Somatic Mutation Profiling of PI3K Pathway in Uterine Carcinosarcoma/ Malignant Mixed Mullerian Tumors
Introduction
Uterine carcinosarcoma (formerly called malignant mixed mullerian tumor) is a rare tumor of the gynecologic tract. It is classified as a mixed epithelial and mesenchymal tumor of the uterus in the 2003 World Health Organization classification. It accounts for <5% of uterine malignancies and typically arises in the uterine corpus or in the cervix.1 Rare cases arising in the ovary and fallopian tube have also been described.2,3 It is an aggressive neoplasm that has an extremely poor prognosis: 5-year survival is about 35%. Therapeutic options are limited and no specific targeted therapy is available.
These unusual tumors are a mixture of carcinoma (cancer of epithelial tissue) and sarcoma (cancer of mesenchymal /connective tissue). Targeted therapies aim to exploit known mutations specific to an individual patient’s tumor, rather than a generic targeting of rapidly dividing cells as it done by traditional chemotherapies.
Who gets uterine carcinosarcoma?
Uterine carcinosarcoma affect postmenopausal women, with a higher incidence among African American women. Although it is a rare tumor with an incidence of 2 per 100,000 women per year, it accounts for a disproportionate percentage of uterine cancer-associated deaths. Risk factors associated with this disease are similar to those encountered in endometrial carcinoma, and include obesity and prolonged estrogen exposure. Some cases have been associated with prior radiotherapy.4
How do patient's present with this disease?
The clinical symptoms are essentially non-specific. The most common symptom is abnormal vaginal bleeding, or a bloody or watery discharge. Some patients may present with a mass or a bulky tumor which protrudes through the uterine cervix into the vagina. In addition to this, patients may have non-specific systemic symptoms such as fatigue. These are aggressive neoplasms and in 60% of patients the disease will have extended beyond the confines of the uterus at the time of presentation.
How is uterine carcinosarcoma diagnosed?
Once patients present with such symptoms they will undergo a uterine biopsy, and a pathologist will review the biopsy. Occasionally both components may not be sampled on biopsy and the final diagnosis will be rendered on the hysterectomy specimen. These are biphasic tumors showing an admixture of high grade carcinoma and sarcoma, although the exact volume and subtype of each component will vary (see Figures 1 & 2). The epithelial component may be endometrioid, serous and rarely clear cell or undifferentiated carcinoma. The sarcomatous component may be either homologous or heterologous. Homologous refers to the fact that the sarcomatous component is one which is native to the gynecologic tract and these typically include undifferentiated sarcoma, fibrosarcoma or leiomyosarcoma. Heterologous mesenchymal elements include rhabdomyosarcoma and chondrosarcoma with rare cases of liposarcoma, osteosarcoma and neurectodermal differentiation having been reported.4
Classification of Uterine Carcinoma
There are several different subtypes of endometrial cancer which include endometrioid carcinoma, serous carcinoma and clear cell carcinoma. Often endometrioid carcinoma is referred to as type I uterine cancer, and serous and clear cell carcinoma are referred to as type II.
Does the Type I Type II designation work?
The Type I and II designations describe loose ‘clinico-pathological clusters’ rather than firm diagnostic categories. Not all tumors fit neatly into this dualistic model; we have previously shown overlap between these categories.5 We performed clustering analysis of biomarkers on a cohort of endometrial cancers and demonstrated three prognostically-distinct groups. Cluster I consists predominantly of ER-positive low-grade endometrioid carcinomas and corresponds to Type I endometrial carcinoma. Cluster III consists predominantly of high-grade serous, clear cell, and mixed (serous and endometrioid) carcinomas, with a smaller number of high-grade endometrioid carcinomas; it has molecular features associated with Type II endometrial carcinoma and has the least favorable outcome. Cluster II included ER-positive tumors that showed p53, p21, and p27 expression. This group consists mainly of grades 2 and 3 endometrioid carcinomas, with a lesser number of mixed tumors and occasional serous and grade 1 endometrioid carcinomas. Cluster II prognosis was intermediate and, based on their immunoprofiles, these may represent Type I tumors that have progressed and become more aggressive over time. Carcinosarcomas are similar to Type II endometrial carcinoma (the most aggressive form) in many respects.
