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Our previous work showed that in a mouse model of gastric adenocarcinoma with loss of p53 and Cdh1 that adding oncogenic Kras (a.k.a. Tcon mice) accelerates tumorigenesis and metastasis. Here, we sought to examine KRAS activation in epithelial-to-mesenchymal transition (EMT) and generation of cancer stem–like cells (CSC). Transduction of nontransformed HFE-145 gastric epithelial cells with oncogenic KRASG12V significantly decreased expression of the epithelial marker E-cadherin, increased expression of the mesenchymal marker vimentin and the EMT transcription factor Slug, and increased migration and invasion by 15- to 17-fold. KRASG12V also increased expression of self-renewal proteins such as Sox2 and increased spheroid formation by 2.6-fold. In tumor-derived organoids from Tcon mice, Kras knockdown decreased spheroid formation, expression of EMT-related proteins, migration, and invasion; similar effects, as well as reversal of chemoresistance, were observed following KRAS knockdown or MEK inhibition in patient tumor-derived gastric adenocarcinoma cell lines (AGS and KATOIII). KRAS inhibition in gastric adenocarcinoma spheroid cells led to reduced AGS flank xenograft growth, loss of the infiltrative tumor border, fewer lung metastases, and increased survival. In a tissue microarray of human gastric adenocarcinomas from 115 patients, high tumor levels of CD44 (a marker of CSCs) and KRAS activation were independent predictors of worse overall survival. In conclusion, KRAS activation in gastric adenocarcinoma cells stimulates EMT and transition to CSCs, thus promoting metastasis.Implications:This study provides rationale for examining inhibitors of KRAS to block metastasis and reverse chemotherapy resistance in gastric adenocarcinoma patients.

Supplementary Table 1. Clinicopathologic characteristics of patients whose tumor samples were included in the tissue microarray (Figure 2).

Supplementary Figure 1. KRAS knockdown inhibits EMT in tumor organoids from a GA GEMM. Supplementary Figure 2. Oncogenic KRAS promotes EMT and acquisition of CSC phenotypes in gastric epithelial cells. Supplementary Figure 3. RTK-RAS pathway controls CSC phenotypes in human GA-derived spheroids. Supplementary Figure 4. MEK inhibition of the RTK-RAS pathway. Supplementary Figure 5. CD44(+) vs. CD44(-) spheroid cells. Supplementary Figure 6. Chemoresistance. Supplementary Figure 7. KRAS knockdown inhibits EMT and infiltrative behavior of human GA-derived cells.

Supplementary Figure Legends 1-7

Our previous work showed that KRAS activation in gastric cancer cells leads to activation of an epithelial-to-mesenchymal transition (EMT) program and generation of cancer stem-like cells (CSCs). Here we analyze how this KRAS activation in gastric CSCs promotes tumor angiogenesis and metastasis. Gastric cancer CSCs were found to secrete pro-angiogenic factors such as vascular endothelial growth factor A (VEGF-A), and inhibition of KRAS markedly reduced secretion of these factors. In a genetically engineered mouse model, gastric tumorigenesis was markedly attenuated when both KRAS and VEGF-A signaling were blocked. In orthotropic implant and experimental metastasis models, silencing of KRAS and VEGF-A using shRNA in gastric CSCs abrogated primary tumor formation, lymph node metastasis, and lung metastasis far greater than individual silencing of KRAS or VEGF-A. Analysis of gastric cancer patient samples using RNA sequencing revealed a clear association between high expression of the gastric CSC marker CD44 and expression of both KRAS and VEGF-A, and high CD44 and VEGF-A expression predicted worse overall survival. In conclusion, KRAS activation in gastric CSCs enhances secretion of pro-angiogenic factors and promotes tumor progression and metastasis.

