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BACKGROUND: Circulating tumour DNA (ctDNA) has been spotlighted as an attractive biomarker because of its easy accessibility and real-time representation of tumour genetic profile. However, the clinical utility of longitudinal ctDNA monitoring has not been clearly defined. METHODS: Serial blood samples were obtained from metastatic colorectal cancer patients undergoing first-line chemotherapy. ctDNA was sequenced using a targeted next-generation sequencing platform which included 106 genes. Changes in ctDNA profile and treatment outcome were comprehensively analysed. RESULTS: A total of 272 samples from 62 patients were analysed. In all, 90.3% of patients had detectable ctDNA mutation before treatment. ctDNA clearance after chemotherapy was associated with longer progression-free survival which was independent of radiological response (adjusted hazard ratio 0.22, 95% confidence interval 0.10–0.46). Longitudinal monitoring was able to detect ctDNA progression which preceded radiological progressive disease (PD) in 58.1% (median 3.3 months). Diverse resistant mutations (34.9%) and gene amplification (7.0%) at the time of PD were discovered. For 16.3% of the PD patients, the newly identified mutations could be potential candidates of targeted therapy or clinical trial. CONCLUSION: ctDNA profile provided a more accurate landscape of tumour and dynamic changes compared to radiological evaluation. Longitudinal ctDNA monitoring may improve personalised treatment decision-making.

Patients with hematuria are commonly given an invasive cystoscopy test to detect bladder cancer (BC). To avoid the risks associated with cystoscopy, several urine-based methods for BC detection have been developed, the most prominent of which is the deep sequencing of urine DNA. However, the current methods for urine-based BC detection have significant levels of false-positive signals. In this study, we report on uAL100, a method to precisely detect BC tumor DNA in the urine without tumor samples. Using urine samples from 43 patients with BC and 21 healthy donors, uAL100 detected BC with 83.7% sensitivity and 100% specificity. The mutations identified in the urine DNA by uAL100 for BC detection were highly associated with BC tumorigenesis and progression. We suggest that uAL100 has improved accuracy compared to other urine-based methods for early BC detection and can reduce unnecessary cystoscopy tests for patients with hematuria.

Traditional tissue-based assessments of genomic alterations in castration-resistant prostate cancer (CRPC) can be challenging. To evaluate the real-world clinical utility of liquid biopsies for the evaluation of genomic alterations in CRPC, we preemptively collected available plasma samples and archival tissue samples from patients that were being treated for clinically confirmed CRPC. The cell-free DNA (cfDNA) and tumor tissue DNA were analyzed using the AlphaLiquid®100-HRR panel. Plasma samples from a total of 87 patients were included in this study. Somatic mutations from cfDNA were detected in 78 (89.7%) patients, regardless of the presence of overt metastasis or concomitant treatment given at the time of plasma sample collection. Twenty-three patients were found to have known deleterious somatic or germline mutations in HRR genes from their cfDNA. Archival tissue samples from 33 (37.9%) patients were available for comparative analysis. Tissue sequencing was able to yield an NGS result in only 51.5% of the tissue samples. The general sensitivity of cfDNA for detecting somatic mutations in tissues was 71.8%, but important somatic/germline mutations in HRR genes were found to have a higher concordance (100%). Liquid biopsies can be a reasonable substitute for tissue biopsies in CRPC patients when evaluating genomic alterations.

PDF file - 114K, Chemical structures of investigated molecules in this article

PDF file - 135K, Treatment of saracatinib in LS174T and SKBR3 cells

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PDF file - 173K, Induction of Bim by saracatinib treatment

A PARP inhibitor is a rationally designed targeted therapy for cancers with impaired DNA repair abilities. RAD51C is a paralog of RAD51 that has an important role in the DNA damage response. We found that cell lines sensitive to a novel oral PARP inhibitor, olaparib, had low levels of RAD51C expression using microarray analysis, and we therefore hypothesized that low expression of RAD51C may hamper the DNA repair process, resulting in increased sensitivity to olaparib. Compared with the cells with normal RAD51C expression levels, RAD51C-deficient cancer cells were more sensitive to olaparib, and a higher proportion underwent cell death by inducing G2–M cell-cycle arrest and apoptosis. The restoration of RAD51C in a sensitive cell line caused attenuation of olaparib sensitivity. In contrast, silencing of RAD51C in a resistant cell line enhanced the sensitivity to olaparib, and the number of RAD51 foci decreased with ablated RAD51C expression. We also found the expression of RAD51C was downregulated in cancer cells due to epigenetic changes and RAD51C expression was low in some gastric cancer tissues. Furthermore, olaparib significantly suppressed RAD51C-deficient tumor growth in a xenograft model. In summary, RAD51C-deficient cancer cells are highly sensitive to olaparib and offer preclinical proof-of-principle that RAD51C deficiency may be considered a biomarker for predicting the antitumor effects of olaparib. Mol Cancer Ther; 12(6); 865–77. ©2013 AACR.

