Biomarker For Pancreatic Ductal Adenocarcinoma Research Articles

Pancreatic juice-derived microRNA-4516 and microRNA-4674 as novel biomarkers for pancreatic ductal adenocarcinoma

Background and AimsPrecise diagnostic biomarkers are urgently required for pancreatic ductal adenocarcinoma (PDAC). Therefore, the aim of this study was to identify PDAC-specific exosomal microRNAs (Ex-miRs) from pancreatic juice (PJ) and evaluate their diagnostic potential. MethodsExosomes in PJ and serum were extracted using ultracentrifugation and confirmed morphologically and biochemically. PDAC-specific Ex-miRs were identified using our original miR arrays, in which "Ex-miRs derived from the PJ of patients with chronic pancreatitis (CP)" were subtracted from Ex-miRs commonly expressed in both "human PDAC cell lines" and "the PJ of patients with PDAC." We verified the expression of these miRs using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Changes in serum Ex-miR levels were assessed in two patients with PDAC who underwent curative resection. In situ hybridization (ISH) was performed to directly visualize PDAC-specific miR expression in cancer cells. ResultsWe identified novel Ex-miR-4516 and Ex-miR-4674 from the PJ of patients with PDAC, and they showed 80.0% and 81.8% sensitivity, 80.8% and 73.3% specificity, and 90.9% and 80.8% accuracy, respectively. The sensitivity, specificity, and accuracy of a triple assay of Ex-miR-4516/4674/PJ cytology increased to 93.3%, 81.8%, and 88.5%, respectively. In serum samples (n = 88), the sensitivity, specificity, and accuracy of Ex-miR-4516 were 97.5%, 34.3%, and 68%, respectively. Presurgical levels of serum-derived Ex-miR-4516 in two patients with relatively early disease stages declined after curative resection. ISH demonstrated that Ex-miR-4516 expression exclusively occurred in cancer cells. ConclusionsLiquid assays using the in situ-proven Ex-miR-4516 may have a high potential for detecting relatively early-stage PDAC and monitoring its clinical course.

Keratin 17 is a prognostic and predictive biomarker in pancreatic ductal adenocarcinoma.

To determine the role of keratin 17 (K17) as a predictive biomarker for response to chemotherapy by defining thresholds of K17 expression based on immunohistochemical tests that could be used to optimize therapeutic intervention for patients with pancreatic ductal adenocarcinoma (PDAC). We profiled K17 expression, a hallmark of the basal molecular subtype of PDAC, by immunohistochemistry in 2 cohorts of formalin-fixed, paraffin-embedded PDACs (n = 305). We determined a K17 threshold of expression to optimize prognostic stratification according to the lowest Akaike information criterion and explored the potential relationship between K17 and chemoresistance by multivariate predictive analyses. Patients with advanced-stage, low K17 PDACs treated using 5-fluorouracil (5-FU)-based chemotherapeutic regimens had 3-fold longer survival than corresponding cases treated with gemcitabine-based chemotherapy. By contrast, PDACs with high K17 did not respond to either regimen. The predictive value of K17 was independent of tumor mutation status and other clinicopathologic variables. The detection of K17 in 10% or greater of PDAC cells identified patients with shortest survival. Among patients with low K17 PDACs, 5-FU-based treatment was more likely than gemcitabine-based therapies to extend survival.

Integration of single-nucleus and exosome RNA sequencing dissected inter-cellular communication and biomarkers in pancreatic ductal adenocarcinoma

BackgroundMounting evidence underscores the importance of cell communication within the tumor microenvironment, which is pivotal in tumor proliferation, invasion, and metastasis. Exosomes play a crucial role in cell-to-cell communication. Although single-cell RNA sequencing (scRNA-seq) provides insights into individual cell transcriptional characteristics, it falls short of comprehensively capturing exosome-mediated intercellular communication. MethodWe analyzed Pancreatic Ductal Adenocarcinoma (PDAC) tissues, separating supernatant and precipitate for exosome purification and single-cell nucleus suspension. We then constructed Single-nucleus RNA sequencing (snRNA-seq) and small RNA-seq libraries from these components. Our bioinformatic analysis integrated these sequences with ligand-receptor analysis and public miRNA data to map the cell communication network. ResultsWe established intercellular communication networks using bioinformatic analysis to track exosome miRNA effects and ligand-receptor pairs. Significantly, hsa-miR-1293 emerged as a prognostic biomarker for pancreatic cancer, linked to immune evasion, increased myeloid-derived suppressor cells, and poorer prognosis. Targeting this miRNA may enhance anti-tumor immunity and improve outcomes. ConclusionOur study offers a novel approach to constructing intercellular communication networks using snRNA-seq and exosome-small RNA sequencing. By integrating miRNA tracing with ligand-receptor analysis, we illuminate the complex interactions in the pancreatic cancer microenvironment, highlighting the pivotal role of miRNAs and identifying potential biomarkers and therapeutic targets.

