Prognostic value of PIK3CA and phosphorylated AKT expression in ovarian cancer

Disrupted phosphatidylinositol 3-kinase (PI3K) activity and its effect on the downstream target AKT plays an important role in malignant diseases. Gain and/or amplification of PIK3CA gene, encoding the catalytic subunit of phosphatidylinositol 3-kinase (p110α) and its increased expression are associated with enhanced PI3K activity in ovarian cancer cell lines. In this study, ovarian carcinomas with documented clinical outcome were assessed for genetic aberrations at the 3q26.3 locus, including PIK3CA, by fluorescence in situ hybridization. PIK3CA amplification was evaluated by quantitative real-time PCR with respect to a control gene situated at 3q13. The expression of p110α, phosphorylated AKT (pAKT) and the proliferation marker Ki-67 were immunohistochemically investigated. PIK3CA amplification and Ki-67 index were strong predictors for an early tumour-associated death. p110α expression correlated with 3q26.3 gain and Ki-67 index but not with the patient outcome. No relationship could be observed between p110α and pAKT or between pAKT and disease outcome. It is interesting to note that cases with a nuclear pAKT immunoreactivity showed a trend of improved overall survival. Our results underline the prognostic significance of PIK3CA in ovarian carcinoma and argue against a simple linear model of PIK3CA gain/amplification followed by PI3K activation and consecutive AKT phosphorylation in ovarian carcinoma.     

PIK3CA gene mutations in endometrial carcinoma: correlation with PTEN and K-RAS alterations

Alterations in the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway are common in endometrial carcinoma. Inactivation of the tumor suppressor gene PTEN leads to a constitutively active PI3K pathway, which plays a role in the early steps of endometrial tumorigenesis. Other alterations in the PI3K/AKT pathway are mutations in the PIK3CA gene, which encode the p110alpha catalytic subunit of PI3K. PIK3CA mutations cluster to the helical (exon 9) and the kinase (exon 20) domains of the gene. In endometrial carcinomas, PIK3CA mutations have been found to coexist frequently with PTEN mutations, but it is not clear whether they occur in cells with monoallelic or biallelic inactivation of PTEN. In the present study we have evaluated PIK3CA mutational status in a series of 33 endometrial carcinomas, previously screened for microsatellite instability and mutations in PTEN, K-RAS, and CTNNB-1. The tumors were also evaluated for loss of heterozygosity on 10q23 and hypermethylation of the promoter region of PTEN/psiPTEN to assess the monoallelic or biallelic inactivation status of PTEN. PIK3CA mutations were detected in 8 (24%) of the 33 cases. Seven mutations were located in exon 20 and 1 in exon 9. PTEN alterations were found in 19 cases (57%). Biallelic inactivation of PTEN was demonstrated in 11 tumors, whereas 8 tumors exhibited alteration in only 1 of the 2 alleles. PIK3CA mutations coexisted with monoallelic alterations of PTEN in 4 cases (2 mutations and 2 allelic imbalances), with biallelic PTEN inactivation in 1 case (mutation and promoter methylation), and 3 tumors showed PIK3CA mutations in association with wild-type PTEN. PIK3CA mutations did not correlate with microsatellite instability or mutations in CTNNB-1. However, PIK3CA and K-RAS mutations (8 cases) were mutually exclusive alterations. In summary, the results confirm that PIK3CA mutations are frequent in endometrial carcinoma and support the hypothesis that PIK3CA mutations may have an additive effect to PTEN monoallelic inactivation in endometrial carcinoma.     

