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Gottlieb et al. J Cancer Metastasis Treat 2018;4:37 I http://dx.doi.org/10.20517/2394-4722.2018.26 Page 3 of 14

possibility that different mutations of an individual gene can exist in a single person’s tissues. The assump-
tion being that finding multiple variants of a single gene within an individual’s tissues is highly unlikely and
therefore if identified is likely the result of either PCR or sequencing errors. Indeed, almost all NGS analyses
[13]
rely on the use of filters and other techniques such as sequence alignment tools to remove such variants .
These techniques further reduce the possibility of finding multiple mutations within an individual gene, as
some are likely to be at very low frequencies, and will be present in only a small minority of cells within an
[14]
individual tumor, as noted in a recent review of post-zygotic somatic mosaicism . Therefore, one of the
challenges of the study was to develop a sequencing analysis approach that allows for the identification of
CSGV. Further, an important practical consideration for identifying CSGV is that it is increasingly becom-
ing apparent that every driver gene mutation does not produce a cancer phenotype, with some driver muta-
tions even being present in non-cancer tissues [15,16] . In the present study, we have used a sequencing approach
that makes it easier to detect multiple mutations of the androgen receptor gene (AR) within individual breast
tumors.

Androgen receptor and breast cancer
In the case of breast cancer (BC), the AR is more widely expressed than either estrogen receptor (ER) alpha
or progesterone receptor (PR) genes, and so it is not surprising that the AR has become a significant marker
[17]
in defining BC subtypes . The AR has therefore started to be singled out as a possible therapeutic target,
particularly in triple-negative [ER-/PR-/herceptin receptor (HER) 2-] BC (TNBC) [18,19] . Indeed, a large cohort
[20]
study reported AR expression in 32% of TNBC cases . In another study examining cases of ER-positive
breast carcinoma, tumor cells changed after treatment from ER-dependent to AR-dependent, possibly ex-
[21]
plaining why such cells become resistant to aromatase inhibitor treatment . At present, most studies have
focused on AR expression during different BC stages, and, indeed, AR expression has been identified as a
[22]
possible critical marker in predicting BC survival . While androgen-based therapeutics have been used for
[23]
over 50 years to treat BC . The authors believe that to truly exploit potential AR related mechanisms to pro-
vide clinical therapeutic benefits, a more detailed understanding of AR variant distribution and frequency in
BC tissues, i.e., AR CSGV, both before and throughout carcinogenesis, will be required.

Further, examining CSGV occurrence in other critical driver genes may help resolve the genotype-pheno-
type disconnects between the mutational status of putative cancer-associated genes and the occurrence and
progression of cancer. For, if it is assumed that somatic clonal evolution is the mechanism driving carcino-
genesis, then tissue microenvironments need to be able to select from different variants of individual genes.
As the presence of a single variant would not allow cells and tissues sufficient flexibility to adapt to different
selection pressures produced by different tissue microenvironments. Further, the ability to collect such data
about all potential driver genes may well provide new insights into resistance to treatment as well as to treat-
ment failures.

METHODS
Laser capture microdissection and DNA extraction
Frozen tumors were obtained from a breast cancer tissue bank [Table 1] that had been set up with all the
required experimental permissions and vetted by the Jewish General Hospital’s ethics board. Histological
slides of 5-7 µm thick were prepared and stained using a standard hematoxylin/eosin protocol. To ensure
the maximum purity of the cancer samples, following histo-pathological characterization by an expert pa-
thologist, cells from cancer tumor areas were dissected by LCM using an AutoPix 100 (Molecular Devices,
Sunnyvale, CA). An average of 2500 cells was dissected from each different section. Genomic DNA was ex-
tracted from the cells using a QIAamp DNA Micro kit (QIAGEN, Germantown, MD) following the manu-
facturer’s directions.