Somatic Mutations In PIK3CA Research Articles

Upper limb muscle overgrowth with hypoplasia of the index finger: a new over-growth syndrome caused by the somatic PIK3CA mutation c.3140A>G

BackgroundScientists have previously described an overgrowth syndrome in Saudi patients and named it ‘Upper limb muscle overgrowth with hypoplasia of the index finger’ syndrome.Case presentationWe describe a new case and document that the syndrome is caused by the somatic PIK3CA mutation c.3140A>G, p.His1047Arg. We also recruited one of the previously reported cases and found the same somatic mutation in the affected muscles. A wider classification of ‘PIK3CA-related pathology spectrum’ is presented which includes cancer, benign skin lesions/tumors, Cowden syndrome, isolated vascular malformations and various overgrowth syndromes. The latter entity is sub-divided into 3 sub-groups: overgrowth with brain involvement, overgrowth with multiple lipomatosis, and overgrowth without brain involvement/multiple lipomatosis.ConclusionOur literature review indicated that “upper limb muscle overgrowth with hypoplasia of the index finger” is not as rare as previously thought to be. This overgrowth syndrome is unique and is caused by the somatic PIK3CA mutation c.3140A>G.

Genetic testing for lymphatic malformations with or without primary lymphedema

Abstract Lymphatic malformations (LMs) show phenotypic variability, as well as clinical and genetic heterogeneity. Inheritance is autosomal dominant, recessive or X-linked and major genes involved in predisposition for LMs are continuously being discovered. The literature also indicates that somatic mutations play an important role in the development of LMs. In fact, activating somatic mutations in PIK3CA have been reported in lymphatic endothelial cells obtained from patients with different kinds of LM. This Utility Gene Test was developed on the basis of an analysis of the literature and existing diagnostic protocols. It is useful for confirming diagnosis, as well as for differential diagnosis, couple risk assessment and access to clinical trials.

Targeted DNA and RNA Sequencing of Paired Urothelial and Squamous Bladder Cancers Reveals Discordant Genomic and Transcriptomic Events and Unique Therapeutic Implications

BackgroundIntegrated molecular profiling has identified intrinsic expression-based bladder cancer molecular subtypes. Despite frequent histological diversity, robustness of subtypes in paired conventional (urothelial) and squamous components of the same bladder tumor has not been reported. ObjectiveTo assess the impact of histological heterogeneity on expression-based bladder cancer subtypes. Design, setting, and participantsWe performed clinically applicable, targeted DNA and/or RNA sequencing (multiplexed DNA and RNA sequencing [mxDNAseq and mxRNAseq, respectively]) on 112 formalin-fixed paraffin-embedded (FFPE) bladder cancer samples, including 12 cases with paired urothelial/squamous components and 21 bladder cancer cell lines. Outcome measurements and statistical analysisUnsupervised hierarchical and consensus clustering of target gene expression enabled derivation of basal/luminal molecular subtyping. Results and limitationAcross 21 bladder cancer cell lines, our custom mxRNAseq panel was highly concordant with whole transcriptome sequencing, and assessed targets robustly determined expression-based basal/luminal subtypes from The Cancer Genome Atlas data (in silico) and internally sequenced FFPE tissues. Frequent deleterious TP53 (56%) and activating hotspot PIK3CA (30%) somatic mutations were seen across 69 high-quality tissue samples. Potentially targetable focal ERBB2 (6%) or EGFR (6%) amplifications were also identified, and a novel subgene copy-number detection approach is described. Combined DNA/RNA analysis showed that focally amplified samples exhibit outlier EGFR and ERBB2 expression distinct from subtype-intrinsic profiles. Critically, paired urothelial and squamous components showed divergent basal/luminal status in three of 12 cases (25%), despite identical putatively clonal prioritized somatic genomic alterations. Limitations include lack of profiled paired normal tissues for formal somatic alteration determination, and the need for formal analytical and clinical validation. ConclusionsOur results support the feasibility of clinically relevant integrative bladder cancer profiling and challenge the intrinsic nature of expression subtypes in histologically diverse bladder cancers. Patient summaryA targeted RNA sequencing assay is capable of assessing gene expression-based subtypes in individual components of clinical bladder cancer tissue specimens. Different histological components of the same tumor may yield divergent expression profiles, suggesting that expression-based subtypes should be interpreted with caution in heterogeneous cancers.

