Phenotype-guided targeted therapy based on functional signal transduction pathway activity in recurrent ovarian cancer patients: The STAPOVER study protocol.

Objective: Ovarian cancer is the fifth cause of cancer-related death among women. The benefit of targeted therapy for ovarian cancer patients is limited even if treatment is stratified by molecular signature. There remains a high unmet need for alternative diagnostics that better predict targeted therapy, as current diagnostics are generally inaccurate predictors. Quantitative assessment of functional signal transduction pathway (STP) activity from mRNA measurements of target genes is an alternative approach. Therefore, we aim to identify aberrantly activated STPs in tumour tissue of patients with recurrent ovarian cancer and start phenotype-guided targeted therapy to improve survival without compromising quality of life. Study design: Patients with recurrent ovarian cancer and either 1) have platinum-resistant disease, 2) refrain from standard therapy or 3) are asymptomatic and not yet eligible for standard therapy will be included in this multi-centre prospective cohort study with multiple stepwise executed treatment arms. Targeted therapy will be available for patients with aberrantly high functional activity of the oestrogen receptor, androgen receptor, phosphoinositide 3-kinase or Hedgehog STP. The primary endpoint of this study is the progression-free survival (PFS) ratio (PFS2/PFS1 ratio) according to RECIST 1.1 determined by the PFS on matched targeted therapy (PFS2) compared to PFS on prior therapy (PFS1). Secondary endpoints include among others best overall response, overall survival, side effects, health-related quality of life and cost-effectiveness. Conclusion: The results of this study will show the clinical applicability of STP activity in selecting recurrent ovarian cancer patients for effective therapies.

Functional estrogen receptor signal transduction pathway activity and antihormonal therapy response in low-grade ovarian carcinoma.

BACKGROUND: Advanced low-grade ovarian carcinoma (LGOC) is difficult to treat. In several studies, high estrogen receptor (ER) protein expression was observed in patients with LGOC, which suggests that antihormonal therapy (AHT) is a treatment option. However, only a subgroup of patients respond to AHT, and this response cannot be adequately predicted by currently used immunohistochemistry (IHC). A possible explanation is that IHC only takes the ligand, but not the activity, of the whole signal transduction pathway (STP) into account. Therefore, in this study, the authors assessed whether functional STP activity can be an alternative tool to predict response to AHT in LGOC. METHODS: Tumor tissue samples were obtained from patients with primary or recurrent LGOC who subsequently received AHT. Histoscores of ER and progesterone receptor (PR) were determined. In addition, STP activity of the ER STP and of six other STPs known to play a role in ovarian cancer was assessed and compared with the STP activity of healthy postmenopausal fallopian tube epithelium. RESULTS: Patients who had normal ER STP activity had a progression-free survival (PFS) of 16.1 months. This was significantly shorter in patients who had low and very high ER STP activity, with a median PFS of 6.0 and 2.1 months, respectively (p < .001). Unlike ER histoscores, PR histoscores were strongly correlated to the ER STP activity and thus to PFS. CONCLUSIONS: Aberrant low and very high functional ER STP activity and low PR histoscores in patients with LGOC indicate decreased response to AHT. ER IHC is not representative of functional ER STP activity and is not related to PFS.

The progression-free survival ratio as outcome measure in recurrent ovarian carcinoma patients: Current and future perspectives.