Abstract 3514: A deep learning model for detection and characterization of tertiary lymphoid structures in H&E-stained images from pancreatic ductal adenocarcinoma

Abstract Tertiary lymphoid structures (TLSs) are ectopic aggregates of a number of immune cells in nonlymphoid tissues under chronically inflamed environments and cancer. Emergence evidence suggested that TLSs were found in diverse cancers and TLSs have been referred as an independently predictive biomarker for immunotherapy response, especially immune checkpoint inhibitors. Moreover, the density and mature status of TLSs often positively associated with the favorable outcomes in cancers. Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related mortality with low survival rate in the whole world. Here, we developed a multi-resolution machine learning model for detection of TLSs from hema-toxylin and eosin (H&E) stained digital pathology slides as a potential clinical biomarker in PDAC. We selected 120 H&E slides (patients) with a total of 1,896 TLSs manually annotated as immature or mature TLSs by two pathologists. We also used immunohistochemistry (IHC) for CD20 and CD23 markers as a reference standard for H&E-based manual annotations. Our model mainly included detection and segmentation of individual TLSs. First, we used a transformer convolutional neural network to build a classification task to detect the presence of immature or mature TLSs. Then the instance segmentation model was applied to quantify the number of lymphocytes per unit square to validate the presence and maturation of TLSs. F1-score, calculated by the harmonic mean of precision (positive predictive value) and recall (sensitivity), was finally used to quantitatively evaluate the performance of model for each H&E slide. In validation dataset with 250 H&E slides (patients), we selected 50 slides to calculate F1-score, suggesting that our machine learning model achieved high F1-scores for detection and classification of immature TLSs (mean = 0.87, range from 0.79 to 0.95), and mature TLSs (mean = 0.92, range from 0.86 to 0.97). We further stratified 370 PDAC patients into three groups with mature TLSs (23%), immature TLSs (16%), and no TLSs (61%), respectively. Combined the clinical information, survival analysis revealed that patients with mature TLS had better survival outcomes than those without TLS (HR = 0.63; 95% CI, 0.44 - 0.90; P = 0.010). We developed a machine learning model that can accurately detect and classify TLSs in PDAC, and also demonstrated the prognosis value of predictive TLSs in H&E slides by automatedly machine learning model. These data highlight the promise of machine learning model for automated identification and quantification of the TLS on H&E slides in PDAC pathology. Citation Format: Chaoxian Zhao, Jidong Jia, Mingxi Lv, Jiayi Feng, Yijun Wang, Yingbin Liu. A deep learning model for detection and characterization of tertiary lymphoid structures in H&E-stained images from pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3514.

Abstract 4153: PET/CT imaging of pancreatic ductal adenocarcinoma with a claudin-4 binding peptide in preclinical mouse model

Abstract There is a critical need to develop new imaging and therapeutic strategies for pancreatic cancer. Current positron emission tomography (PET) imaging assessments of pancreatic ductal adenocarcinoma (PDAC) rely on 18F-FDG as a radiopharmaceutical. To increase the sensitivity of PDAC detection, we identified Claudin-4 (CLDN4), a tight junction transmembrane protein, as a PDAC biomarker through spatial transcriptomic and proteomic analyses. CLDN4 expression has previously been reported to be enhanced in 99% of primary and 100% of metastatic pancreatic cancer tissue samples and in 10 of 11 precancerous pancreatic intraepithelial neoplasia (PanIN) specimens. A small fragment of naturally occurring Clostridium perfringens enterotoxin (CPE) was previously reported to bind to the extracellular loop domain of CLDN4 protein (PDB:7kp4) with nanomolar (nM) affinity (Kd). A CLDN4 binding peptide (C4BP) was developed as a radioligand by modifying the n-terminus of C4BP which does not impact receptor binding. Here, we designed and synthesized a DOTA-PEG1-C4BP ligand on a rink amide resin using a microwave-assisted peptide synthesizer. Radiolabeling of DOTA-PEG1-C4BP (2 nmol) with 64CuCl2 (70 MBq) conferred 64Cu-C4BP (28 MBq/nmol) with 80% non-decay corrected yield and >99% radiochemical purity. PET/CT imaging of 64Cu-C4BP (~0.1 μg/mouse) performed in an inducible PDAC (KrasLSL-G12D/+;Rosa26LSL-tdTomato/LSL-tdTomato(KT);Ptf1aCreER;Trp53fl/fl) model at 84 day after tamoxifen injection resulted in ~25% injected activity per cubic centimeter (IA/cc) accumulation in metastases and at ~18% IA/cc retention in pancreatic tumors within 30 minutes of tail vein injection with minimal background accumulation. Ex vivo PET imaging confirmed the localized accumulation of 64Cu-C4BP in transgenic mice within the colon, pancreas, and liver. Blocking of 64Cu-C4BP accumulation with the cold compound in a xenograft model (NOD/SCID) bearing Capan-1 PDAC tumors demonstrated the specificity of 64Cu-C4BP binding. Here we report that CLDN4 is a promising target and 64Cu-C4BP is a promising ligand for the detection of pancreatic cancer. Citation Format: Jai Woong Seo, James Wang, Aris J. Kare, Spencer K. Tumbale, Bo Wu, Marina N. Raie, Mallesh Pandrala, Katherine W. Ferrara. PET/CT imaging of pancreatic ductal adenocarcinoma with a claudin-4 binding peptide in preclinical mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4153.