Breast cancer in women with neurofibromatosis type 1 (NF1): a comprehensive case series with molecular insights into its aggressive phenotype.

The purpose of the study was to improve the understanding of NF1-associated breast cancer, given the increased risk of breast cancer in this tumour predisposition syndrome and the limited data. We identified 18 women with NF1 and breast cancer at our institution. Clinical and pathologic characteristics of NF1-associated breast cancers were compared with 7132 breast cancers in patients without NF1 from our institutional database. Next generation sequencing was performed on DNA from blood and breast cancer specimens available. Blood specimens negative for NF1 mutation were subjected to multiplex ligation-dependent probe amplification (MLPA) to identify complete/partial deletions or duplications. Expression of neurofibromin in the NF1-associated breast cancers was evaluated using immunohistochemistry. There was a higher frequency of grade 3 (83.3% vs 45.4%, p = 0.005), oestrogen receptor (ER) negative (66.7% vs 26.3%, p < 0.001) and human epidermal growth factor receptor 2 (HER2)-positive (66.7% vs 23.4%, p < 0.001) tumours among NF1 patients compared to non-NF1 breast cancers. Overall survival was inferior in NF1 patients in multivariable analysis (hazard ratio 2.25, 95% CI 1.11-4.60; p = 0.025). Apart from germline NF1 mutations (11/16; 69%), somatic mutations in TP53 (8/10; 80%), second-hit NF1 (2/10; 20%), KMT2C (4/10; 40%), KMT2D (2/10; 20%), and PIK3CA (2/10; 20%) were observed. Immunohistochemical expression of neurofibromin was seen in the nuclei and/or cytoplasm of all specimens, but without any consistent pattern in the intensity or extent. This comprehensive series of NF1-associated breast cancers suggests that their aggressive features are related to germline NF1 mutations in cooperation with somatic mutations in TP53, KMT2C and other genes.

Evaluation of circulating tumor DNA in patients with endometrial cancer harboring somatic PIK3CA or KRAS mutations: A potential high-risk factor for recurrence

Evaluation of circulating tumor DNA in patients with endometrial cancer harboring somatic PIK3CA or KRAS mutations: A potential high-risk factor for recurrence

Clinico-pathological Features of PIK3CA Mutation in HER2-Positive Breast Cancer of Indian Population.

PIK3CA pathway is one of the important signaling pathways in cells, which is involved in cell proliferation, cell survival, motility, and growth. Mutation in PIK3CA gene negatively effects to anti-HER2 therapy in breast cancer patients. PIK3CA gene of HER2-positive breast cancers associated with reduced sensitivity to neoadjuvant therapy. In this study, we assessed the frequency of PIK3CA mutations and influence of PIK3CA mutations on patient survival in a series of HER2-positive breast cancer patients. PIK3CA mutations were assessed by pyrosequencing and next generation sequencing in 107 HER2-positive breast cancer patients of a tertiary Cancer Centre of India from Jan 2012 to Jun 2013 with minimum follow-up of 3years. We found PIK3CA mutations in 26 tumors (24.2%) of which 5 were in exon 9, 20 were in exon 20, and 1 was in both exon 9 and 20. In exon 9, the mutation c.1634A>G was found in 4 cases and mutation c.1636C>A was found in 2 cases. In exon 20, the mutation c.3140A>G was found in 15 cases and c.3140A>T was found in 6 cases. The outcome between PIK3CA mutated versus PIK3CA wild type was significant showing p value 0.014. Overall survival of mutation and treatment with herceptin, mutation with other chemotherapy treatment in both early breast cancer (EBC), and locally advanced breast cancer (LABC) showed significant p value 0.037 and 0.044 respectively. In conclusion, we identified 24.2% somatic mutation of PIK3CA in HER2-positive breast cancer patients. PIK3CA mutation is significantly associated with ER-positive tumors. The frequency and distribution pattern reported in this study is similar to the global report. Overall survival of PIK3CA mutation is slightly lower but in patients who received herceptin with PIK3CA mutation showed better clinical outcome.

An investigation of PIK3CA mutations in isolated macrodactyly.

