Author response: Diagnostic potential of tumor DNA from ovarian cyst fluid (2016)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract We determined whether the mutations found in ovarian cancers could be identified in the patients' ovarian cyst fluids. Tumor-specific mutations were detectable in the cyst fluids of 19 of 23 (83%) borderline tumors, 10 of 13 (77%) type I cancers, and 18 of 18 (100%) type II cancers. In contrast, no mutations were found in the cyst fluids of 18 patients with benign tumors or non-neoplastic cysts. Though large, prospective studies are needed to demonstrate the safety and clinical utility of this approach, our results suggest that the genetic evaluation of cyst fluids might be able to inform the management of the large number of women with these lesions. https://doi.org/10.7554/eLife.15175.001 eLife digest More than a third of women develop ovarian cysts during their lifetimes. The vast majority of these cysts are harmless, but a small number are caused by ovarian cancers. These cancers often produce no symptoms until the disease has spread throughout the abdomen or to other organs, so many women go undiagnosed until their chances of being successfully treated are low. Currently, there is no reliable way to determine whether an ovarian cyst is cancerous without performing surgery. As a result, many women undergo unnecessary, invasive surgeries for harmless ovarian cysts. Tumors shed cells and cell fragments into any fluid that surrounds them. Fluids from cysts in the pancreas, kidney, and thyroid are routinely examined to identify whether they contain cancerous cells. Now, Wang, Sundfeldt et al. show that ovarian cancers also shed DNA into the surrounding cyst fluid. Furthermore, mutations found in this DNA can provide valuable information about whether the cysts are cancerous. The study was performed by extracting DNA from the fluid in ovarian cysts that had been surgically removed from 77 women. Of these cysts, 10 were harmless cysts, 12 were benign tumors, 31 were invasive cancers, and 24 were so-called borderline tumors, which fall somewhere between the benign tumors and invasive cancers. Only cysts associated with the borderline tumors and invasive cancers need to be surgically removed. Here, Wang, Sundfeldt et al. report that DNA mutations that are characteristic of ovarian cancers were found in 87% of the cysts associated with borderline tumors and invasive cancers. In contrast, these mutations were not found in any of the cysts that do not require surgery. Fluid can be extracted from an ovarian cyst with a needle during an outpatient visit. Therefore, the results presented by Wang, Sundfeldt et al. suggest a relatively straightforward way of testing the DNA from ovarian cysts before deciding whether surgery is really necessary. First, however, larger studies that follow women with cysts over time will be necessary to confirm that this type of testing is effective and safe. https://doi.org/10.7554/eLife.15175.002 Introduction Ovarian cancer is the most lethal gynecologic malignancy, with 21,290 estimated new cases and 14,180 estimated deaths in the United States in 2015. Approximately 1.3% of women will be diagnosed with ovarian cancer during their lifetime (Howlader et al., 2014). These cancers commonly present as an adnexal mass with cystic components, but are not associated with specific symptoms. As a result, two-thirds of ovarian cancers are diagnosed at late stage (Stage III or IV), when the 5-year survival is less than 30% (Howlader et al., 2014) Complicating the diagnosis of ovarian cancer is the fact that ovarian cysts are common in women of all ages, with a prevalence of 35% and 17% in pre- and post-menopausal women, respectively (Pavlik et al., 2013). These cysts are frequently benign and found incidentally on routine imaging (Pavlik et al., 2013). Though malignancy is an unusual cause of the cysts, 30% of the cysts exhibit radiographic features suspicious for malignancy, such as solid areas or mass (Pavlik et al., 2013). In addition to the anxiety that such findings provoke, many women undergo unnecessary surgery for cysts that are not malignant and may not be responsible for the symptoms they have. For example, only 5% of 570 women in a large ovarian cancer screening randomized trial who underwent surgical evaluation actually had a malignancy (Buys et al., 2005). Similarly in another study of symptomatic women, only 4% of 197 women who had concerning features on transvaginal ultrasound were ultimately diagnosed with ovarian cancer (Gilbert et al., 2012). Compounding this issue is the fact that surgery for ovarian cysts requires general anesthesia and is associated with significant morbidity, causing complications in 15% of women (Buys et al., 2011). These complications include damage to nerves and ureters, bleeding, infection, and perforation of adjacent viscera. Furthermore, the procedure often results in hormonal and fertility loss (in the case of bilateral oophorectomy) (Buys et al., 2011). Even minimal procedures such as ovarian cystectomy can affect fertility in premenopausal women by decreasing follicular response