ctDNA Sequencing Research Articles

Abstract P5-05-08: Dual ctDNA and tissue sequencing improves detection of actionable variants in breast cancer patients

Abstract Background: Next-generation sequencing of circulating tumor DNA (ctDNA) and solid-tissue can identify clinically actionable genomic variants that may be used for both treatment selection and disease surveillance. Due to differences in tumor biology and assay design, ctDNA and solid biopsies may identify unique variants. Here, we investigate a real-world dataset of breast cancer patients to determine whether clinically actionable variant detection is enhanced by dual ctDNA and solid tissue testing. Methods: We used the deidentified Tempus Lens database to retrospectively analyze stage IV breast cancer patients with known hormonal subtype. Each patient had dual testing defined as Tempus xF (ctDNA) and Tempus xT (tumor tissue)—which resulted in clinical reports for both tests. Patients were further stratified according to the timing of ctDNA biopsy relative to tissue biopsy. Concurrent dual testing was defined as samples collected ≤30 days apart and longitudinal dual testing was defined as liquid >30 days after solid. Variants were included in analyses if they met the limit of detection criteria of both assays. Clinical actionability was defined by indication-matched OncoKB Level 1-3. Fisher exact test was used to calculate significance. Results: Of the 1,341 breast cancer patients with dual ctDNA and tissue sequencing, at least one actionable variant was identified in 61% (n=823) of patients. In the subset of concurrent tested patients (n=782), 60% (n=473) had one or more actionable findings: 54% (n=257/473) of patients with actionable variants had perfectly concordant variants, 29% (n=136/473) had at least one unique variant detected only by solid tumor testing, and 20% (n=93/473) had at least one unique variant detected only by ctDNA testing. Similarly, in the longitudinal set (n=559), 63% (n=350) had one or more actionable findings: 34% (n=118/350) were concordant, 43% (n=150/350) were unique to solid, and 27% (n=96/350) were unique to ctDNA. When stratifying concurrent patients by OncoKB levels of evidence, 72% (n=98/136) of patients with variants unique in solid had at least one level 1-2 variant, while 39% (n=53/136) contained unique level 3 variants. Level 1-2 variants in PIK3CA were the most frequent variants seen uniquely in solid tumors, occurring in 54% (n=73/136) of patients. In contrast, in patients with unique ctDNA variants, 37% (n=34/93) of patients had at least one level 1-2 variants and 72% (n=67/93) had level 3 variants. Level 3 variants in ESR1 were the most frequent variants seen uniquely in ctDNA, occurring in 57% (n=53/93) of patients. The proportion of concurrent patients with actionable variants found exclusively in ctDNA significantly differed by subtype (p=0.04): Luminal A (22%) and Luminal B (23%) contained the most patients with unique ctDNA variants. This ability to detect additional variants in ctDNA remained true even if profiling occurred over time. Indeed, in patients with ESR1 variants tested with ctDNA > 1 year after tissue, 78% (n=43/55) had ESR1 variants only detected in blood. Conclusions: We show that dual testing in breast cancer patients improves the identification of clinically actionable variants which may be missed by either ctDNA or solid tissue biopsy alone. Adoption of dual testing should be considered as standard practice to provide a comprehensive view of actionable molecular alterations. Citation Format: Matthew Mackay, Kabir Manghnani, Adam Hockenberry, Joshua Drews, James Chen, Rotem Ben-Shachar, Justin Guinney. Dual ctDNA and tissue sequencing improves detection of actionable variants in breast cancer patients [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-05-08.

Molecular residual disease: A new clue for individualized approach in non‐small cell lung cancer

AbstractThe prognostic value of molecular residual disease (MRD) in non‐small cell lung cancer (NSCLC) cases using high‐depth circulating tumor DNA (ctDNA) sequencing has been well documented. The utility of MRD to direct individualized therapy has increasingly emerged in the clinical trial design of solid tumors, such as escalation or de‐escalation of adjuvant therapy based on MRD. And the efficiency of MRD assay is a key determinant to the success of clinical trials, especially the limitation of detection and predictive value. Here, we review the progress made in evaluating the clinical validity of ctDNA‐MRD test and provide insight into exploiting these developments to future clinical scenarios for improving the individualized therapy of NSCLC.

Abstract OT1-05-02: Efficacy and safety of sintilimab in combination with anlotinib plus metronomic chemotherapy in advanced triple negative breast cancer (SPACE): preliminary results of a single-arm, multicenter phase II trial

