PIK3CA Amplification Associates with Aggressive Phenotype but Not Markers of AKT-MTOR Signaling in Endometrial Carcinoma.

PURPOSE: Amplification of PIK3CA, encoding the PI3K catalytic subunit alpha, is common in uterine corpus endometrial carcinoma (UCEC) and linked to an aggressive phenotype. However, it is unclear whether PIK3CA amplification acts via PI3K activation. We investigated the association between PIK3CA amplification, markers of PI3K activity, and prognosis in a large cohort of UCEC specimens. EXPERIMENTAL DESIGN: UCECs from 591 clinically annotated patients including 83 tumors with matching metastasis (n = 188) were analyzed by FISH to determine PIK3CA copy-number status. These data were integrated with mRNA and protein expression and clinicopathologic data. Results were verified in The Cancer Genome Atlas dataset. RESULTS: PIK3CA amplifications were associated with disease-specific mortality and with other markers of aggressive disease. PIK3CA amplifications were also associated with other amplifications characteristic of the serous-like somatic copy-number alteration (SCNA)-high subgroup of UCEC. Tumors with PIK3CA amplification also demonstrated an increase in phospho-p70S6K but had decreased levels of activated phospho-AKT1-3 as assessed by Reverse Phase Protein Arrays and an mRNA signature of MTOR inhibition. CONCLUSIONS: PIK3CA amplification is a strong prognostic marker and a potential marker for the aggressive SCNA-high subgroup of UCEC. Although PIK3CA amplification associates with some surrogate measures of increased PI3K activity, markers for AKT1-3 and MTOR signaling are decreased, suggesting that this signaling is not a predominant pathway to promote cancer growth of aggressive serous-like UCEC. Moreover, these associations may reflect features of the SCNA-high subgroup of UCEC rather than effects of PIK3CA amplification itself.

PIK3CA exon9 mutations associate with reduced survival, and are highly concordant between matching primary tumors and metastases in endometrial cancer.

Mutations of the phosphoinositide-3-kinase (PI3K) catalytic subunit alpha gene (PIK3CA) are frequent in endometrial cancer. We sequenced exon9 and exon20 of PIK3CA in 280 primary endometrial cancers to assess the relationship with clinicopathologic variables, patient survival and associations with PIK3CA mRNA and phospho-AKT1 by gene expression and protein data, respectively. While PIK3CA mutations generally had no impact on survival, and were not associated with clinicopathological variables, patients with exon9 charge-changing mutations, providing a positive charge at the substituted amino acid residue, were associated with poor survival (p = 0.018). Furthermore, we characterized PIK3CA mutations in the metastatic setting, including 32 patients with matched primary tumors and metastases, and found a high level of concordance (85.7%; 6 out of 7 patients), suggesting limited heterogeneity. PIK3CA mRNA levels were increased in metastases compared to the primary tumors (p = 0.031), independent of PIK3CA mutation status, which rather associated with reduced PIK3CA mRNA expression. PIK3CA mutated tumors expressed higher p-AKT/AKT protein levels, both within primary (p < 0.001) and metastatic lesion (p = 0.010). Our results support the notion that the PI3K signaling pathway might be activated, both dependent- and independently of PIK3CA mutations, an aspect that should be considered when designing PIK3 pathway targeting strategies in endometrial cancer.

Molecular profiling of endometrial carcinoma precursor, primary and metastatic lesions suggests different targets for treatment in obese compared to non-obese patients.

Obesity is linked to increased incidence of endometrioid endometrial cancer (EEC) and complex atypical hyperplasia (CAH). We here explore pattern and sequence of molecular alterations characterizing endometrial carcinogenesis in general and related to body mass index (BMI), to improve diagnostic stratification and treatment strategies. We performed molecular characterization of 729 prospectively collected EEC and CAH. Candidate biomarkers were identified in frozen samples by whole-exome and Sanger sequencing, oligonucleotide gene expression and Reverse Phase Protein Arrays (investigation cohort) and further explored in formalin fixed tissues by immunohistochemistry and Fluorescent in Situ Hybridization (validation cohort). We here demonstrate that PIK3CA mutations, PTEN loss, PI3K and KRAS activation are early events in endometrial carcinogenesis. Molecular changes related to KRAS activation and inflammation are more common in obese CAH patients, suggesting different prevention and systemic treatment strategies in obese and non-obese patients. We also found that oncoprotein Stathmin might improve preoperative diagnostic distinction between premalignant and malignant endometrial lesions.

High phospho-Stathmin(Serine38) expression identifies aggressive endometrial cancer and suggests an association with PI3K inhibition.