and oocyte number (Loh et al., 1999; Demirol et al., 2006). If a preoperative test could help determine whether the cystic lesion was benign or malignant, unnecessary surgery and its associated complications could be avoided in many patients. This would be particularly helpful for women of reprodutive age who wish to preserve their fertility, as well as women whose medical comorbidities or functional status makes anesthesia and surgery hazardous. Ovarian cysts and tumors are classified as non-neoplastic, benign, borderline, or malignant based on microscopic examination after surgical removal (Figure 1). Non-neoplastic cysts are by far the most common class of ovarian cysts. They are frequently found in pre-menopausal women, arising when an egg is not released properly from either the follicle or corpus luteum and usually resolve spontaneously within several months (Christensen et al., 2002). Benign cystic tumors, such as cystadenomas and cystadenofibromas, rarely progress to malignancy (Cheng et al., 2004; Levine et al., 2010). No genetic alterations have yet been identified in either non-neoplastic cysts or in benign cystic tumors (Cheng et al., 2004). Neither of these cyst types requires surgery unless they are symptomatic or large (Levine et al., 2010). These cysts can be easily sampled with ultrasound-guided fine-needle aspiration within minutes in an outpatient setting without the need for anesthesia (Duke et al., 2006). Figure 1 Download asset Open asset Schematic showing classes of ovarian cysts and the diagnostic potential of the cyst fluid. Ovarian cysts and tumors are currently classified according to microscopic evaluation after surgical removal. The majority of ovarian cysts are non-neoplastic (often 'functional' in premenopausal women). Ovarian tumors with combined cystic and solid components are either benign tumors, borderline tumors, or malignant cancers (type I or II). Only cysts associated with borderline tumors and cancers require surgical excision. We show here that the DNA purified from cyst fluid can be analyzed for somatic mutations commonly found in their associated tumors. The type of mutation detected not only could indicate the type of tumor present but also could potentially inform management. https://doi.org/10.7554/eLife.15175.003 At the other end of the spectrum are epithelial ovarian cancers, which are potentially lethal and unequivocally require surgery. A dualistic model has been proposed to classify these neoplasms (Kurman and Shih, 2010). Type I tumors are composed of low-grade serous, low-grade endometrioid, clear cell, and mucinous carcinomas. They are generally indolent, frequently diagnosed at early stage (Stage I or II), and develop from well-established precursor lesions ('borderline' or 'atypical proliferative' tumors, as described below) (Kurman and Shih, 2011). Type I cancers commonly exhibit mutations in KRAS, BRAF, CTNNB1, PIK3CA, PTEN, ARID1A, or PPP2R1A (Kurman and Shih, 2010). In contrast, type II tumors are generally high-grade serous carcinomas. They are highly aggressive, most often diagnosed in late stage (Stage III or IV), and have suggested origins from the distal fallopian tube (Lee et al., 2007). Type II cancers are the most clinically important group of ovarian cancers, comprising 75% of all ovarian carcinomas and responsible for 90% of ovarian cancer deaths (Kurman and Shih, 2011). They almost always harbor TP53 mutations (Cancer Genome Atlas Research Network. 2011). Also unlike type I cancers, which are relatively chemo-resistant and more often treated only with surgical excision, type II cancers respond to conventional chemotherapy, particularly after maximal debulking to reduce tumor burden (Bristow et al., 2002; Schmeler et al., 2008). 'Borderline' or 'atypical proliferative' tumors lie in the middle of this spectrum, between the malignant cancers and the generally harmless non-neoplastic or benign lesions. They are distinguished from carcinomas by the absence of stromal invasion and are precursors of type I cancers. In light of their potential for malignancy, the standard of care for borderline tumors is surgical excision. Following surgery, the prognosis is excellent compared to ovarian cancers, with 10-year survival rates over 94% (Sherman et al., 2004). A minor but significant portion of borderline tumors recur after surgery, however, and a subset of the recurrences are found to have advanced to type I cancers (Shih et al., 2011). This progression is consistent with molecular findings: serous borderline tumors typically exhibit mutations in BRAF or KRAS, like their malignant counterparts (low-grade serous carcinoma) (Mayr et al., 2006; Jones et al., 2012). The presence of a BRAF mutation in a borderline tumor is associated with better prognosis and a low probability of progression to carcinoma (Grisham et al., 2013). In contrast, KRAS mutations are associated with the progression to type I cancers (Tsang et al., 2013). The examination of fluids from pancreatic, renal, and thyroid cysts is routinely used in clinical management (Frossard et al., 2003; Lin et al., 2005; Volpe et al., 2007). The fluids