PTPRO promoter methylation is predictive of poorer outcome for HER2-positive breast cancer: indication for personalized therapy

A total of 221 breast tissue specimens were harvested and preserved from patients between June 1998 and December 2010 at the Affiliated Cancer Hospital of Shantou University Medical College. The median age of the patients was 51 years (range 21–89 years). Among the 221 cases, 123 women were premenopausal and 98 were postmenopausal. Histologically, all cases were invasive ductal carcinomas. At the time of operation, 41 cases (18.5%) were grade I tumors, 79 (35.7%) cases were grade II, and 83 cases (37.5%) were grade III. The pT stage of patients at initial diagnosis was stage 1 in 12 patients, 2 in 118, 3 in 22, and 4 in 18; in 5 cases the pT stage was not available. The pathological stage was assessed by surgical clinicians based on pathological reports and according to the 2003 TNM classification criteria by the International Union Against Cancer. A total of 74 cases (48.8%) were lymph node-negative, 41 cases (21.6%) were N1, 43 (10.0%) were N2, and 16 cases (19.6%) were N3. All patients, unless deceased, were followed up for at least 15 months and up to 124 months (mean 45.81 ± 17.91). All patients received conventional postoperative treatments, depending on the extent of the disease. The patients with ER+/PR + tumors were treated for 2–5 years with tamoxifen. The outcome was defined by the months of overall survival (OS) post-surgery. Preoperative peripheral blood (5 ml) from each patient was collected into an EDTA tube for the isolation of plasma. Control blood samples were obtained from 10 healthy volunteers. The use of human tissues in this study was approved by the Academic Committee of Shantou University Medical College. Patients who died of causes unrelated to the disease were not included in the study.

Paraffin blocks were cut to 8 μm-thick sections. In order to avoid cross contamination, a special procedure was employed in section cutting whereby new blades were used for each sample, and apparati, such as microtomes and tweezers, were carefully cleaned and disinfected between sample processing[21, 22]. Two sections were collected into 1.5-ml microcentrifuge tubes. After xylene deparaffination and treatment with absolute ethanol, the sections were digested at 55°C overnight with proteinase K (0.1 mg/mL) in 200 mL of DNA extraction buffer. Bisulfite treatment was then performed with 20 μl of digestion supernatant, using an EZ DNA Methylation-Direct™ Kit (Zymo, Beijing) to convert unmethylated cytosine to uracil.

Paraffin blocks of PTPRO-methylated MCF-7 human cancer cell lines and PTPRO-unmethylated NE-2 immortalized normal cell lines were used as positive and negative controls, respectively, throughout the procedure (including FFPE block slicing, DNA extraction, bisulfite modification, and methylation-specific PCR).

Blood samples were centrifuged at 3000 × g, and plasma was carefully transferred into plain polypropylene tubes and stored at -70°C until further processing. DNA from plasma samples was extracted using a TIANamp Blood DNA Kit (TIANGEN, Beijing), following the blood and body fluid protocol as recommended by the manufacturer (8). The plasma samples (400 μl/column) were used for DNA extraction. A final elution volume of 50 μl was used. DNA (1 μg) from each sample was subjected to bisulfite modification through the use of an EZ DNA Methylation-Gold Kit (Zymo Research) following the manufacturer’s instructions.

Methylation-specific PCR (MSP) was performed with bisulfite-converted genomic DNA isolated from 221 FFPE specimens, using primers designed by Motiwala[17]: nonmethylation-specific primers hPTP-UF (5′- ATGTTTTTGGAGGATTTTGGGT-3′) and hPTP-UR (5′- ATACCCCATCACTACACAAACA-3′) and methylation-specific primers hPTP-MF (5′- CGTTTTTGGAGGATTTCGGGC-3′) and hPTP-MR (5′- AAAACACGACTACGCTAACG-3′) amplify 201- and 170-bp products respectively. Thermocycling conditions were modified according to Motiwala′s method and carried out in a 50-μl reaction containing ≈ 100 ng of bisulfite-converted DNA, PCR buffer, 10 pmol each of forward and reverse primers, 0.2 mM dNTPs, and 0.75 units of HotStarTaq® Plus DNA Polymerase (Qiagen, Valencia, CA). After an initial incubation at 94°C for 3 min, a touch-down PCR with 10 cycles of denaturation at 94°C for 30 sec, annealing at 55°C (Δ –0.4°C per cycle) for 1 min (Δ –0.04 sec per cycle), and extension at 72°C for 30 sec was performed, followed by an additional 35 cycles of denaturation for 30 sec at 94°C, annealing for 15 sec at 50.4°C, and extension for 30 sec at 72°C. The reactions ended with a 3 min final extension at 72°C, and then 10-μl aliquots of the PCR reaction were subjected to electrophoresis on a 2.5% agarose gel. DNA was visualized by ethidium bromide staining and captured by a BioRad Gel Doc™ XR imaging system.