Abstract 4156: Monitoring early chemotherapy response in murine pancreatic ductal adenocarcinoma using non-invasive hyperpolarized magnetic resonance spectroscopic imaging with 13C-pyruvate

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is a rarely operable cancer with a very poor prognosis. Nearly 100% of affected patients receive one of two alternative first-line chemotherapies during the course of their disease: 1.) Gemcitabine-Abraxane (G/A) or 2.) FOLFIRINOX. Treatment success varies widely between the patients due to individual molecular, structural tumor and metabolic heterogeneity. In this project, we use hyperpolarized 13C-pyruvate magnetic resonance spectroscopic imaging (HP-MRSI) to non-invasively characterize early metabolic changes in murine endogenous PDAC in response to the mentioned chemotherapies. Methods: 18 KPC (Ptf1awt/cre(C)Kraswt/G12D(K)Trp53fl/fl(P)) mice with 74 distinct tumour nodules, visually identified based on T2-weighted anatomical imaging, were used in this study. The conversion of pyruvate to lactate was detected by HP-MRSI, performed on 7 T preclinical system using modified 3D balanced steady-state free precession (bSSFP) sequence on day 0 pre-therapy and on day 3 post-therapy. 100μg/g G (intraperitoneally) and 30μg/g A (intravenously) were applied twice on day 0 post-scan and day 2 (n=6). FOLFIRINOX (60μg/g 5-Fluoruracil, 27μg/g Irinotecan, 12.8μg/g Oxaliplatin, 60μg/g Leucovorin) was injected intravenously once on day 0 post-scan (n=6). Vehicle mice (n=6) were left untreated. Area under the curves (AUC) of lactate to pyruvate time-courses (AUCl/AUCp) were calculated. Primary murine PDAC cell lines were established from biopsies of 58 imaging region, sequenced using 3’RNA-Seq (Poly-ASeq) method and analysed using Dseq2 R package. Results: G/A and FOLFIRINOX-treated tumors exhibited heterogeneous metabolic responses in distinct nodules, while non-treated group tumors remained stable. A significant difference in metabolic changes (p = 0.01, ANOVA) from day 0 to day 3 was observed between FOLFIRINOX, G/A, and non-treated groups. Low glycolytic tumors showed a positive trend, more pronounced with FOLFIRINOX than G/A treatment. Metabolically active nodules with high AUCl/AUCp on day 0 reduced lactate turnover under therapy. These findings suggest that metabolic HP-MRSI can detect treatment-induced changes in tumor composition and subtype-specific epithelial/mesenchymal plasticity, correlating with the metabolic phenotype. Further investigation will include histology, subtype-specific biomarkers, transcriptional signatures, and regional transcriptome analysis to explain molecular changes in metabolism. Conclusions: We have applied a non-invasive approach for detection of metabolic changes under chemotherapy in murine PDAC. These findings will contribute in the long term to the establishment of predictive imaging biomarkers for PDAC. Citation Format: Irina Heid, Anna-Maria Schmidmüller, Simon Baller, Jason G. Skinner, Geoffrey J. Topping, Wolfgang Gottwald, Hussein Trabulssi, Rupert Öllinger, Katja Steiger, Roland Rad, Franz Schilling, Rickmer F. Braren. Monitoring early chemotherapy response in murine pancreatic ductal adenocarcinoma using non-invasive hyperpolarized magnetic resonance spectroscopic imaging with 13C-pyruvate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4156.

Intratumoral T-cell receptor repertoire composition predicts overall survival in patients with pancreatic ductal adenocarcinoma

ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy that is refractory to immune checkpoint inhibitor therapy. However, intratumoral T-cell infiltration correlates with improved overall survival (OS). Herein, we characterized the diversity and antigen specificity of the PDAC T-cell receptor (TCR) repertoire to identify novel immune-relevant biomarkers. Demographic, clinical, and TCR-beta sequencing data were collated from 353 patients across three cohorts that underwent surgical resection for PDAC. TCR diversity was calculated using Shannon Wiener index, Inverse Simpson index, and “True entropy.” Patients were clustered by shared repertoire specificity. TCRs predictive of OS were identified and their associated transcriptional states were characterized by single-cell RNAseq. In multivariate Cox regression models controlling for relevant covariates, high intratumoral TCR diversity predicted OS across multiple cohorts. Conversely, in peripheral blood, high abundance of T-cells, but not high diversity, predicted OS. Clustering patients based on TCR specificity revealed a subset of TCRs that predicts OS. Interestingly, these TCR sequences were more likely to encode CD8+ effector memory and CD4+ T-regulatory (Tregs) T-cells, all with the capacity to recognize beta islet-derived autoantigens. As opposed to T-cell abundance, intratumoral TCR diversity was predictive of OS in multiple PDAC cohorts, and a subset of TCRs enriched in high-diversity patients independently correlated with OS. These findings emphasize the importance of evaluating peripheral and intratumoral TCR repertoires as distinct and relevant biomarkers in PDAC.

MicroRNA-20a in extracellular vesicles derived from duodenal fluid is a possible biomarker for pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) has a high mortality rate owing to its late diagnosis and aggression. In addition, there are relatively few minimally invasive screening methods for the early detection of PDAC, making the identification of biomarkers for this disease a critical priority. Recent studies have reported that microRNAs in extracellular vesicles (EV-miRs) from bodily fluids can be useful for the diagnosis of PDACs. Given this, we designed this study to evaluate the utility of cancer EVs extracted from duodenal fluid (DF) and their resident EV-miRs as potential biomarkers for the detection of PDAC. EV-miRs were evaluated and identified in the supernatants of various pancreatic cancer cell lines (Panc-1, SUIT2, and MIAPaca2), human pancreatic duct epithelial cells, and the DF from patients with PDAC and healthy controls. EVs were extracted using ultracentrifugation and the relative expression of EV-miR-20a was quantified. We collected a total of 34 DF samples (27 PDAC patients and seven controls) for evaluation and our data suggest that the relative expression levels of EV-miR-20a were significantly higher in patients with PDAC than in controls (p = 0.0025). In addition, EV-miR-20a expression could discriminate PDAC from control patients regardless of the location of the tumor with an area under the curve values of 0.88 and 0.88, respectively. We confirmed the presence of EVs in the DF and suggest that the expression of EV-miR-20a in these samples may act as a potential diagnostic biomarker for PDAC.

