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Supplementary Data from Branching Copy-Number Evolution and Parallel Immune Profiles across the Regional Tumor Space of Resected Pancreatic Cancer

The outlook for patients with pancreatic cancer remains dismal. Treatment options are limited and chemotherapy remains standard of care, leading to only modest survival benefits. Hence, there is a great need to further explore the mechanistic basis for the intrinsic therapeutic resistance of this disease, and to identify novel predictive biomarkers. RNA‐binding motif protein 3 (RBM3) has emerged as a promising biomarker of disease severity and chemotherapy response in several types of cancer, including pancreatic cancer. The aim of this study was to unearth RBM3‐regulated genes and proteins in pancreatic cancer cells in vitro, and to examine their expression and prognostic significance in human tumours. Next‐generation RNA sequencing was applied to compare transcriptomes of MIAPaCa‐2 cells with and without RBM3 knockdown. The prognostic value of differentially expressed genes (DEGs) was examined in The Cancer Genome Atlas (TCGA). Top deregulated genes were selected for further studies in vitro and for immunohistochemical analysis of corresponding protein expression in tumours from a clinically well‐annotated consecutive cohort of 46 patients with resected pancreatic cancer. In total, 19 DEGs (p

Background Periampullary cancer is a term for cancers arising in or in close proximity to the pancreas. Pancreatic cancer is the 3rd leading cause of cancer death for both sexes and while surgery is the only option for cure, chemotherapy is given in both the adjuvant and palliative settings. The aim of this study was to investigate any sex and gender differences in patients with pancreatic and other periampullary adenocarcinomas enrolled in a prospective, observational trial. Methods The study cohort consists of the first 100 patients, 49 women and 51 men, enrolled in the Chemotherapy, Host Response and Molecular dynamics in Periampullary cancer (CHAMP) study, an ongoing study of patients undergoing neoadjuvant, adjuvant or first-line palliative chemotherapy treatment. Twenty-five patients had surgery with curative intent and subsequent adjuvant treatment, and 75 patients were treated with palliative chemotherapy. Data regarding health-related quality of life (HRQoL, EORTC-QLQ-C30) at baseline, demographic and clinicopathological factors were examined and stratification by treatment intention according to sex. Overall survival (OS) was calculated through Kaplan–Meier analysis. Results There was a statistically significant difference between male and female patients treated with curative intent, with fewer women having undergone surgery (18 vs 7, p = 0.017), also after adjustment for age, tumor location and performance status. No statistical differences were found between the sexes regarding age, comorbidities, or clinicopathological factors. Before start of chemotherapy treatment, health-related quality of life (HRQoL) was lower in female than in male patients. However, HRQoL was not associated with performance status in female patients, whereas in male patients several HRQoL indicators were significantly positively associated with poorer performance status at baseline. Conclusions This study shows no clear differences between the sexes regarding biological factors concluding that gender bias might be responsible for the discrepancy between men and women being offered curative surgery. The observed difference between women and men regarding the association between HRQoL and performance status is unprecedented. Altogether these findings underline the importance of taking gender into consideration when assessing eligibility for curative surgery in order to improve biological outcome and decrease suffering in both sexes. Trial registration NCT03724994.

Background Pancreatic cancer is a devastating disease with a dismal prognosis. Despite profound medical advances in systemic therapies for other types of aggressive tumours during recent years, a diagnosis of pancreatic cancer is still often synonymous with a fatal outcome. The term periampullary cancer includes pancreatic cancer and applies to the group of tumours found in proximity to the ampulla of Vater. Molecular events and immune response in the host during chemotherapy remain largely unexplored in this group of tumours. Therefore, the “Chemotherapy, Host Response and Molecular Dynamics in Periampullary Cancer (CHAMP)” study aims to monitor these processes to gain new insight into this perplexing disease. Methods The CHAMP study is a prospective, single-arm observational study. All patients diagnosed with pancreatic or other periampullary adenocarcinoma undergoing adjuvant or palliative chemotherapy treatment in the Department of Oncology, Skåne University Hospital, are invited to participate. Clinical and pathological data will be compiled at study entry. A single tissue microarray (TMA) block is constructed for each patient with a resected tumour and blood samples are drawn before, during and after chemotherapy in order to sample peripheral blood mononuclear cells (PBMC), cytokines and circulating tumour DNA (ctDNA). Next generation sequencing will be performed on tumour tissue and ctDNA to detect changes in the clonal landscape over space and time. Discussion Despite the recent emergence of some promising biomarkers for periampullary cancer, there has been a lack of success in clinical implementation. Cancer cells continuously adapt and become resistant to treatment during chemotherapy. To be able to keep pace with and hopefully overtake this rapid evolution we must, with the help of new diagnostic tools, be ready to adapt and alter treatment accordingly. It seems to us that the only way forward is to gain a better understanding of the dynamics of the disease during treatment. With insights gained from the CHAMP study we hope to find answers to key questions in this largely unexplored territory. Trial registration This study has been registered 30th October 2018 at clinicaltrials.gov as NCT03724994.