The association between methylation status and other molecular and clinicopathological parameters was calculated using contingency table methods and tested for significance using the chi-square test for categorical variables. Overall survival curves were calculated using the Kaplan-Meier method and the log-rank test was used to compare survival curves. Multivariate analysis was carried out using the Cox proportional hazard regression with a confidence interval of 95% to examine whether PTPRO methylation status was an independent prognostic factor for survival. All calculations were performed with SPSS 17.0 software (SPSS Inc., Chicago, IL, USA) for Windows. P-values less than 0.05 were considered to be statistically significant (two-sided).

MSP analysis resulted in an identifiable product in 175 cases. Among these cases, methylation of the PTPRO promoter was detected in 130 (74.3%) of the 175 tumors. Among the tumors positive for promoter methylation, 35 (27%) were positive only for the methylated reaction and 95 (73%) were positive for both the unmethylated and methylated reactions. For the 20 adjacent normal tissues, only 1 (5%) displayed the methylation amplicon. A chi-square test suggested hypermethylation of PTPRO promoter occurs specifically and frequently in primary breast cancer (P < 0.000). Figure 1 shows a representative assay for PTPRO promoter methylation by methylation-specific PCR.

The clinical characteristics of the 175 breast cancer patients at the time of surgery are summarized in Table 1. Chi-square testing revealed a statistically significant association between the PTPRO promoter methylation and tumor grade (P = 0.028), whereas there was no significant correlation between PTPRO promoter methylation and age, pT, pN, stage, ER, PR, HER2 or menopausal status. Patients with lymph node metastasis (pN), younger age, premenopausal status, loss of ER and PR in tumors, larger tumor size (pT), and advanced stage showed a trend toward greater hypermethylation, but the trend was not significant (P = 0.051, 0.103, 0.123, 0.119, 0.118, 0.146, 0.250, respectively).

Using a univariate Cox proportional hazard analysis, we examined clinicopathologic parameters, PTPRO methylation, and their association with overall survival end points. As summarized in Table 2, there were trends but no significant prognostic effects for PTPRO methylation in the 175 cohorts (HR, 3.273, CI 95%, 0.754-14.209; P = 0.113). Concerning the biological role of the PTPRO in the ER pathway and HER2 signaling, we stratified the entire patient cohort into subpopulations according to ER and HER2 status. In ER-positive patients, the unmethylated-PTPRO group did not show a trend for a more favorable outcome (n = 95; HR, 2.149; CI 95%, 0.463-9.983; P = 0.329). When tumor were HER2-positive, patients with methylated-PTPRO had a tendency for poor overall survival compared to those of unmethylated-PTPRO, but the differences were not significant (n = 78, HR, 5.112; CI 95%, 0.658-39.723; P = 0.119). Further analysis according to histological grade could not find additional significant difference (see Additional file1: Figure S1). Kaplan-Meier curves for the above subpopulations according to PTPRO methylation are shown in Figure 2b,2c and Additional file1: Figure S1. These inconclusive results may be due to the limited sample size and restricted follow-up period. We then compared patients with combined methylated-PTPRO and HER2-positivity to the remaining patients in the 175 cohorts (unmethylated-PTPRO/HER2-positive patients and patients with HER2-negative tumors irrespective of the PTPRO methylation status). Significantly higher risk was observed for the methylated-PTPRO&HER2-positive group of patients (HR, 2.749, CI 95%, 1.065-7.097; P = 0.037). To confirm the significance of this finding, we performed multivariate analysis, treating methylated-PTPRO/HER2-positivity as a factor with tumor size, lymph node metastasis, histological grade and HER2 status for their impact on overall survival. After adjustment for these covariates, methylated-PTPRO/HER2-positivity was identified as an independent predictor for overall survival (HR, 3.663; CI 95%, 1.371-9.784; P = 0.010). Similarly, tumor size (pT) and lymph node metastasis (pN) also had an independent association with overall survival in this patient cohort. Kaplan-Meier survival curves showed that promoter methylation of the PTPRO gene plus HER2-positivity was associated with poor overall survival (P = 0.029; Figure 2d).

We further explored the feasibility of detecting PTPRO methylation in the plasma of matched peripheral blood samples from breast cancer patients (Figure 3). Among 24 matched plasma samples, PTPRO was aberrantly methylated in 11 (45.8%) (Table 3), and detected only in plasma from patients whose corresponding primary tumors were also methylated. Additionally, no methylation of PTPRO was observed in any normal control plasma samples from 10 healthy individuals.