Chromosome X aneusomy and androgen receptor gene copy number aberrations in apocrine carcinoma of the breast

Consecutive cases were retrieved from the files of the Surgical Pathology Units of the Department of Biomedical and Neuromotor Sciences at Bellaria Hospital in Bologna (Italy), St. Vincent’s University Hospital in Dublin (Ireland) and Morgagni Hospital in Forlì (Italy).

Cases were retained for the present study when they showed (a) ER and PR negativity and AR positivity; (b) the morphological and immunohistochemical profile consistent with the diagnosis of CAD according to recent guidelines [1]; (c) sufficient tissue to perform in situ hybridization and molecular tests. All cases had been diagnosed in the period January 2000–December 2016. All cases with their immunohistochemical profile were reviewed, classified and graded according to currently available criteria [1]. All tissue specimens had been fixed in buffered formalin for 24 h and then paraffin-embedded (FFPE) according to routine procedures.

Immunohistochemistry was performed on an automated stainer (Ventana BenchMark, Ventana Medical Systems Inc., Tucson, AZ, USA) applying a pre-diluted monoclonal anti-androgen receptor (Cell Marque, clone SP 107) and anti-GCDFP-15 (Thermo Fisher Scientific, clone 23A3) antibodies.

Dual-colour FISH was carried out according to a standard protocol as previously described [10, 11], shortly summarized as follows: Five micron sections were obtained from each tumour block. One specific probe kit for the X chromosome (ON AR (Xq12)/SE X, Kreatech Diagnostics, Amsterdam, The Netherlands), added with the Smart-ISH hybridization buffer (OACP IE LTD, Cork, IE), was applied. The AR gene-specific probe length is stated of 340Kb (product-specific datasheet from Kreatech Diagnostics). The X chromosome centromeric region-specific probe length has not been disclosed by the producer. The two regions tested are schematically illustrated in Fig. 1.

FISH for HER2 gene amplification status was routinely performed, at the time of diagnosis, applying specific probes (ON HER2/CE 17, Kreatech Diagnostics, Amsterdam, The Netherlands) with the same method cited above.

FISH analysis was carried out using an Olympus BX61 epifluorescence microscope (Olympus, Melville, NY) equipped with a 100-planar objective. For each case, a minimum of 60 non-overlapping nuclei was scored. Scoring was performed as previously described [10‐12]: total number of X chromosome centromeric probe signals (spectrum green), average number of green signals, total number of AR signals (spectrum orange), average number of orange signals and ratio between green (X chromosome) and orange (AR) signals. In the non-neoplastic ductal cells, utilized as internal control, two signals for both the AR gene (spectrum orange) and the centromeric X chromosome region (spectrum green) identified the normal chromosome asset.

HER2 results were scored following currently available guidelines [13].

Methylation analysis was performed as previously described [11]. Briefly, DNA obtained from formalin-fixed paraffin-embedded tissues was purified using the QuickExtract™ FFPE DNA extraction kit (Epicentre, Madison, WI). Two hundred to 500 ng of genomic DNA underwent bisulfite treatment applying the EZ DNA Methylation-Lightning™ Kit (Zymo Research Europe, Freiberg, Germany) according to the manufacturer’s protocol. DNA methylation was analysed by bisulfite next-generation sequencing (bisulfite-SEQ) following an internal protocol [14] using a two-step library preparation approach: the first step comprises multiplex PCR amplification for target enrichment and a second PCR amplification for specimen barcoding (primers and region details are available on Supplementary Table 1). Libraries were loaded on the MiSeq (Illumina, San Diego, CA) according to the manufacturer’s protocol. Each NGS experiment was designed to allocate 1,000 reads for any region of interest, in order to have a depth of coverage of at least 1000×. FASTQ files were processed for quality control (> Q30), reads length (> 100 bp) and converted into FASTA format in galaxy project environment [14]. In order to evaluate the methylation ratio of each CpG, a single specific file for every case and every gene was created by Perl, which was then visualized using KISMETH [15]. In parallel, reads were mapped by BWAmeth generating a bam file which was then processed by MethylDackel using hg38 as a reference; this tool created an excel file assigning at each CpG position the exact methylation level.

