Driver gene mutations in micro-invasive cervical squamous cancers have no prognostic significance.

OBJECTIVE: To evaluate the prevalence of somatic gene mutations in different stages of cervical carcinogenesis placing special emphasis on micro-invasive pT1a cervical squamous cell cancers (SCC). METHODS: Micro-dissected samples of 32 micro-invasive pT1a and 55 ≥ pT1b SCC were evaluated by next generation sequencing of 50 cancer genes (cancer hot spot panel). RESULTS: At primary diagnosis, 8/32 (25%) pT1a SCC, 10/28 (36%) pT1b SCC and 15/27 (56%) pT2/3 SCC carried somatic gene mutations. The most commonly affected gene was the PIK3CA gene in hot spot regions E545K and E453K in 5/8 (62%) pT1a SCC, 7/15 (70%) pT1b SCC and 10/15 (66%) pT2/3 SCC followed by FBXW7 (n = 4), KRAS and RB1 (n = 2 each). ERBB2, APC, ATM, MLP gene mutations occurred only once. Solitary activating oncogenic somatic mutations dominated over tumor suppressor mutation in 88% pT1a, 80% pT1b and 60% pT2/3 SCC. Concomitant mutations involved typically an activating oncogenic mutation and an inactivating tumor-suppressor gene mutation. All patients with pT1a SCC are alive without evidence of disease after surgical treatment, independent of mutational status or lympho-vascular space invasion. CONCLUSIONS: Activating oncogenic gene mutations, in particular in the PIK3CA gene, occur early in cervical carcinogenesis. Although driver gene mutations bestow tumor cells with a growth advantage, early detection and complete removal of all cancer cells - with or without somatic gene mutations - are essential for cure. In contrast to advanced inoperable SCC, where PIK3CA driver gene mutations carry an adverse prognosis, the mutational status in surgically treated micro-invasive SCC is prognostically irrelevant.

PIK3CA hotspot mutations p. H1047R and p. H1047L sensitize breast cancer cells to thymoquinone treatment by regulating the PI3K/Akt1 pathway.

BACKGROUND: Nigella sativa (N. sativa) exhibits anti-inflammatory, antioxidant, antidiabetic, antimetastatic and antinociceptive effects and has been used to treat dozens of diseases. Thymoquinone (TQ) is an important and active component isolated from N. sativa seeds. Inhibition of cancer-associated activating PIK3CA mutations is a new prospective targeted therapy in personalized metastatic breast cancer (MBC). TQ is reported to be an effective inhibitor of the PI3K/Akt1 pathway in MBC. This study aimed to evaluate the in vitro antitumor effect of TQ in the context of two PIK3CA hotspot mutations, p. H1047R and p. H1047L. METHODS AND RESULTS: Molecular dynamics, free energy landscapes and principal component analyses were also used to survey the mechanistic effects of the p. H1047R and p. H1047L mutations on the PI3K/Akt1 pathway. Our findings clearly confirmed that the p. H1047R and p. H1047L mutants could reduce the inhibitory effect of ΔNp63α on the kinase domain of PIK3CA, resulting in increased activity of PI3K downstream signals. Structurally, the partial disruption of the interaction between the ΔNp63α DNA binding domain and the PIK3CA kinase domain at residues 114-359 and 797-1068 destabilizes the conformation of the activation loop and modifies the PIK3CA/ΔNp63α complex. Alongside these structural changes, we found that TQ treatment resulted in high PI3K/Akt1 pathway inhibition in p. H1047R and p. H1047L-expressing cells versus wild-type cells. CONCLUSIONS: These two PIK3CA hotspot mutations therefore not only contribute to tumor progression in patients with MBC but may also serve as targets for the development of novel small molecule therapeutic strategies.

Targeting the mutant PIK3CA gene by DNA-alkylating pyrrole-imidazole polyamide in cervical cancer.

