733984 research-article20172017
TUB0010.1177/1010428317733984Tumor BiologySadlecki et al.
Original Article
KRAS mutation testing in borderline ovarian tumors and low-grade ovarian carcinomas with a rapid, fully integrated molecular diagnostic system
Tumor Biology October 2017: 1–8 © The Author(s) 2017 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/1010428317733984 https://doi.org/10.1177/1010428317733984 journals.sagepub.com/home/tub
Pawel Sadlecki1, Paulina Antosik2, Dariusz Grzanka2, Marek Grabiec1 and Malgorzata Walentowicz-Sadlecka1
Abstract Epithelial ovarian neoplasms are a heterogeneous group of tumors, including various malignancies with distinct clinicopathologic and molecular features. Mutations in BRAF and KRAS genes are the most frequent genetic aberrations found in low-grade serous ovarian carcinomas and serous and mucinous borderline tumors. Implementation of targeted therapeutic strategies requires access to highly specific and highly sensitive diagnostic tests for rapid determination of mutation status. One candidate for such test is fully integrated, real-time polymerase chain reaction–based Idylla™ system for quick and simple detection of KRAS mutations in formaldehyde fixed-paraffin embedded tumor samples. The primary aim of this study was to verify whether fully integrated real-time polymerase chain reaction–based Idylla system may be useful in determination of KRAS mutation status in patients with borderline ovarian tumors and low-grade ovarian carcinomas. The study included tissue specimens from 37 patients with histopathologically verified ovarian masses, operated on at the Department of Obstetrics and Gynecology, Nicolaus Copernicus University Collegium Medicum in Bydgoszcz (Poland) between January 2009 and June 2012. Based on histopathological examination of surgical specimens, 30 lesions were classified as low-grade ovarian carcinomas and 7 as borderline ovarian tumors. Seven patients examined with Idylla KRAS Mutation Test tested positive for KRAS mutation. No statistically significant association was found between the incidence of KRAS mutations and histopathological type of ovarian tumors. Mean survival of the study subjects was 48.51 months (range 3–60 months). Presence of KRAS mutation did not exert a significant effect on the duration of survival in our series. Our findings suggest that Idylla KRAS Mutation Test may be a useful tool for rapid detection of KRAS mutations in ovarian tumor tissue. Keywords Low-grade ovarian cancer, borderline ovarian tumor, KRAS mutation Date received: 13 July 2017; accepted: 7 September 2017
Introduction Epithelial ovarian neoplasms are a heterogeneous group of tumors, including various malignancies with distinct clinicopathologic and molecular features. For many years, epithelial ovarian cancer was considered to be a single disease originating from ovarian surface epithelium or epithelium of cortical inclusion cysts, and having potential for divergent differentiation resulting in common occurrence of mixed tumors. Based on differences in clinical phenotype and genetic background, ovarian neoplasms are classified
1Department
of Obstetrics and Gynecology, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Torun, Bydgoszcz, Poland 2Department of Clinical Pathomorphology, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Torun, Bydgoszcz, Poland Corresponding author: Pawel Sadlecki, Department of Obstetrics and Gynecology, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Torun, ul. Ujejskiego 75, 85-168 Bydgoszcz, Poland. Email: [email protected]
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2 as low-grade and high-grade tumors.1 A large body of evidence suggests that the most common subtype of ovarian malignancy, high-grade serous carcinoma, usually originates from the epithelium of the distal fallopian tube.2,3 In turn, the majority of clear-cell and endometrioid carcinomas arise from endometriosis, and most low-grade serous cancers originate from serous borderline tumors.4 Better understanding of ovarian cancer risk factors and pathogenesis will likely contribute to earlier detection and better prevention of this malignancy and is fundamental for development of more effective therapies. The need for accurate histologic classification is particularly important in the current era of personalized therapy, when histologic type of a malignancy is frequently listed as an inclusion criterion of many clinical trials. Molecular testing still did not become a routine component in ovarian cancer management; however, this may soon change, as molecular biomarkers of this malignancy are a subject of extensive research. Mutations in BRAF and KRAS genes are the most frequent genetic aberrations found in low-grade serous ovarian carcinomas, serous borderline tumors, and mucinous cancers.5 Low-grade serous carcinomas with KRAS mutation seem to be more aggressive and more likely to recur than BRAF-positive malignancies.6,7 Both KRAS and BRAF are involved in RAS-RAF-mitogen/extracellular signal-regulated kinase (MEK), extracellular signal-regulated kinase (ERK), and mitogen-activated protein kinase (MAPK) pathways that regulate cell proliferation.8 Determination of molecular profile of the ovarian tumor requires access to highly specific and highly sensitive diagnostic tests for rapid determination of mutation status. One candidate for such test is the fully integrated real-time polymerase chain reaction (RT-PCR)-based Idylla™ system for quick and simple detection of KRAS mutations in formaldehyde fixed-paraffin embedded (FFPE) tumor samples.9 Idylla KRAS Mutation Test can detect 21 KRAS mutations: 7 mutations in codons 12 and 13 (exon 2), 9 mutations in codons 59 and 61 (exon 3), and 5 mutations in codons 117 and 146 (exon 4). The results are obtained quickly, and the risk of PCR contamination is limited to a minimum due to the closed nature of the cartridge. To the best of our knowledge, none of the previous studies analyzed application of the Idylla system in detection of KRAS mutations in patients with borderline ovarian tumors and ovarian carcinomas. The primary aim of this study was to verify whether, fully integrated RT-PCR-based Idylla system can be used for rapid determination of KRAS mutation status in patients with borderline ovarian tumors and low-grade ovarian carcinomas.
Material and methods The study included tissue specimens from 37 patients with histopathologically verified ovarian masses, operated on at
Tumor Biology Table 1. Clinicopathological characteristics of the study subjects.