子宫内膜癌中磷脂酰肌醇3激酶/蛋白激酶通路活化及临床意义

目的 探讨磷脂酰肌醇3激酶/蛋白激酶( PI3 K/AKT)信号通路的重要组分PTEN、PIK3CA和p-AKT在子宫内膜癌组织中的改变及其预后意义.方法 免疫组织化学EnVision二步法检测PTEN、p-AKT、雌激素受体(ER)和孕激素受体在71例子宫内膜癌组织中的表达,聚合酶链反应测序法分析其中34例PIK3CA基因外显子9和20的突变情况.结果 (1)71例子宫内膜癌组织中,子宫内膜样癌( EEC) 65例,非子宫内膜样癌(NEEC)6例.PTEN失表达率为63.4％( 45/71),在EEC中的比例(66.2％,43/65)高于NEEC( 2/6;P=0.18).PTEN失表达患者(45例)预后优于PTEN正常表达者(26例;P=0.07).进一步分组分析显示在ER阴性病例中,PTEN失表达者(12例)的预后优于PTEN正常表达者(7例),差异有统计学意义(P=0.04).(2)本组34例中PIK3CA突变率为41.2％ (14/34),突变热点为外显子9的T544.PIK3CA突变在EEC中的发生比例(44.8％,13/29)高于NEEC( 1/5;P ＞0.05).外显子9突变全部见于Ⅰ期EEC病例中,且在高、中分化的EEC中发生率高于低分化肿瘤(P＞0.05).外显子20突变在早期病例组(14.3％,4/28)的发生率显著低于晚期病例组(4/6;P=0.0l).(3)p-AKT的阳性率为59.2％ (42/71),在EEC中的阳性率(60.0％,39/65)高于NEEC(3/6;P=0.68);但高、中分化组EEC的阳性比例(75.0％,21/28;53.6％,15/28)高于低分化组(3/9),差异有统计学意义(P=0.02).p-AKT阳性与ER阳性相关(r=0.339,P=0.00).结论 PI3K/AKT信号通路的关键分子,如PTEN失表达、p-AKT阳性提示预后好.PIK3CA基因外显子9和20的突变对子宫内膜癌的预后影响可能不同.子宫内膜癌中PTEN和p-AKT的功能可能受到ER状态的影响.     

Expression of down stream molecules of RET (p-ERK, p-p38 MAPK, p-JNK and p-AKT) in papillary thyroid carcinomas

To evaluate the roles of 4 putative downstream molecules (ERK, p38 MAPK, JNK and AKT) of the RET signal pathway in the tumorigenesis of papillary carcinomas, the expression patterns of RET and phosphorylated forms of ERK, p38 MAPK, JNK and AKT were evaluated in 115 cases of papillary thyroid carcinomas by 3 mm-core tissue microarray based immunohistochemical staining. The prevalence of RET protein expression was 62.6%. No distinct expression of p-ERK and p-p38 MAPK was demonstrated in tumor cells of papillary carcinomas. All papillary carcinomas except 5 cases expressed nuclear p-JNK and p-JNK expression was increased in tumors compared with paired normal tissues (p < 0.05). There was no difference in the p-JNK expression between RET protein-positive and RET protein-negative papillary carcinomas (p > 0.05). Unequivocal nuclear staining for p-AKT was demonstrated only in 10 cases of papillary carcinomas, and all of them showed focal staining. Our results showing constitutive expression of p-JNK in most cases of surgically excised papillary thyroid carcinomas irrespective of RET protein expression status suggest that JNK activation may play a role in the tumorigenesis or survival of sporadic papillary thyroid carcinoma.     

PIK3CA mutations in advanced ovarian carcinomas

PIK3CA belongs to the phosphatidylinositol 3-kinases (PI3Ks) family, which play an important role in proliferation, adherence, transformation and cell survival through the PI3K/AKT signaling pathway. Somatic activating mutations of this gene have recently been detected in several types of cancers. In the present study, 109 advanced ovarian carcinomas were analyzed for PIK3CA mutations in exon 9 and exon 20 by direct sequencing. Activating missense mutations were observed in 4 of the 109 tumors in addition to one variant leading no change of the PIK3CA protein. Two of the cases with mutations were mucinous and clear cell tumors, suggesting that PIK3CA mutations are more common in these rare histological types.     

