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Altered RNA expression of repetitive sequences and retrotransposition are frequently seen in colorectal cancer, implicating a functional importance of repeat activity in cancer progression. We show the nucleoside reverse transcriptase inhibitor 3TC targets activities of these repeat elements in colorectal cancer preclinical models with a preferential effect in p53-mutant cell lines linked with direct binding of p53 to repeat elements. We translate these findings to a human phase II trial of single-agent 3TC treatment in metastatic colorectal cancer with demonstration of clinical benefit in 9 of 32 patients. Analysis of 3TC effects on colorectal cancer tumorspheres demonstrates accumulation of immunogenic RNA:DNA hybrids linked with induction of interferon response genes and DNA damage response. Epigenetic and DNA-damaging agents induce repeat RNAs and have enhanced cytotoxicity with 3TC. These findings identify a vulnerability in colorectal cancer by targeting the viral mimicry of repeat elements.Significance:Colorectal cancers express abundant repeat elements that have a viral-like life cycle that can be therapeutically targeted with nucleoside reverse transcriptase inhibitors (NRTI) commonly used for viral diseases. NRTIs induce DNA damage and interferon response that provide a new anticancer therapeutic strategy.This article is highlighted in the In This Issue feature, p. 1397

Supplementary Figure from Reverse Transcriptase Inhibition Disrupts Repeat Element Life Cycle in Colorectal Cancer

Supplementary Data from Reverse Transcriptase Inhibition Disrupts Repeat Element Life Cycle in Colorectal Cancer

Purpose:Patient-derived organoids (PDO) are a promising technology to support precision medicine initiatives for patients with pancreatic ductal adenocarcinoma (PDAC). PDOs may improve clinical next-generation sequencing (NGS) and enable rapid ex vivo chemotherapeutic screening (pharmacotyping).Experimental Design:PDOs were derived from tissues obtained during surgical resection and endoscopic biopsies and studied with NGS and pharmacotyping. PDO-specific pharmacotype is assessed prospectively as a predictive biomarker of clinical therapeutic response by leveraging data from a randomized controlled clinical trial.Results:Clinical sequencing pipelines often fail to detect PDAC-associated somatic mutations in surgical specimens that demonstrate a good pathologic response to previously administered chemotherapy. Sequencing the PDOs derived from these surgical specimens, after biomass expansion, improves the detection of somatic mutations and enables quantification of copy number variants. The detection of clinically relevant mutations and structural variants is improved following PDO biomass expansion. On clinical trial, PDOs were derived from biopsies of treatment-naïve patients prior to treatment with FOLFIRINOX (FFX). Ex vivo PDO pharmacotyping with FFX components predicted clinical therapeutic response in these patients with borderline resectable or locally advanced PDAC treated in a neoadjuvant or induction paradigm. PDO pharmacotypes suggesting sensitivity to FFX components were associated with longitudinal declines of tumor marker, carbohydrate-antigen 19–9 (CA-19–9), and favorable RECIST imaging response.Conclusions:PDOs established from tissues obtained from patients previously receiving cytotoxic chemotherapies can be accomplished in a clinically certified laboratory. Sequencing PDOs following biomass expansion improves clinical sequencing quality. High in vitro sensitivity to standard-of-care chemotherapeutics predicts good clinical response to systemic chemotherapy in PDAC.See related commentary by Zhang et al., p. 3176

Supplementary Figure from Precision Medicine in Pancreatic Cancer: Patient-Derived Organoid Pharmacotyping Is a Predictive Biomarker of Clinical Treatment Response

Supplementary Table from Precision Medicine in Pancreatic Cancer: Patient-Derived Organoid Pharmacotyping Is a Predictive Biomarker of Clinical Treatment Response

Supplementary Data from Precision Medicine in Pancreatic Cancer: Patient-Derived Organoid Pharmacotyping Is a Predictive Biomarker of Clinical Treatment Response

Altered RNA expression of repetitive sequences and retrotransposition are frequently seen in colorectal cancer, implicating a functional importance of repeat activity in cancer progression. We show the nucleoside reverse transcriptase inhibitor 3TC targets activities of these repeat elements in colorectal cancer preclinical models with a preferential effect in p53-mutant cell lines linked with direct binding of p53 to repeat elements. We translate these findings to a human phase II trial of single-agent 3TC treatment in metastatic colorectal cancer with demonstration of clinical benefit in 9 of 32 patients. Analysis of 3TC effects on colorectal cancer tumorspheres demonstrates accumulation of immunogenic RNA:DNA hybrids linked with induction of interferon response genes and DNA damage response. Epigenetic and DNA-damaging agents induce repeat RNAs and have enhanced cytotoxicity with 3TC. These findings identify a vulnerability in colorectal cancer by targeting the viral mimicry of repeat elements.Significance:Colorectal cancers express abundant repeat elements that have a viral-like life cycle that can be therapeutically targeted with nucleoside reverse transcriptase inhibitors (NRTI) commonly used for viral diseases. NRTIs induce DNA damage and interferon response that provide a new anticancer therapeutic strategy.This article is highlighted in the In This Issue feature, p. 1397

Overcoming intrinsic resistance to immune checkpoint blockade for microsatellite stable (MSS) colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) remains challenging. We conducted a single-arm, non-randomized, phase II trial (NCT03104439) combining radiation, ipilimumab and nivolumab to treat patients with metastatic MSS CRC (n = 40) and PDAC (n = 25) with an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. The primary endpoint was disease control rate (DCR) by intention to treat. DCRs were 25% for CRC (ten of 40; 95% confidence interval (CI), 13–41%) and 20% for PDAC (five of 25; 95% CI, 7–41%). In the per-protocol analysis, defined as receipt of radiation, DCR was 37% (ten of 27; 95% CI, 19–58%) in CRC and 29% (five of 17; 95% CI, 10–56%) in PDAC. Pretreatment biopsies revealed low tumor mutational burden for all samples but higher numbers of natural killer (NK) cells and expression of the HERVK repeat RNA in patients with disease control. This study provides proof of concept of combining radiation with immune checkpoint blockade in immunotherapy-resistant cancers. Ting and colleagues report the safety and efficacy of combined radiation and immunotherapy in a phase II trial on patients with MSS colorectal or pancreatic cancer and observe that disease control correlates with higher repetitive RNA transcription.

