Somatic Mutations In PIK3CA Research Articles

P.19.6 PIK3CA somatic mutation in congenital monomelic muscular hypertrophy of the upper extremity. Case report

Congenital isolated unilateral hypertrophy of upper extremity secondary to muscular hyperplasia is a rare phenomenon of unknown etiology. There are only few reports variably defining it as an aberrant muscle syndrome or congenital monomelic muscular hypertrophy. We report the case of a 6-year old girl with a congenital enlargement of the whole left arm, and hand deformity. MRI showed a generalized enlargement given by homogenous increase in muscle bulk with no fat replacement together with aberrant muscles in the hand. No dysfunctions of the shoulder, elbow or wrist were detected. The patient presented normal development, with no cutaneous stigma nor additional features usually seen in Proteus syndrome. The left hand was wider with ulnar drift of the index finger, increased muscle bulk of the first dorsal interosseous and widening of the 2nd and 3rd metacarpal space. A muscle biopsy of the prominent tissue in the dorsal face of the hand revealed dystrophic features with abnormally increased variability of fibres together with many hypertrophic fibres associated with increased perimysial and endomysial fibrosis. Type 1 fiber predominance, and disorganization of the intermyofibrilar network was evident at histochemistry for NADH, COX and SDH. Mosaic activating mutation in the PI3K–AKT cell signaling pathway have been described in overgrowth syndromes such as Proteus syndrome, CLOVES syndrome, megalencephaly–capillary malformation, fibroadipose hyperplasia and macrodactyly. Given the relationship of mosaic activating mutations in the PI3K–AKT cell signaling pathway with other overgrowth syndromes, Sanger sequencing for AKT, PIK3CA, and PTEN was performed. A mutation c.3140A > G, p.H1047R in PIK3CA was detected in DNA from the affected muscle but not from blood leucocytes. Isolated congenital upper limb hypertrophy with aberrant hand muscles is another condition related genetically to overgrowth syndromes.

PIK3CA mutations are frequently observed in BRCAX but not BRCA2-associated male breast cancer.

IntroductionAlthough a substantial proportion of male breast cancers (MBCs) are hereditary, the molecular pathways that are activated are unknown. We therefore examined the frequency and clinicopathological associations of the PIK3CA/mammalian target of rapamycin (mTOR) and mitogen-activated protein kinase (MAPK) pathways and their regulatory genes in familial MBC.MethodsHigh resolution melting analysis and confirmatory sequencing was used to determine the presence of somatic mutations in PIK3CA (exon 9 and 20), AKT1 (exon 4), KRAS (exon 2) and BRAF (exon 15) genes in 57 familial MBCs. Further analysis of the PIK3CA/mTOR pathway was performed using immunohistochemistry for the pAKT1, pS6 and p4EBP1 biomarkers.ResultsPIK3CA somatic mutations were identified in 10.5% (6 of 57) of cases; there were no AKT1, KRAS or BRAF somatic mutations. PIK3CA mutations were significantly more frequent in cancers from BRCAX patients (17.2%, 5/29) than BRCA2 (0%, 0/25) carriers (P = 0.030). Two BRCAX patients had an E547K mutation which has only been reported in one female breast cancer previously. PIK3CA mutation was significantly correlated with positive pS6 (83.3% vs. 32.0%, P = 0.024) and negative p4EBP1 (100% vs. 38.0%, P = 0.006) expression, but not pAKT expression. Expression of nuclear p4EBP1 correlated with BRCA2 mutation carrier status (68.0% vs. 38.7%, P = 0.035).ConclusionsSomatic PIK3CA mutation is present in familial male breast cancer but absent in BRCA2 carriers. The presence of two of the extremely rare E547K PIK3CA mutations in our cohort may have specific relevance in MBCs. Further study of PIK3CA in MBCs, and in particular BRCAX patients, may contribute to further establishing the relevance of specific PIK3CA mutations in MBC aetiology and in the identification of particular patient groups most likely to benefit from therapeutic targeting with the novel PIK3CA inhibitors that are currently in development.