Abstract Background: Anti-PD-1 antibody combined with antiangiogenic drugs have demonstrated antitumor activity in advanced triple negative breast cancer (TNBC). Anlotinib, an oral multi-targeted tyrosine kinase inhibitor (TKI), has synergistic effect with anti-PD-1 antibody. Preclinical studies showed that metronomic chemotherapy inhibited angiogenesis and enhanced immunotherapy efficacy in TNBC via modulation of tumor immune microenvironment. We hereby conducted a single-arm, multicenter, phase II trial to investigate the efficacy and safety of sintilimab (anti-PD-1 antibody) in combination with anlotinib plus metronomic chemotherapy as a potential novel therapeutic strategy in advanced TNBC and explore potential biomarkers. Methods: Forty-three cases were planning to be included in this trial. The eligible patients who had received no more than two lines of chemotherapy for metastatic disease were enrolled and received sintilimab (200 mg iv q3w) in combination with anlotinib (12 mg po d1-14 q3w) plus capecitabine (500 mg po, tid) or vinorelbine (40 mg po, tiw) until disease progression or intolerable toxicity. The primary endpoint is objective response rate (ORR) and secondary endpoints are disease control rate (DCR), progression free survival (PFS), and overall survival (OS). The safety profile has also been assessed. Blood samples collected at different time points of the baseline, first and second cycle post-treatment, and disease progression were used for next-generation sequencing of ctDNA containing 654 tumor-related genes. Results: As of July 2022, a total of 32 patients were enrolled, and 29 patients were evaluable for efficacy. 2 patients (6.9%) achieved complete response (CR). 6 patients (20.7%) achieved partial response (PR). The ORR is 27.6% (8/29) and DCR is 79.3% (23/29). The median PFS was not reached. The most common grade 1 or 2 adverse events (AEs) include elevated thyroid stimulating hormone (37.0%, 10/27), hand-foot syndrome (18.5%, 5/27), elevated aspartate aminotransferase (14.8%, 4/27), elevated bilirubin (11.1%, 3/27) and hypertension (11.1%, 3/27). Grade 3 AEs include elevated bilirubin (3.7%, 1/27) and hypertension (3.7%, 1/27). No grade 4 or 5 AEs occurred. By analyzing ctDNA mutations of blood samples in 10 patients at baseline, we found that genes with high mutation frequency were HLA-DRB5 (8/10, 80%), TP53 (7/10, 70%), HLA-DRB1 (5/10, 50%) and PIK3CA (4/10, 40%). Among these 10 patients, 2, 3 and 5 patients achieved PR, SD and PD, respectively. The number of gene mutations in patients with PD was higher than that in patients with PR or SD at baseline. This indicate that mutations in ctDNA may be associated with poor efficacy in advanced TNBC. But this still needs further verification. Dynamic analysis of gene mutations at different time points showed that the amplification of HLA-DRB5 or the elimination of KMT2D, RELN and TP53 occurred in patients with PR and SD, but not in patients with PD. Conclusions: Sintilimab in combination with anlotinib plus metronomic chemotherapy has shown favorable efficacy and acceptable safety profile in patients with advanced TNBC. The clinical significance of ctDNA dynamic monitoring needs further validation. Clinical trial information: ChiCTR2100044725 Citation Format: Huihui Li, Qiaorui Tan, Shujuan Sun, Dongdong Zhou, Bo Yu, Mu Su, Baojiang Li, Shu Fang, Ling Qiang, Guohua Ren, Bing Bu, Sha Yin, Xiaochu Man, Pengfei Qiu, Xinzhao Wang, Chao Li, Fangli Cao, Qian Shao, Dali Han, Lihua Song, Bingjie Fan, Baoxuan Zhang, Liang Xu, Xianguang Zhao, Yuqian Liao, Xuemei Xie, Lanping Liu. Efficacy and safety of sintilimab in combination with anlotinib plus metronomic chemotherapy in advanced triple negative breast cancer (SPACE): preliminary results of a single-arm, multicenter phase II trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT1-05-02.

Abstract PD17-01: Genomic analysis of circulating tumor DNA (ctDNA) from patients with HR+, HER2-mutant metastatic breast cancer (MBC) enrolled in SUMMIT: mechanisms of acquired resistance to neratinib + fulvestrant + trastuzumab (N+F+T)

Abstract Background: In the SUMMIT trial, original cohorts of patients with HR+, HER2-negative (locally assessed), HER2-mutant MBC received N alone or N+F, with promising clinical response rates but abbreviated duration. Clinical progression coincided with emergence of additional HER2 mutations and/or amplification of the mutant allele [Smyth et al. Cancer Discov 2020;10:198–213]. Addition of T to the combination was postulated to prolong response; the combination of N+F+T in heavily pretreated patients with HR+, HER2-mutant MBC who had received CDK4/6 inhibitors (n=51) yielded a confirmed overall response rate (ORR) of 35.3%, median duration of response (DOR) of 14.3 months, clinical benefit rate (CBR) of 47.1%, and median progression-free survival (PFS) of 8.2 months [Jhaveri et al. J Clin Oncol 2022;40:1028]. Seven of these 51 patients were part of a randomized (1:1:1) cohort who received N+F+T, F+T, or F alone. Patients randomized to F+T or F could crossover to N+F+T upon progression. No patients responded to F or F+T; however, one of four patients who crossed over to N+F+T upon progression on F+T responded to the triplet, as did two of six who crossed over upon progression on F. We undertook ctDNA sequencing in patients who responded to N+F+T upfront and after crossover. Methods: Patients with HR+, HER2-negative MBC with activating HER2 mutation(s) and prior CDK4/6i received N+F+T (oral N 240 mg/d with loperamide prophylaxis, im F 500 mg d1, d15, and d29 of cycle 1 then q4w, iv T 8 mg/kg initially then 6 mg/kg q3w) or F+T or F alone. Efficacy endpoints included investigator-assessed ORR and CBR (RECIST v1.1), DOR, and best overall response. ctDNA was collected at baseline, every third cycle during treatment, and at the end of treatment and analyzed by next-generation sequencing. Samples were analyzed using the TEMPUS xF assay. Results: Sequencing results from ctDNA analysis are pending at the time of this abstract submission. Baseline HER2 mutations and co-alterations will be reported and compared with those found in tissue samples. Genomic spectrum and variant allele frequencies in samples taken at baseline, on treatment, and at the end of treatment from patients who experienced complete or partial response to N+F+T and then progressed (n=25) will be sequenced and mechanisms of acquired resistance will be posited. ctDNA genomic profiles of serial time points from patients randomized to F or F+T before and after crossover to N+F+T (n=10) will also be evaluated. Conclusions: Similarities and differences between the mechanisms of acquired resistance to N+F+T, and those previously reported to be associated with progression on N or N+F, will be discussed. Citation Format: Cynthia Ma, James Waisman, Adam M. Brufsky, Eddy S. Yang, Hans Wildiers, John P. Crown, Sarina A. Piha-Paul, Jennifer M. Suga, José Ángel García-Sáenz, Valentina Gambardella, Angel Guerrero, Salomon Stemmer, Ron Bose, Tonya Novara-Demgen, Daniel DiPrimeo, Lisa D. Eli, Komal Jhaveri. Genomic analysis of circulating tumor DNA (ctDNA) from patients with HR+, HER2-mutant metastatic breast cancer (MBC) enrolled in SUMMIT: mechanisms of acquired resistance to neratinib + fulvestrant + trastuzumab (N+F+T) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD17-01.

