Cohort Of Pancreatic Ductal Adenocarcinoma Patients Research Articles

Longitudinal tissue-based evaluation of TIGIT expression in patients with pancreatic cancer: Effect of expression with advancing clinical stages and across racial groups.

e16314 Background: While ground has been gained, pancreatic ductal adenocarcinoma (PDAC) continues to have a low 5-year survival of 13%. This is owed partially to a lack of early detection biomarkers and resistance to standard therapeutic options. TIGIT, an immune checkpoint receptor, is a marker of T-cell exhaustion and plays a key role in the inhibition of anti-tumor immune responses. TIGIT inhibitors are being explored in clinical trials in PDAC. Here we evaluate TIGIT expression in a cohort of PDAC patients and correlate level and intensity of expression with clinical parameters. We also examine changes in expression as the disease progresses from primary to metastatic disease. Methods: We performed RNAscope in situ hybridization (ISH) with a probe specific for human TIGIT mRNA on 82 formalin-fixed paraffin-embedded (FFPE) tissue samples. The cohort of tissue samples included 9 biopsies, 67 primary resections and 6 metastatic lesions. We evaluated the total TIGIT expression (%) as well as the intensity of TIGIT expression (% of cells with 3+ punctae, signifying putative immune cells), and compared these values between samples. Utilizing linear regression for continuous outcomes and logistic regression for binary outcomes, we tested for associations between TIGIT expression and clinical covariates. Results: Staining analysis showed that TIGIT expression did not differ significantly between racial groups. The mean percentage of TIGIT positive cells was 64.0%. High expression of TIGIT was associated with more advanced clinical stage (p < 0.05). Evaluation of three longitudinal samples from the same patient revealed decreased TIGIT expression from the initial biopsy (41.6%) to resection (33.4%) and metastasis (2.8%). In these specimens, 3+ TIGIT expression also declined (2.1%, 1.2% and 0.02%, respectively). Conclusions: Anti-TIGIT therapy has potential to reverse immune suppression and, with other therapeutic modalities, may provide survival benefit. Here we demonstrate that increased TIGIT expression correlates with more advanced stage at diagnosis and also present data demonstrating that overall TIGIT expression, as detected by RNAscope ISH, may decrease as PDAC progresses to metastasis. As such, anti-TIGIT therapy may have important implications for evading an important mechanism of cancer progression.

ADRA2A promotes the classical/progenitor subtype and reduces disease aggressiveness of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) manifests diverse molecular subtypes, including the classical/progenitor and basal-like/squamous subtypes, with the latter known for its aggressiveness. We employed integrative transcriptome and metabolome analyses to identify potential genes contributing to the molecular subtype differentiation and its metabolic features. Transcriptome analysis in PDAC patient cohorts revealed downregulation of adrenoceptor alpha 2A (ADRA2A) in the basal-like/squamous subtype, suggesting its potential role as a candidate suppressor of this subtype. Reduced ADRA2A expression was significantly associated with a high frequency of lymph node metastasis, higher pathological grade, advanced disease stage, and decreased survival among PDAC patients. In vitro experiments demonstrated that ADRA2A transgene expression and ADRA2A agonist inhibited PDAC cell invasion. Additionally, ADRA2A-high condition downregulated the basal-like/squamous gene expression signature, while upregulating the classical/progenitor gene expression signature in our PDAC patient cohort and PDAC cell lines. Metabolome analysis conducted on the PDAC cohort and cell lines revealed that elevated ADRA2A levels were associated with suppressed amino acid and carnitine/acylcarnitine metabolism, which are characteristic metabolic profiles of the classical/progenitor subtype. Collectively, our findings suggest that heightened ADRA2A expression induces transcriptome and metabolome characteristics indicative of classical/progenitor subtype with decreased disease aggressiveness in PDAC patients. These observations introduce ADRA2A as a candidate for diagnostic and therapeutic targeting in PDAC.

Circulating autoantibodies to alpha-enolase (ENO1) and far upstream element-binding protein 1 (FUBP1) are negative prognostic factors for pancreatic cancer patient survival

Pancreatic ductal adenocarcinoma (PDA) has a dismal prognosis due to a lack of early diagnostic markers and effective therapy. In PDA patients, the glycolytic enzyme and plasminogen receptor alpha-enolase (ENO1) and the transcription factor far upstream element-binding protein 1 (FUBP1) are upregulated and elicit the production of autoantibodies (aAb) that discriminate healthy subjects from PDA patients, with the latter mostly directed to post-translational phosphorylated isoforms. Here, the correlation of prognosis with circulating ENO1 and FUBP1aAb, and their protein tissue expression was analyzed in PDA patients. Circulating ENO1 and FUBP1 aAb was analyzed in two cohorts of PDA patients by ELISA (n = 470), while tissues expression was observed by immunohistochemistry (n = 45). Overall survival (OS) was estimated using the Kaplan–Meier method, while the Cox model was used to estimate the hazard ratios (HR) adjusted for the main prognostic factors. Logistic models were applied to assess associations between death and its risk indicators. All statistical analyses were performed with Stata version 15. Unlike ENO1 aAb, there was a significant correlation between FUBP1 aAb and FUBP1 expression in tumors (p = 0.0268). In addition, we found that high ENO1 (p = 0.016) and intermediate FUBP1 aAb levels (p = 0.013) were unfavorable prognostic factors. Notably, it was found that high anti-FUBP1 aAb level is a good prognostic marker for tail-body PDA (p = 0.016). Our results suggest that different levels of circulating aAb to ENO1 and FUBP1 predict a poor outcome in PDA patients and can be used to improve therapeutic strategies.

