Kasi, Pashtoon Murtaza

Clinical trials reporting the robust antitumor activity of immune checkpoint inhibitors (ICIs) in microsatellite instability-high (MSI-H) solid tumors have used tissue-based testing to determine the MSI-H status. This study assessed if MSI-H detected by a plasma-based circulating tumor DNA liquid biopsy test predicts robust response to ICI in patients with pancreatic ductal adenocarcinoma (PDAC). Retrospective analysis of patients with PDAC and MSI-H identified on Guardant360 from October 2018 to April 2021 was performed; clinical outcomes were submitted by treating providers. From 52 patients with PDAC +MSI-H, outcomes were available for 10 (19%) with a median age of 68 years (range: 56–82 years); the majority were male (80%) and had metastatic disease (80%). Nine of 10 patients were treated with ICI. Eight out of nine patients received single-agent pembrolizumab (8/9), while one received ipilimumab plus nivolumab. The overall response rate by Response Evaluation Criteria in Solid Tumors was 77% (7/9). The median progression-free survival and overall survival were not reached in this cohort. The median duration of treatment with ICI was 8 months (range: 1–24), and six out of seven responders continued to show response at the time of data cut-off after a median follow-up of 21 months (range: 11–33). Tissue-based MSI results were concordant with plasma-based G360 results in five of six patients (83%) who had tissue-based test results available, with G360 identifying one more patient with MSI-H than tissue testing. These results suggest that detecting MSI-H by a well-validated liquid biopsy test could predict a robust response to ICI in patients with PDAC. The use of liquid biopsy may expand the identification of PDAC patients with MSI-H tumors and enable treatment with ICI resulting in improved outcomes.

3601 Background: Two cfDNA assays are FDA-approved for use in advanced cancer pts across solid tumors. While several NCCN guidelines recommend cfDNA as a valid and useful method for molecular testing, the utility varies by cancer type. Despite the growing number of molecular alterations pertinent to first-line treatment in mCRC, current guidelines offer little guidance on the use of cfDNA in mCRC. Here we assess the ability of cfDNA to detect guideline-relevant actionable biomarkers informative for first-line therapy selection and beyond in patients with mCRC. Methods: We queried consecutive samples from advanced cancer pts with a diagnosis of CRC who underwent testing via a commercially available cfDNA assay (Guardant360) from September 2018 to January 2022. Validation and content of this assay has been previously described. To enrich for less heavily treated pts, we limited analysis to those whose first cfDNA test was done during the study period and only included the pt’s first test. The maximum variant allele fraction (maxVAF) for each test was used as a proxy for tumor shed and was compared between cancers with a Mann-Whitney test. The frequency of suspected germline alterations was assessed only in pts who received testing via a panel version with expanded mismatch repair (MMR) gene coverage. Tissue frequencies for select biomarkers were obtained from the MSK-IMPACT Clinical Sequencing Cohort in cBioPortal. Results: In total, 14,345 pts with mCRC received their first cfDNA test during the study period, with 92% of pts (13,190) having >1 cfDNA alteration detected. Median age was 62 and 44% of pts were female. The frequencies of key biomarkers are listed in Table 1. The median maxVAF was 5.6%, compared to 2.5% and 1.8% for pts with advanced gastric and NSCLC, respectively (p = < 0.0001 for both). The average turnaround time (TAT) for tests was 7 calendar days. Conclusions: cfDNA results from pts with mCRC demonstrated detection of biomarkers essential to first-line treatment decisions at a frequency comparable to what has been reported via tissue genotyping. mCRC pts had significantly higher tumor shed compared to other cancer types with guidelines that recommend cfDNA as an option for molecular testing, suggesting a potential gap in coverage. Tissue availability in mCRC does not guarantee comprehensive testing is completed quickly (compared to the 7-day TAT seen for cfDNA here), making it an attractive alternative strategy for first-line therapy selection in mCRC. [Table: see text]