PDF file - 96K, Downregulation of thymidylate synthase (TS) by saracatinib

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PDF file - 3250K, Figure S1 Olaparib has variable levels of anti-tumor activity in human cancer cell lines. Figure S2 Olaparib sensitive cell lines have different patterns of gene expression compared to insensitive cell lines. Figure S3 PTEN expression does not affect olaparib sensitivity. Figure S4 RAD51C expression affects olaparib sensitivity in human cancer cell lines. Figure S5 RAD51C over-expression decreases sensitivity to olaparib in the BT-549 cell line. Figure S6 Olaparib induces G2/M cell cycle arrest and apoptosis in sensitive cell lines. Figure S7 RAD51C depletion increases the accumulation of DNA damage. Figure S8 RAD51C expression decreases in tumor tissues. Figure S9 RAD51C expression is down-regulated in gastric tumor tissues via DNA methylation. Table S1. Sequences of the primers for PCR and real-time PCR analyses. Table S2. BRCA1 and BRCA2 mutations in the gastric cancer cell lines.

PDF file - 215K, mRNA and protein expression level of integrin �8 in gastric cancer cell lines

EGFR tyrosine kinase inhibitors have shown promising efficacy in the treatment of tumors with EGFR mutations and amplifications. However, tyrosine kinase inhibitors have also proven ineffective against most tumors with EGFR wild-type (WT) alleles. Although some genetic changes, including the KRAS mutation, have been shown to confer resistance to tyrosine kinase inhibitors, novel strategies for the treatment of cancer patients with tumors harboring EGFR WT alleles have yet to be thoroughly delineated. The principal objective of this study was to improve our current understanding of drug interactions between EGFR and MAP/ERK kinase (MEK) inhibitors in an effort to gain insight into a novel therapeutic strategy against EGFR WT tumors. Using a panel of human EGFR WT gastric cancer cell lines, we showed that gastric cancer cells harboring the KRAS mutation were selectively sensitive to MEK inhibition as compared with those cells harboring KRAS and PI3K mutations and KRAS WT alleles. However, all cell lines were found to be resistant to EGFR inhibition. The results from Western blots and phosphoprotein arrays showed that, in MEK inhibitor resistant cell lines, AKT was activated through the EGFR/HER3/PI3K pathway following AZD6244 (ARRY-142886) treatment. Blockade of this feedback mechanism through the targeting of MEK and EGFR resulted in detectable synergistic effects in some cell lines in vitro and in vivo. Our results provide the basis for a rational combination strategy against human EGFR WT gastric cancers, predicated on the understanding of cross-talk between the MEK and EGFR pathways. [Mol Cancer Ther 2009;8(9):2526–36]

Supplementary Data from Combination of EGFR and MEK1/2 inhibitor shows synergistic effects by suppressing EGFR/HER3-dependent AKT activation in human gastric cancer cells

Supplementary Figures 1-2 from Enzastaurin, a Protein Kinase Cβ Inhibitor, Suppresses Signaling through the Ribosomal S6 Kinase and Bad Pathways and Induces Apoptosis in Human Gastric Cancer Cells

Activation of protein kinase C (PKC) has been implicated in gastric carcinogenesis. Enzastaurin is an oral ATP-competitive inhibitor of the PKCβ isozyme. Although enzastaurin was initially advanced to the clinic based on its antiangiogenic activity, it is also known to have a direct effect on a variety of human cancer cells, inducing apoptosis by inhibiting the Akt signal pathway. However, data on enzastaurin for gastric cancer are limited. Therefore, this study was performed to assess the antitumor activity of enzastaurin on gastric cancer cells and to investigate the underlying antitumor mechanisms. Enzastaurin suppressed the proliferation of cultured gastric cancer cells and the growth of gastric carcinoma xenografts. Enzastaurin did not have an effect on gastric cancer cell cycle progression; however, it had a direct apoptosis-inducing effect through the caspase-mediated mitochondrial pathway. Glycogen synthase kinase 3β phosphorylation, a reliable pharmacodynamic marker of enzastaurin activity, and Akt phosphorylation were both decreased after treatment with enzastaurin. Although the p90 ribosomal S6 kinase (Rsk) was also dephosphorylated, Erk phosphorylation was not affected in the enzastaurin-treated gastric cancer cells. Enzastaurin activated Bad, one of the Bcl-2 proapoptotic proteins, through dephosphorylation at Ser112, and depletion of Bad activity resulted in resistance to enzastaurin-induced apoptosis and cytotoxicity in gastric cancer cells. These data suggest that enzastaurin induces apoptosis through Rsk-mediated and Bad-mediated pathways, besides inhibiting the Akt signal cascade. Furthermore, enzastaurin had synergistic or additive effects when combined with 5-fluorouracil, cisplatin, paclitaxel, or irinotecan. These results warrant further clinical investigation of enzastaurin for gastric cancer treatment. [Cancer Res 2008;68(6):1916–26]