Down regulation of Cathepsin W is associated with poor prognosis in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is associated with a very poor prognosis. Therefore, there has been a focus on identifying new biomarkers for its early diagnosis and the prediction of patient survival. Genome-wide RNA and microRNA sequencing, bioinformatics and Machine Learning approaches to identify differentially expressed genes (DEGs), followed by validation in an additional cohort of PDAC patients has been undertaken. To identify DEGs, genome RNA sequencing and clinical data from pancreatic cancer patients were extracted from The Cancer Genome Atlas Database (TCGA). We used Kaplan–Meier analysis of survival curves was used to assess prognostic biomarkers. Ensemble learning, Random Forest (RF), Max Voting, Adaboost, Gradient boosting machines (GBM), and Extreme Gradient Boosting (XGB) techniques were used, and Gradient boosting machines (GBM) were selected with 100% accuracy for analysis. Moreover, protein–protein interaction (PPI), molecular pathways, concomitant expression of DEGs, and correlations between DEGs and clinical data were analyzed. We have evaluated candidate genes, miRNAs, and a combination of these obtained from machine learning algorithms and survival analysis. The results of Machine learning identified 23 genes with negative regulation, five genes with positive regulation, seven microRNAs with negative regulation, and 20 microRNAs with positive regulation in PDAC. Key genes BMF, FRMD4A, ADAP2, PPP1R17, and CACNG3 had the highest coefficient in the advanced stages of the disease. In addition, the survival analysis showed decreased expression of hsa.miR.642a, hsa.mir.363, CD22, BTNL9, and CTSW and overexpression of hsa.miR.153.1, hsa.miR.539, hsa.miR.412 reduced survival rate. CTSW was identified as a novel genetic marker and this was validated using RT-PCR. Machine learning algorithms may be used to Identify key dysregulated genes/miRNAs involved in the disease pathogenesis can be used to detect patients in earlier stages. Our data also demonstrated the prognostic and diagnostic value of CTSW in PDAC.

ATG4B and ATG4A loss result in two-stage cell cycle defects in pancreatic ductal adenocarcinoma cells.

Pancreatic ductal adenocarcinoma (PDAC) exhibits elevated levels of autophagy which promote tumor progression and treatment resistance. ATG4B is an autophagy-related cysteine protease under consideration as a potential therapeutic target, but is largely unexplored in PDAC. Here, we investigated the clinical and functional relevance of ATG4B expression in PDAC. Using two PDAC patient cohorts, we found that low ATG4B mRNA or protein expression is associated with worse patient survival outcomes, poorly differentiated PDAC tumors, and lack of survival benefit from adjuvant chemotherapy. In PDAC cell lines, ATG4B knockout reduced proliferation, abolished LC3B processing, reduced GABARAP and GABARAPL1 levels but increased ATG4A levels. ATG4B and ATG4A double knockout lines displayed a further reduction in proliferation, characterized by delays in G1/S-phase transition and mitosis. Pro-LC3B accumulated aberrantly at the centrosome with a concomitant increase in centrosomal proteins PCM1 and CEP131, which was rescued by exogenous ATG4B. The two-stage cell cycle defects following ATG4B and ATG4A loss have important therapeutic implications for PDAC.

Impact of preoperative statin medication on long-term outcomes after pancreatoduodenectomy for ductal adenocarcinoma: an international multicentric cross-sectional study

PurposeStatin treatment has been shown in certain population studies and meta-analyses to improve survival of patients with pancreatic ductal adenocarcinoma (PDAC). This study assessed if patients with statin treatment had better overall survival (OS) and disease-free survival (DFS) after upfront pancreatoduodenectomy for PDAC.MethodsConsecutive PDAC patients were retrospectively collected from three centers in Europe and USA (study period: 2000–2017). Adult patients who underwent upfront pancreatoduodenectomy and survived the first 90 postoperative days were included. Patients with metastasis at diagnosis or with macroscopic incomplete resection were excluded. Patients were considered under statin if started at least one month before pancreatoduodenectomy. Survival rates were calculated using Kaplan–Meier method and compared with log-rank test.ResultsA total of 496 patients were included. Median age was 67 years (IQR 59–75), 48% (n = 236) were women, and 141 patients (28%) received statin treatment already preoperatively. Patients with and without statin treatment were comparable in terms of demographics and pre-/intraoperative characteristics, except for age and pre-existing diabetes. Median OS and DFS were similar in patients with and without statin treatment (OS: 29, 95% CI 22–36 vs. 27 months, 95% CI 22–32, p = 0.370, DFS: 18, 95% CI 14–22 vs. 16 months, 95% CI 14–18, p = 0.430). On multivariable Cox regression, lymph node involvement (HR 1.9, 95% CI 1.6–2.2, p < 0.001), tumor differentiation (HR 1.3, 95% CI 1.1–1.6, p = 0.003), and postoperative chemotherapy (HR 0.5, 95% CI 0.4–0.7, p < 0.001) were predictors of OS, whereas statin treatment was not a prognostic factor (HR 0.9, 95% CI 0.7–1.2, p = 0.376).ConclusionIn this international cohort of PDAC patients, statin treatment did not influence survival after upfront pancreatoduodenectomy. Nodal involvement, tumor differentiation, and postoperative chemotherapy were independent predictors of OS.

