Citations

Abstract Background: Cholangiocarcinoma is an aggressive malignancy with limited systemic therapeutics. We have recently identified LCK, a non-receptor tyrosine kinase, as a potential therapeutic target in select subtypes of cholangiocarcinoma. Proteomic profiling in other cancer types has proven beneficial in predicting response to systemic therapies. Herein, we present proteomic profiling of cholangiocarcinoma can predict sensitivity to a novel LCK inhibitor in patient derived xenograft models. Methods: Proteins from 28 unique cholangiocarcinoma patient derived xenograft models (PDX) were isolated, labeled with tandem mass tags (TMT) and then analyzed by liquid chromatography and mass spectroscopy (LC/MS). Phosphopeptides were separately evaluated both by Immobilized Metal Affinity Chromatography and pY-enrichment techniques. Following normalization, the global proteome, serine/threonine phosphoproteome, and tyrosine phosphoproteome were analyzed and a proteomic signature was created for each tumor. These signatures were compared to PDX models with known response profiles to NTRC0652-0, an LCK inhibitor (1 resistant and 1 sensitive). The top 200 differentially abundant proteins (log2FC 2.6) were then utilized to develop a sensitive and a resistant signature. Correlation indices were then calculated across the 28 PDX model library. An additional predicted resistant and predicted sensitive model was expanded. Tumor bearing mice were randomized based on tumor volume to either treatment with NTRC (30mg/kg PO daily), n=5, or vehicle, n=4, (10% Kolliphor, 10% DMSO, 80% Water, 2 mol equivalents HCl). Tumors were measured weekly with calipers and then weighed at the end of the study. Results: In the top 200 differentially abundant proteins that represented the proteomic signature there was over representation of proteins with decreased abundance. There were only 12 proteins with higher abundance in the sensitive model as compared to the resistant. Utilizing the signature developed from the global proteomics data, nine tumors were predicted to be sensitive while 17 were predicted to be resistant to NTRC. PDX115 is a an intrahepatic CCA PDX known to contain an IDH1 mutation and was predicted to be resistant by modeling. The model was resistant to single agent NTRC with no demonstrable effect on tumor growth in vivo. PDX175 is a distal CCA PDX and was predicted to be sensitive by modeling. In vivo growth was inhibited by NTRC as compared to vehicle treated mice following two weeks of treatment (983% vs 435%, p=0.03). Additionally, final tumor weight was significantly lower in NTRC treated mice (0.651 g vs 0.317 g, p=0.04). Conclusion: Cholangiocarcinoma proteomic signatures can be used to predict therapeutic response to a novel LCK inhibitor in preclinical models. Whether or not this could be applied to other treatment modalities in cholangiocarcinoma remains to be determined. Citation Format: Ryan Watkins, Roberto Alva-Ruiz, Caitlin Conboy, Dong-Gi Mun, Erik Jessen, Diep Vu, Jos de Man, Rogier Buijsman, Akhilesh Pandey, Mitesh Borad, Gregory Gores, Rory Smoot. Proteomic profiling of cholangiocarcinoma predicts response to a novel small molecule inhibitor in preclinical models [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 3094.

PURPOSE This investigation sought to evaluate the prognostic value of pretreatment of circulating tumor DNA (ctDNA) in metastatic biliary tract cancers (BTCs) treated with platinum-based first-line chemotherapy treatment. MATERIALS AND METHODS We performed a retrospective analysis of 67 patients who underwent ctDNA testing before platinum-based chemotherapy for first-line treatment for metastatic BTC. For analysis, we considered the detected gene with highest variant allele frequency as the dominant clone allele frequency (DCAF). Results of ctDNA analysis were correlated with patients' demographics, progression-free survival (PFS), and overall survival (OS). RESULTS The median age of patients was 67 (27-90) years. Fifty-four (80.6%) of 67 patients evaluated had intrahepatic cholangiocarcinoma; seven had extrahepatic cholangiocarcinoma, and six gallbladder cancers. Forty-six (68.6%) of the patients were treated with cisplatin plus gemcitabine, and 16.4% of patients received gemcitabine and other platinum (carboplatin or oxaliplatin) combinations, whereas 15% of patients were treated on a clinical trial with gemcitabine and cisplatin plus additional agents (CX4945, PEGPH20, or nab-paclitaxel). TP53, KRAS, FGFR2, ARID1A, STK11, and IDH1 were the genes with highest frequency as DCAF. The median DCAF was 3% (0%-97%). DCAF > 3% was associated with worse OS (median OS: 10.8 v 18.8 months, P = .032). Stratifying DCAF in quartiles, DCAF > 10% was significantly related to worse PFS (median PFS: 3 months, P = .014) and worse OS (median OS: 7.0 months, P = .001). Each 1% increase in ctDNA was associated with a hazard ratio of 13.1 in OS when adjusting for subtypes, metastatic sites, size of largest tumor, age, sex, and CA19-9. CONCLUSION DCAF at diagnosis of advanced BTC can stratify patients who have worse outcomes when treated with upfront platinum-based chemotherapy. Each increase in %ctDNA decreases survival probabilities.

