Absence of hotspot mutations in exons 9 and 20 of the PIK3CA gene in human oral squamous cell carcinoma in the Greek population

Absence of hotspot mutations in exons 9 and 20 of the PIK3CA gene in human oral squamous cell carcinoma in the Greek population George C. Kostakis, MD, DDS, MSc,a Nikos Papadogeorgakis, MD, DDS, PhD,b Vasiliki Koumaki, MD, MPH, PhD,c Smaragda Kamakari, PhD,d Dimitra Koumaki, MD,e and Constantinos Alexandridis, DDS, PhD,f Athens, Greece UNIVERSITY OF ATHENS, PAPADIMITRIOU HOSPITAL, AND BIOGENOMICA LABORATORY

Objective. Phosphatidylinositol-3 kinases (PI3K) are a group of heterodimeric lipid kinases that regulate many cellular processes. Recent studies have reported high frequencies of somatic hotspot mutations in the phosphatidylinositol-3 kinase catalytic ␣ (PIK3CA) gene, which encodes for one of these kinases, in several human solid tumors, including oral squamous cell carcinoma (OSCC). The aim of this study was to determine the frequency of hotspot mutations in exons 9 and 20 of the PIK3CA gene in OSCC in the Greek population. Study design. Eighty-six formalin-fixed and paraffin-embedded primary tumor specimens were analyzed by direct genomic DNA sequencing. Chi-square was used for statistical analysis. Results. No hotspot mutations were detected in any of the samples. Two intronic polymorphisms IVS8 and IVS9 were detected, mainly in patients with cancer of the buccal mucosa and lower gingival and alveolus respectively. Conclusions. PIK3CA hotspot mutations are unlikely to play a major role in the pathogenesis of OSCC in the Greek population. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;109:e53-e58)

Squamous cell carcinoma of the oral cavity (OSCC) is a subset of head and neck squamous cell carcinoma (HNSCC) and involves the oral tongue, upper gingival, lower gingival and alveolus, floor of the mouth, buccal mucosa, retromolar triangle, lip mucosa, and hard palate. The incidence of oral cancer is increasing worldwide. It is estimated to be 11th in frequency and 13th in cancerspecific mortality worldwide.1 HNSCC is strongly related to smoking and alcohol consumption but infection by human papillomavirus has also emerged as a risk factor for HNSCC.2 Oncogenesis of these types of tumors is considered to involve progressive accumulation of genetic alterations, although to date, the underlying genetic mechanisms remain unclear and complex.3,4 a

Oral and Maxillofacial Surgeon, Department of Oral and Maxillofacial Surgery, Evagelismos Hospital, Dental School, University of Athens, Athens, Greece. b Associate Professor, Department of Maxillofacial Surgery, Evagelismos Hospital, Dental School, University of Athens, Athens, Greece. c Resident in Microbiology, Department of Microbiology, Papadimitriou Hospital, Athens, Greece. d Chief Scientific, Biogenomica Laboratory, Centre for Genetic Research and Analysis, Athens, Greece. e Resident in Internal Medicine, Department of Internal Medicine, Papadimitriou Hospital, Athens, Greece. f Professor, Department of Oral and Maxillofacial Surgery, Evagelismos Hospital, Dental School, University of Athens, Athens, Greece. Received for publication Jul 8, 2009; returned for revision Dec 14, 2009; accepted for publication Jan 25, 2010. 1079-2104/$ - see front matter © 2010 Mosby, Inc. All rights reserved. doi:10.1016/j.tripleo.2010.01.015

Phosphatidylinositol-3 kinases (PI3Ks) constitute a large family of heterodimeric lipid kinases composed of catalytic and adaptor regulatory subunits encompassing 3 classes.5 PIKs are important regulators of cellular growth, transformation, adhesion, apoptosis, survival, and motility.6 Once activated by growth factor tyrosine kinases, the PI3K complex is recruited on the inner cell membrane, where PIK3CA phosphorylates its substrate phosphatidylinositol (4,5) biphosphate (PIP2) at the 3-position of the inositol ring, generating the second messenger phosphatidylinositol (3,4,5) triphosphate (PIP3), which in turn recruits the serine-threonine kinase AKT and 3-phosphoinositide-dependant kinase (PDK) to the plasma membrane.7 PDK phosphorylates and activates AKT, which then regulates a range of target downstream proteins that control a variety of intracellular proteins. PIK3CA is located on chromosome 3q26.3 and encodes for the catalytic subunit p110␣ of class IA PI3K.8 Gene amplifications, deletions, and, more recently, somatic missense mutations in the PIK3CA gene have been reported in many human cancer types including cancers of the colon, breast, brain, liver, stomach, and lung.9-13 These somatic missense mutations were proposed to increase the kinase activity of PIK3CA. More than 75% of these mutations harbour in the helical (exon 9) and kinase domains (exon 20) of the gene.14 Moreover, the 3 hotspot mutations, namely E542K, E545K, and H1047R, were proven to elevate the enzyme’s lipid kinase activity, to phosphorylate AKT and activate the PI3K-AKT signaling pathway, resulting in e53