Early diagnosis of brain metastases using cerebrospinal fluid cell‐free DNA‐based breakpoint motif and mutational features in lung cancer

Early diagnosis of brain metastases using cerebrospinal fluid cell‐free DNA‐based breakpoint motif and mutational features in lung cancer

Abstract P5-02-02: Genomic Landscape of ER+/HER2- metastatic breast cancer as a function of prior treatment with a CDK4/6 inhibitor

Abstract Background CDK4/6 inhibitors (CDK4/6i), like palbociclib, ribociclib, and abemaciclib, along with antiestrogens, have revolutionized treatment for ER+/HER2- metastatic breast cancer (MBC). Although most patients initially respond, almost all eventually progress, and ER+ HER2- MBC remains incurable. There is an urgent need to understand the molecular processes that drive resistance in order to improve survival. The landscape of acquired somatic alterations causal to CDK4/6i resistance remains unknown. Here we report differences in mutational landscapes between ER+ HER2- MBC patients treated with and without CDK4/6i. Methods Deidentified data from 780 and 1073 ER+ HER2- MBC patients (solid tumor or ctDNA liquid biopsy sequencing respectively) with at least 6 months between diagnosis of Stage 4 disease and biopsy were analyzed. Patients were divided into either treated or untreated with CDK4/6i prior to biopsy. Sequencing was performed using the Tempus xT tumor assay (DNA sequencing of 595-648 genes at 500x coverage) and Tempus xF liquid biopsy (ctDNA sequencing of 105-523 genes). Gene alterations (consisting of pathogenic/likely pathogenic short variants and copy number alterations) were compared between groups by Chi-squared/Fisher’s Exact tests and p-values adjusted for false-discovery. Results We first analyzed sequencing data of both solid tumor and liquid ctDNA from ER+/HER2- MBC patients. ESR1 mutations were significantly more frequent in those that received CDK4/6i than those that did not (Solid tumor 33% vs 16%, p < 0.001, q = 0.001; Liquid biopsy 32% vs 16%, p < 0.001 and q < 0.001). We also saw more frequent mutations/amplifications in the following genes in the CDK4/6i treated cohort vs. those that were not. These results trended towards significance in our solid tumor, but not in our liquid biopsy cohort: CCND1 (18% vs 11% p = 0.028 q =0 .3); FGF3 (17% vs 9.5% p = 0.010 q = 0.2); FGF4 (17% vs 11% p = 0.035 q = 0.3), GATA3 (17% vs 8.9% p = 0.008 q = 0.2), PTEN (12% vs 6.1% p = 0.030 q = 0.3) and FGF19 (8.2% vs 1.7% p = 0.002 q = 0.12). Interestingly, 96-98% of CCND1, FGF3, FGF4 and FGF19 alterations were copy number amplifications. Conversely, we saw a trend towards significance for more mutations in TP53 (37% vs 27% p=0.008 and q=0.2) in those that had not received a CDK4/6i than those that did. Conclusions Here we present the landscape of somatic alterations in ER+/HER2- MBC patients with and without prior CDK4/6i therapy from our large real world de-identified data set. Patients with prior CDK4/6i therapy harbored significantly more ESR1 somatic alterations, demonstrated in both solid tissue and liquid biopsies. In solid tissue biopsies, patients with prior CDK4/6i therapy harbored more CCND1, FGF3, FGF4, and GATA3 alterations and less TP53 alterations. These trends were not significant after adjustment for multiple testing. CCND1, FGF3, FGF4 and FGF19 alterations were copy number amplifications, which may be consistent with 11q13 amplification. Further studies will provide insights into how these trends translate towards our understanding of CDK4/6i related resistance mechanisms. Citation Format: M Rosario Chica-Parrado, Chang-Ching Lin, Timothy Mahoney, Elizabeth Mauer, Ariella Hanker, Carlos Arteaga. Genomic Landscape of ER+/HER2- metastatic breast cancer as a function of prior treatment with a CDK4/6 inhibitor. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-02.

Role of adenomatous polyposis coli (APC) gene in SPOP-mutant prostate cancer and clinical outcomes.