Objective: Clinical efficacy of cytostatic anticancer agents can be determined with the progression-free survival (PFS) ratio. This outcome measure compares PFS achieved by a new treatment (PFS2) to the PFS of the most recent treatment on which the patient has experienced progression (PFS1). Clinical benefit has been defined as a PFS-ratio (PFS2/PFS1) > 1.3. However, in order to demonstrate significant benefit, trial designs require an assumption on the proportion of patients who reach this ratio during palliative options. For ovarian carcinoma, data is lacking to support this assumption. Therefore in this study, we assess the PFS-ratio in recurrent ovarian carcinoma patients treated with current palliative options. Methods: We included 67 patients with recurrent high-grade serous (HGSC, 73.1%) or low-grade (LGOC, 26.9%) ovarian carcinoma. We determined the median PFS-ratio and investigated the association with clinicopathological characteristics. Results: Overall, we observed a median PFS-ratio of 0.69. The proportion of patients with a PFS-ratio > 1.3 was 22.4%. For HGSC patients, the median PFS-ratio was significantly lower than for LGOC patients (respectively, 0.58 and 1.26, p = 0.007). Multivariate logistic regression analysis revealed that the LGOC subtype and CA125 tumor marker concentration were independent factors related to a PFS-ratio > 1.3. Conclusions: Although the PFS-ratio represents a meaningful outcome measure in studies investigating cytostatic anticancer agents, we conclude that it is influenced by tumor histology and biological behavior. In future research, these factors should be taken into account when determining thresholds for clinical benefit in trial designs.

Signal transduction pathway activity in high-grade serous carcinoma, its precursors and Fallopian tube epithelium.

OBJECTIVE: To determine the activity of key signal transduction pathways in serous tubal intraepithelial carcinoma (STIC) and concurrent high-grade serous carcinoma (HGSC) and compare this to pathway activity in normal Fallopian tube epithelium (FTE). METHODS: We assessed mRNA expression levels of pathway-specific target genes with RT-qPCR in STIC and concurrent HGSC (n = 8) and normal FTE (n = 8). Subsequently, signal transduction pathway assays were used to assess functional activity of the androgen (AR) and estrogen receptor (ER), phosphoinositide-3-kinase (PI3K), Hedgehog (HH), transforming growth factor beta (TGF-ß) and canonical wingless-type MMTV integration site (Wnt) pathways. RESULTS: There were no statistically significant differences in pathway activity between STIC and HGSC, but STIC and HGSC demonstrated significantly lower ER and higher PI3K and HH pathway activity in comparison to normal FTE, suggesting these pathways as putative early drivers. In addition, we determined FOXO3a protein expression by immunohistochemistry and found loss of FOXO3a protein expression in STIC and HGSC compared to normal FTE. This observation confirmed that activation of PI3K signaling by loss of FOXO is an early hallmark of serous carcinogenesis. Furthermore, HGSC demonstrated significant loss of AR and Wnt pathway activity in relation to FTE, suggesting these pathways contribute to disease progression. CONCLUSION: Our observations, together with the previously described associations between p53 signaling and both PI3K and HH pathway activity, provide evidence that increased PI3K and HH pathway activity and loss of ER pathway activity may be underlying events contributing to neoplastic transformation of FTE into STIC.

Cyclic activity of signal transduction pathways in fimbrial epithelium of the human fallopian tube.

INTRODUCTION: The local environment of the fallopian tube represents the optimal conditions for reproductive processes. To maintain tissue homeostasis, signal transduction pathways are thought to play a pivotal role. Enhancing our understanding of functional signal transduction pathway activity is important to be able to clarify the role of aberrant signal transduction pathway activity leading to female subfertility and other tubal diseases. Therefore, in this study we investigate the influence of the hormonal cycle on the activity of key signal transduction pathways in the fimbrial epithelium of morphologically normal fallopian tubes. MATERIAL AND METHODS: We included healthy pre- (n = 17) and postmenopausal (n = 8) patients who had surgical interventions for benign gynecologic conditions. Histologic sections of the fallopian tubes were reviewed by two pathologists and, for the premenopausal patients, hormone serum levels and sections of the endometrium were examined to determine the hormonal phase (early follicular [n = 4], late follicular [n = 3], early luteal [n = 5], late luteal [n = 5]). After laser capture microdissection, total mRNA was extracted from the fimbrial epithelium and real-time quantitative reverse transcription-PCR was performed to determine functional signal transduction pathway activity of the androgen receptor (AR), estrogen receptor (ER), phosphoinositide-3-kinase (PI3K), Hedgehog (HH), transforming growth factor-beta (TGF-ß) and canonical wingless-type MMTV integration site (Wnt) pathways. RESULTS: The early luteal phase demonstrated high AR and ER pathway activity in comparison with the late luteal phase (p = 0.016 and p = 0.032, respectively) and low PI3K activity compared with the late follicular phase (p = 0.036), whereas the late luteal phase showed low activity of HH and Wnt compared with the early follicular phase (both p = 0.016). Signal transduction pathway activity in fimbrial epithelium from postmenopausal patients was most similar to the early follicular and/or late luteal phase with regard to the AR, ER and PI3K pathways. Wnt pathway activity in postmenopausal patients was comparable to the late follicular and early luteal phase. We observed no differences in HH and TGF-ß pathway activity between pre- and postmenopausal samples. The cyclic changes in signal transduction pathway activity suggest a stage-specific function which may affect the morphology and physiology of the human fallopian tube. CONCLUSIONS: We demonstrated cyclic changes in activity of the AR, ER, PI3K, HH and Wnt pathways throughout the hormonal cycle.