PIK3CA is the most frequently mutated oncogene in cervical cancer, and somatic mutations in the PIK3CA gene result in increased activity of PI3K. In cervical cancer, the E545K mutation in PIK3CA leads to elevated cell proliferation and reduced apoptosis. In the present study, we designed and synthesized a novel pyrrole-imidazole polyamide-seco-CBI conjugate, P3AE5K, to target the PIK3CA gene bearing the E545K mutation, rendered possible by nuclear access and the unique sequence specificity of pyrrole-imidazole polyamides. P3AE5K interacted with double-stranded DNA of the coding region containing the E545K mutation. When compared with conventional PI3K inhibitors, P3AE5K demonstrated strong cytotoxicity in E545K-positive cervical cancer cells at lower concentrations. PIK3CA mutant cells exposed to P3AE5K exhibited reduced expression levels of PIK3CA mRNA and protein, and subsequent apoptotic cell death. Moreover, P3AE5K significantly decreased the tumor growth in mouse xenograft models derived from PIK3CA mutant cells. Overall, the present data strongly suggest that the alkylating pyrrole-imidazole polyamide P3AE5K should be a promising new drug candidate targeting a constitutively activating mutation of PIK3CA in cervical cancer.

Effects of miR-202-5p silencing PIK3CA gene expression on proliferation, invasion, and epithelial-mesenchymal transition of cervical cancer SiHa cells through inhibiting PI3K/Akt/mTOR signaling pathway activation.

To explore the mechanism of miR-202-5p targeting the expression of PIK3CA and mediating the activation of PI3K/Akt/mTOR signaling pathway on the proliferation, invasion, and epithelial-mesenchymal transition (EMT) of cervical cancer. The objects of study were 105 cases of cervical cancer and their corresponding normal tissues. qRT-PCR was used to detect the expression of miR-202-5p and PIK3CA in adjacent normal tissue and cervical cancer tissue. Dual luciferase reporter assay was used to verify the targeting relationship between miR-202-5p and PIK3CA gene. Human cervical cancer cell lines HPV-16E6, SiHa, HeLa, and CaSki were purchased for our cell experiments. The expression levels of PIK3CA in the cells were detected by qRT-PCR. The cell line with higher expression levels was selected to complete the follow-up experiment. The cultured cells were transfected and divided into the miR-202-5p mimic NC group, miR-202-5p mimic group, miR-202-5p inhibitor NC group, miR-202-5p inhibitor group, siRNA-PIK3CA NC group, siRNA-PIK3CA group, miR-202-5p inhibitor NC + siRNA-PIK3CA NC group, miR-202-5p inhibitor + siRNA-PIK3CA NC group, and miR-202-5p inhibitor + siRNA-PIK3CA group. QRT-PCR was used to detect the expression of miR-202-5p. Western blot and qRT-PCR were applied to detect the mRNA and protein expression levels of related pathway proteins (PIK3CA, PI3K, PTEN, p-Akt1, and p-mTOR) and epithelial-mesenchymal transition-related factors (N-cadherin, E-cadherin, and vimentin). Cell proliferation was detected by plate colony formation assay. Transwell assay was used to detect the invasion ability of each group. When compared with the adjacent tissues, PIK3CA mRNA expression level was significantly increased and miR-202-5p expression level was significantly decreased in cervical cancer tissues (all P < 0.05). PIK3CA was a target gene of miR-202-5p. The mRNA expression level of PIK3CA in SiHa cervical cancer cells was significantly higher than that in CaSki, HeLa, and HPV-16E6 cells (all P < 0.05), and SiHa cervical cancer cells were selected to complete the follow-up experiments. When compared with the corresponding NC group, the expression of miR-202-5p in miR-202-5p mimic group was increased. In addition, the mRNA and protein expression levels of E-cadherin and PTEN in miR-202-5p mimic and siRNA-PIK3CA groups were increased, and the protein expression of p-Akt1 and p-mTOR was decreased, and also, the mRNA and protein expression levels of PIK3CA, PI3K, N-cadherin, and vimentin were decreased (all P < 0.05); in miR-202-5p inhibitor group, the expression levels of miR-202-5p, E-cadherin, and PTEN decreased, the protein expression of p-Akt1 and p-mTOR increased, and the mRNA and protein expression of PIK3CA, PI3K, N-cadherin, and vimentin increased in miR-202-5p inhibitor group (all P < 0.05); in miR-202-5p inhibitor + siRNA-PIK3CA group, the expression of miR-202-5p decreased (P < 0.05), but the mRNA and protein expression of PIK3CA, PI3K, p-Akt1, p-mTOR, N-cadherin, E-cadherin, and vimentin had no significant changes (all P > 0.05). When compared with the corresponding NC group, the number of cell clones in miR-202-5p mimic group and siRNA-PIK3CA group was decreased, and the invasion ability of miR-202-5p inhibitor group was increased, and the invasion ability was enhanced (all P < 0.05); miR-202-5p inhibitor + siRNA-PIK3CA group showed no significant change in the number of cell clones and the rate of invasion (P > 0.05). In conclusion, the overexpression of miR-202-5p can suppress PIK3CA gene expression and the activation of PI3K/Akt/mTOR signaling pathway to suppress the proliferation, invasion, and EMT of cervical cancer.