Routine Molecular Profiling in Both Resectable and Unresectable Pancreatic Adenocarcinoma: Relevance of Cytologic Samples

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease, for which it is crucial to promptly detect actionable and prognostic alterations to drive specific therapeutic decisions, regardless of tumor resectability status. Endoscopic ultrasonography-guided fine-needle aspiration (EUS-FNA) is of key importance for PDAC diagnosis and can contribute significantly to tumor molecular profiling. Comprehensive genomic profile by targeted next-generation sequencing (NGS) was performed on 2 independent PDAC patient cohorts. Cohort 1 consisted of 77 patients with resectable PDAC for whom the histologic sample at the time of resection was available; for 56 patients cytologic specimens at the time of diagnosis also were obtained by EUS-FNA. Cohort 2 consisted of 20 patients with unresectable PDAC, for whom only the EUS-FNA cytologic sample was available. In cohort 1, a complete concordant mutational profile between the cytologic sample at diagnosis and the corresponding histologic specimen after surgery was observed in 88% of the cases, proving the ability to detect potential clinically relevant alterations in cytologic samples by NGS analysis. Notably, clinically actionable mutations were identified in 20% of patients. In cohort 2, comprehensive mutational profiling was obtained successfully for all samples. Consistent with the findings of cohort 1, KRAS, TP53, CDKN2A, and SMAD4 were the most altered genes. Most importantly, 15% of the patients harbored actionable mutations. Our findings show the feasibility of an NGS approach using both surgical specimens and cytologic samples. The model proposed in this study can be included successfully in the clinical setting for comprehensive molecular profiling of all PDAC patients irrespective of their surgical eligibility.

A CT Radiomics-Based Risk Score for Preoperative Estimation of Intraoperative Superior Mesenteric-Portal Vein Involvement in Pancreatic Ductal Adenocarcinoma.

The current radiologic criteria for assessing intraoperative superior mesenteric-portal vein (SMPV) involvement (i.e., presence of tumor-SMPV contact >180° or venous deformity) in pancreatic ductal adenocarcinoma (PDAC) are highly specific but insufficiently sensitive. Therefore, development of improved markers for a more accurate prediction is essential. This study aimed to develop a risk score model to estimate SMPV involvement in PDAC using radiomics analysis of computed tomography (CT) images. Data from two institution-based cohorts of PDAC patients undergoing preoperative CT scans were used to develop (n = 173) and validate (n = 156) a radiomics-based risk score of SMPV involvement using clinical and imaging variables. A radiomics signature was developed based on 2436 radiomic features extracted from the semi-automatic three-dimensional segmentation ofn CT images. The SMPV involvement risk score was built using multivariate logistic regression and compared with the current radiologic criteria. The study surgically identified SMPV involvement in 59 (34.1%) and 57(36.5 %) patients with PDAC in the development and validation cohorts, respectively. A 12-feature-based radiomics signature achieved areas under receiver operating characteristics curves (AUCs) of 0.89 or greater for estimating SMPV involvement. Multivariate regression identified the radiomics signature and SMPV deformity as associated with SMPV involvement. The risk score model had significantly improved AUC (0.928 vs. 0.768; P < 0.001) and sensitivity (84.2% vs. 66.7%; P = 0.025) in the radiologic evaluation. The novel risk score in this study, combining radiomics signature and venous deformity, demonstrated promising performance for estimating SMPV involvement preoperatively for patients with PDAC.

Multimodal survival prediction in advanced pancreatic cancer using machine learning

BackgroundExisting risk scores appear insufficient to assess the individual survival risk of patients with advanced pancreatic ductal adenocarcinoma (PDAC) and do not take advantage of the variety of parameters that are collected during clinical care.MethodsIn this retrospective study, we built a random survival forest model from clinical data of 203 patients with advanced PDAC. The parameters were assessed before initiation of systemic treatment and included age, CA19-9, C-reactive protein, metastatic status, neutrophil-to-lymphocyte ratio and total serum protein level. Separate models including imaging and molecular parameters were built for subgroups.ResultsOver the entire cohort, a model based on clinical parameters achieved a c-index of 0.71. Our approach outperformed the American Joint Committee on Cancer (AJCC) staging system and the modified Glasgow Prognostic Score (mGPS) in the identification of high- and low-risk subgroups. Inclusion of the KRAS p.G12D mutational status could further improve the prediction, whereas radiomics data of the primary tumor only showed little benefit. In an external validation cohort of PDAC patients with liver metastases, our model achieved a c-index of 0.67 (mGPS: 0.59).ConclusionsThe combination of multimodal data and machine-learning algorithms holds potential for personalized prognostication in advanced PDAC already at diagnosis.

