Cancer Biology & Therapy ISSN: 1538-4047 (Print) 1555-8576 (Online) Journal homepage: https://www.tandfonline.com/loi/kcbt20 Sequence mutations and amplification of PIK3CA and AKT2 genes in purified ovarian serous neoplasms Kentaro Nakayama, Naomi Nakayama, Robert J. Kurman, Leslie Cope, Gudrun Pohl, Yardena Samuels, Victor E. Velculescu, Tian-Li Wang & Ie-Ming Shih To cite this article: Kentaro Nakayama, Naomi Nakayama, Robert J. Kurman, Leslie Cope, Gudrun Pohl, Yardena Samuels, Victor E. Velculescu, Tian-Li Wang & Ie-Ming Shih (2006) Sequence mutations and amplification of PIK3CA and AKT2 genes in purified ovarian serous neoplasms, Cancer Biology & Therapy, 5:7, 779-785, DOI: 10.4161/cbt.5.7.2751 To link to this article: https://doi.org/10.4161/cbt.5.7.2751 Published online: 01 Jul 2006. Submit your article to this journal Article views: 414 View related articles Citing articles: 114 View citing articles Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=kcbt20 [Cancer Biology & Therapy 5:7, 779-785, July 2006]; ©2006 Landes Bioscience Research Paper Sequence Mutations and Amplification of PIK3CA and AKT2 Genes in Purified Ovarian Serous Neoplasms Kentaro Nakayama1,† Naomi Nakayama1,† Robert J. Kurman1-3 Leslie Cope2 Gudrun Pohl1 Yardena Samuels2 Victor E. Velculescu2 Tian-Li Wang2,3,* Ie-Ming Shih1-3,* ABSTRACT Departments of 1Pathology, 2Oncology and 3Gynecology and Obstetrics; Johns Hopkins Medical Institutions; Baltimore, Maryland USA †These authors contibuted equally to this manuscript. *Correspondence to:Ie-Ming Shih; Johns Hopkins Medical Institutions; 550 Orleans Street, Room 305; Baltimore, Maryland 21231 USA; Tel.: 410.502.7774; Fax: 410.502.7943; Email: ishih@jhmi.edu/ Tian-Li Wang; Johns Hopkins Medical Institutions; 1503 E. Jefferson Street, Room: B-315; Baltimore, Maryland 21231 USA; Tel.: 410.502.0863; Fax: 410.502.7943; Email: tlw@jhmi.edu .D ON OT DI ST RIB UT E . Sequence mutations and gene amplifications lead to activation of the PIK3CA-AKT2 signaling pathway and have been reported in several types of neoplasms including ovarian cancer. Analysis of such genetic alterations, however, is usually complicated by contamination of normal cell DNA, artifacts associated with formalin-fixed tissues and the sensitivity of the techniques employed. In this study, we analyzed the sequence mutations in PIK3CA and AKT2 genes using purified tumor cells that were isolated from high-grade ovarian serous carcinomas and serous borderline tumors (SBTs) and assessed gene amplification using a dual-color FISH on tissue microarrays. Somatic sequence mutations in the kinase domain of AKT2 were not detected in any of the 65 ovarian tumors analyzed. Mutations of PIK3CA were rare, occurring only in one (2.3%) of 44 high-grade serous carcinomas and in only one (4.8%) of 21 SBTs. Dual-color FISH demonstrated that PIK3CA and AKT2 were not amplified in SBTs but amplified in 13.3% and 18.2% high-grade carcinomas, respectively. High-level amplification (>3 fold) was more frequently observed in AKT2 than in PIK3CA. Unlike mutations in ERBB2, KRAS and BRAF which are mutually exclusive in SBTs, coamplification of PIK3CA and AKT2 was present in five high-grade carcinomas including the OVCAR3 cells. Amplification in either of the genes occurred in 27% high-grade serous carcinomas. In conclusion, the methods we employed provide unambiguous evidence that somatic sequence mutations of PIK3CA and ATK2 are rare in ovarian serous tumors but amplification of both genes may play an important role in the development of high-grade ovarian serous carcinoma. Previously published online as a Cancer Biology & Therapy E-publication: http://www.landesbioscience.com/journals/cbt/abstract.php?id=2751 KEY WORDS ACKNOWLEDGEMENTS LA ND ES This study was supported by the US Department of Defense grant (OC04-0060) and the National Cancer Institute grant (RO1 CA103937). The authors thank Dr. May J. Yen for preparation of the figure