Analysis of PIK3CA Mutations and Activation Pathways in Triple Negative Breast Cancer

Paolo Cossu-Rocca, Sandra Orrù, Maria Rosaria Muroni, Francesca Sanges, Giovanni Sotgiu, Sara Ena, Giovanna Pira, Luciano Murgia, Alessandra Manca, Maria Gabriela Uras, Maria Giuseppina Sarobba, Silvana Urru, Maria Rosaria De Miglio, Paolo Cossu-Rocca, Sandra Orrù, Maria Rosaria Muroni, Francesca Sanges, Giovanni Sotgiu, Sara Ena, Giovanna Pira, Luciano Murgia, Alessandra Manca, Maria Gabriela Uras, Maria Giuseppina Sarobba, Silvana Urru, Maria Rosaria De Miglio

Abstract

Background: Triple Negative Breast Cancer (TNBC) accounts for 12-24% of all breast carcinomas, and shows worse prognosis compared to other breast cancer subtypes. Molecular studies demonstrated that TNBCs are a heterogeneous group of tumors with different clinical and pathologic features, prognosis, genetic-molecular alterations and treatment responsivity. The PI3K/AKT is a major pathway involved in the regulation of cell survival and proliferation, and is the most frequently altered pathway in breast cancer, apparently with different biologic impact on specific cancer subtypes. The most common genetic abnormality is represented by PIK3CA gene activating mutations, with an overall frequency of 20-40%. The aims of our study were to investigate PIK3CA gene mutations on a large series of TNBC, to perform a wider analysis on genetic alterations involving PI3K/AKT and BRAF/RAS/MAPK pathways and to correlate the results with clinical-pathologic data.

Materials and methods: PIK3CA mutation analysis was performed by using cobas® PIK3CA Mutation Test. EGFR, AKT1, BRAF, and KRAS genes were analyzed by sequencing. Immunohistochemistry was carried out to identify PTEN loss and to investigate for PI3K/AKT pathways components.

Results: PIK3CA mutations were detected in 23.7% of TNBC, whereas no mutations were identified in EGFR, AKT1, BRAF, and KRAS genes. Moreover, we observed PTEN loss in 11.3% of tumors. Deregulation of PI3K/AKT pathways was revealed by consistent activation of pAKT and p-p44/42 MAPK in all PIK3CA mutated TNBC.

Conclusions: Our data shows that PIK3CA mutations and PI3K/AKT pathway activation are common events in TNBC. A deeper investigation on specific TNBC genomic abnormalities might be helpful in order to select patients who would benefit from current targeted therapy strategies.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. Morphologic and immunohistochemical features of…
Fig 1. Morphologic and immunohistochemical features of Triple Negative Breast Cancer.
(A) Haematoxylin & Eosin stain illustrates a Triple Negative variant with features of high grade invasive ductal carcinoma (original magnification 100X); (B) Immunohistochemistry for EGFR displaying diffuse and moderate membranous and membranous-cytoplasmic immunoreactivity (original magnification 100X); (C) Immunohistochemistry for CK5/6 showing diffuse and intense cytoplasmic immunoreactivity (original magnification 100X); (D) Immunohistochemistry for p- AKT showing diffuse and intense nuclear immunoreactivity (original magnification 100X); (E) Immunohistochemistry for p-p44/42 MAPK displaying diffuse and intense nuclear-cytoplasmic immunoreactivity (original magnification 100X); (F) Immunostaining for PTEN showing diffuse and intense nuclear immunoreactivity (original magnification 100X).