Clinical Outcome and Prognostic Factors of Pancreatic Adenosquamous Carcinoma Compared to Ductal Adenocarcinoma-Results from the German Cancer Registry Group.

Simple SummaryAbout 0.5% of pancreatic malignancies are adenosquamous carcinomas. The pathophysiology of these carcinomas is poorly understood and clinical data is sparse. We compared characteristics and prognostic factors of adenosquamous carcinoma and ductal adenocarcinoma, which represent the most common type of pancreatic cancer based on data from the German cancer registry group. Adenosquamous carcinoma showed poorer differentiation and higher frequency of blood vessel invasion indicative of a more aggressive tumor biology. Adenosquamous tumor differentiation was a strong negative prognostic factor. Survival of patients with adenosquamous carcinoma was shorter compared to patients with a ductal adenocarcinoma after surgical tumor resection. This study suggests that distinct multimodal treatment protocols should be considered for adenosquamous carcinomas of the pancreas.Background: Adenosquamous carcinoma of the pancreas (ASCP) is a rare malignancy and its pathophysiology is poorly understood. Sparse clinical data suggest that clinical outcome and overall survival is worse in comparison to common pancreatic ductal adenocarcinoma (PDAC). Methods: We evaluated clinical outcome and prognostic factors for overall survival of patients with ASCP in comparison to patients with PDAC recorded between 2000 and 2019 in 17 population-based clinical cancer registries at certified cancer centers within the Association of German Tumor Centers (ADT). Results: We identified 278 (0.5%) patients with ASCP in the entire cohort of 52,518 patients with pancreatic cancer. Significantly, more patients underwent surgical resection in the cohort of ASCP patients in comparison to patients with PDAC (p < 0.001). In the cohort of 142 surgically resected patients with ASCP, the majority of patients was treated by pancreatoduodenectomy (44.4%). However, compared to the cohort of PDAC patients, significantly more patients underwent distal pancreatectomy (p < 0.001), suggesting that a significantly higher proportion of ASCP tumors was located in the pancreatic body/tail. ASCPs were significantly more often poorly differentiated (G3) (p < 0.001) and blood vessel invasion (V1) was detected more frequently (p = 0.01) in comparison with PDAC. Median overall survival was 6.13 months (95% CI 5.20–7.06) for ASCP and 8.10 months (95% CI 7.93–8.22) for PDAC patients, respectively (p = 0.094). However, when comparing only those patients who underwent surgical resection, overall survival of ASCP patients was significantly shorter (11.80; 95% CI 8.20–15.40 months) compared to PDAC patients (16.17; 95% CI 15.78–16.55 months) (p = 0.007). ASCP was a highly significant prognostic factor for overall survival in univariable regression analysis (p = 0.007) as well as in multivariable Cox regression analysis (HR 1.303; 95% CI 1.013–1.677; p = 0.039). Conclusions: In conclusion, ASCP showed poorer differentiation and higher frequency of blood vessel invasion indicative of a more aggressive tumor biology. ASCP was a significant prognostic factor for overall survival in a multivariable analysis. Overall survival of resected ASCP patients was significantly shorter compared to resected PDAC patients. However, surgical resection still improved survival significantly.

Abstract 2244: Video-based germline testing for individuals with pancreatic ductal adenocarcinoma: Influence of COVID-19 pandemic

Abstract Purpose: National guidelines recommend universal germline genetic testing (GT) for patients with Pancreatic Ductal Adenocarcinoma (PDAC), but rates of testing remain low. Given the aggressiveness of PDAC, the window of opportunity for GT is short and often overshadowed by treatment initiation and other clinical milestones. Thus, there is an unmet need for a model that streamlines GT and makes it available to a wider audience in a rapid fashion. Moreover, in pandemic times, video-based alternatives for medical care are increasingly relevant. Methods: We implemented a novel care delivery model in which a seven-minute educational video describing the benefits, risks, and implications of GT was shown to PDAC patients. The video was shown in lieu of an initial consult with a genetic counselor. Only patients who had not undergone GT or previously met with a genetic counselor were included. After watching the video, patients could elect to pursue GT and get tested on-side or remotely (at home). Genetic counselors disclosed results and provided post-test counseling by phone. Clinical and germline data were collected through medical records on a cohort of PDAC patients seen at the Gastrointestinal Center-MD Anderson during a 2-year enrollment period (May 2019-July 2021), which included the COVID-19 pandemic period. Results: A total of 286 PDAC patients watched the educational video. From 175 patients that watched the video pre-pandemic, 12 declined testing, whereas in the post-pandemic period, none of the 111 patients declined testing (6.9% vs 0%; p&amp;lt;0.004). We excluded data from 29 patients who elected to undergo GT but declined to participate in the registry. From the 241 patients with successfully collected samples, 21 patients (8.7%) had a pathogenic variant (PV), 38 patients (15.8%) had a Variant of Uncertain Significance (VUS), and 182 patients (75.5%) tested negative. The pathogenic variants detected included: BRCA2 (most frequent), ATM, BRCA1, CDKN2A, PALB2 and APC. Conclusions: GT can have tremendously beneficial effects, such as qualifying for targeted treatment options and facilitating cancer prevention in probands’ at-risk family members. Comparing uptake of GT pre- versus post-pandemic suggests that patients were more willing to trust information from a video platform, likely due to the global effect of living in a “virtual“ society as a result of the pandemic. We suggest an approach in which every PDAC patient is shown a genetics educational video and given the choice to undergo GT and post-result counseling, greatly reducing the burden on genetic counselors. We report here the feasibility of implementing video-based germline testing in PDAC patients which resulted in unexpectedly high uptake levels, particularly post-pandemic. Further investigations are needed to explore the feasibility of a fully remote GT model in diverse populations to assess additional barriers to universal GT. Citation Format: Saumya Kasliwal, Seyda Baydogan, Devon Harrison, Mark Hurd, Maureen Mork, Anirban Maitra, Florencia McAllister. Video-based germline testing for individuals with pancreatic ductal adenocarcinoma: Influence of COVID-19 pandemic [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2244.