Alternate molecular genetic pathways in ovarian carcinomas of common histological types

We evaluated alterations in p53, PIK3CA, PTEN, CTNNB1 (beta-catenin), MLH1, and BRAF among common histological subsets of epithelial ovarian tumors to characterize patterns of alterations of different molecular pathways. There were 12 clear cell, 26 endometrioid, and 51 serous carcinomas evaluated by direct DNA sequencing for mutations in p53, PIK3CA, PTEN, BRAF, and CTNNB1. Methylation-specific polymerase chain reaction (PCR) assessed MLH1 promoter methylation status. Quantitative PCR identified PIK3CA amplification in 22 EC/CC and 94 SC. p53 mutations were identified in 25 (49%) of 51 SC, 11 (42%) of 26 EC, and 1 (8.3%) of 12 CC neoplasms and were more common in grade 3 EC (P = .045) and advanced-stage EC/CC (P = .007). PIK3CA mutations were identified in 3 (25%) of 12 CC, 3 (12%) of 26 EC, and 0 of 51 SC. PTEN mutations were significantly more common in EC (8/26, 31%) compared with CC (0/12; P = .04) and SC (2/51, 4%; P = .002). CTNNB1 mutations were identified, 6 (23%) EC and no CC or SC (P = .008). Both PTEN and CTNNB1 mutations were more common in low-grade EC/CC, whereas PIK3CA mutations occurred only in grade 3 cancers. PTEN and PIK3CA mutations were more common in p53 wild-type tumors (P = .003). PIK3CA amplification occurred in fewer EC/CC (0/22) versus SC (19/94, 20%; P = 0.02) and were slightly more common in p53 wild-type compared with p53 mutant SC (P = .08). Of 26 EC, 22 (85%) had a mutation in one of the genes studied compared with 4 33% of 12 CC (P = .003). Women with EC/CC had significantly better overall survival (P = .0008), and this remained significant after accounting for stage (P=.04). Mutations in p53 or in PTEN/PIK3CA are alternative pathways in ovarian carcinogenesis. Activation of PIK3CA occurs by gene amplification in SC but via somatic mutation of PIK3CA or PTEN in EC and CC. PIK3CA mutations are associated with high-grade tumors, whereas PTEN and CTNNB1 mutations are associated with low-grade tumors. Mutations in p53, PIK3CA, PTEN, and CTNNB1 account for most EC tumors; most CC remain unexplained. EC/CC histology is a favorable prognostic factor.     

Loss of ARID1A protein expression occurs as an early event in ovarian clear-cell carcinoma development and frequently coexists with PIK3CA mutations

ARID1A is a recently identified tumor suppressor gene that is mutated in ～50% of ovarian clear-cell carcinomas. This mutation is associated with loss of ARID1A protein expression as assessed by immunohistochemistry. The present study aimed at determining the timing of the loss of ARID1A protein expression during the development of ovarian clear-cell carcinoma and assessing its relevance in correlation to PIK3CA gene mutations. A total of 42 clear-cell carcinoma cases with adjacent putative precursor lesions (endometriosis-associated carcinoma cases (n=28) and (clear-cell) adenofibroma-associated carcinoma cases (n=14)) were selected and subjected to immunohistochemical analysis for ARID1A protein expression and direct genomic DNA sequencing of exons 9 and 20 of the PIK3CA gene. ARID1A immunoreactivity was deficient in 17 (61%) of the 28 endometriosis-associated carcinomas and 6 (43%) of the 14 adenofibroma-associated carcinomas. Among the precursor lesions adjacent to the 23 ARID1A-deficient carcinomas, 86% of the non-atypical endometriosis (12 of 14) and 100% of the atypical endometriosis (14 of 14), benign (3 of 3), and borderline (6 of 6) clear-cell adenofibroma components were found to be ARID1A deficient. In contrast, in the 19 patients with ARID1A-intact carcinomas, all of the adjacent precursor lesions retained ARID1A expression regardless of their types and cytological atypia. Analysis of 22 solitary endometrioses and 10 endometrioses distant from ARID1A-deficient carcinomas showed that all of these lesions were diffusely immunoreactive for ARID1A. Among the 42 clear-cell carcinomas, somatic mutations of PIK3CA were detected in 17 (40%) tumors and majority (71%) of these were ARID1A-deficient carcinomas. These results suggest that loss of ARID1A protein expression occurs as a very early event in ovarian clear-cell carcinoma development, similar to the pattern of PIK3CA mutation recently reported by our group, and frequently coexists (not mutually exclusive) with PIK3CA mutations.     