Age (years) 70 Histotype Serous Mucinous Endometrioid Clear cell Serous borderline tumors FIGO (stage) IA IB IC Other Grade – G1 G2
N
%
5 5 18 4 5
13.5 13.5 48.6 10.9 13.5
13 3 5 9 7
35.1 8.1 13.5 24.4 18.9
23 5 7 2
62.1 13.6 18.9 5.4
7 8 22
18.9 21.7 59.4
FIGO: International Federation of Gynecology and Obstetrics
the Department of Obstetrics and Gynecology, Nicolaus Copernicus University Collegium Medicum in Bydgoszcz (Poland) between January 2009 and June 2012. All patients underwent surgical resection appropriate to the clinical stage of their malignancy and, whenever necessary, received adjuvant platinum–based chemotherapy in line with current Polish guidelines.10 Based on histopathological examination of surgical specimens, 30 lesions were classified as low-grade ovarian carcinomas and 7 as serous borderline ovarian tumors. Probability of 5-year survival was analyzed in relation to clinicopathological features. Clinicopathological characteristics of the study subjects are summarized in Table 1. Molecular studies of FFPE specimens were conducted at the Department of Clinical Pathology, Nicolaus Copernicus University, Collegium Medicum in Bydgoszcz. Idylla system is a fully automated molecular (CE-IVD) diagnostic platform (Biocartis, Mechelen, Belgium). Biocartis’ Idylla KRAS Mutation Test is suitable for detection of KRAS mutations from FFPE tissue sections. The test was shown to detect 21 clinically relevant KRAS mutations: seven mutations in codons 12 and 13 (exon 2), including p.G12C (c.34G>T), p.G12R (c.34G>C), p.G12S (c.34G>A), p.G12A (c.35G>C), p.G12D (c.35G>A), p.G12V (c.35G>T), and p.G13D (c.38G>A); nine mutations in codons 59 and 61 (exon 3), including p.A59E (c.176C>A), p.A59G (c.176C>G), p.A59T (c.175G>A), p.Q61K (c.181C>A; c.180_181TC>AA),
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Sadlecki et al. p.Q61L (c.182A>T), p.Q61R (c.182A>G), and p.Q61H (c.183A>C; c.183A>T); and five mutations in codons 117 and 146 (exon 4), among them p.K117N (c.351A>C; c.351A>T), p.A146P (c.436G>C), p.A146T (c.436G>A), and p.A146V (c.437C>T). Prior to the analysis, each sample was verified by two independent pathologists, to confirm that at least 10% of tumor cells were present in every section. Each test included a single 10 µm section (with at least 50-mm2 tumor area) of FFPE tissue. The specimen, protected by wet filter paper, was transferred to a cartridge and loaded to the Idylla instrument. During the test, isolated nucleic acids were separated to 5 PCR chambers via microfluidic channels inside the cartridge (Idylla KRAS). To provide appropriate RT-PCR amplification and detection, all reagents were used in a dry form. Allele-specific targets were identified with a fluorescence-based detection system. The result was available after approximately 130 min. Idylla software calculated quantification cycle (Cq) value for each valid PCR curve. Presence of a mutant genotype was determined based on the difference between Cq for wild-type KRAS and Cq for the PCR signal(s) of KRAS mutant. KRAS total, that is, wild-type gene, was used as a sample processing control, and colorectal cancer specimen with established KRAS mutation served as a positive control. The results were presented as “KRAS mutation detected,” along with a specific mutation type, or “no mutation detected.” The protocol of the study was approved by the Local Bioethics Committee at the Nicolaus Copernicus University, Collegium Medicum in Bydgoszcz (decision no. KB 413/2016), and written informed consent was sought from each patient or their next of kin. Statistical
analysis of the results was carried out with PQStat ver. 1.6.2.901 package. Regarding potential association between the incidence of KRAS mutations and histopathological type of ovarian tumor, the calculations were carried out with permutation Fisher test. Overall survivals were obtained as life-table and Kaplan–Meier estimates, and compared with log-rank, Wilcoxon–Breslow–Gehan, and Tarone–Ware tests. Potential predictors of survival were identified in Cox proportional-hazards regression model. The results were considered significant at p C). Another two KRAS mutations, in codon 12 G12A (p.Gly12Ala; nucleotide: c35G>C) and in codon 12 G12V (p.Gly12Val; nucleotide: c35G>T) were detected in lowgrade serous ovarian carcinomas. Finally, two patients, one with low-grade mucinous carcinoma and another with low-grade endometrioid carcinoma, carried KRAS mutations in codon 12 G12V (p.Gly12Val; nucleotide: c35G>T). Representative PCR curves and corresponding Cq values are shown in Figures 1–4. No statistically significant association was found between the incidence of KRAS mutations and histopathological type of ovarian tumors (p > 0.05; Table 2).
Figure 1. (a) Microphotograph presenting H&E stained specimens in ovarian tumor, primary objective magnification 4×, (b) PCR KRAS curves showing presence of KRAS-G12V mutation in mucinous carcinoma, and (c) the KRAS mutation results presented by Idylla.
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Tumor Biology
Figure 2. (a). Microphotograph presenting H&E stained specimens in ovarian tumor, primary objective magnification 4×, (b) PCR KRAS curves showing presence of KRAS-G12A mutation in serous carcinoma, and (c) the KRAS mutation result presented by Idylla.
Figure 3. (a) Microphotograph presenting H&E stained specimens in ovarian tumor, primary objective magnification 4×, (b) PCR KRAS curves showing presence of KRAS-G12D mutation in borderline tumor, and (c) the KRAS mutation result presented by Idylla.
Mean survival of the study subjects was 48.51 months (range 3–60 months). Presence of KRAS mutation did not exert a significant effect on the duration of survival in our series. Overall survivals were obtained based on life tables and Kaplan–Meier estimates, and compared with log-rank, Wilcoxon–Breslow–Gehan, and Tarone–Ware tests. The results are shown in Table 3. Irrespective of the statistical tests, presence of KRAS mutation exerted no significant effect on survival time. Statistical power of the log-rank test was estimated at 0.55.
Discussion Biological mechanisms leading to malignant transformation of ovarian surface epithelium are still not completely understood. Mutations in BRAF and KRAS genes are the most frequent genetic aberrations found in low-grade serous ovarian carcinomas and serous and mucinous borderline tumors.11 The RAS oncogene family comprises three principal members, KRAS, HRAS, and NRAS. All these genes have been implicated in the development of human malignancies. KRAS, located on chromosome
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Sadlecki et al.
Figure 4. (a) Microphotograph presenting H&E stained specimens in ovarian tumor, primary objective magnification 4×, (b) PCR KRAS curves showing no KRAS mutation detected in clear-cell carcinoma, and (c) the KRAS mutation results presented by Idylla. Table 2. Relationship between the incidence of KRAS mutations and histopathological type of ovarian tumors. Histopathological type
Serous borderline tumors Clear-cell carcinoma Endometrioid carcinoma Mucinous carcinoma Serous carcinoma p-value
Patients with KRAS mutation Other patients N
%
N
%
3
42.86
4 9 4 2 11
13.33 30.00 13.33 6.67 36.67
1 14.29 1 14.29 2 28.57 0.1835
Table 3. Comparison of survival times depending on the presence of KRAS mutation.