References

    1. Ban KA, Godellas CV. Epidemiology of breast cancer. Surg Oncol Clin N Am. 2014; 23(3):409–422. 10.1016/j.soc.2014.03.011
    1. Herschkowitz JI, Simin K, Weigman VJ, Mikaelian I, Usary J, hu Z, et al. Identification of conserved gene expression features between murine mammary carcinoma models and human breast tumors. Genome Biol. 2007; 8(5):R76
    1. Perou CM, Sorlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA, et al. Molecular portraits of human breast tumours. Nature. 2000; 406(6797):747–752.
    1. Sorlie T, Tibshirani R, Parker J, Hastie T, Marron JS, Nobel A, et al. Repeated observation of breast tumor subtypes in independent gene expression data sets. Proc Natl Acad Sci USA. 2003; 100(14):8418–8423.
    1. Sorlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci USA. 2001; 98(19):10869–10874.
    1. De Abreu FB, Wells WA, Tsongalis GJ. The emerging role of the molecular diagnostics laboratory in breast cancer personalized medicine. Am J Pathol. 2013; 183(4):1075–1083. 10.1016/j.ajpath.2013.07.002
    1. Weigelt B, Horlings HM, Kreike B, Hayes MM, Hauptmann M, Wessels LF, et al. Refinement of breast cancer classification by molecular characterization of histological special types. J Pathol. 2008; 216(2):141–150. 10.1002/path.2407
    1. The-Cancer-Genome-Atlas-Network. Comprehensive molecular portraits of human breast tumours. Nature. 2012; 490(7418):61–70. 10.1038/nature11412
    1. Oakman C, Viale G, Di Leo A. Management of triple negative breast cancer. Breast. 2010; 19(5):312–321. 10.1016/j.breast.2010.03.026
    1. Metzger-Filho O, Tutt A, de Azambuja E, Saini KS, Viale G, Loi S, et al. Dissecting the heterogeneity of triple-negative breast cancer. J Clin Oncol. 2012; 30(15):1879–1887. 10.1200/JCO.2011.38.2010
    1. Lehmann BD, Bauer JA, Chen X, Sanders ME, Chakravarthy AB, Shyr Y, et al. Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J Clin Invest. 2011; 121(7):2750–2767. 10.1172/JCI45014
    1. Carey L, Winer E, Viale G, Cameron D, Gianni L. Triple-negative breast cancer: disease entity or title of convenience? Nat Rev Clin Oncol. 2010; 7(12):683–692. 10.1038/nrclinonc.2010.154
    1. Reis-Filho JS, Tutt AN. Triple negative tumours: a critical review. Histopathology. 2008; 52(1):108–118. 10.1111/j.1365-2559.2007.02889.x
    1. Tokunaga E, Oki E, Egashira A, Sadanaga N, Morita M, Kakeji Y, et al. Deregulation of the Akt pathway in human cancer. Curr Cancer Drug Targets. 2008; 8(1):27–36.
    1. Abramson VG, Cooper Lloyd M, Ballinger T, Sanders ME, Du L, Lai D, et al. Characterization of breast cancers with PI3K mutations in an academic practice setting using SNaPshot profiling. Breast Cancer Res Treat. 2014; 145(2):389–399. 10.1007/s10549-014-2945-3
    1. Bachman KE, Argani P, Samuels Y, Silliman N, Ptak J, Szabo S, et al. The PIK3CA gene is mutated with high frequency in human breast cancers. Cancer Biol Ther. 2004; 3(8):772–775.
    1. Kandula M, Chennaboina KK, Ys AR, Raju S. Phosphatidylinositol 3-kinase (PI3KCA) oncogene mutation analysis and gene expression profiling in primary breast cancer patients. Asian Pac J Cancer Prev. 2013; 14(9):5067–5072.
    1. Stemke-Hale K, Gonzalez-Angulo AM, Lluch A, Neve RM, Kuo WL, Davies M, et al. An integrative genomic and proteomic analysis of PIK3CA, PTEN, and AKT mutations in breast cancer. Cancer Res. 2008; 68(15):6084–6091. 10.1158/0008-5472.CAN-07-6854
    1. Shah SP, Roth A, Goya R, Oloumi A, Ha G, Zhao Y, et al. The clonal and mutational evolution spectrum of primary triple-negative breast cancers. Nature. 2012; 486(7403):395–399. 10.1038/nature10933
    1. Boyault S, Drouet Y, Navarro C, Bachelot T, Lasset C, Treilleux I, et al. Mutational characterization of individual breast tumors: TP53 and PI3K pathway genes are frequently and distinctively mutated in different subtypes. Breast Cancer Res Treat. 2012; 132: 29–39. 10.1007/s10549-011-1518-y
    1. Arsenic R, Lehmann A, Budczies J, Koch I, Prinzler J, Kleine-Tebbe A, et al. Analysis of PIK3CA mutations in breast cancer subtypes. Appl Immunohistochem Mol Morphol. 2014; 22: 50–56.
    1. Millis SZ, Gatalica Z, Winkler J, Vranic S, Kimbrough J, Reddy S, et al. Predictive biomarker profiling of > 6000 breast cancer patients shows heterogeneity in TNBC, with treatment implications. Clin Breast Cancer. 2015. April 28 pii: S1526-8209(15)00098–1. 10.1016/j.clbc.2015.04.008