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transformation in vitro.5,15 This evidence shows that PIK3CA acts as an oncogene in many types of cancers. To date, studies have shown that PIK3CA is overexpressed in squamous cell carcinoma of the oral tongue.16 In addition, hotspot mutations have been reported in HNSCC (10.5%)17 as well as in advanced stages of OSCC in the Japanese population (7.5%).18 More recently, Murugan et al.19 suggested a different frequency of PIK3CA mutations in HNSSC in different ethnic groups (Indian 10.5%, Vietnamese 0%). Absence of hotspot mutations has also been reported in 33 cases of HNSCC in a German study.20 There seems to be a racial disparity in PIK3CA mutation frequency. No analysis of PIK3CA genetic alterations and their clinicopathological significance has ever been performed in the Greek population, which lives in a different geographic location and presents different alcohol and smoking habits. The present study aimed to determine the contribution of PIK3CA hotspot mutations in the pathogenesis of OSCC in the Greek population. The primary working hypothesis was that PIK3CA hotspot mutations appear in later stages of OSCC after the accumulation of mutations in other genetic loci, as suggested by Kozaki et al.18 To test this hypothesis, we performed a large-scale screening of PIK3CA hotspot mutations in exons 9 and 20 by direct genomic DNA sequencing in paraffin-embedded primary tumors of 86 consecutive cases of OSCC, mainly in advanced stages, that were operated for their cancer. MATERIALS AND METHODS Specimens from 86 consecutive patients, who underwent a major operation for their cancer, were collected in and retrieved from the Department of Oral and Maxillofacial Surgery at the Evagelismos Hospital, University of Athens, Athens, Greece, from January 2003 to December 2007. Most of them had advanced stages of the disease. In addition, blood samples from 26 healthy control volunteers, age and sex matched, were also collected from a similar population. We specifically chose samples from the primary tumor of each original specimen where cancer cells were identified by the pathologists. Demographic, preoperative, and postoperative data were available for all patients. The patients were staged according to International Union Against Cancer.21 This work was approved by the ethical committees of Evagelismos Hospital and the University of Athens. Patients gave informed consent to the work. Extraction of genomic DNA All samples were formalin fixed and paraffin embedded, and genomic DNA was extracted using the QIAmp extraction DNA kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s protocols. Concentration of the

