Partial PTEN deletion is linked to poor prognosis in breast cancer

A pre-existing tissue microarray (TMA) was used for the purpose of this study [24]. In short, one 0.6 mm core was taken from a representative tissue block from each patient. The tissues were distributed among 6 TMA blocks, each containing 263 to 522 tumor samples. The TMA contained in total 2,197 human breast cancer samples from paraffin-embedded tissue specimens fixed in 4 % neutral buffered formalin. The median patient’s age was 63 (range 26–101) years. The use of the specimens and data for research purposes was approved by local laws (HmbKHG, §12,1) and the local ethics committee (Ethics commission Ärztekammer Hamburg, WF-049/09 and PV3652). According to local laws, informed consent was not required for this study. Patient records/information was anonymized and de-identified prior to analysis. All work has been carried out in compliance with the Helsinki Declaration. Survival data were either obtained from the cancer registry of Basel or collected from the patients attending physicians. Raw survival data were available from 1,982 patients (713 patients with and 1,508 without event (death)). The mean follow-up time was 63 months (range 1–176 months). Tumor size and nodal status were obtained from the primary pathology reports. All slides from the tumors were reviewed by specialized pathologists to define the histologic grade according to Elston and Ellis [25] and the tumor type according to the WHO classification (WHO 2012). Four μm sections of the TMA blocks were transferred to an adhesive coated slide system (Instrumedics Inc., Hackensack, New Jersey) for FISH analysis. Molecular data used in this study were available from previously published studies. These included amplification data obtained by FISH for HER2, CCND1, and MYC as well as expression data obtained by immunohistochemistry for estrogen receptor (ER), progesteron receptor (PR) and Ki67 [24, 26].

Four micrometer TMA sections were used for fluorescence in-situ hybridization (FISH). For proteolytic slide pretreatment, a commercial kit was used (paraffin pretreatment reagent kit; Abbott, Wiesbaden, Germany). TMA sections were deparaffinized, air-dried, and dehydrated in 70 %, 85 %, and 100 % ethanol, followed by denaturation for 5 min at 74 °C in 70 % formamid 2x SSC solution. The home-made FISH probe set consisted of a spectrum-orange labeled PTEN probe (made from a mixture of BAC RP11-380G5 and BAC RP11-813O3), and a spectrum-green labeled commercial centromere 10 probe (#6J37-10; Abbott, Wiesbaden, Germany) as a reference. Hybridization was performed overnight at 37 °C in a humidified chamber. Slides were subsequently washed and counterstained with 0.2μmol/L 4′-6-diamidino-2-phenylindole in antifade solution. Stained slides were manually interpreted with an epifluorescence microscope (Axio Imager A1, Zeiss, Oberkochen, Germany), and the predominant FISH signal numbers were recorded in each tissue spot. Presence of fewer PTEN signals than centromere 10 probe signals in at least 60 % tumor nuclei were considered as heterozygous deletion. Complete absence of PTEN signals in the tumor cells, but presence of centromere 10 signals and PTEN signals in adjacent normal cells, was considered homozygous deletion (Fig. 1). Tissue spots lacking any detectable PTEN signals in all (tumor and normal cells) or lack of any normal cells as an internal control for successful hybridization of the FISH probe were excluded from analysis. These thresholds were based on our previous study analyzing PTEN deletions on a prostate cancer TMA [12].

Statistical calculations were performed with JMP 9 software (SAS Institute Inc., NC, USA). Contingency table analysis and Chi square test were used to study the relationship between IHC and FISH results and clinicopathological variables. Kaplan–Meier plots were used to estimate disease-specific and overall survival and the statistical significance was determined by the log rank test. The log-Rank test was applied to test the significance of differences between stratified survival functions.

PTEN deletions were found in 19 % of NST cancers, 9 % of lobular (p = 0.0036 vs NST), 35 % of papillary cancers (p = 0.1150 vs NST) and 46 % of cancers with medullary features (p = 0.0002 vs NST). If all cancers were jointly analyzed, deletion of PTEN were significantly linked to advance tumor stage (p = 0.0054), and high histopathological grade (p < 0.0001) in all cancers. These associations held also true in the largest subset of No Special Type (NST) cancers. In addition, PTEN deletions were strongly linked to the subset of hormone receptor (ER/PR) negative breast cancers: deletion was found in 43 % of ER negative and 24 % of PR negative but only in 11 % of ER negative and 11 % of PR positive breast cancers (p < 0.0001 each). PTEN deletion was unrelated to presence of lymph node metastases. All results are summarized in Table 1.

HER2, CCND1 and MYC amplification results were available from a previous study [24]. In total, FISH results on both PTEN deletions and amplifications of HER2, CCND1 and MYC were available in subsets of 1,047 (HER2), 792 (MYC) and 1,149 (CCND1) cancers. There was a positive association between PTEN deletion and amplification of HER2 and MYC, but an inverse association to amplification of CCND1. PTEN deletion was found in 27 % of 195 HER2 amplified cancers but in only 18 % of 852 tumors with normal HER2 copy numbers (p = 0.0065), as well as in 26 % of 57 MYC amplified cancers but in only 17 % of 735 tumors with normal MYC copy numbers (p = 0.0430). In contrast, PTEN deletion was found in only 12 % of 237 CCND1 amplified cancers, but in 21 % of 912 tumors with normal CCND1 copy numbers (p = 0.0020). These associations are shown in detail in Fig. 2.

Data on the tumor cell proliferation, as determined by immunohistochemical analysis of the Ki67 antigen, were available from a previous study using the same TMA [24]. PTEN deletions were strongly associated with a high Ki67 labeling index (LI) if all cancers were jointly analyzed (p < 0.0001), as well as in subsets of cancers with identical grade (p < 0.05 each, Fig. 3a–d).

Data on raw survival were available from 1,236 cancers with interpretable PTEN FISH results. Presence of PTEN deletion was linked to shortened overall survival if all cancers were jointly analyzed (p = 0.0090, Fig. 4a), as well as in the subsets of NST cancers (p = 0.0629, Fig. 4b), and in the subset of NST cancers with nodal metastases (p < 0.0001, Fig. 4c). To study, whether PTEN deletion has an additional prognostic value in cancers harboring co-amplification of HER2, MYC, or CCND1, we stratified cancers for survival analysis according to the status of PTEN and HER2 (Fig. 5a), PTEN and MYC (Fig. 5b), as well as PTEN and CCND1 (Fig. 5c). These analyses suggested an inferior prognosis in cancers harboring PTEN deletions with co-amplifications of HER2, MYC, or CCND1 as compared to those without co-amplification, but the differences did not reach statistical significance. Since the failure to find significant differences was most likely due to small numbers in the various subsets, we combined all cancers according to the number of alterations, including PTEN deletion and amplification of any of HER2, MYC, and CCND1. In this combined analysis, we found a shortened survival for patients with cancers harboring alterations in 2 or more of these genes as compared to tumors with no or one alteration (p = 0.0002, Fig. 5d).