ABSTRACT
BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma cancer (PDAC) is a highly lethal malignancy requiring efficient detection when the primary tumor is still resectable. We previously developed the MxPancreasScore comprising 9 analytes and serum carbohydrate antigen 19-9 (CA19-9), achieving an accuracy of 90.6%. The necessity for 5 different analytical platforms and multiple analytical runs, however, hindered clinical applicability. We therefore aimed to develop a simpler single-analytical run, single-platform diagnostic signature. METHODS: We evaluated 941 patients (PDAC, 356; chronic pancreatitis [CP], 304; nonpancreatic disease, 281) in 3 multicenter independent tests, and identification (ID) and validation cohort 1 (VD1) and 2 (VD2) were evaluated. Targeted quantitative plasma metabolite analysis was performed on a liquid chromatography-tandem mass spectrometry platform. A machine learning-aided algorithm identified an improved (i-Metabolic) and minimalistic metabolic (m-Metabolic) signatures, and compared them for performance. RESULTS: The i-Metabolic Signature, (12 analytes plus CA19-9) distinguished PDAC from CP with area under the curve (95% confidence interval) of 97.2% (97.1%-97.3%), 93.5% (93.4%-93.7%), and 92.2% (92.1%-92.3%) in the ID, VD1, and VD2 cohorts, respectively. In the VD2 cohort, the m-Metabolic signature (4 analytes plus CA19-9) discriminated PDAC from CP with a sensitivity of 77.3% and specificity of 89.6%, with an overall accuracy of 82.4%. For the subset of 45 patients with PDAC with resectable stages IA-IIB tumors, the sensitivity, specificity, and accuracy were 73.2%, 89.6%, and 82.7%, respectively; for those with detectable CA19-9 >2 U/mL, 81.6%, 88.7%, and 84.5%, respectively; and for those with CA19-9 <37 U/mL, 39.7%, 94.1%, and 76.3%, respectively. CONCLUSIONS: The single-platform, single-run, m-Metabolic signature of just 4 metabolites used in combination with serum CA19-9 levels is an innovative accurate diagnostic tool for PDAC at the time of clinical presentation, warranting further large-scale evaluation.
Subject(s)
Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Pancreatitis, Chronic , Humans , CA-19-9 Antigen , Biomarkers, Tumor , ROC Curve , Case-Control Studies , Carcinoma, Pancreatic Ductal/pathology , Pancreatic Neoplasms/pathology , Pancreatitis, Chronic/diagnosis , Reference Standards , Carbohydrates , Pancreatic NeoplasmsABSTRACT
BACKGROUNDS & AIMS: Fluoropyrimidine c (5-fluorouracil [5FU]) increasingly represents the chemotherapeutic backbone for neoadjuvant, adjuvant, and palliative treatment of pancreatic ductal adenocarcinoma (PDAC). Even in combination with other agents, 5FU efficacy remains transient and limited. One explanation for the inadequate response is insufficient and nonspecific delivery of 5FU to the tumor. METHODS: We designed, generated, and characterized 5FU-incorporated systematic evolution of ligands by exponential enrichment (SELEX)-selected epidermal growth factor receptor (EGFR)-targeted aptamers for tumor-specific delivery of 5FU to PDAC cells and tested their therapeutic efficacy in vitro and in vivo. RESULTS: 5FU-EGFR aptamers reduced proliferation in a concentration-dependent manner in mouse and human pancreatic cancer cell lines. Time-lapsed live imaging showed EGFR-specific uptake of aptamers via clathrin-dependent endocytosis. The 5FU-aptamer treatment was equally effective in 5FU-sensitive and 5FU-refractory PDAC cell lines. Biweekly treatment with 5FU-EGFR aptamers reduced tumor burden in a syngeneic orthotopic transplantation model of PDAC, in an autochthonously growing genetically engineered PDAC model (LSL-KrasG12D/+;LSL-Trp53flox/+;Ptf1a-Cre [KPC]), in an orthotopic cell line-derived xenograft model using human PDAC cells in athymic mice (CDX; Crl:NU-Foxn1nu), and in patient-derived organoids. Tumor growth was significantly attenuated during 5FU-EGFR aptamer treatment in the course of follow-up. CONCLUSIONS: Tumor-specific targeted delivery of 5FU using EGFR aptamers as the carrier achieved high target specificity; overcame 5FU resistance; and proved to be effective in a syngeneic orthotopic transplantation model, in KPC mice, in a CDX model, and in patient-derived organoids and, therefore, represents a promising backbone for pancreatic cancer chemotherapy in patients. Furthermore, our approach has the potential to target virtually any cancer entity sensitive to 5FU treatment by incorporating 5FU into cancer cell-targeting aptamers as the delivery platform.