Concordance between PIK3CA mutations in endoscopic biopsy and surgically resected specimens of esophageal squamous cell carcinoma.

BACKGROUND: PIK3CA mutations are expected to be potential therapeutic targets for esophageal squamous cell carcinoma (ESCC). We aimed to clarify the concordance between PIK3CA mutations detected in endoscopic biopsy specimens and corresponding surgically resected specimens. METHODS: We examined five hotspot mutations in the PIK3CA gene (E542K, E545K, E546K, H1047R, and H1047L) in formalin-fixed and paraffin-embedded tissue sections of paired endoscopic biopsy and surgically resected specimens from 181 patients undergoing curative resection for ESCC between 2000 and 2011 using a Luminex technology-based multiplex gene mutation detection kit. RESULTS: Mutation analyses were successfully performed for both endoscopic biopsy and surgically resected specimens in all the cases. A PIK3CA mutation was detected in either type of specimen in 13 cases (7.2%, 95% confidence interval: 3.9-12.0). The overall concordance rate, positive predictive value, and negative predictive value were 98.3% (178/181), 90.9% (10/11), and 98.8% (168/170), respectively. Among patients with a PIK3CA mutation detected in both types of specimens, the concordance between PIK3CA mutation genotypes was 100%. There were three cases with a discordant mutation status between the types of specimens (PIK3CA mutation in surgically resected specimen and wild-type in biopsy specimen in two cases, and the opposite pattern in one case), suggesting possible intratumoral heterogeneity in the PIK3CA mutation status. CONCLUSIONS: The PIK3CA mutation status was highly concordant between endoscopic biopsy and surgically resected specimens from the same patient, suggesting that endoscopic biopsy specimens can be clinically used to detect PIK3CA mutations in patients with ESCC.

Association between the Prevalence of Somatic Mutations in PIK3CA Gene in Tumors and Clinical and Morphological Characteristics of Breast Cancer Patients.

The presence of activating somatic mutations in codons 542 and 545 of exon 9 (p.E542K c.1624G>A and p.E545K c.1633G>A) and in codon 1047 of exon 20 (p.H1047R c.3140A>G and p.H1047L c.3140A>T) of PIK3CA gene encoding catalytic p110α-subunit of phosphatidylinositol-3-kinase was studied in tumors of 473 breast cancer patients by multiplex allele-specific real-time PCR. Fifty-eight (12.3%) different mutations were found. An increase in the frequency of PIK3CA gene mutations with disease progression (from 2.4 to 28.7% with tumor progression from I-IIa to III-IV stage; p=0.0001) and a trend towards its increase in the tumors with unfavorable prognostic characteristics (high histological grade, triple negative phenotype) were demonstrated. The presence of the studied PIK3CA gene mutations in tumors significantly reduces relapse-free survival in the total group and in stage III cancer patients.

PIK3CA-related overgrowth spectrum (PROS): diagnostic and testing eligibility criteria, differential diagnosis, and evaluation.