Inhibition of the Alternative Complement Pathway May Cause Secretion of Factor B, Enabling an Early Detection of Pancreatic Cancer.

This study aims to elucidate the cellular mechanisms behind the secretion of complement factor B (CFB), known for its dual roles as an early biomarker for pancreatic ductal adenocarcinoma (PDAC) and as the initial substrate for the alternative complement pathway (ACP). Using parallel reaction monitoring analysis, we confirmed a consistent ∼2-fold increase in CFB expression in PDAC patients compared with that in both healthy donors (HD) and chronic pancreatitis (CP) patients. Elevated ACP activity was observed in CP and other benign conditions compared with that in HD and PDAC patients, suggesting a functional link between ACP and PDAC. Protein-protein interaction analyses involving key complement proteins and their regulatory factors were conducted using blood samples from PDAC patients and cultured cell lines. Our findings revealed a complex control system governing the ACP and its regulatory factors, including Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation, adrenomedullin (AM), and complement factor H (CFH). Particularly, AM emerged as a crucial player in CFB secretion, activating CFH and promoting its predominant binding to C3b over CFB. Mechanistically, our data suggest that the KRAS mutation stimulates AM expression, enhancing CFH activity in the fluid phase through binding. This heightened AM-CFH interaction conferred greater affinity for C3b over CFB, potentially suppressing the ACP cascade. This sequence of events likely culminated in the preferential release of ductal CFB into plasma during the early stages of PDAC. (Data set ID PXD047043.).

Emerging Horizons in the Diagnosis of Pancreatic Cancer: The Role of Circulating microRNAs as Early Detection Biomarkers for Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with a poor prognosis, primarily due to a late diagnosis. Recent studies have focused on identifying non-invasive biomarkers for early detection, with microRNAs (miRNAs) emerging as promising candidates. This systematic review aims to evaluate the potential of circulating miRNAs as biomarkers for the early detection of PDAC, analyzing their diagnostic accuracy, specificity, and sensitivity. Following Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, a comprehensive search across PubMed, Embase, and the Cochrane Library was conducted. Studies published from January 2013 to October 2023 focusing on miRNA biomarkers for early PDAC detection were included. Data synthesis was performed through a narrative approach due to the heterogeneity of the studies. Nine studies met the inclusion criteria. Key findings include the elevated levels of specific miRNAs, such as miR-18a, miR-106a, and miR-25, in early-stage PDAC patients compared to controls. The integration of miRNA profiles with traditional biomarkers like CA19-9 showed improved diagnostic performance. However, challenges in the standardization of miRNA evaluation methodologies were noted. Circulating miRNAs demonstrate significant potential as non-invasive biomarkers for early PDAC detection. Despite promising results, further research and standardization are necessary for clinical application.

Prospective Evaluation of Circulating Tumor DNA using Next Generation Sequencing as a Biomarker during Neoadjuvant Chemotherapy in Localized Pancreatic Cancer.

In this prospective study, we aim to characterize the prognostic value of circulating tumor DNA (ctDNA) by next-generation-sequencing (NGS) in patients undergoing neoadjuvant chemotherapy (NAC) for pancreatic ductal adenocarcinoma (PDAC). Circulating tumor DNA is a promising blood-based biomarker that is prognostic in several malignancies. Detection of ctDNA by NGS may provide insights regarding the mutational profiles in PDAC to help guide clinical decisions for patients in a potentially curative setting. However, the utility of ctDNA as a biomarker in localized PDAC remains unclear. Patients with localized PDAC were enrolled in a prospective study at Northwestern Medicine between October 2020 and October 2022. Blood samples were collected to perform targeted tumor agnostic NGS utilizing the Tempus x|F 105 gene panel at three timepoints: pre-therapy (at diagnosis), post-NAC, and after local therapy, including surgery. The relationship between ctDNA detection and CA19-9, and the prognostic significance of ctDNA detection were analyzed. 56 patients were included in the analysis. ctDNA was detectable in 48% at diagnosis, 33% post-NAC, and 41% after local therapy. After completion of NAC, patients with detectable ctDNA had higher CA19-9 levels versus those without (78.4 vs. 30.0, P=0.02). The presence of baseline ctDNA was associated with a CA19-9 response; those without ctDNA had a significant CA19-9 response following NAC (109.0U/mL vs. 31.5U/mL; P=0.01), while those with ctDNA present at diagnosis did not (198.1U/mL vs. 113.8U/mL; P=0.77). In patients treated with NAC, the presence of KRAS ctDNA at diagnosis was associated with and independently predicted worse progression-free-survival. This report demonstrates the prognostic value of ctDNA analysis with NGS in localized PDAC. NGS ctDNA is a biomarker of treatment response to NAC. KRAS ctDNA at diagnosis independently predicts worse survival in patients treated with NAC.

New cutoff of CA19-9 values for predicting pancreatic cancer using liquid biopsy.