Purpose: Patient-derived organoids (PDO) are a promising technology to support precision medicine initiatives for patients with pancreatic ductal adenocarcinoma (PDAC). PDOs may improve clinical next-generation sequencing (NGS) and enable rapid ex vivo chemotherapeutic screening (pharmacotyping). Experimental Design: PDOs were derived from tissues obtained during surgical resection and endoscopic biopsies and studied with NGS and pharmacotyping. PDO-specific pharmacotype is assessed prospectively as a predictive biomarker of clinical therapeutic response by leveraging data from a randomized controlled clinical trial. Results: Clinical sequencing pipelines often fail to detect PDAC-associated somatic mutations in surgical specimens that demonstrate a good pathologic response to previously administered chemotherapy. Sequencing the PDOs derived from these surgical specimens, after biomass expansion, improves the detection of somatic mutations and enables quantification of copy number variants. The detection of clinically relevant mutations and structural variants is improved following PDO biomass expansion. On clinical trial, PDOs were derived from biopsies of treatment-naïve patients prior to treatment with FOLFIRINOX (FFX). Ex vivo PDO pharmacotyping with FFX components predicted clinical therapeutic response in these patients with borderline resectable or locally advanced PDAC treated in a neoadjuvant or induction paradigm. PDO pharmacotypes suggesting sensitivity to FFX components were associated with longitudinal declines of tumor marker, carbohydrate-antigen 19–9 (CA-19–9), and favorable RECIST imaging response. Conclusions: PDOs established from tissues obtained from patients previously receiving cytotoxic chemotherapies can be accomplished in a clinically certified laboratory. Sequencing PDOs following biomass expansion improves clinical sequencing quality. High in vitro sensitivity to standard-of-care chemotherapeutics predicts good clinical response to systemic chemotherapy in PDAC. See related commentary by Zhang et al., p. 3176

Purpose: Oncogenic Kirsten rat sarcoma virus (KRAS) mutations are the most prevalent cancer mutations in all human tumors. Also, genotyping the KRAS gene has become increasingly important with the emergence of new evidence highlighting differences in downstream signaling pathways, tumor microenvironment composition, treatment responses and prognoses linked to specific single nucleotide polymorphisms (SNPs) in the G12 or G13 codons. FDA approval of G12C-specific anti-KRAS drugs highlights the importance of the development of reliable SNP assays to interrogate the mutation status in a tumor sample. Currently available genotyping assays are based on NGS, PCR, or ddPCR. These methods can be time-consuming, costly, suffer from amplification bias, or require follow-up testing or bioinformatics skills to analyze. Building on Meso Scale Discovery’s (MSD) existing technology, we aimed to develop a method for the identification of eight KRAS SNPs located on the G12-G13 codons in a single reaction. Methods: Ten-spot, 96-well N-PLEX plates were used for the study. SNP-specific upstream probes carrying unique 5′ leader sequences complementary to spot-specific captures on the N-PLEX plates were designed for eight KRAS genotypes (WT, G12R, G12C, G12S, G12A, G12D, G12V, G13D). Locus-specific downstream probes were 5’ phosphorylated for ligation and 3’ biotinylated for detection. Synthetic DNA targets were used as assay calibrators, and commercially available FFPE reference samples were tested for validation. DNA (10 ng) was PCR amplified with primers flanking the KRAS mutation sites. A multiplexed mixture of upstream and downstream probes was hybridized to the amplicons, and 30 cycles of ligation was performed using DNA ligase in a thermal cycler. Samples were subsequently hybridized to spot-specific capture oligonucleotides on the surface of the assay plates and detected with SULFO-TAG labeled streptavidin. Electrochemiluminescence readout was collected on an MSD imager. Results: Optimization of probe concentrations and ligation temperature was performed to maximize signal-to-background ratios and assay specificity. Spike recovery experiments using artificial target DNA showed successful identification of KRAS mutant genotypes at spike levels as low as 0.1% over wild-type background. Validation experiments with gDNA extracted from FFPE reference samples confirmed that the multiplex SNP assay can accurately differentiate eight KRAS genotypes in samples with >0.4% tumor burden. Conclusion: These data highlight a novel approach to simultaneously identify eight KRAS genotypes from 10 ng of DNA input in a single reaction within 4-5 hours. The assay is capable of identifying double mutants and can provide a semiquantitative assessment of tumor burden without the need for follow-up testing. Citation Format: Annamaria Szabolcs, Timothy J. Break, Isaac H. Shin, Seth B. Harkins, Jacob N. Wohlstadter. Multiplex detection of G12/G13 KRAS mutations with an electrochemiluminescent hybridization assay. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4351.

Blood-borne metastasis to the brain is a major complication of breast cancer, but cellular pathways that enable cancer cells to selectively grow in the brain microenvironment are poorly understood. We find that cultured circulating tumor cells (CTCs), derived from blood samples of women with advanced breast cancer and directly inoculated into the mouse frontal lobe, exhibit striking differences in proliferative potential in the brain. Derivative cell lines generated by serial intracranial injections acquire selectively increased proliferative competency in the brain, with reduced orthotopic tumor growth. Increased Hypoxia Inducible Factor 1A (HIF1A)-associated signaling correlates with enhanced proliferation in the brain, and shRNA-mediated suppression of HIF1A or drug inhibition of HIF-associated glycolytic pathways selectively impairs brain tumor growth while minimally impacting mammary tumor growth. In clinical specimens, brain metastases have elevated HIF1A protein expression, compared with matched primary breast tumors, and in patients with brain metastases, hypoxic signaling within CTCs predicts decreased overall survival. The selective activation of hypoxic signaling by metastatic breast cancer in the brain may have therapeutic implications., The mechanisms underlying the growth of breast cancer metastasis in the brain are unclear. Here, the authors use an intracranial injection mouse model and single-cell analysis of patient circulating tumour cells to demonstrate that increased hypoxic and HIF1A signalling promotes tumour growth in the brain.