Pancreatic ductal adenocarcinoma (PDAC) remains a highly lethal disease. The only option for curative treatment is resection of the tumor followed by standard adjuvant chemotherapy. Yet, early relapse due to chemoresistance is almost inevitable. Herein, we delineated the genetic intratumor heterogeneity in resected PDAC, with the aim to identify evolutionary patterns that may be associated with overall survival (OS) following treatment with curative intent. Potential relationships with the adjacent immune microenvironment were also examined. The genetic and immune landscapes of the regional tumor space were analyzed in nine patients with resected PDAC. Targeted deep sequencing and genome wide SNP array were followed by clonal deconvolution and phylogenetic analysis. A mathematical complexity score was developed to calculate the network extent of each phylogeny. Spatial variation in abundancy and tumor nest infiltration of immune cells was analyzed by double IHC staining. Copy-number heterogeneity was denoted as the major contributing factor to the branching architectures of the produced phylogenetic trees. Increased tree complexity was significantly inversely associated with OS, and larger regional maximum aberrations (higher treetops) were associated with increased PD-L1 expression on tumor cells. Contrastingly, an FREM1 gene amplification, found in one patient, coincided with a particularly vigorous immune response. Findings from this limited case series suggest that complex evolutionary patterns may be associated with a shorter survival in surgically treated patients with PDAC. Some hypothesis-generating associations with the surrounding immune microenvironment were also detected.Evolutionary copy-number patterns may be associated with survival in patients with resected PDAC.

PURPOSE Periampullary adenocarcinomas encompass a heterogeneous group of tumors with dismal prognosis and limited treatment options. Emerging evidence shows that tumor morphology (ie, intestinal type [I-type] or pancreatobiliary type [PB-type]) is a more relevant prognostic factor than tumor origin. Knowledge is sparse, however, on whether key mutations differ according to morphology. MATERIALS AND METHODS Next-generation sequencing was applied to assess the mutational status of 70 genes in 102 tumors from a retrospective cohort of 175 patients with resected periampullary adenocarcinoma. Brahma-related gene 1 protein expression was examined by immunohistochemistry on tissue microarrays with primary tumors from the original cohort. RESULTS APC mutations were significantly more common in I-type than in PB-type tumors (27.5% v 0%; P < .001), as were ERBB3 mutations (20.8% v 4.8%; P = .016), whereas CDKN2A mutations were more common in PB-type than in I-type tumors (19.4% v 2.5%; P = .013). KRAS mutation was an independent factor of poor prognosis in I-type tumors (hazard ratio, 3.73; 95% CI, 1.10 to 12.67). In PB-type tumors, SMARCA4 mutation was an adverse prognostic factor in patients not receiving adjuvant chemotherapy, and there was a significant treatment interaction between expression of Brahma-related gene 1 protein, the protein encoded by SMARCA4, and adjuvant chemotherapy ( Pinteraction = .007). CONCLUSION To our knowledge, this is the first description of the mutational landscape in the full spectrum of periampullary adenocarcinoma that demonstrates that the distribution and prognostic and predictive significance of commonly mutated genes differ by morphology. The results emphasize that morphology is an important factor to consider in the search for novel biomarkers and targeted personalized treatment of these patients. In addition, the findings support the concept that molecular profiling of these tumors could be of clinical benefit.