Subject(s)
Antimetabolites, Antineoplastic/administration & dosage , Aptamers, Nucleotide/administration & dosage , Carcinoma, Pancreatic Ductal/drug therapy , Drug Delivery Systems , ErbB Receptors/metabolism , Fluorouracil/administration & dosage , Pancreatic Neoplasms/drug therapy , Animals , Antimetabolites, Antineoplastic/metabolism , Aptamers, Nucleotide/metabolism , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma, Pancreatic Ductal/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Resistance, Neoplasm , Endocytosis , ErbB Receptors/genetics , Female , Fluorouracil/metabolism , Humans , Male , Mice, Inbred C57BL , Mice, Transgenic , Organoids , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , SELEX Aptamer Technique , Tumor Burden/drug effects , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Chemotherapy based on platinum compounds is the standard treatment for NSCLC patients with EGFR wild type, and is also used as second line in mutated EGFR patients. Nevertheless, this therapy presents poor clinical outcomes. ERCC1, ERCC2, XRCC1, MDM2, MTHFR, MTR, and SLC19A1 gene polymorphisms may contribute to individual variation in response and survival to platinum-based chemotherapy. The aim of this study was to investigate the influence of these polymorphisms on response and survival of NSCLC patients treated with platinum-based chemotherapy. A retrospective-prospective cohorts study was conducted, including 141 NSCLC patients. Polymorphisms were analyzed by PCR real-time with Taqman® probes. Patients with ERCC1 rs3212986-GG (p = 0.0268; OR = 2.50; CI95% = 1.12-5.69) and XRCC1 rs25487-GG (p = 0.0161; OR = 2.99; CI95% = 1.26-7.62) genotype showed significantly better ORR. Cox survival analysis revealed that patients carrying the MDM2 rs1690924-GG genotype (p = 0.0345; HR = 1.99; CI95% = 1.05-3.80) presented higher risk of death. Furthermore, carriers of MTR rs1805087-A alleles (p = 0.0060; HR = 8.91; CI95% = 1.87-42.42) and SLC19A1 rs1051266-AA genotype (p = 0.0130; HR = 1.74; CI95% = 1.12-2.68) showed greater risk of progression. No influence of ERCC1 rs11615, ERCC2 rs13181, ERCC2 rs1799793, XRCC1 rs1799782, MDM2 rs1470383, MTHFR rs1801131, and MTHFR rs1801133 on platinum-based chemotherapy clinical outcomes was found. In conclusion, our results suggest that ERCC1 rs3212986, XRCC1 rs25487, MDM2 rs1690924, MTR rs1805087, and SLC19A1 rs1051266 gene polymorphisms may significantly act as predictive factors in NSCLC patients treated with platinum-based chemotherapy.