Chi-square statistics Degrees of freedom P-value
Log-rank test
Wilcoxon– Breslow– Gehan test
Tarone– Ware test
0.1066
0.1466
0.1257
1
1
1
0.7441
0.7018
0.7229
12p12, encodes a 21-kD protein (p21RAS) involved in MAP-kinase signal transduction pathway, responsible for control of cellular proliferation and differentiation. Presence of KRAS mutation contributes to constitutive activation of this signal transduction pathway and resultant uncontrolled proliferation and differentiation of cells.12,13 A plethora of previous studies documented presence of activated forms of KRAS in human malignancies from various locations, inter alia pancreas (60%), biliary tract (32%),
large and small intestine (32% and 20%, respectively), gastrointestinal tract (indeterminate location, 19%), thymus (15%), and endometrium (14%).14 The incidence of KRAS point mutations in epithelial ovarian carcinomas is estimated at 15% to 39%.14,15 KRAS and BRAF mutations have not been found in early-stage serous cystadenomas,16 but were detected in borderline serous tumors, as well as in adjacent cystadenoma epithelium of serous cystadenomas associated with small borderline tumors.17 While these mutations are present in serous borderline tumors and in either noninvasive or invasive micropapillary serous carcinomas, they are absent in high-grade serous carcinomas; this implies that at least two distinct pathways exist in serous ovarian carcinogenesis.18,19 KRAS/NRAS mutations are relatively frequent in advanced/recurrent low-grade serous cancers.20 Presence of KRAS mutations was also documented in mucinous, clear-cell, and endometrioid ovarian carcinomas.21–23 In this study, we found KRAS mutations in 7/37 patients with borderline ovarian tumors and low-grade ovarian carcinomas. No statistically significant relationship was observed between the incidence of KRAS mutations and histopathological type of ovarian malignancies. According to literature, KRAS and BRAF mutations are detected in approximately a third of borderline ovarian tumors and in a third of low-grade ovarian serous carcinomas.24 However, KRAS and BRAF mutations never coexist within the same tumor, which suggests that they are mutually exclusive. This hypothesis is also supported by the results of our present and previous studies; while we found BRAF or KRAS mutations in 6 out of 7 serous borderline tumors examined with the Idylla Mutation Test, these genetic defects were never present within the same specimen.25 Although the impact of KRAS mutation on survival
6 in ovarian malignancies is yet to be established, several previous studies found no correlation between these two parameters,26 and only few authors demonstrated a significant inverse relationship between them on multivariate analysis.27 Similar to most previous studies, we did not find a statistically significant association between the presence of KRAS mutation and 5-year survival rate in our group of patients with borderline ovarian tumors and lowgrade ovarian carcinomas. Point mutations in cancer cells can be detected with a number of various methods, including PCR, direct sequencing, multiplex PCR and array hybridization, and amplification-refractory mutation systems. The most popular method for molecular characterization of genetic variants is direct sequencing, which can detect all potential variations, including base substitutions, insertions, and deletions. However, direct sequencing has some limitations when applied to clinical samples. First, it is not sensitive enough (10%–30% level) to detect specific point mutations.28 Analytic sensitivity of this method can be improved by pyrosequencing, high-resolution melting analysis, real-time, or allele-specific PCR assays.29,30 Next-generation sequencing methods have high analytic sensitivity, offer broad reportable range of mutation spectrum, and provide adequate capacity in quantitative measurement of mutant allele frequencies and simultaneous detection of concomitant mutations.31 One of such novel methods suitable for detection of KRAS mutations is peptide nucleic acid–locked nucleic acid–mediated loop-mediated isothermal amplification (PNA-LNA mediated LAMP). Advantages of this nonPCR DNA amplification technique include high sensitivity and short reaction time, no longer than 50 min.32,33 Direct sequencing is still the best method to detect changes in codons 12 and 13 of KRAS gene, providing accurate information about the type of genetic anomalies, including nucleotide variant of the mutation. However, the quality of tests involving paraffin-embedded tissue specimens may be very low. Potential drawbacks of such tests include lack of amplification or ambiguous outcome of the sequencing associated with abundance of nonspecific background, resulting from DNA degradation. Another potential problem is the small quantity of cell clones in codons 12 and 13, resulting in the presence of low fluorographic peaks, hardly distinguished from the background.34 Information about molecular profile of the tumor, including its KRAS mutation status, is currently used to optimize treatment of various malignancies, among them colorectal cancer,35,36 non-small-cell lung cancer,37 and pancreatic cancer.38 Selection of an adequate targeted therapy on the basis of the result of molecular testing stimulated progress in treatment of various malignancies. Molecular testing has helped to identify patients who will most or least likely benefit from a
Tumor Biology given therapy, and accelerated the process of novel drug development. Application of direct sequencing as a routine method for cytological diagnosis in a hospital setting requires implementation of complicated procedures and purchase of expensive equipment. Another limitation of this method in everyday clinical practice is long analytical times. This stimulated a search for a simple, rapid, specific, and sensitive methodology to detect point mutations. Recently, some new molecular assays for detection of KRAS mutations have become available. These are fully automated molecular diagnostic systems for quantitative allele-specific RT-PCR-based analyses. Unlike currently available technologies for KRAS mutation detection, Idylla KRAS Mutation Test does not require manual preprocessing of the sample, that is, deparaffinization, digestion of FFPE tissue, or DNA extraction, since all these procedures are integrated within a single-use cartridge.39 Instead, whole FFPE tissue sections or macro-dissected FFPE specimens are inserted into the cartridge and processed by the Idylla system; also further stages, that is, RT-PCR-based mutation detection and result reporting, are fully integrated and automated.40 Without the need for highly skilled staff, mutational tests approved by the European Community for in vitro diagnostic use (labeled as CE-IVD) can genotype KRAS with a 5% detection limit, which allows for their use as a standardized method, even at diagnostic centers without a developed molecular infrastructure.41 Idylla KRAS Mutation Test can be used for detection of KRAS mutations directly from FFPE tissue specimens in approximately 2 h, with less than 2-min hands-on time. While the cost of a single Idylla cartridge is relatively high, this method does not require preprocessing of the sample, necessary in the case of other conventional techniques, and frequently representing a hidden cost.42 According to Colling et al.,43 Idylla System is a simple, time- and costeffective method for molecular testing, but has an important limitation, namely that it detects only the dominant mutation for a given specimen.44If the result of the test is ambiguous, in line with the rules of good laboratory practice, the presence of the mutation or lack thereof should be confirmed with two independent, adequately sensitive and accurate methods.34 Our findings suggest that Idylla KRAS Mutation Test may be a useful tool for rapid detection of KRAS mutations in ovarian tumor tissue. Introduction of personalized therapies has changed standards of care in selected cancer patients; presence of a critical somatic mutation in cancer cells improves accuracy of the diagnosis and prognosis, which eventually produces both health and economic benefits. This study demonstrated that the fully automated molecular diagnostic system Idylla is a promising tool for detection of KRAS mutations in histological specimens. Using this instrument, even pathologists from less experienced laboratories can easily integrate morphological
Sadlecki et al. findings with molecular data, which is crucial for further diagnostic and therapeutic decisions. Declaration of conflicting interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The author(s) received no financial support for the research, authorship, and/or publication of this article.
References 1. Bodurka DC, Deavers MT, Tian C, et al. Reclassification of serous ovarian carcinoma by a 2-tier system: a Gynecologic Oncology Group study. Cancer 2012; 118: 3087–3094. 2. Kurman RJ and Shih leM. Molecular pathogenesis and extraovarian origin of epithelial ovarian cancer—shifting the paradigm. Hum Pathol 2011; 42: 918–931. 3. Hollis RL and Gourley C. Genetic and molecular changes in ovarian cancer. Cancer Biol Med 2016; 13: 236–247. 4. Ramalingam P. Morphologic immunophenotypic, and molecular features of epithelial ovarian Cancer. Oncology 2016; 30(2): 166–176. 5. Grisham RN, Iyer G, Garg K, et al. BRAF mutation is associated with early stage disease and improved outcome in patients with low-grade serous ovarian cancer. Cancer 2013; 119(3): 548–554. 6. Sieben NL, Macropoulos P, Roemen GM, et al. In ovarian neoplasms, BRAF, but not KRAS, mutations are restricted to low-grade serous tumours. J Pathol 2004; 202: 336–340. 7. Singer G, Oldt R, Cohen Y, et al. Mutations in BRAF and KRAS characterize the development of low-grade ovarian serous carcinoma. J Natl Cancer Inst 2003; 95: 484–486. 8. Kolch W. Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions. Biochem J 2000; 351(Pt 2): 289–305. 9. Solassol J, Vendrell J, Märkl B, et al. Multi-center evaluation of the fully automated PCR-based Idylla™ KRAS mutation assay for rapid KRAS mutation status determination on formalin-fixed paraffin-embedded tissue of human colorectal cancer. PLoS One 2016; 11(9): e0163444. 10. Basta A, Bidziński M, Bieńkiewicz A, et al. Rekomendacje Polskiego Towarzystwa Ginekologii Onkologicznej dotyczące diagnostyki i leczenia raka jajnika, http://ptgo.pl/ dla-lekarza/rekomendacje-i-zalecenia/, 2015. 11. Nakamura K, Nakayama K, Ishibashi T, et al. KRAS/BRAF analysis in ovarian low-grade serous carcinoma having synchronous all pathological precursor regions. Int J Mol Sci 2016; 17: 625. 12. Keller JW, Haigis KM, Franklin JL, et al. Oncogenic K-RAS subverts the antiapoptotic role of N-RAS and alters modulation of the N-RAS: gelsolin complex. Oncogene 2007; 26(21): 3051–3059. 13. Patra SK. Ras regulation of DNA-methylation and cancer. Exp Cell Res 2008; 314(6): 1193–1201. 14. Karnoub AE and Weinberg RA. Ras oncogenes: split personalities. Nat Rev Mol Cell Biol 2008; 9(7): 517–531.