Abstract 3389: An exosomal miRNA-based liquid biopsy signature for the noninvasive early detection of pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second leading cause of cancer-related deaths in the United States by 2030. Most of the PDAC patients are diagnosed with advanced disease, and less than 20% of patients are resectable at the time of diagnosis. The current imaging tools and blood markers (e.g. CA 19-9), are inadequate for early disease detection due to their poor specificity and sensitivity. This highlights the need to develop robust, noninvasive biomarkers for early detection of PDAC. While a large body of literature supports the use of cell-free miRNAs (cf-miRNAs) as potential diagnostic biomarkers in cancer, their tumor specificity is often debatable. Given the emerging evidence that exosomal cargo is a more robust representation of individual tumor types, in this study we sought to explore the diagnostic potential of cf-miRNAs along with exosomal miRNAs (exo-miRNAs), to establish a non-invasive miRNA signature for the early detection of PDAC. Methods: As part of the NCI’s Pancreatic Cancer Detection Consortium (PCDC) funded project, in this study, small RNA-sequencing was performed in exosome and cell-free (cf) samples from a cohort of 57 PDAC cases and 57 non-disease controls. Using rigorous bioinformatic and biostatistical approaches, we prioritized a panel of cf- and exo-miRNAs and evaluated its diagnostic performance in the sequencing-based discovery and validation cohorts. Subsequently, the performance of the discovered miRNA panel was validated using qRT-PCR assays in an independent clinical validation cohort of PDAC patients and non-disease controls (n=48/each group). The results were examined by ROC curve analysis to determine the diagnostic power of the biomarker panel individually and in combination for their ability to discriminate PDAC from controls. Results: The genomewide transcriptomic analyses led to the identification of a panel of 13 cf-miRNA and 17 exo-miRNA candidates. Sequencing validation in an independent cohort revealed that a combined panel of cf and exo-miRNAs exhibited an area under curve (AUC) of 0.89. Subsequent risk score analysis demonstrated that our biomarker signature was also robust in the identification of PDAC patients with early-stage cancers (stage I &amp; II) vs. controls (p &amp;lt;0.001). Moreover, when we combined the miRNA biomarker panel with CA19-9 values, the diagnostic performance was significantly superior when compared to the biomarker panel alone. Finally, the validation efforts in clinical cohorts by qRT-PCR revealed that the combined miRNA panel yielded an impressive accuracy with an AUC of 0.91, and a sensitivity of 0.88 and specificity of 0.87. Conclusions: In conclusion, we report a novel, exosome-based miRNA signature for the early detection of patients with PDAC; which could potentially improve early-detection efforts for this fatal malignancy. Citation Format: Kota Nakamura, Souvick Roy, Zhongxu Zhu, Eunsung Jun, Haiyong Han, Ruben M. Munoz, Satoshi Nishiwada, Geeta Sharma, Derek Cridebring, Frederic Zenhausern, Seungchan Kim, Denise Roe, Sourat Darabi, In Woong Han, Douglas Evans, Suguru Yamada, Michael Demure, Scott A. Celinski, Erkut Borazanci, Susan Tsai, John Bolton, Yasuhiro Kodera, Joon Oh Park, Song Cheol Kim, Xin Wang, Daniel Von Hoff, Ajay Goel. An exosomal miRNA-based liquid biopsy signature for the noninvasive early detection of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3389.

Oncogenic KRAS-Induced Protein Signature in the Tumor Secretome Identifies Laminin-C2 and Pentraxin-3 as Useful Biomarkers for the Early Diagnosis of Pancreatic Cancer.