Subject(s)
Carcinoma, Non-Small-Cell Lung/drug therapy , DNA Repair/genetics , Folic Acid/genetics , Pharmacogenetics , 5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase/genetics , Aged , Biomarkers, Tumor/genetics , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , DNA-Binding Proteins/genetics , Endonucleases/genetics , Female , Folic Acid/metabolism , Humans , Male , Methylenetetrahydrofolate Reductase (NADPH2)/genetics , Middle Aged , Platinum/administration & dosage , Platinum/adverse effects , Prognosis , Proportional Hazards Models , Proto-Oncogene Proteins c-mdm2/genetics , Reduced Folate Carrier Protein/genetics , Survival Analysis , X-ray Repair Cross Complementing Protein 1/genetics , Xeroderma Pigmentosum Group D Protein/geneticsABSTRACT
OBJECTIVE: Lung cancer, particularly the non-small-cell lung cancer (NSCLC) subtype, is the leading cause of cancer-related death worldwide. Several functional polymorphisms in inflammatory cytokine genes, such as IL1B, IL6, IL12A, IL13 and IL16, have been associated with the risk of NSCLC. The aim of this study was to evaluate the association between ILs gene polymorphisms and the risk of developing NSCLC. PARTICIPANTS AND METHODS: A retrospective case-control study was carried out, including 174 NSCLC cases and 298 controls of Spanish origin. IL1B (rs1143634), IL1B (rs12621220), IL1B (rs1143623), IL1B (rs16944), IL1B (rs1143627), IL12A (rs662959), IL13 (rs1881457), IL6 (rs1800795) and IL16 (rs7170924) gene polymorphisms were analysed by TaqMan. RESULTS: The genotypic logistic regression model adjusted by smoking status showed that the IL1B rs1143634-TT genotype was associated with a lower risk of NSCLC (P=0.04312; odds ratio=0.226; 95% confidence interval=0.044-0.840). No other gene polymorphisms showed an association with NSCLC in any of the models tested. CONCLUSION: In conclusion, IL1B rs1143634 was significantly associated with a higher risk of NSCLC. No influence of IL1B rs12621220, rs1143623, rs16944, rs1143627, IL12A rs662959, IL13 rs1881457 and IL16 rs7170924 on the risk of developing NSCLC was found in our study.
Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , Genetic Predisposition to Disease , Interleukins/genetics , Lung Neoplasms/genetics , Polymorphism, Single Nucleotide , Case-Control Studies , Female , Humans , Male , Middle Aged , Retrospective StudiesABSTRACT
Platinum-based chemotherapy is the standard treatment for NSCLC patients with EGFR wild-type, and as alternative to failure to EGFR inhibitors. However, this treatment is aggressive and most patients experience grade 3-4 toxicities. ERCC1, ERCC2, ERCC5, XRCC1, MDM2, ABCB1, MTHFR, MTR, SLC19A1, IL6 and IL16 gene polymorphisms may contribute to individual variation in toxicity to chemotherapy. The aim of this study was to evaluate the effect of these polymorphisms on platinum-based chemotherapy in NSCLC patients. A prospective cohorts study was conducted, including 141 NSCLC patients. Polymorphisms were analyzed by PCR Real-Time with Taqman(®) probes and sequencing. Patients with ERCC1 C118T-T allele (p=0.00345; RR=26.05; CI95%=4.33, 515.77) and ERCC2 rs50872-CC genotype (p=0.00291; RR=4.06; CI95%=1.66, 10.65) had higher risk of general toxicity for platinum-based chemotherapy. ERCC2 Asp312Asn G-alelle, ABCB1 C1236T-TT and the IL1B rs12621220-CT/TT genotypes conferred a higher risk to present multiple adverse events. The subtype toxicity analysis also revealed that ERCC2 rs50872-CC genotype (p=0.01562; OR=3.23; CI95%=1.29, 8.82) and IL16 rs7170924-T allele (p=0.01007; OR=3.19; CI95%=1.35, 7.97) were associated with grade 3-4 hematological toxicity. We did not found the influence of ERCC1 C8092A, ERCC2 Lys751Gln, ERCC2 Asp312Asn, ERCC5 Asp1104His, XRCC1 Arg194Trp, MDM2 rs1690924, ABCB1 C3435T, ABCB1 Ala893Ser/Thr, MTHFR A1298C, MTHFR C677T, IL1B rs1143623, IL1B rs16944, and IL1B rs1143627 on platinum-based chemotherapy toxicity. In conclusion, ERCC1 C118T, ERCC2 rs50872, ERCC2 Asp312Asn, ABCB1 C1236T, IL1B rs12621220 and IL16 rs7170924 polymorphisms may substantially act as prognostic factors in NSCLC patients treated with platinum-based chemotherapy.