Circulating cell-free DNA (ccfDNA) is used increasingly as a cancer biomarker for prognostication, as a correlate for tumor volume, or as input for downstream molecular analysis. Determining optimal blood processing and ccfDNA quantification are crucial for ccfDNA to serve as an accurate biomarker as it moves into the clinical realm. Whole blood was collected from 50 subjects, processed to plasma, and used immediately or frozen at -80°C. Plasma ccfDNA was extracted and concentration was assessed by real-time quantitative PCR (qPCR), fluorimetry, and droplet digital PCR (ddPCR). For the 24 plasma samples from metastatic pancreatic cancer patients, the variant allele fractions (VAF) of KRAS G12/13 pathogenic variants in circulating tumor DNA (ctDNA) were measured by ddPCR. Using a high-speed (16,000 × g) or slower-speed (4100 × g) second centrifugation step showed no difference in ccfDNA yield or ctDNA VAF. A two- versus three-spin centrifugation protocol also showed no difference in ccfDNA yield or ctDNA VAF. A higher yield was observed from fresh versus frozen plasma by qPCR and fluorimetry, whereas a higher yield was observed for frozen versus fresh plasma by ddPCR, however, no difference was observed in ctDNA VAF. Overall, our findings suggest factors to consider when implementing a ccfDNA extraction and quantification workflow in a research or clinical setting.

Our previous work showed that KRAS activation in gastric cancer cells leads to activation of an epithelial-to-mesenchymal transition (EMT) program and generation of cancer stem-like cells (CSCs). Here we analyze how this KRAS activation in gastric CSCs promotes tumor angiogenesis and metastasis. Gastric cancer CSCs were found to secrete pro-angiogenic factors such as vascular endothelial growth factor A (VEGF-A), and inhibition of KRAS markedly reduced secretion of these factors. In a genetically engineered mouse model, gastric tumorigenesis was markedly attenuated when both KRAS and VEGF-A signaling were blocked. In orthotropic implant and experimental metastasis models, silencing of KRAS and VEGF-A using shRNA in gastric CSCs abrogated primary tumor formation, lymph node metastasis, and lung metastasis far greater than individual silencing of KRAS or VEGF-A. Analysis of gastric cancer patient samples using RNA sequencing revealed a clear association between high expression of the gastric CSC marker CD44 and expression of both KRAS and VEGF-A, and high CD44 and VEGF-A expression predicted worse overall survival. In conclusion, KRAS activation in gastric CSCs enhances secretion of pro-angiogenic factors and promotes tumor progression and metastasis. Citation Format: Changhwan Yoon, Jun Lu, Yukyung Jun, Bang- Jin Kim, Jacob Till, Jong Hyun Kim, Sara Keshavjee, Sandra Ryeom, Sam Yoon. KRAS activation in gastric cancer stem-like cells promotes tumor angiogenesis and metastasis [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 2451.

We have previously demonstrated an independent association between high levels of plasma circulating cell-free DNA (ccfDNA) concentration and poor survival outcomes in patients with newly diagnosed glioblastoma. Given that somatic mutations are rarely detected in glioblastoma patient plasma, we reasoned that DNA shed by tumor (circulating tumor DNA, ctDNA) was not likely to be a driver of prognostic increases in ccfDNA. To investigate the tissue of origin for this prognostic ccfDNA, we analyzed the plasma ccfDNA methylation signatures of 23 patients with newly diagnosed glioblastoma using the Illumina Infinium EPIC array, a genome-wide DNA methylation technique covering over 850,000 CpG sites. Deconvolution of the ccfDNA methylation signatures based on published reference methylomes revealed an increased proportion of ccfDNA derived from granulocytes in glioblastoma specimens compared to healthy controls (p < 0.0001). Further, this granulocyte proportion was increased in patients with high ccfDNA levels (above median value) compared to patients with low ccfDNA (p = 0.0001). Granulocyte proportion correlated with ccfDNA concentration (Spearman r = 0.64, p = 0.001). The top two gene sets identified by differential methylation analysis followed by gene set enrichment analysis (methylGSA) between high- and low-ccfDNA specimens were “neutrophil activation involved in immune response” (GO:0002283, p = 3.4E-23) and “neutrophil degranulation” (GO:0043312, p = 3.4E-23). Additional analysis of ccfDNA fragment size identified larger fragments ( > 700 bp) as the major source of the prognostic signal (log-rank p = 0.002 for progression-free survival) compared to traditionally sized ccfDNA fragments (50-700 bp, log-rank p = 0.12). Taken together, these data suggest that granulocytes are the primary contributing source of prognostic ccfDNA in glioblastoma. Future studies are needed to determine the mechanism through which granulocyte-derived ccfDNA is associated with clinical outcomes in glioblastoma and to explore related therapeutic opportunities.