illustrations. NOTES INTRODUCTION Ovarian cancer is the most lethal gynecologic malignancy and serous tumors are the most common type of ovarian cancer.1,2 Ovarian serous neoplasms are heterogeneous and can be divided into high- and low-grade tumors that are characterized by distinctive molecular, histopathological and clinical features.3-6 High-grade tumors are the usual type of ovarian serous carcinoma. They are aggressive and have a high mortality rate. Low-grade tumors are composed mainly of serous borderline tumors (SBTs) and invasive low-grade serous carcinomas. SBTs may progress to invasive low-grade carcinomas which are indolent neoplasms with a better outcome than high-grade serous carcinomas. Mutations of KRAS and BRAF occur in approximately two thirds of low-grade serous tumors but are rare in high-grade serous carcinomas.5-8 In contrast, somatic TP53 mutations are detected in greater than 60% of high-grade serous carcinoma and only rarely (8%) in low-grade serous tumors.9 Furthermore, both high-grade carcinomas and low-grade tumors are characterized by distinctive gene expression profiles.10-12 Based on these findings, a dualistic model of ovarian serous carcinogenesis in which high-grade and low-grade ovarian serous tumors develop along distinctly different molecular pathways has been proposed.3 Activating mutations and amplification of genes in kinase signaling pathways play a critical role in tumorigenesis. Moreover, the mutated kinase proteins can potentially provide new targets for a kinase inhibitor or antibody-based therapy. In this study, we focused on somatic mutations and gene amplifications of the phosphoinositide 3-kinase (PIK3CA)-AKT2 signaling pathway because PIK3CA gene and its downstream gene, AKT2, are thought to be important in ovarian cancer development and therefore are potential molecular targets for new therapeutics.8,13-16 The PI3K-AKT2 signaling pathway regulates diverse cellular functions including cellular proliferation, survival and migration.17-19 Somatic mutations within the PIK3CA kinase domains have been reported in colorectal, brain, ovarian and breast cancers.20-24 In addition, increased PIK3CA and AKT2 gene copy numbers have been detected in pancreatic, ovarian, cervical, head and neck, and lung BIO SC mutation, amplification, FISH, digital karyotyping, ovarian cancer IEN CE Received 02/27/06; Accepted 03/26/06 © 20 06 Supplemental information can be found at: http://www.landesbioscience.com/journals/cbt/ supplement/nakayama5-7-supp.pdf www.landesbioscience.com Cancer Biology & Therapy 779 Mutation Profiles of PIK3CA and AKT2 in Ovarian Cancer carcinomas.13,14,25-27 Although molecular genetic analysis has been performed in ovarian cancer,23,28,29 none of the reports employed purified tumor samples to determine sequence mutations and fluorescence in situ hybridization (FISH) analysis on surgical specimens to assess gene copy number. Finally, simultaneous analyses of copy number changes in both PIK3CA and AKT2 have not been performed on the same tumor tissues. In order to clarify the mutational profiles of PIK3CA and AKT2 genes in surgical specimens, we obtained genomic DNA from purified high-grade serous carcinomas and SBTs in which the high purity of tumor cells was confirmed by cytokeratin staining and identification of heterozygous somatic mutations in control genes. We also analyzed DNA copy number changes of PIK3CA and AKT2 genes in the same archival tumor specimens using a dual-color FISH which is an accurate method of assessing gene amplification especially for those with a low-level gain. MATERIALS AND METHODS Tissue samples and tumor cell isolation. High-grade (conventional) ovarian serous carcinomas and ovarian serous borderline tumors (SBTs) [atypical proliferative serous tumors and intraepithelial (micropapillary) low-grade serous carcinomas] were obtained from the Department of Pathology at the Johns Hopkins Hospital between 2000 and 