7 15. Tammela J and Odunsi K. Gene expression and prognostic significance in ovarian cancer. Minerva Ginecol 2004; 56(6): 495–502. 16. Cheng EJ, Kurman RJ, Wang M, et al. Molecular genetic analysis of ovarian serous cystadenomas. Lab Invest 2004; 84: 778–784. 17. Ho CL, Kurman RJ, Dehari R, et al. Mutations of BRAF and KRAS precede the development of ovarian serous borderline tumors. Cancer Res 2004; 64: 6915–6918. 18. Fujita M, Enomoto T, Murata Y, et al. Genetic alterations in ovarian carcinoma: with specific reference to histological subtypes. Mol Cell Endocrinol 2003; 202: 97–99. 19. Sugiyama T, Kamura T, Kigawa J, et al. Clinical characteristics of clear cell carcinoma of the ovary: a distinct histologic type with poor prognosis and resistance to platinum-based chemotherapy. Cancer 2000; 88: 2584–2589. 20. Della Pepa C, Tonini G, Santini D, et al. Low grade serous ovarian carcinoma: from the molecular characterization to the best therapeutic strategy. Cancer Treat Rev 2015; 41: 136–143. 21. Okuda T, Otsuka J, Sekizawa A, et al. P53 mutations and overexpression affect prognosis of ovarian endometrioid cancer but not clear cell cancer. Gynecol oncol 2003; 88: 318–325. 22. Mayr D, Hirschmann A, Löhrs U, et al. KRAS and BRAF mutations in ovarian tumors: a comprehensive study of invasive carcinomas, borderline tumors and extraovarian implants. Gynecol oncol 2006; 103(3): 883–887. 23. Dobrzycka B, Terlikowski SJ, Kowalczuk O, et al. Mutations in the KRAS gene in ovarian tumors. Folia Histochem Cytobiol 2009; 47(2): 221–224. 24. Singer G, Shih IM, Truskinovsky A, et al. Invasive mpsc (low-grade tumor) and conventional serous carcinoma (high-grade tumor). Int J Gynecol Pathol 2003; 22: 37–41. 25. Sadlecki P, Walentowicz P, Bodnar M, et al. Determination of BRAF V600E (VE1) protein expression and BRAF gene mutation status in codon 600 in borderline and low-grade ovarian cancers. Tumor Biology 2017; 39(5): 1010428317706230. 26. Varras MN, Sourvinos G, Diakomanolis E, et al. Detection and clinical correlations of ras gene mutations in human ovarian tumors. Oncology 1999; 56(2): 89–96. 27. Scambia G, Masciullo V, Benedetti Panici P, et al. Prognostic significance of ras/p21 alterations in human ovarian cancer. Br J Cancer 1997; 75(10): 1547–1553. 28. Anderson SM. Laboratory methods for KRAS mutation analysis. Expert Rev Mol Diagn 2011; 11: 635–642. 29. Dudley JC, Gurda GT, Tseng LH, et al. Tumor cellularity as a quality assurance measure for accurate clinical detection of BRAF mutations in melanoma. Mol Diagn Ther 2014; 18: 409–418. 30. Harbison CT, Horak CE, Ledeine JM, et al. Validation of companion diagnostic for detection of mutations in codons 12 and 13 of the KRAS gene in patients with metastatic colorectal cancer: analysis of the NCIC CTG CO.17 trial. Arch Pathol Lab Med 2013; 137: 820–827. 31. Haley L, Tseng L, Zheng G, et al. Performance characteristics of next-generation sequencing in clinical mutation detection of colorectal cancers. Mod Pathol 2015; 28: 1390–1399. 32. Notomi T, Okayama H, Masubuchi H, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 2000; 28: E63.
8 33. Itonaga M, Matsuzaki I, Warigaya K, et al. Novel methodology for rapid detection of KRAS mutation using PNA-LNA mediated loop-mediated isothermal amplification. PLoS One 2016; 11(3): e0151654. 34. Tysarowski A, Fabisiewicz A, Kolasa I, et al. Validation of selected molecular methods for the mutations determination in codons 12 and 13 of K-RAS gene in five Polish oncological research centers. Onkol Prak Klin 2008; 4(6): 232–244. 35. Khan SA, Zeng Z, Shia J, et al. EGFR gene amplification and KRAS mutation predict response to combination targeted therapy in metastatic colorectal cancer. Pathol Oncol Res 2017; 23(3): 673–677. 36. Allegra CJ, Jessup JM, Somerfield MR, et al. American Society of Clinical Oncology provisional clinical opinion: testing for KRAS gene mutations in patients with metastatic colorectal carcinoma to predict response to anti-epidermal growth factor receptor monoclonal antibody therapy. J Clin Oncol 2009; 27(12): 2091–2096. 37. Park KS, Yang H, Choi J, et al. The HSP90 inhibitor, NVPAUY922, attenuates intrinsic PI3K inhibitor resistance in KRAS-mutant non-small cell lung cancer. Cancer Lett 2017; 406: 47–53. 38. Zhao X, Wang X, Fang L, et al. A combinatorial strategy using YAP and pan-RAF inhibitors for treating KRASmutant pancreatic cancer. Cancer Lett 2017; 402: 61–70.
Tumor Biology 39. Sam SS, Lebel KA, Bissaillon CL, et al. Automation of genomic DNA isolation from formalin-fixed, paraffin-embedded tissues. Pathol Res Pract 2012; 208: 705–707. 40. Janku F, Claes B, Huang HJ, et al. BRAF mutation testing with a rapid, fully integrated molecular diagnostics system. Oncotarget 2015; 6(29): 26886–26894. 41. De Biase D, De Luca C, Gragnano G, et al. Fully automated PCR detection of KRAS mutations on pancreatic endoscopic ultrasound fine-needle aspirates. J Clinpathol 2016; 69: 986–991. 42. De Luca C, Vigliar E, d’Anna M, et al. KRAS detection on archival cytological smears by the novel fully automated polymerase chain reaction-based Idylla mutation test. Cytojournal 2017; 14: 5. 43. Colling R, Wang LM, Soilleux E, et al. Validating a fully automated real-time PCR-based system for use in the molecular diagnostic analysis of colorectal carcinoma: a comparison with NGS and IHC. J Clin Pathol 2017; 70: 610–614. 44. Weyn C, Van Raemdonck S, Dendooven R, et al. Clinical performance evaluation of a sensitive, rapid low-throughput test for KRAS mutation analysis using formalin-fixed, paraffin-embedded tissue samples. BMC Cancer 2017; 17: 139.