607 Background: Immunotherapy (IO) is known to have robust anti-tumor activity in patients with MSI-H solid tumors. However, clinical trials investigating IO activity have used tissue-based testing to determine MSI-H status. Pancreatic tumor biopsy often does not provide sufficient tumor tissue for MSI testing. We investigated if the MSI-H status detected by plasma-based circulating tumor DNA (ctDNA) testing predicts robust response to IO in patients with PC. Methods: Genomic results from a well-validated plasma-based ctDNA assay (Guardant360[G360]) performed as part of routine clinical care between October 1, 2018 and September 7, 2021 in patients with PC were queried to identify patients with MSI-H tumors. Patient characteristics, tumor characteristics, treatment details, and outcomes were reported by ordering clinicians where available. The data cut-off date was September 1, 2021. Results: A total of 52 patients with PC who had MSI-H tumors on G360 were identified. Clinical outcomes data were available for 10/52 (19%) patients who were included for analysis. This patient cohort had a median age of 68 years (range: 56-82); 80% were male and 80% of patients had metastatic disease. 9/10 patients received IO: 3 in the first-line, 3 in the second-line, 3 in the third-line setting; most received pembrolizumab (8/9) while 1 received ipilimumab plus nivolumab. The median duration of IO was 8 months (range: 1-24). The overall response rate was 77% (7/9) and 6 of the 7 responders continue to show response at the time of data cut-off after a median follow-up of 21 months (range:11-33). The median progression-free survival and overall survival were not reached in the IO-treated cohort. Tissue-based MSI testing results were concordant with plasma-based G360 results in 5 of 6 patients (83%) who had tissue-based test results available. The patient with the discordant result was MSI-H by G360 but had intact mismatch repair protein expression by immunohistochemistry. This patient received neoadjuvant IO followed by surgery and the resected specimen confirmed pathological complete response. Conclusions: The detection of MSI-H status by plasma-based ctDNA testing is highly concordant to tissue-based testing and predicts robust and durable response to IO in patients with PC. The use of a well-validated plasma-based ctDNA analysis may expand the identification of MSI-H tumors in patients with PC and enable treatment with IO resulting in improved outcomes.

3040 Background: Pembrolizumab was recently FDA approved across solid tumors for TMB scores ≥ 10mut/Mb as assessed by next-generation sequencing (NGS) of tissue (tTMB). A prior study of advanced cancer patients treated with immunotherapy found that higher somatic TMB, as defined by the 80th percentile in each histology, was associated with better overall survival. Previously, bTMB assessed by ctDNA from patients with newly diagnosed advanced NSCLC at a score of 16 mut/MB correlated with a tTMB score of 10 mut/MB. TMB levels vary by cancer type, line of treatment, and therapy received; the distribution of bTMB scores across solid tumor types has not been well characterized. Here we report the distribution of bTMB scores in patients with advanced malignancies. Methods: We queried 5,610 samples from patients with different cancer types undergoing clinical cell-free DNA testing (Guardant360; Redwood City, CA) and assessed bTMB scores from October 2020 - January 2021. bTMB score was derived via a previously described computational algorithm examining the total number of synonymous and non-synonymous SNVs and indels across a 1.0MB genomic footprint. We assessed the success rate of bTMB evaluation, overlap with microsatellite instability (MSI) status, and defined the distribution of bTMB levels across indications in this dataset. Results: bTMB score was successfully assessed in 4,275/5,610 (76.3%) samples (Table). The majority of samples (58%) were tested at disease progression as compared to initial diagnosis (42%). The median turnaround time from sample receipt to clinical reporting was 11 days and decreased to 9 days over the course of the study. For the majority of cancer types the 80th percentile TMB was ≥ 16 mut/MB tissue equivalency. Conclusions: Our analysis demonstrates the feasibility of measuring bTMB using a commercially available liquid biopsy assay. bTMB scores trended higher than tTMB previously reported in these cancer types, reflecting the ability of ctDNA to better capture tumor heterogeneity. cfDNA may allow for exploration of bTMB evolution throughout treatment. TMB should be interpreted in the context of disease, treatment, and method; these data establish a pan-cancer benchmark for bTMB which will serve as a resource for further studies.[Table: see text]

