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Lung cancer is the leading cause of cancer-related deaths in the United States, with estimates of 236,740 new cases and 118,830 deaths in 2022 secondary to the disease. Blood-based liquid biopsies hold promise to reduce morbidity and mortality from lung cancer by enabling early detection to downstage disease at diagnosis, theragnostic identification of patients most likely to be helped or harmed by therapy, monitoring of therapeutic efficacy, and detection of residual disease. PrognomiQ’s multi-omics platform comprehensively profiles proteins, metabolites, lipids, mRNA, and cfDNA in blood samples which can be used for the development of liquid biopsy tests with high sensitivity and specificity for lung cancer. We conducted a case-control study comprising 1031 subjects: 361 subjects with untreated non-small-cell lung cancer (NSCLC) and 670 matched controls which included 340 subjects with salient pulmonary and gastrointestinal co-morbidities. Blood samples from each subject were processed to provide 7 different `omics readouts. LCMS was used to detect and quantify proteins, metabolites, and lipids. In addition, cfDNA and mRNA were assayed using next-generation sequencing. cfDNA reads were analyzed to estimate fragment-lengths, copy-number variation, and CpG site methylation. All molecular data were normalized using standard methods specific to each assay. Univariate analyses of cases vs controls were performed to identify differentially abundant features on all available samples per assay. We detected 9,868 proteins, 605 lipids, 329 metabolites, and 109,070 mRNA transcripts. Of these, 3,098 proteins, 210 lipids, 57 metabolites, and 30,236 mRNA transcripts were significantly different (FWER < 0.05) in cases versus controls. Gene set enrichment analysis on statistically significant transcripts and proteins identified multiple gene-ontology terms associated with cancer including the Wnt signaling process and IgA immunoglobulin complex, respectively. From cfDNA data, we identified 234 non-contiguous genomic regions associated with the fragment-length disorder, 4,790 with copy-number variation, and 74 differentially methylated genomic regions spanning 184 CpG sites (FWER < 0.05). With the premise that deviations from copy number neutrality are more likely to indicate a tumor contribution, we then focused our examination on those differentially expressed proteins that overlap with differentially expressed mRNA transcripts as well as CNV genomic regions. We identified 52 protein coding genes including E-cadherin (associated with EMT) and related binding proteins such as RAB11B, CAPZB, EPS15, FLNB, MYH9, STK24 and YWHAE. Ongoing machine-learning-based classifier training to distinguish between cancer and non-cancer can serve as the basis for the development of high-sensitivity liquid-biopsy tests for lung cancer. Citation Format: Jinlyung Choi, Ajinkya Kokate, Ehdieh Khaledian, Manway Liu, Preethi Prasad, John Blume, Jessica Chan, Rea Cuaresma, Kevin Dai, Manoj Khadka, Thidar Khin, Yuya Kodama, Joon-Yong Lee, Hoda Malekpour, Megan Mora, Nithya Mudaliar, Sara Nouri Golmaei, Madhuvanthi Ramaiah, Saividya Ramaswamy, Peter Spiro, Dijana Vitko, Kavya Swaminathan, James Yee, Brian Young, Chinmay Belthangady, Bruce Wilcox, Brian Koh, Philip Ma. Biomarker discovery in non-small-cell lung cancer enabled by deep multi-omics profiling of proteins, metabolites, transcripts, and genes in blood. [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 6606.

Pancreatic ductal adenocarcinoma (PDAC) is currently the 3rd leading cause of cancer-related deaths in the US. Although the all-stage 5-year survival rate is ~10%, early-stage 5-year survival is markedly superior and in excess of 40%. Hence, early detection of PDAC via blood-based liquid biopsies holds promise to reduce morbidity and mortality. PrognomiQ’s multi-omics platform performs deep and unbiased molecular profiling of blood samples to detect proteins, metabolites, lipids, mRNA, miRNA, cfDNA fragmentation and copy-number, and CpG methylation. Here we report results from training and validation of a classifier on a subset of that multi-omic data with the potential to enable the development of high sensitivity and specificity tests for early detection of PDAC.We conducted a case-control study comprising 146 subjects across 16 clinical sites, including 63 pathology-confirmed, untreated PDAC cases (12 stage I, 8 stage II, 4 stage III, 36 stage IV, and 3 stage unknown) and 83 age- and gender- matched controls without any known cancer. For each subject, venous blood samples including plasma were collected. Unbiased LCMS was used to detect and quantify proteins, and targeted, multiplexed MRM-LCMS assays were used for both metabolites and lipids. After data processing, we detected 54,114 proteomic features, 898 lipids, and 373 metabolites. 