have historically been studied by cytology to identify malignant cysts. Ovarian cysts share many features with these other types of cysts, in that they are common, often diagnosed incidentally, and are nearly always benign. However, aspiration of ovarian cyst fluid for cytology is not standard-of-care. From a historical perspective, the difference in diagnostic management partly lies in the fact that cytology has not proven to be very informative for ovarian cysts, particularly for distinguishing benign vs. borderline tumors (Moran et al., 1993; Martínez-Onsurbe et al., 2001). There have also been concerns raised about the safety of ovarian cyst aspiration (see Discussion), and these concerns have not often been raised for other types of cysts. More recently, genetic analysis of specific types of cyst fluids has been considered as an aid to cytology, given that conventional cytology often has limited sensitivity and specificity (Wu et al., 2011b). Based on the emerging success of the molecular genetic evaluation of other types of cysts, we reasoned that a similar approach could be applied to ovarian cysts. Evaluation of DNA from cells and cell fragments shed into the cyst fluid would presumably allow the identification of tumor-specific mutations. Unlike other, conventional markers of neoplasia such as CA-125, cancer gene mutations are exquisitely specific indicators of a neoplastic lesion (Vogelstein et al., 2013). Moreover, the type of mutation can in some cases indicate the type of neoplastic lesion present (Wu et al., 2011a). Yamada et al. have demonstrated that mutations can be detected in the cystic fluid of ovarian tumors by querying exons 4 to 9 of TP53, achieving sensitivities of 12.5% and 10%, for borderline and malignant tumors, respectively (Yamada et al., 2013). Recently developed, extremely sensitive methods for mutation detection, capable of identifying one mutant template allele among thousands of normal templates in a panel of genes, could potentially increase this sensitivity (Kinde et al., 2011; Murtaza et al., 2013; Newman et al., 2014). In this study, we have applied one of these technologies to assess mutations in ovarian cyst fluids and to inform the development of tests that could eventually be applied to patients. Results Characteristics of the tumors and cyst fluid samples DNA was isolated from surgically excised ovarian cysts of 77 women. Ten of them had non-neoplastic cysts, 12 had benign tumors, 24 had borderline tumors, and 31 had cancers (13 Type I and 18 Type II). Age, histopathologic diagnosis, stage, and other clinical information are provided in Supplementary file 1. The median amount of DNA recovered from the cysts was 222 ng (interquartile range (IQR) of 53 to 3120 ng) (Supplementary file 2). There was no significant difference in the amounts of DNA between borderline tumors and type I or type II cancers. However, the borderline tumors and cancers contained significantly more DNA than the non-neoplastic cysts or benign tumors (4453 ± 6428 ng vs. 62 ± 64 ng; p<0.001, Wilcoxon rank-sum test). A multiplex PCR-based assay to identify tumor-specific mutations in cyst fluid samples We designed a multiplex PCR-based assay that could simultaneously assess the regions of 17 genes frequently mutated in ovarian tumors. The amount of DNA shed from neoplastic cells was expected to be a minor fraction of the total DNA in the cyst fluid, with most DNA emanating from normal cells. We therefore used a sensitive detection method, called Safe-SeqS (Safe-Sequencing System), to identify mutations in cyst fluid samples (Kinde et al., 2011). In brief, primers were designed to amplify 133 regions, covering 9054 distinct nucleotide positions within the 17 genes of interest (Supplementary file 3). Three multiplex PCR reactions, each containing non-overlapping amplicons, were then performed on each sample. One primer in each pair included a unique identifier (UID) for each template molecule, thereby drastically minimizing the error rates associated with PCR and sequencing, as described previously (Kinde et al., 2011). Under the conditions used in the current experiments, mutations present in >0.1% of template molecules could generally be reliably determined (Kinde et al., 2011; 2013; Bettegowda et al., 2014). We could not perform sequencing on five cysts (four non-neoplastic cysts and one cyst associated with a borderline tumor) because there was insufficient DNA (<3 ng recovered), and these were scored as uninterpretable. When this assay was applied to cyst fluid samples with sufficient DNA, no mutations were identified in the 18 cysts obtained from patients with simple cysts (n = 6) or benign tumors (n = 12) (Table 1). This was in stark contrast to the fluids obtained from the 18 patients with type II cancers, all of which were found to contain a mutation (Table 1). Ten (77%) of the 13 cyst fluids from patients with type I cancers and 19 (83%) of the 23 cyst fluids from patients with borderline tumors contained at least one detectable mutation. When categorized by the need for surgery (i.e., presence of a borderline tumor or a type I or type II cancer), the