Chasing Hippos: Implications of YAP1 and TAZ Expression in Pregnancy-associated Breast Cancer Tumorigenesis

Abstract Background/Aim: The Hippo pathway is a molecular pathway recently associated with tumorigenesis, metastasis, and drug resistance. Pregnancy-associated breast cancer (PABC) is the most common malignancy diagnosed during gestation; however, the molecular mechanisms underlying PABC are largely unknown. The aim of the present study was to evaluate Hippo pathway transducers TAZ and YAP1 expression in PABC in relation to the clinicopathological characteristics of the disease. Patients and Methods: Formalin-fixed paraffin-embedded (FFPE) tissues from 21 PABC patients treated at Alexandra Hospital in Athens, Greece, were analyzed with immunohistochemistry. Results: Strong nuclear TAZ/YAP1 stanning was found in 48% of the PABC patients analyzed. Hormone receptor negative patients had a statistically significant correlation with strong positive expression of TAZ/YAP1 co-transcription factors. No association was observed with overall and disease-free survival. Conclusion: The Hippo pathway is de-regulated in a subset of PABC patients, highlighting the complex molecular background of the disease, which certainly requires further investigation.

diagnosed with PABC, which represents the most frequently diagnosed pregnancy-related malignancy (12). As women tend to defer childbearing to a later age, PABC incidence is expected to increase substantially in the upcoming years. Additionally, the application of non-invasive prenatal testing (NIPT) in all pregnant women, aiming to detect fetal abnormalities, has unquestionably increased the identification of asymptomatic PABC patients (13,14). The genomic profile of PABC has recently been addressed in a systematic review, but the underlying mechanisms of the disease still require further investigation in order to explain PABC aggressiveness; advanced T stage in diagnosis, nodal involvement, high histologic grade, negative estrogen receptor (ER) and progesterone receptor (PR) status, and HER-2 over-expression (15,16).
Toward this effort, the study of the Hippo pathway transducers TAZ and YAP1 in PABC aimed to further elucidate the oncogenic mechanism of PABC and to detect signal molecules that may serve as novel diagnostic biomarkers and therapeutic targets of anticancer drugs.

Patients and Methods
This cohort study of patients diagnosed with PABC was conducted at the Alexandra Hospital that is affiliated with the National and Kapodistrian University of Athens in Greece. All participants were required to sign the informed consent form according to the principles of the Declaration of Helsinki, to have completed the 18 th year of age, and to have attended the Breast Unit of the Department of Obstetrics and Gynecology or the Department of Clinical Therapeutics at Alexandra Hospital in Athens, Greece. The study protocol was approved by the Institutional Review Board (IRB) of Alexandra Hospital (Protocol code: 908/15.11.2019; Date of approval: 15.11.2019).
Participants. By the time the written informed consent was granted, all medical records of women diagnosed with PABC in our Institution according to the inclusion criteria were retrospectively reviewed for the period 2000-2019. Additionally, all women diagnosed with the disease from January 2020 until December 2020 were also prospectively included in our study. The following clinicopathological data regarding PABC, and the patients' demographic characteristics were extracted from the medical files of each eligible patient; date of birth, gestational age at diagnosis, family cancer history, histopathologic evaluation (tumor size, stage, grade, lymph node status, hormone receptor and HER-2 expression), genetic testing, PABC treatment (e.g., surgery, chemotherapy, immunotherapy, hormonotherapy, radiotherapy), and follow-up data. Furthermore, for each patient enrolled in the study, a sample of formalin-fixed paraffin-embedded (FFPE) tissue was obtained from the Department of Pathology in Alexandra Hospital to evaluate Hippo pathway transducers expression.
Immunohistochemistry. TAZ and YAP1 transducers expression in PABC was detected on FFPE tissue using the rabbit anti-YAP and anti-TAZ antibodies, clone D24E4 of the ImmPress REAGENT KIT, UNIVERSAL, Anti-mouse/rabbit Ig, VECTOR, at the dilution 1:50 and according to the manufacturer's protocol (Cell Signaling Technology, Danvers, MA, USA). Their expression was reported both in terms of percentage of tumor-expressing cells and staining intensity (0=absent, 1+=weak, 2+=moderate, 3+=strong). The topographic TAZ and YAP1 expression were evaluated in both the cytoplasm and the nucleus of tumor cells, and in the main cellular components, namely endothelial cells, non-lymphocytic stromal cells, and tumor-infiltrating lymphocytes (TILS) that were morphologically identified (9). Nuclear staining, which demonstrated variable expression, was further classified as negative/weak positive or strong positive following the study by Rodríguez-Núñez et al.; tissue was given a score which resulted from multiplying the nuclear staining intensity from 0 (no staining) to 3 (strong staining) by the extension based on the percentage of positive cells (from 0-3). Thus, samples were grouped as negative or weak positive (scores 0-2), and strong positive (scores 3-9) (17).
Statistical analysis. Descriptive statistics were used to summarize study participants' characteristics. The Pearson's Chi-Squared and Fisher Exact Tests, when appropriate, for categorical variables were used to investigate the correlation among the immunohistochemical YAP1/TAZ expression and the clinicopathological characteristics. Survival curves were estimated with the Kaplan-Meier method. All the statistical analyses were performed using the SPSS v24 (IBM, Armonk, NY, USA, SPSS Statistics) software. p-value<0.05 was considered statistically significant.

