Chapter 9 SUMMARIZING DISCUSSION AND FUTURE PERSPECTIVES WELMOED REITSMA

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1 Chapter 9 SUMMARIZING DISCUSSION AND FUTURE PERSPECTIVES WELMOED REITSMA 163

2 CHAPTER 9 SUMMARIZING DISCUSSION Epithelial ovarian cancer is the second most common gynecological malignancy and the deadliest in terms of absolute figure. In 2010, 1400 patients were diagnosed and 1065 patients died of epithelial ovarian cancer in the Netherlands (Dutch Cancer Registry). The disease is often diagnosed in an advanced stage, resulting in an overall five-year survival of 30%. Although there are many subtypes of epithelial ovarian cancer, high-grade serous cancer is the most frequently diagnosed histological type, which corresponds to 75% of all cases and 90% of all deaths due to epithelial ovarian cancer. 1, 2 Epithelial ovarian cancer with a high-grade serous histology is currently known as pelvic high-grade serous cancer (PHGSC). Although in PHGSC the bulk of the tumor is often located in the ovary, there is accumulating evidence that the majority (if not all) of the PHGSC cases originates from (malignant) epithelium of the tubal fimbriae instead of the ovary. 3-5 Women with a mutation in BReast CAncer susceptibility gene (BRCA)1 6 or BRCA2 7 have an increased lifetime risk of 18% to 54% and 2.4 to 23% respectively, at the age of 70. 8, 9 BRCA1/2 mutation carriers account for up to 24% of the PHGSC patients. 10 To reduce their PHGSC risk, BRCA1/2 mutation carriers are advised to undergo risk-reducing salpingooophorectomy (RRSO), as gynecological screening has yet proven to be ineffective in reducing ovarian cancer-specific mortality. 11 If RRSO is performed at a young age, before the epithelial ovarian cancer incidence rises, RRSO has been estimated to be effective in over 96% for BRCA-associated PHGSC (HR 0.21; 95%-CI ). 12 Although effective in preventing PHGSC, RRSO has several physical and psychological consequences following surgery due to the iatrogenic induced menopause The knowledge about early development of PHGSC is still incomplete, since most PHGSCs are detected at a late stage and precursor lesions are not recognizable anymore or are overgrown with tumor. Research on prophylactically removed ovaries and fallopian tubes gives more insight into prevalence of (pre)invasive PHGSC and early development of the disease. Also investigating tumor-induced angiogenesis can provide insight in early development of PHGSC. Research should focus on finding sensitive enough screening and detection methods that accurately detect PHGSC in its early (pre-invasive) stage, which makes RRSO unnecessary. MicroRNAs (mirnas) are short non-coding RNA molecules that have been recognized as important regulators of gene expression and deregulation of mirnas has been functionally linked to the development of cancer. The purpose of this thesis was to gain more insight in the tumor and survival characteristics of epithelial ovarian cancer in women with a BRCA1 or BRCA2 mutation, and in the prevalence and early development of PHGSC in BRCA1 and BRCA2 mutation carriers. A general introduction on PHGSC is provided in chapter 1. This chapter emphasizes the incidence of PHGSC, outlines the fallopian tube hypothesis which assumes that the fallopian tube is the most probable primary site of origin, illustrates the background of early development and highlights the need for new biomarkers for potential early diagnosis. 164