TUB0010.1177/1010428317733984Tumor BiologySadlecki et al.
Original Article
KRAS mutation testing in borderline ovarian tumors and low-grade ovarian carcinomas with a rapid, fully integrated molecular diagnostic system
Tumor Biology October 2017: 1–8 © The Author(s) 2017 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/1010428317733984 https://doi.org/10.1177/1010428317733984 journals.sagepub.com/home/tub
Pawel Sadlecki1, Paulina Antosik2, Dariusz Grzanka2, Marek Grabiec1 and Malgorzata Walentowicz-Sadlecka1
Abstract Epithelial ovarian neoplasms are a heterogeneous group of tumors, including various malignancies with distinct clinicopathologic and molecular features. Mutations in BRAF and KRAS genes are the most frequent genetic aberrations found in low-grade serous ovarian carcinomas and serous and mucinous borderline tumors. Implementation of targeted therapeutic strategies requires access to highly specific and highly sensitive diagnostic tests for rapid determination of mutation status. One candidate for such test is fully integrated, real-time polymerase chain reaction–based Idylla™ system for quick and simple detection of KRAS mutations in formaldehyde fixed-paraffin embedded tumor samples. The primary aim of this study was to verify whether fully integrated real-time polymerase chain reaction–based Idylla system may be useful in determination of KRAS mutation status in patients with borderline ovarian tumors and low-grade ovarian carcinomas. The study included tissue specimens from 37 patients with histopathologically verified ovarian masses, operated on at the Department of Obstetrics and Gynecology, Nicolaus Copernicus University Collegium Medicum in Bydgoszcz (Poland) between January 2009 and June 2012. Based on histopathological examination of surgical specimens, 30 lesions were classified as low-grade ovarian carcinomas and 7 as borderline ovarian tumors. Seven patients examined with Idylla KRAS Mutation Test tested positive for KRAS mutation. No statistically significant association was found between the incidence of KRAS mutations and histopathological type of ovarian tumors. Mean survival of the study subjects was 48.51 months (range 3–60 months). Presence of KRAS mutation did not exert a significant effect on the duration of survival in our series. Our findings suggest that Idylla KRAS Mutation Test may be a useful tool for rapid detection of KRAS mutations in ovarian tumor tissue. Keywords Low-grade ovarian cancer, borderline ovarian tumor, KRAS mutation Date received: 13 July 2017; accepted: 7 September 2017
Introduction Epithelial ovarian neoplasms are a heterogeneous group of tumors, including various malignancies with distinct clinicopathologic and molecular features. For many years, epithelial ovarian cancer was considered to be a single disease originating from ovarian surface epithelium or epithelium of cortical inclusion cysts, and having potential for divergent differentiation resulting in common occurrence of mixed tumors. Based on differences in clinical phenotype and genetic background, ovarian neoplasms are classified
1Department
of Obstetrics and Gynecology, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Torun, Bydgoszcz, Poland 2Department of Clinical Pathomorphology, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Torun, Bydgoszcz, Poland Corresponding author: Pawel Sadlecki, Department of Obstetrics and Gynecology, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Torun, ul. Ujejskiego 75, 85-168 Bydgoszcz, Poland. Email: [email protected]
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2 as low-grade and high-grade tumors.1 A large body of evidence suggests that the most common subtype of ovarian malignancy, high-grade serous carcinoma, usually originates from the epithelium of the distal fallopian tube.2,3 In turn, the majority of clear-cell and endometrioid carcinomas arise from endometriosis, and most low-grade serous cancers originate from serous borderline tumors.4 Better understanding of ovarian cancer risk factors and pathogenesis will likely contribute to earlier detection and better prevention of this malignancy and is fundamental for development of more effective therapies. The need for accurate histologic classification is particularly important in the current era of personalized therapy, when histologic type of a malignancy is frequently listed as an inclusion criterion of many clinical trials. Molecular testing still did not become a routine component in ovarian cancer management; however, this may soon change, as molecular biomarkers of this malignancy are a subject of extensive research. Mutations in BRAF and KRAS genes are the most frequent genetic aberrations found in low-grade serous ovarian carcinomas, serous borderline tumors, and mucinous cancers.5 Low-grade serous carcinomas with KRAS mutation seem to be more aggressive and more likely to recur than BRAF-positive malignancies.6,7 Both KRAS and BRAF are involved in RAS-RAF-mitogen/extracellular signal-regulated kinase (MEK), extracellular signal-regulated kinase (ERK), and mitogen-activated protein kinase (MAPK) pathways that regulate cell proliferation.8 Determination of molecular profile of the ovarian tumor requires access to highly specific and highly sensitive diagnostic tests for rapid determination of mutation status. One candidate for such test is the fully integrated real-time polymerase chain reaction (RT-PCR)-based Idylla™ system for quick and simple detection of KRAS mutations in formaldehyde fixed-paraffin embedded (FFPE) tumor samples.9 Idylla KRAS Mutation Test can detect 21 KRAS mutations: 7 mutations in codons 12 and 13 (exon 2), 9 mutations in codons 59 and 61 (exon 3), and 5 mutations in codons 117 and 146 (exon 4). The results are obtained quickly, and the risk of PCR contamination is limited to a minimum due to the closed nature of the cartridge. To the best of our knowledge, none of the previous studies analyzed application of the Idylla system in detection of KRAS mutations in patients with borderline ovarian tumors and ovarian carcinomas. The primary aim of this study was to verify whether, fully integrated RT-PCR-based Idylla system can be used for rapid determination of KRAS mutation status in patients with borderline ovarian tumors and low-grade ovarian carcinomas.
Material and methods The study included tissue specimens from 37 patients with histopathologically verified ovarian masses, operated on at
Tumor Biology Table 1. Clinicopathological characteristics of the study subjects.