Discovery of colorectal cancer PIK3CA mutation as potential predictive biomarker: power and promise of molecular pathological epidemiology

Regular use of aspirin reduces incidence and mortality of various cancers, including colorectal cancer. Anticancer effect of aspirin represents one of the 'Provocative Questions' in cancer research. Experimental and clinical studies support a carcinogenic role for PTGS2 (cyclooxygenase-2), which is an important enzymatic mediator of inflammation, and a target of aspirin. Recent 'molecular pathological epidemiology' (MPE) research has shown that aspirin use is associated with better prognosis and clinical outcome in PIK3CA-mutated colorectal carcinoma, suggesting somatic PIK3CA mutation as a molecular biomarker that predicts response to aspirin therapy. The PI3K (phosphatidylinositol-4,5-bisphosphonate 3-kinase) enzyme has a pivotal role in the PI3K-AKT signaling pathway. Activating PIK3CA oncogene mutations are observed in various malignancies including breast cancer, ovarian cancer, brain tumor, hepatocellular carcinoma, lung cancer and colon cancer. The prevalence of PIK3CA mutations increases continuously from rectal to cecal cancers, supporting the 'colorectal continuum' paradigm, and an important interplay of gut microbiota and host immune/inflammatory reaction. MPE represents an interdisciplinary integrative science, conceptually defined as 'epidemiology of molecular heterogeneity of disease'. As exposome and interactome vary from person to person and influence disease process, each disease process is unique (the unique disease principle). Therefore, MPE concept and paradigm can extend to non-neoplastic diseases including diabetes mellitus, cardiovascular diseases, metabolic diseases, and so on. MPE research opportunities are currently limited by paucity of tumor molecular data in the existing large-scale population-based studies. However, genomic, epigenomic and molecular pathology testings (for example, analyses for microsatellite instability, MLH1 promoter CpG island methylation, and KRAS and BRAF mutations in colorectal tumors) are becoming routine clinical practices. In order for integrative molecular and population science to be routine practice, we must first reform education curricula by integrating both population and molecular biological sciences. As consequences, next-generation hybrid molecular biological and population scientists can advance science, moving closer to personalized precision medicine and health care.

PIKHER2: A phase Ib/II study evaluating safety and efficacy of oral BKM120 in combination with lapatinib in HER2-positive, PI3K-activated, trastuzumab-resistant advanced breast cancer.

TPS663 Background: Phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR)-pathway is frequently activated in HER2-positive breast cancer and may play a major role in resistance to trastuzumab. BKM120 is an oral pan-class I PI3K inhibitor with potent and selective activity against wild-type and mutant PI3K p110α. PIKHER2 study will evaluate safety and efficacy of oral, daily lapatinib-BKM120 combination in HER2-positive, trastuzumab-resistant advanced breast cancer (ABC) with activated PI3K/AKT/mTOR pathway. Methods: This open label, single arm, multi-center study includes a phase Ib dose-escalation part and a phase II expansion part at the recommended dose (RP2D). Primary objectives include determination of the maximum-tolerated dose (MTD) of BKM120 in combination with lapatinib in trastumuzab-resistant HER2-positive ABC (phase Ib part) and evaluation of the activity of BKM120-lapatinib combination at RP2D as measured by objective response rate (ORR) in patients with activated PI3K/AKT/mTOR pathway, as defined by PTEN-negative by IHC and/or somatic mutations (exons 9 and 20) of PIK3CA and/or overexpression of phospho-AKT by IHC (phase II part). Secondary objectives include safety and tolerability of the combination, clinical benefit and progression-free survival, pharmacokinetic profiles, biological and pharmacodynamic correlates. Main eligibility criteria are PS ≤ 1, HER2-positive ABC resistant to trastuzumab, documented activated PI3K/AKT/mTOR pathway (phase II part only). A modified CRM using an adaptive Bayesian model guides the dose escalation of both agents with a maximum of 24 patients planned to be enrolled in phase Ib part. Efficacy will be tested according to a standard 2-stage Simon design under the following unacceptable and desirable hypotheses: H0, ORR <= 10% (unacceptable rate) vs. H1, ORR >=30% (desirable rate). A maximum of 35 patients with activated PI3K/AKT/mTOR pathway is planned to be enrolled in phase II part with an interim look after 18 evaluable patients will have been recruited. Clinical trial information: NCT01589861.

Safety, pharmacokinetics, and preliminary activity of the α-specific PI3K inhibitor BYL719: Results from the first-in-human study.