    1. Kriegsmann M, Endris V, Wolf T, Pfarr N, Stenzinger A, Loibl S, et al. Mutational profiles in triple-negative breast cancer defined by ultradeep multigene sequencing show high rates of PI3K pathway alterations and clinically relevant entity subgroup specific differences. Oncotarget. 2014; 5(20):9952–9965.
    1. Hashimoto K, Tsuda H, Koizumi F, Shimizu C, Yonemori K, Ando M, et al. Activated PI3K/AKT and MAPK pathways are potential good prognostic markers in node-positive, triple-negative breast cancer. Ann Oncol. 2014; 25(10):1973–1979. 10.1093/annonc/mdu247
    1. Colditz G, Chia KS, Wilson C, Britton P, Marraw M, Rutgers E, et al. Invasive breast carcinoma: introduction and general features In: Lakhani SR, Ellis IO, Schnitt SJ, Tan PH, van de Vijver MJ, editors. World Health Organization Classification of Tumours WHO Classification of Tumours of the Breast, 4th Edition. Lyon: IARC; 2012. pp. 14–31.
    1. De Miglio MR, Mura A, Uras MG, Manca A, Contini M, Murgia L, et al. High sensitivity of reverse-hybridization methodology in the detection of KRAS mutations from formalin-fixed paraffin-embedded colorectal cancer samples. Diagn Mol Pathol. 2010; 19(4):201–208. 10.1097/PDM.0b013e3181db67d5
    1. Sar M, Lubahn DB, French FS, Wilson EM. Immunohistochemical localization of the androgen receptor in rat and human tissues. Endocrinology. 1990; 127(6):3180–3186.
    1. Ordonez NG. Value of cytokeratin 5/6 immunostaining in distinguishing epithelial mesothelioma of the pleura from lung adenocarcinoma. Am J Surg Pathol. 1998; 22(10):1215–1221.
    1. Nicholson KM, Anderson NG. The protein kinase B/Akt signalling pathway in human malignancy. Cell Signal. 2002; 14(5):381–395.
    1. Baccarini M. Second nature: biological functions of the Raf-1 "kinase". FEBS Lett, 2005; 579(15):3271–3277.
    1. Mutter GL, Lin MC, Fitzgerald JT, Kum JB, Baak JP, Lees JA, et al. Altered PTEN expression as a diagnostic marker for the earliest endometrial precancers. J Natl Cancer Inst. 2000; 92(11):924–930.
    1. Carpenter G, Cohen S. Epidermal growth factor. J Biol Chem. 1990; 265(14):7709–7712.
    1. Hammond ME, Hayes DF, Wolff AC, Mangu PB, Temin S, Badve S, et al. American society of clinical oncology/college of american pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer. J Oncol Pract. 2010; 6: 195–197. 10.1200/JOP.777003
    1. Dupont Jensen J, Laenkholm AV, Knoop A, Ewertz M, Bandaru R, Liu W, et al. PIK3CA mutations may be discordant between primary and corresponding metastatic disease in breast cancer. Clin Cancer Res. 2011; 17: 667–677. 10.1158/1078-0432.CCR-10-1133
    1. Kalinsky K, Jacks LM, Heguy A, Patil S, Drobnjak M, Bhanot UK, et al. PIK3CA mutation associates with improved outcome in breast cancer. Clin Cancer Res. 2009; 15(16):5049–5059. 10.1158/1078-0432.CCR-09-0632
    1. Cizkova M, Susini A, Vacher S, Cizeron-Clairac G, Andrieu C, Driouch K, et al. PIK3CA mutation impact on survival in breast cancer patients and in ERalpha, PR and ERBB2-based subgroups. Breast Cancer Res. 2012; 14(1):R28
    1. Saal LH, Holm K, Maurer M, Memeo L, Su T, Wang X, et al. PIK3CA mutations correlate with hormone receptors, node metastasis, and ERBB2, and are mutually exclusive with PTEN loss in human breast carcinoma. Cancer Res. 2005; 65(7):2554–2559.
    1. Maruyama N, Miyoshi Y, Taguchi T, Tamaki Y, Monden M, Noguchi S, et al. Clinicopathologic analysis of breast cancers with PIK3CA mutations in Japanese women. Clin Cancer Res. 2007; 13(2Pt1):408–414.
    1. Gonzalez-Angulo AM, Chen H, Karuturi MS, Chavez-MacGregor M, Tsavachidis S, Meric-Bernstam F, et al. Frequency of mesenchymal-epithelial transition factor gene (MET) and the catalytic subunit of phosphoinositide-3-kinase (PIK3CA) copy number elevation and correlation with outcome in patients with early stage breast cancer. Cancer. 2013; 119(1):7–15. 10.1002/cncr.27608
    1. Buttitta F, Felicioni L, Barassi F, Martella C, Paolizzi D, Fresu G, et al. PIK3CA mutation and histological type in breast carcinoma: high frequency of mutations in lobular carcinoma. J Pathol. 2006; 208(3): 350–355.
