The first two authors contributed equally to this paper.
Patterns of PIK3CA alterations in familial colorectal and endometrial carcinoma
Article first published online: 30 APR 2007
Copyright © 2007 Wiley-Liss, Inc.
International Journal of Cancer
Volume 121, Issue 4, pages 915–920, 15 August 2007
How to Cite
Ollikainen, M., Gylling, A., Puputti, M., Nupponen, N. N., Abdel-Rahman, W. M., Butzow, R. and Peltomäki, P. (2007), Patterns of PIK3CA alterations in familial colorectal and endometrial carcinoma. Int. J. Cancer, 121: 915–920. doi: 10.1002/ijc.22768
- Issue published online: 22 JUN 2007
- Article first published online: 30 APR 2007
- Manuscript Accepted: 8 MAR 2007
- Manuscript Received: 9 FEB 2007
- Sigrid Juselius Foundation
- The Academy of Finland
- The Finnish Cancer Foundation
- The Finnish Cultural Foundation
- The Finnish Cultural Foundation Kymenlaakso Fund
- The Paulo Foundation
- The K Albin Johansson Foundation
- The Ida Montin Foundation
- Helsinki University Science Foundation
- The Helsinki University Funds
- familial colorectal cancer;
- familial endometrial cancer;
- hereditary nonpolyposis colorectal cancer;
While the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is known to be activated in multiple sporadic cancers, the role of this pathway in familial tumors is mostly unknown. We searched for alterations in the catalytic domain of PI3K (PIK3CA), PTEN and KRAS, all of which may contribute to PI3K/AKT pathway activation, in a total of 160-familial colorectal (CRC) and endometrial carcinomas (EC), stratified by the presence vs. absence of germline mutations in DNA mismatch repair (MMR) genes. PIK3CA alterations (consisting of point mutations or low-level amplification, which were mutually exclusive with 1 exception) occurred in 10/70 (14%) of CRCs and 19/90 (21%) of ECs. Within ECs, amplification was significantly associated with the subgroup lacking germline mutations in MMR genes (familial site-specific endometrial cancer) (p = 0.015). Decreased or lost PTEN expression was characteristic of endometrial tumourigenesis (51/81, 63%, in EC compared with 24/62, 39%, in CRC, p = 0.004) and KRAS mutations of colorectal tumourigenesis (19/70, 27% in CRC vs. 9/89, 10%, in EC, p = 0.006) regardless of the MMR gene mutation status. PIK3CA alterations frequently coexisted with PTEN or KRAS changes. Combined with published studies on sporadic tumors, our data broaden the understanding of the role for PI3K pathway genes in human tumorigenesis. © 2007 Wiley-Liss, Inc.
The phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is activated in multiple cancers, leading to oncogenic transformation.1, 2, 3 Activation may result from activating mutations in PI3-kinase genes or inactivating mutations in the tumor suppressor gene PTEN (phosphatase and tensin homolog); additionally, Ras may stimulate PI3-kinase activity. PI3-kinases constitute a large family of lipid kinases, with phosphorylation of the inositol phospholipid PIP2 to form a tumor promoting second messenger, PIP3, as a main function. PIP3 causes AKT to translocate to the plasma membrane, where it is activated by phosphorylation allowing it to mediate many of the biological consequences of PI3K activation. For proliferation and tumorigenesis, the most important PI3K proteins are those belonging to class IA, which are heterodimeric proteins consisting of a catalytic domain (p110α), encoded by the PIK3CA gene on 3q26.3, and an associated regulatory subunit (p85). The PIK3CA gene is mutated in 25–40% of sporadic cancers of the colon, rectum and stomach,4 endometrium5 and breast.6
Since most previous studies have focused on sporadic cancers, we set out to determine the significance of the PI3K/AKT signaling pathway in the development of familial tumors. Colorectal cancer (CRC) and endometrial cancer (EC) are the most common tumors in hereditary nonpolyposis colorectal cancer (HNPCC), which is associated with germline mutations in DNA mismatch repair (MMR) genes and consequent microsatellite instability (MSI) in tumor tissue; moreover, 15–25% of sporadic CRCs or ECs arise through deficient MMR.7 A significant fraction of families with nonpolypotic CRC show no germline mutations in MMR genes (“familial colorectal cancer type X” FCCX) and their predisposing genes are unknown at present.8 A majority of families with the sole or predominant clustering of EC (“familial site-specific endometrial cancer”, FSSEC),9, 10 likewise remain to be molecularly characterized. By evaluating the PI3K/AKT pathway status in relation to the presence vs. absence of MMR gene germline mutations and MSI, we aimed to investigate the role of the MMR system in the PI3K/AKT pathway alterations and to better define those groups of familial cancer that are molecularly unexplained.