Statistical analyses were performed using commercially available software: QuickCalcs, which is an online tool for linear regression by GraphPad, and Excel, from Microsoft Corporation.

All investigations were conducted according to the principles expressed in the Declaration of Helsinki; the study was approved by the local Ethical Committee (Code: n. CE17133).

FISH analysis of the X chromosome was performed on all tumours included in the study and informative results were obtained in 13 tumours. In all cases, normal ducts served as an internal control. Tumours were eliminated from the study when the two signals (red and green) were not clearly evaluable.

X chromosome copy number variation was observed in 13 of 13 tested tumours. Specifically, all except one (case 3) showed a proportion of neoplastic cells with loss of one X chromosome copy (monosomy) (Fig. 6). The percentage of neoplastic cells displaying X chromosome monosomy (Fig. 7) ranged from 4.63 to 73.10% (average 31.3%).

In three tumours (cases 3, 4 and 10), a neoplastic population with an additional X chromosome copy (polysomy) (Fig. 8) was identified. X chromosome polysomy affected 7.89% (case 10) to 23.66% (case 3) of the neoplastic population.

Not all additional copies of the X chromosome carried the AR gene leading to AR gene relative deletion. AR gene deletion was detected in 9/13 cases and affected from 2.43 to 56.17% (average 28.62%) of the neoplastic cells.

Overall, AR gene loss (due to either X chromosome monosomy or AR gene deletion) was observed in all 13 cases tested. The neoplastic cell population showing loss of one AR gene copy varied from 5 to 84% (mean 48.93%). Minimal differences were observed between HER2-positive and HER2-negative cases (Table 3). HER2-positive cases showed a tendency towards a higher level of X chromosome monosomy.

In situ duct carcinoma, present in 1 case, showed AR gene monosomy in 23.8% (case 10) of the neoplastic cells.

Non-neoplastic, non-apocrine cells present around the tumour showed AR monosomy in two cases, in 15.73% and 4.17% of the cell population respectively. The related tumours showed AR monosomy in 25.19% and 57.14% of the neoplastic cells (cases 4 and 14).

All 20 tumours were tested, but informative DNA was obtained in 9 cases only due to DNA over-fixation problems. The methylation status of the AR, FLNA and UXT genes, and of the MAGE family genes (MAGEA1, MAGEA2, MAGEA3, MAGEA9, MAGEA11, MAGEC1, MAGEC2), all present on the X chromosome, was evaluated. MAGEG1, a member of the MAGE family that maps to chromosome 15, served as a control as it is not affected by X chromosome inactivation in females. It was consistently non-methylated, as expected, as chromosome 15 is not subject to lyonization.

In all tumours, the tested genes showed variation in methylation status, with respect to the methylation condition of a single chromosome (50% of methylation).

The AR gene showed a mean methylation value in the CpG islands lower than 50% in all but one case (case 20) (Table 2 in Supplementary files shows the data related to the methylation of each CpG island of AR).

MAGE family members that mapped on chromosome X were hypermethylated, with methylation values varying from 42.8 to 100% (average 81.43%) (the mean methylation values obtained for each gene are shown in Table 3 Supplementary files).

FLNA gene displayed a variable methylation pattern ranging from 0 to 70% (Table 4, Supplementary files).

UXT gene also displayed a variable methylation pattern ranging from 4 to 50% (Table 5, Supplementary files).

A complete immunohistochemical and molecular profile was obtained in 6 tumours. In all 6 tumours, AR immunohistochemical expression was high (positivity in > 95% of the neoplastic cells in 5/6 cases) in spite of X chromosome monosomy and AR gene loss in a variable percentage of neoplastic cells. All cases showed variable levels of hypomethylation of the tested genes. Even if a statistically significant correlation was not detected, methylation levels were lower in cases with higher AR gene loss, thus suggesting that the residual AR gene was transcriptionally active.