Results
Patient characteristics. Overall, 21 women diagnosed with PABC were enrolled in this study and screened for the expression of the Hippo signaling transducers TAZ and YAP1. Detailed data regarding the demographic variables and pregnancy characteristics of the patients including age, gestational age at diagnosis, ethnicity, and family cancer history are demonstrated in Table I. The age at diagnosis ranged from 28 to 42 years, with a mean age of 35.1 years (SD=3.83; range=28.0-42.0). Six patients (38%) were diagnosed with PABC within the first year after delivery, whereas 10 patients (63%) were diagnosed during pregnancy. Most of the participants reported a negative family cancer history (76%), whereas only 5 patients (24%) had a positive family history of lung, prostate, breast, or colorectal cancer among first degree relatives.
No association was observed between YAP1/TAZ nuclear expression, the gestational age at diagnosis, and the family cancer history.
Histopathological characteristics. The histopathological features of the 21 PABC patients examined in this study are summarized in Table II. The most frequently diagnosed histopathological type was invasive ductal carcinoma (IDC) (81%), whereas a few cases of invasive lobular (ILC) and metaplastic carcinoma were also identified. The vast majority of tumors were of high grade (81%) and the mean value of tumor size was 4.21 cm (SD=3.01; range=0. 8-12.0 in vivo 36: 2869-2874 (2022) cm). In most cases analyzed, high levels of Ki-67 >20% were detected (75%). Only two women (10%) were diagnosed with primary metastatic PABC. Nineteen patients received adjuvant chemotherapy (90%). The majority had a diagnosis of stage II disease (52.4%).
The nuclear YAP1/TAZ staining was classified as negative/weak positive in 52% (11/21) and as strong positive in 48% (10/21) of the patients analyzed. As far as the expression of hormone receptors (HRs) is concerned, HR negative status was associated with strong nuclear expression of the Hippo pathway transducers TAZ and YAP1 in tumor cells (p-value=0.006). Furthermore, when evaluating separately the ER and the PR expression, a statistically significant correlation was identified solely among the PR negative status and the strong positive nuclear YAP1/TAZ expression (p-value=0.007). Representative patterns of TAZ and YAP1 immunohistochemical staining are illustrated in Figure 1. No significant association was observed with the histopathological type, tumor grade and size, HER-2 and Ki-67 expression, molecular subtype (Luminal A, Luminal B -HER-2 positive/negative, TNBC, HER-2 positive) and stage of the disease. YAP1/TAZ nuclear expression and prognosis. We next investigated the impact of TAZ and YAP1 expression on the overall survival (OS) and disease-free survival (DFS) rate of PABC patients. Follow-up data were retrieved from the medical files of 15 out of 21 PABC patients enrolled in this study; 2 deaths were recorded, and the remaining 13 patients demonstrated a mean OS rate of 172.348 months (Figure 2A, 95%CI=134.305-210.392). Furthermore, as far as progressionfree disease is concerned, the following events were reported; local breast relapse was observed in four patients, liver and bone metastases developed in three patients, lung metastasis in two, and brain metastasis in a single patient. In total, 6 patients were characterized as metastatic; 2 primary metastatic PABC patients, one of which showed further signs of relapse with bone and lung metastases 5 months after the initial diagnosis, and 4 secondary metastatic patients. The progression-free survival (PFS) rate was not estimated as the cohort was too small to draw any conclusions. Additionally, the mean DFS rate was estimated to be 86.453 months ( Figure  2B, 95%CI=54.283-118.622). Even though most patients characterized by strong positive nuclear YAP1/TAZ staining relapsed or passed away, no statistically significant correlation was identified with the OS and DFS rates.