Background:Assessment of genomic alterations in castration-resistant prostate cancer (CRPC) patients has become an important process for treatment decisions, but traditional tissue-based evaluation can be challenging. Methods:To evaluate the clinical utility of liquid biopsy for the evaluation of genomic alterations in CRPC, we have prospectively collected plasma samples from patients being treated for clinically confirmed CRPC. The cell-free DNA (cfDNA) extracted from the plasma samples were analyzed using AlphaLiquid®100-HRR panel. Available archival tumor tissue samples were also collected and analyzed using the same panel. Results:Plasma samples from a total of 87 patients were included in this study. Somatic mutations from cfDNA were detected in 78 (89.7%) of all 87 CRPC patients. Somatic mutations were detectable regardless of the presence of overt metastasis, or concomitant treatment given at the time of plasma sample collection. 23 patients were found to have known deleterious mutations in HRR genes from their cfDNA. 11 patients were additionally found to have possibly pathogenic mutations calculated by pathogenicity prediction algorithm. Archival tissue samples were available in 33 (37.9%) of the patients, 31 from prostate and 2 from metastatic bone lesions, collected a median of 2 years before the blood sample collection. Tissue sequencing was able to yield an NGS result in only 51.5% of the tissue samples. The general sensitivity of cfDNA for detecting somatic mutations discovered from tissues was 71.8%, but important somatic/germline mutations in HRR genes were found in higher concordance (100%). Conclusions:Liquid biopsy can be a reasonable substitute for tissue biopsy in CRPC patients for evaluating genomic alterations.

Circulating tumor DNA (ctDNA) may reveal dynamic tumor status during therapy. We conducted serial ctDNA analysis to investigate potential association with clinical outcome in metastatic colorectal cancer (mCRC) patients receiving chemotherapy. Tissue KRAS/NRAS wild-type mCRC patients were enrolled and treated with first-line cetuximab-containing chemotherapy. ctDNA isolated from plasma were analyzed by next generation sequencing (NGS) with 16 targeted gene panel. Among 93 patients, 84 (90.3%) had at least 1 somatic mutation in baseline ctDNA samples (average 2.74). Five patients with KRAS or NRAS hotspot mutation in the ctDNA showed significantly worse progression-free survival (PFS) (p = 0.029). Changes in average variant allele frequency (VAF) in ctDNA showed significant correlation with tumor size change at the time of first response evaluation (p = 0.020) and progressive disease (PD) (p = 0.042). Patients whose average VAF decreased below cutoff (p p = 0.018). At the time of PD, 54 new mutations including KRAS and MAP2K1 emerged in ctDNA. ctDNA sequencing can provide mutation profile that could better reflect tumor mutation status and predict treatment outcome.

Tumor heterogeneity is one of the ongoing huddles in the field of colon cancer therapy. It is evident that there are countless clones which exhibit different phenotypes and therefore, single cell analysis is inevitable. Cancer stem cells (CSCs) are rare cell population within tumor which is known to function in cancer metastasis and recurrence. Although there have been trials to prove intra-tumoral heterogeneity using single cell sequencing, that of CSCs has not been clearly elucidated. Here, we articulate the presence of heterogeneous subclones within CD133 positive cancer stem cells through single cell sequencing. As a proof of principle, we performed phenotype-based high-throughput laser isolation and single cell sequencing (PHLI-seq) of CD133 positive cells in a frozen tumor tissue obtained from a patient with colorectal cancer. The result proved that CD133 positive cells were shown to be heterogeneous both in copy number and mutational profiles. Single cancer stem cell specific mutations such as RNF144A, PAK2, PARP4, ADAM21, HYDIN, KRT38 and CELSR1 could be also detected in liver metastatic tumor of the same patient. Collectively, these data suggest that single cell analysis used to spot subclones with genetic variation within rare population, will lead to new strategies to tackle colon cancer metastasis.