Coronavirus disease-19 (COVID-19) is caused by a newly discovered coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although SARS-CoV-2 is visualized on electron microscopy, there is an increasing demand for widely applicable techniques to visualize viral components within tissue specimens. Viral protein and RNA can be detected on formalin-fixed paraffin-embedded (FFPE) tissue using immunohistochemistry (IHC) and in situ hybridization (ISH), respectively. Herein, we evaluate the staining performance of ISH for SARS-CoV-2 and an IHC directed at the SARS-CoV nucleocapsid protein and compare these results to a gold standard, tissue quantitative real-time polymerase chain reaction (qRT-PCR). We evaluated FFPE sections from 8 COVID-19 autopsies, including 19 pulmonary and 39 extrapulmonary samples including the heart, liver, kidney, small intestine, skin, adipose tissue, and bone marrow. We performed RNA-ISH for SARS-CoV-2 on all cases with IHC for SARS-CoV and SARS-CoV-2 qRT-PCR performed on selected cases. Lungs from 37 autopsies performed before the COVID-19 pandemic served as negative controls. The ISH and IHC slides were reviewed by 4 observers to record a consensus opinion. Selected ISH and IHC slides were also reviewed by 4 independent observers. Evidence of SARS-CoV-2 was identified on both the IHC and ISH platforms. Within the postmortem lung, detected viral protein and RNA were often extracellular, predominantly within hyaline membranes in patients with diffuse alveolar damage. Among individual cases, there was regional variation in the amount of detectable virus in lung samples. Intracellular viral RNA and protein was localized to pneumocytes and immune cells. Viral RNA was detected on RNA-ISH in 13 of 19 (68%) pulmonary FFPE blocks from patients with COVID-19. Viral protein was detected on IHC in 8 of 9 (88%) pulmonary FFPE blocks from patients with COVID-19, although in 5 cases the stain was interpreted as equivocal. From the control cohort, FFPE blocks from all 37 patients were negative for SARS-CoV-2 RNA-ISH, whereas 5 of 13 cases were positive on IHC. Collectively, when compared with qRT-PCR on individual tissue blocks, the sensitivity and specificity for ISH was 86.7% and 100%, respectively, while those for IHC were 85.7% and 53.3%, respectively. The interobserver variability for ISH ranged from moderate to almost perfect, whereas that for IHC ranged from slight to moderate. All extrapulmonary samples from COVID-19-positive cases were negative for SARS-CoV-2 by ISH, IHC, and qRT-PCR. SARS-CoV-2 is detectable on both RNA-ISH and nucleocapsid IHC. In the lung, viral RNA and nucleocapsid protein is predominantly extracellular and within hyaline membranes in some cases, while intracellular locations are more prominent in others. The intracellular virus is detected within pneumocytes, bronchial epithelial cells, and possibly immune cells. The ISH platform is more specific, easier to analyze and the interpretation is associated with the improved interobserver agreement. ISH, IHC, and qRT-PCR failed to detect the virus in the heart, liver, and kidney.

Pancreatic ductal adenocarcinoma (PDAC) lethality is due to metastatic dissemination. Characterization of rare, heterogeneous circulating tumor cells (CTCs) can provide insight into metastasis and guide development of novel therapies. Using the CTC-iChip to purify CTCs from PDAC patients for RNA-seq characterization, we identify three major correlated gene sets, with stemness genes LIN28B/KLF4, WNT5A, and LGALS3 enriched in each correlated gene set; only LIN28B CTC expression was prognostic. CRISPR knockout of LIN28B—an oncofetal RNA-binding protein exerting diverse effects via negative regulation of let-7 miRNAs and other RNA targets—in cell and animal models confers a less aggressive/metastatic phenotype. This correlates with de-repression of let-7 miRNAs and is mimicked by silencing of downstream let-7 target HMGA2 or chemical inhibition of LIN28B/let-7 binding. Molecular characterization of CTCs provides a unique opportunity to correlated gene set metastatic profiles, identify drivers of dissemination, and develop therapies targeting the “seeds” of metastasis., Metastatic dissemination contributes to the lethality in pancreatic ductal adenocarcinoma (PDAC). Here, the authors perform RNA-sequencing on patient derived circulating tumor cells (CTCs) and identify three major CTC subgroups, and show the therapeutic potential of targeting LIN28B/let-7 pathway to halt cancer metastasis.