How do we treat patients with uterine carcinosarcoma?
These patients will be managed with complete surgical staging undergoing a total abdominal hysterectomy, bilateral salpingo-oophrectomy, pelvic and para-aortic lymphadenectomy, omentectomy biopsies of peritoneal surfaces and tumor debulking.1 If the tumor has spread widely at the time of diagnosis, surgical excision may not be possible. In addition to surgery, adjuvant radiation therapy and chemotherapy may be used. Radiation therapy reduces local pelvic recurrence but it is unclear whether there is improvement in overall survival.6,7,8 9 Since many of these patients will have disseminated disease and recur in a metastatic site, chemotherapy is used but there is uncertainty as to the best treatment.10,11,12,13 Multimodality therapy is often used with patients receiving both chemotherapy and radiation therapy.
The Clinical Problem
Despite extensive surgery and multimodality adjuvant therapy, these tumors have an extremely poor prognosis: 5-year survival is about 35%. There is no standard second line therapy and no specific targeted therapy is available. Traditional chemotherapies also have multiple side effects since they are targeting dividing cells, including normal cells. It is hoped that targeted therapeutics would be more effective in treating the tumor and cause less side effects. Given the limitations of surgery and radiotherapy, the only realistic hope for improving outcomes is improving chemotherapy.
Difficulties in Conducting Uterine Carcinosarcoma Research
Given the relatively low frequency of this disease within the population, intelligent trial design and drug selection is crucial when seeking to improve the outcome for women with this disease. Obtaining additional biological information that can guide selection of agents for investigation in this underserviced patient population is essential.
The Origin of Uterine Carcinosarcoma
A number of theories as to the cell of origin of such tumors has been proposed.14 The collision theory posits that these are biclonal synchronous tumors arising from separate tumour-initiating cells (TICs) that subsequently merge. The conversion theory posits that these are monoclonal tumors that arise from a single primitive TIC which undergoes divergent or metaplastic differentiation.
Molecular evidence suggests that most uterine carcinosarcomas are monoclonal.15, 16, 17 Accordingly, supported by molecular, epidemiologic, genetic, and histologic data, uterine carcinosarcomas are now regarded as high-grade carcinomas with sarcomatous/stromal differentiation, similar to other organ systems.18, 19 This is reflected in the use of platinum based treatment regimens similar to those used for uterine carcinomas.
Why is it so interesting that uterine carcinosarcoma may be a uterine carcinoma?
If uterine carcinosarcomas are closely related to high-grade endometrial carcinomas, insight into the molecular pathogenesis of the latter can be exploited to identify therapeutic targets in the former. Knowledge of such targets provides an opportunity for a directed but comprehensive analysis of the genetics of carcinosarcomas with the possibility of identifying similar targets.
So what can we learn from endometrial cancer?
We are developing a better understanding of the molecular development of endometrial cancer. Increasingly both type I and type II endometrial carcinoma are being found to have genetic alterations affecting the AKT/PI3K pathway, a pathway which may be targeted by therapeutic agents.20-25
Targeting the PI3K Pathway
Deletion of Phosphatase and tensin homologue on chromosome 10 (PTEN) is the most common genetic defect in endometrial cancer and is seen in up to 83% of tumours.26-28 PTEN is part of the PI3k/AKT pathway which is an important signaling pathway in cancers. mTOR is a kinase which functions downstream of PI3K/Akt and inhibition of this is seen as an important therapeutic target in cancers.29-31 Loss of PTEN causes deregulated PI3K/Akt/mTOR signalling which may provide neoplastic cells with a selective survival advantage by enhancing angiogenesis, protein translation and cell cycle progression.