Somatic PIK3CA mutations may relate to pathogenesis of isolated macrodactyly. We set up to test the association between PIK3CA mutations with isolated macrodactyly in order to establish a more accurate and molecular mechanism-based diagnosis and classification. DNA extracted from affected tissues in 12 individuals with isolated macrodactyly was tested for PIK3CA mutation using targeted Sanger DNA sequencing. Ten patients had macrodactyly in the foot and two in the hand. Nine of the 12 patients were found to carry a low-level, mosaic PIK3CA mutation. The mutations identified, p.His1047Arg, p.His1047Leu, p.Glu545Lys, and p.Glu542Lys, are codons frequently mutated in cancers. Among all tissues tested, adipose had the highest mutation detection rate, followed by nerve and skin. Our results indicate that a high proportion of isolated macrodactyly patients carry a pathogenic PIK3CA mutation. Affected adipose, nerve and skin tissues are ideal for PIK3CA mutation analysis.

Characterization of PIK3CA and PIK3R1 somatic mutations in Chinese breast cancer patients

Deregulation of the phosphoinositide 3-kinase (PI3K) pathway contributes to the development and progression of tumors. Here, we determine that somatic mutations in PIK3CA (44%), PIK3R1 (17%), AKT3 (15%), and PTEN (12%) are prevalent and diverse in Chinese breast cancer patients, with 60 novel mutations identified. A high proportion of tumors harbors multiple mutations, especially PIK3CA plus PIK3R1 mutations (9.0%). Next, we develop a recombination-based mutation barcoding (ReMB) library for impactful mutations conferring clonal advantage in proliferation and drug responses. The highest-ranking PIK3CA and PIK3R1 mutations include previously reported deleterious mutations, as well as mutations with unknown significance. These PIK3CA and PIK3R1 impactful mutations exhibit a mutually exclusive pattern, leading to oncogenesis and hyperactivity of PI3K pathway. The PIK3CA impactful mutations are tightly associated with hormone receptor positivity. Collectively, these findings advance our understanding of PI3K impactful mutations in breast cancer and have important implications for PI3K-targeted therapy in precision oncology.

Intraductal Tubulopapillary Neoplasm of the Pancreas: An Overview.

Intraductal tubulopapillary neoplasm is a rare tumor that the World Health Organization recognized in 2010 as a subtype of premalignant pancreatic neoplasms. It is important to distinguish it from other intraductal neoplasms, including intraductal papillary mucinous neoplasm, pancreatic ductal adenocarcinoma, and intraductal variant of acinar cell carcinoma, because intraductal tubulopapillary neoplasm has a favorable prognosis. Histopathologically, intraductal tubulopapillary neoplasms are characterized by tubulopapillary growth, uniform high-grade cytologic atypia, frequent necrotic foci, evident ductal differentiation, and absence of mucin. Intraductal tubulopapillary neoplasms show distinct immunohistochemical and molecular findings, with positive cytokeratin 7, cytokeratin 19, MUC1, and MUC6, and somatic PIK3CA mutations (2 of 11; 18%), and low rates of KRAS (2 of 20; 10%), TP53 (5 of 22; 23%), and BRAF (2 of 13; 15%) mutations. These differences also highlight the fact that intraductal tubulopapillary pancreatic neoplasm is distinct from other similar neoplasms.

Affinity-purified DNA-based mutation profiles of endometriosis-related ovarian neoplasms in Japanese patients.

