 while invasive compartments were graded using the Elston & Ellis system. 17 Estrogen receptor (ER) and progesterone receptor (PR) status was determined by immunohistochemistry, and were previously published. 18 Samples were considered ER/PR positive if >10% of the cells showed positive nuclear staining. HER2-status was determined using Silver-enhanced in situ hybridization and scored as previously described. 18

| Laser capture microdissection
Invasive, in situ and normal cell areas were microdissected using laser capture microdissection on a Zeiss inverted microscope PALM Laser MicroBeam System (Carl Zeiss, Germany) as previously described. 8 Frozen 14 μm-thick sections were mounted on polyethylene membrane (PALM) covered slides and stained with hematoxylin (60 μL) mixed with RNasin for 1 minute, incubated in Zincfix (60 μL) for 30 seconds, followed by a series of 30-seconds incubation steps in 75%, 95%, and 100% ethanol, respectively. Adjacent 4 μm-thick sections were cut and stained by a routine hematoxilin and eosin protocol to locate the areas to be microdissected. Cells were captured into collecting caps and preserved in 50 μL Trizol at −80 C for DNA extraction. The number of cells obtained was estimated by the operator during microdissection and between 100 and 4000 cells were obtained for each sample. Pure DCIS samples were not microdissected; for these samples, whole FFPE tumor sections were used for DNA isolation.

| Variant calling
Data was analyzed using the AmpliSeq Variant Caller plug-in within the Ion Torrent Suite software (version 5.0.4, Thermo Fisher Scientific) with Human genome assembly build 37 (GRCh37) as reference.
In total, 57 samples were sequenced. Three samples were excluded from further analysis after quality assessment and altogether were 44 microdissected samples from 26 mixed tumors and 10 pure DCIS successfully sequenced. Due to low input and varying sample quality for the microdissected samples, a strict cut-off was applied; only variants with maximum allele frequency >10% and quality >100 across all microdissected samples were included. For the pure DCIS samples, variants with allele frequency <10% were also included since these data were of high quality. Variants were manually assessed in Integrative Genomics Viewer 19

| Statistical analysis
Fisher's exact tests were performed to test whether there was any statistically significant association between variants in genes and ER or PR status. in the invasive compartment. Across the cohort, nine normal breast epithelium samples were sequenced, and amongst these, three carried potentially pathogenic variants ( Figure 2). However, since all normal tissue samples sequenced in this study were obtained adjacent to tumor areas, they should not be considered entirely normal.

| RESULTS AND DISCUSSION
We found a significant association between PIK3CA variants and positive PR status (P = .039, Fisher's exact test), which has been previously noted. [21][22][23] A similar association was not seen for ER (P = .44), however; the low number of samples in this study may have prevented the identification of any such association.
In addition to microdissected tissue from mixed tumors, we  cells from the in situ compartment. 9 In one tumor in our study, we found