2005. All high-grade ovarian tumors were advanced stage (FIGO stage III and IV). Acquisition of tissue specimens was approved by the institutional review board at the Johns Hopkins Hospital. For sequencing analysis, tumor cells from 65 serous tumors (44 high-grade serous carcinomas and 21 serous borderline tumors) were isolated using the following protocol illustrated in Figure 1. Frozen section examination was performed by a surgical pathologist (IS) on all specimens to confirm the diagnosis before tissue harvesting. For high-grade carcinomas, fresh tumor tissues were washed in cold phosphate buffered saline (PBS), minced to ~1 mm3 fragments and digested with collagenase A (10 mg/ml) with mild agitation at 37˚C for 40 min. Single tumor cells or small tumor cell clusters (<10 cells) were collected from the top portion of centrifuge tubes after the large incompletely digested tissue fragments descended to the bottom of the tube. The tumor cells were washed with PBS and then isolated using magnetic beads coated with an Ep-CAM antibody (Dynal, Oslo, Norway). The tumor cells were either directly harvested for genomic DNA isolation or cultured in RPMI1640 containing 10% fetal bovine serum for three days to expand the tumor cell population for those samples with only limited amounts of tumor tissue. For SBTs, the fresh tumor fragments were harvested directly into centrifuge tubes containing 0.05% trypsin and 200 µg/ml EDTA in HBSS (Invitrogen) without mincing and collagenase digestion. This approach allowed epithelial (tumor) cells that covered the surface of papillary structures of borderline tumors to detach from the basement membrane without digesting the underlying stromal cells, thus minimizing possible stromal cell contamination. After incubation at 37˚C for 20 min, the SBT fragments were agitated at room temperature for 1 min to allow complete separation of epithelial cells from the tissue fragments. The epithelial cells were washed with culture medium twice and cultured for 3 days. Purity of the tumor cells was confirmed by immunostaining with an anti-cytokeratin antibody, CAM 5.2 (Becton Dickinson, San Jose, CA). The genomic DNA from normal uterus or colonic mucosa from the same patient was also obtained for all cases. For dual-color FISH analysis, formalin-fixed, paraffin-embedded tissues were used. A total of 124 specimens consisting of 74 high-grade 780 Figure 1. Tumor cell purification from surgical specimens in a serous borderline tumor SBT) and a high-grade (HG) ovarian serous carcinoma. serous carcinomas, 37 SBTs and 13 normal ovaries were arranged onto tissue microarrays for FISH analysis. Three representative cores (1.5 mm diameter) from each tumor block were placed on the tissue microarrays. Mutational analysis. Nucleotide sequencing was used to analyze the mutational status of PIK3CA and AKT2 in tumor cells isolated from the ovarian serous tumors. In addition, KRAS, BRAF, ERBB2 and TP53 genes were also analyzed in the same panel for comparison. In this study, we focused on analyzing the exons that have been reported to harbor the majority of mutations for each of the genes. The primer sequences and the PCR protocol have been previously described.6,9,20,30-35 Supplement Table 1 listed PCR and sequencing primers of all the exons that were sequenced in this study. PCR products were purified and sequenced at Agencourt Bioscience (Beverly, MA). Fluorescence in situ hybridization (FISH). For the PIK3CA locus, bacterial artificial chromosome clones containing the target (RP11-245C23) and referenced (RP11-69N24) chromosomal regions hybridized to 3q26.32 and 3q13.11, respectively. For the AKT2 locus, bacterial artificial chromosome clones containing the target (CTC-425O23) and referenced (RP11-75H6 and CTD-3195E18) chromosomal regions hybridized to 19q13.2 and the reference probe to 19p13.13, respectively. The bacterial artificial chromosome clones were purchased from Bacpac Resources (Children’s Hospital Oakland, CA) and Invitrogen. Cancer Biology & Therapy 2006; Vol. 5 Issue 7 Mutation Profiles of PIK3CA and AKT2 in Ovarian Cancer Table 1 Mutational status of kinase genes in ovarian serous tumors Case No. Tumor PIK3CA AKT2 ERBB2 KRAS BRAF 1 SBT WT WT 12 bp ins** WT WT 2 SBT WT WT WT WT T1976A:V600E 3 SBT WT WT WT G35T:G12V WT 4 SBT WT WT WT G35A:G12D WT 5 SBT WT WT WT G35A:G12D WT 6 SBT WT WT WT WT T1976A:V600E 7 SBT WT WT WT WT WT 8 SBT WT WT WT WT WT 9 SBT WT WT WT WT WT 10 SBT WT WT WT WT T1976A:V600E 11 SBT A3140G:H1047R WT WT G35T:G12V WT 12 SBT WT WT WT G38T:G13V WT 13 SBT WT WT WT WT WT 14 SBT WT WT WT G35T:G12V WT 15 SBT WT WT WT WT WT 16 SBT WT WT WT WT T1976A:V600E 17 SBT WT WT 12 bp ins WT WT 18 SBT WT WT WT WT T1976A:V600E 19 SBT WT WT WT WT WT 20 SBT WT WT WT WT WT 21 SBT WT WT WT G35T:G12V WT 22–63 HG1-42 WT WT WT WT WT 64 HG43 WT WT WT WT T1976A:V600E 65 HG44 A3140G:H1047R WT WT WT WT SBT, serous borderline tumor; HG, high-grade serous carcinoma. *12 bp insertion at 2313–2324. The method for FISH on tissue sections has been detailed in a previous report.36 Target and reference probes were labeled with biotin and digoxigenin, respectively. To detect biotin-labeled and digoxigenin-labeled signals, slides were first incubated with FITC-avidin (Vector, Burlingame, CA) and anti-digoxigenin sheep Fab fragment (Roche, Indianapolis, IN); then incubated with a biotinylated anti-avidin antibody (Vector, Burlingame, CA) and TRITC-conjugated rabbit anti-sheep F(ab)2 (Jackson ImmunoResearch, West Grove, PA); followed by incubation with FITC-avidin and TRITC-conjugated goat anti-rabbit F(ab)2 (Jackson ImmunoResearch). Two investigators (TLW and IS) who were not aware of the tumor grade and clinical information evaluated the FISH signals. Approximately 100 tumor cells were examined for each specimen. Gain of DNA copy number of a gene was defined as the ratio of the gene probe signal to the control probe signal exceeding 1.5. High-level of amplification was defined as a signal ratio greater than 3. www.landesbioscience.com RESULTS Our method for tumor cell purification yielded a sample in which tumor cells comprised greater than 99% of the sample for all high-grade carcinomas and SBTs based on immunostaining for cytokeratin. Both high-grade carcinoma and SBT cells formed cohesive epithelial nests in short term primary cultures (Fig. 1). The mutational status of PIK3CA, AKT2, ERBB2, KRAS and BRAF in all 65 purified ovarian serous tumors is summarized in Table 1. Somatic mutations of PIK3CA were identified in one (2.3%) of 44 high-grade serous carcinomas and in one (4.8%) of 21 SBTs. Both mutations were heterozygous and were located at A3140G of a kinase domain (Fig. 2 and Table 1). Neither SBTs nor high-grade serous carcinomas demonstrated a somatic mutation in the AKT2 kinase domain. Since mutations of both kinase genes in either high-grade carcinomas or SBTs were so infrequent, we used TP53 gene as a positive control for high-grade serous carcinomas because TP53 mutation is the most frequent molecular genetic change known so far in high-grade ovarian Cancer Biology & Therapy 781 Mutation Profiles of PIK3CA and AKT2 in Ovarian Cancer Table 2 DNA copy number changes of the PIK3CA locus in ovarian tumors based on FISH analysis PIK3CA Locus OSE SBT HG No gain or amplified Low gain (1.5–3 fold) High gain (> 3 fold) polyploidy 13 0 0 0 37 0 0 0 52 7 1 14 Total 13 37 74 OSE, ovarian surface epithelium from normal ovaries; SBT, serous borderline tumor; HG, high-grade serous carcinoma. Figure 2. Chromatograms of PIK3CA and ERBB2 mutational status in two representative serous borderline tumors (case number 11 and 17). Case 11 shows a heterozygous somatic mutation at the nucleotide 3140 (A to G). Case 17 demonstrates a heterozygous 12 bp in-frame insertion mutation at the nucleotide of 2313–2324. serous carcinomas.3,9,35 Two kinase genes including KRAS and BRAF were used as controls for