AimEndometriosis-related ovarian neoplasms (ERONs) have recently attracted considerable attention; however, the prevalence and patterns of ARID1A and POLE mutations in ERONs have not been studied in detail. The aim of this study was to investigate not only the carcinogenesis of ERONs, but also the prognostic significance of several gene mutations in this cohort. We used DNA purified from only tumor epithelial cells, from which fibroblasts were removed, using a specific method we called “liquid microdissection”.MethodsTissue samples from 22 ovarian carcinomas (13 endometrioid, and nine clear cell) were used. Tumor cells were isolated using a cell sorting system and DNA was purified from tumor epithelial cells. Nucleotide sequencing was conducted to analyze the mutational status of ARID1A, p53, PTEN, POLE, PIK3CA, and KRAS.ResultsIn ERONs, the frequencies of somatic mutations in ARID1A, p53, POLE, PTEN, PIK3CA, and KRAS were 19/20 (95.0%), 7/19 (36.8%), 9/22 (40.9%), 13/19 (68.4%), 3/19 (15.8%), and 1/9 (11.1%). The frequency of ARID1A mutations was significantly higher than that reported previously. Kaplan-Meier survival analysis revealed that mutations in all genes, including POLE, were not associated with patient prognosis in our Japanese cohort.ConclusionsOur results suggest that the frequency of ARID1A mutations in ERONs may be higher than that previously reported. In addition, the “liquid microdissection” method that we chose for DNA purification could be used to obtain high-quality sequencing results. The findings suggest that ARID1A mutations represent the basis of ERON carcinogenesis; other subsequent gene mutations may result in the progression of carcinogenesis.

Abstract P3-04-11: Implications of somatic TP53 and PIK3CA mutations in patients with metastatic breast cancer who underwent germline BRCA testing

Abstract Background: TP53 and PIK3CA are the most frequently mutated genes in breast cancer. However, there is limited data evaluating the impact of somatic mutations in TP53 and/or PIK3CA patients (pts) with metastatic breast cancer (MBC) who have undergone germline testing for BRCA mutations. Here, we report the frequency of somatic TP53 and PIK3CA mutations in MBC pts who previously underwent germline BRCA (gBRCA) testing and evaluate their impact on long-term outcomes. Methods: We identified pts with MBC from our prospectively maintained high risk genetics database who underwent gBRCA testing and had somatic mutation testing performed for TP53 and/or PIK3CA through hotspot sequencing of single genes or as part of a next generation sequencing panel. Univariable logistic regression was used to evaluate associations between clinicopathological characteristics, including gBRCA status, and the presence of somatic TP53 and PIK3CA mutations. Overall survival (OS) was defined as the time from diagnosis to death from any cause. Multivariable cox regression analysis was used to identify independent predictors of OS. Results: 104 pts met all inclusion criteria. Somatic mutations in TP53 and PIK3CA were found in 46% (39/84) and 23% (24/104) of tested pts, respectively. Associations between clinicopathological characteristics and somatic TP53 and PIK3CA mutation status are summarized in Table 1. Pts with hormone receptor positive (HR+) disease were less likely to have TP53 mutations (odds ratio [OR]: 0.28, 95% confidence interval [CI]: 0.11-0.70, p=0.007) but more likely to harbor PIK3CA mutations (OR: 3.27, 95% CI: 1.11-9.62, p=0.031). There were no significant associations between gBRCA mutation status and somatic TP53 or PIK3CA mutations. On multivariable analysis, somatic TP53 mutations (adjusted hazard ratio [aHR]: 1.98, 95% CI: 1.10-3.58, p=0.023) and de novo metastatic disease (aHR=2.88, 95% CI: 1.26-6.58, p=0.012) independently predicted poorer OS, while HR+ disease (aHR: 0.40, 95% CI: 0.21-0.74, p=0.004) and HER2+ disease (aHR: 0.33, 95% CI: 0.13-0.83, p=0.019) were significantly associated with improved OS. PIK3CA mutations (aHR: 1.20, 95% CI: 0.59-2.44, p=0.62) and gBRCA mutations (aHR: 0.78, 95% CI: 0.33-1.83, p=0.57) did not significantly impact OS in this study. Conclusion: In this study, somatic TP53 mutations independently predicted worse OS in pts with MBC after adjusting for significant covariates, including gBRCA mutation status. These findings should be validated in a larger cohort of pts. Table 1: Association between patient characteristics and somatic TP53 and PIK3CA mutation status TP53 PIK3CA TP53 mutant (n=39)TP53 wild type (n=45)p valuePIK3CA mutant (n=24)PIK3CA wild type (n=80)p valueMedian Age – years (Interquartile Range)37 (33-47)39 (36-44)0.93739 (34-46)38 (34-43)0.494gBRCA mutant – n(%)3 (8)11 (24)0.0511 (4)15 (19)0.116ER/PR positive - n(%)17 (44)33 (73)0.00719 (79)43 (54)0.031HER2 positive - n(%)8 (21)6 (13)0.3827 (29)10 (13)0.060De Novo Metastatic – n(%)6 (15)8 (18)0.7693 (13)16 (20)0.409Visceral Disease – n(%)23 (59)26 (58)0.91215 (63)47 (59)0.743 Citation Format: Yam C, Gutierrez Barrera A, Huang D, Lin X, Litton JK, Arun B. Implications of somatic TP53 and PIK3CA mutations in patients with metastatic breast cancer who underwent germline BRCA testing [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-04-11.