    1. Kim Y, Kim J, Lee HD, Jeong J, Lee W, Lee W, et al. Spectrum of EGFR gene copy number changes and KRAS gene mutation status in Korean triple negative breast cancer patients. PLoS One. 2013; 8(10):e79014 10.1371/journal.pone.0079014
    1. Sanchez-Munoz A, Gallego E, de Luque V, Perez-Rivas LG, Vicioso L, Ribelles N, et al. Lack of evidence for KRAS oncogenic mutations in triple-negative breast cancer. BMC Cancer. 2010; 10:136–144. 10.1186/1471-2407-10-136
    1. Tilch E, Seidens T, Cocciardi S, Reid LE, Byrne D, Simpson PT, et al. Mutations in EGFR, BRAF and RAS are rare in triple-negative and basal-like breast cancers from Caucasian women. Breast Cancer Res Treat. 2014; 143(2): 385–392. 10.1007/s10549-013-2798-1
    1. Secq V, Villeret J, Fina F, Carmassi M, Carcopino X, Garcia S, et al. Triple negative breast carcinoma EGFR amplification is not associated with EGFR, Kras or ALK mutations. Br J Cancer. 2014; 110(4):1045–1052. 10.1038/bjc.2013.794
    1. Martin V, Botta F, Zanellato E, Molinari F, Crippa S, Mazzucchelli L, et al. Molecular characterization of EGFR and EGFR-downstream pathways in triple negative breast carcinomas with basal like features. Histol Histopathol. 2012; 27(6):785–792.
    1. Grob TJ, Heilenkotter U, Geist S, Paluchowski P, Wilke C, Jaenicke F, et al. Rare oncogenic mutations of predictive markers for targeted therapy in triple-negative breast cancer. Breast Cancer Res Treat. 2012; 134(2):561–567. 10.1007/s10549-012-2092-7
    1. Tanaka Y, Kanai F, Tada M, Asaoka Y, Guleng B, Jazag A, et al. Absence of PIK3CA hotspot mutations in hepatocellular carcinoma in Japanese patients. Oncogene. 2006; 25(20):2950–2952.
    1. Perez-Tenorio G, Alkhori L, Olsson B, Waltersson MA, Nordenskjold B, Rutqvist LE, et al. PIK3CA mutations and PTEN loss correlate with similar prognostic factors and are not mutually exclusive in breast cancer. Clin Cancer Res. 2007; 13(12):3577–3584.
    1. Loi S, Michiels S, Baselga J, Bartlett JM, Singhal SK, Sabine VS, et al. PIK3CA genotype and a PIK3CA mutation-related gene signature and response to everolimus and letrozole in estrogen receptor positive breast cancer. PLoS One. 2013; 8(1):e53292 10.1371/journal.pone.0053292
    1. Li SY, Rong M, Grieu F, Iacopetta B. PIK3CA mutations in breast cancer are associated with poor outcome. Breast Cancer Res Treat. 2006; 96(1):91–95.
    1. Barbareschi M, Buttitta F, Felicioni L, Cotrupi S, Barassi F, Del Grammastro M, et al. Different prognostic roles of mutations in the helical and kinase domains of the PIK3CA gene in breast carcinomas. Clin Cancer Res. 2007; 13(20):6064–6069.
    1. Lai YL, Mau BL, Cheng WH, Chen HM, Chiu HH, Tzen CY. PIK3CA exon 20 mutation is independently associated with a poor prognosis in breast cancer patients. Ann Surg Oncol. 2008; 15: 1064–1069. 10.1245/s10434-007-9751-7
    1. Higgins MJ, Baselga J. Targeted therapies for breast cancer. J Clin Invest. 2011; 121(10):3797–3803. 10.1172/JCI57152
    1. Miller TW, Rexer BN, Garrett JT, Arteaga CL. Mutations in the phosphatidylinositol 3-kinase pathway: role in tumor progression and therapeutic implications in breast cancer. Breast Cancer Res. 2011; 13(6):224–235. 10.1186/bcr3039
    1. Gordon V, Banerji S. Molecular pathways: PI3K pathway targets in triple-negative breast cancers. Clin Cancer Res. 2013; 19(14):3738–3744. 10.1158/1078-0432.CCR-12-0274
    1. Janku F, Wheler JJ, Naing A, Falchook GS, Hong DS, Stepanek VM, et al. PIK3CA mutation H1047R is associated with response to PI3K/AKT/mTOR signaling pathway inhibitors in early-phase clinical trials. Cancer Res. 2013; 73(1):276–284. 10.1158/0008-5472.CAN-12-1726
    1. Moulder SL. Does the PI3K pathway play a role in basal breast cancer? Clin Breast Cancer. 2010; 10 Suppl 3:S66–71. 10.3816/CBC.2010.s.014
    1. Janku F, Wheler JJ, Westin SN, Moulder SL, Naing A, Tsimberidou AM, et al. PI3K/AKT/mTOR inhibitors in patients with breast and gynecologic malignancies harboring PIK3CA mutations. J Clin Oncol. 2012; 30(8):777–782. 10.1200/JCO.2011.36.1196
    1. Fink LS, Beatty A, Devarajan K, Peri S, Peterson JR. Pharmacological Profiling of Kinase Dependency in Cell Lines across Triple-Negative Breast Cancer Subtypes. Mol Cancer Ther. 2015; 14(1):298–306. 10.1158/1535-7163.MCT-14-0529

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