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.     

Clinicopathological characteristics of PIK3CA and HBx mutations in Korean patients with hepatocellular carcinomas

Hepatocellular carcinoma (HCC) is the fourth most common form of cancer in the Korean population, caused primarily by infection with either the Hepatitis B or C virus. Progression of this disease is frequently associated with mutations in either phosphoinositide‐3‐kinase, catalytic, alpha (PIK3CA) or hepatitis B virus X (HBx) gene. Previous studies have examined the frequency of PIK3CA mutations in HCC, although the clinical significance of these mutations has not been studied in a Korean population. In addition, HBx appears to play a key role in modulating a wide range of cellular functions, leading to HCC. In this study, we examined microdissected tumor samples from 50 HCC patients who underwent hepatectomy at Keimyung University Dongsan Medical Center. These patients were screened for mutations in PIK3CA and HBx to identify the clinical outcomes associated with these mutations. Exons 9 and 20 of PIK3CA and the entirety of HBx were screened for mutations by polymerase chain reaction and direct DNA sequencing. PIK3CA mutations were detected in 7 of 50 patients (14%). Among the 42 patients who were seropositive for hepatitis B, 17 (40.5%) had HBx mutations and 4 (9.52%) had mutations in PIK3CA. PIK3CA mutations were strongly correlated with tumor size. Patients harboring HBx mutations exhibited a longer time to recurrence; this difference was statistically significant not only in comparison with the PIK3CA mutation but also compared with those without any mutations. This result suggests a role for PIK3CA and HBx mutations as prognostic markers in HCC.     

PIK3CA mutation and histological type in breast carcinoma: high frequency of mutations in lobular carcinoma

Mutations in the PIK3CA gene have recently been reported in different human neoplasms, including breast cancer. This paper reports the results of a systematic analysis of PIK3CA mutations in different histological types of breast carcinoma. One hundred and eighty invasive breast carcinomas, comprising 74 ductal, 56 lobular, 22 mucinous, 20 medullary, and eight papillary, were selected on the basis of their histological type in a consecutive series of 780 breast cancers. Exons 1-20 of the PIK3CA gene were subjected to SSCP analysis followed by direct sequencing. PIK3CA mutations were observed in 46 (26%) of the 180 tumours examined: 23 (50%) mutations were located in exon 9, and 23 (50%) in exon 20. Mutations were frequent in lobular (46%), less frequent in ductal (22%), and uncommon in medullary (10%), mucinous (5%), and papillary tumours (12%) (p = 0.0002). Mutations in exon 9 were more frequent in lobular carcinomas (30% of cases) than in the other histological types (less than 5% of cases) (p = 0.00014). No significant differences were observed in the distribution of mutations in exon 20. There was no significant correlation between PIK3CA mutations and other clinicopathological and biological variables, including age, tumour size, lymph node metastases, oestrogen receptor (ER) status, progesterone receptor (PgR) status, p53 gene mutations, and p53 protein expression. The findings indicate that in invasive breast carcinomas, PIK3CA alterations are mainly present in lobular and ductal tumours, whereas the other histological types, known to be associated with a favourable prognosis, show a very low incidence of PIK3CA mutations.     

Correlation of DLC1 gene methylation with oncogenic PIK3CA mutations in extramammary Paget's disease

Extramammary Paget's disease is a rare cutaneous malignant neoplasm. The genetic and epigenetic mechanisms underlying its pathology remain unknown. In this study, we investigated the expression levels, and mutation and methylation status of a common tumor suppressor gene, deleted in liver cancer 1 (DLC1), and an oncogene, PIK3CA, in tumor (n=132) and normal tissues (n=20) from unrelated patients. The presence of epigenetic and genetic lesions was then correlated to the patient pathology data to determine the potential role of these genes in extramammary Paget's disease etiology and progression. The DLC1 gene was found to be downregulated in 43 (33%) tumors, as compared with immunohistochemistry results from normal tissues. Methylation-sensitive, high-resolution melting analysis indicated that the DLC1 promoter was hypermethylated in 51 (39%) extramammary Paget's disease tumors. This hypermethylation was associated with significantly decreased DLC1 levels (P=0.011), and had a strong positive correlation with advanced age (P=0.002). PIK3CA mutations were detected by direct sequencing in 32 (24%) tumors, the majority of which were invasive. Furthermore, PIK3CA mutations significantly correlated with DLC1 hypermethylation. Thus, aberrant DLC1 methylation and PIK3CA mutations may have important roles in extramammary Paget's disease pathogenesis, and may represent potential molecular targets for therapy.     