2531 Background: BYL719 is an oral small-molecule inhibitor of the p110α catalytic subunit of phosphatidylinositol 3-kinase (PI3K), which is encoded by the PIK3CA gene, one of the most commonly mutated genes in human cancers. BYL719 inhibits proliferation of PI3Kα-driven cancer cell lines in vitro and causes regression of PIK3CA-mutant tumor models in vivo. Methods: This Ph I study was performed in patients (pts) with advanced solid tumors carrying a somatic mutation of PIK3CA. Dose escalation used an adaptive Bayesian logistic regression model with overdose control. Following determination of the maximum tolerated dose (MTD), an expansion cohort was opened at the MTD to evaluate safety, pharmacokinetics (PK), and clinical activity in pts with PIK3CA-mutant advanced solid tumors, including estrogen receptor-positive (ER+) metastatic breast cancer (mBC). Results: During dose escalation 36 pts received doses up to 450 mg/d, where 4/9 pts had dose-limiting toxicities (DLTs). The MTD for once-daily dosing was declared as 400 mg/d. As of Nov 20 2012, DLTs were hyperglycemia, nausea, vomiting, and diarrhea. The most common BYL719-related adverse events (all grades, all cohorts, >25%) were hyperglycemia (49%), nausea (45%), diarrhea (40%), decreased appetite (38%), vomiting (30%), and fatigue (27%). 39 pts are enrolled in the MTD dose-expansion cohort. Investigation of a twice-daily regimen is also ongoing. BYL719 has a favorable, approximately dose-proportional PK profile with a Tmax of 2h and a T½ of 11h at the MTD. Partial responses were seen in 7 pts (in ER+ breast [2], cervical, trichilemmal, endometrial, ovarian, and head &neck cancer [1 each]); 17 pts stayed on study for >24 weeks. For 67 pts (76%) treated at doses of ≥270 mg/d, the median progression-free survival (mPFS) was 3.6 months (mo; 95% CI: 3.5–5.5 mo). mPFS in 15 ER+ HER2– mBC pts treated at ≥270 mg/d was 5.5 mo (95% CI: 3–7 mo). Conclusions: BYL719 displays dose-proportional and predictable PK. The safety profile is favorable, with mostly manageable on-target toxicities. At doses of ≥270 mg/d, tumor regression and prolonged disease control were observed in heavily pretreated pts with various tumor types carrying a PIK3CA mutation. Clinical trial information: NCT01219699.

Abstract 4764: The pathogenesis of ovarian Brenner tumors.

Abstract Benign Brenner tumors (BTs) are composed of nests of epithelial cells resembling urothelium, which are surrounded by a dense fibromatous stroma and are considered the benign counterpart of ovarian transitional cell tumors. Their origin remains enigmatic. We have previously described the junction between the fallopian tubal epithelium and the mesothelium of the tubal serosa, and reported that transitional cell metaplasia (TM) is frequently found in this location. We hypothesize that BTs arise from TM epithelium that, when embedded in the ovary as Walthard cell nests (WCs), may develop into BTs, some of which may progress to so-called “atypical proliferative BTs”. The aim of the present study was to evaluate the immunohistochemical (IHC) and molecular genetic features of BTs in an attempt to confirm this hypothesis. A total of 17 ovaries, 25 peritoneal mesothelia, 38 fallopian tubes, 10 TMs, 56 WCs, 48 BTs and 7 atypical proliferative BTs were included in this study. IHC for AKR1C3, alpha-inhibin (INH), AR, β-catenin, calretinin (CRT), CEA, CYP19, ER, p16, PAX2, PAX8, PR, SF1, and WT1, and fluorescence in situ hybridization (FISH) for CDKN2A (p16 encoding gene) were performed. Genomic DNA from 20 BTs was extracted for sequence analysis of BRAF, CTNNB1, ERBB2, FOXL2, KRAS, PIK3CA, and PPP2R1A mutations. A somatic mutation of PIK3CA was detected in the stromal component in one BT. No other mutations were detected in either the epithelial or the stromal component in the remaining BTs. IHC analysis revealed that fallopian tubal secretory cells, TM, WC and epithelial component of BTs and atypical proliferative BTs shared the same IHC profile, consistently expressing AKR1C3, AR, but not CRT, as opposed to mesothelium and ovarian surface epithelium. The stromal component of the tumors showed weak expression of AKR1C3 in 23/26 (88%), AR in 26/29 (90%), ER in 32/42 (74%), PR in 38/43 (88%), and WT1 in 13/41 (32%), and strong expression of CRT in 34/41 (83%), INH in 33/41 (80%), and SF1 in 38/40 (95%) BTs. Notably, CRT, INH, and SF1 showed particularly strong staining in the stromal cells that immediately surrounded the epithelial nests. In contrast, expression of these biomarkers was absent in the BT epithelium. p16 was intensely and diffusely positive in the epithelial component of 9/13 (69%), patchy in 3/13 (23%), and negative in 1/13 (8%) benign BTs. In contrast all 7 atypical proliferative BTs were completely negative for p16 in the epithelial component. FISH identified homozygous deletion of CDNK2A in all atypical proliferative BTs, but CDKN2A was retained in benign BTs (Fisher exact test two sided p-value<0.0001). In summary, our findings suggest: 1) tubal origin of BTs through TM and WC, based on similar IHC profile, 2) an active role of the ovarian stroma in the pathogenesis of BT, based on the periepithelial IHC pattern of CRT, INH, and SF1, markers of steroidogenic cells, along with the epithelial expression of AR, 3) the role of CDKN2A loss in the progression of BT to atypical proliferative BT. Citation Format: Elisabetta Kuhn, Ayse Ayhan Ayhan, Jeffrey D. Seidman, Robert J. Kurman. The pathogenesis of ovarian Brenner tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4764. doi:10.1158/1538-7445.AM2013-4764 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Novel Method for PIK3CA Mutation Analysis: Locked Nucleic Acid–PCR Sequencing