sensitivity of this assay was 87% (47 of 54 cysts; 95% confidence interval of 75% to 95%) and its specificity was 100% (95% confidence interval of 74% to 100%; Table 1). Table 1 Detection of tumor-specific mutations in cyst fluid. The fraction of samples detected and the median fraction of mutant alleles are indicated, grouped by cyst type, cancer stage, and the need for surgery. https://doi.org/10.7554/eLife.15175.004 Fraction of samples detected (95% confidence interval)Median fraction of mutant alleles (IQR)Total # of samplesTypeNon-neoplastic0% (0–46%)0% (0–0%)6Benign tumor0% (0–26%)0% (0–0%)12Borderline tumor83% (61–95%)2.4% (1.5–10.8%)23Type I cancer77% (46–95%)7.8% (3.3–28.7%)13Type II cancer100% (81–100%)60.3% (31.3–70.8%)18Cancer stageEarly (I and II)82% (48–97%)7.4% (3.0–30.9%)11Late (III and IV)95% (75–100%)51.2% (30.2–69.5%)20Cysts requiring surgeryNo0% (0–26%)0% (0–0%)18Yes87% (75–95%)12.6% (2.7–40.2%)54 Ovarian cancers are generally detected only late in the course of disease, perhaps explaining the poor prognosis of patients. Accordingly, only 11 of the 31 cysts associated with cancers in our study had early (Stage I or II) disease (Supplementary file 1). As expected, most of these were type I carcinomas (n = 8). Nevertheless, it was encouraging that mutant DNA could be detected in nine (82%) of these 11 patients (Table 1). Mutations could be detected in 95% of the 20 patients with Stage III or IV cancers (Table 1). A variety of control experiments were performed to confirm the integrity of these results. One informative positive control was provided by the analysis of DNA from the tumors, using the identical method used to analyze DNA from the cyst fluids. Fifty-three of the 55 borderline and malignant cases had tumors available for this purpose. Every mutation identified in a tumor was found in its cyst fluid, and vice versa. As expected, the mutant allele frequencies in the tumors were often, but not always, higher than in the cyst fluids (Supplementary file 2). As another positive control, we used an independent PCR and sequencing reaction to confirm each of the cyst fluid mutations listed in Supplementary file 2. This validated not only the presence of a mutation, but also confirmed its fractional representation. The median relative difference between the fractions of mutant alleles in replicate experiments was 7.0% (IQR of 3.5% to 8.9%). Finally, four patients were found to have two independent mutations (Supplementary file 2). For example, the cyst fluid of patient OVCYST 081, who had a high-grade endometrioid carcinoma, harbored a missense mutation (R280K) in TP53 plus an in-frame deletion of PIK3R1 at codons 458 and 459. The TP53 mutation was found in 3.0% of alleles while the PIK3R1 mutation was found in 3.7% of the alleles analyzed. Similar mutant allele frequencies among completely different mutations in the cyst fluid of three other patients provided further indicators of reproducibility. All genetic assays were performed in a blinded manner, with the operator unaware of the diagnoses of the patients from whom the cyst fluids were obtained. In addition to DNA from normal individuals used as controls, additional negative controls were provided by the simple cysts and benign tumors. Using the identical assay, none of the DNA from their cyst fluids contained detectable mutations. A final control was provided by the borderline and malignant tumors themselves. In general, only one or two of the 9054 base-pairs (bp) queried were mutated in any one tumor (Supplementary file 2). The other ~9000 bp could then be independently queried in the corresponding cyst fluid, and none of these positions were found to be mutated. Relationship between the type of tumor present and the type of mutation found in the associated cyst fluid sample The mutant allele fractions in the cyst fluids tended to be higher in the type II cancers (median of 60.3%) than the type I cancers (median of 7.8%) or borderline tumors (median of 2.4%), though there was considerable overlap (Table 1; Figure 2A). With respect to stage, the DNA from cyst fluids of late-stage cancers had higher median mutant allele fractions (51.2%) than of cancers or borderline tumors but with considerable overlap (Table 1; Figure Figure Download asset Open asset allele by tumor No mutations were found in the DNA of non-neoplastic or benign cysts. Of the cysts that surgery, the median mutant allele fraction was higher in the cyst fluids associated with type II cancer than type I or borderline tumors by tumor The DNA from cyst fluids of late-stage cancers had a higher median mutant allele fraction (51.2%) than of cancers or borderline tumors mutant allele is on a median and the other the type of mutation among these cysts (Figure 3). In type I tumors, the genes mutated were BRAF (n = KRAS (n = (n = PIK3R1 (n = PPP2R1A (n = (n = or TP53 (n = 3). distinct mutations were found sample in three type I cancers. The BRAF mutation was unusual that it from an in-frame than the characteristic of the vast majority of BRAF mutations in the This mutation has been in a thyroid cancer and a et al., 2003; et al., 2014). The