Material and methods
Patients and specimens
This study was performed on 160-familial tumors including 70 colorectal carcinomas (CRC) and 90 endometrial (endometrioid) carcinomas (EC) (see Table I below). Altogether 48 CRC tumors were from families with mismatch repair (MMR) gene germline mutations (HNPCC) and 22 CRC tumors were from families with unknown genetic predisposition (FCCX), which had screened negative for MLH1, MSH2, MSH6, PMS1 and PMS2 mutations.12, 13, 14, 15 Sixty EC samples were derived from HNPCC families with MMR gene germline mutations, and another 30 samples were from families with site-specific EC with no demonstrable MMR gene germline alterations (FSSEC).11
|Total number of tumors||48||22||60||30|
|Mean age at onset (years)||471||571||492||652|
|Fulfilment of Amsterdam or Bethesda criteria||48/48||21/22||60/60||0/303|
|MMR gene germline mutation in|
|MSI status||46/48 (96%)||1/22 (4.5%)||38/60 (63%)||7/30 (23%)|
|1||7/28 (25%)||2/15 (13%)||21/41 (51%)||14/29 (48%)|
|2||11/28 (39%)||11/15 (73%)||11/41 (27%)||10/29 (34%)|
|3||10/28 (36%)||2/15 (13%)||9/41 (22%)||5/29 (17%)|
|A/I||9/30 (30%)||4/12 (33%)||13/24 (54%)||25/28 (89%)|
|B/II||13/30 (43%)||6/12 (50%)||5/24 (21%)||1/28 (3.6%)|
|C/III||8/30 (27%)||2/12 (17%)||4/24 (17%)||2/28 (7.1%)|
|D/IV||0/30 (0%)||0/12 (0%)||2/24 (8.3%)||0/28 (0%)|
|Proximal: Distal ratio for CRC4||27:7 (3.86)5||7:12 (0.58)5||–||–|
Paraffin-derived specimens of tumor and matching normal tissues were collected from pathology departments and used for immunohistochemistry (IHC) and DNA extraction. DNA was prepared according to the method of Isola et al.16 Areas with high tumor percentages or with pure normal cells were selected and verified histologically and subsequently dissected out. The appropriate institutional review boards of the Helsinki University Central Hospital approved this study.
Microsatellite instability analysis
Microsatellite instability (MSI) status was determined using the Bethesda panel, BAT25, BAT26, D5S346, D2S123 and D17S250.17 Tumors were classified as having MSI if at least 2 markers out of 5 were unstable and being microsatellite-stable if only 1 marker or none of the markers was unstable.
PIK3CA and KRAS mutations were analyzed from genomic PCR products of CRC and EC samples by single strand conformation polymorphism (SSCP) analysis. Samples were separated on polyacrylamide gels with 1× MDE Gel Solution (Cambrex Bio Science Rockland, Rockland, ME) at 3W for 17–20 hr. SSCP gels were silver stained after the run. Changes observed in SSCP were confirmed by direct sequencing. For SSCP and sequencing exons 1 (p85 binding domain), 9 (helical domain) and 20 (kinase domain) representing known mutational hotspots of PIK3CA4 were amplified using primers complementary to surrounding sequences as follows; PIK3CA exon 1A forward 5′GTTTCTGCTTTGGGACAACCAT 3′, 1A reverse 5′-CTGCTTCTTGAGTAACACTTACG-3′, exon 1B forward 5′-CCCCTCCATCAACTTCTTC-3′, 1B reverse 5′-GATTACGAAGGTATTGGTTTAGACAG-3′, exon 20A forward 5′-GGTATTAACATCATTTGCTCCAA-3′, 20A reverse 5′-TCCAAAGCCTCTTGCTCAGT-3′, exon 20B forward 5′-GAATGCCAGAACTACAATCTTTT-3′ and 20B reverse 5′-GGTCTTTGCCTGCTGAGAGT-3′. Exons 1 and 20 of PIK3CA were divided into 2 overlapping fragments to improve PCR amplification. PIK3CA primers for exon 9 were obtained from Li et al.18 and were particularly designed to avoid amplification of pseudogenes. All mutation detection methods used were based on PCR amplification. PCR consisted of initial heating at 94°C for 1 min, followed by an additional 35 cycles with 1 min denaturation at 94°C, 1 min annealing at primer specific temperature (58–62°C) and 2 min elongation at 72°C. Final extension was achieved by 3 min incubation at 72°C. KRAS exon 2 was studied using primers from Deng et al.19 The KRAS data for CRC were taken from our previous study20 and generated for EC in the present investigation.