Discussion
PABC is the most common malignancy accompanying pregnancy (16). The distinct clinical and genetic data of PABC are not fully elucidated (15). Herein, we have conducted a translational study of Hippo pathway transducers in relation to the clinicopathological characteristics of PABC.   The Hippo pathway was found to be de-regulated in 10/21 cases (48%), implying that almost half of PABC patients may present with this abnormality. When we further studied the correlation between HR status and Hippo pathway activity, we observed a statistically significant result for HR negative patients and YAP1/TAZ strong nuclear positivity (p=0.006). This result was also prominent for PR negative cases (p=0.007). These findings are in accordance with previous publications showing that PABC are commonly tumors with low HR status (16,18). Additionally, our data confer a possibly crucial molecular mechanism for the development of a subset of PABC cases, which are generally shown to have a complex genetic and molecular background (8,15).
When we tested the histopathological type, tumor grade and size, HER-2 status and Ki-67 expression, molecular subtype, and stage of the disease, we did not find any statistically significant results. Possibly, these findings depict the molecular complexity of the disease (15,(19)(20)(21). Hippo signaling seems not to be present and meaningfully active in all PABC cases. Furthermore, Hippo signaling positivity did not have a statistically significant correlation with the gestational age at diagnosis of PABC during or after pregnancy. Here, it should be highlighted that our data show a higher percentage of patients being diagnosed during pregnancy than postpartum, as is published by others (16).
Our study did not reveal any correlations between OS, DFS, and Hippo pathway. Even though most patients with Hippo pathway de-regulation experienced relapse or died from the disease, no statistically significant correlations were found. However, a longer follow up may be needed to extract more accurate conclusions regarding these clinical parameters.
The characteristics of our study population are representative of PABC patients (16,18). The majority harbor an aggressive disease with high Ki-67 levels, low HR status, and grade 3 tumors. Only 2 (9.5%) patients were primarily metastatic, 4 developed metastases at a later course of their disease, while 4 patients experienced local relapse. Stage II and III patients were the majority, in agreement with previous publications. Furthermore, luminal B and TNBC patients were most commonly found in our cohort, also in accordance with previous publications (16).
The present study had several limitations that need to be addressed. Meaningful information was not reported in 6 cases, thus decreasing the number of our patients with clinical data to 15 included cases and as a result, the statistical power was limited and anticipated associations were not observed. Even though our study is limited by the retrospective nature of the analysis and its small number of patients, it adds an important amount of clinical data regarding the clinical characteristics and outcome of PABC, as well as the significance of the Hippo pathway in the development of this rare disease.

Conclusion
To conclude, Hippo pathway signaling is de-regulated in a subset of PABC patients. We are the first to report that almost half of PABC patients had strong nuclear immune-positivity for YAP1/TAZ co-transcription factors, implying high activity and de-regulation of the Hippo pathway. HR negative tumors were associated with the Hippo pathway, depicting an important role of this molecular signaling in the development of these PABC tumors. PABC remains a rare tumor entity with distinct clinical and molecular characteristics, with the Hippo pathway playing a significant role in the molecular complexity of these tumors.

Conflicts of Interest
The Authors declare the following financial interests/personal relationships, which may be considered as potential competing interests: FZ has received honoraria for lectures and served in an advisory role for Astra-Zeneca, Daiichi, Eli-Lilly, Merck, Novartis, Pfizer, and Roche. MAD has received honoraria from participation in advisory boards from Amgen, Bristol-Myers-Squibb, Celgene, Janssen, Takeda. The remaining Authors (AK, AMK, MAP, KA, AN, EZ, GB, DT) declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.