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Somatic Mutations of PI3K in Early and Advanced Gallbladder Cancer

2016; Elsevier BV; Volume: 18; Issue: 3 Linguagem: Inglês

10.1016/j.jmoldx.2015.12.003

1943-7811

Iván Roa, Hernán García, Anakaren Game, Gonzalo de Toro, Xabier de Aretxabala, Milind Javle,

Chronic Lymphocytic Leukemia Research

Gallbladder cancer (GBC) is the second-leading cause of death from malignant tumors in Chilean women. The phosphatidylinositol 3-kinase (PI3K) pathway is involved in proliferation, cell survival, and growth. We investigated mutations in exons 9 and 20 of the PI3K gene in GBC. Mutations in exons 9 (E542K, E545G, E545K) and 20 (H1047L and H1047R) of PI3K were determined by direct sequencing in 130 cases of GBC. The patient group consisted of 110 women and 20 men, and mutations were found in 22 cases (16.9%). Of these, 14 cases had mutations in exon 9 (63.6%) (E542K, 64%; E545K, 29%; and E545G, 7%) and 8 in exon 20 (37.4%; H1047L, 50%; H1047R, 50%). No differences were noted in the frequency and type of mutations analyzed by sex, age, or histologic features. We observed mutations in 22% of the early-stage GBC and 14.6% of the advanced cases. In this series of GBC, 17% of cases were noted as having mutations in either exons 9 or 20 of PI3K. These results suggest that therapeutic testing of inhibitors of the PI3K/AKT pathway may be of benefit in advanced GBC patients. Gallbladder cancer (GBC) is the second-leading cause of death from malignant tumors in Chilean women. The phosphatidylinositol 3-kinase (PI3K) pathway is involved in proliferation, cell survival, and growth. We investigated mutations in exons 9 and 20 of the PI3K gene in GBC. Mutations in exons 9 (E542K, E545G, E545K) and 20 (H1047L and H1047R) of PI3K were determined by direct sequencing in 130 cases of GBC. The patient group consisted of 110 women and 20 men, and mutations were found in 22 cases (16.9%). Of these, 14 cases had mutations in exon 9 (63.6%) (E542K, 64%; E545K, 29%; and E545G, 7%) and 8 in exon 20 (37.4%; H1047L, 50%; H1047R, 50%). No differences were noted in the frequency and type of mutations analyzed by sex, age, or histologic features. We observed mutations in 22% of the early-stage GBC and 14.6% of the advanced cases. In this series of GBC, 17% of cases were noted as having mutations in either exons 9 or 20 of PI3K. These results suggest that therapeutic testing of inhibitors of the PI3K/AKT pathway may be of benefit in advanced GBC patients. Chile has the highest incidence and mortality rate from gallbladder cancer (GBC) in the world for both men and women. GBC represents the second-leading cause of cancer death in women in Chile.1Eslick G.D. Epidemiology of gallbladder cancer.Gastroenterol Clin North Am. 2010; 39 (ix): 307-330Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 2Andia M.E. Hsing A.W. Andreotti G. Ferreccio C. Geographic variation of gallbladder cancer mortality and risk factors in Chile: a population-based ecologic study.Int J Cancer. 2008; 123: 1411-1416Crossref PubMed Scopus (77) Google Scholar Next in incidence are Latin American countries such as Bolivia, Mexico, and Ecuador and Asian countries such as India and Pakistan.3Hundal R. Shaffer E.A. 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Aloia T.A. Vauthey J.N. Curley S. Mills G. Roa I. Molecular characterization of gallbladder cancer using somatic mutation profiling.Hum Pathol. 2014; 45: 701-708Crossref PubMed Scopus (56) Google Scholar The PI3K pathway is a highly evolutionarily conserved intracellular signaling pathway seen in all species from yeasts to mammals.14Engelman J.A. Luo J. Cantley L.C. The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism.Nat Rev Genet. 2006; 7: 606-619Crossref PubMed Scopus (2508) Google Scholar In the eukaryotic cells, the PI3K pathway regulates numerous cell processes, including metabolism, survival, proliferation, apoptosis, growth, and migration.15Martelli A.M. Evangelisti C. Chiarini F. Grimaldi C. Cappellini A. Ognibene A. McCubrey J.A. 