Survival Is Related to Estrogen Signal Transduction Pathway Activity in Postmenopausal Women Diagnosed with High-Grade Serous Ovarian Carcinoma.

High-grade serous ovarian carcinoma (HGSC), the most common subtype of ovarian cancer, has a high mortality rate. Although there are some factors associated with survival, such as stage of disease, there are remarkable differences in survival among women diagnosed with advanced stage disease. In this study, we investigate possible relations between survival and signal transduction pathway (STP) activity. We assessed the functional activity of the androgen receptor (AR), estrogen receptor (ER), phosphoinositide-3-kinase (PI3K), Hedgehog (HH), transforming growth factor beta (TGF-ß) and canonical wingless-type MMTV integration site (Wnt) pathway in 85 primary tumor samples of patients with FIGO stage IIIC to IVB HGSC and disease-free survival (DFS) below 12 (n = 52) or over 24 months (n = 33). There were no significant differences in median pathway activity between patients with a short and long DFS. In univariate Cox proportional hazards analysis, ER pathway activity was related to a favorable DFS and overall survival (OS) in postmenopausal women (p = 0.033 and p = 0.041, respectively), but not in premenopausal women. We divided the postmenopausal group into subgroups based on ER pathway activity quartiles. Survival analysis revealed that postmenopausal women in the lowest ER quartile had a shorter DFS and OS (log-rank p = 0.006 and p < 0.001, respectively). Furthermore, we were able to form subgroups of patients based on an inverse relation between ER and PI3K pathway activity. In conclusion, in postmenopausal patients with advanced stage HGSC, a poorer survival outcome was associated with low functional ER pathway activity.

The effectiveness of monotherapy with PI3K/AKT/mTOR pathway inhibitors in ovarian cancer: A meta-analysis.

OBJECTIVE: To determine the clinical benefit of monotherapy with PI3K/AKT/mTOR inhibitors in patients diagnosed with advanced or recurrent ovarian cancer and to investigate the predictive value of current PI3K/AKT/mTOR biomarkers on therapy response. METHODS: A systematic search was conducted in PubMed, Embase and the Cochrane Library for articles reporting on treatment with PI3K/AKT/mTOR inhibitors in ovarian cancer. The primary endpoint was defined as the clinical benefit rate (CBR), including the proportion of patients with complete (CR) and partial response (PR) and stable disease (SD). Secondary endpoints included the overall response rate (ORR, including CR and PR) and drug-related grade 3 and 4 adverse events. RESULTS: We included 233 patients from 19 studies and observed a pooled CBR of 32% (95% CI 20-44%) and ORR of 3% (95% CI 0-6%) in advanced or recurrent ovarian cancer patients treated with PI3K/AKT/mTOR inhibitors. Subgroup analysis tended to favor the studies who selected patients based on current PI3K/AKT/mTOR biomarker criteria (e.g. genomic alterations or loss of PTEN protein expression), but the difference in CBR was not statistically significant from studies with unselected populations (respectively, CBR of 42% (95% CI 23-62%) and 27% (95% CI 14-42%), P = 0.217). To better reflect true patient benefit, we excluded SD <6 months as a beneficial outcome which resulted in a pooled CBR of 7% (95% CI 2-13%). The overall proportion of patients with drug-related grade 3 and 4 adverse events was 36%. CONCLUSIONS: The efficacy of monotherapy with PI3K/AKT/mTOR inhibitors in advanced recurrent ovarian cancer patients is limited to a small subgroup and selection of patients with the use of current biomarkers did not improved the CBR significantly. Given the toxicity profile, we suggest that current treatment with PI3K/AKT/mTOR inhibitors should not be initiated unless in clinical trials. Furthermore, improved biomarkers to measure functional PI3K/AKT/mTOR pathway activity are needed to optimize patient selection.