Somatic mutations in uterine carcinosarcoma
Mutations of PIK3CA have recently been found in 36-39% of uterine endometrioid carcinomas and 15% of serous carcinomas.32, 33 Such mutations would collude with PTEN in promoting tumor. Other genetic alterations in endometrial cancer which would affect this pathway include K-ras mutations and these are found in 10-30% of type I endometrial cancers.34, 35
Thus targeting the PI3K/MTOR pathway alone and in combination in endometrial cancer is being actively exploited as a treatment strategy in this disease.36
Clinical Trials of Targeted Therapies in Endometrial Cancer
A recent clinical trial conducted by the National Cancer Institute of Canada (NCIC) investigating the MTOR inhibitor Temsirolimus, showed promising single agent activity in women with advanced and metastatic endometrial cancer.37 Deforolimus, is an intravenous mTOR inhibitor and in a phase II trial for women with uterine malignancies (endometrial cancer and uterine carcinosarcoma); 28% of patients demonstrated either a complete or partial response to this drug.38 Thus mTOR inhibitors do exhibit activity in women with advanced or recurrent endometrial cancer and carcinosarcoma.39-46
Using Sequenom Mass ARRAY System to Analyze the PI3K Pathway in Uterine Carcinosarcoma
New sequencing and profiling technologies are allowing us to unlock the paraffin archive of tumor samples stored in hospital pathology departments.47 One such technology is Sequenom matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) MassArray system.
The "pathology archive" is a term used to refer to the countless tissue samples which have been used for diagnostic purposes and are stored in pathology laboratories. Such samples are fixed in formalin and stored in paraffin blocks, hence they are sometimes referred to as the "paraffin archive." DNA in such samples tends to degrade, making it suboptimal for genetic analysis. New technologies such as the Sequenom Mass ARRAY system are able to overcome this and are able to analyze small quantities of degraded DNA derived from such archives. This allows analysis of some tumor types, by looking at samples collected over many years.
Sequenom MassARRAY: This system assays 20 different nucleotide variants (NVs) per well in a 384 well format. The MassARRAY can detect a NV even if it accounts for only 5% of the DNA. The Sequenom MassArray requires very little DNA, and can utilize degraded DNA like that obtained from formalin-fixed, paraffin-embedded tissues.
In this project we will be analyzing mutation profile of the genes involved in the PI3K pathway in an annotated cohort of 70 uterine carcinosarcomas.
Specific genes to be targeted: We will perform comprehensive genomic mutation analysis of the PI3K/Akt/mTOR pathway and other genes implicated in endometrial carcinogenesis using a customized Sequenom panel. The panel will include hot-spot mutations in members of the PI3k pathway such as AKT1, AKT2, AKT3, PDPK1, Rictor, FRAP, PHLPP2, PIK3R1 and PIK3CA. Other genes to be analysed include ALK, BRAK, CDK4, CTNNB1, EGFR, ERα, GNAS, HIF1A, IDH1, IGF1R, JAK2, KIT, KRAS, MEK1, MET, NRAS, PDGFR, RET, TNK, and STK11.
Significance
Uterine carcinosarcomas have poor prognosis and there are limited therapeutic options. This study will allow assessment of potential therapeutic targets in these cancers. Two recent publications provide strongly supportive data for this hypothesis. First, a group at Harvard has identified PIK3CA mutations in 19% of gynaecologic carcinosarcomas (uterine, ovarian, fallopian tube and vaginal) with mutations in the phosphatidylinositol 3-kinase pathway predominating in the uterine carcinosarcomas.48 Second, another group has identified a 20% rate of PIK3CA mutations in uterine MMMTs using Sequenom technology.49 This study will build on these observations by a comprehensive analysis of the PI3K pathway. If successful this will allow design of rational clinical trials for new treatments designed to target those abnormalities. In addition the study will contribute fundamental insights into the genetic alterations of these understudied tumors.
Grant Funding
The Liddy Shriver Sarcoma Initiative announced the funding of this $50,000 grant in June 2011. The study was made possible by a generous donation from Dr. Laura Somerville, by generous donations made in memory of Adrianne Pfeiffer (through several MMMT awareness events organized by her husband, Robert Pfeiffer), and by generous donations made in memory of Mary Anne Voeglie.