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genomic DNA was assessed by the GenQuant spectrometer (Pharmacia LNK Biotechnology Inc., Piscataway, NJ). PCR analysis and DNA sequencing Two sets of primers were used for the detection of any point mutations in exons 9 and 20 of the PIK3CA gene, followed by direct sequencing. The sequences of the 2 oligonucleotide primer pairs were as follows: (1) exon 9 forward 5=-GATTGGTTCTTTCCTGTCTCTG-3=, exon 9 reverse 5=-CCACAAATATCAATTTACAACCATTG-3=, and exon 9 nested 5=-CCTGTCTCTGAAAATAAAGTCTTGCA-3=; (2) exon 20 forward 5=TGGGGTAAAGGGAATCAAAAG-3=, exon 20 reverse 5=-CCTATGCAATCGGTCTTTGC-3=, and exon 20 nested 5=-ATGTTGGTAAGAGAAGTGAGAGAGGA-3=, both generating fragments of about 500 base pairs. The seminested polymerase chain reaction (PCR) reaction was carried out in a total volume of 30 ␮L in a PTC-200 Peltier Thermal Cycler (MJ Research, Inc., Waltham, MA). The mixture of the PCR reaction contained 3 ␮L 10⫻ Reaction Buffer, 1 ␮L dNTP (10 mM of each dNTP), 1 ␮L forward primer (25 pmol), 1 ␮L reverse primer (25 pmol), 1.8 ␮L MgCl2 (25 mM), 1 ␮L Taq DNA polymerase (5 units/mL), and 5 ng/mL DNA template. Amplification was carried out with 5 minutes of initial denaturation at 95°C followed by 35 cycles of denaturation at 95°C for 1 minute, exon 9 primary primer annealing at 58°C and semi-nested PCR at 55°C for 1 minute, exon 20 primary primer annealing at 60°C and semi-nested PCR at 55°C, and extension at 72°C for 1 minute. The PCR products were subsequently analyzed on a 4200 Two-Dye DNA Analysis System (LI-COR Biosciences, Lincoln, NE). Statistical analysis All the available data were entered into a database using SPSS statistical software (SPSS 15.0 for Windows; SPSS Inc, Chicago, IL). The Mann-Whitney U test was used to compare the medians of 2 different groups of patients. If more than 2 groups were compared, Kruskal-Wallis analysis of variance test was used. The chi-square test was also used for comparison of qualitative variables. The P value was found to be significant when it was less than .05. RESULTS Out of the 86 patients, 17 (19.8%) were stage I, 12 (14.0%) were stage II, 14 (16.3%) were stage III, and 43 (50.0%) were stage IV, according to pTNM stage. Thirty-one patients (36.0%) had cancer of the oral tongue, 8 (9.3%) of the floor of the mouth, 25 (29.1%) of the lower gingival and alveolus, 4 (4.7%) of the upper gingival, 9 (10.5%) of the buccal mucosa, 6 (7.0%) of the retromolar triangle, and 3 (3.5%) patients had cancer of the lip mucosa. The histological

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Table I. Clinical characteristics and PIK3CA genetic alterations in patients with oral squamous cell carcinoma Patient no.

Sex

Age, y

Tumor location

Histological grade

Stage

PIK3CA genetic alterations

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62

M F F F F M F M F F M F F M F F F M M F M F M M F F M F M F M M F F F M M M M M F M M F M F F M F M M M M F M F F F F F M M

56 54 54 71 69 66 56 43 70 78 80 26 48 41 91 65 72 45 45 79 67 30 65 65 75 — 74 54 69 76 55 51 61 69 87 64 24 62 57 73 74 31 55 — 58 63 73 44 78 61 61 73 37 79 46 72 72 48 26 47 46 57

Floor of mouth Floor of mouth Buccal mucosa Upper gingiva Lower gingiva Floor of mouth Lower gingiva Retromolar triangle Buccal mucosa Oral tongue Lower gingiva Oral tongue Oral tongue Buccal mucosa Lip Lip Lower gingiva Floor of mouth Lower gingiva Oral tongue Upper gingiva Oral tongue Retromolar triangle Retromolar triangle Oral tongue Lower gingiva Retromolar triangle Buccal mucosa Lower gingiva Retromolar triangle Lip Lower gingiva Oral tongue Oral tongue Buccal mucosa Upper gingiva Oral tongue Oral tongue Oral tongue Floor of mouth Lower gingiva Oral tongue Floor of mouth Buccal mucosa Floor of mouth Buccal mucosa Retromolar triangle Oral tongue Oral tongue Lower gingiva Lower gingiva Lower gingiva Lower gingiva Lower gingiva Oral tongue Oral tongue Lower gingiva Lower gingiva Oral tongue Oral tongue Oral tongue Oral tongue

Moderate Moderate Moderate — Poor Moderate Moderate Well Moderate Moderate Moderate Well Moderate Moderate Moderate Moderate Moderate Well — Poor Moderate Well Moderate Moderate Moderate Moderate Moderate — Moderate Moderate Moderate Well Moderate Poor Well Well Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Moderate Well Moderate Moderate Moderate Moderate Moderate Well Poor Poor Moderate Poor Well Moderate Moderate Poor

IV IV IV II IV IV IV I III II IV III III III III I I I IV IV IV IV III III IV IV IV IV IV IV I IV IV III I I IV III III IV IV I III I IV II II IV IV IV IV IV IV IV IV II I IV III II II IV