Quantitative real-time PCR
Primers and Taqman probes were designed for PIK3CA exon 21 and glucokinase gene (GCK) exon 2. GCK was used as reference because this region (7p15.3) is typically not associated with copy number changes in CRC or EC. Primers were chosen using Assays-by-Design File Builder software (Applied Biosystems, Foster City, CA) as follows. PIK3CA exon 21 forward primer 5′-AAATGAAAGCTCACTCTGGATTCCA-3′, reverse primer 5′-GCAATTCCTATGCAATCGGTCTTTG-3′ and Taqman probe 3′-ACTGCACTGTTAATAACTC-5′, GCK exon 2 forward primer 5′-CGGATGCAGAAGGAGATGGA-3′, reverse primer 5′-CATCTTCACACTGGCCTCTTCA-3′ and Taqman probe 5′-CTGAGGCTGGAGACCCA-3′. The Taqman probes were labeled with 5′FAM as a reporter and 3′TAMRA as a quencher. The concentration of primer and probe were based on optimization preruns by Applied Biosystems. The final volume in each real-time PCR reaction was 20 μl, and the reaction mix consisted of 1× Taqman Universal PCR Master Mix, 0.9 μM forward primer, 0.9 μM reverse primer and 0.25 μM probe, and 9 ng of template DNA. All samples were subjected to PicoGreen (Invitrogen, Carlsbad, CA) measurement prior to real-time PCR analysis. The prerun thermal cycling conditions were 2 min at 50°C, 10 min at 95°C, followed by 40 cycles consisting of 15 sec at 95°C and 1 min at 60°C. In each assay (96 wells), a no-template background control, and a positive control were included; furthermore a normal colorectal tissue specimen was included as a calibrator sample. Target and reference genes for each sample were run in parallel during the same run, in separate wells. The amount of PIK3CA was normalized to GCK and relative to normal calibrator DNA. The PCR was performed in the ABI 7500 Sequence Detection System (Applied Biosystems, Foster City, CA). The threshold and baseline were set automatically, and all values were reviewed after analysis of the study data as recommended by the manufacturer.
A validation experiment with 3 different normal samples, including 1 blood DNA sample and 2 paraffin-extracted DNA samples, was carried out according to guidelines “Real-time PCR Systems Chemistry Guide” (Applied Biosystems). The practically identical slopes of the target gene and reference gene demonstrated equal efficiencies of amplification over a range of DNA concentrations, making it possible to calculate PIK3CA copy number using the comparative CT method. Furthermore, low standard deviation values were considered critical for the reliability of the assay.
Multiplex ligation-dependent probe amplification
SALSA Gain probemix (P173) multiplex ligation-dependent probe amplification (MLPA) kit (MRC Holland, Amsterdam, The Netherlands) was used to detect PIK3CA amplifications according to the manufacturer's instructions (www.mrc-holland.com). The SALSA P173 contains 43 probe pairs from 28 tumorigenesis related genes including 3 probe pairs for the PIK3CA gene; 1 for the p85 binding domain (exon 1), 1 for the C2 domain (exon 6) and 1 for the kinase domain (exon 18). Normal DNA specimens derived from lymphocytes from healthy controls were included in every assay. For each MLPA reaction 100–150 ng of paraffin-derived DNA was denatured and subsequently hybridized with the MLPA probes. Ligase-65 enzyme was used to ligate the annealed probe pairs. The ligation products were amplified by PCR using a fluorescently labeled primer. The PCR products were separated by capillary electrophoresis (on ABI 3730 Automatic DNA sequencer, Applied Biosystems) and analyzed using Genemapper v3.0 (Applied Biosystems). Relative peak values were calculated for each sample by dividing the peak area of a given probe pair by the sum of the peak areas of all probes in that sample. This value was then divided by the mean relative peak value of normal DNAs from healthy controls, to obtain a dosage ratio. Genes that have been reported to show frequent gains or losses in CRC or EC21 were excluded from the calculations, resulting in an EC panel of 25 probes, and a CRC panel of 19 probes that were used for calculations in the respective tumors. In addition to tumor tissues, dosage ratios were determined for the paired normal tissues. For sequences present in 2 copies/diploid genome, a dosage ratio of 1 is expected. In the literature, values of 1.3 or higher have been interpreted to indicate increased dosage (www.mrc-holland.com).22 Taking the tumor cell percentages (average 58%) and values observed in paired normal tissues into account, our definition for increased dosage (amplification) in tumor tissue was a dosage ratio >1.7 in at least 2 out of 3 studied PIK3CA exons.