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Alvarado-Kristensson M. Gonzalez-Garcia A. Pulido R. Carrera A.C. PTEN regulation, a novel function for the p85 subunit of phosphoinositide 3-kinase.Sci STKE. 2006; 2006: pe49Crossref PubMed Scopus (36) Google Scholar The members of class I are heterodimers with a regulatory unit (with three isoforms: p85α, p85β, and p55γ) and a catalytic subunit of p110 (also with three isoforms: α, β, and δ). Although signaling through all of the PI3K classes is connected to key growth regulatory processes, to date a central role in cancer has been demonstrated mainly for PI3K class IA. PI3K class IA transmits signals downstream from pro-oncogenic receptor tyrosine kinases, and PIK3CA encodes the catalytic subunit class IA PI3K p110α, which is to date the only PI3K gene noted to have somatic mutations in human malignancies.19Brzezianska E. Pastuszak-Lewandoska D. 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Take your PIK: phosphatidylinositol 3-kinase inhibitors race through the clinic and toward cancer therapy.Mol Cancer Ther. 2009; 8: 1-9Crossref PubMed Scopus (128) Google Scholar This could enable a targeted therapy and provide powerful molecular tools for diagnosis and treatment, particularly in GBC. Our goal was to determine the frequency and type of somatic mutations in exons 9 and 20 of the PI3K gene in GBC and to establish relations with clinical and morphologic variables and patient survival. The presence of mutations of exons 9 (E542K, and E545K E545G) and exon 20 (H1047R and H1057L) of the PI3K gene was determined by direct capillary sequencing of 130 cases of GBC. All were archived, anonymized tissue samples from GBC cases that were fixed in formalin and embedded in paraffin. This study was approved by the Ethics Committee of the National Fund for Science and Technology, Faculty of Medicine of Clinica Alemana-Universidad del Desarrollo of Santiago, Chile, and the MD Anderson Cancer Center (Houston, TX). From each case, we selected a representative sample that included at least 30% tumor cells. Samples were cut to yield 10 slices of 10 μm thickness and a final cut that was 4 μm thick. This last slice was stained with hematoxylin and eosin to delineate the tumor areas that were then dissected under a stereomicroscope. Samples were deparaffinized in xylene and hydrated in decreasing concentrations of alcohol. The fragments obtained were resuspended and centrifuged for 5 minutes at 25,200 × g. DNA extraction was performed with the AxyPrep Multisource Genomic DNA Miniprep Kit (Axygen, Union City, CA) according to the manufacturer's instructions. DNA integrity was determined by amplification of the β-globin gene with expected amplification products of 110 and 268 pb. The mixture used for the reaction contained 0.2 mm dNTP, 1.5 mmol/L magnesium chloride, 12.5 pmol of each primer, 1 U of Taq polymerase (Promega Gotaq Flexi DNA polymerase), and 5 μL of the simple DNA in a reaction volume of 50 μL. Verification of the products was performed by 2% agarose gel electrophoresis. We amplified exons 9 and 20, including the sites E542K, E545K, E545G, and H1057L and H1047R. The primer sequences are shown in Table 1. Each PCR reaction was prepared in a total volume of 50 μL and contained 0.32 mmol/L dNTP (Thermo Scientific, Waltham, MA), 3 mmol/L MgCl2, 0.32 μmol/L primer mix, 1× PCR buffer, and 1 U of Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA). As a template, we used 4 ng of genomic DNA. The thermocycler program consisted of initial denaturation at 95°C for 5 minutes, followed by 35 cycles that consisted of 95°C for 45 seconds, 55°C for 45 seconds, and 72°C for 45 seconds. The last elongation was at 72°C for 10 minutes. PCR products were run on 2% agarose gel and stained with ethidium bromide.Table 1Primer Sequences