In research settings, circulating tumor DNA (ctDNA) shows promise as a tumor-specific biomarker for pancreatic ductal adenocarcinoma (PDAC). This study aims to perform analytical and clinical ...validation of a
ctDNA assay in a Clinical Laboratory Improvement Amendments (CLIA) and College of American Pathology-certified clinical laboratory.
Digital-droplet PCR was used to detect the major PDAC-associated somatic
mutations (G12D, G12V, G12R, and Q61H) in liquid biopsies. For clinical validation, 290 preoperative and longitudinal postoperative plasma samples were collected from 59 patients with PDAC. The utility of ctDNA status to predict PDAC recurrence during follow-up was assessed.
ctDNA was detected preoperatively in 29 (49%) patients and was an independent predictor of decreased recurrence-free survival (RFS) and overall survival (OS). Patients who had neoadjuvant chemotherapy were less likely to have preoperative ctDNA than were chemo-naïve patients (21% vs. 69%;
< 0.001). ctDNA levels dropped significantly after tumor resection. Persistence of ctDNA in the immediate postoperative period was associated with a high rate of recurrence and poor median RFS (5 months). ctDNA detected during follow-up predicted clinical recurrence sensitivity 90% (95% confidence interval (CI), 74%-98%), specificity 88% (95% CI, 62%-98%) with a median lead time of 84 days (interquartile range, 25-146). Detection of ctDNA during postpancreatectomy follow-up was associated with a median OS of 17 months, while median OS was not yet reached at 30 months for patients without ctDNA (
= 0.011).
Measurement of
ctDNA in a CLIA laboratory setting can be used to predict recurrence and survival in patients with PDAC.
Intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystic neoplasms (MCNs) are non-invasive neoplasms that are often observed in association with invasive pancreatic cancers, but their ...origins and evolutionary relationships are poorly understood. In this study, we analyze 148 samples from IPMNs, MCNs, and small associated invasive carcinomas from 18 patients using whole exome or targeted sequencing. Using evolutionary analyses, we establish that both IPMNs and MCNs are direct precursors to pancreatic cancer. Mutations in SMAD4 and TGFBR2 are frequently restricted to invasive carcinoma, while RNF43 alterations are largely in non-invasive lesions. Genomic analyses suggest an average window of over three years between the development of high-grade dysplasia and pancreatic cancer. Taken together, these data establish non-invasive IPMNs and MCNs as origins of invasive pancreatic cancer, identifying potential drivers of invasion, highlighting the complex clonal dynamics prior to malignant transformation, and providing opportunities for early detection and intervention.
Background
Intraductal papillary mucinous neoplasms (IPMNs) are the most common cystic precursor lesions of invasive pancreatic cancer. The recent identification of activating
GNAS
mutations at codon ...201 in IPMNs is a promising target for early detection and therapy. The purpose of this study was to explore clinicopathological correlates of
GNAS
mutational status in resected IPMNs.
Methods
Clinical and pathologic characteristics were retrieved on 54 patients in whom
GNAS
codon 201 mutational status was previously reported (“historical group”, Wu et al. Sci Transl Med 3:92ra66,
2011
). In addition, a separate cohort of 32 patients (validation group) was included. After microdissection and DNA extraction,
GNAS
status was determined in the validation group by pyrosequencing.
Results
GNAS
activating mutations were found in 64 % of the 32 IPMNs included in the validation group, compared with a previously reported prevalence of 57 % in the historical group. Overall, 52 of 86 (61 %) of IPMNs demonstrated
GNAS
mutations in the two studies combined. Analysis of both groups confirmed that demographic characteristics, tumor location, ductal system involvement, focality, size, grade of dysplasia, presence of an associated cancer, and overall survival were not correlated with
GNAS
mutational status. Stratified by histological subtype, 100 % of intestinal type IPMNs demonstrated
GNAS
mutations compared to 51 % of gastric IPMN, 71 % of pancreatobiliary IPMNs, and 0 % of oncocytic IPMNs.
Conclusions
GNAS
activating mutations can be reliably detected in IPMNs by pyrosequencing. In terms of clinicopathological parameters, only histological subtype was correlated with mutational frequency, with the intestinal phenotype always associated with
GNAS
mutations.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
To validate next-generation sequencing (NGS) technology for clinical diagnosis and to determine appropriate read depth.
We validated the KRAS, BRAF, and EGFR genes within the Ion AmpliSeq Cancer ...Hotspot Panel using the Ion Torrent Personal Genome Machine (Life Technologies, Carlsbad, CA).
We developed a statistical model to determine the read depth needed for a given percent tumor cellularity and number of functional genomes. Bottlenecking can result from too few input genomes. By using 16 formalin-fixed, paraffin-embedded (FFPE) cancer-free specimens and 118 cancer specimens with known mutation status, we validated the six traditional analytic performance characteristics recommended by the Next-Generation Sequencing: Standardization of Clinical Testing Working Group. Baseline noise is consistent with spontaneous and FFPE-induced C:G→T:A deamination mutations.