Background: Pancreatic cancer remains one of the most devastating malignancies due to lack of target therapeutic options for advanced disease. Liquid biopsy which analyzes tumor-related biomarkers in blood, has emerged as an innovative technology. The aim of this study was to identify circulating tumor DNA (ctDNA) mutations in pancreatic adenocarcinoma(PDAC) patients using multi-gene panels. Methods: Total of 45 patients (27 male, median age 63 years) blood samples were obtained at baseline then follow-up after 2 months samples were acquired in 16 patients at National Cancer Center, Korea from November 2021 to April 2022. CtDNA were analyzed by next-generation-sequencing (NGS) panel of 118 genes and the varient allele frequency (VAF) of detected genes were analyzed. Then the association between outcomes of 5FU treated response and survival (overall survival and progression-free survival; OS and PFS) and VAF reduction at follow-up were analyzed. Results: Total of 35 genes with 114 genetic alterations were detected and frequent mutations were as follows: KRAS (29.9%), TP53 (14.9%), GNAS(4.4%), SMAD4(4.4%), AR(3.5%), RNF434(3.5%), ATM(3.5%), BRCA2(2.6%) and PIK3CA(2.6%). The mean VAF level considering all pathogenic variants of 45 patients was 8.9%(range, 0 - 87.2) including 8 patients who were not detected ctDNA mutations in baseline, and the mean VAF was 4.2% (0 - 50.0) at follow-up in 16 patients. With association of clinical response, patients with partial response (PR) and stable disease (SD) were decreased VAF from 11% at baseline to 10% after 2 months, and three (60%) of patients showed clearance of ctDNA. Progressive disease (PD) patients were also decreased VAF 67% after 2 months, however, clearance of ctDNA was only observed in one patient (12.5%). Reduction of ctDNA more than 50% were associated with longer survival as PFS (> 50% reduction groups vs. no reduction group; 5 vs. 3 months, p value=0.903, OS (undefined vs. 6 months, p value =0.180). Conclusions: This study showed most common mutation was KRAS with several other mutations of ctDNA in PDAC, and outcomes represented association with ctDNA. Citation Format: SooBeen Heo, Sunhwa Park, Jung Won Chun, Yun-Hee Kim, Jun-Kyu Kang, Hwang-Phill Kim, Sang Myung Woo, Sun-Young Kong. Genetic alterations of circulating tumor DNA in advanced pancreatic cancer patients receiving 5-fluorouracil based chemotherapy [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 6683.

433 Background: Serial circulating tumor DNA (ctDNA) analysis enables us to detect minimal residual disease, to estimate recurrence and to trace evolution of cancer. However, the role of ctDNA in patients with advanced gastric cancer (AGC) who received palliative chemotherapy is still unclear. We performed this study to evaluate if serial ctDNA can predict clinical outcomes of chemotherapy and propose evolutionary findings during chemotherapy. Methods: The patients with histopathologically confirmed AGC and who received palliative chemotherapy were enrolled. Serial blood sample for ctDNA sequencing were obtained at baseline (BL) (before first-line chemotherapy), after first cycle and every 2-3 months until disease progression. ctDNA sequencing was performed for all samples using the next-generation sequencing (NGS) platform with a targeted gene panel including 106 genes. Single nucleotide variants (SNVs), insertions and deletions (Indels), and copy number variants (CNVs) were identified by ctDNA sequencing while serial ctDNA were monitored by tracing changes in the variants. ctDNA clearance was defined as no detection of any kind of variants or variant allele frequency (VAF) less than 0.1%. Results: A total of 50 patients were registered and 248 samples were collected. Median age was 62. HER2-positive GC were 5 (10.0%) and 49 received fluoropyrimidine plus platinum (1 did not receive chemotherapy). Response rate and median progression-free survival (PFS) were 42.9% and 10.7 months (95% CI, 7.9-13.6). Among 40 who were available for BL ctDNA, patients who were detected with ctDNA variant (D group) were 21 (52.5%). TP53 mutation was the most frequently observed genetic alteration in BL ctDNA (76.2%) and median copy numbers of ERBB2 in HER2-positive GC were 28 (range 2-42). D group were more frequently observed in tubular or papillary adenocarcinoma than in poorly cohesive carcinoma (69.0% vs. 9.1%, p = 0.001). Patients who were not detected with any kind of ctDNA variant (ND group) showed a trend in longer PFS, compared to D group, however it was not significant (10.5 vs 7.9 months, p = 0.29). In serial analysis during first-line chemotherapy, ctDNA clearance was observed in 14 (70.0%) out of 20 in D group. Patients with ctDNA clearance showed significantly longer PFS, compared with patients without ctDNA clearance (9.8 vs. 3.6 months, p < 0.001). Among 32 patients who had disease progression, increased VAF or re-development of previous BL ctDNA variants were observed in 16 (50.0%). Increased VAF or re-development of BL ctDNA were detected in 2.1 months earlier than clinical or radiologic deterioration. Conclusions: Serial ctDNA could predict clinical outcomes of chemotherapy and detect disease progression earlier than clinical or radiological findings in AGC. Further study for serial ctDNA is needed to find out the resistant mechanism and evolutionary process in cancer.