PIK3CA exon 20 mutations are associated with poor prognosis in breast cancer patients

OBJECTIVES:

The phosphatidylinositol 3-kinase/AKT axis is an important cell-signaling pathway that mediates cell proliferation and survival, two biological processes that regulate malignant cell growth. The phosphatidylinositol 3-kinase CA gene encodes the p110α subunit of the phosphatidylinositol 3-kinase protein. There are phosphatidylinositol 3-kinase CA mutations in several types of human tumors, and they are frequently observed in breast cancer. However, these mutations have not been investigated in Brazilian breast cancer patients.

METHODS:

PCR-SSCP and direct DNA sequencing were performed to identify phosphatidylinositol 3-kinaseCA exon 9 and exon 20 mutations in 86 patients with sporadic breast cancer. The relationships between PIK3CA mutations and patient clinicopathological characteristics and survival were analyzed. The presence of the TP53 mutation was also examined.

RESULTS:

Twenty-three (27%) of the 86 primary breast tumors contained PIK3CA mutations. In exons 9 and 20, we identified the hotspot mutations E542K, E545K, and H1047R, and we identified two new missense mutations (I1022V and L1028S) and one nonsense (R992X) mutation. Phosphatidylinositol 3-kinase CA exon 20 mutations were associated with poor overall survival and TP53 gene mutations.

CONCLUSIONS:

Phosphatidylinositol 3-kinase CA mutations are common in tumors in Brazilian breast cancer patients, and phosphatidylinositol 3-kinase CA and TP53 mutations are not mutually exclusive. Phosphatidylinositol 3-kinase CA exon 20 mutations are associated with poor survival, and they may be useful biomarkers for identifying breast cancer patients with aggressive tumors and for predicting the response to treatment with PI3K pathway inhibitors.     

MRX93 syndrome (BRWD3 gene): five new patients with novel mutations

Overgrowth syndromes (OGS) comprise a heterogeneous group of disorders whose main characteristic is that either the weight, height, or head circumference are above the 97th centile or 2 to 3 SD above the mean for age and sex. Additional features, such as facial dysmorphism, developmental delay or intellectual disability (ID), congenital anomalies, neurological problems and an increased risk of neoplasia are usually associated with OGS. Genetic analysis in patients with overlapping clinical features is essential, to distinguish between two or more similar conditions, and to provide appropriate genetic counseling and recommendations for follow up. In the present paper, we report five new patients (from four unrelated families) with an X-linked mental retardation syndrome with overgrowth (XMR93 syndrome), also known as XLID-BRWD3-related syndrome. The main features of these patients include ID, macrocephaly and dysmorphic facial features. XMR93 syndrome is a recently described disorder caused by mutations in the Bromodomain and WD-repeat domain-containing protein 3 (BRWD3) gene. This article underscores the importance of genetic screening by exome sequencing for patients with OGS and ID with unclear clinical diagnosis, and expands the number of reported individuals with XMR93 syndrome, highlighting the clinical features of this unusual disease.     

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.     

胃癌组织中PIK3CA基因热点突变筛查及临床意义

目的 检测中国人群胃癌组织中PIK3 CA基因突变频率及热点突变模式,探讨PIK3 CA基因突变的临床意义.方法 应用荧光PCR法筛查胃癌PIK3 CA基因突变阳性病例,Sanger DNA测序法检测PIK3CA基因热点突变模式;统计分析PIK3 CA基因突变与胃癌临床病理特征之间的关系.结果 400例肿瘤标本中检测出PIK3 CA基因突变率为7.5％ (30/400),突变位点主要集中在第9外显子(E542K,E545K)和第20外显子(H1047R,H1047L);且PIK3 CA基因突变与肿瘤预后相关(P＜0.05),而与肿瘤大小、组织学分级、淋巴结状态、患者年龄等因素无关.结论 中国人群胃癌组织中PIK3CA基因突变具独特的突变频率与突变模式;PIK3 CA基因突变对于胃癌预后判断及基因靶向治疗具潜在临床价值.     