Abstract P3-04-08: Genomic analysis of breast papillomas

Abstract Background: Papillomas are often found co-existing with breast carcinoma yet they are not considered to be a true precursor of the disease. Previous studies have shown that some cases may carry copy number alterations (CNA) or mutations in AKT1/PIK3CA (Troxell et al., 2010, Modern Pathology 23: 27-37) suggesting this lesion may have malignant potential. To date, a detailed study of both pure papillomas (not associated with cancer) and those seen in the same breast as a carcinoma has not been undertaken. Therefore, we set out to investigate the molecular changes associated with this lesion and whether papillomas can be clonally related to synchronous breast carcinoma. Method: Papilloma cases were identified from a hospital database and independently reviewed by consultant pathologists followed by micro-dissection of formalin-fixed paraffin-embedded tumour tissue and DNA extraction. For CNA detection either Affymetrix Molecular inversion Probe (MIP) 330K arrays were used or low-coverage whole genome sequencing using 5-10 ng of DNA (Kader et al., 2016, Genome Medicine 8: 121) where there was insufficient DNA for MIP arrays. We applied either of these 2 methods to 24 cases of pure papilloma as well as 20 papilloma with synchronous ductal carcinoma in situ (DCIS) and/or invasive ductal carcinoma (IDC). Additionally, targeted exon sequencing of breast cancer driver genes was performed for a subset of cases. Results: Among the pure papillomas 31% (5/16) showed CN change with, the most frequent change being 16q loss (2/16). Of the papillomas synchronous with DCIS/IDC analysed to date, 2/5 were shown to be clonal with the co-existing carcinoma. Final CNA analysis will be presented for 24 pure papilloma cases and 20 synchronous cases. Targeted sequencing revealed that all for pure papillomas analysed to date harboured somatic mutations in PIK3CA (3/4 cases) and PIK3R1 (1/4,) suggesting that most papillomas are driven by alterations in the PI3-kinase/AKT pathway. The final sequencing data to be presented will include an additional 5 pure papillomas and 10 synchronous cases. Conclusion: Our observation that 40% of papillomas are clonal to breast carcinoma suggests that DCIS or IDC can arise from a common ancestor as co-existing papillomas, however, most papillomas co-existing with carcinoma are likely to be independent in our cohort. Citation Format: Elder KJ, Kader T, Hill P, Opeskin K, Goode DL, Pang J-M, Fox SB, Mann GB, Campbell IG, Gorringe KL. Genomic analysis of breast papillomas [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-04-08.

Somatic Mutations Activating the mTOR Pathway in Dorsal Telencephalic Progenitors Cause a Continuum of Cortical Dysplasias

Focal cortical dysplasia (FCD) and hemimegalencephaly (HME) are epileptogenic neurodevelopmentalmalformations caused by mutations in mTOR pathway genes. Deep sequencing of these genes in FCD/HME brain tissue identified an etiology in 27 of 66 cases (41%). Radiographically indistinguishable lesions are caused by somatic activating mutations in AKT3, MTOR, and PIK3CA and germline loss-of-function mutations in DEPDC5, NPRL2, and TSC1/2, including TSC2 mutations in isolated HME demonstrating a "two-hit" model. Mutations in the same gene cause a disease continuum from FCD to HMEto bilateral brain overgrowth, reflecting the progenitorcell and developmental time when the mutation occurred. Single-cell sequencing demonstrated mTOR activation in neurons in all lesions. Conditional Pik3ca activation in the mouse cortex showed that mTOR activation in excitatory neurons and glia, but not interneurons, is sufficient for abnormal cortical overgrowth. These data suggest that mTOR activation in dorsal telencephalic progenitors, in some cases specifically the excitatory neuron lineage, causes cortical dysplasia.