Background: Alpelisib, a PI3Kα inhibitor, was recently approved by the FDA for the treatment of metastatic PIK3CA-mutated HR+ breast cancer in combination with fulvestrant. This therapeutic breakthrough came after years of disappointing efforts to develop solid tumor drugs targeting PIK3CA, one of the most commonly altered oncogenes in cancer, including 40% of HR+ breast and 30% of head and neck squamous cell cancer. The limited success of PI3Kinhibitors outside of breast cancer may be partly because cancers may harbor heterogeneous PIK3CA mutations and/or co-occurring mutations that confer varying degrees of sensitivity to PI3Kα inhibition. Methods: To identify biomarkers of sensitivity and resistance to PI3Kinhibitors, we assessed all plasma samples sent to a CLIA-certified commercial laboratory (Guardant Health) for a targeted NGS assay (Guardant360) from November 2016 to March 2019. The assay included 73 genes for single-nucleotide variants, 23 genes for insertions / deletions, 18 genes for amplifications, and 6 genes for fusions. In total, 53,118 cancer patients were sequenced, of whom 8,464 patients (15.9%) had a PIK3CA point mutation or amplification. This PIK3CA-mutated subset served as our analysis cohort. Results: The four most common tumor types in our PIK3CA-mutated cohort were breast (n=2060), non-small cell lung (n=2667), colorectal (n=999), and prostate cancer (n=713); head and neck cancer patients (n=129) were also included given the high frequency of PIK3CA mutations in this disease. Breast cancer patients had a median of 6 plasma mutations detected (range 1-92) and median maximum variant allele frequency (VAF) of 7.6% (range 0.05-96.5%). PIK3CA mutations were clonal in 68.2% of breast cancer patients (as defined by a VAF>50% of the maximum VAF), compared to 18-44% in patients of other tumor types (p Conclusions: PIK3CA-mutated breast cancer patients were more likely to have clonal PIK3CA mutations and multiple PIK3CA and PI3K pathway mutations compared to patients with other tumor types, which may suggest greater PI3Kpathway dependency and sensitivity to treatment with PI3Kα inhibitors. Ongoing whole exome and RNA sequencing of 21 pairs of pre- and post-treatment tissue biopsies of breast cancer patients treated with PI3Kα inhibitors will provide additional genomic and transcriptomic context to these plasma-based results. Breast (n=2060)NSCLC (n=2667)Colorectal (n=999)Prostate (n=713)Head and neck (n=129)Median age (range)60 (25-101)68 (25-97)62 (17-94)71 (39-97)60 (21-88)% male0%49%56%100%72.7%Median # mutations (range)6 (1-92)6 (1-51)8 (1-207)7 (1-76)6 (1-66)Median max VAF (range)7.6% (0.05-96.5)9.2% (0.04-96.1)17.7% (0.08-90.1)12.2% (0.01-94.5)3.7% (0.08-84.5)% PIK3CA clonal mutation a68.2% (1404)27.1% (724)44.1% (441)18.2% (130)40.3% (52)5 most frequent co-mutated genes, among patients with clonal PIK3CA mutationsTP53 (50.7%)TP53 (66.3%)APC (81.0%)AR (48.5%)TP53 (36.6%)ESR1 (31.6%)EGFR (34.9%)TP53 (70.5%)TP53 (40%)ARID1A (25%)EGFR (24.7%)KRAS (21.7%) bKRAS (67.3%) bEGFR (21.5%)NF1 (21.2%)ERBB2 (20.8%)NF1 (18.0%)EGFR (31.1%)MYC (19.2%)EGFR (21.2%)FGFR1 (18.7%)ARID1A(15.0%)BRAF (23.4%)CDK6 (18.5%)TERT (17.3%)5 most frequent co-mutated genes, among patients with subclonal PIK3CA mutationsTP53 (60.7%)TP53 (80.6%)APC (81.6%)TP53 (63.9%)TP53 (60%)ESR1 (28.1%)EGFR (43.3%)TP53 (76.2%)AR (63.0%)NOTCH1 (36.7%)ERBB2 (24.7%)KRAS (24.0%)KRAS (64.7%)EGFR (30.3%)EGFR 33.3%)NF1 (24.0%)NF1 (18.3%)EGFR (41.3%)BRAF (26.9%)MTOR (26.7%)EGFR (23.0%)MET (18.3%)BRAF (32.0%)APC (26.0%)ERBB2 (26.7%)>1 PIK3CA mutation c 29.7% (612)14.2% (378)17.3% (173)10.8% (77)31.0% (40)>1 PI3K pathway mutation c38.2% (787)21.0% (561)26.6% (266)20.9% (149)36.4% (47)% helical mutations36.8% (1079)16.1% (504)19.9% (245)10.7% (87)12.9% (25)% kinase mutations a15.1% (444)16.0% (501)24.1% (296)9.5% (77)16.0% (31)% other point mutations15.1% (444)16.0% (501)24.1% (296)9.5% (77)16.0% (31)% amplifications18.4% (538)37.7% (1182)14.6% (180)64.9% (526)37.6% (73)a p Citation Format: Christopher Chen, Avinash Sahu, Kristin Price, Christopher Pinto, Read Allen, Xiaoman Wang, Annamaria Szabolcs, Lipika Goyal, David Ting, Shirley Liu, Dejan Juric. Plasma sequencing demonstrates that breast cancer patients have a higher prevalence of clonal and multiple PIK3CA mutations than other solid tumor patients [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-10-34.

Transcriptional profiling has defined pancreatic ductal adenocarcinoma (PDAC) into distinct subtypes with the majority being classical epithelial (E) or quasi-mesenchymal (QM). Despite clear differences in clinical behavior, growing evidence indicates these subtypes exist on a continuum with features of both subtypes present and suggestive of interconverting cell states. Here, we investigated the impact of different therapies being evaluated in PDAC on the phenotypic spectrum of the E/QM state. We demonstrate using RNA-sequencing and RNA-in situ hybridization (RNA-ISH) that FOLFIRINOX combination chemotherapy induces a common shift of both E and QM PDAC toward a more QM state in cell lines and patient tumors. In contrast, Vitamin D, another drug under clinical investigation in PDAC, induces distinct transcriptional responses in each PDAC subtype, with augmentation of the baseline E and QM state. Importantly, this translates to functional changes that increase metastatic propensity in QM PDAC, but decrease dissemination in E PDAC in vivo models. These data exemplify the importance of both the initial E/QM subtype and the plasticity of E/QM states in PDAC in influencing response to therapy, which highlights their relevance in guiding clinical trials.

Immune checkpoint blockade has limited efficacy in microsatellite stable (MSS) colorectal (CRC) and pancreatic (PDAC) cancer. Preclinical models have demonstrated the use of radiation to activate the innate immune response and stimulate responsiveness to immune checkpoint blockade. Here, we describe a Phase 2 trial of radiation therapy combined with combined anti-CTLA4 (ipilimumab) and anti-PD1 (nivolumab) antibodies in MSS CRC and PDAC. In the per protocol analysis disease control rate was 37% (10/27) in CRC and 29% (5/17) in PDAC with an overall response rate of 15% (4/27) and 18% (3/17), respectively. Whole exome and RNA sequencing of biopsies from 17 patients revealed low tumor mutational burden in all tumors, but a notable upregulation of interferon stimulated genes with concordant high expression of multiple repeat RNA transcripts in responders. Altogether, this study provides foundational human proof of concept of radiation with combination immune checkpoint blockade therapy in otherwise immunotherapy resistant cancers.

Overcoming intrinsic resistance to immune checkpoint blockade for microsatellite stable (MSS) colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) remains challenging. We conducted a single-arm, non-randomized, phase II trial (NCT03104439) combining radiation, ipilimumab and nivolumab to treat patients with metastatic MSS CRC (n = 40) and PDAC (n = 25) with an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. The primary endpoint was disease control rate (DCR) by intention to treat. DCRs were 25% for CRC (ten of 40; 95% confidence interval (CI), 13-41%) and 20% for PDAC (five of 25; 95% CI, 7-41%). In the per-protocol analysis, defined as receipt of radiation, DCR was 37% (ten of 27; 95% CI, 19-58%) in CRC and 29% (five of 17; 95% CI, 10-56%) in PDAC. Pretreatment biopsies revealed low tumor mutational burden for all samples but higher numbers of natural killer (NK) cells and expression of the HERVK repeat RNA in patients with disease control. This study provides proof of concept of combining radiation with immune checkpoint blockade in immunotherapy-resistant cancers.