202 Background: Speckle-type POZ protein (SPOP) mutations are a frequent initiating event in prostate cancer (PC) and are associated with increased androgen receptor transcriptional output. While SPOP-Mutant (MT) PCs may be more sensitive to hormonal therapy, they are still common amongst castration-resistant PCs (CRPCs) and frequently lethal. It remains unknown which SPOPMT PCs will respond well to hormonal therapy, and which have the potential to develop early castration resistance and cause patient death. Methods: With IRB approval, we retrospectively analyzed NGS data obtained via Tempus|xT tissue assay (DNA sequencing of 648 genes in tumor and matched normal samples at 500x depth) and/or Tempus|xF liquid biopsy assay (ctDNA sequencing of 105 genes in peripheral blood samples at 5,000x depth) for germline and/or somatic mutations in 234 consecutive patients with high-risk localized, locally advanced or metastatic PC followed at Ben Taub Hospital (BTH), a public hospital serving a racially/ethnically diverse patient population (>50% of PC patients are African American). For confirmation, we examined de-identified NGS data from a nationwide cohort of 8812 PC patients sequenced with xT and/or xF by Tempus Labs (Chicago, IL), as well as publicly available data from four additional PC cohorts (PMIDs: 26544944, 34667026, 31061129 and 26855148). Results: We found SPOP mutations in 31/234 BTH PC patients (13.2%). Eleven of these 31 patients (35.5%) also exhibited loss-of-function (LOF) mutations or deletions in the tumor suppressor gene Adenomatous polyposis coli (APC). APC LOF events were found only in 15/203 (7.4%) of the SPOP-wild type (WT) PCs, and the enrichment of SPOPMT PCs for APC LOF events was highly significant (Chi square with Yates correction: P<0.00001). The co-mutation pattern between these two genes was observed even when the analysis was limited specifically to the tissue NGS (133 patients) or the liquid biopsy NGS subsets of the dataset, respectively. This enrichment was confirmed in the nationwide TEMPUS cohort and in the four publicly available clinical PC datasets (APC LOF found in 23.4, 27.0, 21.5, 32.0 and 30.8% of SPOPMT PCs, respectively; and only in 5.8, 2.4, 6.0, 6.7 and 1.0% of SPOP-Wild Type (WT) PCs, respectively (P<0.001 in each cohort). In the BTH PC cohort, SPOPMT PCs harboring APC LOF mutations were more likely to present with metastatic disease (M1) at diagnosis and to develop castration-resistance within 1 year of first-line hormonal therapy than SPOPMT PCs with WT APC. Conclusions: In SPOPMT PCs, inactivation of APC is a common event and associated with more aggressive disease (higher metastatic potential and faster progression to castration resistance). This observation may help identify SPOPMT PC patients who are at higher risk for early castration resistance and could benefit from more intensive treatment.

Assessing the utility of a cell-free tumor (ct)DNA assay (MSK-ACCESS) in patients (pts) with node-positive (N+) muscle-invasive bladder cancer (MIBC) undergoing neoadjuvant chemotherapy (NAC).

544 Background: Circulating ctDNA is associated with disease progression, worse overall survival, and recurrence in pts with bladder cancer. This study correlated targeted exome ctDNA sequencing with pathologic response to NAC and metastatic recurrence in pts with N+ MIBC undergoing radical cystectomy (RC). Methods: We prospectively identified pts with cT2-3N1-2M0 bladder cancer who underwent NAC prior to RC. Node positivity was determined radiographically and/or by node biopsy. Plasma samples were collected pre-NAC, mid-treatment, after NAC completion, and 3 months after RC. Samples were analyzed using MSK-ACCESS, an ultrasensitive ctDNA platform designed to identify somatic mutations in 129 cancer associated genes. Primary bladder tumors were sequenced using targeted exome sequencing. Results: Samples (31) from 9 pts (6 men) were analyzed. Median age was 63 years (IQR 58-69). NAC regimens included gemcitabine and cisplatin alone (78%) or with paclitaxel (22%). Seven (78%) pts had >1 detectable mutation pre-NAC. The most altered genes detected in both tissue and ctDNA included: TERT (88% vs 28%), TP53 (76% vs 40%), ARID1A (43% vs 17%), RB1 (43% vs 15%), KDM6A (43% vs 15%), and ATM (17% vs 17%). Four (45%) pts had complete response (ypT0N0), 1 (11%) had a partial response (ypT1N0), and 4 (33.3%) were non-responders (ypT2N0-3). All non-responders had disease recurrence after RC (median 3 months, range 2-11 months). Of 7 pts with on-treatment MSK-ACCESS, 2 (29%) had detectable ctDNA (1 ypT2N0, 1 ypT0N0). The pt with a PR had detectable ctDNA post-NAC with 1 mutation identified that was not detected at 3 months. All pts with >ypT2N0 had detectable ctDNA post-NAC. Pts with recurrence/non-responders had a significantly higher mutation count 3 months after RC compared to responders, with a median of 7 vs. 0 mutations, respectively. Two pts with CR had detectable ctDNA at 3 months post-RC but have not recurred (Table). Conclusions: Clearance of ctDNA post-NAC correlated with pathologic complete response. Approximately 70% of pts had ctDNA clearance on NAC. All pts with residual disease at RC had detectable ctDNA post-NAC. [Table: see text]

Association of emergent neuroendocrine prostate cancer detected by liquid biopsies with survival and treatment resistance.