Somatic activating mutations in the phosphatidylinositol-3-kinase/AKT/mTOR pathway underlie heterogeneous segmental overgrowth phenotypes. Because of the extreme differences among patients, we sought to characterize the phenotypic spectrum associated with different genotypes and mutation burdens, including a better understanding of associated complications and natural history. Historically, the clinical diagnoses in patients with PIK3CA activating mutations have included Fibroadipose hyperplasia or Overgrowth (FAO), Hemihyperplasia Multiple Lipomatosis (HHML), Congenital Lipomatous Overgrowth, Vascular Malformations, Epidermal Nevi, Scoliosis/Skeletal and Spinal (CLOVES) syndrome, macrodactyly, Fibroadipose Infiltrating Lipomatosis, and the related megalencephaly syndromes, Megalencephaly-Capillary Malformation (MCAP or M-CM) and Dysplastic Megalencephaly (DMEG). A workshop was convened at the National Institutes of Health (NIH) to discuss and develop a consensus document regarding diagnosis and treatment of patients with PIK3CA-associated somatic overgrowth disorders. Participants in the workshop included a group of researchers from several institutions who have been studying these disorders and have published their findings, as well as representatives from patient-advocacy and support groups. The umbrella term of "PIK3CA-Related Overgrowth Spectrum (PROS)" was agreed upon to encompass both the known and emerging clinical entities associated with somatic PIK3CA mutations including, macrodactyly, FAO, HHML, CLOVES, and related megalencephaly conditions. Key clinical diagnostic features and criteria for testing were proposed, and testing approaches summarized. Preliminary recommendations for a uniform approach to assessment of overgrowth and molecular diagnostic testing were determined. Future areas to address include the surgical management of overgrowth tissue and vascular anomalies, the optimal approach to thrombosis risk, and the testing of potential pharmacologic therapies.

Clinical delineation and natural history of the PIK3CA-related overgrowth spectrum.

Somatic mutations in the phosphatidylinositol/AKT/mTOR pathway cause segmental overgrowth disorders. Diagnostic descriptors associated with PIK3CA mutations include fibroadipose overgrowth (FAO), Hemihyperplasia multiple Lipomatosis (HHML), Congenital Lipomatous Overgrowth, Vascular malformations, Epidermal nevi, Scoliosis/skeletal and spinal (CLOVES) syndrome, macrodactyly, and the megalencephaly syndrome, Megalencephaly-Capillary malformation (MCAP) syndrome. We set out to refine the understanding of the clinical spectrum and natural history of these phenotypes, and now describe 35 patients with segmental overgrowth and somatic PIK3CA mutations. The phenotypic data show that these previously described disease entities have considerable overlap, and represent a spectrum. While this spectrum overlaps with Proteus syndrome (sporadic, mosaic, and progressive) it can be distinguished by the absence of cerebriform connective tissue nevi and a distinct natural history. Vascular malformations were found in 15/35 (43%) and epidermal nevi in 4/35 (11%) patients, lower than in Proteus syndrome. Unlike Proteus syndrome, 31/35 (89%) patients with PIK3CA mutations had congenital overgrowth, and in 35/35 patients this was asymmetric and disproportionate. Overgrowth was mild with little postnatal progression in most, while in others it was severe and progressive requiring multiple surgeries. Novel findings include: adipose dysregulation present in all patients, unilateral overgrowth that is predominantly left-sided, overgrowth that affects the lower extremities more than the upper extremities and progresses in a distal to proximal pattern, and in the most severely affected patients is associated with marked paucity of adipose tissue in unaffected areas. While the current data are consistent with some genotype-phenotype correlation, this cannot yet be confirmed.

A novel PIK3CA hot-spot mutation in breast cancer patients detected by HRM-COLD-PCR analysis.

BACKGROUND: The PI3K protein is involved in the PI3K/AKT/mTOR pathway. Deregulation of this pathway through PIK3CA mutation is common in various tumors. The aim of this work is to identify hotspot mutation at exons 9 and 20 in Tunisian patients with sporadic or hereditary breast cancer. METHODS: Hotspot mutations in exon 9 and exon 20 of the PIK3CA gene were identified by QPCR-High Resolution Melting followed by COLD-PCR and sequencing in 63 (42 sporadic cases and 21 hereditary cases) tumor tissues collected from Tunisian patient with breast cancer. MCF7, and BT20 breast cancer cell lines harboring the PIK3CA hotspot mutations E545K and H1047R in exon 9 and exon 20 respectively, were used as controls in HRM experiments. RESULTS: PIK3CA hotspot mutations were detected in 66.7% (28 out of 42) of sporadic BC cases, and in 14.3% (3 out of 21) of hereditary BC. The E545K and the H1048Y were the most prevalent mutations identified in patients with sporadic and hereditary BC, whereas the H1047R hotspot mutation was not found in our patients. Statistical analysis showed that PIK3CA mutation associated with an aggressive behavior in patients with sporadic BC, while it's correlated with age, tumor stage and tumor size in the group patients with hereditary breast cancer. CONCLUSIONS: Our results showed a novel PIK3CA hotspot mutation in Tunisian breast cancer patients detected by HRM-COLD-PCR. Moreover, the absence of PIK3CA hotspot mutation associated with good prognosis.