PIK3CA mutations and amplification in endometrioid endometrial carcinomas: relation to other genetic defects and clinicopathologic status of the tumors

Alterations of the PIK3CA gene occur in endometrial carcinomas, but their role in the carcinogenesis of those malignancies is still poorly understood. In this study, PIK3CA mutations and amplification in 196 endometrioid endometrial carcinomas and 20 endometrial hyperplasias were assessed by single-strand conformation polymorphism (PCR-SSCP), sequencing, and quantitative polymerase chain reaction. Results were correlated with mutations in the PTEN, KRAS, and CTNNB1 genes and with the clinicopathologic parameters of the tumors. PIK3CA mutations were found in 39 (20%) carcinomas. Six new mutations were identified. No PIK3CA mutations were found in endometrial hyperplasias. PIK3CA amplification was observed in 24 (12.2%) carcinomas and in 2 (10%) hyperplasias. The PIK3CA mutations and amplifications (with the exception of 6 cases) occurred independently. PIK3CA mutations were significantly associated with PTEN mutations (P = .0414) and tended to be associated with CTNNB1 (P = .0833), but not with KRAS mutations. Conversely, the PIK3CA amplifications significantly negatively correlated with PTEN mutations (P = .0038) and did not coexist with CTNNB1 and KRAS mutations. The PIK3CA mutations were significantly associated with poorly differentiated tumors (P = .0423). Interestingly, PIK3CA amplifications, but not mutations, were strongly associated with older age (≥63 years, P = .0018). Our data show that mutations and amplification of PIK3CA are significant genetic alterations in endometrioid endometrial carcinomas associated with adverse clinicopathologic parameters (grade and stage). These data also demonstrate that PIK3CA mutations cooperate with PTEN mutations, suggesting an additive effect to PTEN, whereas PIK3CA amplification can, as an isolated event, enable the development of those tumors. Moreover, for the first time, a possible role of PIK3CA amplification in initiation and progression of endometrial carcinomas in older women is suggested, but this preliminary suggestion requires further research.     

Mutations in FGFR3 and PIK3CA, singly or combined with RAS and AKT1, are associated with AKT but not with MAPK pathway activation in urothelial bladder cancer

Different members of the phosphoinositide 3 kinase - serine threonine protein kinase (PI3K-AKT) pathway are altered in bladder cancer. Fibroblast growth factor receptor 3 (FGFR3) mutations characterize the low-grade tumors, and RAS genes are mutated in approximately 13% of all bladder tumors. Interestingly, a percentage of bladder tumors have alterations in more than 1 PI3K-AKT or rat sarcoma viral oncogene homolog-RAF mitogen activated protein kinase (RAS-MAPK) pathway gene or their upstream regulators, but some combinations are mutually exclusive. We analyzed mutations in FGFR3, phosphoinositide 3 kinase catalytic alpha polypeptide (PIK3CA), v-akt murine thymoma viral oncogene homolog 1 (AKT1), v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), v-Ha-ras Harvey rat sarcoma viral oncogene homolog (HRAS), and v-raf murine sarcoma viral oncogene homolog B1 (BRAF) in 88 urothelial cell carcinomas and the immunohistochemical expression of phospho-v-akt murine thymoma viral oncogene homolog (AKT) and mitogen-activated protein kinase 1 and 2 (pERK1/2) in 80 and 77 urothelial cell carcinomas, respectively. Approximately 43% and 20.5% of tumors presented 1 and 2 mutated genes, respectively. FGFR3 mutations were more frequent alone, whereas PIK3CA mutations were associated with another mutated gene (FGFR3 and KRAS). Overall, mutated FGFR3 (FGFR3(mut)) and mutated FGFR3 (FGFR3(mut))-mutated PIK3CA (PIK3CA(mut)) genotypes were associated with low-grade bladder tumors and mutated PIK3CA (PIK3CA(mut))-mutated KRAS (KRAS(mut)) and mutated AKT1 (AKT1(mut)) were only present in high-grade tumors. There are no mutated FGFR3 (FGFR3(mut))-mutated RAS (RAS(mut)) nor mutated PIK3CA (PIK3CA(mut))-mutated AKT1 (AKT1(mut)) combinations. Fifty percent and 56% of tumors showed high levels of pAKT and pERK1/2, respectively. High levels of pAKT were associated with total mutations, FGFR3(mut), and PIK3CA(mut) tumors but not with tumor grade or stage. Wild-type tumors presented significantly higher pERK1/2 expression. Mutations in FGFR3 and FGFR3-PIK3CA but not single PIK3CA mutations characterize low-grade bladder tumors. Single FGFR3 or PIK3CA mutations and the different mutation combinations FGFR3-PIK3CA/AKT1 and PIK3CA-RAS can activate the AKT but not the MAPK pathway. Other genes different from FGFR3 may be related with the pERK activation in bladder tumors.     

