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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.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.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 CA19-92 U/mL, 81.6%, 88.7%, and 84.5%, respectively; and for those with CA19-937 U/mL, 39.7%, 94.1%, and 76.3%, respectively.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.

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.

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.

The results of this investigation are part of the doctoral thesis presented by Cristina Pérez-Ramírez at the University of Granada., 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., This work was partly supported by a research grant for Cristina Pérez-Ramírez (FPU12/04722), from Ministerio de Educación, Cultura y Deporte.

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.

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.

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.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.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.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.

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 naive 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; CI 95% = 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; CI 95% = 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.