Redundant bioinformatic pipelines are essential, since a single analysis pipeline gave false-negative and false-positive results. NGS is sufficiently robust for the clinical detection of gene mutations, with attention to potential artifacts.
Pancreatic imaging can identify neoplastic cysts but not microscopic neoplasms. Mutation analysis of pancreatic fluid after secretin stimulation might identify microscopic neoplasias in the ...pancreatic duct system. We determined the prevalence of mutations in KRAS and guanine nucleotide-binding protein α-stimulating genes in pancreatic juice from subjects undergoing endoscopic ultrasound for suspected pancreatic intraepithelial neoplasia, intraductal papillary mucinous neoplasms, or pancreatic adenocarcinoma.
Secretin-stimulated juice samples were collected from the duodenum of 272 subjects enrolled in Cancer of the Pancreas Screening studies; 194 subjects were screened because of a family history of, or genetic predisposition to, pancreatic cancer, and 78 subjects were evaluated for pancreatic cancer (n = 30) or other disorders (controls: pancreatic cysts, pancreatitis, or normal pancreata, n = 48). Mutations were detected by digital high-resolution melt-curve analysis and pyrosequencing. The number of replicates containing a mutation determined the mutation score.
KRAS mutations were detected in pancreatic juice from larger percentages of subjects with pancreatic cancer (73%) or undergoing cancer screening (50%) than controls (19%) (P = .0005). A greater proportion of patients with pancreatic cancer had at least 1 KRAS mutation detected 3 or more times (47%) than screened subjects (21%) or controls (6%, P = .002). Among screened subjects, mutations in KRAS (but not guanine nucleotide-binding protein α-stimulating) were found in similar percentages of patients with or without pancreatic cysts. However, a greater proportion of patients older than age 50 years had KRAS mutations (54.6%) than younger patients (36.3%) (P = .032); the older subjects also had more mutations in KRAS (P = .02).
Mutations in KRAS are detected in pancreatic juice from the duodenum of 73% of patients with pancreatic cancer, and 50% of asymptomatic individuals with a high risk for pancreatic cancer. However, KRAS mutations were detected in pancreatic juice from 19% of controls. Mutations detected in individuals without pancreatic abnormalities, based on imaging analyses, likely arise from small pancreatic intraepithelial neoplasia lesions. ClinicalTrials.gov no: NCT00438906 and NCT00714701.
Manganese superoxide dismutase-2 (
) plays a crucial role in cells' protection against mitochondrial oxidative damage. A genetic polymorphism in the mitochondrial targeting sequence of the
gene has ...been implicated in various diseases, including prostate cancer. Paller et al. have shown an increase in prostate-specific antigen (PSA) doubling time in patients with the Ala/Ala (wildtype) genotype when treated with pomegranate/grape extract antioxidants. We developed and validated a pyrosequencing assay that detects the common germline
SNP (rs_4880) with the aim of identifying men with castrate-resistant prostate cancer eligible for an antioxidant therapy clinical trial. We first selected 37 samples from the 1000 genomes study with known genotypes determined using Illumina-based sequencing and confirmed them by Sanger sequencing. In a blinded design, we then performed the new pyrosequencing assay on these samples and assigned genotypes. Genotypes for all 37 samples (13 homozygous Ala, 12 heterozygous Ala/Val, and 12 homozygous Val) were all concordant by pyrosequencing. The pyrosequencing assay has been live since May 2018 and has proven to be robust and accurate.
Aims
Gastric pyloric gland adenomas (PGAs) are rare epithelial polyps that are found more commonly in autoimmune atrophic gastritis and familial adenomatous polyposis (FAP). Little is known about the ...morphology and genetics of PGAs in FAP. PGAs in FAP are studied morphologically and genetically. Findings in FAP‐associated PGAs are compared to sporadic PGAs and related lesions such as oxyntic gland adenoma (OGA) to increase our understanding of these rare polyps.
Methods and results
Seven PGAs and 18 fundic gland polyps (FGPs) from FAP patients were collected. KRAS and GNAS mutations were determined in six PGAs and 18 FGPs. Immunohistochemistry was applied on five PGAs to provide further confirmation of the histological subtypes and genetic alterations. Morphology of all PGAs was studied and compared to literature on sporadic PGAs and related lesions. All successfully sequenced PGAs (six of six) carried GNAS mutations and half of the successfully sequenced PGAs carried a KRAS mutation (three of six). Nuclear β‐catenin was seen only in one PGA with focal high‐grade dysplasia. Morphologically, PGAs in FAP showed overlapping features with OGA.