Genetic alterations and aberrant expression of genes related to the phosphatidyl-inositol-3'-kinase/protein kinase B (Akt) signal transduction pathway in glioblastomas

Glioblastomas frequently carry mutations in the PTEN tumor suppressor gene on 10q23.3. The tumor suppressor properties of Pten are closely related to its inhibitory effect on the phosphatidyl-inositol-3'-kinase (Pi3k)-dependent activation of protein kinase B (Akt) signalling. Here, we report on the analysis of 17 genes related to the Pi3k/Akt signalling pathway for genetic alteration and aberrant expression in a series of 103 glioblastomas. Mutation, homozygous deletion or loss of expression of PTEN was detected in 32% of the tumors. In contrast, we did not find any aberrations in the inositol polyphosphate phosphatase like-1 gene (INPPL1), whose gene product may also counteract Pi3k-dependent Akt activation. Analysis of genes encoding proteins that may activate the pathway upstream of Pi3k revealed variable fractions of tumors with EGFR amplification (31%), PDGFRA amplification (8%), and IRS2 amplification (2%). The protein tyrosine kinase 2 (PTK2/FAK1) gene was neither amplified nor overexpressed at the mRNA level. Investigation of three genes encoding catalytic subunits of Pi3k (PIK3CA, PIK3CD, and PIK3C2B) revealed amplification of PIK3C2B (1q32) in 6 tumors (6%). Overexpression of PIK3C2B mRNA was detected in 4 of these cases. PIK3CD (1p36.2) and PIK3CA (3q26.3) were not amplified but PIK3CD mRNA was overexpressed in 6 tumors (6%). Amplification and overexpression of AKT1 was detected in a single case of gliosarcoma. The IRS1, PIK3R1, PIK3R2, AKT2, AKT3, FRAP1, and RPS6KB1 genes were neither amplified nor overexpressed in any of the tumors. Taken together, our data indicate that different genes related to the Pi3k/Akt signalling pathway may be aberrant in glioblastomas.     

Rare PIK3CA hotspot mutations in carcinomas of the biliary tract

Somatic mutations of the PIK3CA gene, which encodes the p110alpha catalytic subunit of phosphatidylinositol 3-kinase (PI3K), are frequent in various cancer types. The majority of mutations cluster at hotspots within exons 9 and 20, which encode the helical and kinase domains of p110alpha. PIK3CA mutations in bile duct and gallbladder carcinomas have not been reported yet. In this study, we analysed 118 carcinomas of the biliary tract and the liver (45 intra- and extrahepatic cholangiocarcinomas (CCA), 23 gallbladder carcinomas, 50 hepatocellular carcinomas) for PIK3CA hotspot mutations using polymerase chain reaction and direct DNA sequencing. PIK3CA missense mutations were found in one of 11 intrahepatic CCA (E545K, 9%), one of 23 gallbladder carcinomas (E542K, 4%), and one of 50 hepatocellular carcinomas (H1047R, 2%). All three mutations represent hotspot mutations, which also occur in other cancer types. PI3K pathway activation in hepato-biliary carcinomas was analyzed using immunohistochemistry for the downstream targets eIF4-E and phosphorylated 4E-BP1 on tissue microarrays. eIF4-E expression was found in 3/13 intrahepatic CCA (23%), 9/38 extrahepatic CCA (24%), 12/34 gallbladder carcinomas (35%), and 9/61 hepatocellular carcinomas (15%). 4E-BP1 phosphorylation was observed in 1/13 intrahepatic CCA (8%), 8/38 extrahepatic CCA (21%), 15/34 gallbladder carcinomas (44%), and 16/61 hepatocellular carcinomas (26%). These results indicate that somatic PIK3CA mutations contribute to the frequent activation of the PI3K/AKT pathway in carcinomas of the biliary tract and liver.     

