PTEN expression as a predictor for the response to trastuzumab-based therapy in Her-2 overexpressing metastatic breast cancer

Background Even though trastuzumab is an effective therapy in early stage Her-2+ breast cancer, 40–50% of advanced Her-2+ breast cancer patients develop trastuzumab resistance. A potential resistance mechanism is aberrant downstream signal transmission due to loss of phosphatase and tensin homologue (PTEN). This study investigated the relationship between the expression of PTEN and trastuzumab response in Her-2 overexpressing metastatic breast cancer patients. Methods Between 2000 and 2007, 164 patients with Her-2+ metastatic breast cancer received trastuzumab-based therapy in our institution. We analyzed PTEN status by immunohistochemistry of 115 available tumor tissues and analyzed associations with other histopathological parameters, response rate, progression free survival (PFS) and overall survival (OS) with a median follow-up of 60 months. Results Eighty patients were PTEN positive (69.6%) and 35 patients PTEN negative (30.4%). We found a significant association of the expression of PTEN and p53 (p = 0.041), while there was no association with grading, hormone receptor status, IGFR or MIB. We found significantly more cases with progressive disease under trastuzumab-based therapy in patients with PTEN positive breast cancers (p = 0.018), while there was no significant correlation with PFS or OS. Conclusion In Her-2-positive metastatic breast cancers, PTEN positivity was significantly associated with progressive disease, but not with PFS or OS.


Introduction
Overexpression of Her-2 is found in approximately 25% of human breast cancers leading to an aggressive phenotype and poor patient survival [1,2,3,4]. Trastuzumab, a humanized monoclonal antibody against the extracellular domain of Her-2, has been shown to be very effective in combination with chemotherapy for the treatment of early stages [5,6] or metastatic breast cancer [7,8] and even as a single-agent for the later group [9] with substantial decrease in breast cancer recurrence and mortality [10,11,12,13]. However, 40-50% have shown resistance to trastuzumab administered as a single agent [9] or in association with taxanes [10,14] within one year. However, the exact mechanism of the development of trastuzumab resistance is not completely clarified yet.
One of the known mechanisms underlying trastuzumab's antitumor activity is the downregulation of p185 ErbB2 and the subsequent inhibition of its downstream PI3K-Akt and MAPK signalling pathways. Molecules located in these pathways are thought to be associated with unresponsiveness to trastuzumab. PTEN (phosphatase and tensin homologue) is a dual phosphatase with membrane localization, which antagonizes PI3K function and inhibits Akt activities and tumor growth [15]. Consequently, PTEN loss leads to hyperactivation of the PI3K pathway and drives tumorigenesis. It has been shown that loss of PTEN, which occurs in about 20-40%, is associated with resistance to trastuzumab-based therapy [16,17,18,19].
Two reported mechanisms how PTEN loss promotes trastuzumab resistance are the transformation of Her-2 positive breast cancer into a triple negative subtype through induction of the epithelial-mesenchymal transition (EMT) [20] and the development of autophagy defects [21].
Nevertheless, the results of existing studies looking at the role of PTEN expression in the development of trastuzumab resistance are conflicting [22].
We therefore investigated the role of PTEN as prognostic marker in 115 metastatic Her-2 positive breast cancer patients who underwent trastuzumab-based therapy in the palliative setting, and then examined PTEN status and its association with clinical and histopathological parameters such as hormone receptor status, p53, MIB-1, IGFR, grading and clinical outcome (response rate, progression free survival (PFS), overall survival (OS)).

Study population
Between March 2000 and March 2007, 164 patients with metastatic Her-2 grade 2+ or 3+ overexpressing breast cancer received trastuzumab (Herceptin 1 , Roche Pharmaceuticals, Vienna, Austria) with or without chemotherapy at our institution.
Before initiation of trastuzumab-based treatment, all patients had and were required to have bi-dimensionally measurable disease (with both diameters > 1.0cm and at least one lesion with both diameters > 1.5cm excluding CNS lesions as the only site of measurable disease) with clearly defined margins and radiologically (CT and/or MRI and/or ultrasound) documented tumor progression. In accordance with the Southwest Oncology Group response criteria and endpoint definitions [23], response evaluation was performed by independent review of patients' records and radiology reports. This study was approved by the ethics committee of the Medical University of Vienna and patients had to sign an informed consent prior to inclusion into the study.
Of 164 patients with Her-2 positive metastatic breast cancer who received trastuzumab, 115 formalin fixed paraffin embedded tumor tissues were available for this study. Tumor grade, age at diagnosis and histopathological parameters such as Her-2 status, estrogen receptor (ER), progesterone receptor (PgR), p53, MIB-1 and insulin like growth factor receptor (IGFR) were extracted from clinical records.