Identification of mutations in the PI3K-AKT-mTOR signalling pathway in patients with macrocephaly and developmental delay and/or autism

BackgroundMacrocephaly, which is defined as a head circumference greater than or equal to + 2 standard deviations, is a feature commonly observed in children with developmental delay and/or autism spectrum disorder. Although PTEN is a well-known gene identified in patients with this syndromic presentation, other genes in the PI3K-AKT-mTOR signalling pathway have also recently been suggested to have important roles. The aim of this study is to characterise the mutation spectrum of this group of patients.MethodsWe performed whole-exome sequencing of 21 patients with macrocephaly and developmental delay/autism spectrum disorder. Sources of genomic DNA included blood, buccal mucosa and saliva. Germline mutations were validated by Sanger sequencing, whereas somatic mutations were validated by droplet digital PCR.ResultsWe identified ten pathogenic/likely pathogenic mutations in PTEN (n = 4), PIK3CA (n = 3), MTOR (n = 1) and PPP2R5D (n = 2) in ten patients. An additional PTEN mutation, which was classified as variant of unknown significance, was identified in a patient with a pathogenic PTEN mutation, making him harbour bi-allelic germline PTEN mutations. Two patients harboured somatic PIK3CA mutations, and the level of somatic mosaicism in blood DNA was low. Patients who tested positive for mutations in the PI3K-AKT-mTOR pathway had a lower developmental quotient than the rest of the cohort (DQ = 62.8 vs. 76.1, p = 0.021). Their dysmorphic features were non-specific, except for macrocephaly. Among the ten patients with identified mutations, brain magnetic resonance imaging was performed in nine, all of whom showed megalencephaly.ConclusionWe identified mutations in the PI3K-AKT-mTOR signalling pathway in nearly half of our patients with macrocephaly and developmental delay/autism spectrum disorder. These patients have subtle dysmorphic features and mild developmental issues. Clinically, patients with germline mutations are difficult to distinguish from patients with somatic mutations, and therefore, sequencing of buccal or saliva DNA is important to identify somatic mosaicism. Given the high diagnostic yield and the management implications, we suggest implementing comprehensive genetic testing in the PI3K-AKT-mTOR pathway in the clinical evaluation of patients with macrocephaly and developmental delay and/or autism spectrum disorder.

Actionable mutations in canine hemangiosarcoma.

BackgroundAngiosarcomas (AS) are rare in humans, but they are a deadly subtype of soft tissue sarcoma. Discovery sequencing in AS, especially the visceral form, is hampered by the rarity of cases. Most diagnostic material exists as archival formalin fixed, paraffin embedded tissue which serves as a poor source of high quality DNA for genome-wide sequencing. We approached this problem through comparative genomics. We hypothesized that exome sequencing a histologically similar tumor, hemangiosarcoma (HSA), that occurs in approximately 50,000 dogs per year, may lead to the identification of potential oncogenic drivers and druggable targets that could also occur in angiosarcoma.MethodsSplenic hemangiosarcomas are common in dogs, which allowed us to collect a cohort of archived matched tumor and normal tissue samples suitable for whole exome sequencing. Mapping of the reads to the latest canine reference genome (Canfam3) demonstrated that >99% of the targeted exomal regions were covered, with >80% at 20X coverage and >90% at 10X coverage.Results and conclusionsSequence analysis of 20 samples identified somatic mutations in PIK3CA, TP53, PTEN, and PLCG1, all of which correspond to well-known tumor drivers in human cancer, in more than half of the cases. In one case, we identified a mutation in PLCG1 identical to a mutation observed previously in this gene in human visceral AS. Activating PIK3CA mutations present novel therapeutic targets, and clinical trials of targeted inhibitors are underway in human cancers. Our results lay a foundation for similar clinical trials in canine HSA, enabling a precision medicine approach to this disease.