Evaluation of AKT phosphorylation and PTEN loss and their correlation with the resistance of rituximab in DLBCL

Background: Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of lymphoma with quite high mortality. PTEN/PI3K/AKT signal pathway is constitutively activated and plays an oncogenic role in most tumors including non-Hodgkin’s lymphoma (NHL). Since rituximab used in chemotherapy has been proved to improve the survival of DLBCL patients, rituximab resistance is a common clinical challenge in the treatment of DLBCL. The aims of the present study are to determine the different levels of several important biomarkers of PTEN/PI3K/AKT pathway in DLBCL patients who are resistant or sensitive to rituximab treatment, and investigate the potential clinical application of these biomarkers. Methods: 48 patients with DLBCL who were treated by rituximab-based chemotherapy were divided into 2 groups according to their reactions to rituximab. The expression of p-AKT, PTEN, and Ki-67 protein in 48 DLBCL tissues were detected using immunohistochemistry and analyzed for the clinical pathological significance and the resistance to rituximab. Meanwhile, PTEN gene deletion was detected also by FISH, and mutation of PIK3CA was performed by sequencing analysis. Results: Activation of p-AKT in 12 of 48 (25.0%) and loss expression of PTEN in 15 of 48 (31.3%) DLBCL species were observed. P-AKT activation (P<0.05) and loss of PTEN expression (P<0.05) were significantly associative with high Ki-67 index. P-AKT and PTEN expression showed a significant negative correlation in all 48 DLBCL patients (r=-0.450, P<0.05), and the Spearman correlation coefficient in the resistant group (r=-0.769, P<0.05) was greater than in the sensitive group (r=-0.691, P<0.05). Conclusion: Regulation of PTEN/PI3K/AKT signal pathway participates in the progression of DLBCL, and may be involved in the development of the resistance to rituximab for some DLBCL patients.     

Frequent genetic abnormalities of the PI3K/AKT pathway in primary ovarian cancer predict patient outcome

Identification and characterization of underlying genetic aberrations could facilitate diagnosis and treatment of ovarian cancer. Copy number analysis using array Comparative Genomic Hybridization (aCGH) on 93 primary ovarian tumors identified PI3K/AKT pathway as the most frequently altered cancer related pathway. Furthermore, survival analyses to correlate gene copy number and mutation data with patient outcome showed that copy number gains of PIK3CA, PIK3CB, and PIK3R4 in these tumors were associated with decreased survival. To confirm these findings at the protein level, immunohistochemistry (IHC) for PIK3CA product p110α and p-Akt was performed on tissue microarrays from 522 independent serous ovarian cancers. Overexpression of either of these two proteins was found to be associated with decreased survival. Multivariant analysis from these samples further showed that overexpression of p-AKT and/or p110α is an independent prognostic factor for these tumors. siRNAs targeting altered PI3K/AKT pathway genes inhibited proliferation and induced apoptosis in ovarian cancer cell lines. In addition, the effect of the siRNAs in different cell lines seemed to correlate with the particular genetic alterations that the cell line carries. These results strongly support the utilization of PI3K pathway inhibitors in ovarian cancer. They also suggest identifying the specific component in the PI3K pathway that is genetically altered has the potential to help select the most effective therapy. Both mutation as well as copy number changes can be used as predictive markers for this purpose.     