AbstractThrough our involvement in KEYNOTE‐059, we unexpectedly observed durable responses in two patients with metastatic gastroesophageal adenocarcinoma (mGEA) who received ramucirumab (anti‐VEGFR‐2)/paclitaxel after immune checkpoint inhibition (ICI). To assess the reproducibility of this observation, we piloted an approach to administer ramucirumab/paclitaxel after ICI in more patients, and explored changes in the immune microenvironment. Nineteen consecutive patients with mGEA received ICI followed by ramucirumab/paclitaxel. Most (95%) did not respond to ICI, yet after irRECIST‐defined progression on ICI, all patients experienced tumor size reduction on ramucirumab/paclitaxel. The objective response rate (ORR) and progression‐free survival (PFS) on ramucirumab/paclitaxel after ICI were higher than on the last chemotherapy before ICI in the same group of patients (ORR, 58.8% vs 11.8%; PFS 12.2 vs 3.0 months; respectively). Paired tumor biopsies examined by imaging mass cytometry showed a median 5.5‐fold (range 4‐121) lower frequency of immunosuppressive forkhead box P3+ regulatory T cells with relatively preserved CD8+ T cells, post‐treatment versus pre‐treatment (n = 5 pairs). We then compared the outcomes of these 19 patients with a separate group who received ramucirumab/paclitaxel without preceding ICI (n = 68). Median overall survival on ramucirumab/paclitaxel was longer with (vs without) immediately preceding ICI (14.8 vs 7.4 months) including after multivariate analysis, as was PFS. In our small clinical series, outcomes appeared improved on anti‐VEGFR‐2/paclitaxel treatment when preceded by ICI, in association with alterations in the immune microenvironment. However, further investigation is needed to determine the generalizability of these data. Prospective clinical trials to evaluate sequential treatment with ICI followed by anti‐VEGF(R)/taxane are underway.

Abstract Purpose Recent advances in molecular diagnostic technologies allow for the evaluation of solid tumor malignancies through noninvasive blood sampling, including circulating tumor DNA profiling (ctDNA). Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis, often because of late presentation of disease. Diagnosis is often made using endoscopic ultrasound or endoscopic retrograde cholangiopancreatography, which often does not yield enough tissue for next-generation sequencing. With this study, we sought to characterize the ctDNA genomic alteration landscape in patients with advanced PDAC with a focus on actionable findings. Materials and Methods From December 2014 through October 2019, 357 samples collected from 282 patients with PDAC at Mayo Clinic underwent ctDNA testing using a clinically available assay. The majority of samples were tested using the 73-gene panel which includes somatic genomic targets, including complete or critical exon coverage in 30 and 40 genes, respectively, and in some, amplifications, fusions, and indels. Clinical data and outcome variables were available for 165 patients; with 104 patients at initial presentation. Results All patients included in this study had locally advanced or metastatic PDAC. Samples having at least one alteration, when variants of unknown significance (VUS) were excluded, numbered 266 (75%). After excluding VUS, therapeutically relevant alterations were observed in 170 (48%) of the total 357 cohort, including KRAS (G12C), EGFR, ATM, MYC, BRCA, PIK3CA, and BRAF mutations. KRAS, SMAD, CCND2, or TP53 alterations were seen in higher frequency in patients with advanced disease. Conclusion Our study is the largest cohort to date that demonstrates the feasibility of ctDNA testing in PDAC. We provide a benchmark landscape upon which the field can continue to grow. Future applications may include use of ctDNA to guide treatment and serial monitoring of ctDNA during disease course to identify novel therapeutic targets for improved prognosis. Implications for Practice Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis often due to late presentation of disease. Biopsy tissue sampling is invasive and samples are often inadequate, requiring repeated invasive procedures and delays in treatment. Noninvasive methods to identify PDAC early in its course may improve prognosis in PDAC. Using ctDNA, targetable genes can be identified and used for treatment.

AbstractPurpose:To analytically and clinically validate microsatellite instability (MSI) detection using cell-free DNA (cfDNA) sequencing.Experimental Design:Pan-cancer MSI detection using Guardant360 was analytically validated according to established guidelines and clinically validated using 1,145 cfDNA samples for which tissue MSI status based on standard-of-care tissue testing was available. The landscape of cfDNA-based MSI across solid tumor types was investigated in a cohort of 28,459 clinical plasma samples. Clinical outcomes for 16 patients with cfDNA MSI-H gastric cancer treated with immunotherapy were evaluated.Results:cfDNA MSI evaluation was shown to have high specificity, precision, and sensitivity, with a limit of detection of 0.1% tumor content. In evaluable patients, cfDNA testing accurately detected 87% (71/82) of tissue MSI-H and 99.5% of tissue microsatellite stable (863/867) for an overall accuracy of 98.4% (934/949) and a positive predictive value of 95% (71/75). Concordance of cfDNA MSI with tissue PCR and next-generation sequencing was significantly higher than IHC. Prevalence of cfDNA MSI for major cancer types was consistent with those reported for tissue. Finally, robust clinical activity of immunotherapy treatment was seen in patients with advanced gastric cancer positive for MSI by cfDNA, with 63% (10/16) of patients achieving complete or partial remission with sustained clinical benefit.Conclusions:cfDNA-based MSI detection using Guardant360 is highly concordant with tissue-based testing, enabling highly accurate detection of MSI status concurrent with comprehensive genomic profiling and expanding access to immunotherapy for patients with advanced cancer for whom current testing practices are inadequate.See related commentary by Wang and Ajani, p. 6887

