The prognostic impact of Akt isoforms, PI3K and PTEN related to female steroid hormone receptors in soft tissue sarcomas

Primary tumor tissue from anonymized patients diagnosed with non-GIST STS at the University Hospital of North Norway (UNN) 1973-2006 and The Hospitals of Arkhangelsk region, Russia, were used in this retrospective study. In total, 496 patients were registered from the hospital databases. Of these, 247 patients were excluded due to missing clinical data (n = 86) or inadequate material for histological examination (n = 161). Thus, 249 STS patients with full clinical records and adequate paraffin-embedded tissue blocks were eligible.

This report includes follow-up data as of September 2009. The median follow-up was 38 (range 0.1 - 392) months. Formalin-fixed and paraffin-embedded tumor specimens were obtained from the archives of the Departments of Pathology at UNN and the Arkhangelsk hospitals. The tumors were graded according to the French Fèdèration Nationales des Centres de Lutte Contre le Cancer (FNCLCC)[27].

All sarcomas were histologically reviewed by two trained pathologists (S.S. and A.V.) and the most representative areas of viable tumor cells (neoplastic cells) were carefully selected and marked on the hematoxylin and eosin (H&E)-stained slides and sampled for the tissue microarray blocks (TMAs). The TMAs were assembled using a tissue-arraying instrument (Beecher Instruments, Silver Springs, MD). The Detailed methodology has been previously reported [28]. Briefly, we used a 0.6 mm diameter stylet, and the study specimens were routinely sampled with two replicate core samples (different areas) of neoplastic tissue. To include all core samples, 12 tissue array blocks were constructed. Multiple 4-μm sections were cut with a Micron microtome (HM355S) and stained using specific antibodies for immunohistochemistry (IHC) analyses.

The applied antibodies were subjected to in-house validation by the manufacturer for IHC analysis on paraffin-embedded material. The applied antibodies had been subjected to in-house validation by the manufacturer for IHC analysis on paraffin-embedded material. The antibodies used in the study were as follows: Phospho-Akt (Ser473) (1:5; Rabbit monoclonal, clone 736E11; #3787; Cell Signalling Technology, Danvers, U.S.A.), detects Akt 1 only when phosphorylated at serine 473, and Akt2 and Akt3 only when phosphorylated at equivalent sites. Phospho-Akt (Thr308) (1:50; Rabbit monoclonal, clone 244F9; #4056; Cell Signalling Technology), recognizes all three Akt isoforms when phosphorylated at this site. Akt2 (1:18; Rabbit monoclonal, clone 54G8; #4057; Cell Signalling Technology), preferentially binds to non-phosphorylated endogenous levels of Akt2. It does not cross-react with recombinant Akt1 or Akt3. Akt3 (1:8; Rabbit polyclonal, #4059; Cell Signalling Technology), detects endogenous levels of total Akt3, but does not recognize the truncated form of rat Akt3. The antibody does not cross-react with recombinant Akt1 or Akt2. PTEN (1:10, Rabbit monoclonal; #9559; Cell Signalling Technology), detects endogenous levels of total PTEN protein. PI3K (1:25; Rabbit polyclonal; #4254; Cell Signalling Technology), detects endogenous levels of total PI3K.

Sections were deparaffinised with xylene and rehydrated with ethanol. Antigen retrieval was performed by placing the specimen in 0.01mol/l citrate buffer at pH 6.0 and exposed to two repeated microwave heatings of 10 minutes at 450W. The DAKO EnVision + System-HRP (DAB) kit was used as endogen peroxidase blocking. Primary antibodies were incubated overnight at 4°C (except PI3K, for 32 minutes at room temperature). The DAKO EnVision+ System-HRP (DAB) kit was used to visualize the antigens for all stains. This yielded a brown reaction product at the site of the target antigen. As negative staining controls, the primary antibodies were replaced with the primary antibody diluent. Finally, all slides were counterstained with hematoxylin to visualize the nuclei. For each antibody, including negative controls, all TMA staining were performed in one single experiment. The immunohistochemical staining for ER and PgR was performed as described earlier[25].