Immunohistochemical detection of PTEN expression
PTEN IHC analyses for this study were conducted using UltraVision LP Detection System. In brief, 4-μm formalin-fixed paraffin embedded tumoral tissue sections were deparaffinised and rehydrated. For antigen unmasking, sections were pre-treated by digestion with Target Retrieval Solution Citrate pH 6 (DakoCytomation). Endogenous peroxidase activity was blocked by incubation in hydrogen peroxide block. Sections were then blocked with Protein Block (DAKO X0909) and incubated with the primary mouse monoclonal anti-PTEN antibody (Clone 6H2.1, Cascade Biosciences, diluted 1:300) over night at 4˚C. After incubation with the secondary anti-mouse IgG antibody (Vector Laboratories cat. # PK6102) for 30 min and washing with PBS buffer, slides were incubated with ABC reagent for 30 min at room temperature. The immunoreaction was visualized by using Liquid DAB-PLUS Substrate Kit (Zymed Laboratories; cat# 00-2020) according to the manufacturer's protocol. Slides were then counterstained with haematoxylin and cover-slipped.
In all subjects, invasive area, in situ area and surrounding breast tissue were separately evaluated for presence of PTEN antibody. Cells were rated as showing nuclear staining or cytoplasmic staining for PTEN. Due to the variations in the intensity of staining, a scoring system was used [16]. The percentage of cells that were semi-quantitatively positively stained for PTEN and the intensities of staining were multiplied and the result was recorded as immunoreactive score (IRS). Intensities of staining were graded as 0 (negative), 1 (weakly positive), 2 (moderately positive) and 3 (strongly positive). Staining was graded as 0 (<5% cells), 1 (5-25% cells), 2 (26-50% cells), 3 (51-75% cells) and 4 (>75% cells). Based on these results, IRS 0-3 was recorded as 0, IRS 4-6 as 1 positive, IRS 7-9 as 2 positive and IRS 10-12 as 3 positive. PTEN positivity was defined as IRS 2 or 3 and PTEN negativity as IRS 0 or 1. Negative control slides without primary antibody were included for each staining. Normal tonsil epithelium known to express normal PTEN was used as positive control. Expression levels were assessed independently by two experienced pathologists from two different institutions (A.R. and J.A). Photomicrographs of immunohistochemical analysis for PTEN expression are depicted in

Statistical analysis
Chi Square and Fisher's Exact test were used to evaluate associations between PTEN expression and clinicopathological parameters. Logistic regression was used to predict if any of the clinicopathological parameters predicts PTEN positivity. Complete, partial remission, stable and progressive disease were defined by World Health Organization (WHO) criteria [24].
Progression free survival was defined as the time from the start of trastuzumab-based treatment to disease progression or death. Overall survival (OS) was defined as the time from initial diagnosis of breast cancer (OS1), from the occurrence of metastatic disease (OS2) and from the start of therapy with trastuzumab (OS3) to death from any cause. Median years for PFS and OS and 95% confidence interval (95%CI) were determined for all patients and were estimated according to the Kaplan-Meier product limit method. Log rank test was used to compare survival distributions of patients with PTEN positivity versus patients with PTEN loss. For all analyses, a p-value <0.05 was considered statistically significant. SPSS statistical software system (SPSS Inc., Chicago, IL, version 23.0) was used for all calculations.

Study population
A total of 115 patients with Her-2/neu overexpressing metastatic breast cancer, who received trastuzumab-based treatment at our institution between March 2000 and March 2007 were included in this study. Patients, tumor characteristics and types and lines of trastuzumabbased therapy are shown in Table 1. These patients were followed up for a median observation   (Table 2).

PTEN expression and its association with other tumor characteristics and clinical outcomes
Distribution of histopathological characteristics by immunohistochemistry of 115 breast cancer specimens is shown in Table 3. PTEN expression was positive in 80 (69.6%) and negative in 35 (30.4%) cases. Looking at the PTEN expression status and possible associations with other immunohistochemical tumor characteristics, we found a statistically significant association between PTEN positivity and p53 (p = 0.041, Chi-square test). There was no significant association between PTEN expression and hormone receptor status, MIB-1 or IGFR (Table 4). While we did not observe any statistically significant difference regarding objective response rates to trastuzumab based therapy (ie. complete response (CR) and partial response (PR)) and rates of clinical benefit (CR, PR and stable disease (SD)) between patients with tumors expressing PTEN as compared to those with PTEN loss, we found a significant association between PTEN positivity and progressive disease (p = 0.018). Log rank test did not show     [40].
Because of these heterogenous results we conducted the present study and looked at the predictive and prognostic value of PTEN in Her-2 positive metastatic breast cancer patients treated with trastuzumab-based therapy with a long term follow up of median 60 months.  In our series of 115 metastatic breast cancer patients we have shown a similar distribution to previous studies with PTEN positivity in 69.6% of cases and loss of PTEN expression in 30.4% of cases [18,22,28,32,35,41].
Regarding the predictive and prognostic value of PTEN expression, we have found a statistically significant association of PTEN positivity with progressive disease under trastuzumabbased therapy, but not with PFS or OS.
When we analyzed associations of PTEN with other histopathological parameters, we found a significant positive association of PTEN positivity with p53. In contrast to our results, other studies have reported that high expression of p53 is associated with PTEN loss in basallike tumors [41,42]. There are no studies showing an association of p53 and PTEN expression in Her-2 positive disease. When we looked at other immunohistochemical parameters like hormone receptor status, MIB or IGFR, we did not find a significant association with the expression of PTEN, which is in line with previous reports [31,32].
Although our study is limited by the heterogeneity of therapeutic approaches in this relatively small sample size and by the fact that we concentrated on PTEN expression without additional information about the PI3K mutation status, we have shown a predictive value of PTEN positivity for the response to trastuzumab-based therapy in metastatic breast cancer patients with a long term follow up of five years. Our results encourage further investigation to define the role of PTEN in the pathogenesis of breast cancer and its role in trastuzumab resistance in larger trials.
In conclusion, we observed more cases with disease progression under trastuzumab-based therapy in patients whose breast cancers expressed PTEN. This underlines the predictive value of PTEN expression for the response under trastuzumab-based therapy. Further studies are warranted to validate these results to be able to further individualize Her-2-targeted therapy and its combinations with PI3K inhibitors and divide this group of patients with aggressive disease as early as possible in responders and non responders.