High Frequency of Mutations in the PIK3CA Gene Helical and Kinase Coding Regions in a Group of Iranian Patients with High-Grade Glioblastomas: Five Novel Mutations

Abstract: Glioblastoma multiform (GBM; World Health Organization (WHO) grade IV) and anaplastic astrocytomas (AA; WHO grade III) are highly aggressive and lethal astrocytic brain tumors. To detect cancer-specific somatic mutations in two hot-spot regions of PIK3CA gene, the helical and kinase domains (encoded by exons 9 and 20, respectively) in GBM and AA, the authors examined the respective sequences 31 paraffin-embedded samples (23 GBM and 8 AA). The samples were obtained from a group of Iranian patients affected with high-grade glioblastoma (HGG). The overall prevalence of PIK3CA mutations was 23% (7/31) for both tumor types (22% in GBM, and 25% in AA). Five mutations were detected in exon 20, p.Arg992Gln (c.2976G→A), p.Met1005Val (c.3014A→G), p.Ile1019→Val (c.3056A→G), p.Ser1008Cys (c.3024C→G), and p.Asn1044Asp (c.3130A→G), and one mutation in exon 9, p.Glu545Ala (c.1634A→C). Additionally exons 4–8 of P53 gene in four unrelated young patients, who showed no mutations in PIK3CA exons 9 and 20, were analyzed. Three mutations were identified: p.Pro72Ala (c.214C→G), g.11608G→T (homozygote splice mutation), and p.Thr170Thr (c.510G→A silent mutation). In conclusion, mutation detection in PIK3CA in patients with a high degree of malignancy and early age at diagnosis should be included in molecular diagnostic protocols, also with regard to possible upcoming therapies.     

Mutational analysis of the AGL gene: five novel mutations in GSD III patients

Total or partial lack of glycogen debranching enzyme (GDE or AGL, amylo-1,6-glucosidase, 4-alpha-glucanotransferase) is responsible for Glycogen Storage Disease type III (GSDIII), a rare autosomal recessive disorder of glycogen metabolism. The clinical and biochemical features of GSDIII subjects are quite heterogeneous, and this mirrors the genotype-phenotype heterogeneity among patients. In this paper, we report the molecular characterisation of five unrelated subjects, four Italian and one Tunisian. The following new mutations are described and confirm the genetic heterogeneity of this disease: p.R864X, p.R428K, c.3911 insA, p.G1087R and c.3512_3549dup+c.3512_3519del. The functional relevance of these mutations is discussed on the basis of the recently acquired knowledge about the boundaries and structures of the two catalytic domains.     

High rate of PIK3CA mutations but no TP53 mutations in low‐grade adenosquamous carcinoma of the breast

Aims

Low‐grade adenosquamous carcinoma of the breast (LGASC) is a rare variant of metaplastic carcinoma characterised by a favourable outcome and histologically composed of glandular and squamous elements in a spindle cell background typically associated with a lymphocytic stromal reaction. Because of its rarity, the immunophenotypic and genetic profile of LGASC has not been sufficiently characterised. The aim of this study was to gain insights into the molecular and phenotypic characteristics of LGASC.

Methods and results

We reviewed the clinical and morphological features and detailed the immunohistochemical characteristics of a retrospective series of 13 LGASCs. Targeted sequencing of 50 genes was performed in 10 of 13 cases. Identified mutations were further assessed by Sanger sequencing in a validation series of 11 additional cases. All tumours showed a triple‐negative immunophenotype, expressed ‘basal’ keratins, showed variable levels of epidermal growth factor receptor expression, and did not express androgen receptor. Sequencing analysis of the screening set of LGASCs revealed a high rate (seven of 10 cases) of PIK3CA mutations, whereas no TP53 mutations were found. All PIK3CA mutations were missense mutations located either in exon 20 (n = 6) or in exon 9 (n = 1). The global PIK3CA mutation rate, including the validation series, was 52% (11 of 21 cases). No disease recurrences were observed. [Correction added on 11 June 2018, after first online publication: The percentage of mutation rate was corrected to 52%]

Conclusions

Our results indicate that LGASC of the breast is a low‐grade triple‐negative breast cancer that harbours a basal‐like phenotype with no androgen receptor expression, and shows a high rate of PIK3CA mutations but no TP53 mutations.     

High frequency of TARDBP gene mutations in Italian patients with amyotrophic lateral sclerosis