463 Background: Molecularly targeted therapy is gaining traction in cholangiocarcinoma with the first FDA approval of a targeted agent in 2020, the FGFR inhibitor pemigatinib. Yet response rates and efficacy are modest, and most cholangiocarcinoma patients still lack targeted therapy options. The hippo pathway effector, YAP, is an oncogene that is activated in the majority of cholangiocarcinoma, yet attempts to develop YAP inhibitors have not yet been clinically successful. Recently, we described a novel mechanism of YAP regulation, via tyrosine phosphorylation by the Src-family kinase LCK. Therefore we hypothesized that LCK inhibition by may be therapeutic in cholangiocarcinoma through suppression of YAP activity. Methods: NTRC 0652-0 is a novel tyrosine kinase inhibitor with specificity for LCK. In vitro pharmacodynamics were defined. In cholangiocarcinoma cells, specificity for LCK inhibition was determined by mass spectrometry-based unbiased analysis of the tyrosine phosphoproteome following treatment with NTRC 0652-0 or genetic deletion of LCK. YAP phosphorylation and cotranscriptional activity, cell viability, and apoptosis were assessed. A panel of eight cholangiocarcinoma patient derived organoids (PDO) were characterized and tested for sensitivity to NTRC 0652-0. Two patient-derived xenograft (PDX) models bearing FGFR2-rearrangements were utilized for in vivo assessment of pharmacokinetics, toxicity, and efficacy. Results: NTRC 0652-0 demonstrated selectivity for LCK inhibition in vitro and in cholangiocarcinoma cells. NTRC 0652-0 treatment led to YAP inhibition and apoptotic cell death in cholangiocarcinoma cell lines, associated with inhibition of MCL1 expression. PDOs demonstrated variable sensitivity to NTRC 0652-0, correlated with basal YAP tyrosine phosphorylation and drug-induced suppression of YAP co-transcriptional activity. FGFR2-altered cholangiocarcinoma was also identified as a subset with enrichment of YAP target genes. Cells from an FGFR2-altered PDX were sensitive to NTRC 0652-0 despite being primarily resistant to pemigatinib. In two PDX models of FGFR2-altered cholangiocarcinoma, daily oral treatment with NTRC 0652-0 inhibited YAP tyrosine phosphorylation, and resulted in stable plasma and tumor drug levels, acceptable toxicity, and significantly decreased tumor growth. Conclusions: A novel LCK inhibitor, NTRC 0652-0, reduced YAP signaling and demonstrated preclinical efficacy in multiple patient-derived models of cholangiocarcinoma. LCK is a novel therapeutic target in cholangiocarcinoma, and YAP activation or FGFR2-alteration are potential biomarkers for response.

AXL and its corresponding ligand growth arrest–specific 6 (GAS‐6) are critically involved in hepatic immunomodulation and regenerative processes. Pleiotropic inhibitory effects on innate inflammatory responses might essentially involve the shift of macrophage phenotype from a pro‐inflammatory M1 to an anti‐inflammatory M2. We aimed to assess the relevance of the AXL/GAS‐6‐pathway in human liver regeneration and, consequently, its association with clinical outcome after hepatic resection. Soluble AXL (sAXL) and GAS‐6 levels were analyzed at preoperative and postoperative stages in 154 patients undergoing partial hepatectomy and correlated with clinical outcome. Perioperative dynamics of interleukin (IL)‐6, soluble tyrosine‐protein kinase MER (sMerTK), soluble CD163 (sCD163), and cytokeratin (CK) 18 were assessed to reflect pathophysiological processes. Preoperatively elevated sAXL and GAS‐6 levels predicted postoperative liver dysfunction (area under the curve = 0.721 and 0.722; P < 0.005) and worse clinical outcome. These patients failed to respond with an immediate increase of sAXL and GAS‐6 upon induction of liver regeneration. Abolished AXL pathway response resulted in a restricted increase of sCD163, suggesting a disrupted phenotypical switch to regeneratory M2 macrophages. No association with sMerTK was observed. Concomitantly, a distinct association of IL‐6 levels with an absent increase of AXL/GAS‐6 signaling indicated pronounced postoperative inflammation. This was further supported by increased intrahepatic secondary necrosis as reflected by CK18M65. sAXL and GAS‐6 represent not only potent and easily accessible preoperative biomarkers for the postoperative outcome but also AXL/GAS‐6 signaling might be of critical relevance in human liver regeneration. Refractory AXL/GAS‐6 signaling, due to chronic overactivation/stimulation in the context of underlying liver disease, appears to abolish their immediate release following induction of liver regeneration, causing overwhelming immune activation, presumably via intrahepatic immune regulation.

