Hotspot mutation profiles of AKT1 in Asian women with breast and endometrial cancers

We obtained 118 frozen tumour surgical specimens and 193 formalin-fixed paraffin-embedded (FFPE) surgical or biopsy tissues that had been preserved in a biobank at the National Cancer Centre Hospital between May 1989 and December 2012. We extracted DNA from the frozen tumour specimens and FFPE tissues using QIAamp DNA Mini Kits and QIAamp DNA FFPE Tissue Kits, respectively (Qiagen GmbH, Hilden, Germany) as described by the manufacturer [9]. Mutations at the AKT1 hotspot detected using peptide nucleic acid-locked nucleic acid (PNA-LNA) clamp methods in samples collected and stored at the National Cancer Center Hospital were analysed by LSI Medience Corporation (Tokyo, Japan) as described previously [10].

We extracted DNA from 142 DNA FFPE surgical tissue samples that had been preserved at the National Cancer Centre Hospital between May 1989 and November 2012 using QIAamp DNA FFPE Tissue Kits (Qiagen) as described by the manufacturer [9]. We detected the AKT1 hotspot mutation using real-time PCR as described [8] with the forward and reverse primer sequences of exon 4 of AKT1 (5′-CACACCCAGTTCCTGCCTG-3′ and 5′-CCTGGTGGGCAAAGAGGGCT-3′, respectively). The PCR cycling program comprised activation at 94 °C for 5 min, followed by 35 cycles at 94 °C for 30 s, 55 °C for 30 s, 72 °C for 60 s, and 72 °C for 10 min. The PCR products were sequenced using the BigDye terminator method and an auto-sequencer (Applied Biosystems, Foster City, CA, USA).

Oestrogen (ER) or progesterone (PgR) receptors were classified as positive or negative based on ASCO/CAP Guideline [11]. In all cases, at least 1% of cells staining was considered positive according to the criteria. Human epidermal growth factor receptor-2 (HER2) positivity was classified according to the guidelines of the American Society of Clinical Oncology/College of American Pathologists [12]. Hormone receptor-positive status was defined as positive for either ER or PgR. The histological and nuclear grade of breast cancer and endometrioid uterine carcinoma were graded as described in previous studies [9, 13] and in a previous report [14], respectively. Pathological evaluations are always performed by at least two expert pathologists (including HY and MY), although the results of diagnoses in institutional practice are used. ND in our Tables is because there was a time when it was not evaluated in the old days, and we could not re-evaluate it sufficiently because of the deterioration of pathological specimens.

Differences in outcomes between patients with and without AKT1 mutations were assessed using data from 311 and 143 patients in breast and endometrial cancer subgroups. Differences between proportions for categorical variables were analysed using chi-square tests. Postoperative relapse-free (RFS) and overall (OS) survival was analysed using Kaplan–Meier curves. We defined RFS as elapsed time between the day of surgery and disease progression or the last follow-up for operable lesions. We defined OS as elapsed time between the first day of diagnosis and the date of death or the last follow-up. Significant prognostic factors associated with RFS and OS were determined by multivariate analysis using Cox hazard models. Clinically relevant factors for breast (initial stage, histologic grade, ER status, PgR status, HER2 status, and AKT1 mutation) and endometrial (diagnostic stage, lymphovascular invasion, and AKT1 mutations) cancers were included as covariates in multiple Cox proportional hazards models. All data were statistically analysed using JMP version 11 (SAS Institute, Cary, NC, USA). Statistical significance was set at P < 0.05.