The ARIOL imaging system (Genetix, San Jose, CA) was used to scan the slides with immunohistochemically stained TMAs. The specimens were scanned at a low resolution (1.25×) and high resolution (20×) using Olympus BX 61 microscope with an automated platform (Prior). The slides were loaded in the automated slide loader (Applied Imaging SL 50). Representative and viable tissue sections were scored manually on computer screen, semiquantitatively for cytoplasmic staining for PI3K/Akt pathway components and for nuclear staining for ER and PgR. The dominant staining intensity in neoplastic cells was scored subjectively as: 0 = negative; 1 = weak; 2 = intermediate; 3 = strong (Figure 1). For ER and PgR, the modified All Red scoring system [25] was used. All samples were anonymized and independently scored by two pathologists (A.V. and S.S.). In cases where score difference was equal to or exceeding 2, the slides were re-examined and a consensus was reached by the observers. When assessing a score for a given core, the observers were blinded to the scores of the other variables and to outcome. Mean score for duplicate cores from each individual was calculated.

All statistical analyses were done using the statistical package SPSS (Chicago, IL), version 16. The IHC scores from each observer were compared for interobserver reliability by use of a two-way random effect model with absolute agreement definition. The intraclass correlation coefficient (reliability coefficient) was obtained from these results. The Chi-square test and Fishers Exact test were used to examine the association between molecular marker expression and various clinicopathological parameters. Univariate analyses were done by using the Kaplan-Meier method, and statistical significance between survival curves was assessed by the log rank test. Disease-specific survival (DSS) was determined from the date of histological confirmed STS diagnosis to the time of STS death. To assess the independent value of different pretreatment variables on survival, in the presence of other variables, multivariate analysis was performed using the Cox proportional hazards model. Only variables with value 0.10 or less from the univariate analysis were entered into the Cox regression analysis. The significance level used in both univariate and multivariate analyses was P < 0.05, but in the post hoc subgroup analysis the significance level was moved from P = 0.05 to P = 0.01 due to risk of false positivity.

The National Cancer Data Inspection Board and The Regional Committee for Research Ethics approved the study. The Regional Committee approved that written consent from the patients for their information to be stored in the hospital database and used for research was not needed because most of the material was more than 20 years old and most of the patients are now dead. The material was collected from our approved biobank for paraffin-embedded material and slides. All material was anonymously collected. The data were analyzed anonymously.

The clinicopathological variables are summarized in Table 1. Median age was 59 (range, 0-91) years and 56% were female. The non-GIST STS comprised 249 tumors including pleomorphic sarcoma (n = 68), leiomyosarcoma (n = 67), liposarcoma (n = 34), malignant fibroblastic/myofibroblastic tumors (n = 20), rhabdomyosarcoma (n = 16), synovial sarcoma (n = 16), angiosarcoma (n = 13), malignant peripheral nerve sheath tumor (MPNST) (n = 11) and other types of sarcoma (n = 4). The tumors were localized in the extremities (n = 89), viscera (n = 58), trunk (n = 47), retroperitoneum (n = 37) and head/neck (n = 18). The treatment option of choice was surgery (n = 228), 120 patients received surgery alone, 55 patients received surgery and radiotherapy, 40 patients received surgery and chemotherapy and13 patients received surgery, radiotherapy and chemotherapy. Of the non-operated patients (inoperable, n = 11; advanced age/other serious disease, n = 5, STS diagnosis confirmed post mortem, n = 3; patient refusal, n = 2) seven received chemotherapy and/or radiotherapy. Fourteen patients did not obtain any treatment.

Interobserver scoring agreement was tested for all markers. The intraclass correlation coefficients were as follows: 0.89 for p-Akt Ser⁴⁷³ (p < 0.001), 0.94 for p-Akt Thr³⁰⁸ (p < 0.001), 0.91 for Akt2 (p < 0.001), 0.95 for Akt3 (p < 0.001), 0.88 for PI3-K (p < 0.001) and 0.89 for PTEN (p < 0.001).

In the immunohistochemical analyses, we used antibodies against all Akt isoforms, including Akt phosphorylated at Ser⁴⁷³ and at Thr³⁰⁸, non-phosphorylated Akt2 and total (both phosphorylated and non-phosphorylated) Akt3. Besides, we investigated expression of total PI3K and PTEN. The p-Akt Ser⁴⁷³, p-Akt Thr³⁰⁸, Akt2, Akt3, PI3K and PTEN showed expression in the cytoplasm or both in the cytoplasm and in the nuclei of tumor cells in the majority of cases, while pure nuclear staining was demonstrated in a smaller proportion of the tumors, varying from 7% of all immunohistochemically positive tumors for PTEN to 19% for p-Akt Thr³⁰⁸ and Akt3.