Association of preoperative and postoperative circulating tumour DNA (ctDNA) with PIK3CA gene mutation with risk of recurrence in patients with non-metastatic breast cancer.

BACKGROUND: Circulating tumour DNA (ctDNA), contains tumour-specific gene mutation in blood circulation and could aid in postoperative risk stratification of non-metastatic breast cancer. In this study, we investigated the feasibility of detecting PIK3CA gene mutations in ctDNA in the preoperative (preop) and postoperative period (postop), and its prognostic significance in patients with breast cancer. METHODS: A cohort of patients with breast cancer undergoing curative surgery with available blood samples preoperatively and postoperatively (Post op) at either Post op time period; week 1-2, week 3-4 or weeks 5-12 were enrolled. PIK3CA gene mutations at exons 9 and 20 were detected in ctDNA with High resolution melting (HRM) PCR and Allele specific fluorescence probe-based PCR. RESULTS: A total of 62 patients (age, median (IQR), 51.50 (45.0-65.0) years), with a median follow-up of 90 months (interquartile range (IQR),60-120 months) were enrolled. In total, 25 (40.3%) and 22 (35%) patients with breast cancer had detectable PIK3CA gene mutations in ctDNA in preoperative and postoperative period, respectively. PIK3CA gene mutations in ctDNA in postoperative period (hazard ratio (H.R: 18.05, p = 0.001) were a negative prognostic factor for recurrencefree survival (RFS) and overall survival (OS) (H.R: 11.9, p = 0.01) in patients with breast cancer. Subgroup analysis of ctDNA indicate that positive ctDNA in both preoperative/postoperative period and post op period only were found to have prognostic effect on RFS and OS (RFS; p < 0.0001, O·S; p = 0.0007). Moreover, ctDNA-based detection preceded clinical detection of recurrence in patients with an average lead time of 12 months (IQR:20-28.5 months) across all the breast cancer subtypes. CONCLUSION: We highlighted the prognostic ability of ctDNA in patients with breast cancer in perioperative period. However, future prospective studies are needed to assess the utility of ctDNA in clinical practice.

A rare case of Klippel-Trenaunay syndrome presenting with chronic myeloid leukemia.

BACKGROUND: Klippel-Trenaunay syndrome (KTS) is an overgrowth syndrome associated with capillary/venous/ lymphatic malformations with limb hypertrophy and cancer risk. Various cancers, mostly Wilms tumor, have been reported in patients with KTS, but not leukemia. Chronic myeloid leukemia (CML) is also a rare disease in children, where there is no known disease or syndrome to predispose to CML. CASE: We report a case of CML incidentally diagnosed in a child with KTS when he was bleeding from surgery of the left groin for vascular malformation. CONCLUSIONS: This case reflects the variety of cancer types that may accompany KTS and provides information about CML prognosis in such patients.

Novel Method for Detection of PIK3CA Mutations in Circulating Tumor DNA of Patients with Colorectal Cancer.

The PIK3CA mutation is considered a potential target for treatment of colorectal cancer. We evaluated a PIK3CA mutation assay on plasma cell-free DNA (cfDNA) using a newly developed PCR with restriction digestion integrated and followed by Sanger's sequencing. We analyzed PIK3CA mutation in plasma with our newly developed assays and in matching tumor tissues by routine methods. We detected the PIK3CA gene mutation status by both methods in samples from 40 colorectal cancer patients. Three H1047R mutations of PIK3CA gene were detected in the cfDNA of the 40 patients by restriction digestion PCR. Neither E545K nor H1047R mutations were detected in the cfDNA by routine PCR/sequencing. The PIK3CA H1047R and E545K mutations in cfDNA can be sensitively detected with our newly developed assays. The colorectal cancer has been used as a clinical example in testing our new assays, which indicates that the new assays may have wider applications in detecting mutations in precision oncology. Trial registration: Current Controlled Trials ChiCTR-DDT-12002848, 8 October 2012.