Formalin-fixed, paraffin-embedded tissue sections were immunohistochemically stained with anti-PTEN antibody, clone 6H2.1 (Cascade Biosciences, Winchester, MA). The DAKO EnVision+ System (DakoCytomation, Glostrup, Denmark) was applied according to manufacturer's instructions with antigen retrieval step by microwave boiling for 20 min in citrate buffer pH 6.0. Two pathologists (W.A.-R. and R.B.) scored the staining patterns and intensities independently. The staining pattern of normal (endothelial) cells included in each section was used as reference for evaluation of the tumor staining results. Part of the IHC data for tumors from MMR gene germline mutation carriers were derived from previous reports23, 24 and were generated for the remaining MMR gene germline mutation carriers and the germline mutation negative cohorts in the present study.
Statistical significance between groups was determined using Fisher's test. All reported p-values were two-tailed and values less than 0.05 were considered significant.
Clinicopathological features of studied cohorts
This study focused on a total of 160 familial CRC and EC tumors falling into four subcategories depending on whether germline mutations in MMR genes were present (HNPCC-CRC and HNPCC-EC) vs. absent (FCCX and FSSEC) (see Material and Methods and Table I). The mean age at diagnosis was significantly lower for CRC tumors from HNPCC patients compared with those representing FCCX (p = 0.0006), and for EC tumors from HNPCC patients compared with those representing FSSEC (p < 0.0001). The family histories of patients with HNPCC-CRC, FCCX and HNPCC-EC met the Amsterdam I25 or II26 criteria, or Bethesda criteria27 with just 1 exception whereas FSSEC fulfilled the criterion of at least 2 first degree relatives with EC in the absence of other cancers.11 As a rule, HNPCC-CRCs had high-degree MSI17 whereas FCCX tumors were microsatellite-stable (MSS) using the Bethesda panel of 5 markers. Unlike CRCs, ECs showed a less straightforward correlation between MMR gene germline mutation status and MSI since a significant fraction of tumors from MMR gene germline mutation carriers28 or those from FSSEC families with aberrant MMR protein expression11 failed to show MSI. The Bethesda markers were originally developed to detect MSI in CRC and might not be ideal for EC; different growth properties of ECs and CRCs might also play a role.28 A majority (70–80%) of CRCs and ECs from the present series were well or moderately differentiated (grade 1 or 2) and local (Dukes stage A or B or FIGO stage I or II, respectively). HNPCC CRCs were more often located in the proximal bowel compared with FCCX tumors (p = 0.003).
All tumors were screened for possible activating alterations (mutations and amplifications) in PIK3CA, possible inactivating alterations in PTEN, and for KRAS mutations, all proposed players in the activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway. The frequency of different changes observed in this series is summarized in Table II according to tissue type, germline MMR gene mutation status and MSI status of tumors.