OBJECTIVE: Patients with pancreatic cancer (PDAC) who undergo surgical resection and receive effective chemotherapy have the best chance of long-term survival. Unfortunately, we lack predictive biomarkers to guide optimal systemic treatment. Ex-vivo generation of patient-derived organoids (PDO) for pharmacotyping may serve as predictive biomarkers in PDAC. The goal of the current study was to demonstrate the clinical feasibility of a PDO-guided precision medicine framework of care. METHODS: PDO cultures were established from surgical specimens and endoscopic biopsies, expanded in Matrigel, and used for high-throughput drug testing (pharmacotyping). Efficacy of standard-of-care chemotherapeutics was assessed by measuring cell viability after drug exposure. RESULTS: A framework for rapid pharmacotyping of PDOs was established across a multi-institutional consortium of academic medical centers. Specimens obtained remotely and shipped to a central biorepository maintain viability and allowed generation of PDOs with 77% success. Early cultures maintain the clonal heterogeneity seen in PDAC with similar phenotypes (cystic–solid). Late cultures exhibit a dominant clone with a pharmacotyping profile similar to early passages. The biomass required for accurate pharmacotyping can be minimized by leveraging a high-throughput technology. Twenty-nine cultures were pharmacotyped to derive a population distribution of chemotherapeutic sensitivity at our center. Pharmacotyping rapidly-expanded PDOs was completed in a median of 48 (range 18–102) days. CONCLUSIONS: Rapid development of PDOs from patients undergoing surgery for PDAC is eminently feasible within the perioperative recovery period, enabling the potential for pharmacotyping to guide post-operative adjuvant chemotherapeutic selection. Studies validating PDOs as a promising predictive biomarker are ongoing.

Metastasis: A matter of translation? Solid tumors shed a small number of cancer cells into the bloodstream, some of which are believed to contribute to metastasis. The molecular features that confer these circulating tumor cells (CTCs) with metastatic potential are poorly understood. Ebright et al. studied CTCs from breast cancer patients and found that cells with increased expression levels of certain ribosomal proteins and regulators of translation had greater metastatic capacity in a mouse model (see the Perspective by Ma and Jeffrey). Consistent with this finding, patients with higher levels of this subset of CTCs tended to have a poorer prognosis. Science , this issue p. 1468 ; see also p. 1424

Women with metastatic hormone receptor positive (HR+) breast cancer benefit from therapies targeting the Estrogen Receptor (ER), but the tumors eventually acquire resistance leading to disease progression. Endocrine resistance involves multiple mechanisms, including deregulation of ER signaling due to activating mutations in the ligand-binding domain of the ESR1 gene. In addition, recent reports describe specific contexts of ESR1 mutations in the preclinical setting where tamoxifen and fulvestrant have demonstrated limited activity. These data together with the frequency of ESR1 mutations (~20-60%) observed in mBC patients in the clinic suggest a need for more effective agents. The activity of elacestrant (RAD1901), an oral selective estrogen receptor degrader (SERD) with demonstrated activity against tumors harboring ESR1 mutations, both in preclinical models and in patients with previously treated advanced breast cancer, was assessed in ex vivo cultures of circulating tumor cells (CTCs) derived from women with metastatic HR+ breast cancer who had received multiple prior hormone therapies. Cultured breast CTCs underwent apoptosis following exposure to elacestrant, and CTCs harboring mutant ESR1 were significantly growth inhibited (IC50: 17.65 ± 3.32 nM) by elacestrant compared to the standard-of-care (SOC) agent fulvestrant. Furthermore, elacestrant eliminated the fulvestrant-resistant CTC populations in vitro. Elacestrant treatment led to significant suppression of ER target genes, PGR and GREB1. Elacestrant is currently being investigated versus standard of care endocrine monotherapy in a Phase 3 clinical trial (EMERALD) in patients with ER+/HER2- advanced/metastatic breast cancer. A co-primary endpoint of the EMERALD trial is evaluation of progression-free survival in patients whose tumors harbor ESR1 mutations. Citation Format: Taronish Dorab Dubash, Aditya Bardia, Brittany A Reeves, Brian Chirn, Annamaria Szabolcs, Ben S Wittner, John Iafrate, David Ting, Hitisha K Patel, Teeru Bihani, Mehmet Toner, Daniel A Haber, Shyamala Maheswaran. Elacestrant (RAD1901) inhibits growth of ex vivo cultured circulating tumor cells derived from hormone receptor-positive metastatic breast cancer (mBC) patients including those harboring ESR1 mutations [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-01-06.

149 Background: Non-coding repeat RNAs in cancers are pervasive and “mimic” viruses with activation of pattern recognition receptors and the innate immune response. Many repeat RNAs replicate in cancer genomes through a reverse transcriptional intermediate analogous to retroviruses. Nucleoside reverse transcriptase inhibitors (NRTIs) block this retroviral life cycle to increases repeat RNAs in p53 mutant colon cancer cell cancer lines. We initiated a Phase 2 study of lamivudine (3TC) in TP53 mutant mCRC. Methods: Two-stage design with target accrual of 20 patients (pts) in stage 1 and total of 32. Eligibility: pts with p53 mutant refractory mCRC with progression on or intolerance to 5FU, oxaliplatin and irinotecan and anti-EGFR if RAS WT. RNA sequencing was performed on pre-treatment (tx) and on tx biopsy to evaluate for repeat RNA expression and expression of other genes linked to 3TC response/resistance. Radiation was allowed. 9 pts were treated with 3TC 150 mg po bid for 28-day cycles, the maximum FDA approved dose of 3TC in HIV. Subsequent pts were treated at 600 mg po bid, previously tested in P1 trials. Tumor assessments were performed every 8 weeks until documented disease progression by RECIST 1.1 criteria or drug intolerance. Results: 29/32 pts have been treated. Median age: 60 yrs. (27-82) 18 males, 11 females. 2/ 9 (22%) pts on standard 3TC dosing had stable disease (SD) on single agent 3TC with a duration of tx of 169 and 167 days, respectively. Both pts had an initial drop in CEA upon initiation of 3TC. Of the next 20 pts on high dose 3TC, 19 were evaluable. 4 had SD, for 110, 159, 130 and 228+ days. 14 pts had tx-related adverse events (TRAE). 1 pt with a definite Grade 1 TRAE (fatigue). No pts with Grade ≥3 TRAEs. We obtained pre-tx fresh frozen biopsies on 24/29 pts. Of those with SD, 4 had biopsies and differential expression identified significantly higher HSATII repeat RNA in pts with SD compared to PD. There was an association of decreased epigenetic gene expression in HSATII repeat RNA high tumors. Conclusions: This proof-of-concept study demonstrates the safety and activity of single-agent 3TC. Repeat RNA levels appear to correlate with clinical benefit and can be measured in biopsies. Further combination studies and correlatives are planned. Clinical trial information: NCT03144804.