Abstract Purpose: To compare the clinical characteristics and overall survival (OS) of germline mutation carriers in homologous recombination repair (HRR) genes and noncarriers with pancreatic ductal adenocarcinoma (PDAC). Experimental Design: Germline DNA from 3,078 patients with PDAC enrolled in a prospective registry at Mayo Clinic between 2000 and 2017 was analyzed for mutations in 37 cancer predisposition genes. Characteristics and OS of patients with mutations in eight genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, PALB2, RAD51C, and RAD51D) involved in HRR were compared with patients testing negative for mutations in all 37 genes. Results: The 175 HRR mutation carriers and 2,730 noncarriers in the study had a median duration of follow-up of 9.9 years. HRR mutation carriers were younger (median age at diagnosis: 63 vs. 66 years, P &lt; 0.001) and more likely to have metastatic disease at diagnosis (46% vs. 36%, P = 0.004). In a multivariable model adjusting for sex, age at diagnosis, and tumor staging, patients with germline HRR mutations had a significantly longer OS compared with noncarriers [HR, 0.83; 95% confidence interval (CI), 0.70–0.97; P = 0.02]. Further gene-level analysis demonstrated that germline ATM mutation carriers had longer OS compared with patients without germline mutations in any of the 37 genes (HR, 0.72; 95% CI, 0.55–0.94; P = 0.01). Conclusions: This study demonstrates that germline mutation carrier status in PDAC is associated with longer OS compared with noncarriers. Further research into tumor biology and response to platinum-based chemotherapy in germline mutation carriers with PDAC are needed to better understand the association with longer OS.

4541 Background: Anti-VEGFR2 therapy (ramucirumab/paclitaxel [RAM/TAX]) and ICI are approved as 2nd- and 3rd-line therapy (Tx), respectively, for pts with mGEA. We unexpectedly saw durable responses in 2 pts on RAM/TAX after progression on an ICI trial (KN-059; PMID 29674442). We performed a pilot to examine the clinical activity of ICI followed by RAM/TAX. Then we retrospectively compared the outcomes of pts who received this serial Tx to pts who received RAM/TAX without prior ICI. Methods: All pts with mGEA at Mayo Clinic who received RAM/TAX (2014-19) were included (N = 87). Outcomes were best objective response rate (ORR: complete [CR] or partial response) per RECIST1.1, progression-free survival (PFS), duration of response (DOR), and overall survival (OS). Chi square and multivariate (MV) logistic and Cox regression were used. Results: 15 consecutive pts with measurable mGEA received ICI immediately followed by RAM/TAX after irRECIST progression. Most pts (95%) did not respond to ICI. Yet on RAM/TAX, 100% (15/15) had tumor reduction (range -8% to -100%) with an ORR of 73% (11/15), including 3 CRs. In these pts (who received ICI followed by RAMTAX), PFS on RAMTAX was longer than on last chemotherapy before ICI (12.3 vs 3.0 m, P < .001). Outcomes on RAM/TAX in these pts were significantly better than in pts who received RAM/TAX alone (see Table). Associations were strengthened after adjusting for total lines of Tx, line of Tx of RAM/TAX, age, and ECOG PS. Exploratory analysis of paired tumor biopsies collected pre-ICI and on RAM/TAX in a small subset revealed that the frequency of intratumoral immunosuppressive FOXP3+ Tregs decreased on RAM/TAX, whereas the frequency of antitumor CD8+ T cells was preserved. Conclusions: RAM/TAX immediately preceded by ICI was associated with significantly higher OS, ORR, and DOR than RAM/TAX alone, suggesting ICI may enhance efficacy of subsequent anti-VEGFR/taxane therapy. This novel sequence of therapy will be tested prospectively in a new randomized phase 2 trial (NCT04069273). [Table: see text]