Somatic PIK3CA Mutations are Present in Multiple Tissues of Facial Infiltrating Lipomatosis

BackgroundFacial infiltrating lipomatosis (FIL) is a congenital disorder that causes overgrowth of one side of the face. The purpose of this study was to determine whether PIK3CA mutations are present in tissues outside of the subcutaneous adipose.MethodsFIL tissues from three patients were dissected to enrich for cells from skin, subcutaneous tissue, orbicularis oris muscle, buccal fat, zygomatic bone, and mucosal neuroma. Endothelial cells within the affected tissue also were enriched using CD31 microbeads. Laser capture microdissection on formalin-fixed paraffin-embedded histologic sections was performed to collect specific cell types. DNA was extracted from each tissue and cell type, and measured for the abundance of mutant PIK3CA alleles using droplet digital PCR.ResultsWe detected mutant PIK3CA alleles in every tissue and cell type tested from each overgrown face; frequencies ranged from 1.5 to 53%. There were fewer mutant endothelial cells compared with nonendothelial cells, and the stromal cell compartment had the highest frequency of mutant cells in each tissue.ConclusionsPIK3CA mutations are not restricted to a single tissue or cell type in FIL. Overgrowth in this condition is likely due to the mutation arising in a cell that contributes to several different facial structures during embryogenesis.

945P - Evaluation of circulating tumor DNA in patients with ovarian cancer harboring somatic PIK3CA or KRAS mutations

945P - Evaluation of circulating tumor DNA in patients with ovarian cancer harboring somatic PIK3CA or KRAS mutations

A Systematic Investigation of PIK3CA Mutations in Isolated Macrodactyly: Indication for Accurate Classification, Diagnosis and Potential Novel Therapeutics

Grant received from: Beijing Talent Funding There is no financial information to disclose. Macrodactyly, a rare congenital digital anomaly, has been challenging the hand surgery society. The recent discovery of somatic PIK3CA mutations in a few isolated macrodactyly patients shed light on the fundamental understanding in pathogenesis of this disabling deformity. We set up to systematically test the association between PIK3CA mutations with isolated macrodactyly in order to establish a molecular pathophysiology-based diagnosis, classification and possibly a novel therapeutic strategy. Overgrowth tissues including skin, nerve, adipose tissues from clinically diagnosed isolated macrodactyly patients (N = 12) were removed during routine surgical debulking or correction procedures and preserved as formalin-fixed-paraffin-embedded (FFPE) blocks. DNA recovered from these FFPE samples were tested for PIK3CA mutation status using a modified and targeted Sanger DNA sequencing method with greatly improved sensitivity for detecting low level somatic mosaic mutations. PIK3CA mutation level, tissue(s) of occurring, type of mutations and their genomic locations were analyzed. PIK3CA mutations were detected from affected tissues in 9 out of the 12 patients studied, with mutation level ranging from 8 to 27%. The mutations detected include p.His1047Arg (N = 4), p.His1047Leu (N = 2), p.545Glu > Lys (N = 2) and p.542Glu > Lys (N = 1). These are codons in the PIK3CA gene that are frequently mutated in cancers. In terms of the tissue sources in which a mutation was found, adipose tissue has the highest mutation detection rate (100%), followed by nerve (83%) and skin (71%). No mutations found in bone tissues from two patients with PIK3CA mutations detected in other tissues of the same patients. Patients and/or tissues negative for PIK3CA mutations are subjected to downstream analysis using high throughput next generation DNA sequencing. (Figs. 40-1, 40-2) •A high proportion (75%) of isolated macrodactyly patients harbors activating PIK3CA mutations that lead to digital overgrowth as a result of abnormally activated Akt-PI3K signaling.•Adipose and nerve tissues provide the highest PIK3CA mutation detection yield among all the sources of affected tissues.•A modified Sanger DNA sequencing method is a cost-effective way to exam PIK3CA mutations in isolated macrodactyly patients clinically.•Patients negative for PIK3CA mutations may harbor other PIK3CA mutations beyond the detection of our assay or due to different molecular mechanism.•To our knowledge, our study is the largest study linking PIK3CA mutations to isolated macrodactyly.Figure 40-2PIK3CA mutations in isolated macrodactyly patients. PIK3CA gene structure with critical domains, targeted regions, and mutations identified in isolated macrodactyly patients are summarized. Red arrows indicate three targeted regions (p.418-435, p.527-555, and p.1009-1058) by Sanger DNA sequencing. Mutations previously reported and numbers of patients are depicted above the gene structure and those found in our patients are shown under. Cancer hotspot mutations are highlighted in red.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Acquired somatic TP53 or PIK3CA mutations are potential predictors of when polyps evolve into colorectal cancer.