3 SUMMARIZING DISCUSSION AND FUTURE PERSPECTIVES In Part I we described the clinicopathological characteristics and survival of epithelial ovarian cancer patients with a BRCA1 or a BRCA2 mutation. In both studies, all histological subtypes of epithelial ovarian cancer were included, and not only the high-grade serous cancers. Therefore chapter 2 and 3 refer to epithelial ovarian cancer instead of PHGSC. Previous studies showed that epithelial ovarian cancer in BRCA1 and BRCA2 mutation carriers differs from sporadic cases: BRCA-associated epithelial ovarian cancers are often diagnosed in a more advanced stage, respond better to platinum-based chemotherapy, and show a better overall survival when compared to sporadic cases. However, the distinct disease penetrance between BRCA1 and BRCA2 epithelial ovarian cancer and the histopathological differences between BRCA1- and BRCA2-associated breast cancer suggest that there might be clinicopathological and/or survival differences in epithelial ovarian cancer between BRCA1 and BRCA2 patients. Therefore, possible differences in clinicopathological characteristics and survival between BRCA1- and BRCA2-associated epithelial ovarian cancers gained our interest. We conducted a prospective cohort study of a consecutive in-house series of 55 BRCA1- and 16 BRCA2-associated epithelial ovarian cancers, as described in chapter 2. In these small series, no significant clinicopathological differences were found between both mutational groups, except for lower age at diagnosis and a higher prevalence of peritoneal cancer in BRCA1 patients. Regarding survival, a non-significant trend was noted for BRCA2 patients having fewer relapses, a longer time to first relapse, and a longer disease-free and overall survival, which might suggest that BRCA2 mutation carriers with epithelial ovarian cancer have a more favorable outcome than BRCA1 mutation carriers. This beneficial trend in survival for BRCA2- associated epithelial ovarian cancer patients was not significant, indicating that our sample might has been too small to demonstrate such differences. To determine whether there is an actual survival benefit for BRCA2 over BRCA1 patients, a nationwide study was performed in which the outcome of BRCA2- was compared with BRCA1-associated epithelial ovarian cancer (chapter 3). In this large cohort of epithelial ovarian cancer patients we further explored clinicopathological characteristics, progression-free survival, treatment-free interval, and overall survival of BRCA1 compared to BRCA2 mutation carriers. Additionally, response to first-line chemotherapy was investigated to assess if a possible survival difference might be explained by a difference in treatment response. Complete response to primary therapy, including chemotherapy, was observed in 86% of BRCA1 and 90% of BRCA2 patients. Progression-free survival was significantly longer in BRCA2- (2.2 years (95%-CI ) compared to BRCA1-associated epithelial ovarian cancer patients (3.9 years (95%-CI ; P=0.006). Also overall survival was significantly improved in BRCA2 (9.7 years (95%-CI ) compared to BRCA1 patients (6.0 years (95%-CI ; P=0.04). However, response to primary therapy was similarly high in both groups and differences could not be explained by age at diagnosis, FIGO-stage or type of treatment. From Part I of this thesis it can be concluded that there is a survival benefit for BRCA2-associated epithelial ovarian cancer patients over BRCA1-patients, which might be explained by a longer duration of response to chemotherapy. We postulate that DNA repair after chemotherapy might be less efficient in BRCA2 compared to BRCA1 mutation carriers. 165

4 CHAPTER 9 In Part II we aimed to obtain more insight in the primary site of origin of PHGSC. We concentrated on pelvic cancer of high-grade serous histology, since this is the most frequently diagnosed subtype in BRCA1 and BRCA2 mutation carriers. 10, 16, 17 In addition, PHGSC corresponds to 75% of all epithelial ovarian cancer cases and 90% of all deaths due to ovarian cancer. 1, 2 The genesis of PHGSC is not fully elucidated. Despite extensive clinical, histopathological and fundamental research, the cell of origin of PHGSC remains subject of debate. This is partly due to the fact that most epithelial ovarian cancers are diagnosed in an advanced stage, in which the tumor has spread through the abdomen. Both the ovaries and fallopian tubes and other pelvic organs are then involved in the tumor process and a precursor lesion responsible for the disease cannot be found anymore. Furthermore, it is not possible to differentiate between primary ovarian and tubal cancer under the microscope, since their morphology is practically identical. To make it even more complex, also uterine papillary serous cancer shares histopathological, genetic, and clinical features with PHGSC. Hence, some researchers raised the hypothesis that uterine papillary serous cancer might also be associated with BRCA1 and BRCA2 mutations and that serous endometrial intraepithelial cancer is the candidate precursor of PHGSC. Consequently, an increased incidence of uterine papillary serous cancers would be expected in BRCA1 and BRCA2 mutation carriers. To further investigate this, we performed a study on the occurrence of uterine papillary serous cancer, as well as other histological types of endometrial cancer, following RRSO in women with a BRCA1 or BRCA2 mutation (chapter 4). A total number of 345 women with a BRCA1 or BRCA2 mutation were followed after RRSO without hysterectomy, and after a median follow-up period of 6 years (total: 2062 women years) the incidence of endometrial cancer following RRSO in those women was not increased (Standardized Incidence Ratio 2.13; 95%-CI ; P=0.27). Therefore, the hypothesis of serous endometrial intraepithelial carcinoma being an important precursor lesion of PHGSC seems unlikely. The most supported theory about the possible origin of PHGSC appoints the fimbrial ends of the fallopian tube as tissue of origin, least because a possible (pre-invasive) precursor lesion has for the first time been identified. Serous tubal intraepithelial cancers (STICs) have been detected in the distal fallopian tube as the earliest morphologic manifestation of highgrade serous cancer discovered so far, and are assumed to subsequently spread to the pelvic organs. Chapter 5 describes a prospective study in asymptomatic, screen-negative women who underwent RRSO. The prevalence, localization and type of occult (non-)invasive PHGSC were determined in the RRSO-specimens of 188 BRCA1 and 115 BRCA2 mutation carriers. Also RRSO-specimens of 57 women with a suggestive family history of breast and/or PHGSC, but who were tested negative for BRCA mutations, were included. A series of 360 RRSO specimens of these high-risk women underwent extensive histopathologic examination. Only asymptomatic, screen-negative women were included, to make an unbiased estimate of the prevalence of early PHGSC. When compared to other studies, a low prevalence of occult invasive cancer (1.1%) was found in these high-risk women. Four occult invasive cancers were detected (1.3%; 95%-CI ), all in BRCA1 mutation carriers >40 years of age. All 166