for PIK3CAExonMutationForwardReversepb9E542K5′-AGTAACAGACTAGCTAGAGA-3′5′-ATTTTAGCACTTACCTGTGAC-3′1399E545K9E545G20H1047R5′-CTCAATGATGCTTGGCTCTG-3′5′-TGGAATCCAGAGTGAGCTTTC-3′24120H1057L Open table in a new tab Purification of DNA was performed with the MinElute PCR Purification Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Sequencing was performed with the ABI PRISM BigDye Terminator Cycle Sequencing Kits with AmpliTaq DNA polymerase (FS enzyme) (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. As a positive control for the mutations, we used three cell lines with constitutive mutations in exon 9 (bladder cancer TCCSUP HTB-5 pE545K c1633G>A) and exon 20 (HCC1954 CRL-2338 c3140A>G breast cancer H1047R) (ATCC, Manassas, VA). The electropherograms were aligned and read on the ClustalW platform (EMBL-EBI) Blast (EMBL-EBI COSMIC version 75, http://www.ebi.ac.uk/Tools/msa/clustalw2, last accessed December 2, 2015; software currently retired). Statistical analysis was conducted with the use of a χ2 test and Fisher's exact test for the contingency tables (P < 0.05), analysis of variance for the mean, and Kaplan-Meier actuarial survival curves with a log-rank significance test. Patient characteristics are summarized in Table 2. Of the 130 patients included in this study, 84.6% were women with a mean age of 65.1 years (SD ± 12.7 years) and men with an average age of 66.9 years (SD ± 8.7 years). All samples corresponded to biopsies (cholecystectomies). A history of gallstones was present in 93% of the GBC cases, and 41.5% were underwent operations for suspected acute cholecystitis. Only 22% of cases were suspected as having cancer in the preoperative period. In 38.8% (51 cases), it was not possible to establish with any degree of certainty the presence of a tumor on the mucosa surface. One hundred percent of the GBC were adenocarcinomas. The infiltration of lymphatic vessels was observed in 65% of the advanced carcinomas, and the involvement of blood vessels was observed in 32%. The level of tumor infiltration of the gallbladder wall was shown to be the most significant independent prognostic factor in the univariate and multivariate analyses (P < 0.001).Table 2General Features of Gallbladder Cancer PatientsFeatureValuePatient age, means ± SD, years Female (n = 110)65.1 ± 12.7 Male (n = 20)66.9 ± 8.7 Total (n = 130)65.3 ± 12.1Location, n (%) Fundus31 (23.5) Body24 (19.2) Neck8 (6.2) Diffuse16 (12.3) Unapparent51 (38.8)Histologic type, n (%) Adenocarcinoma130 (100.0) Differentiation degreeWell28 (21.4)Moderate72 (55.4)Poor30 (23.2)Infiltration level, n (%) Mucosa18 (13.8) Muscular23 (17.7) Subserosal34 (26.2) Serosal29 (22.3) Beyond serosa26 (20) Total130 (100) Open table in a new tab The subserosal GBC patients had a 5-year survival of 42%, and those with serosal GBC had 14%; however, patients with unresectable tumors were associated with a survival of 5.5% at 3 years with none surviving at 5 years. We found 22 point mutations in the 130 cases examined (16.9%) (Figure 1). All missense mutations found were substitution types. In 63.6%14Engelman J.A. Luo J. Cantley L.C. The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism.Nat Rev Genet. 2006; 7: 606-619Crossref PubMed Scopus (2508) Google Scholar of cases, the mutations were located in exon 9, with the following distribution: E542K (64% glutamic acid to lysine), E545K (29% glutamic acid to lysine), and E545G (7% glutamic acid to glycine). The remaining eight cases showed mutations in exon 20 (37.4%) and had the following distribution: H1047L (50% histidine to lysine) and H1047R (50% histidine to arginine). Table 3 shows all cases with the type of mutation, the amino acid change, location, and type of lesion.Table 3Summary of PI3K MutationNCase No.ExonMutationAmino Acid ChangePositionCosmid IDBase Change199GAA/AAAGlutamic acid/lysineE542K7601624 G>A2149GAA/AAAGlutamic acid/lysineE542K7601624 G>A32720CAT/CTTHistidine/leucineH1047L7763140 