Simple SummaryPancreatic ductal adenocarcinoma is an aggressive neoplasia that responds poorly to treatments and is frequently recognized at advanced stages. Early diagnosis and the possibility to undergo resective surgery would increase the rate of patient survival and the chance of a definitive cure. In search of candidate biomarkers to improve laboratory tests for early diagnosis, we have characterized the proteins secreted by pancreatic cells expressing the oncogene KRASG12V. Of several upregulated proteins, the expression of seven proteins was quantified in tumors of surgically resected patients. For two of these proteins, Laminin-C2 and Pentraxin-3, the plasma levels were significantly higher in patients than in healthy donors, and their good laboratory performance makes them two promising biomarkers of pancreatic cancer.KRAS mutations characterize pancreatic cell transformation from the earliest stages of carcinogenesis, and are present in >95% of pancreatic ductal adenocarcinoma (PDAC) cases. In search of novel biomarkers for the early diagnosis of PDAC, we identified the proteins secreted by the normal human pancreatic cell line (HPDE) recently transformed by inducing the overexpression of the KRASG12V oncogene. We report a proteomic signature of KRAS-induced secreted proteins, which was confirmed in surgical tumor samples from resected PDAC patients. The putative diagnostic performance of three candidates, Laminin-C2 (LAMC2), Tenascin-C (TNC) and Pentraxin-3 (PTX3), was investigated by ELISA quantification in two cohorts of PDAC patients (n = 200) eligible for surgery. Circulating levels of LAMC2, TNC and PTX3 were significantly higher in PDAC patients compared to the healthy individuals (p < 0.0001). The Receiver Operating Characteristics (ROC) curve showed good sensitivity (1) and specificity (0.63 and 0.85) for LAMC2 and PTX3, respectively, but not for TNC, and patients with high levels of LAMC2 had significantly shorter overall survival (p = 0.0007). High levels of LAMC2 and PTX3 were detected at early stages (I–IIB) and in CA19-9-low PDAC patients. In conclusion, pancreatic tumors release LAMC2 and PTX3, which can be quantified in the systemic circulation, and may be useful in selecting patients for further diagnostic imaging.

Reconstructing the tumor microenvironment to unlock therapeutic options in pancreatic cancer.

589 Background: Spatiotemporal heterogeneity, paucity of actionable targets, and complexity of the tumor microenvironment (TME) are major barriers to therapeutic advances in pancreatic ductal adenocarcinoma (PDAC). We reconstructed the transcriptomic data from a heterogeneous cohort of PDAC patients (pts) to examine the TME and identify putative therapeutic strategies. Methods: Transcriptomic profiling and targeted gene sequencing data (Tempus) on primary or metastatic specimens from PDAC pts treated at the Medical College of Wisconsin (MCW) between 2015-2020 were analyzed. Mutation calling, expression analysis, cell type deconvolution from the transcriptome, and TME reconstruction were performed using BostonGene’s automated pipelines. Mann-Whitney U test and Fisher's exact test were used to assess statistical significance. Results: The cohort (N = 79) comprised of resectable (19%), borderline resectable (37%), locally advanced (24%) and metastatic (20%) PDAC pts. The most frequently used tumor sites for transcriptomic profiling were pancreas primary (59%), liver (16%), lung (10%) and peritoneum (10%). Four distinct subtypes were identified based on the BostonGene classification of the transcriptomic TME– Immune Enriched (IE; 14%), Fibrotic (F; 28%), Immune Enriched &amp; Fibrotic (IEF; 36%), and Immune Depleted (ID; 22%). Analyses of the cellular composition of the TME subtypes with RNA-seq-based deconvolution showed that T-cell fractions (CD4, CD8) were higher in the IE/IEF subtypes compared to the F/ID subtypes (CD8 means: 6.4% vs 2.9%, p &lt; 0.001; CD4 means: 15.1% vs. 7.6%, p &lt; 0.001), while fibroblast content was higher in the F/IEF subtypes compared to the IE/ID subtypes (37.4% vs 18.4%; p &lt; 0.001). KRAS wild-type (WT) tumors were enriched in the IEF subtype (58%), while KRAS mutated tumors comprised all four transcriptomic subtypes. Primary PDACs that underwent radiotherapy were significantly more enriched in fibroblasts compared to samples from the TCGA cohort that did not undergo radiotherapy (means: 30%(MCW) vs. 20% (TCGA), p &lt; 0.001). Primary PDACs were enriched in the IEF subtype (46%), while liver and lung metastases were enriched in the ID (74%) and IE subtypes (70%), respectively. When pts were dichotomized to short (&lt; 400 days) versus long (&gt; 800 days) survivors, tumors from pts with longer survival demonstrated a trend towards enrichment in CD4/CD8 T cells and IE subtype that did not meet statistical significance. Conclusions: Lung metastases and KRAS WT PDACs harbor an immunogenic TME while liver metastases harbor an immune-cold TME, highlighting the biologic heterogeneity of PDAC. The efficacy of immunotherapeutic strategies in PDAC pts who demonstrate an IE/IEF transcriptomic subtype merits prospective evaluation. The four distinct subtypes identified by TME transcriptomic classification highlight the possibility of personalized immunotherapeutic strategies in PDAC.

Actin gamma 1 is a critical regulator of pancreatic ductal adenocarcinoma

Background:Pancreatic ductal adenocarcinoma (PDAC) accounts for about 90% of pancreatic cancers, which represents one of the most lethal malignancies with a 5-year overall survival less than 10%. Identifying molecular biomarkers is invaluable in helping to predict clinical outcomes and developing targeted chemotherapies. Actin gamma 1 (ACTG1) is a kind of actin isoform that exists in almost all cell types as a component of the cytoskeleton, thus mediating cell viability. Although there have been studies revealing the prognostic significance of ACTG1 in several malignancies such as glioblastoma and hepatocellular carcinoma, its involvement and function in pancreatic cancer needs to be elucidated.Methods:We retrospectively enrolled a cohort of PDAC patients after surgical resection (n = 149) and conducted immunohistochemistry experiments to explore the expression profile of ACTG1. Univariate and multivariate analyses were performed to investigate the clinical relevance of ACTG1. The functional role of ACTG1 in PDAC progression was further validated via both in vitro and in vivo studies.Results:ACTG1 presented a higher expression in PDAC tissues than in nontumorous pancreatic tissues. ACTG1 level positively correlated with tumor stage, implying its potential role as a tumor promoter. Univariate and multivariate analyses identified that patients with lower ACTG1 showed a better overall survival compared to those with higher ACTG1 expression. Cellular and xenograft experiments confirmed the role of ACTG1 on facilitating tumor proliferation both in vitro and in vivo.Conclusions:Our study revealed a pro-oncogenic role of ACTG1 in PDAC, which may help predict prognosis and serve as a novel therapeutic target.