Conclusion
Familial adenomatous polyposis‐associated PGAs have a similar genetic background as sporadic PGAs, i.e. KRAS and GNAS mutation. Based on morphological findings in FAP associated PGAs, it is hypothesized that PGAs and OGAs are closely related lesions.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
Epithelial-mesenchymal transition (EMT), a key event in cancer metastasis, allows polarized epithelial cells to assume mesenchymal morphologies, enhancing invasiveness and migration, and can be ...induced by reactive oxygen species (ROS). Val16A (Ala) SOD2 polymorphism has been associated with increased prostate cancer (PCa) risk. We hypothesized that SOD2 Ala single nucleotide polymorphism (SNP) may promote EMT. We analyzed SOD2 expression and genotype in various prostate cell lines. Stable overexpression of Ala-SOD2 or Val-SOD2 allele was performed in Lymph Node Carcinoma of the Prostate (LNCaP) cells followed by analysis of intracellular ROS and EMT marker protein expression. Treatments were performed with muscadine grape skin extract (MSKE) antioxidant, with or without addition of H
O
to provide further oxidative stress. Furthermore, MTS cell proliferation, cell migration, and apoptosis assays were completed. The results showed that SOD2 expression did not correlate with tumor aggressiveness nor SOD2 genotype. We demonstrated that the Ala-SOD2 allele was associated with marked induction of EMT indicated by higher Snail and vimentin, lower E-cadherin, and increased cell migration, when compared to Val-SOD2 allele or Neo control cells. Ala-SOD2 SNP cells exhibited increased levels of total ROS and superoxide and were more sensitive to co-treatment with H
O
and MSKE, which led to reduced cell growth and increased apoptosis. Additionally, MSKE inhibited Ala-SOD2 SNP-mediated EMT. Our data indicates that treatment with a combination of H
O
-generative drugs, such as certain chemotherapeutics and antioxidants such as MSKE that targets superoxide, hold promising therapeutic potential to halt PCa progression in the future.
Abstract Background Activating point mutations of GNAS at codon 201 have been detected in approximately two thirds of intraductal papillary mucinous neoplasms (IPMNs) of the pancreas. Intraductal ...papillary neoplasms of the bile ducts (IPNBs) morphologically resemble pancreatic IPMNs. This study sought to assess the mutational status of GNAS at codon 201 in IPNBs. Methods Thirty-four patients were included. DNA from microdissected IPNBs was subjected to a polymerase chain reaction and ligation method for the detection of GNAS mutations at codon 201 and of KRAS mutations at codon 12. Mutational status was compared with clinical and pathologic data. Results The IPNBs had a median diameter of 3.5 cm and were located intrahepatically ( n = 6), extrahepatically ( n = 13), both intra- and extrahepatically ( n = 4) or in the gallbladder (intracystic papillary neoplasms, n = 11). Most exhibited pancreatobiliary differentiation ( n = 20), high-grade dysplasia ( n = 26) and an associated adenocarcinoma ( n = 20). Analysis of GNAS codon 201 identified only one mutant sample in a multifocal intestinal subtype intrahepatic IPNB with high-grade dysplasia. Six lesions harboured a KRAS codon 12 mutation. Conclusions GNAS codon 201 mutations are uncommon in IPNBs, by contrast with pancreatic IPMNs. More comprehensive molecular profiling is needed to uncover the pathways involved in IPNB development.
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BFBNIB, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Metanephric adenoma (MA) has historically been considered to represent a differentiated form of epithelial Wilms tumor (WT), based in part upon cases that morphologically overlap these 2 neoplasms. ...More recently, BRAF V600E mutations have been demonstrated in the majority of MAs but not in unselected or even epithelial-predominant WTs, suggesting 2 genetically distinct entities. However, no prior study has examined BRAF status in neoplasms with overlapping histologic features of epithelial WT and MA. We studied a cohort of 11 such overlapping lesions, 2 of which we considered morphologically to be otherwise typical MAs with unusually prominent mitotic activity and 9 of which we classified as epithelial WTs with areas resembling MA. Both mitotically active MAs demonstrated the BRAF V600E mutation. While the majority (5/9) of epithelial WTs with areas resembling MA were negative for BRAF V600E mutation, 4 such cases were positive. Two BRAF V600E mutation-positive WTs occurred in children. One case in a 6-year-old male was morphologically similar to the BRAF V600E mutation-positive adult cases and subsequently metastasized to the lungs; remarkably, the metastases then completely resolved on Braf targeted therapy. A second occurred in a 3-year-old girl whose posttherapy nephrectomy specimen’s tumor was encapsulated and mitotically active like a typical WT, but also had more differentiated areas resembling MA. Immunohistochemistry for Braf V600E paralleled the molecular findings, demonstrating immunoreactivity in both the WT and MA-like areas of all 4 of these neoplasms. In summary, we demonstrate that BRAF V600E mutations are not entirely restricted to typical MA, as they may be seen in MAs showing mitotic activity along with a subset of epithelial-predominant WTs in adults and children that have foci which overlap morphologically with MA.