Functional estrogen receptor signaling pathway activity in high-grade serous ovarian carcinoma as compared to estrogen receptor protein expression by immunohistochemistry.

PURPOSE: Anti-estrogen therapy may be used as a palliative treatment option in high-grade serous ovarian carcinomas (HGSC). However, clinical implementation is limited as the use of estrogen receptor (ER) protein expression by immunohistochemistry remains insufficient in predicting therapy response. To determine the accuracy of ER protein expression as a marker for ER signaling pathway activity, we aimed to correlate ER protein expression to functional ER signaling pathway activity in HGSC. METHODS: Immunohistochemical ER protein expression was visually scored using total percentages of stained tumor cells and histoscores. Subsequently, mRNA was extracted, and RT-qPCR analysis was performed. Functional ER pathway activity was assessed by a computational Bayesian model inferring ER signaling pathway activity from mRNA levels of ER-specific target genes. RESULTS: Our analysis of 29 HGSCs shows that neither total percentage of ER protein expression, nor ER histoscores are significantly correlated to ER signaling pathway activity (respectively, p = 0.473 and p = 0.606). Classification of HGSC into three groups based on ER histoscores 0-100 (n = 6), 101-200 (n = 15) and 201-300 (n = 8) resulted in comparable mean ER signaling pathway activity among the groups (p = 0.356). Several samples in the higher ER histoscore groups had low ER signaling pathway activity, indicating that nuclear ER protein expression is not sufficient to describe transcriptional ER activation. CONCLUSION: Positive immunohistochemical ER staining is not always indicative of an active ER signaling pathway and is, therefore, a poor predictor of anti-estrogen response. Further research is needed to prove the predictive value of ER signaling pathway activity regarding anti-estrogen sensitivity in HGSC patients.

Signal Transduction Pathway Activity in High-Grade, Serous Ovarian Carcinoma Reveals a More Favorable Prognosis in Tumors with Low PI3K and High NF-κB Pathway Activity: A Novel Approach to a Long-Standing Enigma.

We investigated signal transduction pathway (STP) activity in high-grade serous ovarian carcinoma (HGSC) in relation to progression-free survival (PFS) and overall survival (OS). We made use of signal transduction pathway activity analysis (STA analysis), a novel method to quantify functional STP activity. Activity of the following pathways was measured: androgen receptor (AR), estrogen receptor (ER), phosphoinositide 3-kinase (PI3K), Hedgehog (Hh), Notch, nuclear factor-kappa B (NF-κB), transforming growth factor beta (TGF-ß), and Wnt. We selected HGSC samples from publicly available datasets of ovarian cancer tissue, and used repeated k-means clustering to identify pathway activity clusters. PFS and OS of the clusters were analyzed. We used a subset of publicly available dataset GSE9891 (n = 140), where repeated k-means clustering based on PI3K and NF-κB pathway activity in HGSC samples resulted in two stable clusters. The cluster with low PI3K and high NF-κB pathway activity (n = 72) had a more favorable prognosis for both PFS (p = 0.004) and OS (p = 0.001) compared to the high-PI3K and low-NF-κB pathway activity cluster (n = 68). The low PI3K and high NF-κB pathway activity of the favorable prognosis cluster may indicate a more active immune response, while the high PI3K and low NF-κB pathway activity of the unfavorable prognosis cluster may indicate high cell division.