A Mosaic PIK3CA Mutation in a Moroccan Female: Exploring the Diagnostic Challenges of PIK3CA-Related Overgrowth Spectrum.

The PIK3CA-related overgrowth spectrum (PROS) encompasses a group of rare disorders characterized by the overgrowth of various body parts, driven by mutations in the PIK3CA gene. This study presents a case of a Moroccan female patient with PROS, demonstrating a phenotype associated with genetic mosaicism in the PIK3CA gene. A multidisciplinary approach, involving clinical examination, radiological assessment, and genetic and bioinformatic analyses, was employed for diagnosis and management. Next-generation sequencing and Sanger sequencing identified a rare variant, c.353G>A, in exon 3 of the PIK3CA gene, not detected in leukocyte DNA but confirmed in tissue biopsy samples. The comprehensive analysis of this case furthers our understanding of PROS and highlights the importance of a multidisciplinary approach to the diagnosis and management of this rare disorder.

The Klippel-Trénaunay Syndrome in 2022: Unravelling Its Genetic and Molecular Profile and Its Link to the Limb Overgrowth Syndromes.

The Klippel-Trénaunay syndrome is an unusual syndrome of vascular and dermatologic manifestation in which patients demonstrate hemihypertrophy of the soft tissue and bones of one limb, cutaneous haemangiomas and varicosities in anatomically abnormal positions. Described in 1900 by two French physicians, the etiology remained unclear until recently, when evidence emerged that there was a genetic basis for this sporadic disorder. Genes that encoded pathological angiogenic factors and caused vascular dysmorphogenesis, explaining the molecular bases of this syndrome, were identified. Several angiogenic genes were identified but one gene, the AGGF1 (formerly VG5Q) gene, was seen in mutations involving patients diagnosed with Klippel-Trénaunay syndrome. Furthermore, this syndrome was also noted to have overlapping clinical features linked with the "overgrowth syndromes," in which genetic mutations along somatic lines were identified. These involved The PI3K enzyme which forms part of the phosphoinositide 3-kinase pathway which is encoded by the PIK3CA-gene. This enzyme mediates embryonic cellular growth in-utero and diseases involved in this pathway are classified as members of the PIK3CA-related overgrowth syndrome. This paper reviews the status of what is now known about the molecular genetics of this unusual, but clinically challenging disorder and its differentiation from similar diseases, linked with the PIK3CA-gene and the related overgrowth syndromes.

An Analysis of the Pathogenic Genes and Mutation Sites of Macrodactyly.

OBJECTIVE: This study aimed to explore the pathogenic genes and mutation sites of macrodactyly. METHODS: Whole-exome sequencing was performed on the pathological tissue and peripheral blood of 12 patients with macrodactyly who were operated in our hospital between June 2018 and May 2020. In order to conduct comprehensive bioinformatics analysis and screen the pathogenic genes of macrodactyly, the patients were divided into four groups: macrodactyly of finger group, macrodactyly of foot group, macrodactyly and syndactyly of finger group, and macrodactyly and syndactyly of foot group. The results of the whole-exome sequencing were verified using Sanger sequencing in order to clarify the pathogenic genes and mutation sites of macrodactyly, and immunohistochemical analysis of the protein signaling pathways encoded by the pathogenic genes was performed to observe the protein expression and further verify the mutant genes. RESULTS: In the comprehensive bioinformatics analysis and Sanger verification of the whole-exome sequencing, the PIK3CA gene mutation was screened as the pathogenic gene of macrodactyly. The mutation sites were identified as the p.E542K (c.G1624A) and p.E545K (c.G1633A) sites of exon10 and the p.H1047R (c.A3140G) and p.G1049R (c.G3145C) sites of exon21. Among these, the p.G1049R (c.G3145C) locus was found in macrodactyly for the first time. The mutation of the PIK3CA gene was also found to lead to increased expression of serine-threonine kinase (AKT) in adipocytes in the PI3K-AKT-mTOR signaling pathway. CONCLUSION: Mutation of the PIK3CA gene leads to the enhancement of the PI3K-AKT-mTOR signaling pathway, which is the cause of macrodactyly. There is also some diversity in PIK3CA gene mutation sites.