692 Background: Carbohydrate antigen 19-9 (CA19-9) is the biomarker most commonly used as a diagnostic aid for treatment efficacy and survival prediction for pancreatic ductal adenocarcinoma (PDAC). However, the cutoff value of CA19-9 is still being discussed. Instead, circulating tumor DNA (ctDNA) has been reported as a new biomarker for PDAC. Our previous study showed a correlation between CA19-9 levels and KRAS mutated ctDNA in the blood of PDAC patients. Based on this correlation, we validated an appropriate cutoff value for CA19-9 before surgery. Methods: We monitored continuously CA19-9 values and KRAS-mutated ctDNAs in unresectable PDAC patients (N=22) who underwent chemotherapy in 2015 to 2017. KRAS mutation in tumor tissues and mutated circulating tumor DNA (MctDNA) in plasma was analyzed for mutations by digital polymerase chain reaction (ddPCR) in 22 patients with pancreatic cancer. KRAS point mutation in plasma was analyzed, according to KRAS point mutation in tissue (ex. 12D, 12V). Receiver operating characteristics (ROC) curve analysis was plotted to determine new cutoff of CA19-9 value that emerged KRAS-mutated ctDNA in the longitudinal monitoring. Recurrence-free survival (RFS) and overall survival (OS) of 60 patients who underwent surgery for resectable PDAC in 2005 to 2013 were analyzed according to many survival variables including new cutoff of CA19-9 value and median CA19-9 before operation. Results: Receiver operating characteristic curve analysis identified 949.7 U/mL of CA19-9 as the cutoff value corresponding to the presence of KRAS mutated ctDNA. The median value of CA19-9 was 221.1 U/mL. Subsequently, these values were verified for their prognostic values of RFS and OS in 60 patients who underwent surgery in 2005 to 2013. Multivariate analysis revealed that 949.7 U/mL of CA19-9 was an independent risk factor for RFS and OS in these patients (P = 0.010 and = P = 0.001, respectively), along with lymph node metastasis( P = 0.017 and P = 0.008), unlike the median CA19-9 level (P = 0.210 and P = 0.150). Conclusions: The results of this study demonstrate that preoperative 949.7 U/mL ≥ CA19-9 may be associate with poor prognosis in PDAC. In summary, our study demonstrated, for the first time, that the adjusted cutoff value of CA19-9 was an important biomarker for the prediction of recurrence and prognosis in patients with resected PDAC. Patients with high levels of CA19-9 before surgery had a higher risk of recurrence and poorer prognosis than those with low levels of CA19-9, therefore they may have benefitted more from drug treatment than surgical intervention. Nevertheless, appropriate clinical decision-making for patients with high CA19-9 levels should be confirmed in future clinical trials. Although our study results should be interpreted within the study limitations and further examinations are required to draw a definitive conclusion, we believe that it clarifies the selection of patients with PDAC.

Abstract A072: Keratin 17-signature for stratification of chemotherapy in pancreatic adenocarcinoma

Abstract Introduction: Keratin 17 (K17) is a hallmark of the most aggressive molecular subtype of pancreatic ductal adenocarcinoma (PDAC). However, it is still unknown if K17 can predict the response to the two standard-of care treatments for PDAC: 1) 5-fluorouracil, irinotecan, leucovorin, and oxaliplatin (FFX) and 2) Gemcitabine/nab-paclitaxel (Gem/Nab). Thus, we hypothesized that K17 expression in patient tumors could be utilized as a biomarker to guide clinical decision-making in predicting the most effective standard-of-care treatment for patients with PDAC. Given that 85% of patients do not undergo resection, to circumvent this limitation we tested the predictive value of K17 using the tissue collected during endoscopic biopsies, which are routine in PDAC diagnosis. Methods: Our pilot study included 68 patients diagnosed with PDAC from Yale-New Haven Hospital treated with either FFX or Gem/Nab. Endoscopic biopsies were evaluated for tumor cellularity and then underwent an immunohistochemical (IHC)-based K17 test using the ABC detection method. Samples were scored by two independent pathologists using the PathSQ method. Results: Of the 68 patients included in the study, 20 (29.4%) were treated with Gem/Nab and 48 (70.6%) were treated with FFX. Based on Akaike’s Information Criterion, a K17 threshold of 45% and 60% were found to maximize stratification of patients by therapy response. At the 45% cutoff, 17 (25.0%) patients were found to have high K17 expression. At the 60% cutoff, 12 (17.6%) patients were found to have high K17 expression. Patients with low K17 expression were found to have similar overall survival (OS) despite treatment. However, patients with high K17 expression both at the 45% cutoff (p=0.031 and HR = 2.68 (CI 0.8, 8.99)) and 60% cutoff (p=0.006, HR = 6.79 (1.20, 38.32)) had decreased survival when treated with Gem/Nab compared to FFX. Conclusions: Patients with high K17 expression had lower overall survival than those with low K17 expression, validating our previous findings of K17 as a negative prognostic biomarker in PDAC. High K17 expression may be a promising predictive biomarker in PDAC, with an observed increase in OS in high K17-expressing patients treated with FFX compared to patients treated with Gem/Nab, indicating a potential biomarker to best determine response to standard-of-care treatments. Citation Format: Sumedha Chowdhury, Deanne Yugawa, Robert Tseng, Mauricio Mejia, Jill Lacy, Timil Patel, Luisa Escobar-Hoyos, Kenneth Shroyer, Guoping Cai, Marie Robert, Michael Cecchini, John Kunstman, James Farrell, Kimberly Johung. Keratin 17-signature for stratification of chemotherapy in pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A072.

