Abstract Introduction: Immune checkpoint inhibitors (ICI) are an important therapeutic option for patients with triple negative breast cancer (TNBC). However, identification of patients most likely to respond is challenging. PD-L1 positivity by immunohistochemistry is the standard biomarker used for ICI therapy selection in TNBC. However, other biomarkers, such as analysis of the tumor microenvironment (TME) may be more accurate in predicting response. The XernaTM TME Panel uses RNA sequencing data and machine learning to analyze the TME, utilizing the angiogenic and immunogenic biology of the TME to classify tumors into four TME subtypes. In this study, the distribution of Xerna TME subtypes and associated genomic alterations in TNBC were investigated for their potential use in therapy selection. Methods: A total of 203 TNBC patient samples underwent tumor-normal whole-exome, whole-transcriptome sequencing testing with the OncoExTraTM assay. The whole-transcriptome expression data were analyzed using the Xerna TME Panel to assign each sample to one of four subtypes: Immune Active (IA), Immune Suppressed (IS), Immune Desert (ID) and Angiogenic (A). The IA and IS subtypes both have high immune scores that may be particularly sensitive to ICI therapy. Actionable alterations, defined as those with FDA-approved matched therapies in any cancer, with matched clinical trials, or with evidence in cancer guidelines or the literature for possible matched therapies, were also identified and associations across Xerna subtypes were explored. Results: Approximately half (100 of 203; 49.3%) of the patient samples had high (IA+IS) immune subtypes (Table 1). Targetable alterations associated with an FDA-approved therapy were present in 114 (56.2%) patients. No biomarkers were significantly associated (p < 0.05) with high (IA+IS) versus low (ID+A) immune scores. Biomarkers associated with ICI response, namely mismatch repair gene alterations (MSH2/3/6, MLH1/3, PMS1/2), high tumor mutational burden (TMB-high) and microsatellite instability were detected in only 6 (3.0%), 3 (1.5%) and 1 (0.5%) patient samples respectively, and all but 1, an MSH6 alteration, were in high immune subtype samples. Conclusions: The Xerna TME Panel classified 49.3% of TNBC patient tumors to IA or IS, suggesting they may respond to ICI therapy. Many (56.2%) patient tumors harbored alterations associated with FDA-approved therapies, providing the potential for novel combination therapies. These findings warrant further study and clinical validation in TNBC patients treated with ICI therapy. Table 1. Frequency of actionable biomarkers that were present in at least 10 (5%) TNBC patient samples. Citation Format: Gargi Basu, Janine Lobello, Snehal Thakkar, Jessica Aldrich, Matthew Halbert, Patrick Eimerman, Cynthia Flannery, Nishitha Therala, David Hall, Daniel Pointing, Lea Vohar, Roman Luštrik, Luka Ausec, Mark Uhlik, Seema Iyer, Laura Benjamin, Frederick Baehner. Prevalence of genomic alterations in Xerna tumor microenvironment subtypes in triple negative breast cancer patients [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-06-10.