Introduction Pancreatic cancer is a lethal disease with an incidence little higher than the mortality rate. Great advances in oncology have been made in recent years with new treatment strategies for many cancers, leading to substantial improvement in patient outcome. The prognosis for pancreatic cancer patients, however, remains dismal and chemotherapy is still standard of care. An increased understanding of the temporal evolution and resistance mechanisms of pancreatic cancer during chemotherapy is necessary to improve patient outcome. To this end, the Chemotherapy, Host response And Molecular dynamics in Periampullary cancer study (CHAMP) was initiated in 2018. It is an ongoing, prospective observational trial in which blood samples are taken from patients at each chemotherapy cycle. Recently, the CHAMP- Extended Tissue Analysis (ETA) study was initiated, wherein selected patients enrolled in the CHAMP-study undergo targeted autopsies. The aim of this study was to compare the expression of selected immunohistochemical (IHC) markers and the quality of DNA in tumor tissue from an ante-mortem resected metastasis to the ileum and post-mortem collected tumor tissue in the first patient enrolled in the CHAMP-ETA study. Methods The autopsy was performed by a senior pathologist in accordance with standard procedure guidelines and all visible tumor was collected for analysis. The post-mortem interval (PMI) was 44 hours. Multiple tissue cores from formalin-fixed paraffin-embedded tumor tissue from the surgical resection and different tumor sites from the autopsy were assembled into a single tissue microarray (TMA) block. DNA was extracted from adjacent tissue cores obtained in parallel with the TMA-construction, and the quality examined by qPCR. IHC staining of standard diagnostic markers CK7, CK20, CDX2 and the proliferation marker Ki67 was performed on the TMA. Results In line with the expected, both normal and tumor tissue in the pancreas displayed a higher degree of autolysis compared to the metastases. Tumor areas surrounded by a more dense stroma tended to be less autolytic. CK20 and CDX2 expression was negative in all samples, both ante-mortem and post-mortem. CK7 was highly expressed in all tumor samples with an ever-stronger intensity in the post-mortem samples. Of note, CK7 was also strongly expressed in the tumor stroma of the post-mortem samples, but not in the pre-mortem samples. Ki67 expression was significantly higher in ante-mortem than in post-mortem samples. The extracted DNA was of sufficient quality in all tested post-mortem samples. Conclusion The results demonstrate that the DNA quality is sufficient after a PMI of 44 hours. The conditions for IHC staining are less optimal but may be sufficient for some markers. Access to post-treatment tumor tissue is an important complement to on-treatment biomarker studies, and of vital importance to understand the molecular events leading to terminal disease. Citation Format: Sofie Olsson Hau, Jasmine Webster, Alexandra Petersson, Jakob Eberhard, David Gisselsson, Karin Jirström. Exploring the temporal dynamics of pancreatic cancer through post-mortem tumor analysis: A pilot case study [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr B024.

Pancreatic cancer (PC) has become one of the leading causes of cancer related deaths. Late detection and resistance to therapy are two reasons for the dismal prognosis of this disease. Hence, there is an immense need for novel treatment options as well as predictive biomarkers, to prolong the life of these patients. The Chemotherapy, Host Response and Molecular Dynamics in Periampullary Cancer, the CHAMP study (clinical trial nr NCT03724994), is an ongoing observational, prospective clinical study that includes newly diagnosed patients with PC or other periampullary cancers receiving adjuvant or first-line palliative chemotherapy. The present study aims to investigate the peripheral immune-cellome in serial blood samples obtained during treatment from 24 patients, of whom 8 had resectable tumors. Buffy coat was isolated from blood samples taken at baseline (before treatment), after one month and after three to four months of treatment. Flow cytometry with two panels of antibodies was applied to detect major immune populations in both the innate and the adaptive immune system, as well as subpopulations of T cells. Immune cell frequencies were compared between patients with progression and no progression during treatment, and the associations of different immune cell subsets with overall survival were examined. As B cells have been highlighted as being of particular clinical relevance, single-cell transcriptomics and proteomics will be applied to further delineate the dynamics and prognostic impact of individual subtypes within this lineage. The results will provide important real-world data on the longitudinal dynamics of circulating immune cells under the pressure of chemotherapy in patients with PC. These results will in the future be related to analyses of circulating tumor DNA in matched blood samples, and together they will provide a foundation for forthcoming adaptive treatment trials based on results from the CHAMP study. Citation Format: Maja Svensson, Alexandra Petersson, Viktor Sincic, Sofie Olsson Hau, Jakob Eberhard, David Gisselsson Nord, Karin Leandersson, Kristina Lundberg, Karin Jirström. Effects of chemotherapy on the peripheral immune-cellome in patients with pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A022.