Colorectal cancer (CRC) develops from accumulated mutations. However, which gene determines the malignant transformation from adenoma to carcinoma is still uncertain. Fifty-three formalin fixed paraffin-embedded polyps that had pathological findings from patients with hyperplasia, adenomatous, and tubular adenoma < 1 cm (non-neoplasia polyps, NNP, n = 27) or tubular adenoma ≥ 1 cm, tubulovillous and villous adenoma (neoplastic polyps, NP, n = 26) were recruited. Six paired synchronous polyps and cancer tissues and 50 independent fresh CRC tumors were also collected. All tissues were analyzed for their mutation genomes using next generation sequencing with a 50-gene panel. There were 40 types of somatic variants found in 7 genes, APC (43%), KRAS (28%), TP53 (11%), FBXW7 (8%), GNAS (4%), SMAD4 (2%), and BRAF (2%), and they were detected in 32 (60%) polyps. If combined with the mutation spectrum found in CRC tissues, a significant increase in the mutation rate in TP53 and PIK3CA from NNP, NP, early and late stage carcinoma (7%, 15%, 33.3% and 65% for TP53, p < 0.001; 0%, 0%, 23.3% and 25% for PIK3CA, p = 0.002) were noticed. Furthermore, distinct molecular features can be found in five pairs of synchronous polyps and tumors. However, TP53 or PIK3CA mutations can be found in tumor tissues but not in polyps. By systematically investigating the genome from polyps to tumor tissues, we demonstrated that acquired TP53 or PIK3CA somatic mutations are potential predictors for malignancy development. These results may aid in the identification of high risk individuals with tissues harboring mutations in these two genes.

De novo EGFR T790M, KRAS and PIK3CA somatic mutations in cell free DNA in non-small cell lung cancer patients and clinical characteristics.

e20561 Background: Several mechanisms for acquired drug resistance EGFR-TKIs in NSCLC have been suggested. These include EGFR T790M mutation and mutations in KRAS, and PIK3CA. Rarely, a de novoT790M mutation has been reported. However, the clinical significance of these mutations remains controversial. Here, we used highly sensitive methods to detect T790M, KRAS and PIK3CA mutations in cell free DNA (cfDNA) and evaluate the clinical significance of those mutations. Methods: The blood samples were collected from 125 patients with NSCLC. Ion AmpliSeq Cancer Hotspot Panel (ICP) using a targeted ultra-deep sequencing (mean depth 22,868x) was performed in cfDNA, which were validated with droplet digital PCR (ddPCR). Results: ICP detected EGFR T790M mutation in 30 of 125 cfDNA (24%), KRAS mutations in 18 (14.4%) and PIK3CA in 33 (26.4%). ddPCR confirmed concordant T790M results in 22 (73.3%) and KRAS in 13 (72.2%). The 8 of 22 (36.4%) in T790M and 2 of 13 (15.4%) in KRAS had simultaneous activating EGFR mutations in cfDNA or tumor tissue. The most single KRAS mutations appeared in codon 12 (n = 7, 63.6%) (3 in G12C, 2 in G12S, 1 in G12V and 1 in G12D) with three mutations in codon 13 (27.3%) and one mutation in codon 61 (9.1%). About 20% to 50% had bone or brain metastases at diagnosis in patients with these mutations. The 5 (62.5%) (4 PR and 1 SD) of 8 patients in T790M and 8 (61.5%) (All PR) of 13 in PIK3CA showed response or stabilization. The median time to disease progression after EGFR-TKIs treatment was 13.5 months in PIK3CA and not reached in T790M. Conclusions: The de novo T790M, KRAS and PIK3CA mutation was detected in 24%, 14.4% and 26.4% using ICP in cfDNA. In our study, the KRAS and EGFR mutation may not be mutually exclusive and PIK3CA mutations may be a factor associated with metastatic sites rather than predictive factor for EGFR-TKIs. In addition, we suggested that although the clinical presentation is more aggressive in patients with de novo T790M and PIK3CA mutation, the EGFR-TKIs could be used in patients who present with both activating EGFR mutations and those mutations.