247 Background: The mainstay of therapy in metastatic prostate cancer is androgen receptor (AR) signaling inhibition. However, the emergence of early castration resistance or neuroendocrine transformation is associated with poor prognosis. Reliable biomarkers are needed to identify these patients and guide selection of clinical therapy. Methods: mRNA was isolated from EpCAM-positive circulating tumor cells (CTCs) isolated from patients with CSPC, CRPC, or NEPC to measure expression of KLK2, KLK3 (PSA), TMPRSS2, FOLH1 (PSMA), synaptophysin ( SYP), and chromogranin ( CHGA). Post-hoc retrospective analysis of an institutional review board–approved prospective cohort (N = 98) was performed to identify patterns of gene expression. Samples were considered AR+ if 3 of 4 AR pathway genes ( TMPRSS2, KLK2, KLK3, and FOLH1) were positive, and were considered NE+ if either or both SYP or CHGA were positive. Blood samples from two prospective clinical trials of men with mCRPC treated with abiraterone and enzalutamide, respectively, were analyzed to confirm results. Longitudinal samples were collected from 17 patients (6 NEPC and 11 Adenocarcinoma) and cell free DNA was isolated and sequenced using a novel targeted exon panel. Results: AR and/or NE positive patients were found to have a median overall survival (OS) of 8.58 months as compared to a median OS of 29.6 months in the AR and NE negative population (p<0.0001; HR=2.75 [1.60-4.56]). In the subset of castrate resistant prostate cancer (CRPC) patients, AR+ and/or NE+ patients (n=31) were found to have a median OS of 6.74 months vs 18.79 months in the AR- and NE- group (n=39) (p= 0.0009; HR 2.38 [1.36-4.18]). We also tested samples from a phase II ARSI trials with abiraterone and enzalutamide, respectively. None of the baseline samples from these two trials met the above criteria for NEPC (AR- NE+) on their CTCs or histologically. Three of 48 total patients were identified with expression in their baseline blood samples without loss of AR target gene expression (AR+/NE+). Preliminary analysis of this small patient cohort in comparison to patients who were NE- shows that NE+ patients had worse OS (HR= 5.5906 [1.143-27.36), as would be expected by patients with emerging neuroendocrine differentiation. Integrated ctDNA sequencing identified mutations in genes associated with NEPC. Conclusions: The expression of NE genes in liquid biopsies while retaining AR target gene expression is associated with worse OS and may indicate the transition to neuroendocrine differentiation, with clinical characteristics consistent with this phenotype. Early identification of these patients may improve therapeutic decisions and improved patient outcomes. Pairing genomic alteration with changes in gene expression may additionally offer the basis for a new mechanism to assess efficacy of novel therapeutics in future clinical trials.

Preliminary clinical study of personalized neoantigen vaccine therapy for microsatellite stability (MSS)-advanced colorectal cancer

Immunotherapy based on immune checkpoint inhibitors (ICIs) has provided revolutionary results in treating various cancers. However, its efficacy in colorectal cancer (CRC), especially in microsatellite stability-CRC, is limited. This study aimed to observe the efficacy of personalized neoantigen vaccine in treating MSS–CRC patients with recurrence or metastasis after surgery and chemotherapy. Candidate neoantigens were analyzed from whole-exome and RNA sequencing of tumor tissues. The safety and immune response were assessed through adverse events and ELISpot. The clinical response was evaluated by progression-free survival (PFS), imaging examination, clinical tumor marker detection, circulating tumor DNA (ctDNA) sequencing. Changes in health-related quality of life were measured by the FACT-C scale. A total of six MSS–CRC patients with recurrence or metastasis after surgery and chemotherapy were administered with personalized neoantigen vaccines. Neoantigen-specific immune response was observed in 66.67% of the vaccinated patients. Four patients remained progression-free up to the completion of clinical trial. They also had a significantly longer progression-free survival time than the other two patients without neoantigen-specific immune response (19 vs. 11 months). Changes in health-related quality of life improved for almost all patients after the vaccine treatment. Our results shown that personalized neoantigen vaccine therapy is likely to be a safe, feasible and effective strategy for MSS–CRC patients with postoperative recurrence or metastasis.

The role of serial circulating tumor DNA for predicting clinical outcomes of chemotherapy in patients with advanced gastric cancer.

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.

Analysis of plasma circulating tumor DNA in borderline resectable pancreatic ductal adenocarcinoma treated with neoadjuvant modified FOLFIRINOX: Clinical relevance of DNA damage repair gene alterations.

737 Background: There is no reliable biomarker to guide treatment for patient with borderline resectable pancreatic ductal adenocarcinoma (PDAC) treated with neoadjuvant modified (m)FOLFIRINOX. We investigated the role of plasma circulating tumor DNA (ctDNA) sequencing to discover potential biomarkers for patients treated with neoadjuvant mFOLFIRINOX in our phase 2 clinical trial (ClinicalTrials.gov identifier: NCT02749136). Methods: Among 44 patients enrolled in the trial, patients with plasma ctDNA analysis performed at baseline or post-operative period were included in the analysis. Plasma cell-free DNA isolation and sequencing of ctDNA were performed using the Guardant 360 assay. Association of clinical outcomes with germline or somatic mutations of DNA damage repair (DDR) genes was analyzed. Somatic alterations other than DDR genes were also analyzed to find potential biomarkers of neoadjuvant mFOLFIRINOX. Progression free-survival (PFS) was defined as time from the initiation of mFOLFIRINOX to disease progression or any cause of death. Overall survival (OS) was defined as the time from the initiation of mFOLFIRINOX to any cause of death. Results: Among 44 patients, 28 patients had ctDNA sequencing data qualified for the analysis and were included in this study (15 patients with baseline data only, 3 patients with post-operative data only, and 10 patients with both baseline and post-operative data). Median age was 59 (range, 41-73) years and 15 patients (53.6%) were male. Among 25 patients with baseline plasma ctDNA data, 11 patients (44%) had somatic or germline alteration of DDR genes detected at baseline including ATM, BRCA1, BRCA2 and MLH1, and showed significantly better PFS than those without such DDR gene alterations (median 26.6 vs. 13.5 months, log-rank p = 0.004). Although there was a trend for better OS in patients with DDR gene alterations, this was not statistically significant (log-rank p = 0.29). The most observed baseline somatic alteration was TP53 (n = 7, 28%), followed by KRAS (n = 6, 24%). Patients with somatic KRAS mutation at baseline had significantly worse OS (median 8.5 months vs. not applicable, log-rank p = 0.003) than those without. Among 13 patients with post-operative plasma ctDNA data, 8 patients (61.5%) had detectable somatic alterations. Conclusions: Detection of germline or somatic mutations of DDR genes from plasma ctDNA at baseline was associated with better survival outcomes of patients with borderline resectable PDAC treated with neoadjuvant mFOLFIRINOX. Detection of DDR gene alterations from plasma ctDNA may have a potential role as biomarker for prognosis in patients with borderline resectable PDAC treated with mFOLFIRINOX.