Electrochemical Biosensor Based on l-Arginine and rGO-AuNSs Deposited on the Electrode Combined with DNA Probes for Ultrasensitive Detection of the Gastric Cancer-Related PIK3CA Gene of ctDNA.

Gene biomarkers of circulating tumor DNA (ctDNA) in liquid biopsies have been explored for use in the precise diagnosis of tumors. There is a great clinical need to realize the ultrasensitive detection of gene biomarkers in ctDNA. Here we reported that an ultrasensitive label-free biosensor was developed for the detection of the gastric cancer-related PIK3CA gene of ctDNA in peripheral blood. The polymeric l-arginine and graphene oxide-wrapped gold nanostars (rGO-AuNSs) were prepared and deposited on the glass electrode. The capturing DNA probes for the PIK3CA gene were prepared and successfully immobilized on the rGO-AuNS-modified electrode surface via π-π interaction among the rGO-AuNS composites and DNA probes. The resultant electrochemical sensor was effectively applied to detect the PIK3CA gene of ctDNA via the hybridization between the capturing DNA probe and ctDNA, the result of which showed that the biosensor exhibited desirable sensitivity, stability, and a wider dynamic response in a ctDNA concentration range from 1.0 × 10-20 to 1.0 × 10-10 M (R2 = 0.997). Moreover, the low limit of detection of 1.0 × 10-20 M (S/N = 3) indicates the biosensor owns satisfactory detection sensitivity. Fourteen PIK3CA genes and two PIK3CA gene mutations were detected in 60 clinical ctDNA samples of gastric cancer patients by using the developed biosensor. In conclusion, this ultrasensitive label-free electrochemical biosensor possesses a significant application prospect in the detection of the PIK3CA gene in ctDNA and in early screening for gastric cancer in the near future.

MicroRNA let-7f-5p regulates PI3K/AKT/COX2 signaling pathway in bacteria-induced pulmonary fibrosis via targeting of PIK3CA in forest musk deer.

Background: Recent studies have characterized that microRNA (miRNA) is a suitable candidate for the study of bleomycin/LPS-induced pulmonary fibrosis, but the knowledge on miRNA in bacteria-induced pulmonary fibrosis (BIPF) is limited. Forest musk deer (Moschus berezovskii, FMD) is an important endangered species that has been seriously affected by BIPF. We sought to determine whether miRNA exist that modulates the pathogenesis of BIPF in FMD. Methods: High-throughput sequencing and RT-qPCR were used to determine the differentially expressed miRNAs (DEmiRNAs) in the blood of BIPF FMD. The DEmiRNAs were further detected in the blood and lung of BIPF model rat by RT-qPCR, and the targeting relationship between candidate miRNA and its potential target gene was verified by dual-luciferase reporter activity assay. Furthermore, the function of the candidate miRNA was verified in the FMD lung fibroblast cells (FMD-C1). Results: Here we found that five dead FMD were suffered from BIPF, and six circulating miRNAs (miR-30g, let-7f-5p, miR-27-3p, miR-25-3p, miR-9-5p and miR-652) were differentially expressed in the blood of the BIPF FMD. Of these, let-7f-5p showed reproducibly lower level in the blood and lung of the BIPF model rat, and the expression levels of PI3K/AKT/COX2 signaling pathway genes (PIK3CA, PDK1, Akt1, IKBKA, NF-κB1 and COX2) were increased in the lung of BIPF model rats, suggesting that there is a potential correlation between BIPF and the PI3K/AKT/COX2 signaling pathway. Notably, using bioinformatic prediction and experimental verification, we demonstrated that let-7f-5p is conserved across mammals, and the seed sequence of let-7f-5p displays perfect complementarity with the 3' UTR of PIK3CA gene and the expression of the PIK3CA gene was regulated by let-7f-5p. In order to determine the regulatory relationship between let-7f-5p and the PI3K/AKT/COX2 signaling pathway in FMD, we successfully cultured FMD-C1, and found that let-7f-5p could act as a negative regulator for the PI3K/Akt/COX2 signaling pathway in FMD-C1. Collectively, this study not only provided a study strategy for non-invasive research in pulmonary disease in rare animals, but also laid a foundation for further research in BIPF.