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/adverse effects , Carboplatin/adverse effects , Carcinoma, Non-Small-Cell Lung/drug therapy , Cisplatin/adverse effects , Lung Neoplasms/drug therapy , Pharmacogenomic Variants , Polymorphism, Genetic , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Female , Gene Frequency , Genetic Predisposition to Disease , Humans , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Male , Middle Aged , Pharmacogenetics , Pharmacogenomic Testing , Phenotype , Predictive Value of Tests , Prospective Studies , Risk Factors , Treatment OutcomeABSTRACT
Chemotherapy, the standard treatment for pancreatic ductal adenocarcinoma (PDAC), has only a modest effect on the outcome of patients with late-stage disease. Investigations of the genetic features of PDAC have demonstrated a frequent occurrence of mutations in genes involved in homologous recombination (HR), especially in the breast cancer susceptibility gene 2 (BRCA2). Olaparib, a poly(ADP-ribose) polymerase (PARP) inhibitor, is approved as a maintenance treatment for patients with advanced PDAC with germline BRCA1/2 mutations following a platinum-containing first-line regimen. Limitations to the use of PARP inhibitors are represented by the relatively small proportion of patients with mutations in BRCA1/2 genes and the modest capability of these substances of inducing objective response. We have previously shown that pancreatic cancer with BRCA2 mutations exhibits a remarkably enhanced sensitivity towards tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL) receptor-stimulating agents. We thus aimed to investigate the effect of combined treatment with PARP inhibitors and TRAIL receptor-stimulating agents in pancreatic cancer and its dependency on the BRCA2 gene status. The respective effects of TRAIL-targeting agents and the PARP inhibitor olaparib or of their combination were assessed in pancreatic cancer cell lines and patient-derived organoids. In addition, BRCA2-knockout and -complementation models were investigated. The effects of these agents on apoptosis, DNA damage, cell cycle, and receptor surface expression were assessed by immunofluorescence, Western blot, and flow cytometry. PARP inhibition and TRAIL synergized to cause cell death in pancreatic cancer cell lines and PDAC organoids. This effect proved independent of BRCA2 gene status in three independent models. Olaparib and TRAIL in combination caused a detectable increase in DNA damage and a concentration-dependent cell cycle arrest in the G2/M and S cell cycle phases. Olaparib also significantly increased the proportion of membrane-bound death receptor 5. Our results provide a preclinical rationale for the combination of PARP inhibitors and TRAIL receptor agonists for the treatment of pancreatic cancer and suggest that the use of PARP inhibitors could be extended to patients without BRCA2 mutations if used in combination with TRAIL agonists.
ABSTRACT
Genetically engineered mouse models (GEMMs) transformed the study of organismal disease phenotypes but are limited by their lengthy generation in embryonic stem cells. Here, we describe methods for rapid and scalable genome engineering in somatic cells of the liver and pancreas through delivery of CRISPR components into living mice. We introduce the spectrum of genetic tools, delineate viral and nonviral CRISPR delivery strategies and describe a series of applications, ranging from gene editing and cancer modeling to chromosome engineering or CRISPR multiplexing and its spatio-temporal control. Beyond experimental design and execution, the protocol describes quantification of genetic and functional editing outcomes, including sequencing approaches, data analysis and interpretation. Compared to traditional knockout mice, somatic GEMMs face an increased risk for mouse-to-mouse variability because of the higher experimental demands of the procedures. The robust protocols described here will help unleash the full potential of somatic genome manipulation. Depending on the delivery method and envisaged application, the protocol takes 3-5 weeks.
Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats , Neoplasms , Animals , CRISPR-Cas Systems/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Gene Editing/methods , Liver , Mice , Mice, Knockout , Neoplasms/genetics , PancreasABSTRACT
Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers. Developing biomarkers for early detection and chemotherapeutic response prediction is crucial to improve the dismal prognosis of PDAC patients. However, molecular cancer signatures based on transcriptome analysis do not reflect intratumoral heterogeneity. To explore a more accurate stratification of PDAC phenotypes in an easily accessible matrix, plasma metabolome analysis using MxP® Global Profiling and MxP® Lipidomics was performed in 361 PDAC patients. We identified three metabolic PDAC subtypes associated with distinct complex lipid patterns. Subtype 1 was associated with reduced ceramide levels and a strong enrichment of triacylglycerols. Subtype 2 demonstrated increased abundance of ceramides, sphingomyelin and other complex sphingolipids, whereas subtype 3 showed decreased levels of sphingolipid metabolites in plasma. Pathway enrichment analysis revealed that sphingolipid-related pathways differ most among subtypes. Weighted correlation network analysis (WGCNA) implied PDAC subtypes differed in their metabolic programs. Interestingly, a reduced expression among related pathway genes in tumor tissue was associated with the lowest survival rate. However, our metabolic PDAC subtypes did not show any correlation to the described molecular PDAC subtypes. Our findings pave the way for further studies investigating sphingolipids metabolisms in PDAC.
Subject(s)
Adenocarcinoma/blood , Carcinoma, Pancreatic Ductal/blood , Metabolome , Metabolomics , Pancreatic Neoplasms/blood , Cohort Studies , Fatty Acids/metabolism , Humans , Lipid Metabolism , Sphingolipids/metabolism , Transcriptome/genetics , Pancreatic NeoplasmsABSTRACT
Lung cancer, particularly non-small cell lung cancer (NSCLC) subtype, is the leading cause of cancer-related death related worldwide. Numerous gene polymorphisms in DNA repair, folate and glutathione pathways have been associated with susceptibility of NSCLC. We conducted this study to evaluate the effects of ERCC1, ERCC2, ERCC5, XRCC1, XRCC3, MTHFR, MTR, MTHFD1, SLC19A1 and GSTP1 gene polymorphisms on risk of NSCLC. No association between these gene polymorphisms and susceptibility of NSCLC were found in our patients, suggesting that genetic variations in genes involved in DNA repair, folate and glutathione metabolism pathways may not influence the risk of NSCLC.
Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , DNA Damage , DNA Repair/genetics , Glutathione/genetics , Lung Neoplasms/genetics , Polymorphism, Single Nucleotide/genetics , Aged , Aged, 80 and over , DNA Damage/genetics , Female , Genotype , Humans , Male , Middle Aged , RiskABSTRACT
BACKGROUND: Surgery is the standard treatment for early-stage NSCLC, and platinum-based chemotherapy remains as the treatment of choice for advanced-stage NSCLC patients with naïve EGFR status. However, overall 5-years relative survival rates are low. Interleukins (ILs) are crucial for processes associated with tumor development. In NSCLC, IL1B, IL6, IL12A, IL13 and IL16 gene polymorphisms may contribute to individual variation in terms of patient survival. The purpose of this study was to evaluate the association between IL gene polymorphisms and survival in NSCLC patients. METHODS: A prospective cohorts study was performed, including 170 NSCLC patients (114 Stage IIIB-IV, 56 Stage I-IIIA). IL1B (C > T; rs1143634), IL1B (C > T; rs12621220), IL1B (C > G; rs1143623), IL1B (A > G; rs16944), IL1B (C > T; rs1143627), IL6 (C > G; rs1800795), IL12A (C > T; rs662959), IL13 (A > C; rs1881457) and IL16 (G > T; rs7170924) gene polymorphisms were analyzed by PCR Real-Time. RESULTS: Patients with IL16 rs7170924-GG genotype were in higher risk of death (p = 0.0139; HR = 1.82; CI95% = 1.13-2.94) Furthermore, carriers of the TT genotype for IL12A rs662959 presented higher risk of progression in the non-resected NSCLC patient subgroup (p = 0.0412; HR = 4.49; CI95% = 1.06-18.99). The rest of polymorphisms showed no effect of on outcomes. CONCLUSIONS: Our results suggest that IL16 rs7170924-GG and IL12A rs662959-TT genotypes predict higher risk of death and progression, respectively, in NSCLC patients. No influence of IL1B rs12621220, IL1B rs1143623, IL1B rs16944, IL1B rs1143627, IL6 rs1800795, IL13 rs1881457 on NSCLC clinical outcomes was found in our patients.