Somatic mutations in PIK3CA are commonly seen in invasive breast cancer and several other carcinomas, occurring in three hotspots: codons 542 and 545 of exon 9 and in codon 1047 of exon 20. We designed a locked nucleic acid (LNA)-PCR sequencing assay to detect low levels of mutant PIK3CA DNA with attention to avoiding amplification of a pseudogene on chromosome 22 that has >95% homology to exon 9 of PIK3CA. We tested 60 FFPE breast DNA samples with known PIK3CA mutation status (48 cases had one or more PIK3CA mutations, and 12 were wild type) as identified by PCR-mass spectrometry. PIK3CA exons 9 and 20 were amplified in the presence or absence of LNA-oligonucleotides designed to bind to the wild-type sequences for codons 542, 545, and 1047, and partially suppress their amplification. LNA-PCR sequencing confirmed all 51 PIK3CA mutations; however, the mutation detection rate by standard Sanger sequencing was only 69% (35 of 51). Of the 12 PIK3CA wild-type cases, LNA-PCR sequencing detected three additional H1047R mutations in "normal" breast tissue and one E545K in usual ductal hyperplasia. Histopathological review of these three normal breast specimens showed columnar cell change in two (both with known H1047R mutations) and apocrine metaplasia in one. The novel LNA-PCR shows higher sensitivity than standard Sanger sequencing and did not amplify the known pseudogene.

Type I to Type II Ovarian Carcinoma Progression: Mutant Trp53 or Pik3ca Confers a More Aggressive Tumor Phenotype in a Mouse Model of Ovarian Cancer

A dualistic pathway model of ovarian carcinoma (OvCA) pathogenesis has been proposed: type I OvCAs are low grade, genetically stable, and relatively more indolent than type II OvCAs, most of which are high-grade serous carcinomas. Endometrioid OvCA (EOC) is a prototypical type I tumor, often harboring mutations that affect the Wnt and phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin signaling pathways. Molecular and histopathologic analyses indicate type I and II OvCAs share overlapping features, and a subset of EOCs may undergo type I→type II progression accompanied by acquisition of somatic TP53 or PIK3CA mutations. We used a murine model of EOC initiated by conditional inactivation of the Apc and Pten tumor suppressor genes to investigate mutant Trp53 or Pik3ca alleles as key drivers of type I→type II OvCA progression. In the mouse EOC model, the presence of somatic Trp53 or Pik3ca mutations resulted in shortened survival and more widespread metastasis. Activation of mutant Pik3ca alone had no demonstrable effect on the ovarian surface epithelium but resulted in papillary hyperplasia when coupled with Pten inactivation. Our findings indicate that the adverse prognosis associated with TP53 and PIK3CA mutations in human cancers can be functionally replicated in mouse models of type I→type II OvCA progression. Moreover, the models should represent a robust platform for assessment of the contributions of Trp53 or Pik3ca defects in the response of EOCs to conventional and targeted drugs.

