Surapaneni, Phani Keerthi

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.

PURPOSE Recent advances in molecular diagnostic technologies have allowed for the evaluation of solid tumor malignancies via noninvasive blood sampling, including circulating tumor DNA (ctDNA) profiling. We sought to characterize the ctDNA genomic alteration landscape in patients with biliary tract cancers (BTCs). PATIENTS AND METHODS From January 2015 to February 2018, 124 patients with BTC at the Mayo Clinic Comprehensive Cancer Center underwent ctDNA testing using a clinically available assay. The majority of samples (n = 122) were tested using the 73-gene panel that includes somatic genomic targets, including complete or critical exon coverage in 30 and 40 genes, respectively, and in some, amplifications, fusions, and indels. RESULTS A total of 138 samples were included, with approximately 70% of patients having intrahepatic BTC. All patients had locally advanced or metastatic BTC. Samples with one or more alterations, when variants of unknown significance were excluded, numbered 105 (76%). Each sample contained, on average, three alterations with a median allelic fraction of 0.52%. The overall landscape of alterations is summarized in Figures 1 and 2. After excluding variants of unknown significance, therapeutically relevant alterations were observed in 76 patients (55%), including BRAF mutations, ERBB2 amplifications, FGFR2 fusions, FGFR2 mutations, and IDH1 mutations seen in 21% of patients. A different spectrum of alterations was observed in patients with early-onset BTC (younger than age 50 years) compared with older patients (older than age 50 years). CONCLUSION Data on ctDNA in BTC is currently limited. Our study, the largest cohort reported to date to our knowledge, demonstrates the feasibility of ctDNA testing in this disease. We provide a foundation upon which the field can continue to grow.

451 Background: Gemcitabine plus platinum (GP) is the standard of care first-line treatment for advanced biliary tract cancers (BTC). There is no established second-line therapy, and retrospective reviews report progression-free survival (PFS) for second-line treatment to be < 3 months. 5-Fluorouracil plus irinotecan (FOLFIRI) is a commonly used regimen in patients (pts) with BTC who have progressed on GP, though there is a paucity of data regarding its efficacy in this population. Methods: We retrospectively evaluated pts with advanced BTC who were treated with FOLFIRI at MD Anderson, University of Michigan and Mayo Clinic in Jacksonville. Data were obtained on pt demographics, type of BTC, PFS, and overall survival (OS). Results: Ninety-eight pts were included of which 74 (76%) had metastatic disease at the time of treatment with FOLFIRI. The median age was 59 (range, 22 to 86) years. The number of pts with extrahepatic cholangiocarcinoma (CCA)/gall bladder (GB)/intrahepatic CCA were 10, 17, and 71. FOLFIRI was used as 1st, 2nd, 3rd or 4th – Nth lines in 8, 50, 36, and 4 pts, respectively. Of the 65 pts whose best responses were documented, 23 (35%) had stable disease and 7 (11%) had a partial response per RECIST v1.1. Median duration on FOLFIRI was 2.2 months. The median PFS and OS were 2.4 (95% CI 1.7 to 3.1) and 6.6 (95% CI 4.7 to 8.4) months, respectively. Median PFS for pts treated with FOLFIRI in 1st, 2nd, 3rd or 4th – Nth lines were 3.1, 2.5, 2.3 and 1.5 months, respectively. Eighteen pts received concurrent bevacizumab (13) or EGFR-targeted therapy (5) with FOLFIRI, and both of groups exhibited a median PFS of 2.7 months. Eastern Cooperative Oncology Group performance status (PS) of 0-1 was associated with improved OS (P = 0.006) compared to PS of 2-3. Conclusions: In this multi-institution retrospective review of 98 pts with BTC treated with FOLFIRI, efficacy of this regimen appears to be modest. While PFS and OS outcomes were similar to what has been previously reported, the 46% disease control rate in this group of predominantly pretreated pts is encouraging. Given the lack of other standard therapies, FOLFIRI may still have a role in this pt population, but these results emphasize the need for more effective treatment options for pts with advanced, pretreated BTC.

291 Background: Cholangiocarcinoma (CCA) has limited treatment options. Genomic analyses have led to development of targeted therapies now in clinical trials, and may enable discovery of new treatment options. However, biopsy often yields limited tissue, thus hampering tissue-based profiling opportunities. Comparative data regarding circulating tumor DNA (ctDNA) analysis and tissue based profiling in CCA are limited. Methods: We performed ctDNA NGS analysis along with tissue based profiling in pts with advanced CCA (January 2015- February 2018). ctDNA analysis was performed using Guardant 360 (Guardant Health, CA) which detects single nucleotide variants, amplifications, fusions, and specific insertion/deletion mutations in up to 73 different genes and the majority of tissue based profiling using Foundation One. The mutant allele fraction (MAF) for detected alterations was calculated relative to wild type in ctDNA. Therapeutic relevance was defined as alterations within OncoKB levels 1-3B and R1. The study was conducted in accordance with Mayo Clinic IRB requirements. Results: Among 124 pts and 139 total samples, ctDNA NGS revealed at least one genomic alteration (excluding variants of uncertain significance and synonymous mutations) in 89% of pts. Median number of alterations per pt was 3 [range, 1-15], with a median MAF of 0.42% (range, 0.1% - 94.2%). The total number of unique alterations was 321. The most commonly altered genes: TP53 (31%), KRAS (11%), FGFR2 (7%), APC and PIK3CA (each 5%) and ARID1A (3%). Amplifications were noted in 14 genes: BRAF, CCND1, CCND2, CCNE1, CDK4, CDK6, EGFR, ERBB2, FGFR1, FGFR2, MET, MYC, PDGFRA, and PIK3CA. Tissue-based profiling was available in 57 (46%) pts, with a median of 63 days between liquid and tissue biopsy. IDH1, FGFR2, TP53 and KRAS were most common gene mutations found in pts who had both liquid and tissue biopsy done. (Comparative results to be shown). Conclusions: ctDNA plasma profiling of pts with advanced CCA is a feasible alternative method to gather comprehensive genomic data. Further larger cohort studies comparing landscape of alterations seen on ctDNA versus tissue-based assays are needed.

