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Supplementary Tables

Figure S1: Read alignments from WES-cfDNA, showing somatic mutations in APC and TP53 genes.

Legends for Supplementary Figures 1-6

Table S3: Treatment regimens of the Avatar patient derived xenograft models carrying the VEGFR2 WT and L840F genotypes.

Figure S2: Read alignments from WES-cfDNA, showing somatic mutations in APC and TP53 genes.

Purpose:The tumor microenvironment plays a key role in cancer development and progression and is involved in resistance to chemo- and immunotherapy. Cancer-associated fibroblast expressing fibroblast-activating protein α (FAPα) is one of the predominant stroma cell types and is involved in resistance to immunotherapy.Experimental Design:We generated OMTX705, a novel antibody–drug conjugate from a humanized anti-FAP antibody linked to a new cytolysin. Here, we studied its antineoplastic activity in vitro and in preclinical mouse models alone and in combination with chemotherapy as well as immunotherapy in PD-1–resistant tumors.Results:In Avatar models, OMTX705 showed a 100% tumor growth inhibition and prolonged tumor regressions as single agent and in combination with chemotherapy. Treatment rechallenge following treatment discontinuation induced additional tumor regression, suggesting lack of treatment resistance. In a mouse model with a humanized immune system resistant to PD-1 inhibition, OMTX705 increased tumor infiltration by CD8+ T cells, induced complete regressions, and delayed tumor recurrence.Conclusions:These data suggest that FAP targeting with OMTX705 represents a novel and potent strategy for cancer treatment, including tumors resistant to immunotherapy, and support its clinical development.

Table S2: Custom primers and probes designed based on the Thermofisher online design tool for TaqManÃ,® genotyping assay for KDR c.2518C>T (VEGFR2 p.L840L). TaqManÃ,® MGB (minor groove binder) probes incorporate a 5' reporter (VIC or FAM) and a 3' nonfluorescent quencher (NFQ).

Detailed methods

Figure S4: A) Molecular dynamics simulations with L840F VEGFR2 indicates that most of the adopted F840 conformations during the simulation are not compatible with inhibitor binding. Clashes between VEGFR2 and the sorafenib area are shown as red discs. B) Kinase assays showing impaired kinase activities of VEGFR2 L840F and R1032Q compared to wild-type. C) Effects of the L840F mutation on Y1175 VEGFR2 phosphorylation. HEK293 cells were transiently transfected with increasing levels of a plasmid encoding for WT or L840F VEGFR2. Cells at 70% confluence were starved in 1% BSA/DMEM for 4 h, and then incubated in the absence or presence of 60 ng/ml VEGF165 for 10 min at 37 {degree sign}C. Whole cell lysates were analyzed by western blotting, using antibodies against phosphoY1175 and total VEGFR2. Representative results are shown. D) PAE cells stably expressing WT or L840F VEGFR2 were generated from a PAE cell line that does not normally express VEGFR2 (empty). Cells at 70% confluence were starved in 1% BSA/DMEM for 4h, and then incubated in the absence or presence of 60 ng/ml VEGF165 for 10 min at 37 {degree sign}C. Whole cell lysates were then analyzed by western blotting, using antibodies against phosphoY1175 and total VEGFR2. Representative results are shown, highlighting the significant decrease in VEGF-induced Y1175 VEGFR2 phosphorylation in the presence of the L840F mutation.

Additional Materials and Methods information not included in the article text, and detailed description of the patient tumors from which xenograft models have been derived from

Figure S3: Analyses of interactions between tyrosine kinase receptors and kinase inhibitors, performed with LigProt.

Supplementary Data from Organoid Sensitivity Correlates with Therapeutic Response in Patients with Pancreatic Cancer

Figure S5: A) Levels of phosphorylated ERK following treatment of the MDST8 colorectal cell line, expressing R1032Q VEGFR2, with different kinase inhibitors in the presence. B) Proliferation assays of Colo320 cell lines, stably expressing WT and R1032Q VEGFR2, upon treatment with TKIs. The expression of the VEGFR2 R1032Q hot-spot mutant in Colo320 cell lines (WT to KRAS/NRAS/BRAF/PIK3CA and mutated to TP53 and APC) increased sensitivity to cabozantinib.

Figure S6: Assessment of the expression of phosphorylated ERK (p-ERK) in the WT and L840F VEGFR2 PDX Avatar models by immunohistochemistry (A), confocal microscopy (B), and immunoblotting (C). The MAPK pathway was activated in the L840F VEGFR2 mutant model and was not decreased following treatment with TKIs.

S1-S7 all supplementary figures with their legends: S1:Figure S1. OMTX705 binding and internalization in HT1080-WT, -FAP and CAFs S2:Weight change of mice bearing Panc 007, Lung 024 and Breast 014 tumors. S3: OMTX705 Activity in the Immunodeficient Patient Derived Xenografts (PDX) Models of Ovarian Cancer. S4: FAP immunostaining in frozen tumor samples from humanized mice bearing CTG-0860 NSCLC PDX tumor. S5: Separate fluorescent staining of human IgG, Rab7 and nuclei in OMTX705 and OMTX005 treated HT1080-FAP cells (complements the merged image of figure 4C) S6: Immunohistochemical analysis of payload, OMTX005 and OMTX705 distribution in tumors from Panc 007 model. S7: Schematic summary of findings and proposed mechanism of action of OMTX705.