BACKGROUND GBM genomic profiling relies on sequencing a limited tumor tissue sample, which is invasive and may underrepresent molecular heterogeneity. CSF, which can be obtained less invasively, is in intimate contact with tumor lesions and may better capture the full genomic profile of the tumor. However, datasets with contemporaneously collected CSF and tissue to support this claim have been lacking. To evaluate the performance of CSF NGS, we conducted a pilot study in patients with GBM undergoing a resection for suspected recurrence following first-line chemoradiotherapy. METHODS Paired CSF and tissue samples were sequenced using a hybrid capture-based NGS assay. Clinically meaningful variants were defined as those that are potentially targetable using off-label or clinical trial options, exhibit prognostic value, or may predict response or resistance to specific treatments. RESULTS Eighteen patients were enrolled, and 13 of 18 CSF samples (72.2%) were sequenced successfully. At least one variant was detected in all CSF samples analyzed. A median of 7 variants (range 1—67) was detected per sample across 54 genes. The median variant allele fraction was 0.6% (range 0.2—72.4%). Among 38 clinically meaningful genes, 102 variants were detected; 25 (24.5%) were detected in both tissue and CSF, while 60 (58.8%) were detectable solely in CSF. Hypermutation was detected by CSF in one patient. Of the 82 variants detected in this patient’s tumor, 15 (18.3%) were identified in both tissue and CSF, 15 (18.3%) were identified only in the tissue, and 52 (63.2%) were identified only in the CSF. CONCLUSIONS CSF NGS detects clinically meaningful variants at a substantial rate and frequently identifies mutations not detected by matched tissue NGS. These results suggest that CSF may be a suitable source material for tumor profiling, overcoming the limitations of tissue, and may also provide a more comprehensive tumor profile.

PURPOSE Differentiation of tumor progression (TP) from pseudoprogression (PsP) is a major unmet need in post-treatment glioblastoma (GBM). 18F-Fluciclovine is a synthetic amino acid PET radiotracer with higher uptake in tumor tissue vs. areas of treatment-related change. We investigated the value of 18F-Fluciclovine PET for differentiating PsP from TP independent from and in combination with multi-parametric MRI. METHODS We prospectively enrolled 30 patients with GBM with a new or enlarging contrast-enhancing lesion on MRI after chemoradiotherapy who were planned for surgical resection of the lesion. Patients underwent pre-operative 18F-Fluciclovine PET and multi-parametric MRI. Following surgery, the relative percentages of viable tumor and therapy-related changes observed in histopathology were quantified. Patients were categorized as TP if viable tumor represented ≥ 50% of the specimen, mixed TP if < 50% and > 10%, and PsP if ≤ 10%. RESULTS 18 patients had TP, 4 had mixed TP, and 8 PsP. Patients with TP/mixed TP had a significantly higher 40-50 minutes SUVmax (6.64 + 1.88 vs 4.11± 1.52, p=0.009) and an SUVmax cut-off of 4.66 provided 90% sensitivity and 83% specificity for differentiation of TP/mixed TP from PsP (AUC=0.856). A maximum cerebral blood volume (CBVmax) cut-off of 3.67 provided 90% sensitivity and 71% specificity for differentiation of TP/mixed TP from PsP (AUC=0.779). Combining a 40-50 minutes SUVmax cut-off of 4.662 and a relative CBVmax cut-off of 3.67 provided 100% sensitivity and 80% specificity for differentiating TP/mixed TP from PsP (AUC=0.95). The time activity curve patterns and time to peaks were not different between the groups. Normalization of PET parameters to normal brain parenchyma were not helpful to differentiate the groups due to variability in radiotracer uptake in normal brain between subjects. CONCLUSION 18F-Fluciclovine PET uptake can accurately differentiate PsP from TP in GBM patients, with even more accurate differentiation achieved when combined with MRI.