5 SUMMARIZING DISCUSSION AND FUTURE PERSPECTIVES cancers were FIGO-stage I/II; two cancers were located in the distal fallopian tube and two in the ovary. Three STICs (1.0%; 95%-CI ) and 22 atypical hyperplastic lesions (7.2%; 95%-CI ) were present, only in BRCA mutation carriers. The tubal epithelium was the primary origin in all of the STICs, in all of the atypical hyperplastic lesions and in half of the occult cancers. Chapter 5.1 is a letter commenting to the remarkably high rate of occult cancers in RRSO specimens (9.1%) in a study by Powell et al., compared to literature. We believe that this high rate presented by the authors gives a distorted picture of the actual prevalence, since the authors included both pre-invasive and invasive cancers in their count, which results in a twice as high rate of occult cancer as is really present. But most importantly, they report an ovarian intraepithelial cancer, which would be the ground-breaking news if it were true, for it would be the first reported case to date ever. We seriously doubted whether this diagnosis of a precursor lesion of PHGSC in the ovary was correct, since an ovarian intraepithelial cancer has never been reported in literature before. As a response to our letter, the authors confirmed that the diagnosis of an ovarian intraepithelial cancer was an error. From Part II it can be concluded that the primary site of origin of PHGSC is most probably not located in the ovary or endometrium, but in the distal fallopian tube. Further research is needed to further prove the fallopian tube hypothesis. In Part III we focused on early development of PHGSC and potential future detection methods to accurately detect PHGSC in an early or preinvasive stage. Considerable attention and an enormous amount of resources have been dedicated to cancer biomarker discovery and validation. However, there are still no useful biomarkers available for clinical use sensitive enough to detect PHGSC in an early or premalignant stage. When focusing on early development of PHGSC, it is reasonable to start with studying angiogenesis, which is the formation of new blood vessels. Angiogenesis is necessary for the growth and metastatic spread of solid tumors. Vascular Endothelial Growth Factor (VEGF) is a potent factor involved in tumor-induced angiogenesis, and overexpression of VEGF mediates tumorigenesis. An interaction between the BRCA1 protein and estrogen receptor (ER-)α inhibits the secretion of VEGF in breast cancer patients. This interaction has not been studied in fallopian tube cancer patients so far, and it is unclear if the absence of the BRCA1 protein in BRCA1 mutation carriers influences VEGF expression. Therefore, in chapter 6, we investigated the expression of VEGF in benign en malignant fallopian tubes in 40 tissue samples from BRCA1 mutation carriers in comparison to non-carriers. The expression of VEGF- A was high in both benign and malignant tubal epithelium (80% versus 100%, respectively), and VEGF-A expression was higher in benign fallopian tube tissue of BRCA1 mutation carriers versus non-carriers, although not significantly in this small series. We postulate that this finding could be an early event in the process of carcinogenesis of high-grade serous tubal cancer in BRCA1 mutation carriers. In addition, this finding might be of significance to future studies, investigating the angiogenic process in early development of PHGSC, in BRCA1-associated and/or sporadic cases. Further research is needed to investigate interaction effects between 167