Abstract B065: Biomarker approach to define tumor subtype in pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal tumors with a predicted five-year survival rate in 2023 of 12%. Building on prior observations that molecular subtypes correlate with survival and response to treatment, our goal has been to understand the patterns of expression and prognostic correlations of three reciprocally expressed proteins previously identified as surrogate biomarkers of PDAC subtypes. Keratin 17 (K17) and Keratin 5 (K5) have both been identified as biomarkers of the basal subtype whereas GATA6, a transcription factor necessary for normal pancreatic development, is a surrogate marker of the classical subtype associated with longer-term survival. Here, we explore the expression of these three diagnostic biomarkers in PDAC and their specificity to tumor cells. Methods: Immunohistochemical analyses of K17 (KDx Diagnostics), GATA6 (R&D Systems), and KT5 (Leica) were performed on archival primary PDAC specimens from Stony Brook University Hospital (n= 30), by routine immunoperoxidase methods on serial sections. IHC stained slides were scanned using an Olympus VS200 slide scanner and images were imported into QuPath for digital analysis. Regions of interest (ROIs) were defined by the presence of tumor cells and were identified by histological assessment of representative sections. Digital scoring was performed on identified ROIs using the integrated "positive cell detection" function in QuPath and classified into three different stain intensities (1+, 2+, 3+). The threshold for each intensity is as follows: 1+, 0.2; 2+, 0.4; 3+, 1.0. According to the number of detections of each intensity, tumor cells were categorized into negative (0 and 1+) or positive (2+ and 3+). The percentage positive of analyzed biomarker expression within the tumor ROI was calculated by dividing the total number of positive cells by the total number of detected cells. Results: K17 was detected in both well and poorly differentiated PDAC components and in a few benign proliferative ducts of chronic pancreatitis but no staining was detected in stromal cells, benign ductal epithelium, or acinar cells. K5 was detected in a smaller proportion of cells from glandular components of the tumor as well as in most diffusely infiltrative single cells, benign ductal, and acinar cells but not in stromal cells. By contrast, GATA6 was detected in the nuclei of most cancer epithelial cells and was also detected in benign ductal cells, acinar cells, and stromal cells. The proportion of K17 expressing cells exceeded that of K5 positive cells in the great majority of ROIs and across cases. Conclusions: Keratin 17 detects a higher proportion of tumor cells than K5 and shows the lowest level of expression in benign cell types. By contrast, GATA6 IHC shows variable patterns of expression related to K17 or K5 expression and is expressed in the highest proportion of tumor cells, including both well-differentiated, diffusely infiltrative components, and benign tissue components. Citation Format: Carson Ho, Michael Horowitz, Lyanne Oblein, Shayan Sarkar, Soma Kobayashi, Karen Bai, Natalia Marchenko, Luisa Escobar-Hoyos, Kenneth Shroyer. Biomarker approach to define tumor subtype in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B065.

Abstract A068: Proteomic analysis of keratin 17 positive cells from laser-capture microdissected pancreatic ductal adenocarcinoma

Abstract Introduction. Although patients with pancreatic ductal adenocarcinoma (PDAC) have one of the lowest survival of all carcinomas, there is considerable variation in survival and disease progression, even when accounting for factors such as age and pathologic stage. Our group identified keratin 17 (K17) as a negative prognostic and predictive biomarker in PDAC. Patients with high K17 expression in their tumors have shorter overall survival as well as decreased response to chemotherapeutic agents, including gemcitabine. K17 has also emerged as an established biomarker of the basal molecular subtype of PDAC. In this study, we aim to identify proteins which correlate, directly or indirectly, with K17 expression via a combined approach using immunohistochemistry (IHC) followed by laser capture microdissection (LCM) and mass spectrometry. Methods. We selected 5 PDAC cases of low K17 expression and 5 PDAC cases of high K17 expression from the archival collections from the Stony Brook Cancer Center Biobank. A modified indirect immunoperoxidase approach was performed on sections mounted on LCM-compatible membrane slides. Carcinoma cells that were either K17 positive or negative by IHC were precisely dissected using LMD7 microscope (Leica) from 10-µM thick sections, followed by a modified tissue digestion and protein extraction protocol. Collision-induced fragment mass information and peptide abundance values were obtained by a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS). Spectral cutoffs were analyzed at both <5% and 1% false discovery rates. The human UniProt dataset was used for data alignment. Results. Mass spectrometry yielded over 1600 detected proteins with approximately 50 of which correlated strongly with K17 expression. Our preliminary results show that keratin 5, a known marker of basal-like PDAC, is positively correlated with keratin 17. Genes whose increased expression contribute to the Moffitt classification of basal molecular subtype of PDAC, such as S100A2, keratin 6A and keratin 7, also showed positive correlation with K17 in our study. The remainder of proteins associated with K17 expression include those involved with cell adhesion, cell-extracellular matrix interaction and smooth muscle contraction pathways, with specific types and classes being identified, pending in-depth analysis. Proteins with inverse relationship to K17 expression included those involved in intracellular organelle trafficking and apoptosis pathways. Conclusion. Our findings demonstrate the utility of mass spectrometry-based spatial proteomic analysis of microdissected cell populations from formalin-fixed, paraffin embedded samples. These methods have the potential to uncover complementary biomarkers to K17 that could enhance prognostic accuracy or serve as therapeutic targets and provide mechanistic insight into the mechanisms through which K17 drives tumor aggression. Citation Format: Ji Dong K. Bai, John D. Haley, Natalia Marchenko, Luisa F. Escobar-Hoyos, Kenneth R. Shroyer. Proteomic analysis of keratin 17 positive cells from laser-capture microdissected pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A068.