BACKGROUND We have previously demonstrated that high baseline plasma cfDNA concentration is associated with poor survival in patients with newly diagnosed GBM. The mechanism of this association remains unknown. To explore whether differences in the immune landscape between high- vs. low-cfDNA patients may play a role in their divergent clinical outcomes, we phenotyped tumors from patients with high vs. low cfDNA using mass cytometry by time of flight (CyTOF). METHODS We performed CyTOF on frozen tumor infiltrate suspension from a pilot cohort of patients with previously untreated GBM with known baseline plasma cfDNA concentration (Bagley, Clin Cancer Res 2020). CyTOF was used to simultaneously measure expression of 39 molecules related to immune cell lineage, differentiation state, and function. Differences in immune cell infiltrates between high- and low-cfDNA patients were assessed using Mann-Whitney U tests. RESULTS Four patients with high cfDNA (median 57, range 33-90 ng/mL) were compared to six patients with low cfDNA (median 12, range 7-16 ng/mL). Immune cell infiltrates with increased adaptive cells (high monocytes and T cells, p=0.05) were present in high-cfDNA compared to low-cfDNA patients. While > 70% of the infiltrating T cells were exhausted in both groups, the pattern of exhaustion was significantly different in high- vs. low-cfDNA patients, with less CXCR5+CD69+ and more CXCR5-CD69- (p=0.008) progenitor exhausted T cells in cfDNA-high patients. CONCLUSIONS In this GBM pilot study, we demonstrated differences in the tumor immune infiltrate in patients with high vs. low baseline plasma cfDNA concentration. Preclinical studies will be needed to determine if this explains the association between high plasma cfDNA and poor outcomes previously observed in patients. Our results may have implications for the use of cfDNA concentration as a predictive biomarker for immunotherapy, as tumors with more intermediate progenitor (CXCR5-CD69-) exhausted T cells may respond better to PD-1 checkpoint blockade.

BACKGROUND Plasma circulating tumor DNA (ctDNA) is rarely detectable by traditional methods in patients with GBM. As a result, unlike in lung and other cancers, serial next generation sequencing of ctDNA for monitoring GBM tumor burden has been challenging. In light of the low tumor fraction (TF) of DNA fragments in GBM patient plasma and the urgent need to improve upon MRI for tracking GBM tumor burden, we conducted a pilot study in patients with newly diagnosed GBM using the C2 intelligence platform (C2i Genomics), which leverages genome-wide mutational integration for highly sensitive ctDNA detection. METHODS Plasma was collected pre- and post-operatively in patients with newly diagnosed GBM undergoing surgical resection/biopsy. cfDNA was extracted, quantified, and analyzed for fragment size. Genomic DNA (gDNA) was extracted from matched tumor tissue. Whole genome sequencing (WGS) was performed on both gDNA and cfDNA. A specific copy number alteration (CNA) compendium was created for each patient to generate a readout of TF (Zviran, Nat Medicine 2020). We assessed the association between TF at post-operative day 1 (a surrogate for residual disease) and OS, adjusting for other prognostic factors using Cox regression. RESULTS 37 patients were enrolled. For samples with high tumor fraction (n=5), a statistically significant (p< 1e-4) correlation between CNA profiles of tumor tissue and plasma samples was observed. Post-operative TF above the median value was associated with inferior OS (median 7.7 vs. 19.3 months, p=0.019). This association persisted after adjusting for age, O6-methylguanine-DNA methyltransferase methylation status, extent of resection, and performance status (adjusted HR 2.5, 95% CI 1.1-5.6, p=0.03). CONCLUSION Genome-wide mutational integration enables ultra-sensitive detection of ctDNA in GBM patient plasma. Post-operative TF measured by the C2i test is independently associated with OS in newly diagnosed GBM, providing the foundation to evaluate this technology for personalized prognostication and disease monitoring.