Impaired Pten Expression in Human Malignant Peripheral Nerve Sheath Tumours

Malignant peripheral nerve sheath tumours (MPNST) are aggressive sarcomas that develop in about 10% of patients with the genetic disease neurofibromatosis type 1 (NF1). Molecular alterations contributing to MPNST formation have only partially been resolved. Here we examined the role of Pten, a key regulator of the Pi3k/Akt/mTOR pathway, in human MPNST and benign neurofibromas. Immunohistochemistry showed that Pten expression was significantly lower in MPNST (n = 16) than in neurofibromas (n = 16) and normal nervous tissue. To elucidate potential mechanisms for Pten down-regulation or Akt/mTOR activation in MPNST we performed further experiments. Mutation analysis revealed absence of somatic mutations in PTEN (n = 31) and PIK3CA (n = 38). However, we found frequent PTEN promotor methylation in primary MPNST (11/26) and MPNST cell lines (7/8) but not in benign nerve sheath tumours. PTEN methylation was significantly associated with early metastasis. Moreover, we detected an inverse correlation of Pten-regulating miR-21 and Pten protein levels in MPNST cell lines. The examination of NF1−/− and NF1+/+Schwann cells and fibroblasts showed that Pten expression is not regulated by NF1. To determine the significance of Pten status for treatment with the mTOR inhibitor rapamycin we treated 5 MPNST cell lines with rapamycin. All cell lines were sensitive to rapamycin without a significant correlation to Pten levels. When rapamycin was combined with simvastatin a synergistic anti-proliferative effect was achieved. Taken together we show frequent loss/reduction of Pten expression in MPNST and provide evidence for the involvement of multiple Pten regulating mechanisms.

Moving next-generation sequencing into the clinical realm: Detection of somatic mutations in cancer by targeted amplicon sequencing.

60 Background: Molecular targeted therapies are increasingly important in treating cancer patients; robust analytically validated clinical assays are required for patient selection in early-stage clinical trials. The goal of Molecular Characterization Laboratory (MoCha) is to develop clinical diagnostic assays using next generation sequencing methods to support clinical studies in DCTD (CTEP). Methods: We developed a custom assay for somatic mutation detection using Fluidigm access array technology for amplicon generation followed by sequencing with the Illumina Miseq. A panel of 62 amplicons covering 6 Kb genomic regions was designed to detect 92 DNA loci, including common therapeutically actionable targets, in 37 genes. Analytical studies were performed using genomic DNA samples from fresh or formalin fixed cancer cell-lines and a normal hapmap individual (CEPH). We subsequently applied this assay to characterize DNA samples from both tumor tissues and blood specimens from ovarian cancer patients. Results: The assay detected known variants in both frozen and fixed DNA samples reproducibly with high sensitivity and specificity (<2%). Using a series of positive control plasmid spikes mixed into a normal reference CEPH DNA at pre-defined copy number ratios, we verified the assay is sensitive to detect variants at 5% allelic frequency with a minimum 400 X coverage. We identified somatic mutations in TP53 and PIK3CA in a few patients, and a germ-line variant D1583N in ATM genes occurring in one-third of tested patients. Conclusions: We developed and validated a next generation sequencing assay suitable for patient selection for clinical trials. Plans are to correlate sequencing and clinical results when clinical data are available.

Prevalence of KRAS, BRAF, and PIK3CA somatic mutations in patients with colorectal carcinoma may vary in the same population: clues from Sardinia.

BackgroundRole of KRAS, BRAF and PIK3CA mutations in pathogenesis of colorectal cancer (CRC) has been recently investigated worldwide. In this population-based study, we evaluated the incidence rates and distribution of such somatic mutations in genetically isolated population from Sardinia.MethodsFrom April 2009 to July 2011, formalin-fixed paraffin-embedded tissues (N = 478) were prospectively collected from Sardinian CRC patients at clinics across the entire island. Genomic DNA was isolated from tissue sections and screened for mutations in KRAS, BRAF, and PIK3CA genes by automated DNA sequencing.ResultsOverall, KRAS tumour mutation rate was 30% (145/478 positive cases). Distribution of mutation carriers was surprisingly different within the island: 87/204 (43%) in North Sardinia vs. 58/274 (21%) in Middle-South Sardinia (p<0.001). Among 384 CRC cases whose DNA was available, only one (0.3%) patient carried a mutation in BRAF gene; PIK3CA was found mutated in 67 (17%) patients. A significant inverse distribution of PIK3CA mutation rates was observed within Sardinian population: 19/183 (10%) cases from northern vs. 48/201 (24%) cases from central-southern island (p<0.001). This heterogeneity in frequencies of KRAS/PIK3CA somatic mutations is consistent with already-reported discrepancies in distribution of germline mutations for other malignancies within Sardinian population. Preliminary clinical evaluation of 118 KRAS wild-type patients undergoing anti-EGFR-based treatment indicated lack of role for PIK3CA in predicting response to therapy.ConclusionsOur findings support the hypothesis that differences in patients’ origins and related genetic backgrounds may contribute to even determine the incidence rate of somatic mutations in candidate cancer genes.