Abstract B012: TP53 missense mutations and keratin 17 are negative prognostic biomarkers in pancreatic ductal adenocarcinoma

Abstract Background: Cancer patients with similar clinical and pathologic characteristics may have different responses to treatments and survival, which may be attributable to distinct molecular properties of their tumors. In pancreatic ductal adenocarcinomas (PDACs), there is a pressing need to identify prognostic biomarkers that could help us to predict disease progression and patient survival. Our group previously reported that keratin17 (K17) expression is a hallmark of PDACs of patients with the shortest patient survival. Furthermore, there is some evidence suggesting that missense mutations in TP53 are associated with worse survival in patients with PDAC. To date, however, no studies have addressed the differential prognostic value of molecular characterization of TP53 coupled with K17 expression for predicting survival in patients with PDAC. Objective and methods: To fill this gap, we assessed the effect of different TP53 mutations and K17 expression on patient survival, using a clinically annotated cohort of 128 patients containing molecular profiling on patient tumors. K17 Immunohistochemistry (IHC) staining on patient’s formalin-fixed, paraffin-embedded (FFPE) tissue blocks were performed to assess the expression of this marker in tumor cells. K17 IHC scoring was conducted based on a subjective assessment of the overall proportion of stained cells relative to the overall number of tumor cells present in a single representative section from each case. Kaplan-Meier and Cox proportional hazards regression models were analyzed to determine survival differences of patients with PDACs harboring different TP53 mutations and levels of K17 IHC expression, adjusting for patient demographics (age at diagnosis, sex, race/ethnicity) and tumor characteristics (presence of distant metastasis at diagnosis). Results: Tumors were classified as high for K17 when ≥90% of tumor cells showed strong staining. High-K17 cases (n=16;12.50%) had shorter survival compared with low-K17 cases (n=112; 87.50%) (26.80 vs. 16.77 months; P=0.035). The majority of cases harbored missense mutations in TP53 (42.19%), while about one-third harbored truncating TP53 (27.34%) mutations. We found that PDACs with missense TP53 mutations and high K17 were associated with worse patient survival compared to tumors with missense TP53 and low K17 expression (HR=5.27; 95% CI, 1.76-15.82; P=0.003). Similarly, compared with patients with wild-type TP53 and low K17, patients with both missense TP53 mutations and high K17 had significantly worse survival (HR=3.51, 95% CI, 1.45-8.51; P=0.005). Conclusions: Our findings suggest that molecular characterization of specific TP53 mutations coupled with K17 IHC staining may help to determine differences in patient prognosis and identify subgroups with worse patient survival, therefore, potentially guiding future clinical management of patients. Citation Format: Carlos Mauricio Mejia Arbelaez, Gabriella Baraks, Lyanne Oblein, Michael Horowitz, Lynn Matrisian, Kenneth Shroyer, Luisa Fernanda Escobar-Hoyos. TP53 missense mutations and keratin 17 are negative prognostic biomarkers in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B012.

Abstract C067: Correlation of keratin17 expression with tumor cell proliferation and invasion in PDAC

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) shows variable degrees of differentiation, with both intratumoral, regional, and case to case heterogeneity, including glandular elements, sheet-like aggregates, cell clusters, and single diffusely infiltrative tumor cells. Prior research from our lab and others have established keratin 17 (K17) to be a defining hallmark of the basal molecular subtype of PDAC. In addition to its role as a component of the cytoskeleton, soluble forms of K17 undergo nuclear translocation to drive cell cycle progression and chemoresistance to first line chemotherapy. While K17 is heterogeneously expressed in the gland-forming elements of PDACs, it is most consistently expressed in tumor cells which are either in small groups or are single diffusely infiltrative. The aims of the current study are to define the correlation between K17 expression and tumor cell differentiation, with the focus on glandular versus single diffusely infiltrative components of PDAC, and to relate these findings to established biomarkers of cell proliferation and invasion. Methods: Archival human PDACs were provided by the Stony Brook Cancer Center Biobank and K17 immunohistochemistry was performed by indirect immunoperoxidase method, as previously described, to select cases with both glandular and diffusely infiltrative cell components and heterogeneous K17 expression. Multiplex immunofluorescence with a panel of antibodies to K17 (Cell Signaling Technology), pan-cytokeratin, Ki-67 and vimentin [KS1] (Miltenyi Biotec) was performed on 5-micron FFPE sections using the MACSima system (Miltenyi Biotec). Distribution patterns of K17 were evaluated in glandular and diffusely invasive components and were related to the Ki-67 status. Results: Cytoplasmic staining for K17 was found in the minority of tumor cells in glands and displayed intraglandular heterogeneity but was found in most diffusely infiltrative tumor cells. Also, nuclear K17 was rarely detected in the glandular components but was found in a high proportion of the diffusely infiltrative cells. Ki-67 was detected mainly in glandular cells, unrelated to K17 status, and rarely in single diffusely infiltrative cells, never co-localizing with nuclear K17. Of note, only a small fraction of tumor cells co-expressed K17 and vimentin, suggesting that majority of diffusely infiltrative tumor cells were not engaged in epithelial to mesenchymal transition. Conclusion: While cytoplasmic K17 is heterogeneously expressed in the glandular components of PDAC, strong staining for K17 in both the cytoplasm and the nucleus is a hallmark of diffusely infiltrative, Ki-67-negative tumor cells. These observations support the paradigm that tumor cells are committed to either cell division in the glandular components, or to diffuse single cell invasion, highlighted by increased cytoplasmic and nuclear K17 expression. Therefore, the results emphasize the importance of understanding the expression of PDAC biomarkers in both gland-forming elements and diffusely infiltrative tumor cells. Citation Format: Shayan Sarkar, Lyanne Oblein, Michael Horowitz, Luisa Escobar-Hoyos, Natalia Marchenko, Kenneth Shroyer. Correlation of keratin17 expression with tumor cell proliferation and invasion in PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C067.