Recent studies identified rare missense mutations in amyotrophic lateral sclerosis (ALS) patients in the TARDBP gene encoding TAR DNA binding protein (TDP)‐43, the major protein of the ubiquitinated inclusions (UBIs) found in affected motor neurons (MNs). The aim of this study was to further define the spectrum of TARDBP mutations in a large cohort of 666 Italian ALS patients (125 familial and 541 sporadic cases). The entire coding region was sequenced in 281 patients, while in the remaining 385 cases only exon 6 was sequenced. In 18 patients, of which six are familial, we identified 12 different heterozygous missense mutations (nine novel) all locating to exon 6, which were absent in 771 matched controls. The c.1144G>A (p.A382T) variation was observed in seven patients, thus representing the most frequent TARDBP mutation in ALS. Analysis of microsatellites surrounding the TARDBP gene indicated that p.A382T was inherited from a common ancestor in 5 of the 7 patients. Altogether, the frequency of TARDBP gene mutations appears to be particularly high in Italian ALS patients compared to individuals of mainly Northern European origin (2.7% vs. 1%). Western blot analysis of lymphocyte extracts from two patients carrying the p.A382T and p.S393L TARDBP mutations showed the presence of lower molecular weight TDP‐43 bands, which were more abundant than observed in healthy controls and patients negative for TARDBP mutations. In conclusion, this report contributes to the demonstration of the causative role of the TARDBP gene in ALS pathogenesis and indicates that mutations may affect the stability of the protein even in nonneuronal tissues. Hum Mutat 0, 1–7, 2009. © 2009 Wiley‐Liss, Inc.     

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.     

Characterization of novel Bruton's tyrosine kinase gene mutations in Central European patients with agammaglobulinemia

X-linked agammaglobulinemia (XLA) is an immunodeficiency disorder caused by mutations in the gene coding for Bruton's tyrosine kinase (BTK). In this study we investigated 10 male patients with XLA-compatible phenotype (agammaglobulinemia and undetectable B cells in peripheral blood) from 9 unrelated Central European families. We identified seven different mutations, six of which were novel. One previously described point mutation caused a premature stop codon (p.C464X), two point mutations resulted in amino acid exchanges (p.W588R; p.G419E), and two point mutations affected splice sites (c.305-1G>A; c.391+1G>A). We further detected one deletion (c.1921_1927del CGTCCCA) and one large duplication. The duplication resulted from Alu element-induced unequal homologous recombination, which was only detectable by extended analysis of cDNA, while direct sequencing of genomic DNA gave a false negative result. Western blot analysis revealed that the patients with the p.W588R and the p.G419E amino acid substitutions, respectively, produced full length BTK, but in clearly diminished amounts. The patient with the 7bp deletion expressed low amounts of protein which might represent truncated BTK. All other genomic alterations resulted in complete loss of BTK protein. In two patients from unrelated families BTK protein expression was normal and no Btk gene mutation was detected. The results of this study further substantiate the importance of using elaborate molecular analysis with different detection techniques to obtain an explicit molecular diagnosis in patients with suspected XLA.     

High-resolution melting analysis for rapid detection of KRAS, BRAF, and PIK3CA gene mutations in colorectal cancer

High-resolution melting analysis (HRMA) provides a valid approach to efficiently detect DNA genetic and somatic mutations. In this study, HRMA was used for the screening of 116 colorectal cancers (CRCs) to detect hot-spot mutations in the KRAS and BRAF oncogenes. Mutational hot spots on the PIK3CA gene, exons 9 and 20, were also screened. Direct sequencing was used to confirm and characterize HRMA results. HRMA revealed abnormal melting profiles in 65 CRCs (56.0%), 16 of them harboring mutations in 2 different genes simultaneously. The frequency of mutations was 17.2% for PIK3CA (11.2% in exon 9 and 6.0% in exon 20), 43.1% for KRAS exon 2, and 9.5% in exon 15 of the BRAF gene. We found a significant association between PIK3CA and KRAS mutations (P = .008), whereas KRAS and BRAF mutations were mutually exclusive (P = .001). This report describes a novel approach for the detection of PIK3CA somatic mutations by HRMA.     

CLAPO syndrome: identification of somatic activating PIK3CA mutations and delineation of the natural history and phenotype

PurposeCLAPO syndrome is a rare vascular disorder characterized by capillary malformation of the lower lip, lymphatic malformation predominant on the face and neck, asymmetry, and partial/generalized overgrowth. Here we tested the hypothesis that, although the genetic cause is not known, the tissue distribution of the clinical manifestations in CLAPO seems to follow a pattern of somatic mosaicism.MethodsWe clinically evaluated a cohort of 13 patients with CLAPO and screened 20 DNA blood/tissue samples from 9 patients using high-throughput, deep sequencing.ResultsWe identified five activating mutations in the PIK3CA gene in affected tissues from 6 of the 9 patients studied; one of the variants (NM_006218.2:c.248T>C; p.Phe83Ser) has not been previously described in developmental disorders.ConclusionWe describe for the first time the presence of somatic activating PIK3CA mutations in patients with CLAPO. We also report an update of the phenotype and natural history of the syndrome.