445 proteomic features, 170 lipids, and 37 metabolites were found to be significantly different as determined by Bonferroni-corrected Wilcoxon tests with FWER < 0.05. For classification, the dataset was split into training (37 cases and 37 controls) and validation (26 cases and 46 controls) sets, with control for collection site and date, age, and gender. XGBoost models were constructed for each analyte class using ten repeats of 10-fold cross-validation. To improve specificity to PDAC, all proteomic features which mapped to GOBP terms associated with acute-phase response, inflammation, and immune response were excluded prior to training. The best-performing hyperparameters were used for a final model built on the full training set and then used for inference on the validation set. At 99% specificity, the proteomic classifier had sensitivities of 77%, 57%, and 88% for Stages 1-4, Stages 1-2, and Stages 3-4, respectively, estimated by bootstrap re-sampling of the validation results. Metabolomics had sensitivities of 81%, 71%, and 88%. Lipidomics had sensitivities of 65%, 71%, and 65%. A joint, multi-omic model was constructed by averaging the scaled probabilities of all models. This joint model improved performance at 99% specificity with sensitivities of 92%, 86%, and 94%, highlighting the synergy of multi-omics data, particularly phenotypically related omics such as those described here. Multi-omic classifiers such as these can serve as the foundation for blood-based liquid biopsies for the early detection of PDAC. Citation Format: John Blume, Ghristine Bundalian, Jessica Chan, Connie Chao-Shern, Jinlyung Choi, Rea Cuaresma, Kevin Dai, Sara N. Golmaei, Jun Heok Jang, Manoj Khadka, Ehdieh Khaledian, Thidar Khin, Yuya Kodama, Ajinkya Kokate, Joon-Yong Lee, Manway Liu, Hoda Malekpour, Megan Mora, Nithya Mudaliar, Preethi Prasad, Madhuvanthi Ramaiah, Saividya Ramaswamy, Peter Spiro, Kavya Swaminathan, Dijana Vitko, James Yee, Brian Young, Susan Zhang, Chinmay Belthangady, Bruce Wilcox, Brian Koh, Philip Ma. A multi-omics classifier achieves high sensitivity and specificity for pancreatic ductal adenocarcinoma in a case-control study of 146 subjects [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 6597.

Pancreatic cancer is the third leading cause of cancer-related deaths in the United States. Disease biomarkers quantified from blood-based assays may help reduce mortality by enabling early detection, treatment selection, or response and resistance assessment. PrognomiQ has developed a multi-omics assay and analysis platform that comprehensively profiles blood samples to detect proteins, metabolites, lipids, mRNA, miRNA, cfDNA fragments, and methylation at CpG sites. This platform can provide deep insights into the biology of pancreatic cancer and could enable the development of high sensitivity and specificity tests for the early detection of pancreatic cancer. We conducted a case-control study comprising 196 subjects: 92 with untreated pancreatic cancer and 104 matched controls without pancreatic cancer. For each subject, blood was collected in assay-specific tubes and processed to provide 7 different `omics readouts. cfDNA and mRNA were isolated from samples and assayed following standard NGS protocols. cfDNA fragments were processed to estimate fragment-length disorder and copy-number variation along with CpG site methylation. In addition, targeted and untargeted LCMS were used to detect and quantify proteins, metabolites, and lipids. After normalization, non-parametric univariate analyses of cases versus controls were performed to identify differentially abundant features on all available samples for each assay. Unsupervised learning was used to investigate the separation of subjects into groups based on disease status for the subset of 157 subjects for which complete data on all 7 readouts were available. We detected 2,812 proteins, 811 lipids, 373 metabolites, and 110,864 mRNA transcripts in all samples where data for each assay was available. Of these, 275 proteins, 232 lipids, 49 metabolites, and 3385 mRNA transcripts were significantly different (FWER < 0.05) in cases versus controls. From cfDNA data, we identified 35 non-contiguous genomic regions associated with fragment-length disorder, 557 with copy-number variation, and 5 with multiple, differentially methylated CpGs (FWER < 0.05) that aggregately span 307 protein-coding genes; of these, the overlap with the differentially expressed proteins included E-cadherin (tumor suppressor) and N-cadherin (involved in epithelial-to-mesenchymal transition). Statistically significant genes and proteins were found to be associated with processes including Wnt signaling, regulation of focal adhesion assembly, and actin cytoskeleton organization. Multi-omics, unsupervised learning showed separation of early- and late-stage cases and controls. High-dimensional bioinformatics analyses systematically decomposed each `omics data type into joint and orthogonal components associated with pancreatic cancer. Ongoing multivariate analyses, including supervised machine learning, will further elucidate the biology of pancreatic cancer development, and serve as the foundation for high-sensitivity blood tests for the early detection and monitoring of pancreatic cancer. Citation Format: Ehdieh Khaledian, Preethi Prasad, John Blume, Ghristine Bundalian, Connie Chao-Shern, Jinlyung Choi, Rea Cuaresma, Jared Deyarmin, Jun Heok Jang, Manoj Khadka, Thidar Khin, Yuya Kodama, Ajinkya Kokate, Joon-Yong Lee, Manway Liu, Nithya Mudaliar, Madhuvanthi Ramaiah, Saividya Ramaswamy, Peter Spiro, Kavya Swaminathan, Preston Williams, Mi Yang, James Yee, Brian Young, Robert Zawada, Susan Zhang, Chinmay Belthangady, Bruce Wilcox, Philip Ma. High-dimensional, multi-omics analyses of proteins, metabolites, transcripts, and genes enable biomarker discovery in early- and late-stage pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A038.