A>T44020CAT/CGTHistidine/arginineH1047R7753140 A>G5569GAA/AAAGlutamic acid/lysineE542K7601624 G>A66120CAT/CGTHistidine/arginineH1047R7753140 A>G7649GAA/AAAGlutamic acid/lysineE542K7601624 G>A8679GAA/AAAGlutamic acid/lysineE542K7601624 G>A99120CAT/CTTHistidine/leucineH1047L7763140 A>T109420CAT/CTTHistidine/leucineH1047L7763140 A>T11959GAG/GGGGlutamic acid/glycineE545G7641634A>G12989GAG/AAGGlutamic acid/lysineE545K7631633 G>A1313620CAT/CGTHistidine/arginineH1047R7753140 A>G1414820CAT/CGTHistidine/arginineH1047R7753140 A>G151649GAA/AAAGlutamic acid/lysineE542K7601624 G>A1616520CAT/CTTHistidine/leucineH1047L7763140 A>T171709GAG/AAGGlutamic acid/lysineE545K7631633 G>A181829GAA/AAAGlutamic acid/lysineE542K7601624 G>A191849GAG/AAGGlutamic acid/lysineE545K7631633 G>A201949GAG/AAGGlutamic acid/lysineE545K7631633 G>A211969GAA/AAAGlutamic acid/lysineE542K7601624 G>A222019GAA/AAAGlutamic acid/lysineE542K7601624 G>AAll mutation types were missense substitutions. Open table in a new tab All mutation types were missense substitutions. PI3K mutations were found in 9 of 41 cases (22%) of early-stage carcinomas (mucosal and muscular carcinomas) and in 13 of 89 cases (14.6%) of advanced cancers (subserosal or serosal carcinomas). This might suggest that PI3K mutations appear in the earlier stages of GBC development. No differences were found in the frequency and distribution of mutations in relation to sex, age, tumor location, or degree of histologic differentiation. The actuarial survival of the 130 patients was 42% at the 5-year follow-up (Figure 2). About 50% of patients had died within 20 months of diagnosis. The survival of patients with earlier-stage GBC (mucosal or muscle carcinomas) was 92% at 5 years; however, advanced carcinomas (subserosal, serosal, or beyond serosal carcinoma) had a survival rate of only 22% at the 5-year follow-up. No significant differences in survival were found between patients with or without mutations in the PI3K gene (P = 0.3). Our results show the PI3K gene to be mutated in approximately 17% of GBC cases, which would make it one of the most frequently mutated genes in GBC after TP53 (40%), CDKN2A (29%), KRAS (18%), and SMAD4 (18%) of the 177 cases reported (COSMIC version 75, http://cancer.sanger.ac.uk/cosmic, last accessed November 4, 2015). In addition, these results suggest that GBC may be one of the human malignant tumors with the highest frequency of mutation in PI3K, after prostate (29%), breast (27%), and endometrium (23%), surpassing the urinary tract (17%) and the upper aerodigestive tract (10%) (COSMIC version 75, http://cancer.sanger.ac.uk/cosmic, last accessed November 4, 2015). The frequency of somatic mutations in our report is slightly higher than that reported in other published works as follows: Riener et al32Riener M.O. Bawohl M. Clavien P.A. Jochum W. Rare PIK3CA hotspot mutations in carcinomas of the biliary tract.Genes Chromosomes Cancer. 2008; 47: 363-367Crossref PubMed Scopus (76) Google Scholar (1/23; 4%), Pignochino et al35Pignochino Y. Sarotto I. Peraldo-Neia C. Penachioni J.Y. Cavalloni G. Migliardi G. Casorzo L. Chiorino G. Risio M. Bardelli A. Aglietta M. Leone F. Targeting EGFR/HER2 pathways enhances the antiproliferative effect of gemcitabine in biliary tract and gallbladder carcinomas.BMC Cancer. 2010; 10: 631Crossref PubMed Google Scholar (1/13; 7.6%), Javle et al13Javle M. Rashid A. Churi C. Kar S. Zuo M. Eterovic A.K. Nogueras-Gonzalez G.M. Janku F. Shroff R.T. Aloia T.A. Vauthey J.N. Curley S. Mills G. Roa I. Molecular characterization of gallbladder cancer using somatic mutation profiling.Hum Pathol. 2014; 45: 701-708Crossref PubMed Scopus (56) Google Scholar (2/57; 3.5%), Deshpande et al30Deshpande V. Nduaguba A. Zimmerman S.M. Kehoe S.M. Macconaill L.E. Lauwers G.Y. Ferrone C. Bardeesy N. Zhu A.X. Hezel A.F. Mutational profiling reveals PIK3CA mutations in gallbladder carcinoma.BMC Cancer. 