Identification of Molecular Pathway Aberrations in Uterine Serous Carcinoma by Genome-wide Analyses

Uterine cancer is the fourth most common malignancy in women, and uterine serous carcinoma is the most aggressive subtype. However, the molecular pathogenesis of uterine serous carcinoma is largely unknown. We analyzed the genomes of uterine serous carcinoma samples to better understand the molecular genetic characteristics of this cancer. Whole-exome sequencing was performed on 10 uterine serous carcinomas and the matched normal blood or tissue samples. Somatically acquired sequence mutations were further verified by Sanger sequencing. The most frequent molecular genetic changes were further validated by Sanger sequencing in 66 additional uterine serous carcinomas and in nine serous endometrial intraepithelial carcinomas (the preinvasive precursor of uterine serous carcinoma) that were isolated by laser capture microdissection. In addition, gene copy number was characterized by single-nucleotide polymorphism (SNP) arrays in 23 uterine serous carcinomas, including 10 that were subjected to whole-exome sequencing. We found frequent somatic mutations in TP53 (81.6%), PIK3CA (23.7%), FBXW7 (19.7%), and PPP2R1A (18.4%) among the 76 uterine serous carcinomas examined. All nine serous carcinomas that had an associated serous endometrial intraepithelial carcinoma had concordant PIK3CA, PPP2R1A, and TP53 mutation status between uterine serous carcinoma and the concurrent serous endometrial intraepithelial carcinoma component. DNA copy number analysis revealed frequent genomic amplification of the CCNE1 locus (which encodes cyclin E, a known substrate of FBXW7) and deletion of the FBXW7 locus. Among 23 uterine serous carcinomas that were subjected to SNP array analysis, seven tumors with FBXW7 mutations (four tumors with point mutations, three tumors with hemizygous deletions) did not have CCNE1 amplification, and 13 (57%) tumors had either a molecular genetic alteration in FBXW7 or CCNE1 amplification. Nearly half of these uterine serous carcinomas (48%) harbored PIK3CA mutation and/or PIK3CA amplification. Molecular genetic aberrations involving the p53, cyclin E-FBXW7, and PI3K pathways represent major mechanisms in the development of uterine serous carcinoma.

Mosaic overgrowth with fibroadipose hyperplasia is caused by somatic activating mutations in PIK3CA

The phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway is critical for cellular growth and metabolism. Correspondingly, loss of function of PTEN, a negative regulator of PI3K, or activating mutations in AKT1, AKT2 or AKT3 have been found in distinct disorders featuring overgrowth or hypoglycemia. We performed exome sequencing of DNA from unaffected and affected cells from an individual with an unclassified syndrome of congenital progressive segmental overgrowth of fibrous and adipose tissue and bone and identified the cancer-associated mutation encoding p.His1047Leu in PIK3CA, the gene that encodes the p110α catalytic subunit of PI3K, only in affected cells. Sequencing of PIK3CA in ten additional individuals with overlapping syndromes identified either the p.His1047Leu alteration or a second cancer-associated alteration, p.His1047Arg, in nine cases. Affected dermal fibroblasts showed enhanced basal and epidermal growth factor (EGF)-stimulated phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) generation and concomitant activation of downstream signaling relative to their unaffected counterparts. Our findings characterize a distinct overgrowth syndrome, biochemically demonstrate activation of PI3K signaling and thereby identify a rational therapeutic target.

Sequence analysis of mutations and translocations across breast cancer subtypes.

Breast carcinoma is the leading cause of cancer-related mortality in women worldwide, with an estimated 1.38 million new cases and 458,000 deaths in 2008 alone. This malignancy represents a heterogeneous group of tumours with characteristic molecular features, prognosis and responses to available therapy. Recurrent somatic alterations in breast cancer have been described, including mutations and copy number alterations, notably ERBB2 amplifications, the first successful therapy target defined by a genomic aberration. Previous DNA sequencing studies of breast cancer genomes have revealed additional candidate mutations and gene rearrangements. Here we report the whole-exome sequences of DNA from 103 human breast cancers of diverse subtypes from patients in Mexico and Vietnam compared to matched-normal DNA, together with whole-genome sequences of 22 breast cancer/normal pairs. Beyond confirming recurrent somatic mutations in PIK3CA, TP53, AKT1, GATA3 and MAP3K1, we discovered recurrent mutations in the CBFB transcription factor gene and deletions of its partner RUNX1. Furthermore, we have identified a recurrent MAGI3-AKT3 fusion enriched in triple-negative breast cancer lacking oestrogen and progesterone receptors and ERBB2 expression. The MAGI3-AKT3 fusion leads to constitutive activation of AKT kinase, which is abolished by treatment with an ATP-competitive AKT small-molecule inhibitor.

