Association of Sperm-Associated Antigen 5 and Treatment Response in Patients With Estrogen Receptor–Positive Breast Cancer

Key Points Question Are sperm-associated antigen 5 (SPAG5) transcript or protein expressions associated with treatment response in patients with estrogen receptor–positive breast cancer? Findings In this cohort study including 12 720 patients with estrogen receptor–positive breast cancer, SPAG5 transcript and SPAG5 protein overexpressions were associated with worse outcomes in patients who received endocrine therapy alone. Overexpressions of SPAG5 transcript or SPAG5 protein were associated with resistance to endocrine therapy but sensitivity to anthracycline-based combination chemotherapy, and downregulation of SPAG5 during the course of preoperative systemic therapies was associated with clinical benefit. Meaning These findings suggest that SPAG5 transcript or SPAG5 protein expression could be used as a clinical tool for selecting and monitoring response to neoadjuvant therapies and guide adjuvant therapy in estrogen receptor–positive breast cancer.


Nottingham University Hospital locally advanced breast cancer (NUH-LABC) cohort (n=361):
The relationship between SPAG5-protein expression and response to chemotherapy was evaluated by investigating its expression in pair-matched pre-chemotherapy core biopsies and post-chemotherapy surgical specimens, from 361 female patients with locally-advanced ER+ primary BC (LAP-BC) (stage IIIA-C) that had been treated with Neo-Adjuvant Anthracycline-based Combination Chemotherapy (Neo-Adj-AC-CT) at NUH between 1996 and 2015. All LAP-BC patients were female and their mean age was 50 years (range 25-75 years). Twenty-nine percent of patients received six cycles of an anthracycline-based therapy (FEC: 5-fluorouracil (5-FU) 500 mg m−2, epirubicin 75-100 mg m−2, cyclophosphamide 500 mg m−2, on day 1 of a 21 day cycle), whereas 58% of patients received FEC plus Taxane. All patients underwent mastectomy or breast-conserving surgery and axillary dissection, followed by adjuvant radiation therapy. Patients with ER+ BCs were offered 5 years of adjuvant endocrine therapy.  . The data from each platform were independently normalized and mapped to Ensembl gene identifiers. Affymetrix data were summarized with Ensembl alternative custom chip definition file, normalized with robust multi-array analysis, integrated with use of ComBat to remove data set -specific bias. Illumina probe profiles were quantile normalized using the lumi-package, mapped to Ensembl gene sequences using reMOAT, BioMart, and a custom BLAST sequence search. Two Agilent microarray data were resented in this study: I-SPY1 trial (accession number GSE22226) and TCGA-BRCA (obtained from FIREHOSE Broad GDAC at http://gdac.broadinstitute.org) datasets. The I-SPY1 trial were assayed on catalog 44,000 features using Agilent Human oligonucleotide microarrays (Agilent-014850 platform GPL1708) whereas TCGA-BRCA datasets were generated using RNA-seq platform (Agilent custom 244K whole genome microarrays). For SWEGENE cohort, global gene-expression analysis of BC was performed by using oligonucleotide microarrays (H_v2.1.1 55K platform GPL5345) produced at the SCIBLU Genomics Centre at Lund University, Sweden as previous described [7]. Human Qiagen microarray was used in one dataset (accession number: GSE9893; n= 155) in which gene expression profiling was performed using 70-mer oligonucleotide microarrays (22,680 oligonucleotide probes representing 21,329 human specific genes (Oligo Set 2.1 from Qiagen-Operon) as previously described. In one dataset (accession number: GSE10510; n=93) a customized microarray was constructed using DKFZ OLIG0-ARRAY-35K platform GPL6486 as previously described.
Gene expression data of each database were converted to a common scale (median equal to 0 and standard deviation equal to 1) in order to merge all of the study data that used the same platform and to create combined cohorts. Then the data was median-centred whereby median is 0. Databases using same platform have been merged and the median expression was calculated. The median expression of SPAG5 transcript for each platform has been calculated and values equal to or higher than the median coded as +1 (overexpression). Values of less than the median have been coded 0 or low SPAG5.