The emergence of RAS/RAF mutant clone is the main feature of EGFR inhibitor resistance in KRAS wild-type colon cancer. However, its molecular mechanism is thought to be multifactorial, mainly due to cellular heterogeneity. In order to better understand the resistance mechanism in a single clone level, we successfully isolated nine cells with cetuximab-resistant (CR) clonality from in vitro system. All CR cells harbored either KRAS or BRAF mutations. Characteristically, these cells showed a higher EMT (Epithelial to mesenchymal transition) signature, showing increased EMT markers such as SNAI2. Moreover, the expression level of CXCL1/5, a secreted protein, was significantly higher in CR cells compared to the parental cells. In these CR cells, CXCL1/5 expression was coordinately regulated by SNAI2/NFKB and transactivated EGFR through CXCR/MMPI/EGF axis via autocrine singling. We also observed that combined cetuximab/MEK inhibitor not only showed growth inhibition but also reduced the secreted amounts of CXCL1/5. We further found that serum CXCL1/5 level was positively correlated with the presence of RAS/RAF mutation in colon cancer patients during cetuximab therapy, suggesting its role as a biomarker. These data indicated that the application of serum CXCL1/5 could be a potential serologic biomarker for predicting resistance to EGFR therapy in colorectal cancer.

Analyzing cell-free DNA (cfDNA) as a source of circulating tumor DNA is useful for diagnosing or monitoring patients with cancer. However, the concordance between cfDNA within liquid biopsy and genomic DNA (gDNA) within tumor tissue biopsy is still under debate. To evaluate the concordance in a clinical setting, we enrolled 54 patients with metastatic colorectal cancer and analyzed their plasma cfDNA, gDNA from peripheral blood mononuclear cells (PBMC), and gDNA from available matched tumor tissues using ultra-deep sequencing targeting 10 genes (38-kb size) recurrently mutated in colorectal cancer. We first established a highly reliable cut-off value using reference material. The sensitivity of detecting KRAS hotspot mutations in plasma was calculated as 100%, according to digital droplet PCR. We could selectively detect clinically important somatic alterations with a variant allele frequency as low as 0.18%. We next compared somatic mutations of the 10 genes between cfDNA and genomic DNA from tumor tissues and observed an overall 93% concordance rate between the two types of samples. Additionally, the concordance rate of patients with the time interval between liquid biopsy and tumor tissue biopsy within 6 months and no prior exposure to chemotherapy was much higher than those without. The patients with KRAS mutant fragments in plasma had poor prognosis than those without the mutant fragments (33 months vs. 63 months; p

Purpose This study was designed to identify novel fusion transcripts (FTs) and their functional significance in colorectal cancer (CRC) lines. Materials and methods We performed paired-end RNA sequencing of 28 CRC cell lines. FT candidates were identified using TopHat-fusion, ChimeraScan, and FusionMap tools and further experimental validation was conducted through reverse transcription-polymerase chain reaction and Sanger sequencing. FT was depleted in human CRC line and the effects on cell proliferation, cell migration, and cell invasion were analyzed. Results One thousand three hundred eighty FT candidates were detected through bioinformatics filtering. We selected six candidate FTs, including four inter-chromosomal and two intrachromosomal FTs and each FT was found in at least one of the 28 cell lines. Moreover, when we tested 19 pairs of CRC tumor and adjacent normal tissue samples, NFATC3-PLA2G15 FT was found in two. Knockdown of NFATC3-PLA2G15 using siRNA reduced mRNA expression of epithelial-mesenchymal transition (EMT) markers such as vimentin, twist, and fibronectin and increased mesenchymal-epithelial transition markers of E-cadherin, claudin-1, and FOXC2 in colo-320 cell line harboring NFATC3-PLA2G15 FT. The NFATC3-PLA2G15 knockdown also inhibited invasion, colony formation capacity, and cell proliferation. Conclusion These results suggest that that NFATC3-PLA2G15 FTs may contribute to tumor progression by enhancing invasion by EMT and proliferation.