Significance PDAC epithelial (E) and quasi-mesenchymal (QM) subtypes can be modulated by different therapies demonstrating the plasticity of PDAC cells that contributes to the heterogeneity of PDAC tumors and their intrinsic resistance to a broad spectrum of therapies. Understanding and monitoring the fluidity of PDAC E and QM states are critical to the development of improved clinical trial design to target these different subpopulations., Transcriptional profiling has defined pancreatic ductal adenocarcinoma (PDAC) into distinct subtypes with the majority being classical epithelial (E) or quasi-mesenchymal (QM). Despite clear differences in clinical behavior, growing evidence indicates these subtypes exist on a continuum with features of both subtypes present and suggestive of interconverting cell states. Here, we investigated the impact of different therapies being evaluated in PDAC on the phenotypic spectrum of the E/QM state. We demonstrate using RNA-sequencing and RNA-in situ hybridization (RNA-ISH) that FOLFIRINOX combination chemotherapy induces a common shift of both E and QM PDAC toward a more QM state in cell lines and patient tumors. In contrast, Vitamin D, another drug under clinical investigation in PDAC, induces distinct transcriptional responses in each PDAC subtype, with augmentation of the baseline E and QM state. Importantly, this translates to functional changes that increase metastatic propensity in QM PDAC, but decrease dissemination in E PDAC in vivo models. These data exemplify the importance of both the initial E/QM subtype and the plasticity of E/QM states in PDAC in influencing response to therapy, which highlights their relevance in guiding clinical trials.

The proteosome inhibitor bortezomib is used in the treatment of patients with multiple myeloma. Proteosomes are responsible for the degradation of I-kappaB, the inhibitory protein of transcription factor nuclear factor kappa B (Nf-kappaB). The heat shock protein (HSP) inducing effect of bortezomib is also documented. The aim of our work was to test the anti-inflammatory effect of bortezomib in cholecystokinin-octapeptide (CCK-8)-induced acute pancreatitis. Male Wistar rats were divided into three groups (n=8 in each). Group P received an i.p. injection of physiological saline (p.s.) 60 min. before the induction of acute pancreatitis by three hourly s.c. injections of 100 microg/kg CCK-8. Group BP received 1 mg/kg bortezomib dissolved in p.s. 1 h previous to pancreatitis induction. Group C was treated with the vehicle (p.s.). Animals were exsanguinated 4 h after the last injection of CCK-8. Bortezomib pre-treatment significantly reduced the pancreatic weight/body weight ratio, and improved the histology by decreasing the extent of vacuolization and infiltration. Bortezomib pre-treatment inhibited I-kappaBbeta degradation, and induced the synthesis of HSP72. The results confirmed the anti-inflammatory effect of bortezomib in acute experimental pancreatitis. This effect of the drug is presumably mediated by the inhibition of Nf-kappaB activation and induction of HSP synthesis.

High-mobility group box protein 1 (HMGB1), a late-acting proinflammatory cytokine, is secreted actively by inflammatory cells, and released passively from necrotic cells. From the aspect that both inflammation and necrosis are involved in the pathogenesis in acute pancreatitis, the aim of the study was a joint investigation of the plasma concentrations of HMGB1, its soluble receptor for advanced glycation end-products (sRAGE), and the circulating DNA as a marker of cell death.62 patients with acute pancreatitis (30 mild, 32 severe), 20 patients with sepsis, and 20 healthy controls were enrolled in the study. HMGB1 and sRAGE plasma levels were measured by means of ELISA. Plasma DNA concentrations were estimated by real-time quantitative PCR for the beta-globin gene.The circulating HMGB1 level was significantly higher in patients with severe acute pancreatitis (13.33 +/- 2.11 ng/ml) than in healthy controls (0.161 +/- 0.03 ng/ml) or than in patients with mild pancreatitis (2.64 +/- 0.185 ng/ml). The plasma concentration of sRAGE was highest in patients with sepsis (2,210 +/- 252 pg/ml), while the levels of sRAGE correlated inversely with that of HMGB1 in patients with acute pancreatitis. The plasma DNA level was significantly elevated in patients with severe acute pancreatitis (2,206 +/- 452 ng/ml).A complex study of the plasma levels of HMGB1, sRAGE and circulating DNA can be informative in evaluations of acute pancreatitis with different levels of severity.

Epidemiological data analysis of a tertiary (regional) medical and surgical center. Diagnostic and therapeutic standards of patients with acute pancreatitis have changed significantly in the last few decades. Progress in laboratory and imaging diagnostics and achievements in experimental research resulted in a significant modification of the guidelines related to the care of pancreatitic patients. The aim was to analyse and compare the data of patients with acute pancreatitis treated in 1996 (period I) and 2004 (period II) at the Departments of Internal Medicine and Surgery, University of Szeged, to evaluate the concordance with international guidelines during medical and surgical treatment. Results: The authors analysed the clinical data of 126 and 124 patients, respectively, with acute pancreatitis observed during the two periods. An increase in the incidence of biliary acute pancreatitis, more frequent use of antibiotics, a higher frequency of therapeutic endoscopies (papillotomy and biliary stone extraction), the general application of ultrasonography-guided fine needle aspiration and bacterial culturing in cases of suspected infected necrosis, and higher effectiveness in complex surgical and supportive management of infected necrosis cases were detected in period II. Conclusion: Although most of the achievements suggested in international guidelines on medical/endoscopic and surgical treatment of acute pancreatitis have been implemented during the observation period, no significant changes in the morbidity and mortality data of patients were found.

The aim of the present study was to investigate whether hyperlipidemia can cause acute pancreatitis or alter its severity. Male Wistar rats were fed a 3% cholesterol-enriched diet or a normal diet for 16 weeks. Edematous and necrotizing pancreatitis was induced with 3x75 mug/kg body weight of cholecystokinin s.c. and 2x2 g/kg body weight of L-arginine i.p., respectively, in separate groups of normal and hyperlipidemic rats. The severity of the pancreatitis was assessed. We studied the influence of hyperlipidemia on the formation of oxygen-derived free radicals, endogenous scavengers, nitric oxide synthases (NOS), peroxynitrite (ONOO(-)), heat shock protein 72 (HSP72) and nuclear factor-kappa B (NF-kappaB) activation in the pancreas during acute edematous and necrotizing pancreatitis. Hyperlipidemia did not worsen edematous, but aggravated necrotizing pancreatitis. The cholesterol-enriched diet significantly reduced the catalase and Mn-superoxide dismutase (SOD) and constitutive NOS (cNOS) activities and increased the inducible NOS (iNOS) in the pancreas relative to those in the rats on the normal diet. The pancreatic nitrotyrosine level, as a marker of ONOO(-), and the NF-kappaB DNA-binding activity in the pancreas, were significantly elevated in the cholesterol-fed rats. The pancreatic HSP72 expression during necrotizing pancreatitis was not influenced by the hyperlipidemia. The pancreatic Mn-SOD, Cu, Zn-SOD, glutathione peroxidase, total glutathione and cNOS activities were significantly reduced, while the catalase, iNOS and NF-kappaB DNA-binding activities were significantly increased in the animals with necrotizing pancreatitis on the cholesterol diet as compared with those with pancreatitis and receiving the normal diet. Hyperlipidemia induced with this cholesterol-enriched diet leads to decreases in endogenous scavenger and cNOS activities, results in iNOS and NF-kappaB activation and stimulates ONOO(-) generation in the pancreas, which may be responsible for the aggravation of acute necrotizing pancreatitis.