Background In advanced colorectal cancer (CRC), analysis of the tumor microenvironment (TME) may be useful as a predictive biomarker, particularly supporting the use of immunotherapies and anti-angiogenic therapies.1 The XernaTM TME Panel utilizes RNA sequencing data and machine learning to analyze the angiogenic and immunogenic biology of the TME to classify tumors into four TME subtypes.2 In this study, we investigated the distribution of Xerna TME subtypes and associated genomic alterations in CRC for their potential use in therapy selection. Methods A total of 336 CRC patient samples underwent testing with the OncoExTraTM assay. This assay utilizes whole-exome, whole-transcriptome sequencing to identify actionable alterations, defined as those with FDA-approved matched therapies in any cancer, with matched clinical trials, or with evidence in cancer guidelines or the literature for possible matched therapies. The whole-transcriptome expression data were analyzed using the Xerna TME Panel to assign each sample to one of four subtypes: Immune Active (IA), Immune Suppressed (IS), Immune Desert (ID) and Angiogenic (A). Biomarker associations were explored. Results Approximately half (49.4%) of the patient samples had high (IA+IS) versus low (ID+A) immune subtypes, and 247 (73.5%) harbored targetable alterations associated with an FDA-approved therapy. Several biomarkers were significantly associated (p<0.05) with Xerna subtypes, most of which were over-represented in high immune subtypes (19 of 21), with 13 indicative of defective DNA repair (table 1). Microsatellite instability (MSI-high) and high tumor mutational burden (TMB-high) were detected in 30 (8.9%) and 37 (11.0%) patient samples, with 28 (16.9%) and 33 (19.9%) occurring within high immune subtypes (IA+IS), respectively. Some MSI-high and TMB-high samples occurred in low immune subtypes (ID+A), perhaps indicating a lower propensity for response to ICI therapy. Of note, 138 of 306 (45.1%) MSI-low and 133 of 299 (44.5%) TMB-low samples were in the high immune subtypes, suggestive of possible sensitivity to ICI therapy. Actionable KRAS/NRAS, and BRAF alterations were detected in 162 (48.2%) and 23 (6.8%) patients respectively, though none were significantly associated with TME subtypes. Conclusions The Xerna TME Panel classified 49.4% of CRC patients to IA or IS subtypes who may benefit from ICI therapy, including many lacking biomarkers currently used for this therapy decision. Most (73.5%) patients harbored alterations associated with FDA-approved therapies, providing the potential for novel combination therapies.3 These findings warrant further study and clinical validation in CRC patients treated with ICI therapy. References Huyghe N, Benidovskaya E, Stevens P, Van den Eynde M. Biomarkers of Response and Resistance to Immunotherapy in Microsatellite Stable Colorectal Cancer: Toward a New Personalized Medicine. Cancers (Basel). 2022 Apr 29;14(9):2241. doi: 10.3390/cancers14092241. PMID: 35565369; PMCID: PMC9105843. Uhlik M, Pointing D, Iyer S, Ausec L, Štajdohar M, Cvitkovič R, Žganec M, Culm K, Santos VC, Pytowski B, Malafa M, Liu H, Krieg AM, Lee J, Rosengarten R, Benjamin L. Xerna™ TME Panel is a machine learning-based transcriptomic biomarker designed to predict therapeutic response in multiple cancers. Front Oncol. 2023 May 12;13:1158345. doi: 10.3389/fonc.2023.1158345. PMID: 37251949; PMCID: PMC10213262. Yang Z, Wu G, Zhang X, Gao J, Meng C, Liu Y, Wei Q, Sun L, Wei P, Bai Z, Yao H, Zhang Z. Current progress and future perspectives of neoadjuvant anti-PD-1/PD-L1 therapy for colorectal cancer. Front Immunol. 2022 Sep 9;13:1001444. doi: 10.3389/fimmu.2022.1001444. PMID: 36159842; PMCID: PMC9501688. Ethics Approval The study was approved by WCG IRB Ethics Board, approval number 20181863.

Background Despite immune checkpoint inhibitor (ICI) monotherapy approvals in NSCLC, SOC predominately utilizes combinations of ICI with non-targeted chemotherapy or precision therapies targeting oncogenic drivers. Biomarkers guiding these clinical decisions rely on tumor genotyping to identify actionable mutations, tumor mutational burden (TMB) and on immunohistochemistry for PD-L1 expression. Currently, neither PD-L1 nor TMB perform adequately for ICI patient selection.1 Emerging evidence indicates a more complete profile of the tumor microenvironment (TME) may improve selection of patients likely to respond to ICI.2 The Xerna machine learning-based RNA sequencing biomarker assay classifies tumors into four TME subtypes; Immune Active (IA), Immune Suppressed (IS), Immune Desert (ID) and Angiogenic (A) . This classification identifies tumors likely to benefit from ICI (IA and IS) or anti-angiogenic agents (ID and A).3 We examined the distribution of actionable oncogenic