Modulation of pancreatic cancer cell sensitivity to FOLFIRINOX through microRNA-mediated regulation of DNA damage

FOLFIRINOX, a combination of chemotherapy drugs (Fluorouracil, Oxaliplatin, Irinotecan -FOI), provides the best clinical benefit in pancreatic ductal adenocarcinoma (PDAC) patients. In this study we explore the role of miRNAs (MIR) as modulators of chemosensitivity to identify potential biomarkers of response. We find that 41 and 84 microRNA inhibitors enhance the sensitivity of Capan1 and MiaPaCa2 PDAC cells respectively. These include a MIR1307-inhibitor that we validate in further PDAC cell lines. Chemotherapy-induced apoptosis and DNA damage accumulation are higher in MIR1307 knock-out (MIR1307KO) versus control PDAC cells, while re-expression of MIR1307 in MIR1307KO cells rescues these effects. We identify binding of MIR1307 to CLIC5 mRNA through covalent ligation of endogenous Argonaute-bound RNAs cross-linking immunoprecipitation assay. We validate these findings in an in vivo model with MIR1307 disruption. In a pilot cohort of PDAC patients undergoing FOLFIRONX chemotherapy, circulating MIR1307 correlates with clinical outcome.

Multiplex Quantitative Analysis of Tumor-Infiltrating Lymphocytes, Cancer-Associated Fibroblasts, and CD200 in Pancreatic Cancer.

Simple SummaryPancreatic cancer is one of the most lethal types of cancer, and immunotherapy treatment options for these patients are limited by the characteristically “cold” tumor microenvironment. In this work, we analyze the expression levels and prognostic value of stromal tumor-infiltrating lymphocyte (CD4, CD8, and CD20) and cancer-associated fibroblast (Thy-1, FAP, and SMA) subpopulations in a cohort of pancreatic ductal adenocarcinoma patients. We additionally characterize the expression and prognostic value of CD200, a potential target for immune checkpoint blockade in these patients. CD8 and FAP were found to have prognostic significance for progression-free survival and overall survival after multivariate analysis. CD200 expression was heterogeneous in tumor and stromal cells and did not demonstrate prognostic value in this cohort. Our results point to CD8 and FAP as potential prognostic biomarkers and demonstrate the heterogeneous expression pattern of CD200 in patients with pancreatic ductal adenocarcinoma.Pancreatic cancer is marked by a desmoplastic tumor microenvironment and low tumor immunogenicity, making it difficult for immunotherapy drugs to improve outcomes for patients. Tumor-infiltrating lymphocytes (TILs) and cancer-associated fibroblasts (CAFs) are seen in the tumor microenvironment of patients with pancreatic ductal adenocarcinoma (PDAC). In this work, we sought to characterize the expression levels and potential prognostic value of TILs (CD4, CD8, and CD20) and CAFs (Thy-1, FAP, and SMA) in a large retrospective cohort of PDAC patients. Additionally, we investigated the expression levels and prognostic significance of CD200, an immunoinhibitory protein that has shown interest as a potential target for immune checkpoint blockade. We measured the expression levels of these seven proteins with multiplexed immunofluorescence staining and quantitative immunofluorescence (QIF). We found CD8 and FAP to be independent predictors of progression-free survival and overall survival. CD200 was found to be heterogeneously expressed in both the tumor and stromal compartments of PDAC, with the majority of patients having positive stromal expression and negative tumor expression. This work demonstrates the potential clinical utility of CD8 and FAP in PDAC patients, and it sheds light on the expression patterns of CD200 in pancreatic cancer as the protein is being tested as a target for immune checkpoint blockade.

A systematic review of the prevalence of germline pathogenic variants in patients with pancreatic cancer.

The genetics of pancreatic ductal adenocarcinoma (PDAC) is complex with patients reported to harbor germline pathogenic variants (PVs) in many different genes. PDAC patients with familial pancreatic cancer (FPC) are more likely to carry germline PVs but there is no consensus main gene involved in FPC. We performed a systematic review of publications from PubMed and Scopus reporting PVs in patients with FPC, sporadic pancreatic cancer (SPC) and unselected cohorts of PDAC patients undergoing genetic testing and calculated a cumulative prevalence of PVs for each gene evaluated across these three groups of patients. When available, variants in the selected publications were reclassified according to the American College of Medical Genetics and Genomics classification system and used for prevalence calculations if classified as pathogenic or likely pathogenic. We observed an increased prevalence of PVs in FPC compared to SPC or unselected PDAC patients for most of the 41 genes reported. The genes with the highest prevalence of carriers of PVs in FPC were ATM, BRCA2, and CDKN2A. BRCA2 and ATM showed the highest prevalence of PVs in both SPC and unselected PDAC cohorts. Several genes with the highest prevalence of PVs are involved in breast and ovarian cancer suggesting strong overlap with underlying genetics in these disorders but no single gene was predominant. More research is needed to further understand the risk of PDAC associated with these many diverse genes.