Wild type Polymorphism ivs8 Polymorphism ivs8 Wild type Polymorphism ivs9 Wild type Polymorphism ivs8 Wild type Polymorphism ivs9 Wild type Polymorphism ivs8 Wild type Wild type Wild type Polymorphism ivs8 Wild type Polymorphism ivs9 Wild type Wild type Wild type Wild type Polymorphism ivs8 Wild type Wild type Wild type Polymorphism ivs9 Wild type Polymorphism ivs9 Wild type Wild type Polymorphism ivs9 Wild type Wild type Polymorphism ivs8 Polymorphism ivs9 Polymorphism ivs8 Wild type Polymorphism ivs8 Wild type Wild type Polymorphism ivs9 Wild type Wild type Wild type Wild type Polymorphism ivs8 Wild type Wild type Polymorphism ivs9 Wild type Polymorphism ivs9 Wild type Polymorphism ivs9 Polymorphism ivs8 Wild type Wild type Polymorphism ivs9 Wild type Polymorphism ivs8 Wild type Wild type Polymorphism ivs9

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Table I. Continued Patient no.

Sex

Age, y

63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86

F F M F F F F M F F M M F F F M M M M M M M M M

76 81 65 73 56 68 74 75 68 66 62 62 60 82 75 70 58 50 52 57 64 51 55 69

Tumor location Lower gingiva Oral tongue Lower gingiva Oral tongue Lower gingiva Oral tongue Buccal mucosa Oral tongue Upper gingiva Oral tongue Lower gingiva Floor of mouth Lower gingiva Lower gingiva Oral tongue Buccal mucosa Oral tongue Oral tongue Oral tongue Lower gingiva Lower gingiva Lower gingiva Oral tongue Oral tongue

Histological grade

Stage

PIK3CA genetic alterations

Moderate Moderate Well Moderate Moderate Well Moderate Moderate Moderate Moderate Moderate Well Poor Moderate Well Moderate Moderate Well Well Moderate Moderate Well — —

III I I II IV I IV IV IV II I I IV IV I IV III I II II II IV IV IV

Wild type Wild type Wild type Wild type Polymorphism ivs9 Wild type Polymorphism ivs8 Wild type Wild type Wild type Polymorphism ivs8 Wild type Polymorphism ivs9 Wild type Wild type Polymorphism ivs8 Wild type Wild type Wild type Polymorphism ivs9 Wild type Polymorphism ivs9 Polymorphism ivs8 Wild type

—, no data available.

grade was well differentiated in 17 (21.0%) of the patients, moderately differentiated in 56 (69.1%) of the patients, and poorly differentiated in 8 (9.9%) of the patients. Contrary to expectations, no hotspot mutations in exons 9 and 20 were identified in the specimens tested. The clinical characteristics of the patients studied and the genetic alterations of the PIK3CA gene are summarized in Table I. We detected 2 polymorphisms previously described in the intronic region flanking exon 9, IVS8 –55 C⬎T (rs45455192) and IVS9 ⫹105 T⬎G (rs17849071).22 Polymorphism IVS8 (shown in Fig. 1) was found in a percentage of 18.6% (16 patients) and polymorphism IVS9 (shown in Fig. 2) in a percentage of 19.8% (17 patients). In the control group, the frequencies of IVS8 and IVS9 were 7.7% and 11.5% respectively. All specimens harboring these polymorphisms were heterozygous. No specimen harbored both polymorphisms. No statistically significant difference was found between the frequencies of IVS8 in patients and controls (Pearson chi-square 1.76, P value .18). No statistically significant difference was also found between the frequencies of IVS9 in patients and controls (Pearson chi-square 0.92, P value .34). IVS8 has not been previously reported in HNSCC, whereas IVS9 has been previously reported in HNSCC specimens in 4 (10.5%) of 38.17 An increased frequency of IVS8 was found in cancer of the buccal mucosa (44%, 4 of 9). IVS9 was mainly found in the lower gingival and alveolus (44%, 11 of 25)