6 CHAPTER 9 the BRCA1 protein, ER-α and VEGF in the development of PHGSC, and future studies may concentrate at the angiogenic process in early development of PHGSC. MiRNAs have reported to be essential regulators of gene expression and deregulation of mirnas has been functionally linked to the development of cancer. Therefore, an overview of aberrantly expressed mirnas in tissue samples of PHGSC compared to normal counterparts (ovarian or tubal tissue) would be informative in establishing a panel of mirnas that is able to distinguish malignant from benign tubal tissue. In chapter 7, a systematic review of the literature is given on the mirnas that are altered in expression in PHGSC, and how these mirna profiles relate to mirna expression in normal ovarian or tubal counterparts. A total of 6 studies met the inclusion criteria and were included, of which 5 studies had used benign control tissue (ovarian or tubal tissue or cell line). In this review it was found that the upregulated mir-200 family, mir-183 family, and the mir-143/145 cluster in PHGSC might play an essential role as tumor suppressor mirnas, whether the downregulated let-7 family and mir-34 may be linked to enhanced levels of oncogenes. Most mirna studies investigating the expression of mirnas in PHGSC relative to normal counterparts choose ovarian tissue or ovarian surface epithelium as a control. Given that a significant proportion of PHGSCs are currently believed to be derived from the tubal epithelium, a comparison should be made between PHGSC and benign fallopian tube epithelium. Chapter 8 deals with the mirna expression profile in PHGSC samples of BRCA1 mutation carriers, in comparison to benign fallopian tube tissue sampled from the fimbriated end of healthy BRCA1 mutation carriers. The aim was to find a panel of candidate mirnas that are differentially expressed between fallopian tube cancer and normal tubes from BRCA1 mutation carriers. Global mirna expression profiling was performed by deep sequencing of small RNA libraries generated from total RNA isolated from a series of 7 formalin-fixed paraffin-embedded (FFPE) PHGSC samples and 9 samples of normal fimbrial fallopian tubes, both from BRCA1 mutation carriers. A set of 133 mirnas was significantly (P<0.01) differentially expressed in PHGSC of BRCA1 mutation carriers compared with benign tubal tissue of BRCA1 mutation carriers, of which 99 up- and 34 downregulated (P<0.01, 1.5-fold). The heatmap of these 133 mirnas showed a clear separation between PHGSC and benign fallopian tube tissue. MiR-205-5p, mir-183-5p, mir-224-5p and mir-363-3p were the four highest upregulated mirnas in PHGSC relative to benign fallopian tube tissue, whereas the mir-34 family, including mir-34c-3p, mir-34b-3p, mir-34b-5p, and mir-34c-5p, were among the most downregulated mirnas. Our results indicate that a large number of mirnas are aberrantly expressed in PHGSC of BRCA1 mutation carriers in comparison to benign fallopian tube tissue of BRCA1 mutation carriers. The preliminary data presented in this study will have to be validated in future studies. 168

7 SUMMARIZING DISCUSSION AND FUTURE PERSPECTIVES FUTURE PERSPECTIVES First, this thesis focuses on tumor and survival characteristics of epithelial ovarian cancer patients with a BRCA1 or BRCA2 mutation. The significant longer progression-free and overall survival in epithelial ovarian cancer patients with a BRCA2 mutation compared to a BRCA1 mutation in our nationwide study, indicate that in upcoming studies concerning the efficacy of new chemotherapeutic strategies in BRCA carriers, patients should be stratified for BRCA1 and BRCA2 mutation status respectively. New studies on molecular biological level are warranted to explain the underlying mechanism of survival differences between BRCA1- and BRCA2-associated epithelial ovarian cancer patients. Secondly, this thesis concerns the prevalence of precursor lesions in RRSO specimens and early development of PHGSC. Whether patients with STICs in RRSO specimens should be considered as having an early malignancy with the consequence of staging and treatment like PHGSC has not been established yet; most clinicians do not act upon that assumption yet. To further find out the prognosis of women with a STIC in their RRSO specimen, a nationwide prospective registration study collecting all clinical and follow-up data on these women should be started in order to find out the malignant potential of STICs and prevent over- and under treatment in the future. Thirdly, this thesis showed that a panel of mirnas is aberrantly expressed in PHGSC in comparison to normal fallopian tube tissue, in BRCA1 mutation carriers. It is necessary to validate these results in a larger series of PHGSC tissue specimens, including a series of STICs as being the early non-invasive stage of PHGSC. If it is possible to establish a panel of mirnas that is able to distinguish STIC from benign tubal epithelium, the next step would be to investigate this mirna panel in serum of women with and without (early stage) PHGSC and STICs. In other words, a mirna panel should be investigated as biomarker profile aiming to detect (precursor lesions of) PHGSC. In BRCA1/2 mutation carriers, the current lack of effective biomarkers and screening tools, has led to RRSO as the only preventive strategy to lower the PHGSC-related mortality. However, RRSO is associated with significant adverse effects, which are caused by early and acute menopause. Since it has not (yet) proven safe to abandon RRSO and replace this by tubectomy only, and moreover since it is not certain that STICs are the only site of origin of PHGSC, sensitive and specific biomarkers would be useful in detecting precursor lesions of PHGSC in these high-risk women. When concentrating on finding new biomarkers it is important to keep in mind that an ideal biomarker is stable and reproducible, should be easily assayed with minimally invasive medical procedures and should possess high sensitivity and specificity. MiRNAs might be potent candidates since we demonstrated a differential expression of mirnas in high-grade serous tubal cancer compared to normal epithelium in BRCA1 mutation carriers. MiRNAs could be an ideal diagnostic biomarker since (a) a small mirna profile can yet be representative for solid tumors, (b) they are stable in serum, (c) they circulate in serum and sampling is relatively non-invasive, (d) and it has been shown that mirna profiles are not only disease-specific but can also be stage-specific. Further research into the significance of mirnas for the early detection of PHGSC is therefore potentially promising. 169