Efficacy and safety of XELOX combined with anlotinib and penpulimab vs XELOX as adjuvant therapy in ctDNA-positive gastric and esophagogastric junction adenocarcinoma: A protocol for a randomized, controlled, multicenter phase II clinical trial—EXPLORING study.

TPS486 Background: The clinical benefit of current postoperative adjuvant chemotherapy for gastric adenocarcinoma/gastroesophageal junction adenocarcinoma (GA/GEJA) leaves much to be desired. CtDNA could serve as a potential marker to identify patients (pts) who are most at risk of recurrence. Reinforcing standard adjuvant chemotherapy with immunotherapy has already been indicated to significantly boost clinical benefit, albeit such evidence is rare in GA/GEJA. Here, we intend to prove the clinical benefit of the reinforcement of adjuvant chemotherapy with immunotherapy in patients who are deemed high risk of recurrence by ctDNA analysis, in hope of shedding light on further improvements in adjuvant therapy for GA/GEJA. Methods: This study is designed as a prospective, randomized, controlled, multicenter phase II study in patients histologically or cytologically diagnosed with CG/GEJC (Siewert III), 18-75 years, ECOG PS 0-1, underwent D2 gastrectomy and achieved R0 or R1 resection. Beginning from February 2022, a total of 270 stage III patients will be enrolled and subjected to ctDNA sequencing, and those with positive results will subsequently be randomized 1:1 to arm A or B. CtDNA-negative patients will be assigned to arm C and pts who had ctDNA positive but failed to be randomized will be assigned to arm D. Pts in arm A will receive anlotinib, penpulimab and XELOX for 6-8 cycles, maintained with anlotinib and penpulimab for up to 1 year, while pts in arm B will receive XELOX alone for 6-8 cycles. Pts in arms C&D will receive the investigator’s choice of therapy. Arms A&B will be monitored for ctDNA levels on a 3-month cycle for 1 year. Survival data, laboratory findings, imaging reports and nutritional status will be regularly followed-up in all arms. The primary endpoint is the median disease-free survival (DFS) of arm A versus arm B determined via CT/MRI imaging. Secondary endpoints include the median DFS of arm B versus arm C, the 2- and 3-year DFS rates, overall survival (OS), the impact of hallmark molecules (presumably ctDNA) on the treatment response, adverse events (AEs), and the impact of nutrition status and exercise on recurrence. Clinical trial information: NCT05494060 .

Heterogeneity of [68Ga]Ga-PSMA-11 PET/CT in metastatic castration-resistant prostate cancer: genomic characteristics and association with abiraterone response.

The aim of this study was to evaluate the impact of the spatial heterogeneity of prostate-specific membrane antigen (PSMA) uptake on circulating tumor DNA (ctDNA) characteristics and the response rate to new hormonal agent (NHA) treatment. This retrospective study included 153 patients with metastatic castration-resistant prostate cancer (mCRPC) who underwent gallium-68 [68Ga]Ga-PSMA-11 positron emission tomography/computed tomography (PET/CT) and ctDNA sequencing with a less than 2-week interval. SUVhetero was defined as the variance of SUVmean for each PSMA-positive lesion. SUVmax-mean was obtained by subtracting the SUVmax by the SUVmean. Patients receiving abiraterone treatment after [68Ga]Ga-PSMA-11 PET/CT and ctDNA sequencing and with complete follow-up record were included into prostate-specific antigen (PSA) response rate analysis. PSA response was defined as a reduction of greater than 50% from baseline. The ctDNA detection rate was 65% (100/153). Higher SUVhetero value contributed to higher ctDNA% (Spearman's rho = 0.278, p < 0.002). A total of 60 patients were included in PSA response rate analysis. The median follow-up was 19.3 (IQR 16.2-23.2) months. Compare to patients with higher SUVhetero value, patients with NA SUVhetero had a higher PSA response rate (52% vs. 90%, p = 0.036). A higher SUVmax-mean value was strongly correlated with higher SUVhetero (Spearman's rho = 0.833, p < 0.0001). Patients with higher SUVmax-mean value also had a higher PSA response rate compared to patients with lower SUVmax-mean value (83.3% vs. 53.3%, p = 0.024). An external cohort confirmed baseline SUVmax-mean value was associated with enzalutamide treatment response rate. Patients with alterations in AR, DNA damage repair pathway, TP53, AR-associated pathway, cell cycle pathway, or WNT pathway had higher SUVmax-mean value compared to those without (p < 0.05). Spatial heterogeneity of the PSMA uptake was associated with ctDNA characteristics and response rate to NHA treatment.

Clinical utility of circulating tumor DNA sequencing with a large panel: a National Center for Precision Medicine (PRISM) study