and buccal mucosa (33.3%, 3 of 9). The results were not further statistically processed because of the limitation of the sample. DISCUSSION The mutation frequency of PIK3CA has been reported at 4% in lung cancer, 32% in colon cancer, 5% to 27% in brain cancer, 4% to 25% in gastric cancer, and 4% to 7% in ovarian cancer.9-12,23-25 In HNSCC, hotspot mutations have been reported at 0% to 29.4%19 and specifically in OSCC at 7.4%.18 This frequency variation could be attributed to the small sample size of most studies and to the different ethnicities studied. The different sites included in the HNSCC (pharynx, larynx, oral cavity) or the different stages of the cases studied could also play a role in this disparity. It seems that PIK3CA is mutated in the pharyngeal squamous cell carcinoma more often rather than in other sites. Qui et al.17 reported a PIK3CA mutation frequency of 10.5% in HNSCC and most of them were detected in the pharynx (3/4). A more recent study by the same group conducted exclusively in specimens with pharyngeal squamous cell carcinoma, using a novel mutantenriched sequencing method, revealed a frequency of hotspot mutations of 20.8%.26 Fenic et al.,20 in a study of PIK3CA mutations in HNSCC in Germany, reported no hotspot mutations in 33 cases. Murugan et al.19 reported 10.5% PIK3CA mutations in Indian tumors and 0% in Vietnamese tumors. Kozaki et al.18 performed a study of

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Fig. 1. Polymorphism IVS8. Sequence chromatogram fragment of intron 8 of PIK3CA gene as determined by automated sequence analysis. The arrow indicates the 2 peaks detected (both wild-type thymine and the substitution by a cytosine) in a heterozygous sample.

Fig. 2. Polymorphism IVS9. Sequence chromatogram fragment of intron 9 of PIK3CA gene as determined by automated sequence analysis. The arrow indicates the 2 peaks detected (both wild-type thymine and the substitution by a guanine) in a heterozygous sample.

108 cases with OSCC and reported a frequency of hotspot mutations of 7.5% mainly in advanced stages. In the present study, we analyzed exclusively 86 consecutive patients of Greek origin with OSCC mainly at advanced stages and we found no mutations. This is in contrast with the published data of Kozaki et al.18 It is suggested in other cancers that different racial groups present a different genetic alteration profile.27,28 Mutation rate of some genes is suggested to be associated with ethnicity.29,30 This may be possible for OSCC. Ethnic differences in genetic makeup, smoking and alcohol consumption patterns, and other unidentified cultural factors may be responsible for the disparity. In the regions of exons 9 and 20 that we sequenced, 4 single nucleotide polymorphisms have been reported, 3 in exon 9 and 1 in exon 20.22 We did not find the polymorphism in exon 20 in our population, but we found 2 of the 3 polymorphisms in exon 9. The first polymorphism IVS8 –55 C⬎T (rs45455192) was found in 18.6% of the patients and in 7.7% of the control population. The second polymorphism IVS9 ⫹105 T⬎G (rs17849071) was found in 19.8% of the patients and in 11.5% of the control group. IVS9 in HNSCC has been previously reported at a frequency of 10.5%.17 To our knowledge, IVS8 has not been previously reported

in published data in HNSCC; therefore, IVS8 represents a novel finding in OSCC. It is reported that IVS8 is mainly found in Caucasians and Hispanics, whereas it is a rare finding in other racial groups.31 In the Greek population, we found an IVS8 frequency of 18.6% in patients with OSCC, whereas a lower percentage of IVS8 was found in the control group 7.7%. Although there is no significant difference between patients and controls with the IVS8 polymorphism, it is possible that more precise results could be obtained from a study with a larger sample size. Nevertheless, because the polymorphisms occur in flanking intronic regions, it is unlikely that they would, by themselves, have significant impact on PIK3CA function. IVS8 and IVS9 polymorphisms do not seem to relate with clinicopathological parameters such as stage and histological grade (shown in Table I). IVS8 polymorphism was found in 44.4% of patients with cancer of the buccal mucosa. IVS9 polymorphism was found in 44.0% of patients with cancer of the lower gingival and alveolus and in 33.3% of patients with cancer of the buccal mucosa. In addition, these polymorphisms did not appear in any case of cancer of the retromolar triangle and they appear only once in cancer of the floor of the mouth. It is suggested that genetic alterations in OSCC

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may be site specific,32 as prognosis of OSCC is site dependent.33 Thus, further studies with larger sample sizes are necessary to compare the site-specific frequencies and delineate any potential role of these polymorphisms. Therefore, according to our findings, it is unlikely that in the Greek population PIK3CA hotspot mutations contribute to OSCC or they do but in a much lower percentage. Other mechanisms of PIK3CA activation or mutations of other molecular pathways could be involved in the pathogenesis of the disease.

17.

18.

19.

20.

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Reprint requests: George Kostakis Department of Oral and Maxillofacial Surgery Evagelismos Hospital University of Athens Grammou 1 Str, N. Kifissia 14564 Athens, Greece [email protected]