Age (years) 70 Histotype Serous Mucinous Endometrioid Clear cell Serous borderline tumors FIGO (stage) IA IB IC Other Grade – G1 G2
N
%
5 5 18 4 5
13.5 13.5 48.6 10.9 13.5
13 3 5 9 7
35.1 8.1 13.5 24.4 18.9
23 5 7 2
62.1 13.6 18.9 5.4
7 8 22
18.9 21.7 59.4
FIGO: International Federation of Gynecology and Obstetrics
the Department of Obstetrics and Gynecology, Nicolaus Copernicus University Collegium Medicum in Bydgoszcz (Poland) between January 2009 and June 2012. All patients underwent surgical resection appropriate to the clinical stage of their malignancy and, whenever necessary, received adjuvant platinum–based chemotherapy in line with current Polish guidelines.10 Based on histopathological examination of surgical specimens, 30 lesions were classified as low-grade ovarian carcinomas and 7 as serous borderline ovarian tumors. Probability of 5-year survival was analyzed in relation to clinicopathological features. Clinicopathological characteristics of the study subjects are summarized in Table 1. Molecular studies of FFPE specimens were conducted at the Department of Clinical Pathology, Nicolaus Copernicus University, Collegium Medicum in Bydgoszcz. Idylla system is a fully automated molecular (CE-IVD) diagnostic platform (Biocartis, Mechelen, Belgium). Biocartis’ Idylla KRAS Mutation Test is suitable for detection of KRAS mutations from FFPE tissue sections. The test was shown to detect 21 clinically relevant KRAS mutations: seven mutations in codons 12 and 13 (exon 2), including p.G12C (c.34G>T), p.G12R (c.34G>C), p.G12S (c.34G>A), p.G12A (c.35G>C), p.G12D (c.35G>A), p.G12V (c.35G>T), and p.G13D (c.38G>A); nine mutations in codons 59 and 61 (exon 3), including p.A59E (c.176C>A), p.A59G (c.176C>G), p.A59T (c.175G>A), p.Q61K (c.181C>A; c.180_181TC>AA),
3
Sadlecki et al. p.Q61L (c.182A>T), p.Q61R (c.182A>G), and p.Q61H (c.183A>C; c.183A>T); and five mutations in codons 117 and 146 (exon 4), among them p.K117N (c.351A>C; c.351A>T), p.A146P (c.436G>C), p.A146T (c.436G>A), and p.A146V (c.437C>T). Prior to the analysis, each sample was verified by two independent pathologists, to confirm that at least 10% of tumor cells were present in every section. Each test included a single 10 µm section (with at least 50-mm2 tumor area) of FFPE tissue. The specimen, protected by wet filter paper, was transferred to a cartridge and loaded to the Idylla instrument. During the test, isolated nucleic acids were separated to 5 PCR chambers via microfluidic channels inside the cartridge (Idylla KRAS). To provide appropriate RT-PCR amplification and detection, all reagents were used in a dry form. Allele-specific targets were identified with a fluorescence-based detection system. The result was available after approximately 130 min. Idylla software calculated quantification cycle (Cq) value for each valid PCR curve. Presence of a mutant genotype was determined based on the difference between Cq for wild-type KRAS and Cq for the PCR signal(s) of KRAS mutant. KRAS total, that is, wild-type gene, was used as a sample processing control, and colorectal cancer specimen with established KRAS mutation served as a positive control. The results were presented as “KRAS mutation detected,” along with a specific mutation type, or “no mutation detected.” The protocol of the study was approved by the Local Bioethics Committee at the Nicolaus Copernicus University, Collegium Medicum in Bydgoszcz (decision no. KB 413/2016), and written informed consent was sought from each patient or their next of kin. Statistical
analysis of the results was carried out with PQStat ver. 1.6.2.901 package. Regarding potential association between the incidence of KRAS mutations and histopathological type of ovarian tumor, the calculations were carried out with permutation Fisher test. Overall survivals were obtained as life-table and Kaplan–Meier estimates, and compared with log-rank, Wilcoxon–Breslow–Gehan, and Tarone–Ware tests. Potential predictors of survival were identified in Cox proportional-hazards regression model. The results were considered significant at p C). Another two KRAS mutations, in codon 12 G12A (p.Gly12Ala; nucleotide: c35G>C) and in codon 12 G12V (p.Gly12Val; nucleotide: c35G>T) were detected in lowgrade serous ovarian carcinomas. Finally, two patients, one with low-grade mucinous carcinoma and another with low-grade endometrioid carcinoma, carried KRAS mutations in codon 12 G12V (p.Gly12Val; nucleotide: c35G>T). Representative PCR curves and corresponding Cq values are shown in Figures 1–4. No statistically significant association was found between the incidence of KRAS mutations and histopathological type of ovarian tumors (p > 0.05; Table 2).
Figure 1. (a) Microphotograph presenting H&E stained specimens in ovarian tumor, primary objective magnification 4×, (b) PCR KRAS curves showing presence of KRAS-G12V mutation in mucinous carcinoma, and (c) the KRAS mutation results presented by Idylla.
4
Tumor Biology
Figure 2. (a). Microphotograph presenting H&E stained specimens in ovarian tumor, primary objective magnification 4×, (b) PCR KRAS curves showing presence of KRAS-G12A mutation in serous carcinoma, and (c) the KRAS mutation result presented by Idylla.
Figure 3. (a) Microphotograph presenting H&E stained specimens in ovarian tumor, primary objective magnification 4×, (b) PCR KRAS curves showing presence of KRAS-G12D mutation in borderline tumor, and (c) the KRAS mutation result presented by Idylla.
Mean survival of the study subjects was 48.51 months (range 3–60 months). Presence of KRAS mutation did not exert a significant effect on the duration of survival in our series. Overall survivals were obtained based on life tables and Kaplan–Meier estimates, and compared with log-rank, Wilcoxon–Breslow–Gehan, and Tarone–Ware tests. The results are shown in Table 3. Irrespective of the statistical tests, presence of KRAS mutation exerted no significant effect on survival time. Statistical power of the log-rank test was estimated at 0.55.
Discussion Biological mechanisms leading to malignant transformation of ovarian surface epithelium are still not completely understood. Mutations in BRAF and KRAS genes are the most frequent genetic aberrations found in low-grade serous ovarian carcinomas and serous and mucinous borderline tumors.11 The RAS oncogene family comprises three principal members, KRAS, HRAS, and NRAS. All these genes have been implicated in the development of human malignancies. KRAS, located on chromosome
5
Sadlecki et al.
Figure 4. (a) Microphotograph presenting H&E stained specimens in ovarian tumor, primary objective magnification 4×, (b) PCR KRAS curves showing no KRAS mutation detected in clear-cell carcinoma, and (c) the KRAS mutation results presented by Idylla. Table 2. Relationship between the incidence of KRAS mutations and histopathological type of ovarian tumors. Histopathological type
Serous borderline tumors Clear-cell carcinoma Endometrioid carcinoma Mucinous carcinoma Serous carcinoma p-value
Patients with KRAS mutation Other patients N
%
N
%
3
42.86
4 9 4 2 11
13.33 30.00 13.33 6.67 36.67
1 14.29 1 14.29 2 28.57 0.1835
Table 3. Comparison of survival times depending on the presence of KRAS mutation.