IHC and tissue microarray
Immunohistochemistry (IHC) of SPAG5 protein expression in breast cancer was investigated by IHC analysis in three cohorts at two independent centres: Nottingham University Hospitals (NUH), UK and University of Queensland, Australia. Immunohistochemical staining was performed using anti-SPAG5 antibody (Sigma HPA022479), at a dilution of 1:50 for 30 minutes. Negative and positive (by omission of the primary antibody and IgG-matched serum) controls were included in each run.

Nottingham University Hospital Early Stage Breast cancer (NUH-ESBC)
Tumours were arrayed in tissue microarrays (TMAs) constructed with two duplicate of 0.6mm cores from the periphery of the tumours. The TMAs were immunohistochemically profiled for SPAG5. Immunohistochemical staining was performed using the Thermo Scientific Shandon Sequenza chamber system (REF: 72110017), in combination with the Novolink Max Polymer Detection System (RE7280-K: 1250 tests), and the Leica Bond Primary Antibody Diluent (AR9352), each used according to the manufacturer's instructions (Leica Microsystems). The tissue slides were deparaffinised with xylene and then rehydrated through five decreasing concentrations of alcohol; 100%, 90%, 70%, 50% and 30% for two minutes each. Pretreatment antigen retrieval was performed on the TMA sections using sodium citrate buffer (pH 6.0) and heated for 20 minutes at 95°C in a microwave (Whirpool JT359 Jet Chef 1000W). A set of slides were incubated with the primary anti-SPAG5 antibody (Sigma HPA022479), at a dilution of 1:50 for 30 minutes. Negative and positive (by omission of the primary antibody and IgG-matched serum) controls were included in each run. The negative control ensured that all the staining was produced from the specific interaction between antibody and antigen.
The individual tissue cores were scored by two experienced pathologists, according to the intensity of tumour cell staining: punctate cytoplasmic staining was recorded as 1+ (if it is of mild/moderate granular intensity) or 2+ (if it is with strong granular intensity). No staining, faint or diffuse staining was recorded as 0 (negative or week). Using the maximum score of duplicate tissue cores for each case, the percentage of each category was estimated (0-100%). A score (range 0-200) was calculated by multiplying intensity of staining and percentage staining. A median score of ≥ 1 was taken as the cut-off for high SPAG5 cytoplasmic expression. Not all cores within the TMA were suitable for IHC analysis as some cores were missing or lacked tumour (<15% tumour). The expression of HER2, ER and PR was re-assessed according to the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines. To validate the use of TMAs for immuno-phenotyping, fullface sections of 40 cases were stained and the protein expression levels were compared. The concordance between TMAs and full-face sections was excellent using Cohen's kappa statistical test for categorical variables (kappa=0.8).

Nottingham University Hospital Locally Advanced Breast cancer (NUH-LABC)
Full face sections from the diagnostic pre-chemotherapy core biopsies (n=361) and post-chemotherapy residual tumours in the surgically removed specimens were stained with SPAG5 as mentioned above.

Queensland breast cancers follow up (QLBCFU)
Four u TMA sections were processed in a decloaker for antigen retrieval in sodium citrate buffer (pH 6.0) for 20 mins, and then IHC was performed using an anti-SPAG5 antibody (Sigma HPA022479; 1:50), and the Mach 1 Universal HRP-Polymer Detection kit (Biocare Medical). Haematoxylin-counterstained, mounted sections were then scanned at 40 x magnification on an Aperio AT Turbo slide scanner (Leica Biosystems). Digital images of individual tissue cores were scored by three experienced molecular pathologists according to the intensity of tumour cell staining: punctate cytoplasmic staining was recorded as 1+ (moderate) or 2+ (strong), and multiplied by the percentage of tumour cells stained to derive a final score ranged (0 to 200). Faint, diffuse staining was recorded as 0. Using the maximum score of duplicate tissue cores for each case, associations between SPAG5 expression and clinicopathological variables were investigated.