Phosphatidylinositol-3-kinase pathway mutations are common in breast columnar cell lesions

The phosphatidylinositol-3-kinase pathway is one of the most commonly mutated pathways in invasive breast carcinoma, with PIK3CA mutations in ～25% of invasive carcinomas, and AKT1 mutations in up to 5%. Ductal carcinoma in situ, and benign papillomas harbor similar mutations. However, activating point mutations in breast columnar cell lesions have been infrequently studied. Twenty-three breast resection specimens containing columnar cell lesions were identified; 14 with associated invasive carcinoma or carcinoma in situ. DNA extracts were prepared from formalin-fixed paraffin-embedded tissue and screened for a panel of point mutations (321 mutations in 30 genes) using a multiplex PCR panel with mass-spectroscopy readout. PIK3CA mutations were identified in 13/24 columnar cell lesions (54%) and 3/8 invasive carcinomas (37%). The mutation status of columnar cell lesions and associated carcinoma was frequently discordant. Of the 14 cases, only 5 demonstrated the same genotype in matched samples of columnar cell lesions and carcinoma (4 wild type, 1 PIK3CA H1047R). Interestingly, five patients had mutations in columnar cell lesions with wild-type carcinoma; two patients had different point mutations in columnar cell lesions and carcinoma. Only three cases had wild-type columnar cell lesion and mutated carcinoma. The 50% PIK3CA mutation prevalence in columnar cell lesions is greater than reported in most studies of invasive breast cancer. Further, columnar cell lesions and carcinoma were frequently discordant for PIK3CA/AKT1 mutation status. These findings raise interesting questions about the role of PIK3CA/AKT pathway in breast carcinogenesis, and the biologic/precursor potential of columnar cell lesions.     

Exon 20 PIK3CA mutations decreases survival in aggressive (HER-2 positive) breast carcinomas

PIK3CA mutations at 9 and 20 exons were studied in a series of 56 selected aggressive breast carcinomas (BC): 27 with Her-2 over-expression and negativity for estrogen receptors (ER) and progesterone receptors (PR), and 29 "triple negative" BC (negative for ER, PR and Her-2). Also, immunohistochemical studies of p53, ki-67, Her-1 (EGFR), pIGF-1R, PTEN, p110alpha, and pAkt were performed. Six mutations in exon 20 PIK3CA were identified among the 27 Her-2 positive BC, whereas only one exon 9 PIK3CA mutation was detected in a triple negative tumor (p = 0.035). Furthermore, PIK3CA mutations were associated with p110alpha over-expression (p = 0.001). Overall survival was shorter in cases with PIK3CA mutations (p = 0.015 in all series; and p = 0.041 for Her-2+ tumors), although multivariate analyses did not show statistical differences. No statistical significance was related with disease-free survival. Exon 20 PIK3CA mutations are relatively frequent in Her-2+ tumors and shorten survival, whereas neither exons 9 and 20 mutations seem related with "triple negative" breast carcinomas.     

Mucinous breast carcinomas lack PIK3CA and AKT1 mutations

Activating point mutations in the phosphatidylinositol-3-kinase catalytic subunit (PIK3CA) are among the most common molecular defects in invasive breast cancer. Point mutations in the downstream kinase AKT1 are seen in a minority of carcinomas. These mutations are found preferentially in estrogen receptor-positive and Her2-positive breast carcinomas; however, special morphologic types of breast cancer have not been well studied. Twenty-nine cases of pure invasive mucinous carcinoma and 9 cases of ductal carcinoma with mucinous differentiation were screened for a panel of point mutations (>321 mutations in 30 genes) using a multiplex polymerase chain reaction panel with mass spectroscopy readout. In addition, associated ductal carcinoma in situ, hyperplasia, or columnar cell lesions were separately tested where available (25 lesions). In 3 invasive cases and 15 ductal carcinoma in situ/proliferative lesions, PIK3CA hotspot mutations were, instead, tested by direct sequencing. No point mutations were identified in invasive mucinous breast carcinoma. This contrasts with the 35% frequency of PIK3CA mutations in a comparative group of invasive ductal carcinomas of no special type. Interestingly, PIK3CA hotspot point mutations were identified in associated ductal carcinoma in situ (3/14) and hyperplasia (atypical ductal hyperplasia [2/3], usual ductal hyperplasia [2/3], columnar cell change [1/5]), suggesting that PIK3CA mutations may play a role in breast epithelial proliferation. This series represents the largest study, to date, of PIK3CA genotyping in mucinous carcinoma and supports the unique pathogenetics of invasive mucinous breast carcinoma.     