8 CHAPTER 9 REFERENCES 1. Seidman JD, Horkayne-Szakaly I, Haiba M, Boice CR, Kurman RJ, Ronnett BM. The histologic type and stage distribution of ovarian carcinomas of surface epithelial origin. Int J Gynecol Pathol. 2004; 23(1): Cho KR, Shih Ie M. Ovarian cancer. Annu Rev Pathol. 2009; 4: Crum CP, Drapkin R, Kindelberger D, Medeiros F, Miron A, Lee Y. Lessons from BRCA: the tubal fimbria emerges as an origin for pelvic serous cancer. Clin Med Res. 2007; 5(1): Yates MS, Meyer LA, Deavers MT, Daniels MS, Keeler ER, Mok SC, et al. Microscopic and earlystage ovarian cancers in BRCA1/2 mutation carriers: building a model for early BRCA-associated tumorigenesis. Cancer Prev Res (Phila). 2011; 4(3): Kim J, Coffey DM, Creighton CJ, Yu Z, Hawkins SM, Matzuk MM. High-grade serous ovarian cancer arises from fallopian tube in a mouse model. Proc Natl Acad Sci U S A. 2012; 109(10): Hall JM, Lee MK, Newman B, Morrow JE, Anderson LA, Huey B, et al. Linkage of early-onset familial breast cancer to chromosome 17q21. Science. 1990; 250(4988): Miki Y, Swensen J, Shattuck-Eidens D, Futreal PA, Harshman K, Tavtigian S, et al. A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science. 1994; 266(5182): Antoniou A, Pharoah PD, Narod S, Risch HA, Eyfjord JE, Hopper JL, et al. Average risks of breast and ovarian cancer associated with BRCA1 or BRCA2 mutations detected in case Series unselected for family history: a combined analysis of 22 studies. Am J Hum Genet. 2003; 72(5): Chen S, Parmigiani G. Meta-analysis of BRCA1 and BRCA2 penetrance. J Clin Oncol. 2007; 25(11): Risch HA, McLaughlin JR, Cole DE, Rosen B, Bradley L, Kwan E, et al. Prevalence and penetrance of germline BRCA1 and BRCA2 mutations in a population series of 649 women with ovarian cancer. Am J Hum Genet. 2001; 68(3): The Netherlands Foundation for the Detection of Hereditary Tumours (STOET): Guideline for diagnosis and prevention. Available at: (viewed on November 1st, 2011). 12. Rebbeck TR, Kauff ND, Domchek SM. Meta-analysis of risk reduction estimates associated with riskreducing salpingo-oophorectomy in BRCA1 or BRCA2 mutation carriers. J Natl Cancer Inst. 2009; 101(2): Madalinska JB, Hollenstein J, Bleiker E, van Beurden M, Valdimarsdottir HB, Massuger LF, et al. Quality-of-life effects of prophylactic salpingooophorectomy versus gynecologic screening among women at increased risk of hereditary ovarian cancer. J Clin Oncol. 2005; 23(28): Madalinska JB, van Beurden M, Bleiker EM, Valdimarsdottir HB, Hollenstein J, Massuger LF, et al. The impact of hormone replacement therapy on menopausal symptoms in younger high-risk women after prophylactic salpingo-oophorectomy. J Clin Oncol. 2006; 24(22): Fakkert IE, Jansen L, Meijer K, Kok T, Oosterwijk JC, Mourits MJ, et al. Breast cancer screening in BRCA1 and BRCA2 mutation carriers after risk reducing salpingo-oophorectomy. Breast Cancer Res Treat. 2011; 129(1): Boyd J, Sonoda Y, Federici MG, Bogomolniy F, Rhei E, Maresco DL, et al. Clinicopathologic features of BRCA-linked and sporadic ovarian cancer. JAMA. 2000; 283(17): Shaw PA, McLaughlin JR, Zweemer RP, Narod SA, Risch H, Verheijen RH, et al. Histopathologic features of genetically determined ovarian cancer. Int J Gynecol Pathol. 2002; 21(4):

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