2011; 11: 60Crossref PubMed Scopus (70) Google Scholar (4/32; 12.5%), and Kumari et al48Kumari N. Corless C.L. Warrick A. Beadling C. Nelson D. Neff T. Krishnani N. Kapoor V.K. Mutation profiling in gallbladder cancer in Indian population.Indian J Pathol Microbiol. 2014; 57: 9-12Crossref PubMed Scopus (22) Google Scholar (2/49; 4.1%). Of the 22 mutations reported in this study, 14 (64%) were found in exon 9, with the most frequent ones being E542K (64.2%) and E545K (28.5%). The remaining eight mutations were located in exon 20 and distributed equally between H1047L and H1047R. Mutations in PI3K were observed in both early (22%) and advanced (14.6%) carcinomas, which could indicate that PI3K mutations are an early event in carcinogenesis of the gallbladder. The incidence of PI3K mutations may vary according to geographic location, and it is possible that a higher rate exists in the Chilean population.49Karakas B. Bachman K.E. Park B.H. Mutation of the PIK3CA oncogene in human cancers.Br J Cancer. 2006; 94: 455-459Crossref PubMed Scopus (375) Google Scholar These findings should be complemented by a study of PI3K mutations in preneoplastic lesions of the gallbladder. However, this information is not yet available. The differences between the frequency of PI3K mutations and the immunohistochemical expression of the active or phosphorylated form of this gene are also noteworthy. This is present in about 50.9% of the GBC (55/108 cases) and is observed with a significantly lower frequency in chronic cholecystitis (8.6%; 3/35 cases).31Li Q. Yang Z. Expression of phospho-ERK1/2 and PI3-K in benign and malignant gallbladder lesions and its clinical and pathological correlations.J Exp Clin Cancer Res. 2009; 28: 65Crossref PubMed Scopus (40) Google Scholar The PTEN gene that negatively regulates PI3K is found to be inactive in about 5% of GBC cases (COSMIC version 75, http://cancer.sanger.ac.uk/cosmic, last accessed November 4, 2015). These findings suggest the existence of alternative and redundant pathways capable of activating PI3K in the absence of these alterations. The PTEN-PI3K-AKT-MTOR signaling pathway has been widely studied in other malignancies, and there are a significant number of selective inhibitory molecules of this pathway such as rapamycin CCI-779 (sirolimus) or everolimus (8RAD-001) and other compounds such as phenoxodiol,50Li Y. Huang X. Huang Z. Feng J. Phenoxodiol enhances the antitumor activity of gemcitabine in gallbladder cancer through suppressing Akt/mTOR pathway.Cell Biochem Biophys. 2014; 70: 1337-1342Crossref PubMed Scopus (13) Google Scholar TIMP-2,51Lu X.S. Sun W. Ge C.Y. Zhang W.Z. Fan Y.Z. Contribution of the PI3K/MMPs/Ln-5gamma2 and EphA2/FAK/Paxillin signaling pathways to tumor growth and vasculogenic mimicry of gallbladder carcinomas.Int J Oncol. 2013; 42: 2103-2115Crossref PubMed Scopus (53) Google Scholar and others, some of which are being investigated in the treatment of advanced stages of other cancers. The information available and our results provide evidence to suggest their possible use in patients with advanced GBC. Furthermore, there is a correlation between PI3K mutations and responsiveness to her2/neu–directed therapy, which is an important consideration in GBC in which HER2/NEU amplification occurs up to 15% of the time.12Roa I. de Toro G. Schalper K. de Aretxabala X. Churi C. Javle M. Overexpression of the HER2/neu gene: a new therapeutic possibility for patients with advanced gallbladder cancer.Gastrointest Cancer Res. 2014; 7: 42-48PubMed Google Scholar Finally, we note that there is evidence that mutation in the PI3K gene is a frequent event in GBC. Current evidence shows that between 40% and 45% of GBC patients could benefit from targeted therapy (overexpression of HER2/NEU, inactivation of PTEN, PI3K, IDH1 mutation), which may open opportunities for advanced GBC patients whose poor prognosis has not changed significantly in recent decades.