|Germline MMR gene mutation present||5/48 (10%)||4/47 (9%)||16/44 (36%)||15/48 (31%)|
|Germline MMR gene mutation absent||1/22 (5%)||1/20 (5%)||8/18 (44%)||4/22 (18%)|
|Microsatellite unstable||5/47 (11%)||4/46 (9%)||16/43 (37%)||16/47 (34%)|
|Microsatellite stable||1/23 (4%)||1/21 (5%)||8/19 (42%)||3/23 (13%)|
|Total||6/70 (9%)||5/67 (7%)||24/62 (39%)||19/70 (27%)|
|Germline MMR gene mutation present||7/60 (12%)||3/57 (5%)||37/53 (70%)||6/60 (10%)|
|Germline MMR gene mutation absent||2/30 (7%)||7/29 (24%)||14/28 (50%)||3/29 (10%)|
|Microsatellite unstable||6/45 (13%)||4/42 (10%)||28/39 (71%)||4/45 (9%)|
|Microsatellite stable||3/45 (7%)||6/44 (14%)||23/42 (55%)||5/44 (11%)|
|Total||9/90 (10%)||10/77 (13%)||51/81 (63%)||9/89 (10%)|
PIK3CA was screened for mutations by single strand conformation polymorphism (SSCP) analysis and subsequent sequencing of exons 1 (p85 binding domain), 9 (helical domain) and 20 (kinase domain) that represent known mutational hotspots, originally reported for CRC in particular.4PIK3CA showed 12 different mutations in 15 cancers (Fig. 1), being mutated in 6/70 (9%) CRCs and 9/90 (10%) ECs (Table II). All mutations were verified from independent PCR products. Five mutations were novel (ΔE109, I112N, E545A, L1036S and H1047Q) and 7 had been reported previously.5, 6, 29, 30, 31, 32 None of the mutations was present in normal lymphocyte DNA, confirming their somatic origin. All but 1 (ΔE109, an in-frame deletion) were missense mutations. All mutations were located in evolutionarily highly conserved regions (www.ensembl.org) and most affected the kinase (6/12, 50%) or helical domain (4/12, 25%). A majority of the mutations are therefore likely to increase the kinase activity of PI3K as directly verified for some by functional tests.4, 29, 33
PIK3CA amplifications were investigated by 2 parallel techniques, quantitative real-time PCR (qPCR) and multiplex ligation-dependent probe amplification (MLPA). We defined significant gene amplification as over 1.7-fold and required that the amplification was detectable by both methods simultaneously. In our series, the average tumor cell percentage was 58%, and a copy number ratio of over 1.7 means that the tumor cells should have more than 4 copies of PIK3CA in the presence of 2 copies of reference gene loci. Of the present 160 tumors, 137 yielded an interpretable result by both methods, and among these, both methods concordantly suggested either the presence or absence of amplification in 130 cases (95%) applying the cut-off level specified above. The 7 discrepant cases were omitted from further calculations of amplifications.
PIK3CA amplification was present in 5/67 CRCs (7%) and 10/77 ECs (Table II). With a single HNPCC-CRC as an exception, PIK3CA mutations and amplifications were mutually exclusive. Examples of PIK3CA amplification plots by qPCR are shown in Figure 2. The observed amplification events were generally of rather low level, the average copy number ratios being 2.2 (including no amplifications over 4-fold) for CRC and 4.6 (including 5 amplifications over 4-fold) for EC by qPCR. Among ECs, amplification was significantly more frequent in the subgroup lacking germline mutations in MMR genes (familial site-specific endometrial carcinoma), 7/29 (24%), compared to HNPCC-EC (3/57, 5%) (p = 0.015). Moreover, the degree of amplification was higher in the former vs. the latter group (average copy number ratio 5.0 vs. 3.5 by qPCR).
PTEN and KRAS alterations and coexistence of PI3K pathway changes
PTEN was screened for decreased protein expression (inactivation) by immunohistochemical (IHC) analysis and KRAS for point mutations (activation) by SSCP and sequencing. Unlike PIK3CA that showed comparable overall frequencies for alterations in CRCs and ECs, PTEN protein expression was more often decreased or lost in ECs than CRCs (51/81, 63%, vs. 24/62, 39%, p = 0.0044) whereas KRAS mutations (in codons 12 and 13) occurred more frequently in CRCs than ECs (19/70, 27%, vs. 9/89, 10%, p = 0.006) (Table II).
The coexistence of PIK3CA, PTEN and KRAS changes is shown in Table III. Alteration in at least 1 gene was present in a significant proportion of familial CRCs (41/70, 59%) and ECs (63/90, 70%). For both types of tumor, the most frequent pattern was altered PTEN alone, accounting for 39% and 57% of all presently observed PI3K pathway alterations in CRCs and ECs, respectively. PIK3CA changes (mutations or amplifications) occurred equally often alone and in combination with altered PTEN or KRAS.
|Number of tumors with altered||Number of tumors with at least one altered gene|
|PIK3CA only||PIK3CA + PTEN||PIK3CA + KRAS||PIK3CA + PTEN + KRAS||PTEN only||PTEN + KRAS||KRAS only|
|CRC total||5 (12%)||1 (2%)||2 (5%)||2 (5%)||16 (39%)||5 (12%)||10 (24%)||41|
|EC total||10 (16%)||8 (13%)||1 (2%)||0 (0%)||36 (57%)||7 (11%)||1 (2%)||63|
The PI3K/AKT pathway changes alone or in combination did not correlate with tumor grade or stage, probably reflecting the fact that a majority of all tumors from the present series were well or moderately differentiated and local (Table I).