Purpose:Pancreatic ductal adenocarcinoma (PDAC) remains a significant health issue. For most patients, there are no options for targeted therapy, and existing treatments are limited by toxicity. The HOPE trial (Harnessing Organoids for PErsonalized Therapy) was a pilot feasibility trial aiming to prospectively generate patient-derived organoids (PDO) from patients with PDAC and test their drug sensitivity and correlation with clinical outcomes.Experimental Design:PDOs were established from a heterogeneous population of patients with PDAC including both basal and classical PDAC subtypes.Results:A method for classifying PDOs as sensitive or resistant to chemotherapy regimens was developed to predict the clinical outcome of patients. Drug sensitivity testing on PDOs correlated with clinical responses to treatment in individual patients.Conclusions:These data support the investigation of PDOs to guide treatment in prospective interventional trials in PDAC.

Table S5: List of KDR/VEGFR2 somatic mutations from the Cosmic, TCGA, GENIE and PCA-WGS databases.

Pancreatic cancer has the worst prognosis of all common tumors. Earlier cancer diagnosis could increase survival rates and better assessment of metastatic disease could improve patient care. As such, there is an urgent need to develop biomarkers to diagnose this deadly malignancy earlier. Analyzing circulating extracellular vesicles (cEVs) using ‘liquid biopsies’ offers an attractive approach to diagnose and monitor disease status. However, it is important to differentiate EV-associated proteins enriched in patients with pancreatic ductal adenocarcinoma (PDAC) from those with benign pancreatic diseases such as chronic pancreatitis and intraductal papillary mucinous neoplasm (IPMN). To meet this need, we combined the novel EVtrap method for highly efficient isolation of EVs from plasma and conducted proteomics analysis of samples from 124 individuals, including patients with PDAC, benign pancreatic diseases and controls. On average, 912 EV proteins were identified per 100µL of plasma. EVs containing high levels of PDCD6IP, SERPINA12 and RUVBL2 were associated with PDAC compared to the benign diseases in both discovery and validation cohorts. EVs with PSMB4, RUVBL2 and ANKAR were associated with metastasis, and those with CRP, RALB and CD55 correlated with poor clinical prognosis. Finally, we validated a 7-EV protein PDAC signature against a background of benign pancreatic diseases that yielded an 89% prediction accuracy for the diagnosis of PDAC. To our knowledge, our study represents the largest proteomics profiling of circulating EVs ever conducted in pancreatic cancer and provides a valuable open-source atlas to the scientific community with a comprehensive catalogue of novel cEVs that may assist in the development of biomarkers and improve the outcomes of patients with PDAC.

Background: As of March 2020, governments throughout the world implemented business closures, work from home policies, and school closures due to exponential increase of coronavirus disease 2019 (COVID-19) cases, leaving only essential workers being able to work on site. For most of the children and adolescent school closures during the first lockdown had significant physical and psychosocial consequences. Here, we describe a comprehensive Return to School program based on a behavior safety protocol combined with the use of saliva-based reverse transcriptase-polymerase chain reaction (RT-PCR) pooled screening technique to keep schools opened. Methods: The program had 2 phases: before school (safety and preparation protocols) and once at school (disease control program: saliva-based RT-PCR pooled screening protocol and contact tracing). Pooling: Aliquots of saliva from 24 individuals were pooled and 1 RT-PCR test was performed. If positive, the initial 24-pool was then retested (12 pools of 2). Individual RT-PCR tests from saliva samples from positive pools of 2 were performed to get an individual diagnosis. Results: From August 31 until December 20, 2020 (16-wk period) a total of 3 pools, and subsequent 3 individual diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) disease were reported (2 teachers and 1 staff). Conclusion: Until COVID-19 vaccine can be administered broadly to all-age children, saliva-based RT-PCR pooling testing is the missing piece we were searching for to keep schools opened.

523 Background: Robust biomarkers are urgently needed to assist in diagnosing pancreatic cancer. Earlier cancer diagnosis could increase survival rates by an estimated 5-fold and more reliable and real-time assessment of treatment effects in patients with cancer could improve quality of life and reduce healthcare costs. Isolation of circulating extracellular vesicles (cEVs) as ‘liquid biopsies’ offers an advantageous approach to diagnose and monitor disease status. Methods: We conducted a comprehensive proteomics study of cEVs from plasma samples to identify EV proteins that may be used as biomarkers for the diagnosis and prognosis of pancreatic cancer. Patients with pancreatic ductal adenocarcinoma (PDAC) of various tumor stages, chronic pancreatitis, intraductal papillary mucinous neoplasm (IPMN), and age-matched controls were enrolled. EVs were isolated directly from plasma samples using the affinity-based EVTrap method then subject to quantitation by liquid chromatography-tandem mass spectrometry. Results: A total of 124 patients (93 with PDAC, 12 with chronic pancreatitis, 8 with IPMN and 11 controls) were included in the discovery cohort. The isolation of EVs with EVtrap allowed the identification on average of 912 EV proteins per 100µL of sample. Principal component analysis of the cEV proteome showed clear separation between PDAC and benign pancreatic diseases. Individuals with IPMN were more closely related to controls, whereas chronic pancreatitis cases were more related to PDAC. At the functional level, we noted that cytokeratin, protein folding chaperons, and actin dynamics regulators were among protein clusters more highly altered in the cEV of patients with PDAC. We further identified new cEV markers associated with metastatic disease, such as PSMB4, RUVBL2, and ANKAR, as well as other EV proteins with strong correlation to prognosis, such as CRP, RALB, and CD55. Finally, we validated a 7-protein PDACEV signature in a validation cohort of 36 separate patients (24 with PDAC, 6 with chronic pancreatitis and 6 with IPMN) which yielded an 89% prediction accuracy for the diagnosis of PDAC. Conclusions: This study provides a valuable resource to the scientific community with a comprehensive catalog of novel proteins on circulating EVs that may assist in the development of novel biomarkers and improve the outcomes of patients with pancreatic cancer.