Introduction: Regorafenib is a multikinase inhibitor which showed clinical benefit in patients with treatment refractory metastatic colorectal cancer. However, as only a subset of patients derives clinical benefit from regorafenib, it is essential to identify biomarker to predict therapeutic response. Circulating tumor DNA (ctDNA) is emerging as a valuable non-invasive tool to identify tumor heterogeneity and tumor burden. This study investigated ctDNA dynamics in patients with metastatic colorectal cancer treated with regorafenib. Methods: This is a prospective biomarker study including patients with refractory metastatic colorectal cancer treated with regorafenib (ClinicalTrial.gov Identifier: NCT01996969). Patients with metastatic colorectal cancer who were refractory to standard therapies (fluoropyrimidine, oxaliplatin, and irinotecan) were eligible for the current study. Patients received oral regorafenib 160 mg once daily for the first 3 weeks of each 4-week cycle and were treated until disease progression, death, unacceptable toxicity, or decision by the treating physicians. Blood samples were obtained prior to regorafenib treatment and after every two cycles of regorafenib treatment until disease progression. ctDNA was detected by AlphaLiquid® 100 target capture panel (IMBdx, Inc., Seoul, South Korea). Alphaliquid® 100 is a tumor agnostic panel consist of 106 genes, including 10 gene fusion and MSI. Variant allele frequency (VAF) amount was calculated by adding the VAF value of all altered genes. Results: A total of 110 patients were included in the present study. Baseline blood samples were successfully acquired in 107 patients (97.3%) with a total of 713 genetic alteration. Mutation was most frequently found in TP53 (76.6%) followed by APC (75.7%), KRAS (43.0%), PIK3CA (17.8%), and SMAD4 (17.8%). BRAF mutation was found in 8.4% of patients and NRAS was detected in 3.7% of patients. Blood samples after two cycle of regorafenib was acquired in 106 patients, and was acquired in 95 patients after disease progression. Among 104 patients with baseline and follow-up cfDNA, the mean VAF at baseline was 12.8% and 7.2% in follow-up. This resulted in a mean VAF change of -5.61% (absolute value) and -43.7% (relative change). VAF decreased markedly after 2 cycles of regorafenib in several genes, including CSF1R, JAK3, KIT, ROS1, and TERT. Although, VAF change of specific gene was not associated with regorafenib outcome, VAF change of whole gene was an early predictive marker for regorafenib. Reduction in VAF amount of ≥ 50% after two cycles of regorafenib were associated with a significantly improved PFS (6.1 vs. 2.7 months, p = 0.002), OS (11.3 vs. 5.9 months, p = 0.001), and higher disease control rate (86.3% vs. 51.1%, p < 0.001). Conclusions: Serial ctDNA could be used as an early predictive biomarker in metastatic colorectal cancer treated with regorafenib. Citation Format: Dae-Won Lee, Sae-Won Han, Yoojoo Lim, Hwang-Phill Kim, Hanseong Roh, Min Jung Kim, Seung-Bum Ryoo, Ji Won Park, Seung-Yong Jeong, Kyu Joo Park, Gyeong Hoon Kang, Tae-You Kim. ctDNA change predicts treatment outcome of regorafenib in metastatic colorectal cancer [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 5157.

Introduction: The fragment size of cfDNA derived from tumor cells are known to be shorter than that from normal cells for not yet precisely known mechanisms. We hypothesized that the fragment size of cfDNA can be utilized to reliably estimate tumor burden in patients with mCRC undergoing palliative chemotherapy. Methods: cfDNA sequencing data from mCRC patients receiving first-line palliative chemotherapy at Seoul National University Hospital (Seoul, Korea) were used for this analysis. The patients were enrolled in a main study evaluating the dynamic changes of circulating tumor DNA mutational profiles during chemotherapy. Blood samples were obtained prior to chemotherapy and after every four cycles of chemotherapy until disease progression. cfDNA sequencing data from normal individuals were used as control data. cfDNA sequencing for both cancer patients and normal individuals were performed using AlphaLiquid® 100 target capture panel (IMBdx, Inc., Seoul, South Korea). AlphaLiquid® 100 is a tumor agnostic panel consist of 106 genes, including 10 gene fusion and MSI. Based on the panel sequencing data, the genetic alterations and the fragment size of cfDNA were calculated. The fragmentation ratio was defined by the ratio of the read fragment proportion in size range P1 (100 - 155 bp) and P2 (160 - 180 bp). Results: cfDNA sequencing data from 280 plasma samples from 62 mCRC patients and 50 healthy controls were used for analysis. Compared to cfDNA from healthy controls, the cfDNA fragment sizes from mCRC patients were significantly shorter (169.585 bp vs. 173.964 bp; p < 0.001). Comparing the fragment sizes by the mutational status, the fragment sizes of alleles with detected somatic mutations were significantly shorter than those with reference alleles (mean fragment size 155.853 bp in alleles with somatic mutations vs. 160.613 bp in reference alleles; p < 0.001), but such difference was not observed with germline mutations (mean fragment size 160.911 bp in alleles with germline mutations vs. 159.889 bp in reference alleles; p = 0.992). Further, the clonality inferred from the variant allele frequency (VAF) of somatic mutation was negatively correlated with the size of the cfDNA fragment. The read fragment proportion in size range P1 was significantly associated with the clonality (r = 0.86, p Conclusions: The fragmentation ratio calculated from cfDNA in mCRC patients can represent tumor burden in dynamic samples and potentially can be used as a biomarker. Citation Format: Jun-Kyu Kang, Hwang-Phill Kim, Yoojoo Lim, Su Yeon Kim, Tae-You Kim. cell-free DNA (cfDNA) fragment size as a potential quantitative biomarker for metastatic colorectal cancer (mCRC) [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 5164.