Background: Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease characterized by rapidly occurring chemoresistance due to clonal evolution. In addition, since most cases are unresectable, tissue analyses are limited to diagnostic biopsy specimens which hampers molecular analyses and does not reflect the genetic tumor heterogeneity. Hence, there is an urgency to develop new complementary tools to detect, characterize and monitor PDAC. The aim of the ongoing prospective, observational study “Chemotherapy, Host Response and Molecular Dynamics in Periampullary Cancer (CHAMP)” (NCT03724994) is to gain new insights into the clonal evolution and parallel immune responses, under the pressure of different cytotoxic treatments, in PDAC and other periampullary cancers. Methods: In a pilot study comprising ten CHAMP patients, an in-depth genomic analysis of ctDNA in serial blood samples (3-4 samples/patient) and matched tumor tissue, either from resected specimens or diagnostic biopsies, was performed using a broad targeted pan-cancer panel. Within this small case series, three patients with resectable disease received adjuvant chemotherapy and seven patients received palliative chemotherapy. Detected ctDNA fluctuation patterns were correlated to tumor progression. Results: In the three patients receiving adjuvant treatment, ctDNA could not be detected in the blood at any time point. For the five patients receiving palliative treatment, varying quantities of ctDNA were detected before the start of treatment (baseline), followed by a decrease during the first month of therapy. Quantitative ctDNA fluctuations were also observed to largely follow the clinical disease course as signs of radiological tumor progression were supported by elevated ctDNA levels. The concordance of known driver mutations in ctDNA detected at baseline and matched tumor tissue was overall high. Yet, a few discrepancies were seen, such as an ARID1A mutation detected only in ctDNA at the time of radiological tumor progression in one patient. Of note, a potentially druggable KRAS p.G12C mutation was observed in both tumor tissue and ctDNA in the same patient. In two palliative patients, one of whom did not respond to treatment, no ctDNA could be detected at any time point. Conclusion: The results from this pilot study emphasize the value of ctDNA analyses, both for characterization and on-treatment monitoring of PDAC. This is of particular importance in cases where no tissue biopsies are available, and at disease relapse or progression. Studies on 50 additional CHAMP patients are now ongoing, wherein a novel ultra-sensitive ctDNA profiling assay is utilized to further detect minimal residual disease and clinically actionable targets. Studies comparing the clonal landscape in treatment naïve primary tumors and/or metastases, serial blood samples, and primary and/or metastatic tumor tissue obtained postmortem, are also in progress. Citation Format: Alexandra Petersson, Sofie Olsson Hau, Jakob Eberhard, Markus Heidenblad, David Gisselsson, Karin Jirström. On-treatment ctDNA monitoring of pancreatic cancer holds promise for improved treatment stratification [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A021.

305 Background: Pancreatic cancer has a dismal prognosis and clinical protocols are still lacking predictive biomarkers. RNA-binding motif protein 3 (RBM3) has emerged as a promising biomarker in several solid cancers, including pancreatic cancer. High RBM3 expression in human tumors has been associated with good response to chemotherapy, as well as to confer increased chemosensitivity in vitro. The aim of this study was to identify RBM3-regulated genes in pancreatic cells in vitro, and further interrogate their potential utility as prognostic and predictive biomarkers in a translational setting. Methods: Next generation RNA-sequencing was applied to compare gene expression between MIAPaCa-2 cells with siRNA-downregulated RBM3 and control cells. Single genes with the strongest association to RBM3 were further selected by gene set enrichment analysis, and their prognostic value in pancreatic cancer was examined in The Cancer Genome Atlas (TCGA). The most promising biomarker candidates with well-validated antibodies were then analyzed by immunohistochemistry in tissue microarrays with tumors from a consecutive, retrospective cohort of 175 patients with periampullary and pancreatic adenocarcinoma. Results: MIAPaCa-2 cells with downregulated RBM3 displayed 21 differentially expressed genes (p < 0.01). One of the top downregulated genes was PDS cohesion associated factor A (PDS5A) encoding a protein involved in sister chromatid cohesion. PDS5A protein expression was reduced in siRBM3-treated MIAPaCa-2 cells compared to control cells. High PDS5A mRNA expression was significantly associated with a reduced survival in pancreatic cancer in the TCGA (n = 176, p = 0.026). High PDS5A protein expression in the separate cohort was significantly associated with a poor prognosis but predictive of improved response to adjuvant chemotherapy in KRAS-mutated, but not wild-type, pancreatobiliary-type tumors (p for interaction = 0.043). Conclusions: Here, we provide the first report of the expression of PDS5A in pancreatic and periampullary cancer, suggesting its potential utility as a prognostic and predictive biomarker. Further studies to unravel the underlying mechanisms are encouraged.