SBTs because they are the most common mutations in SBTs.37 As ERBB2 may regulate the KRAS signaling pathway, we also analyzed the mutational status of this gene in SBTs. Among 44 high-grade carcinomas, 34 (77%) tumors harbored TP53 nonsynonymous mutations or deletions and the majority of the mutations were homozygous changes (data not shown). Somatic mutations of either ERBB2, KRAS and BRAF occurred in 9.5%, 33% and 24% of SBTs, respectively. Most KRAS mutations were located at codon 12 and all BRAF mutations at codon 600, the hot spots of mutations for both genes. ERBB2 mutations occurred as a 12-bp insertion at the nucleotide 2313-2324 (Fig. 2 and Table 1). The mutations of ERBB2, KRAS and BRAF mutations were not shared in any of the SBTs. The high frequency of somatic mutations detected in those control genes with an unambiguous chromatogram tracing indicated that the purified samples used in this study had a high fraction of tumor cells and that the rare mutation of PIK3CA and lack of mutation in the AKT2 genes were not likely to be an artifact due to sample preparation or methods of PCR and nucleotide sequencing. In addition to somatic sequence mutations, gene amplification provides another mechanism to activate a kinase oncogenic pathway. Accordingly, we performed dual-color FISH to determine the amplification status of PIK3CA and AKT2 in both high-grade serous carcinomas and SBTs. Dual-color FISH was used because this method provides high sensitivity and specificity in counting gene copy number. PIK3CA and AKT2 gene copy numbers were assessed in the same tumor tissues to determine if there was a correlation of DNA copy number gain between both genes. FISH was first performed in the OVCAR3 ovarian cancer cell line and 6 representative high-grade serous carcinoma tissues in which the DNA copy 782 number changes in PIK3CA and AKT2 had been detected by digital karyotyping.38 The latter is a genome-wide technology to assess gene copy number by counting sequencing tags that represent different genomic loci.39 Digital karyotyping analysis showed that all samples lacked a discrete gain or amplification (< 5 Mb) in the 3q PIK3CA locus including the OVCAR3 cells which demonstrated a two-fold gain along the whole 3q arm (Fig. 3 and supplementary Fig. 1). Dual-color FISH was performed on the metaphase OVCAR3 cells and demonstrated a low level gain (~2 fold) at the PIK3CA locus in OVCAR3 cells (Fig. 3) which was consistent with a previous report.25 We correlated the PIK3CA gene copy number and mRNA expression levels and found only a marginal correlation (p = 0.04). Among those cases with gain of the PIK3CA region, four specimens showed downregulation of PIK3CA mRNA as compared to ovarian surface epithelial cultures. In contrast to this low level gain of PI3KCA, digital karyotyping showed a discrete 10 fold amplification spanning ~3 Mb of the AKT2 locus in the OVCAR3 cells (Fig. 2) and a 5 fold amplification in the other carcinoma tissues (Park et al, unpublished data). FISH analysis of this region showed a homogenously staining region (HSR) in tumor cells in both cases (Fig. 3). In the remainder of the cases, both target and reference probes showed equal signals in both PIK3CA and AKT2 loci. The analysis correlating digital karyotyping and FISH results validated the FISH method to determine DNA copy number of PIK3CA and AKT2 in archival paraffin tissues. Using the same probes, we performed dual-color FISH on a panel of paraffin tissues from different types of ovarian serous tumors and normal ovaries that were arranged in tissue microarrays. Based on the FISH analysis, we did not identify gain of PIK3CA gene copy number in 37 SBTs and 13 cases of surface epithelium from normal ovaries (Table 2). In contrast, PIK3CA amplification was detected in 8 of the 60 (13.3%) high-grade serous carcinomas in addition to 14 other cases with a polyploid pattern (same increased number of signals between PIK3CA and