Chi-square statistics Degrees of freedom P-value
Log-rank test
Wilcoxon– Breslow– Gehan test
Tarone– Ware test
0.1066
0.1466
0.1257
1
1
1
0.7441
0.7018
0.7229
12p12, encodes a 21-kD protein (p21RAS) involved in MAP-kinase signal transduction pathway, responsible for control of cellular proliferation and differentiation. Presence of KRAS mutation contributes to constitutive activation of this signal transduction pathway and resultant uncontrolled proliferation and differentiation of cells.12,13 A plethora of previous studies documented presence of activated forms of KRAS in human malignancies from various locations, inter alia pancreas (60%), biliary tract (32%),
large and small intestine (32% and 20%, respectively), gastrointestinal tract (indeterminate location, 19%), thymus (15%), and endometrium (14%).14 The incidence of KRAS point mutations in epithelial ovarian carcinomas is estimated at 15% to 39%.14,15 KRAS and BRAF mutations have not been found in early-stage serous cystadenomas,16 but were detected in borderline serous tumors, as well as in adjacent cystadenoma epithelium of serous cystadenomas associated with small borderline tumors.17 While these mutations are present in serous borderline tumors and in either noninvasive or invasive micropapillary serous carcinomas, they are absent in high-grade serous carcinomas; this implies that at least two distinct pathways exist in serous ovarian carcinogenesis.18,19 KRAS/NRAS mutations are relatively frequent in advanced/recurrent low-grade serous cancers.20 Presence of KRAS mutations was also documented in mucinous, clear-cell, and endometrioid ovarian carcinomas.21–23 In this study, we found KRAS mutations in 7/37 patients with borderline ovarian tumors and low-grade ovarian carcinomas. No statistically significant relationship was observed between the incidence of KRAS mutations and histopathological type of ovarian malignancies. According to literature, KRAS and BRAF mutations are detected in approximately a third of borderline ovarian tumors and in a third of low-grade ovarian serous carcinomas.24 However, KRAS and BRAF mutations never coexist within the same tumor, which suggests that they are mutually exclusive. This hypothesis is also supported by the results of our present and previous studies; while we found BRAF or KRAS mutations in 6 out of 7 serous borderline tumors examined with the Idylla Mutation Test, these genetic defects were never present within the same specimen.25 Although the impact of KRAS mutation on survival
6 in ovarian malignancies is yet to be established, several previous studies found no correlation between these two parameters,26 and only few authors demonstrated a significant inverse relationship between them on multivariate analysis.27 Similar to most previous studies, we did not find a statistically significant association between the presence of KRAS mutation and 5-year survival rate in our group of patients with borderline ovarian tumors and lowgrade ovarian carcinomas. Point mutations in cancer cells can be detected with a number of various methods, including PCR, direct sequencing, multiplex PCR and array hybridization, and amplification-refractory mutation systems. The most popular method for molecular characterization of genetic variants is direct sequencing, which can detect all potential variations, including base substitutions, insertions, and deletions. However, direct sequencing has some limitations when applied to clinical samples. First, it is not sensitive enough (10%–30% level) to detect specific point mutations.28 Analytic sensitivity of this method can be improved by pyrosequencing, high-resolution melting analysis, real-time, or allele-specific PCR assays.29,30 Next-generation sequencing methods have high analytic sensitivity, offer broad reportable range of mutation spectrum, and provide adequate capacity in quantitative measurement of mutant allele frequencies and simultaneous detection of concomitant mutations.31 One of such novel methods suitable for detection of KRAS mutations is peptide nucleic acid–locked nucleic acid–mediated loop-mediated isothermal amplification (PNA-LNA mediated LAMP). Advantages of this nonPCR DNA amplification technique include high sensitivity and short reaction time, no longer than 50 min.32,33 Direct sequencing is still the best method to detect changes in codons 12 and 13 of KRAS gene, providing accurate information about the type of genetic anomalies, including nucleotide variant of the mutation. However, the quality of tests involving paraffin-embedded tissue specimens may be very low. Potential drawbacks of such tests include lack of amplification or ambiguous outcome of the sequencing associated with abundance of nonspecific background, resulting from DNA degradation. Another potential problem is the small quantity of cell clones in codons 12 and 13, resulting in the presence of low fluorographic peaks, hardly distinguished from the background.34 Information about molecular profile of the tumor, including its KRAS mutation status, is currently used to optimize treatment of various malignancies, among them colorectal cancer,35,36 non-small-cell lung cancer,37 and pancreatic cancer.38 Selection of an adequate targeted therapy on the basis of the result of molecular testing stimulated progress in treatment of various malignancies. Molecular testing has helped to identify patients who will most or least likely benefit from a
Tumor Biology given therapy, and accelerated the process of novel drug development. Application of direct sequencing as a routine method for cytological diagnosis in a hospital setting requires implementation of complicated procedures and purchase of expensive equipment. Another limitation of this method in everyday clinical practice is long analytical times. This stimulated a search for a simple, rapid, specific, and sensitive methodology to detect point mutations. Recently, some new molecular assays for detection of KRAS mutations have become available. These are fully automated molecular diagnostic systems for quantitative allele-specific RT-PCR-based analyses. Unlike currently available technologies for KRAS mutation detection, Idylla KRAS Mutation Test does not require manual preprocessing of the sample, that is, deparaffinization, digestion of FFPE tissue, or DNA extraction, since all these procedures are integrated within a single-use cartridge.39 Instead, whole FFPE tissue sections or macro-dissected FFPE specimens are inserted into the cartridge and processed by the Idylla system; also further stages, that is, RT-PCR-based mutation detection and result reporting, are fully integrated and automated.40 Without the need for highly skilled staff, mutational tests approved by the European Community for in vitro diagnostic use (labeled as CE-IVD) can genotype KRAS with a 5% detection limit, which allows for their use as a standardized method, even at diagnostic centers without a developed molecular infrastructure.41 Idylla KRAS Mutation Test can be used for detection of KRAS mutations directly from FFPE tissue specimens in approximately 2 h, with less than 2-min hands-on time. While the cost of a single Idylla cartridge is relatively high, this method does not require preprocessing of the sample, necessary in the case of other conventional techniques, and frequently representing a hidden cost.42 According to Colling et al.,43 Idylla System is a simple, time- and costeffective method for molecular testing, but has an important limitation, namely that it detects only the dominant mutation for a given specimen.44If the result of the test is ambiguous, in line with the rules of good laboratory practice, the presence of the mutation or lack thereof should be confirmed with two independent, adequately sensitive and accurate methods.34 Our findings suggest that Idylla KRAS Mutation Test may be a useful tool for rapid detection of KRAS mutations in ovarian tumor tissue. Introduction of personalized therapies has changed standards of care in selected cancer patients; presence of a critical somatic mutation in cancer cells improves accuracy of the diagnosis and prognosis, which eventually produces both health and economic benefits. This study demonstrated that the fully automated molecular diagnostic system Idylla is a promising tool for detection of KRAS mutations in histological specimens. Using this instrument, even pathologists from less experienced laboratories can easily integrate morphological
Sadlecki et al. findings with molecular data, which is crucial for further diagnostic and therapeutic decisions. Declaration of conflicting interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The author(s) received no financial support for the research, authorship, and/or publication of this article.
References 1. Bodurka DC, Deavers MT, Tian C, et al. Reclassification of serous ovarian carcinoma by a 2-tier system: a Gynecologic Oncology Group study. Cancer 2012; 118: 3087–3094. 2. Kurman RJ and Shih leM. Molecular pathogenesis and extraovarian origin of epithelial ovarian cancer—shifting the paradigm. Hum Pathol 2011; 42: 918–931. 3. Hollis RL and Gourley C. Genetic and molecular changes in ovarian cancer. Cancer Biol Med 2016; 13: 236–247. 4. Ramalingam P. Morphologic immunophenotypic, and molecular features of epithelial ovarian Cancer. Oncology 2016; 30(2): 166–176. 5. Grisham RN, Iyer G, Garg K, et al. BRAF mutation is associated with early stage disease and improved outcome in patients with low-grade serous ovarian cancer. Cancer 2013; 119(3): 548–554. 6. Sieben NL, Macropoulos P, Roemen GM, et al. In ovarian neoplasms, BRAF, but not KRAS, mutations are restricted to low-grade serous tumours. J Pathol 2004; 202: 336–340. 7. Singer G, Oldt R, Cohen Y, et al. Mutations in BRAF and KRAS characterize the development of low-grade ovarian serous carcinoma. J Natl Cancer Inst 2003; 95: 484–486. 8. Kolch W. Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions. Biochem J 2000; 351(Pt 2): 289–305. 9. Solassol J, Vendrell J, Märkl B, et al. Multi-center evaluation of the fully automated PCR-based Idylla™ KRAS mutation assay for rapid KRAS mutation status determination on formalin-fixed paraffin-embedded tissue of human colorectal cancer. PLoS One 2016; 11(9): e0163444. 10. Basta A, Bidziński M, Bieńkiewicz A, et al. Rekomendacje Polskiego Towarzystwa Ginekologii Onkologicznej dotyczące diagnostyki i leczenia raka jajnika, http://ptgo.pl/ dla-lekarza/rekomendacje-i-zalecenia/, 2015. 11. Nakamura K, Nakayama K, Ishibashi T, et al. KRAS/BRAF analysis in ovarian low-grade serous carcinoma having synchronous all pathological precursor regions. Int J Mol Sci 2016; 17: 625. 12. Keller JW, Haigis KM, Franklin JL, et al. Oncogenic K-RAS subverts the antiapoptotic role of N-RAS and alters modulation of the N-RAS: gelsolin complex. Oncogene 2007; 26(21): 3051–3059. 13. Patra SK. Ras regulation of DNA-methylation and cancer. Exp Cell Res 2008; 314(6): 1193–1201. 14. Karnoub AE and Weinberg RA. Ras oncogenes: split personalities. Nat Rev Mol Cell Biol 2008; 9(7): 517–531.