8569 Background: The current standard of care (SOC) for limited disease (LD) small cell lung cancer (SCLC) is definitive concurrent chemoradiotherapy (CCRT). Despite the high response rate to SOC, up to 70% of the patients potentially experience disease recurrence and fail to show the prolonged clinical benefit. We investigated the feasibility of cell-free DNA (cfDNA) based genomic alteration and fragmentomic analysis using pre-and post-treatment samples to investigate the predictive value of disease recurrence in LD-SCLC. Methods: The blood sample from fifty LD-SCLC who were treated with definitive CCRT were collected before and after the treatment. Target sequencing, AlphaLiquid scan (IMBdx, Korea), composed of 106 target genes, was conducted using cfDNA and peripheral blood mononuclear cells. Based on the recurrence-free survival (RFS) interval of 12 months, patients were categorized into group with persistent response (PeR, n = 29) and non-PeR (n = 21). Fragmentomics analyses were collected using the proportion of cfDNA fragments sized in P1 (100 – 155 bp) and P2 (160 – 180 bp) ranges. Results: Initial analysis was conducted based on the gene of interest. Patients with RB1 mutation (n = 11) detected in follow-up sample demonstrated significant shorter RFS compared to the RB1 wild type (WT) patients (n = 39) (7.9 mon. vs. NR, P = 0.002). Among the patients who were available for our fragmentomics analysis in follow up samples (n = 19), non-PeR (n = 9) group were significantly high in P1 range ( P = 0.003) and low in P2 range ( P = 0.002) compared to PeR group (n = 10). AUCs using the fragment proportion in P1, proportion in P2, and the fragmentation ratio (proportion in P1 / proportion in P2), were 0.889, 0.911, and 0.889, respectively. The survival analysis using fragmentation ratio showed significantly longer RFS in fragmentation ratio low group compared to the high group (7.6 mon. vs. NR, P = 0.002). Integrating RB1 mutation status with fragmentation ratio, patients with both RB1 WT and fragmentation ratio low (n = 9) showed the most favorable outcomes ( P = 0.006). On the contrary, patients with either RB1 mutated or high in fragmentation ratio (n = 10) showed median RFS of rest than 12 months. Conclusions: In this study, we investigated the clinical feasibility of cfDNA detected mutation and size of the fragment in disease recurrence of LD-SCLC using the sample collected before and after definitive CCRT. It is observed that patients with either RB1 mutation or high fragmentation ratio detected from cfDNA after the completion of CCRT are likely to experience early disease recurrence. Our findings suggest that cfDNA could provide supplementary information in predicting early treatment failure and support the clinical decision in selecting high-risk patients who might need intense monitoring and additional consolidative treatment.