control probes) as these tumors were not considered to have amplification specific to PIK3CA. Among the amplified cases, only one showed a high level of amplification manifested as HSR and the other six cases were of low level gains (1.5-3 fold). FISH analysis also demonstrated that AKT2 did not amplify in any of the normal ovarian surface epithelial samples and SBTs (Table 3). In contrast, AKT2 amplification was observed in 12 of 66 (18.2%) high-grade serous carcinomas, the majority of which demonstrated high-level amplification as manifested by HSR. In addition, there were 8 cases showing a polyploid pattern (Table 3). Performing FISH analysis on the same tissues allowed us to assess the relationship of PIK3CA and AKT2 amplification in high-grade ovarian serous carcinomas. Four high-grade serous carcinomas and the OVCAR3 cell line showed coamplification of PIK3CA and AKT2. Cancer Biology & Therapy 2006; Vol. 5 Issue 7 Mutation Profiles of PIK3CA and AKT2 in Ovarian Cancer Figure 3. Digital karyotyping and dual-color FISH analysis of the PIK3CA and AKT2 copy number in OVCAR3 cells. Digital karyotyping of OVCAR3 cell line shows a two-fold increase in the entire 3q arm that harbors PIK3CA. FISH probes are designed to hybridize to the PIK3CA locus (red arrow) and a reference chromosomal region (green arrow). The centromeres are indicated by triangles. FISH analysis demonstrated an increase in the signals of PIK3CA with a ratio of PIK3CA/reference probe signal counts of 2. In contrast, the AKT2 region which is located on chromosome 19q is highly amplified in OVCAR3 cells and FISH shows a homogenously stained region pattern in tumor cells. Amplification in either PIK3CA and/or AKT2 occurred in 16 of 59 (27%) nonpolyploid cases. DISCUSSION In this study, we performed a comprehensive mutational analysis of exons 1, 9 and 20 of PIK3CA and the kinase domain of AKT2 in purified ovarian serous neoplasms including high-grade (conventional) serous carcinomas and serous borderline tumors (SBTs). In contrast to previous studies which analyzed nonpurified tumors,23,28,29 only purified tumor samples from surgical specimens were analyzed for sequence mutations in this study. Purified tumor cells are far superior for mutational analysis as DNA contamination from normal cells and the PCR/sequencing artifacts from formalin-fixed paraffin- embedded tissue are minimized. In addition, dual-color FISH provides a clear assessment of DNA copy number gains of PIK3CA and AKT2 in the same tumor tissues thereby allowing a determination of the relationship of amplification between both genes. Our findings demonstrate that compared to high-grade serous carcinomas, SBTs are characterized by mutations in the ERBB2-KRAS-BRAF signaling pathway while high-grade ovarian serous carcinomas have an increased gene copy number of the PIK3CA-AKT2 signaling pathway. These results shed new light on the pathogenesis of serous carcinoma of the ovary and may have important therapeutic implications. The low frequency (3%) of mutations of PIK3CA in both purified high-grade serous carcinomas and SBTs confirms what has been reported previously.23,28 These data and the lack of somatic mutations in the AKT2 kinase domain in all the samples tested indicates that activating sequence mutations of the PIK3CA-AKT2 pathway most likely do not play a significant role in the development of ovarian serous tumors. We also analyzed the copy number www.landesbioscience.com changes of PIK3CA and AKT2 in ovarian serous tumors using dual-color FISH, a method thought to provide a more sensitive and specific assessment of gene copy number in tissues. We found a low level gain (1.5-3 fold increase) in PIK3CA in the majority of PIK3CA amplified specimens. This is consistent with a previous report using FISH that showed a low copy number gain (1.5–2.5 fold) of PIK3CA in the majority of ovarian cancer cell lines.25 These results differ from another study reporting high