Clinicopathologic and biological analysis of PIK3CA mutation in ovarian clear cell carcinoma

Somatic mutations of PIK3CA (phosphoinositide-3-kinase) have recently been shown playing an important role in the pathogenesis of ovarian clear cell carcinoma. In this study, the frequency of PIK3CA mutations and the relationship of PIK3CA mutations with clinicopathologic and biological variables were investigated in ovarian clear cell carcinomas from Japanese patients. Mutational analysis of PIK3CA was performed in 56 primary ovarian clear cell carcinomas from Japanese women. The relationship of these mutations with various clinicopathologic and biological variables (phosphorylated AKT and phosphorylated mTOR (mammalian target of rapamycin) expression by immunohistochemistry) was determined. To clarify the roles of PI3K/AKT activation in ovarian clear cell carcinomas harboring PIK3CA mutations, we inactivated the PI3K/AKT/mTOR pathway in ovarian carcinoma cells with LY294002, temsirolimus and NVP-BEZ235. Missense mutations of PIK3CA were found in 16 (28.6%) of 56 ovarian clear cell carcinomas, but no mutation was found in 15 ovarian high-grade serous carcinomas. PIK3CA mutations were significantly associated with a favorable overall survival of patients with ovarian clear cell carcinoma (P < .05). There was no significant association between PIK3CA mutations and phosphorylated AKT or phosphorylated mTOR immunointensity status. No relationship was found between PIK3CA mutation status and sensitivity to PI3K/AKT/mTOR inhibitors in ovarian clear cell carcinoma cells. No association of PIK3CA mutations was found between positive phosphorylated AKT and positive phosphorylated mTOR, which suggests that the PI3K/AKT/mTOR pathway may be activated by other molecular mechanisms. Although PIK3CA mutations were associated with a more favorable prognosis, they did not predict the sensitivity of ovarian clear cell carcinoma cells to PI3K/AKT/mTOR inhibitors.

Abstract 985: Dissecting PI3K-AKT pathway by phosphoproteomic profiling of PIK3CA knock-in mutants

Abstract The phosphatidylinositol 3-kinase subunit PIK3CA is frequently mutated in human cancers. PIK3CA mutations are observed in about 30% of breast cancers. Three recurrent oncogenic “hotspot” mutations comprise the majority of somatic PIK3CA mutations. Two of these mutations, E542K and E545K, occur in the helical domain found in exon 9, and the third mutation, H1047R, affects the kinase domain located within exon 20. Although many studies have implicated PIK3CA mutations with features of transformation, definitive mechanisms describing how these mutations promote cell growth and proliferation have not been fully elucidated. Two MCF10A (a normal mammary gland epithelial cell line) knock-in cell lines with E545K or H1047R hotspot mutations were used for our phosphoproteome study. We employed stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative mass spectrometry to study phosphoproteomic alterations caused by PIK3CA onogenic mutations. In order to identify signaling proteins directly involved in PI3K-AKT pathway, a novel PIK3CA specific inhibitor (J124) was used to suppress PIK3CA activity. Phosphopeptides were enriched by TiO2-based strategy after strong cation exchange (SCX) fractionation and analyzed on a high resolution Fourier transform LTQ-Orbitrap Velos mass spectrometer. We identified 5,491 unique phosphopeptides from 1,546 unique proteins - of these, 2,239 peptides were hyperphosphorylated in MCF10A cell with PIK3CA mutants. Notably, the PIK3CA inhibitor, J124, could specifically suppress the phosphorylation levels of 1,187 hyperphosphorylated peptides. These are likely to be effectors of PI3Kalthough only 34 of them were previously known AKT1 substrates. By integrating data from protein microarray-based phosphorylation experiments with AKT1, we found 16 novel hyperphosphorylated proteins that were phosphorylated in vitro by AKT1. In addition to identifying novel AKT substrates, we also identified multiple receptor tyrosine kinases including EGFR, FGFR and EPHA2 to be hyperphosphorylated in PIK3CA mutant knockin cells. This novel discovery of the reverse crosstalk initiated from PI3K-AKT to RTKs could explain oncogenic consequences of cells with PIK3CA mutants such as growth factor-independent growth and invasive ability. In summary, our comprehensive phosphoproteomic study reveals that oncogenic activation of the PI3K-AKT pathway can activate a broad spectrum of signaling molecules. By integrating data from large-scale protein microarray studies, we were able to identify novel AKT substrates. The knowledge gained from these studiescan greatly enhance our understanding of the PI3K-AKT pathway and provide potential therapeutic targets for attenuating oncogenic effects of PIK3CA mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 985. doi:1538-7445.AM2012-985