PURPOSE: High Stathmin expression has recently been associated with clinical progress in endometrial cancers. Stathmin protein activity is modulated by phosphorylation, and the Serine38 site is one of four Stathmin phospho-sites. The presence and significance of pStathmin(S38) is largely unknown in human cancers, and we here examined the associations between this marker and tumor cell proliferation, clinicopathologic phenotype, and survival impact in endometrial cancer. A relationship with possible treatment targets was explored by integrated analysis of transcriptional alterations. EXPERIMENTAL DESIGN: Primary endometrial cancers from two independent patient series (n = 518/n = 286) were analyzed. Biomarkers were assessed by immunohistochemistry, FISH, flow cytometry, DNA oligonucleotide microarray, single-nucleotide polymorphism array, and Sanger sequencing, and related to clinicopathologic annotations and follow-up information. RESULTS: High pStathmin(S38) level was associated with poor prognosis, independent of other features, and correlated to increased tumor cell proliferation as well as high Stathmin levels. On the basis of transcriptional differences between high/low pStathmin(S38) tumors, phosphoinositide 3-kinase (PI3K)/mTOR/HSP90 were suggested as possible targets in pStathmin(S38)-high cases. High pStathmin(S38) was associated with several PI3K pathway alterations: amplification of the 3q26 region, increased PIK3CA copy number (FISH) and a PI3K activation score (all P < 0.05). CONCLUSIONS: High pStathmin(S38) is a novel biomarker of increased tumor cell proliferation and impaired prognosis as reported here for independent cohorts of endometrial cancer and not previously shown in human cancer. Our data support a rationale for further studies exploring effects of drugs inhibiting the PI3K signaling pathway in pStathmin(S38)-high endometrial cancer, including a potential value of pStathmin(S38) in predicting response to PI3K/mTOR/HSP90 inhibitors.

Lack of estrogen receptor-α is associated with epithelial-mesenchymal transition and PI3K alterations in endometrial carcinoma.

PURPOSE: We hypothesized that estrogen receptor-α (ER-α) status in endometrial carcinomas, associated with poor prognosis, is reflected in transcriptional signatures suggesting targets for new therapy. EXPERIMENTAL DESIGN: Endometrial carcinoma samples in a primary investigation cohort (n = 76) and three independent validation cohorts (n = 155/286/111) were analyzed through integrated molecular profiling. Biomarkers were assessed by immunohistochemistry (IHC), DNA oligonucleotide microarray, quantitative PCR (qPCR), single-nucleotide polymorphism (SNP) array, and Sanger sequencing in the cohorts, annotated for comprehensive histopathologic and clinical data, including follow-up. RESULTS: ER-α immunohistochemical staining was strongly associated with mRNA expression of the receptor gene (ESR1) and patient survival (both P < 0.001). ER-α negativity associated with activation of genes involved in Wnt-, Sonic Hedgehog-, and TGF-ß signaling in the investigation cohort, indicating epithelial-mesenchymal transition (EMT). The association between low ER-α and EMT was validated in three independent datasets. Furthermore, phosphoinositide 3-kinase (PI3K) and mTOR inhibitors were among the top-ranked drug signatures negatively correlated with the ER-α-negative tumors. Low ER-α was significantly associated with PIK3CA amplifications but not mutations. Also, low ER-α was correlated to high expression of Stathmin, a marker associated with PTEN loss, and a high PI3K activation signature. CONCLUSION: Lack of ER-α in endometrial cancer is associated with EMT and reduced survival. We present a rationale for investigating ER-α's potential to predict response to PI3K/mTOR inhibitors in clinical trials and also suggest EMT inhibitors to ER-α-negative endometrial carcinomas.

High-throughput mutation profiling of primary and metastatic endometrial cancers identifies KRAS, FGFR2 and PIK3CA to be frequently mutated.

BACKGROUND: Despite being the most common pelvic gynecologic malignancy in industrialized countries, no targeted therapies are available for patients with metastatic endometrial carcinoma. In order to improve treatment, underlying molecular characteristics of primary and metastatic disease must be explored. METHODOLOGY/PRINCIPAL FINDINGS: We utilized the mass spectrometric-based mutation detection technology OncoMap to define the types and frequency of point somatic mutations in endometrial cancer. 67 primary tumors, 15 metastases corresponding to 7 of the included primary tumors and 11 endometrial cancer cell lines were screened for point mutations in 28 known oncogenes. We found that 27 (40.3%) of 67 primary tumors harbored one or more mutations with no increase in metastatic lesions. FGFR2, KRAS and PIK3CA were consistently the most frequently mutated genes in primary tumors, metastatic lesions and cell lines. CONCLUSIONS/SIGNIFICANCE: Our results emphasize the potential for targeting FGFR2, KRAS and PIK3CA mutations in endometrial cancer for development of novel therapeutic strategies.