Circulating tumor DNA (ctDNA) sequencing is a promising approach for tailoring therapy in patients with cancer. We report hereby the results from a prospective study where we investigated the impact of comprehensive molecular profiling of ctDNA in patients with advanced solid tumors. Genomic analysis was performed using the FoundationOne Liquid CDx Assay [324 genes, tumor mutational burden (TMB), microsatellite instability status]. Each individual genomic report was reviewed and discussed weekly by a multidisciplinary tumor board (MTB). Actionable targets were classified by ESMO Scale for Clinical Actionability of Molecular Targets (ESCAT) tier leading to molecular-based treatment suggestions wherever it was possible. Between December 2020 and November 2021, 1772 patients with metastatic solid tumors underwent molecular profiling. Median time to assay results was 12 days. Results were contributive for 1658 patients (94%). At least one actionable target was detected in 1059 patients (64%) with a total of 1825 actionable alterations including alteration of the DNA damage repair response pathway (n= 336, 18%), high TMB (>16 mutations/Mb; n= 243, 13%), PIK3CA mutations (n= 150, 8%), ERBB family pathway alterations (n= 127, 7%), PTEN alterations (n= 95, 5%), FGFR alterations (n= 67, 4%) and MET activations (n= 13, 0.7%). The MTB recommended a matched therapy for 597 patients (56%) with a total of 819 therapeutic orientations: clinical trials (n= 639, 78%), off-label/compassionate use (n= 81, 10%), approved drug (n= 51, 6%), and early access program (n= 48, 6%). In total, 122 patients (21%) were treated. Among the assessable patients (n= 107), 4 (4%) had complete response, 35 (33%) had partial response, 27 (25%) had stable disease, and 41 (38%) a progressive disease as best response. The median progression-free survival and median overall survival were 4.7 months (95% confidence interval 2.7-6.7 months) and 8.3 months (95% confidence interval 4.7-11.9 months) respectively. ctDNA sequencing with a large panel is an efficient approach to match patients with advanced cancer with targeted therapies.

Abstract PR007: Comprehensive ctDNA profiling reveals potential metastatic genomic signatures in treatment-naive early-stage breast cancer patients

Abstract Background: Genomic profiling has revolutionized precision oncology impacting the diagnosis, prognosis, and therapy decisions. Considering high spatiotemporal diversity and heterogenicity of breast tumor-cell genomes, small-gene panels often fail to capture rare but important genomic alterations. Conversely, comprehensive ctDNA sequencing approaches enable the identification of under characterized ‘long tailed driver’ genomic alterations and capture Intra and inter metastatic heterogeneity. Here, we demonstrate the clinical utility of comprehensive genome profiling with higher sensitivity to predict the possibility of metastasis in early-stage breast cancer patients. Methods: We retrospectively analyzed ctDNA and genomic DNA (gDNA) from FFPE samples as well as circulating tumor cells (CTC) in 10 treatment-naive hormone positive and HER2 negative, primary-stage breast cancer patients [GS1] using the OncoIndx comprehensive 600 gene panel. The panel captures all important cancer-relevant genomic alterations including Tumor Mutation Burden (TMB), Micro Satellite Instability (MSI), homologous recombination deficiency (HRD) prediction, and cfDNA tumor fraction (TF). CTCs were enumerated from 1.5 ml of blood using the OncoDiscover platform approved by the Drug Controller General of India having anti-EpCAM antibody-mediated immunomagnetic nanoparticles. CTCs were confirmed for cytokeratin 18+ and DAPI + markers and the absence of CD45. Results: Comprehensive genomic profile obtained from ctDNA and gDNA from FFPE of early-stage breast cancer patients predominantly exhibited the presence of alterations in PIK3CA and ESR1 signaling pathways. PIK3CA mutations were present in 77% and 44% of baseline ctDNA and gDNA samples, while ESR1 mutations were present in 44% and 22% of baseline ctDNA and gDNA, respectively. In addition, we observed about 70% additional driver mutations in ctDNA samples suggesting shedding of ctDNA together with CTC (80% positive), a likely positive biomarker of metastasis. About 50% of the patients showed higher TMB and HRD. Notably, TF representing ctDNA varied between 13% to 27% in blood samples with a corresponding ploidy range of 2.9 to 4.7. Surprisingly, ~50% of the patient population matched the mutation profile of clinically confirmed metastatic patients. All the patients harboring potential metastatic driver alterations showed the presence of CTCs in peripheral blood. Conclusions: Comprehensive ctDNA genomic profiling showed potential metastasis driving alterations suggesting the role of ctDNA-based liquid biopsy to predict metastasis in early breast cancer patients. We observed enhanced TF at the time of diagnosis, possibly due to the presence of distant metastasis, high disease burden, and aggressive tumor biology. Our results suggest that ctDNA dynamics at the time of disease presentation can predict early metastasis, and may demonstrate the divergent response of tumor heterogeneity to treatment in early-stage breast cancer. Citation Format: Gowhar Shafi, Manoj Dongare, Atul Bharde, Moubeen Fauzul, Kanchan Hariramani, Alain D’Souza, Bhagwat Jadhav, Trupti Kad, Sangeeta Prajapati, Vikas Jadhav, ManojKumar Kumaran, Sumit Haldar, Vatsal Mehra, Sujit Joshi, Gourishankar Aland, Richa Dave, Sreeja Jayant, Aravindan Vasudevan, Mohan Uttarwar, Jayant Khandare. Comprehensive ctDNA profiling reveals potential metastatic genomic signatures in treatment-naive early-stage breast cancer patients [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr PR007.

ICGC-ARGO precision medicine: an update on targeted therapy based on longitudinal analysis of tumour heterogeneity and evolution in colorectal cancer

We previously reported two patients with metastatic colorectal adenocarcinoma who, after referral to the precision medicine tumour board, had tissue and longitudinal liquid biopsies and responded to targeted therapy based on their molecular profile. These patients are from the SCRUM-MONSTAR programme, a nationwide genome-screening project in Japan that is part of the International Cancer Genome Consortium–Accelerating Research in Genomic Oncology. Here, we summarise the clinical history of the two patients and report an update on clinical course and longitudinal molecular assessment using circulating-tumour DNA (ctDNA) sequencing.