PIK3CA is a frequently mutated gene in cancer, including about ~15 to 20% of colorectal cancers (CRC). PIK3CA mutations lead to activation of the PI3K/AKT/mTOR signaling pathway, which plays pivotal roles in tumorigenesis. Here, we investigated the mechanism of resistance of PIK3CA-mutant CRC cell lines to gedatolisib, a dual PI3K/mTOR inhibitor. Out of a panel of 29 CRC cell lines, we identified 7 harboring one or more PIK3CA mutations; of these, 5 and 2 were found to be sensitive and resistant to gedatolisib, respectively. Both of the gedatolisib-resistant cell lines expressed high levels of active glycogen synthase kinase 3-beta (GSK3β) and harbored the same frameshift mutation (c.465_466insC; H155fs*) in TCF7, which encodes a positive transcriptional regulator of the WNT/β-catenin signaling pathway. Inhibition of GSK3β activity in gedatolisib-resistant cells by siRNA-mediated knockdown or treatment with a GSK3β-specific inhibitor effectively reduced the activity of molecules downstream of mTOR and also decreased signaling through the WNT/β-catenin pathway. Notably, GSK3β inhibition rendered the resistant cell lines sensitive to gedatolisib cytotoxicity, both in vitro and in a mouse xenograft model. Taken together, these data demonstrate that aberrant regulation of WNT/β-catenin signaling and active GSK3β induced by the TCF7 frameshift mutation cause resistance to the dual PI3K/mTOR inhibitor gedatolisib. Cotreatment with GSK3β inhibitors may be a strategy to overcome the resistance of PIK3CA- and TCF7-mutant CRC to PI3K/mTOR-targeted therapies.

Although regorafenib has demonstrated survival benefits in patients with metastatic colorectal and gastrointestinal stromal tumors, no proven biomarker has been identified for predicting sensitivity to regorafenib. Here, we investigated preclinical activity of regorafenib in gastric and colorectal cancer cells to identify genetic alterations associated with sensitivity to regorafenib. Mutation profiles and copy number assays of regorafenib target molecules indicated that amplification of fibroblast growth factor receptor 2 (FGFR2) was the only genetic alteration associated with in vitro sensitivity to regorafenib. Regorafenib effectively inhibited phosphorylation of FGFR2 and its downstream signaling molecules in a dose-dependent manner and selectively in FGFR2-amplified cells. Regorafenib induced G1 arrest (SNU-16, KATO-III) and apoptosis (NCI-H716); however, no significant changes were seen in cell lines without FGFR2 amplification. In SNU-16 mice xenografts, regorafenib significantly inhibited tumor growth, proliferation, and FGFR signaling compared to treatment with control vehicle. Regorafenib effectively abrogates activated FGFR2 signaling in FGFR2-amplified gastric and colorectal cancer and, therefore, might be considered for integration into treatment in patients with FGFR2-amplified gastric and colorectal cancers.

189 Background: ctDNA is an attractive alternative to tissue for its easy accessibility and real time representation of systemic tumor profile. We explored the utility of ctDNA profiling during the course of mCRC treatment. Methods: Serial blood samples were obtained from mCRC patients before and during first-line palliative chemotherapy at fixed intervals (after every four cycles) until confirmed disease progression. ctDNA was sequenced using targeted next-generation sequencing (NGS) platform with 106 genes. Changes of ctDNA profile and treatment outcome were comprehensively analyzed. Results: A total of 272 samples from 62 patients were analyzed. In the pre-treatment blood samples, 56 (90.3%) of patients had detectable ctDNA mutations including single nucleotide variants, short insertions/deletions and copy number changes (median 4.5 mutations/ patient, range 0 - 133). In 31 (50.0%) patients who had tissue NGS panel results performed in the clinic, overall concordance between mutations from ctDNA and tissue was 86.5%. In three patients, ctDNA mutational profiles were found to be completely different from tissue profiles. At further investigation, these patients were found to have a separate primary cancer in their colon. At the time of the first follow-up, most (98.0%) patients showed decrease of ctDNA from baseline, represented by average variant allele frequency (VAF) changes of all ctDNA mutations found. Clearance of ctDNA was achieved in 40 (78.4%) patients and was associated with longer progression-free survival (median PFS 11.8 moths in ctDNA clearance (+) vs. 4.7 months in ctDNA clearance (-), p < 0.001). The ctDNA clearance at the same time point was able to further discriminate the patients in same category by RECIST 1.1. Serial follow-up monitoring revealed three patterns of ctDNA changes at the time of clinical progressive disease (PD): 1) re-emergence or re-increase of baseline ctDNA mutations, 2) emergence of new resistance mutations, 3) radiologic PD without evidence of ctDNA progression. In the patients with detectable ctDNA at PD, the ctDNA progression preceded radiologic progression in 25 (58.1%) patients by median of 3.3 months. The patients in clinical PD without ctDNA progression showed different patterns of metastasis having mainly extrahepatic spread, while 77.8% of the patients with ctDNA progression had their progression confirmed in liver metastasis. Diverse resistant mutations and gene amplifications in PD patients were discovered by ctDNA sequencing. For seven (16.3%) of the PD patients, the newly identified mutations could be potential candidates of targeted therapy or clinical trial. Conclusions: ctDNA profile provided additional information to conventional evaluation methods and reflected dynamic changes. ctDNA monitoring may improve precise treatment decision-making for individual patients.