289 Background: PDAC has limited treatment options. Genomic analyses have led to development of targeted therapies now in several clinical trials, and may enable the discovery of new treatment options. However, biopsy often yields limited tissue, thus hampering tissue-based profiling opportunities. Data regarding circulating tumor DNA (ctDNA) profiling in PDAC during real time clinical practice is limited. Methods: We performed ctDNA NGS analysis in pts with advanced PDAC (December 2014–August 2018). ctDNA analysis was performed using Guardant 360 (Guardant Health, CA), which detects single nucleotide variants, amplifications, fusions, and specific insertion/deletion mutations in up to 73 different genes. The mutant allele fraction (MAF) for detected alterations was calculated relative to wild type in ctDNA. Therapeutic relevance (TR) was defined as possible treatments within OncoKB levels 1-3B and R1. The study was conducted in accordance with Mayo Clinic Institutional Review Board requirements. Results: Among 171 pts and 206 total samples, ctDNA NGS revealed at least one genomic alteration in 150 pts (88%). Median number of alterations per patient was 3 [range, 1-15]. The total number of unique alterations was 450 with the most commonly altered genes being: TP53 (181 alterations, 40%), followed by KRAS (118 alterations, 26%), CDKN2A (23 alterations, 5%), SMAD4 (15 alterations, 3%), EGFR (11 alterations, 2.4%), PIK3CA (9 alterations, 2%), GNAS (8 alterations, 1.5%). Amplifications were noted in 16 genes, including BRAF, CCND2, CCNE1, CDK4, CDK6, EGFR, ERBB2, FGFR1, FGFR2, KIT, KRAS, MET, MYC, PDGFRA, PIK3CA and RAF1. Therapeutically relevant alterations were seen in 95 pts of the 150 pts (63%). Tissue based profiling (Results to be shown) was done in 56 (33%) pts of total 171 pts with a median of 130 days between ctDNA and tissue biopsy. KRAS and TP53 were the most common gene mutations found in patients with both liquid and tissue biopsy results. Conclusions: ctDNA plasma profiling of pts with advanced PDAC is a feasible alternative method to gather comprehensive genomic data.

288 Background: Obesity is a risk factor for developing cholangiocarcinoma (CCA). However, the effect of obesity on survival of CCA is unclear. The primary aim of this study was to analyze the impact of obesity upon overall survival of CCA patients. Secondary aims were to analyze impact of obesity upon other disease characteristics such as tumor site, stage, age, sex, BMI and Ca 19-9. Methods: A total of 411 unique pts diagnosed with CCA at Mayo Clinic Florida between 2000 and 2018 were retrieved from our collective SDMS database. Variables evaluated included:demographics, Body Mass Index (BMI), AJCC stage, tumor location and Ca 19-9.A total of 185 pts had all data available pertaining to these variables. We further restricted the analysis to pts with intrahepatic CCA classified BMI as per CDC criteria normal (18.5-25kg/m2), overweight (25-29.9kg/m2) and obese (≥30 kg/m2), thus leaving a total of 152 pts. Continuous and categorical variables were compared across BMI groups using Chi-squared or Fisher’s exact test. Overall survival rates after diagnosis at 1, 2 and 3 years were estimated using Kaplan-Meier method. Results: Among 152 pts included in the study, 28% were normal weight, 40% were overweight and 32% were obese. The overall survival rate at 1, 2 and 3 years for normal weight pts with all stages combined was 54.1%, 35%, and 30.7%, respectively. The overall survival rate at 1, 2 and 3 years for overweight pts with all stages combined was 59.7 %, 32.6%, and 25.4%, respectively. The overall survival rate at 1, 2 and 3 years for obese pts with all stages combined was 63.9%, 37.6%, and 26.7%, respectively(p = 0.8766). Multivariate analysis demonstrated is no significant difference in overall survival for obese pts compared to normal or overweight pts.(Table to be shown) However it showed, gender and Ca19-9 were statistically significant predictors of overall survival, with males and pts with Ca19-9≥100 doing worse (HR1.65 (CI = 1.05, 2.61, p = 0.031) and HR 2.31 (CI = 1.49, 3.59, p = < 0.01), respectively). Conclusions: BMI did not make a significant impact on the overall survival, though there may be a trend toward worse OS for ptswith higher BMI. A larger, stage focused evaluation is warranted for further exploration of this trend.