Alpelisib-Induced Diabetic Ketoacidosis in a Non-diabetic Patient.

Alpelisib is a phosphoinositol-3-kinase alpha catalytic subunit (PIK3CA) inhibitor used in patients with PIK3CA mutated breast cancer. The phosphatidylinositol 3-kinase (PI3K) pathway is responsible for activating protein kinase-B (AKT), and activated AKT promotes translation of glucose transporter 4 and glycogen synthesis in insulin-responsive tissues. Therefore, it is perhaps not surprising that hyperglycemia is the most common side effect of alpelisib, though diabetic ketoacidosis (DKA) appears to be a rare complication. This case describes the unique presentation of a patient with no prior history of diabetes who presented with DKA after starting alpelisib, and returned to euglycemia off of insulin just three days after stopping the drug suggesting that alpelisib can cause DKA in patients who did not previously have diabetes, and that the hyperglycemia is completely reversible upon discontinuation of the PIK3CA inhibitor and consequent restoration of the PI3K/AKT pathway.

Detection of PIK3CA Gene Mutation in Head and Neck Squamous Cell Carcinoma Using Droplet Digital PCR and RT-qPCR.

Head and neck squamous cell carcinomas (HNSCC) are the seventh cause of human malignancy with low survival rate due to late diagnosis and treatment. Its etiology is diverse; however genetic factors are significant. The most common mutations in HNSCC were found in the genes: PIK3CA (10-12%), BRCA1 (6%), and BRCA2 (7-9%). In some cases, these biomarkers correlate with recurrences or survival showing a potential of prognostic and predictive value. A total of 113 formalin-fixed paraffin embedded (FFPE) tumor samples were collected from patients with HNSCC (oral cavity: 35 (31.0%); oropharynx: 30 (26.0%); larynx: 48 (43.0%)). We examined PIK3CA H1047R mutation by Real Time PCR (RT-qPCR) and droplet digital PCR (ddPCR). BRCA1 and BRCA2 mutations were analyzed by RT-qPCR while p16 protein expression was assessed by immunohistochemistry. Finally, we identified HPV infection by RT-qPCR. The relationships between genomic alterations and clinical parameters were assessed using the Yates' corrected Chi-squared test or Fisher's exact test for nominal variables. Kaplan Meier plots were applied for survival analysis. Our results revealed 9 PIK3CA H1047R mutations detected by ddPCR: 8 of them were negative in RT-qPCR. Due to the use of different methods to test the presence of the PIK3CA gene mutation, different treatment decisions might be made. That is why it is so important to use the most sensitive methods available. We confirmed the usefulness of ddPCR in the PIK3CA mutation assessment in FFPE samples.

Klipple-Trenaunay Syndrome: A Rare Disorder With Multisystemic Clinical Attributes.

Klippel-Trenaunay syndrome (KTS) is a rare disorder characterized by abnormal development of soft tissues, lymphatic system, and blood vessels. Major features include tissue and bone overgrowth, vein malformation, and port-wine stains with or without lymphatic abnormalities. It is crucial to review this rare syndrome to avoid any diagnostic delay. In addition, it is also vital to follow disease courses with symptomatic treatment for rare complex diseases, which would help clinicians understand and implement a better treatment plan in the future. We present the case of a 19-year-old male eventually diagnosed with KTS who initially presented with swelling of his feet and skin erosion with bloody discharge. Associated findings were bluish skin discoloration, nodularity, and bleeding per rectum, leading to anemia and subsequent heart failure. Colonoscopy/sigmoidoscopy showed vascular malformation and an active bleeding site. Our patient manifested most of the clinical attributes of KTS, with an interesting clinical course of arteriovenous, soft tissue, capillary, lymphatic, and vascular malformations. However, in our case, the patient is receiving only symptomatic treatment (blood transfusion) without any limb amputation or reconstruction surgery, leading to no further deterioration of the quality of life.