PIK3CA mutations in the kinase domain (exon 20) of uterine endometrial adenocarcinomas are associated with adverse prognostic parameters.

Mutations of the oncogene PIK3CA occur frequently in endometrial carcinomas, but their prognostic significance is unclear. To determine the clinicopathological and molecular implications of these mutations, PIK3CA status was investigated in 109 endometrial (102 endometrioid and 7 mixed) carcinomas and the results were compared with clinicopathological parameters associated with prognosis. Tumors were also investigated for microsatellite instability and PTEN, beta-catenin gene (CTNNB1), K-RAS, and B-RAF mutations. We found 35 PIK3CA somatic missense mutations in 32 (29%) endometrial carcinomas. Eighteen mutations occurred in exon 20 (kinase domain), and 17 in exon 9 (helical domain). Almost all mutated tumors were pure endometrioid adenocarcinomas. All tumors with PIK3CA mutations exhibited myometrial invasion (P=0.032). Lymphovascular invasion was found more frequently in mutated (28%) than nonmutated carcinomas (18%). Histological grade varied significantly according to the location of the PIK3CA mutations whether in exon 9 or exon 20 (P=0.033). The frequency of exon 9 mutations was higher in grade 1 carcinomas (57%) than in grade 2 (29%) or grade 3 (14%) tumors. Conversely, mutations in exon 20 were more common in grade 3 (60%) than in grade 2 (20%) or grade 1 (20%) carcinomas. None of the tumors confined to the endometrium (stage IA) had PIK3CA mutations. Furthermore, whereas 64% of adenocarcinomas with exon 9 mutations had invaded < or =(1/2) of the myometrial thickness (stage IB), 73% of tumors with exon 20 mutations had either deeper myometrial invasion (stage IC) or cervical involvement (stage II) (P=0.045). PIK3CA mutations coexisted with microsatellite instability and mutations in PTEN, CTNNB1, K-RAS, and B-RAF genes. These results favor that PIK3CA mutations are associated with myometrial invasion and, moreover, that tumors harboring PIK3CA mutations in exon 20 are frequently high-grade, deeply invasive endometrial carcinomas that tend to exhibit lymphovascular invasion.     

Involvement of the PI3K/Akt pathway in myxoid/round cell liposarcoma

The molecular determinants involved in the progression of myxoid liposarcoma to increased cellularity/round cell change are poorly understood. We studied the PI3K/Akt pathway in myxoid and round cell liposarcomas using a tissue microarray composed of 165 tumors from 111 patients, and mutational analysis of PIK3CA in 44 cases. Activating PIK3CA mutations were found in 6/44 cases, 14%; mutations were more frequent in round cell vs myxoid tumors (5/15, 33% vs 1/29, 3%; P=0.013). Complete loss of PTEN, an alternative mechanism for PI3K/Akt activation, was found in 13/111 (12%) cases and was mutually exclusive with PIK3CA mutation. Strong IGF1R expression was demonstrated in 14/39 (36%) of round cell and 11/58 (19%) of myxoid tumors (P=0.062). Activation of the PI3K pathway was confirmed using immunohistochemical analysis for downstream targets phospho-S6 ribosomal protein and phospho-4EBP1. Phospho-4EBP1 was increased in round cell tumors compared with myxoid tumors (24/30, 80% vs 25/44, 57%; P=0.038) or tumors with treatment effect (10/24, 42%; P=0.02). Phospho-S6 was highly expressed in both myxoid and round cell tumors (29/47, 62% and 14/30, 47%, respectively; P=0.2). In tumors with PIK3CA mutation, any IGF1R expression, or loss of PTEN expression, phospho-4EBP1 was more frequently elevated compared with tumors without a known activating event in the PI3K pathway (55/72; 76% vs 3/8, 38%; P=0.033). These findings suggest that activation of the PI3K/Akt pathway via activating mutation of PIK3CA, loss of PTEN, or IGF1R expression have a role in round cell transformation. The PI3K/Akt pathway may therefore provide a therapeutic target in round cell liposarcoma.