level amplification (>7 fold) in 24.5% of ovarian cancers based on genomic real-time PCR.23 The discrepancy is probably due to the different methods used to measure the PIK3CA copy number. It is generally acknowledged that FISH analysis which is based on directly counting probe signals provides the most sensitive and specific method of assessing gene copy number, especially for samples with low copy number gains.40-42 In contrast, analysis of DNA copy number using genomic real-time PCR can be complicated by the primer selection, the presence of genomic repeats/pseudogenes that can lead to spurious results because of inappropriate primer set, as well as background noise inherent in real-time PCR. The frequency of high copy number gain in the PIK3CA gene is much lower than in the AKT2 gene which is known to be a frequently amplified oncogene in ovarian cancer.13 The molecular genetic findings in this report imply that amplification of the genes in the PIK3CA-AKT2 pathway may play an important role in the development of ovarian high-grade serous carcinoma. However, it is also likely that PIK3CA contributes to tumor development through another downstream mediator, other than AKT2, because amplification of AKT2 and gain of PIK3CA were found in a small set of 4 high-grade carcinomas and in the OVCAR3 cell line. The low level gains of PIK3CA may not have significant biological significance as the increased DNA copy number of PIK3CA only marginally correlates with its RNA copy number. In this study, we have shown overexpression of PIK3CA in some of ovarian high-grade serous carcinomas, a finding similar to a previous report demonstrating that PIK3CA protein (p110α) overexpression could be detected in of ovarian carcinomas.43 The identification of a 12 bp in-frame insertion mutation of ERBB2 in this study is of interest since sequence mutations have not been reported in ovarian neoplasms although amplification of ERBB2 has been extensively studied in ovarian carcinomas. The frequency and type of ERBB2 insertion mutation in SBTs are similar to those reported in lung adenocarcinomas31 but are different from gastric, colorectal and breast carcinomas.44 Although the number of tumors with ERBB2 mutations in this study was small, the mutually Cancer Biology & Therapy 783 Mutation Profiles of PIK3CA and AKT2 in Ovarian Cancer Table 3 DNA copy number changes of the AKT2 locus in ovarian tumors based on FISH analysis AKT2 locus status OSE SBT HG No gain or amplified 13 34 54 Low gain (1.5-3 fold) 0 0 4 8* High gain (> 3 fold) 0 0 Polyploidy 0 3 8 Total 13 37 74 OSE, ovarian surface epithelium from normal ovaries; SBT, serous borderline tumor; HG, high-grade serous carcinoma. *Cases show high-level amplification as manifested by homogenous staining region. exclusive pattern among ERBB2, KRAS and BRAF mutations suggests that each of the kinase genes has a similar effect in the development of SBTs. In conclusion, our findings indicate that amplification of genes in the PIK3CA-AKT2 pathway occurs in high-grade ovarian serous carcinomas and somatic mutations in the ERBB2-KRAS-BRAF pathway occur in SBTs. These findings provide further support for the dualistic model of ovarian serous tumorigenesis which proposes that low-grade serous carcinomas arise from a well characterized precursor, namely SBTs, whereas high grade serous carcinoma arises along an entirely different pathway in which morphologically characterized precursor lesions have not yet been identified.3,37 As small molecule kinase inhibitors show promise for the treatment of tumors with specific kinase activation,15,45,46 the findings in this study have potential clinical application for target-based therapy in patients with different types of ovarian serous neoplasms. References 1. Banks E, Beral V, Reeves G. The epidemiology of epithelial ovarian cancer: A review. Int J Gynecol Cancer 1997; 7:425-38. 2. 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