Abstract 2798: CD73 suppresses anti-cancer immunity and promotes glycolysis, resulting in poor prognosis in pancreatic ductal adenocarcinoma

Abstract CD73 is a cell surface enzyme involved in adenosine metabolism by converting extracellular adenosine-triphosphate to adenosine together with CD39. Accumulated extracellular adenosine is known to play pro-cancer role, by promoting angiogenesis, leading immune suppression, accelerating tumor growth, and metastasis in some types of malignancies. However, its role in pancreatic ductal adenocarcinoma (PDAC) remains unclear. In this study, we investigated clinical significance and possible roles of CD73 in PDAC by bioinformatical using publicly available cohorts (TCGA, E-MTAB, ICGC) and experimental approaches using murine pancreatic and human pancreatic cancer cells. First, we found that PDAC patients with high CD73 expression showed worse prognosis in all three PDAC patient cohorts (overall survival (OS) in TCGA; P=0.002, disease-free survival (DFS) in TCGA; P=0.012, E-MTAB; P&amp;lt;0.001, ICGC; P=0.002). In addition, infiltrated immune cell analysis by xCell algorism revealed lower CD8-positive T cell infiltration in CD73 high expressing PDACs in two cohorts (TCGA; P=0.009, E-MTAB; P&amp;lt;0.001). To confirm the role of CD73 in immune suppression in PDAC, we established Panc02 carcinomatosis mouse model using CD73 overexpressed cells. We found that the mice bearing CD73 overexpressed tumors showed shorter survival in immune competent mice, however, there was no survival difference in immune deficient mice. Consistent with patient samples, infiltrated CD8-positive T cells were significantly lower in CD73 overexpressed tumors compared to control tumors in immune competent mice (P=0.041). There were very weak to no correlations of CD73 expression with CD39 or adenosine receptors, including ADORA1, ADORA2A, ADORA2B, ADORA3 in all three cohorts. On the other hand, we found that CD73 high expressing PDACs were positively correlated with glycolytic pathway in all three patient cohorts (TCGA; P=0.018, E-MTAB; P=0.004, ICGC; P=0.039). Consistently, CD73 knockdown PANC1 cells showed lower baseline glycolysis (P=0.023) and glycolytic capacity (P=0.014) compared to control cells by Seahorse assay. CD73 expression was positively correlated with cancer stem cell markers, CD44 and cMET in all three cohorts. CD73 was also positively correlated with hypoxia-associated genes in patient samples, which is one of cancer stem cell features. In conclusion, we found that PDAC patients with CD73 high expressing tumors have worse prognosis. As a possible mechanism, we observed immune suppressive role of CD73 in PDAC similar to other cancers. By further elucidating CD73 transcriptional regulation in hypoxic condition and how glycolysis is connected to CD73 upregulation, we will demonstrate by experimental approaches. Citation Format: Eriko Katsuta, Mariko Asaoka, Tao Dai, Li Yan, Subhamoy Dasgupta, Kazuaki Takabe. CD73 suppresses anti-cancer immunity and promotes glycolysis, resulting in poor prognosis in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2798.

Pentraxin 3 is a stromally-derived biomarker for detection of pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC), characterized by dense desmoplastic stroma laid down by pancreatic stellate cells (PSC), has no reliable diagnostic biomarkers for timely detection. A multi-center cohort of PDAC patients and controls (chronic pancreatitis, intra-ductal papillary neoplasms, gallstones and otherwise healthy) donated serum in an ethically approved manner. Serum PTX3 above 4.34 ng/mL has a higher sensitivity (86%, 95% confidence interval (CI): 65–97%) and specificity (86%, 95% CI: 79–91%), positive predictive value (97%) and likelihood ratio (6.05), and is superior when compared to serum CA19-9 and CEA for detection of PDAC. In vitro and ex vivo analyses of PTX3, in human PDAC samples, PSCs, cell lines and transgenic mouse model for PDAC, suggest that PTX3 originates from stromal cells, mainly PSC. In activated PSC, PTX3 secretion could be downregulated by rendering PSC quiescent using all-trans-retinoic acid (ATRA). PTX3 organizes hyaluronan in conjunction with tumor necrosis factor-stimulated gene 6 (TSG-6) and facilitates stellate and cancer cell invasion. In SCALOP clinical trial (ISRCTN96169987) testing chemo-radiotherapy without stromal targeting, PTX3 had no prognostic or predictive role. However, in STARPAC clinical trial (NCT03307148), stromal modulation by ATRA even at first dose is accompanied with serum PTX3 response in patients who later go on to demonstrate disease control but not those in whom the disease progresses. PTX3 is a putative stromally-derived biomarker for PDAC which warrants further testing in prospective, larger, multi-center cohorts and within clinical trials targeting stroma.