OBJECTIVE Our experiments were designed to investigate the effects of zerumbone pretreatment on cholecystokinin octapeptide (CCK-8)-induced acute pancreatitis in rats. METHODS Male Wistar rats weighing 240 to 280 g were divided into a control group, a group treated with CCK-8, a group receiving 20 mg/kg zerumbone before CCK-8 administration, and a group treated with zerumbone only. RESULTS The serum amylase and lipase activities and the pancreatic weight-body weight ratio were significantly reduced by zerumbone pretreatment, but the drug failed to influence the histological parameters of pancreatitis. The anti-inflammatory effects of the drug were manifested in decreases in the cytosolic interleukin 6 and tumor necrosis factor alpha concentrations and an elevation in the I-kappaB concentration, whereas the antioxidant ability of zerumbone was demonstrated by reductions in inducible nitric oxide synthase, Mn- and Cu/Zn-superoxide dismutase activities in the zerumbone-treated rats. CONCLUSION Zerumbone ameliorated the changes of several parameters of acute pancreatitis probably by interfering with I-kappaB degradation, but in the applied dose, it failed to influence the histology of the disease.

Penetratin is a cationic cell-penetrating peptide that has been frequently used for the intracellular delivery of polar bioactive compounds. Recent studies have just revealed the major role of polyanionic membrane proteoglycans and cholesterol-enriched lipid rafts in the uptake of the peptide. Both proteoglycans and lipid-rafts influence inflammatory processes by binding a wide array of proinflammatory mediators; thus, we decided to analyze the effect of penetratin on in vitro and in vivo inflammatory responses. Our in vitro luciferase gene assays demonstrated that penetratin decreased transcriptional activity of nuclear factor-kappaB (NF-kappaB) in tumor necrosis factor (TNF)-stimulated L929 fibroblasts and lipopolysaccharide-activated RAW 264.7 macrophages. Penetratin also inhibited TNF-induced intercellular adhesion molecule-1 expression in human endothelial HMEC-1 cells. Exogenous heparan sulfate abolished the in vitro NF-kappaB inhibitory effects of the peptide. Uptake experiments showed that penetratin was internalized by all of the above-mentioned cell lines in vitro and rapidly entered the cells of the lung and pancreas in vivo. In an in vivo rat model of acute pancreatitis, a disease induced by elevated activities of stress-responsive transcription factors like NF-kappaB, pretreatment with only 2 mg/kg penetratin attenuated the severity of pancreatic inflammation by interfering with IkappaB degradation and subsequent nuclear import of NF-kappaB, inhibiting the expression of proinflammatory genes and improving the monitored laboratory and histological parameters of pancreatitis and associated oxidative stress.

Resveratrol is a phytoalexin with strong antioxidant and anti-inflammatory effects reaching high concentrations in red wine. The aim of our study was to test the effects of resveratrol pretreatment on cholecystokinin-octapeptide (CCK-8)-induced acute pancreatitis in rats. Animals were divided into a control group, a group treated with CCK-8 and a group receiving 10 mg/kg resveratrol prior to CCK-8 administration. Resveratrol ameliorated the CCK-8-induced changes in the laboratory parameters, and reduced the histological damage in the pancreas. The drug failed to improve the pancreatic antioxidant state, but increased the amount of hepatic reduced glutathione and prevented the reduction of hepatic catalase activity. Resveratrol-induced inhibition of nuclear factor kappa B (NF-kappaB) activation or reduction of the pancreatic tumor necrosis factor-alpha (TNF-alpha) concentration could not be demonstrated. In conclusion, the beneficial effects of resveratrol on acute pancreatitis seem to be mediated by the antioxidant effect of resveratrol or by an NF-kappaB-independent anti-inflammatory mechanism.

Background Pancreatic cancer is the fourth leading cause of cancer related deaths in America. Monoclonal antibodies are a viable treatment option for inhibiting cancer growth. Tumor specific drug delivery could be achieved utilizing these monoclonal antibodies as targeting agents. This type of designer therapeutic is evolving and with the use of gold nanoparticles it is a promising approach to selectively deliver chemotherapeutics to malignant cells. Gold nanoparticles (GNPs) are showing extreme promise in current medicinal research. GNPs have been shown to non-invasively kill tumor cells by hyperthermia using radiofrequency. They have also been implemented as early detection agents due to their unique X-ray contrast properties; success was revealed with clear delineation of blood capillaries in a preclinical model by CT (computer tomography). The fundamental parameters for intelligent design of nanoconjugates are on the forefront. The goal of this study is to define the necessary design parameters to successfully target pancreatic cancer cells. Methodology/principal findings The nanoconjugates described in this study were characterized with various physico-chemical techniques. We demonstrate that the number of cetuximab molecules (targeting agent) on a GNP, the hydrodynamic size of the nanoconjugates, available reactive surface area and the ability of the nanoconjugates to sequester EGFR (epidermal growth factor receptor), all play critical roles in effectively targeting tumor cells in vitro and in vivo in an orthotopic model of pancreatic cancer. Conclusion Our results suggest the specific targeting of tumor cells depends on a number of crucial components 1) targeting agent to nanoparticle ratio 2) availability of reactive surface area on the nanoparticle 3) ability of the nanoconjugate to bind the target and 4) hydrodynamic diameter of the nanoconjugate. We believe this study will help define the design parameters for formulating better strategies for specifically targeting tumors with nanoparticle conjugates.