Pancreatic cancer is the seventh leading cause of cancer-related death worldwide and the third leading cause of cancer-related death in the USA. The low survival rate of pancreatic cancer is due to the challenges in early detection of disease, highlighting the need for early diagnostic test development. While cancer signatures are less challenging to identify at the localized pancreatic tumor via biopsy, cancer signals found in the bloodstream due to cellular leakage, metastasis, signaling, innate immune response, remain of key interest due to reduced invasive sampling. The key challenges encountered in liquid biopsy cancer biomarker discovery studies are analyte degradation and dilution in a complex biological matrix, which limits high specificity and sensitivity measurements. To overcome these challenges, PrognomiQ has developed a comprehensive multi-omics platform that facilitates uncovering previously untapped information to gain a more holistic biological perspective at unprecedented depths and integrate molecular signatures across complex levels of biology. Implementation of this approach has led to the discovery of new pancreatic cancer-specific biomarkers and a deeper understanding of the integrated pathways of pancreatic cancer. In this case-control study, the plasma proteome and metabolome data were collected from 193 human plasma samples comprising 92 pancreatic cancer and 104 healthy subjects utilizing liquid chromatography-mass spectrometry. Subject samples were collected post-diagnosis, but pre-treatment for cancer subjects versus non-cancer controls. Sample collection and handling were the same for all samples. In our initial analysis, we detected 3,381 proteins in all samples (minimum of 3 samples per class), and utilizing a Bonferroni correction (FDR = 0.05) we showed 124 proteins to be statistically significant. We also determined ~200 lipids out of 678 total lipids and 49 of 299 metabolites present in all samples (minimum of 3 samples per class) to be statistically significant with a Bonferroni correction (FDR = 0.05). The detected analytes (proteins, lipids, and metabolites) are both known, and unknown, to have an association with pancreatic cancer. Analysis of the data collected from the described cohort will continue to determine Analysis is ongoing to integrate the multi-omics datasets and determine multivariate statistical performance to detect pancreatic cancer. Conclusion: The intent of this cohort and study was to detect biological signal for pancreatic cancer, and this preliminary analysis suggests there are significant differences between classes in the samples as collected. It remains to be seen if combining features within and across analyte classes improve detection. This is a case-control study, not an intent to test study but shows promise for the detection of pancreatic cancer across a multitude of analyte classes. Citation Format: Bruce Wilcox, John Blume, Kavya Swaminathan, Preston Williams, Manoj Khadka, Jared Deyarmin, Saividya Ramaswamy, Yuya Kodama, Brian Young, Chinmay Belthangady, Manway Liu, Mi Yang, Philip Ma. Deep, unbiased multi-omics approach for identification of pancreatic cancer biomarkers from blood [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 3924.

Introduction: Comprehensive assessment of the proteome remains elusive because of proteoforms arising from alternative splicing, allelic variation, and protein modifications. Characterization of the variable protein forms, or proteoforms will expand our understanding of the molecular mechanisms underlying diseases, however requires unbiased protein coverage at sufficient scale. Scalable, deep and unbiased proteomics studies have been impractical due to cumbersome and lengthy workflows required for complex samples, like blood plasma. Here, we demonstrate the power of Proteograph in a proof-of-concept proteogenomic analysis of 80 healthy controls and 61 early-stage non-small-cell lung cancer (NSCLC) samples to dissect differences between protein isoforms arising from alternative gene splicing, as well as the identification of novel peptides arising from allelic variation. Materials, Methods and Results: Processing the 141 plasma samples with Proteograph yielded 21,959 peptides corresponding to 2,499 protein groups. Using peptides with significant abundance differences (p < 0.05; Benjamini-Hochberg corrected), we extracted proteins comprised of peptides where at least one peptide had significantly higher plasma abundance, and another significantly lower plasma abundance in controls vs. cancer, resulting in a set of putative proteoforms. For three of these proteins, the abundance variation is possibly explained by underlying protein isoforms. To identify protein variants, we performed exome sequencing on 29 individuals from the NSCLC study, created personalized mass spectrometry search libraries for each individual, and identified 464 protein variants. Conclusions: Proteograph can generate unbiased and deep plasma proteome profiles that enable identification of protein variants and peptides present in plasma, at a scale sufficient to enable population-scale proteomic studies. Citation Format: Margaret Donovan, Henry Huang, John Blume, Marwin Ko, Ryan Benz, Theodore Platt, Juan Cuevas, Serafim Batzoglou, Asim Siddiqui, Omid Farokhzad. Deep, unbiased and peptide-centric plasma proteomics with differential analysis of proteoforms enabling proteogenomic studies of NSCLC at scale [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 6340.