Expression of p-Akt Ser⁴⁷³ (r = 0.179, P = 0.005), p-Akt Thr³⁰⁸ (r = 0.150, P = 0.019), Akt2 (r = 0.250, p < 0.001) and PI3K (r = 0.223, p < 0.001) correlated significantly positive with STS histological grade. PI3K and p-Akt Thr³⁰⁸ positivity in STSs correlated with presence of metastasis at the time of diagnosis. Strong expression of p-Akt Thr³⁰⁸ was observed in 69% of the metastasizing tumors, whereas only 41% of non-metastasizing STSs (r = 0.208, P = 0.001) were strongly positive for this marker. For PI3K, the metastasizing versus non-metastasizing characteristics comprised 78% and 53%, respectively (r = 0.188, P = 0.003). None of the investigated markers correlated significantly with age, gender, tumor location, depth, size or relapse rate.

Data are presented in Table 1. Patient nationality (P = 0.011), tumor size (P = 0.027), malignancy grade (p < 0.001), tumor depth (p < 0.001), metastasis at time of diagnosis (p < 0.001), surgery (p < 0.001) and resection margins (p < 0.001) were all significant prognostic variables for DSS.

The prognostic impact of the investigated molecular factors is shown in Table 2. Among these, p-Akt Thr³⁰⁸ (P = 0.002), Akt2 (P = 0.008) and PI3K (p < 0.001) were significant indicators of shorter DSS, Figure 2, A-C.

In order to find out whether subcellular location of proteins belonging to the Akt/PI3K signaling pathway has impact on survival, we performed a series of univariate analyses to compare the impact of their expression in nucleus, cytoplasm or both. Nuclear expression of p-Akt Thr³⁰⁸ expression showed a significantly favorable prognosis (P = 0.029), compared to cytoplasmic and especially mixed cytoplasmic and nuclear expression, Figure 2, D. The other factors did not show any significant prognostic differences in the subcellular location.

Subgroup analysis based on clinical variables revealed that high expression of both p-Akt Thr³⁰⁸ (P = 0.006) and Akt3 (P = 0.001) were adverse prognostic indicators for STSs located to extremities and for tumors larger than 5 cm in largest dimension (P = 0.001 for both markers). Interestingly, high expression of p-Akt Thr³⁰⁸ was a negative prognostic factor particularly for men (P = 0.009 vs. P = 0.064 for women). In contrast, p-Akt Ser⁴⁷³, which appeared to be a negative prognosticator exclusively for female patients (P = 0.023 vs. P = 0.868 for men), Table 2.

The results of the multivariate analysis are presented in Table 3. Advanced age of the patient (P = 0.038), deep site (P = 0.018), high malignancy grade (p < 0.001), metastasis at time of diagnosis (P = 0.010), lack of surgery (P = 0.031), non-free resection margins (p < 0.001), and PI3K expression by tumor cells (P = 0.042) were significant independent negative prognostic indicators of DSS.

The co-expression profiles of both types of activated Akt and PI3K with female steroid hormone receptors, in the group as a whole and stratified into gender were tested as shown in Table 4. The co-expression phenotypes PgR+/p-Akt Thr³⁰⁸+ among men (P = 0.023, HR = 2.4, 95% CI = 1.1-5.2), ER-/PI3K+ both in whole cohort (P = 0.005, HR = 2.0, 95% CI = 1.2-3.2) and among women (P = 0.014, HR = 2.4, 95% CI = 1.2-4.8), as well as PgR-/PI3K+ (P = 0.007, HR = 1.9, 95% CI = 1.2-3.0) and PgR+/PI3K+ (P = 0.014, HR = 1.9, 95% CI = 1.1-3.2) in the whole cohort of patients were significant independent negative prognostic factors. Interestingly, both steroid hormone receptors and Akt phosphorylation site seem to have opposite prognostic impact depending on the gender. This was further proved by the co-expression of these factors. Indeed, PgR-/p-Akt Ser⁴⁷³+ phenotype tended to have an unfavorable impact in women (P = 0.087) but was favorable in men (P = 0.010). Co-expression of ER and p-Akt Ser⁴⁷³ showed similar results, with significantly adverse influence of -/+ profile on DSS among female patients (P = 0.006). There was no significant difference among the four possible profiles in men, but the -/+ curve demonstrated the best survival rate, Figure 2E and 2F.