Abstract P008: Native Cell-Free DNA sequence analysis reliably infers epigenetically diverse subtypes of prostate cancer

Abstract Background: With the adoption of potent AR signaling inhibitors, there has been a prominent rise in the frequency of treatment-resistant tumor phenotypes. Highly aggressive transdifferentiated tumors may emerge as AR low or AR null prostate cancers (PCa) with or without neuroendocrine signatures. An accurate determination of these epigenetically diverse phenotypic subtypes has direct clinical implications, including therapy choice. Current approaches for disease subtype determination often require tissue biopsies, limiting due to the high risk of comorbidity. For a temporally evolving cancer, a minimally invasive molecular assay is urgently needed to determine disease subtypes accurately to guide clinical decisions. Methods &amp; Results: Cell-free DNA assay emerging as a nextgen tool for cancer early detection and real-time tumor evolution monitoring tool. Cell-free DNA primarily consists of DNA protected by nucleosomes and DNA binding elements and is shed into the bloodstream by cells that undergo apoptosis. We hypothesized that a nucleosome protection map and transcriptional regulation could be reliably determined from circulating tumor DNA (ctDNA) analysis. This analysis can accurately classify advanced prostate cancer into epigenetically divergent subclasses such as adenocarcinoma (ARPC) and neuroendocrine (NEPC). For developing robust phenotype-specific signatures, we performed whole-genome sequencing on plasma cfDNA from 30 PDX lines to achieve moderately deep coverage (&amp;gt;10X) of “pure” human ctDNA sequence data. We applied an analysis framework “Griffin” to determine transcription factor binding sites and transcription start site nucleosome protection signatures, predictive of specific transcription factor activity. We also generated H3K27ac, H3K27me1, and H3K27me3 histone profiles from resected PDX tumors and observed that ctDNA fragmentation and coverage patterns are consistent with histone modifications. Guided by differential nucleosome profiles, we derived a novel ctDNA fragment size variability signature that accurately predicted expression deregulation for phenotype-defining genes. These signatures helped us build an unsupervised statistical model that accurately classified PCa subtypes. We applied our model to 2 clinical cohorts and attained 96% accuracy for classifying patients with well-defined subtypes of advanced PCa, using low coverage sequencing. Conclusion: We have developed a ctDNA analysis method that can accurately classify epigenetically diverse subtypes of advanced prostate cancer, spanning AR active and neuroendocrine subtypes. This scalable and cost-effective approach is also suitable for longitudinal monitoring and prediction of treatment-emergent prostate cancer resulting from therapeutic resistance. Citation Format: Navonil De Sarkar. Native Cell-Free DNA sequence analysis reliably infers epigenetically diverse subtypes of prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P008.

Circulating tumor DNA: toward evolving the clinical paradigm of pancreatic ductal adenocarcinoma.

Over a decade of sequencing-based genomics research has unveiled a diverse somatic mutation landscape across patients with pancreatic ductal adenocarcinoma (PDAC), and the identification of druggable mutations has aligned with the development of novel targeted therapeutics. However, despite these advances, direct translation of years of PDAC genomics research into the clinical care of patients remains a critical and unmet need. Technologies that enabled the initial mapping of the PDAC mutation landscape, namely whole-genome and transcriptome sequencing, remain overly expensive in terms of both time and financial resources. Consequentially, dependence on these technologies to identify the relatively small subset of patients with actionable PDAC alterations has greatly impeded enrollment for clinical trials testing novel targeted therapies. Liquid biopsy tumor profiling using circulating tumor DNA (ctDNA) generates new opportunities by overcoming these challenges while further addressing issues particularly relevant to PDAC, namely, difficulty of obtaining tumor tissue via fine-needle biopsy and the need for faster turnaround time due to rapid disease progression. Meanwhile, ctDNA-based approaches for tracking disease kinetics with respect to surgical and therapeutic interventions offer a means to elevate the current clinical management of PDAC toward higher granularity and accuracy. This review provides a clinically focused summary of ctDNA advances, limitations, and opportunities in PDAC and postulates ctDNA sequencing technology as a catalyst for evolving the clinical decision-making paradigm of this disease.

Cerebrospinal fluid circulating tumor DNA depicts profiling of brain metastasis in NSCLC.

Brain metastasis (BM) genetically diverges from the primary tumor in non-small-cell lung cancer (NSCLC). Hence, accurately capturing clinically relevant alterations is pivotal for the delivery of targeted therapies. Circulating tumor DNA (ctDNA) sequencing has emerged as a promising liquid biopsy in the biomarker-based clinical management of recurrent and extracranial metastatic NSCLC. However, the absence of simultaneous sequencing data from brain metastatic sites prevents the definitive evaluation of the efficacy of ctDNA in representing genetic profiles in BM. Here, we performed parallel genomic comparisons between matched BM and primary tumor DNA, plasma ctDNA, and cerebrospinal fluid (CSF) ctDNA. The results indicated that CSF ctDNA had a greater ability than plasma ctDNA to comprehensively represent the mutational landscape of BM, with CSF ctDNA detecting all BM mutations in 83.33% of patients, while plasma ctDNA was only 27.78%. Mutant allele frequency (MAF) in CSF ctDNA was highly correlated with the tumor size of BM (r= 0.95), and the mean MAF in CSF ctDNA was higher than that in plasma ctDNA (38.05% vs. 4.57%, respectively). MAF and tumor mutational burden in CSF ctDNA were strongly associated with those in BM (r= 0.96 and 0.97, respectively). Of note, CSF ctDNA had significantly higher concordance with BM than plasma ctDNA (99.33% vs. 67.44%), facilitating the identification of clinically relevant mutations. Moreover, we found that plasma ctDNA has stronger profiling performance, with a concordance of 93.01% in multiple brain metastases, equivalent to CSF ctDNA. Collectively, our study indicates that CSF ctDNA is superior to plasma ctDNA in accurately representing the profiling of single BM. Plasma ctDNA could be an alternative liquid biopsy material to be applied in multiple brain metastatic NSCLC.