Undoubtedly ovarian cancer is a vexing, incurable disease for patients with recurrent cancer and therapeutic options are limited. Although the polycomb group gene, Bmi-1 that regulates the self-renewal of normal stem and progenitor cells has been implicated in the pathogenesis of many human malignancies, yet a role for Bmi-1 in influencing chemotherapy response has not been addressed before. Here we demonstrate that silencing Bmi-1 reduces intracellular GSH levels and thereby sensitizes chemoresistant ovarian cancer cells to chemotherapeutics such as cisplatin. By exacerbating ROS production in response to cisplatin, Bmi-1 silencing activates the DNA damage response pathway, caspases and cleaves PARP resulting in the induction apoptosis in ovarian cancer cells. In an in vivo orthotopic mouse model of chemoresistant ovarian cancer, knockdown of Bmi-1 by nanoliposomal delivery significantly inhibits tumor growth. While cisplatin monotherapy was inactive, combination of Bmi-1 silencing along with cisplatin almost completely abrogated ovarian tumor growth. Collectively these findings establish Bmi-1 as an important new target for therapy in chemoresistant ovarian cancer.

The transforming growth factor-beta (TGF-β) superfamily is one of the most diversified cell signaling pathways and regulates many physiological and pathological processes. Recently, neuropilin-1 (NRP-1) was reported to bind and activate the latent form of TGF-β1 (LAP-TGF-β1). We investigated the role of NRP-1 on Smad signaling in stromal fibroblasts upon TGF-β stimulation. Elimination of NRP-1 in stromal fibroblast cell lines increases Smad1/5 phosphorylation and downstream responses as evidenced by up-regulation of inhibitor of differentiation (Id-1). Conversely, NRP-1 loss decreases Smad2/3 phosphorylation and its responses as shown by down-regulation of α-smooth muscle actin (α-SMA) and also cells exhibit more quiescent phenotypes and growth arrest. Moreover, we also observed that NRP-1 expression is increased during the culture activation of hepatic stellate cells (HSCs), a liver resident fibroblast. Taken together, our data suggest that NRP-1 functions as a key determinant of the diverse responses downstream of TGF-β1 that are mediated by distinct Smad proteins and promotes myofibroblast phenotype.

The consequences of the exocrine pancreatic insufficiency are well-known in pancreatic diabetes (DM). Aim: The aim of this study was to determine the frequency of low stool elastase activity in type-1 and type-2 DM patients (pts). The metabolic parameters and the chronic complications were evaluated in pts with normal and abnormal exocrine function and the effects of enzyme substitution were analysed. Patients and Methods: Stool elastase of 146 pts with non-pancreatic DM was measured by ELISA. Autonomic neuropathy (AN), peripheral sensory function, retinopathy, HbA1c, BMI, microalbuminuria (MAU), quality of life (QoL) were measured. Results: 41 pts (28%) had an affected exocrine function (elastase: 417±37 vs. 141±11 ug/g; mean of pts with normal vs. abnormal values, p 0.05). The severity of AN decreased (AN score: from 3.09±0.61 to 2.36±0.66, p>0.05). Conclusions: A moderately impaired exocrine pancreatic function is a characteristic finding in a high number of patients with both types of diabetes. This altered function is more severe in older patients. The enzyme substitution might have an important role in the treatment of diabetic patients with higher age, severe neuropathy and unstable glycemic control.

Bacterial translocation from the intestinal tract plays an important role in severe acute pancreatitis (AP). Human β-defensins are a family of antimicrobial peptides present at the mucosal surface. The aim of this study was to investigate the relevance of single nucleotide polymorphisms (SNPs) in the DEFB1 gene and copy number polymorphisms of the DEFB4 genes in AP.124 AP patients (30 with mild and 94 with severe disease) and 100 healthy controls were enrolled in the study. Three SNPs of the DEFB1 gene [G-20A (c.-20G→A), C-44G (c.-44C→G) and G-52A (c.-52G→A)] were genotyped by Custom TaqMan assay. The DEFB4 gene copy number was determined by means of a TaqMan real-time PCR assay.Significantly higher frequencies of the AA genotype of G-20A (c.-20G→A) and the AA genotype of G-52A (c.-52G→A) were observed among the patients with severe AP (SAP) compared with the healthy controls (38 vs. 20 and 41 vs. 18%, respectively). The GG protective genotype of C-44G (c.-44C→G) SNP was much less frequent (1%) among the patients than among the controls (9%). A higher frequency of a lower (4) copy number of the DEFB4 gene was observed in the patients with SAP compared with the healthy controls (62 vs. 24%, respectively).The variations in the genes encoding human β-defensin-1 and -2 may be associated with the risk of SAP. and IAP.

Cytokine regulation may be an important factor in the susceptibility for the development of chronic pancreatitis; transforming growth factor-beta1 (TGF-beta1) plays a central role in the pathogenesis of pancreatic fibrogenesis. The aim of our study was to analyse the relevance of TGF-beta1, interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-alpha) polymorphisms in patients with chronic pancreatitis.of the 83 patients enrolled in the study, 43 were treated medically and 40 patients underwent surgical intervention. Healthy blood donors (n=75) served as controls.the polymorphisms of TGF-beta1 +869 T--C and IL-8 -251 T--A were determined by the ARMS method, while that of TNF-alpha -308 was investigated using NcoI RFLP.there was a higher frequency (50%) of the TT genotype of TGF-beta1 +869, with a concomitantly higher TGF-beta1 level in the plasma (5.2 +/- 1.7 ng/mL) of patients with chronic pancreatitis than in healthy blood donors (28% and 2.8 +/- 0.9 ng/mL respectively). The number of TT homozygotes differed significantly between the patients who underwent surgical intervention and the controls, and even between the surgical and the non-surgical patients. The frequency of the T/A genotype with higher IL-8 production, was significantly higher in both groups of patients than in the controls (58% and 58% versus 40%). No correlation was found between the TNF-alpha -308 polymorphism and chronic pancreatitis.correlations of the TGF-beta1 and IL-8 single nucleotide polymorphisms (SNPs) with chronic pancreatitis underline the importance of these cytokines in the pathomechanism of the disease. Moreover, it seems that the TT genotype of +869 TGF-beta1 might be a risk factor for the development of a severe form of chronic pancreatitis, and could serve as a prognostic sign for any future surgical intervention or even repeat surgery. Further studies on a larger group of patients, in addition to a follow-up study, are necessary to confirm this preliminary observation.