Abstract A014: Combinations of previously reported biomarkers achieve improved sensitivity and specificity of detection of early stage pancreatic ductal adenocarcinoma

Abstract A promising approach to the early detection of pancreatic ductal adenocarcinoma (PDAC) is surveillance among people at elevated risk for the disease using a blood test. Several groups have reported promising biomarkers, but no single biomarker delivers the rate of positively predicting cancers and of negatively predicting non-cancers needed for routine use. We hypothesized that combinations of previously reported PDAC biomarkers could contribute complementary detection across heterogeneous subgroups of patients and consequently improve biomarker performance. Blood plasma specimens from 427 individuals, grouped into a training set (n = 132) and a validation set (n = 295), were distributed to four different laboratories for analysis using their independently developed biomarker assays. We analyzed the training set to identify biomarker combinations that improved upon CA19-9 for identification of early stage PDAC relative to benign disease conditions, both for improved sensitivity while controlling for high specificity and improved specificity while controlling for high sensitivity. We then applied the biomarker combinations to the validation set to determine their performance. The combination of CA19-9 with the glycan biomarker CA199.STRA improved sensitivity from 0.44 sensitivity with 0.98 specificity for CA19-9 to 0.71 sensitivity with 0.94 specificity (p < 0.001, 1000-fold bootstrap), and CA19-9 combined with the protein biomarker LRG1 and CA199.STRA improved specificity from 0.16 specificity and 0.94 sensitivity for CA19-9 to 0.58 specificity and 0.89 sensitivity (p < 0.001, 1000-fold bootstrap). Notably, cross-laboratory biomarker combinations that did not include CA19-9 in the panel significantly improved upon CA19-9. The combination of CA199.STRA with the protein biomarker MUC5AC improved sensitivity over CA19-9 with 0.61 sensitivity and 0.94 specificity (p = 0.0002, 1000-fold bootstrap), and the combination of CA199.STRA and the protein biomarkers MUC5AC, THSP2, and ANG improved specificity over CA19-9 with 0.66 specificity and 0.88 sensitivity (p < 0.0001, 1000-fold bootstrap). By coordinating this biomarker study across independent laboratories, we have identified novel biomarker combinations that significantly increase the sensitivity and specificity of early stage PDAC detection and that demonstrate the future feasibility of surveillance for PDAC using combinations of serum assays. Citation Format: Brian Haab, Lu Qian, Ben Staal, Maneesh Jain, Johannes Fahrmann, Christine Worthington, Denise Prosser, Liudmila Velokokhatnaya, Camden Lopez, Runlong Tang, Mark Hurd, Gopalakrishnan Natarajan, Sushil Kumar, Lynette Smith, Samir Hanash, Surinder Batra, Anirban Maitra, Anna Lokshin, Ying Huang, Randall E. Brand, Liudmila Velokokhatnaya. Combinations of previously reported biomarkers achieve improved sensitivity and specificity of detection of early stage pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A014.

A Comprehensive Review of the Potential Role of Liquid Biopsy as a Diagnostic, Prognostic, and Predictive Biomarker in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma is one of the most lethal malignant diseases, with a mortality rate being close to incidence. Due to its heterogeneity and plasticity, as well as the lack of distinct symptoms in the early phases, it is very often diagnosed at an advanced stage, resulting in poor prognosis. Traditional tissue biopsies remain the gold standard for making a diagnosis, but have an obvious disadvantage in their inapplicability for frequent sampling. Blood-based biopsies represent a non-invasive method which potentially offers easy and repeated sampling, leading to the early detection and real-time monitoring of the disease and hopefully an accurate prognosis. Given the urgent need for a reliable biomarker that can estimate a patient's condition and response to an assigned treatment, blood-based biopsies are emerging as a potential new tool for improving patients' survival and surveillance. In this article, we discuss the current advances and challenges in using liquid biopsies for pancreatic cancer, focusing on circulating tumour DNA (ctDNA), extracellular vesicles (EVs), and circulating tumour cells (CTCs), and compare the performance and reliability of different biomarkers and combinations of biomarkers.

Integrative Analysis of PAIP2B to Identify a Novel Biomarker for Pancreatic Ductal Adenocarcinoma.

The aim of the study was to evaluate the potential diagnostic and prognostic value of gene, Poly A-Binding Protein Interacting Protein 2B ( PAIP2B ) in pancreatic cancer. We used the gene expression data and clinical information of pancreatic adenocarcinoma patients from The Cancer Genome Atlas database and Gene Expression Omnibus database to analyze the expression of PAIP2B in pancreatic cancer samples, and validated the expression of PAIP2B in tumor tissue, using bioinformatics technology to explore the prognostic value of PAIP2B and its possible biological function. A significantly lower level of PAIP2B was observed in pancreatic cancer patients than in controls, and validated by immunohistochemistry. PAIP2B reduced the proliferation and invasion of cancer cells and had a significantly high expression in early stage. Patients with lower levels of PAIP2B had a significantly shorter median survival time than those with higher levels. DNA demethylation played an important role in PAIP2B expression. In addition, PAIP2B expression was significantly associated with the tumor-infiltrating immune cells, especially T cells CD8, T cells CD4 memory resting, macrophages M0, and dendritic cells resting. Our study also found that PAIP2B regulated miRNA function leading to disease progression in pancreatic cancer patients. Our study explored the potential value of PAIP2B as a biological link between prognosis and pancreatic cancer, and provided reference for the follow-up study on the role of PAIP2B in pancreatic cancer.