The clonal and mutational evolution spectrum of primary triple-negative breast cancers

Primary triple-negative breast cancers (TNBCs), a tumour type defined by lack of oestrogen receptor, progesterone receptor and ERBB2 gene amplification, represent approximately 16% of all breast cancers. Here we show in 104 TNBC cases that at the time of diagnosis these cancers exhibit a wide and continuous spectrum of genomic evolution, with some having only a handful of coding somatic aberrations in a few pathways, whereas others contain hundreds of coding somatic mutations. High-throughput RNA sequencing (RNA-seq) revealed that only approximately 36% of mutations are expressed. Using deep re-sequencing measurements of allelic abundance for 2,414 somatic mutations, we determine for the first time-to our knowledge-in an epithelial tumour subtype, the relative abundance of clonal frequencies among cases representative of the population. We show that TNBCs vary widely in their clonal frequencies at the time of diagnosis, with the basal subtype of TNBC showing more variation than non-basal TNBC. Although p53 (also known as TP53), PIK3CA and PTEN somatic mutations seem to be clonally dominant compared to other genes, in some tumours their clonal frequencies are incompatible with founder status. Mutations in cytoskeletal, cell shape and motility proteins occurred at lower clonal frequencies, suggesting that they occurred later during tumour progression. Taken together, our results show that understanding the biology and therapeutic responses of patients with TNBC will require the determination of individual tumour clonal genotypes.

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.

Mutational analysis of PIK3CA,JAK2,BRAF,FOXL2,IDH1,AKT1 and EZH2 oncogenes in sarcomas

Recent studies have revealed several recurrent mutations in oncogenes that could not only be underlying mechanisms of tumorigenesis, but also be potential targets for cancer therapies. Compared to carcinomas, genetic alterations of sarcomas are relatively unknown. To see whether recurrent oncogenes discovered in non-sarcomatous malignancies are present in sarcomas as well, we analyzed oncogenes with known mutations in various types of sarcomas. We performed mutational analysis of recurrent mutation sites of PIK3CA (exons 9 and 20), JAK2 (exon 14), BRAF (exon 15), FOXL2 (exon 1), IDH1 (exon 4), AKT1 (exon 3), and EZH2 (exon 16) genes in 108 sarcomas by single- strand conformation polymorphism and DNA sequencing. The sarcomas consisted of malignant fibrous histiocytomas, rhabdomyosarcomas, osteosarcomas, malignant peripheral nerve sheath tumors, leiomyosarcomas, synovial sarcomas, liposarcomas, angiosarcomas, chondrosarcomas, and Ewing sarcomas. Overall, we detected the two PIK3CA mutations and one JAK2 mutation (total: 3/108: 2.8%). Two rhabdomyosarcomas (16.7%) and one angiosarcoma (16.7%) harbored the mutations, whereas other sarcomas harbored none. The PIK3CA mutations were novel missense mutations that had not been detected in other cancers. The JAK2 mutation was an intron mutation. This study demonstrated that the somatic mutations of PIK3CA and JAK2 occurred in a small fraction of the sarcomas and that these mutations may not play a principal role in the development of sarcomas.

Somatic mutation analysis of EGFR, KRAS, BRAF and PIK3CA in 861 patients with non-small cell lung cancer

The prevalence of EGFR, KRAS, BRAF and PIK3CA somatic mutations in 861 randomly selected Chinese patients with non-small cell lung cancer (NSCLC) was assayed by the SurPlex®-xTAG70plex platform and analyzed. The results showed that the occurrence rates were 41.0, 8.0, 0.7 and 3.7%, respectively. The mutation rates significantly correlated with gender, histology and smoking history. The EGFR exon 19, 20 and 21 mutations were higher in females compared to males (p< 0.001, exon 19 and 21; p=0.018, exon 20), higher in adenocarcinomas compared to other forms of lung cancers (p< 0.001, exon 19 and 21; p=0.035, exon 20), and higher in non-smokers compared to smokers (p< 0.001, exon 19 and 21; p=0.029, exon 20). Conversely, the KRAS mutations were higher in males compared to females (p=0.004), higher in adenocarcinomas compared to other forms of lung cancers (p< 0.001), and higher in smokers compared to non-smokers (p< 0.001). The PIK3CA mutation rate was lower in adenocarcinomas compared to other forms of lung cancers (p=0.003).