While a lot of published data are available implicating the PI3K/AKT pathway in various sporadic tumors, the role of this pathway in familial tumors is unknown, which prompted us to conduct this investigation. The frequencies we observed for PIK3CA mutations for familial CRC (9%) and EC (10%) are compatible with those reported for sporadic CRC (14–32%)4, 34 and EC (4–39%).5, 32, 35, 36 In our investigation, low-level amplification of PIK3CA was equally common as mutations, occurring in 7% of CRCs and 13% of ECs. For the corresponding sporadic tumors, amplifications have seldom been addressed, but available studies suggest that these events are rare and less frequent than PIK3CA mutations in the same tumor types. For example, amplifications were absent in CRCs studied by Samuels et al.4 and Campbell et al.6 and occurred in 11% in ECs studied by Oda et al.5 In certain other tumors, such as ovarian cancer, (high-level) amplification may be the predominant type of PIK3CA alteration.6
In our study, PIK3CA amplification was characteristic of familial site-specific endometrial cancer, which is featured by the clustering of EC in the absence of other cancers, and lacks germline mutations in MMR genes.11 The observation of PIK3CA amplification is important given the fact that no other molecular features to define this syndrome have been recognized, so far. Moreover, it is noteworthy that whereas amplification of the PIK3CA containing region in EC has primarily been associated with poorly differentiated or serous adenocarcinoma,5 most EC tumors with amplification from the present series were well differentiated (2 were grade 2), and all were local and had endometrioid histology—features that generally correlate with a better prognosis.
Consistent with studies on sporadic tumors,6 we found that PIK3CA mutation and amplification were mutually exclusive except for a microsatellite-unstable CRC tumor from an HNPCC patient. Besides PIK3CA mutation (ΔE109) and amplification (2-fold), PTEN protein was decreased and KRAS mutated (G12V) in the same tumor. The accumulation of multiple genetic changes with apparently overlapping consequences may simply reflect a chaotic tumorigenic process. Alternatively, it is possible that the PIK3CA mutation, which was a novel in-frame deletion affecting an evolutionarily conserved amino acid in the p85 binding domain (Fig. 1) is not sufficiently pathogenic alone.
Apart from PIK3CA, PTEN and KRAS were investigated for changes possibly contributing to PI3K/AKT activation, and at least 1 of these genes was altered in 57% of CRCs and 70% of ECs. The coexistence of different changes was common. This finding differs from published data on many sporadic tumors, in which PIK3CA and PTEN alterations30, 31, 37, 38 or PIK3CA and KRAS alterations36 have been reported to be mutually exclusive. The inverse association between PIK3CA and PTEN changes is consistent with the idea that an alteration in either one of these genes is sufficient to increase the PIP3-pool and direct the PI3K/AKT pathway towards oncogenic activation. On the other hand, recent studies on sporadic EC reported a frequent coexistence of PIK3CA and PTEN alterations, suggesting an additive effect on PI3K activation.5, 32, 36
For sporadic cancers, the role of MSI status in PIK3CA changes has been addressed only rarely and the data are in part conflicting. In regard to CRC, Samuels et al.4 found a significantly higher frequency of PIK3CA mutations in MMR-deficient tumors compared to MMR-proficient tumors (16/33 vs. 58/201, p = 0.028), whereas no correlation of PIK3CA mutations with MSI was observed in another investigation.34 For EC, Velasco et al.36 reported no correlation between PIK3CA mutations and MSI. Our study showed no association between PIK3CA mutation and MSI status in either CRC or EC (Table II). Of the 15 tumors with PIK3CA amplification, 8 were microsatellite-unstable and 7 stable. Out of the 5 tumors (all ECs) with high-level (over 4-fold) amplification, all but 1 were MSS, which is compatible with the idea of amplifications being a manifestation of chromosomal rather than MSI.39
Collectively, the present study shows that the PI3K/AKT pathway is altered in more than half of familial CRCs and ECs in a pattern that depends on the genetic basis (association of PIK3CA amplification with familial site-specific EC) and tissue type (association of PTEN changes with EC and KRAS changes with CRC). Together with studies on sporadic tumors, our data significantly extend the existing knowledge of the PI3K pathway genes in human tumorigenesis.
We thank Ms. Gynel Arifdshan and Ms. Saila Saarinen for expert technical assistance. Ms. Heli Surma-Aho and Ms. Katja Kuosa are acknowledged for assistance with sample collection.
- 17A national cancer institute workshop on microsatellite instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res 1998; 58: 5248–57., , , , , , , , , , .