deletion results in loss of a in the of BRAF, while the of an has been suggested to increase BRAF by an et al., 2002). In contrast, all but one type II cancers of had mutations in the only was OVCYST a high-grade endometrioid The borderline tumors were distinguished by yet a different from that of the either type I or type II cancers. Of the 19 mutations in borderline tumors, 12 were BRAF in type I or type II cancers, and the were at KRAS 12 or (Supplementary file 2). Figure Download asset Open asset genes found in the cyst fluid mutations with mutant allele between and mutations with between 1 and mutations with than patients with insufficient DNA for patients with two detected associated with the need for surgery A analysis was used to identify the most informative molecular features of cyst fluids and to them to the commonly used for ovarian and et al., et al., (Table 2). We as a need for surgery (i.e., borderline tumors or type I or II The amount of DNA in cyst fluids was but not higher in the cysts requiring surgery Table though there were many cysts not requiring surgery that had higher DNA than cysts requiring surgery (Figure the were significantly higher in cysts requiring surgery Table but there were many cysts not requiring surgery that had higher than requiring surgery (Figure were not with cyst type Table Figure the other the presence of a mutation was highly informative for the presence of a cyst requiring surgery in the as no mutations were found in cysts not requiring surgery Table 2). Table analysis for markers associated with need for surgery. The presence of a mutation, cyst DNA and common for ovarian cancer were analyzed for with cysts that require surgical removal DNA Figure 4 Download asset Open asset associated with the need for surgery. DNA amount and of commonly used ovarian cancer are according to the cyst type and need for surgery. The amounts of DNA in cyst fluids was generally higher in cysts requiring surgery than that do not but no significant was found was significantly higher in cysts that surgery than that do not was not with the need for surgery All are on a were using in a model Materials and Discussion Ovarian cancer is the most lethal gynecologic cancer in women. screening is not by the using current diagnostic which frequently to surgical in women who do not have and 2012). study was by the that cancer gene mutations are of neoplasia and in non-neoplastic conditions (Vogelstein et al., 2013). We have demonstrated here that mutations in ovarian tumors are also present in their associated cyst fluids. Moreover, the mutant allele frequencies in the cyst fluids are relatively (median of to their There were no mutations detected in the cyst fluids that were not also present in the tumors, and vice versa. Also no mutation was identified in non-neoplastic cysts or cysts associated with benign tumors. mutations were detected in a fraction of cysts requiring surgery but not in any cyst that not require surgery (Table 1). most of the 54 cysts requiring surgery had detectable mutations in their All of these cysts in borderline tumors or type I cancers, while mutations were always (100%) detectable in type II cancers (Table 1). There are two potential for our to mutations in these cysts. First, it is that the mutant DNA in these cysts was the of sensitivity of our assay mutant allele We this by the tumors no mutations were detected in any of the tumors from these patients. The and therefore more is that our panel of 133 amplicons, containing regions of 17 genes, was not to the mutations that were Unlike type II cancers, which nearly always contain TP53 mutations of the type II cancers we for the of type I cancers and borderline tumors are more and not as well studied (Kurman and Shih, 2010). genetic evaluation of these tumors the of additional in the panel to higher Nevertheless, the 100% sensitivity for type II cancers in our study is highly given that these cancers for over 90% of ovarian cancer One of our study is the number of patients Though of ovarian cysts is one of the most commonly performed surgical of cyst fluids is not common, in we only had relatively small (n = of non-neoplastic cysts and benign tumors available for Even the in genetic alterations among the cyst types were (Table 1). study will and of ovarian cyst fluids that will be able to confidence the sensitivities and in the current A potential clinical of our approach is the by that needle of a malignant ovarian cyst to of the This is based on about the of cyst during surgery and at A study of patients who had ovarian cyst no in all malignant cases with a of two Furthermore, a found no difference in survival for ovarian cancer patients who had cyst vs. no et al., 2013). We current a surgical could a tumor stage to and the patient to with its associated However, the that malignant cysts might shed cancer cells with the of removal of ovarian cysts et al., 2006). removal of a cyst a significant of cyst higher than when a needle is et al., Finally, malignant cysts might be to be as as malignant ovarian cysts (Howlader et al., yet the for cysts of cyst fluid ultrasound over many