driver mutations across Xerna TME subtypes to investigate the potential use for therapy selection. Methods Biomarker prevalence, and Xerna TME subtype classification, were determined for 104 metastatic lung cancer cases previously analyzed using the OncomapTM ExTra test, tumor-normal whole-exome and whole-transcriptome sequencing. DNA variants and high TMB (≥10 mut/Mb) were identified from DNA sequencing, and RNA expression levels were used to assign tumors to Xerna subtypes. Biomarker and associations were compared using Fisher's Exact Test. The study was approved by WCG IRB Ethics Board, approval number 20181863. Results In total, 53% of cases had high (IA+IS) vs. low (ID+A) Xerna immune subtypes and 60% harbored targetable oncogenic driver mutations (table 1). Actionable EGFR and KRAS mutations were detected in 31% and 20% of cases respectively, while high TMB was detected in 27% of cases. High TMB was significantly higher in IA (62%) vs. IS (14%) or A (13%) categories (p<0.05). Although no significant associations between Xerna subtype and oncogenic drivers were observed, EGFR mutations were least frequent in IA tumors (15%) while 33% of the IS subtype contained KRAS mutations (10% G12C). Conclusions The Xerna TME panel identified a high prevalence of patients who may benefit from ICI (IA+IS) and harbored actionable oncogenic drivers. Within this group, the prevalence of targetable oncogenic drivers within the IS phenotype, such as KRAS G12C, may represent the potential for novel ICI combination therapies.4 These findings further highlight the importance of adding TME analysis to comprehensive biomarker testing in NSCLC References Steuer CE, Ramalingam SS. Advances in Immunotherapy and Implications for Current Practice in Non-Small-Cell Lung Cancer. JCO Oncol Pract. 2021 Nov;17(11):662–668. doi: 10.1200/OP.21.00305. Epub 2021 Jun 25. Erratum in: JCO Oncol Pract. 2022 Mar;18(3):244. Horvath L, Thienpont B, Zhao L, Wolf D, Pircher A. Overcoming immunotherapy resistance in non-small cell lung cancer (NSCLC) – novel approaches and future outlook. Mol Cancer. 2020 Sep 11;19(1):141. Iyer, S., Ausec, L., Pointing, D., Zganec, M., Cvitkovic, R., Stajdohar, M., ... & Uhlik, M. T. (2022). Xerna? TME Panel: A pan-cancer RNA-based investigational assay designed to predict patient responses to angiogenic and immune targeted therapies.? Cancer Research,? 82(12_Supplement), 1232–1232. Mugarza E, van Maldegem F, Boumelha J, Moore C, Rana S, Llorian Sopena M, East P, Ambler R, Anastasiou P, Romero-Clavijo P, Valand K, Cole M, Molina-Arcas M, Downward J. Therapeutic KRASG12C?inhibition drives effective interferon-mediated antitumor immunity in immunogenic lung cancers. Sci Adv. 2022 Jul 22;8(29) Ethics Approval The study was approved by WCG IRB Ethics Board, approval number 20181863.

Our novel, enzymatic DNA synthesis (EDS) technology was made through engineering of three critical components: a DNA polymerase, a solid support, and reversible terminators. Terminal deoxynucleotidyl-Transferase (TdT), which performs poly-nucleotide additions in a template-free manner, was engineered for improved expression, stability, and the ability to incorporate non-natural deoxynucleotide triphosphate bases. Novel surface chemistries have been developed to allow TdT to incorporate DNA bases on paramagnetic beads, resins, and glass as solid supports for EDS. Synthesis is enabled by the use of 3â€-O-blocked reversible terminators, which suppresses poly-nucleotide addition by TdT until the 3'OH group on the extended polymer is deprotected. Here, we describe the development and key functionality of a benchtop EDS system for lab use. Unlike phosphoramidite synthesis, EDS produces DNA in the 'biological' orientation (i.e. 5'->3'), with an intact 5'-phosphate group. EDS provides a scalable synthesis system that eliminates the need for solvents, like acetonitrile, minimizing organic waste and decreasing research's carbon footprint, allowing users to make oligos without the need for specialized training and delivering oligos for iterative experiments without having to wait for a centralized oligo manufacturer. Current data indicates that purity levels comparable to those of existing technologies can be achieved, with an average per-position error rate <1%. For oligos up to 280nt long, we have demonstrated a cycle efficiency of 99.4%, which is greater than or equivalent to that of standard phosphoramidite synthesis. Using PCR, qPCR, dPCR, and sequencing assays we have demonstrated comparable performance to the same sequences synthesized using conventional chemistry. EDS is a disruptive technology that enables simple, robust systems capable of supporting same-day production of high quality, custom oligos. On-demand access to oligos revolutionize genomics research.