7 15. Tammela J and Odunsi K. Gene expression and prognostic significance in ovarian cancer. Minerva Ginecol 2004; 56(6): 495–502. 16. Cheng EJ, Kurman RJ, Wang M, et al. Molecular genetic analysis of ovarian serous cystadenomas. Lab Invest 2004; 84: 778–784. 17. Ho CL, Kurman RJ, Dehari R, et al. Mutations of BRAF and KRAS precede the development of ovarian serous borderline tumors. Cancer Res 2004; 64: 6915–6918. 18. Fujita M, Enomoto T, Murata Y, et al. Genetic alterations in ovarian carcinoma: with specific reference to histological subtypes. Mol Cell Endocrinol 2003; 202: 97–99. 19. Sugiyama T, Kamura T, Kigawa J, et al. Clinical characteristics of clear cell carcinoma of the ovary: a distinct histologic type with poor prognosis and resistance to platinum-based chemotherapy. Cancer 2000; 88: 2584–2589. 20. Della Pepa C, Tonini G, Santini D, et al. Low grade serous ovarian carcinoma: from the molecular characterization to the best therapeutic strategy. Cancer Treat Rev 2015; 41: 136–143. 21. Okuda T, Otsuka J, Sekizawa A, et al. P53 mutations and overexpression affect prognosis of ovarian endometrioid cancer but not clear cell cancer. Gynecol oncol 2003; 88: 318–325. 22. Mayr D, Hirschmann A, Löhrs U, et al. KRAS and BRAF mutations in ovarian tumors: a comprehensive study of invasive carcinomas, borderline tumors and extraovarian implants. Gynecol oncol 2006; 103(3): 883–887. 23. Dobrzycka B, Terlikowski SJ, Kowalczuk O, et al. Mutations in the KRAS gene in ovarian tumors. Folia Histochem Cytobiol 2009; 47(2): 221–224. 24. Singer G, Shih IM, Truskinovsky A, et al. Invasive mpsc (low-grade tumor) and conventional serous carcinoma (high-grade tumor). Int J Gynecol Pathol 2003; 22: 37–41. 25. Sadlecki P, Walentowicz P, Bodnar M, et al. Determination of BRAF V600E (VE1) protein expression and BRAF gene mutation status in codon 600 in borderline and low-grade ovarian cancers. Tumor Biology 2017; 39(5): 1010428317706230. 26. Varras MN, Sourvinos G, Diakomanolis E, et al. Detection and clinical correlations of ras gene mutations in human ovarian tumors. Oncology 1999; 56(2): 89–96. 27. Scambia G, Masciullo V, Benedetti Panici P, et al. Prognostic significance of ras/p21 alterations in human ovarian cancer. Br J Cancer 1997; 75(10): 1547–1553. 28. Anderson SM. Laboratory methods for KRAS mutation analysis. Expert Rev Mol Diagn 2011; 11: 635–642. 29. Dudley JC, Gurda GT, Tseng LH, et al. Tumor cellularity as a quality assurance measure for accurate clinical detection of BRAF mutations in melanoma. Mol Diagn Ther 2014; 18: 409–418. 30. Harbison CT, Horak CE, Ledeine JM, et al. Validation of companion diagnostic for detection of mutations in codons 12 and 13 of the KRAS gene in patients with metastatic colorectal cancer: analysis of the NCIC CTG CO.17 trial. Arch Pathol Lab Med 2013; 137: 820–827. 31. Haley L, Tseng L, Zheng G, et al. Performance characteristics of next-generation sequencing in clinical mutation detection of colorectal cancers. Mod Pathol 2015; 28: 1390–1399. 32. Notomi T, Okayama H, Masubuchi H, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 2000; 28: E63.
8 33. Itonaga M, Matsuzaki I, Warigaya K, et al. Novel methodology for rapid detection of KRAS mutation using PNA-LNA mediated loop-mediated isothermal amplification. PLoS One 2016; 11(3): e0151654. 34. Tysarowski A, Fabisiewicz A, Kolasa I, et al. Validation of selected molecular methods for the mutations determination in codons 12 and 13 of K-RAS gene in five Polish oncological research centers. Onkol Prak Klin 2008; 4(6): 232–244. 35. Khan SA, Zeng Z, Shia J, et al. EGFR gene amplification and KRAS mutation predict response to combination targeted therapy in metastatic colorectal cancer. Pathol Oncol Res 2017; 23(3): 673–677. 36. Allegra CJ, Jessup JM, Somerfield MR, et al. American Society of Clinical Oncology provisional clinical opinion: testing for KRAS gene mutations in patients with metastatic colorectal carcinoma to predict response to anti-epidermal growth factor receptor monoclonal antibody therapy. J Clin Oncol 2009; 27(12): 2091–2096. 37. Park KS, Yang H, Choi J, et al. The HSP90 inhibitor, NVPAUY922, attenuates intrinsic PI3K inhibitor resistance in KRAS-mutant non-small cell lung cancer. Cancer Lett 2017; 406: 47–53. 38. Zhao X, Wang X, Fang L, et al. A combinatorial strategy using YAP and pan-RAF inhibitors for treating KRASmutant pancreatic cancer. Cancer Lett 2017; 402: 61–70.
Tumor Biology 39. Sam SS, Lebel KA, Bissaillon CL, et al. Automation of genomic DNA isolation from formalin-fixed, paraffin-embedded tissues. Pathol Res Pract 2012; 208: 705–707. 40. Janku F, Claes B, Huang HJ, et al. BRAF mutation testing with a rapid, fully integrated molecular diagnostics system. Oncotarget 2015; 6(29): 26886–26894. 41. De Biase D, De Luca C, Gragnano G, et al. Fully automated PCR detection of KRAS mutations on pancreatic endoscopic ultrasound fine-needle aspirates. J Clinpathol 2016; 69: 986–991. 42. De Luca C, Vigliar E, d’Anna M, et al. KRAS detection on archival cytological smears by the novel fully automated polymerase chain reaction-based Idylla mutation test. Cytojournal 2017; 14: 5. 43. Colling R, Wang LM, Soilleux E, et al. Validating a fully automated real-time PCR-based system for use in the molecular diagnostic analysis of colorectal carcinoma: a comparison with NGS and IHC. J Clin Pathol 2017; 70: 610–614. 44. Weyn C, Van Raemdonck S, Dendooven R, et al. Clinical performance evaluation of a sensitive, rapid low-throughput test for KRAS mutation analysis using formalin-fixed, paraffin-embedded tissue samples. BMC Cancer 2017; 17: 139.