Prostate‐specific membrane antigen (PSMA) is a validated target for molecular diagnostics and targeted radionuclide therapy. Our purpose was to evaluate PSMA expression in hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA), and hepatic adenoma (HCA); investigate the genetic pathways in HCC associated with PSMA expression; and evaluate HCC detection rate with 68Ga‐PSMA‐11 positron emission tomography (PET). In phase 1, PSMA immunohistochemistry (IHC) on HCC (n = 148), CCA (n = 111), and HCA (n = 78) was scored. In a subset (n = 30), messenger RNA (mRNA) data from the Cancer Genome Atlas HCC RNA sequencing were correlated with PSMA expression. In phase 2, 68Ga‐PSMA‐11 PET was prospectively performed in patients with treatment‐naïve HCC on a digital PET scanner using cyclotron‐produced 68Ga. Uptake was graded qualitatively and semi‐quantitatively using standard metrics. On IHC, PSMA expression was significantly higher in HCC compared with CCA and HCA (P < 0.0001); 91% of HCCs (n = 134) expressed PSMA, which principally localized to tumor‐associated neovasculature. Higher tumor grade was associated with PSMA expression (P = 0.012) but there was no association with tumor size (P = 0.14), fibrosis (P = 0.35), cirrhosis (P = 0.74), hepatitis B virus (P = 0.31), or hepatitis C virus (P = 0.15). Overall survival tended to be longer in patients without versus with PSMA expression (median overall survival: 4.2 vs. 1.9 years; P = 0.273). FGF14 (fibroblast growth factor 14) mRNA expression correlated positively (rho = 0.70; P = 1.70 × 10‐5) and MAD1L1 (Mitotic spindle assembly checkpoint protein MAD1) correlated negatively with PSMA expression (rho = −0.753; P = 1.58 × 10‐6). Of the 190 patients who met the eligibility criteria, 31 patients with 39 HCC lesions completed PET; 64% (n = 25) lesions had pronounced 68Ga‐PSMA‐11 standardized uptake value: SUVmax (median [range] 9.2 [4.9‐28.4]), SUVmean 4.7 (2.4‐12.7), and tumor‐to‐liver background ratio 2 (1.1‐11). Conclusion: Ex vivo expression of PSMA in neovasculature of HCC translates to marked tumor avidity on 68Ga‐PSMA‐11 PET, which suggests that PSMA has the potential as a theranostic target in patients with HCC.

Objective: The aim of this study was to define robust benchmark values for the surgical treatment of perihilar cholangiocarcinomas (PHC) to enable unbiased comparisons. Background: Despite ongoing efforts, postoperative mortality and morbidity remains high after complex liver surgery for PHC. Benchmark data of best achievable results in surgical PHC treatment are however still lacking. Methods: This study analyzed consecutive patients undergoing major liver surgery for PHC in 24 high-volume centers in 3 continents over the recent 5-year period (2014–2018) with a minimum follow-up of 1 year in each patient. Benchmark patients were those operated at high-volume centers (≥50 cases during the study period) without the need for vascular reconstruction due to tumor invasion, or the presence of significant co-morbidities such as severe obesity (body mass index ≥35), diabetes, or cardiovascular diseases. Benchmark cutoff values were derived from the 75th or 25th percentile of the median values of all benchmark centers. Results: Seven hundred eight (39%) of a total of 1829 consecutive patients qualified as benchmark cases. Benchmark cut-offs included: R0 resection ≥57%, postoperative liver failure (International Study Group of Liver Surgery): ≤35%; in-hospital and 3-month mortality rates ≤8% and ≤13%, respectively; 3-month grade 3 complications and the CCI: ≤70% and ≤30.5, respectively; bile leak-rate: ≤47% and 5-year overall survival of ≥39.7%. Centers operating mostly on complex cases disclosed better outcome including lower post-operative liver failure rates (4% vs 13%; P = 0.002). Centers from Asia disclosed better outcomes. Conclusion: Surgery for PHC remains associated with high morbidity and mortality with now the availability of benchmark values covering 21 outcome parameters, which may serve as key references for comparison in any future analyses of individuals, group of patients or centers.

Abstract Background:Biliary tract cancers (BTC) are rare, chemo resistant and are associated with a poor prognosis. Preclinical and early clinical work had demonstrated interesting anti-tumor activity from targeting fibroblast growth factor receptor (FGFR) pathway. We hypothesized that ponatinib, a multi-targeted tyrosine kinase inhibitor with activity against FGFR, would be active in BTC patients with FGFR alterations.Methods:This was a multi-center, single institution pilot study of ponatinib in patients with advanced, refractory BTC with FGFR alterations. The primary end point was overall response rate, with secondary points of overall survival (OS), progression-free survival (PFS) and Health Related Quality of Life (HRQoL) assessment. Results:Twelve patients were enrolled prior to early termination of the trial. Clinical benefit (defined as complete/partial response + stable disease) of 45.5% was observed. All observed toxicities were manageable and reversible. Toxicities were mild, with lymphopenia (75%), rash (63%) and fatigue (50%) being the most frequent. No significant detriment in global QoL was observed.Conclusions:Ponatinib as a single agent in FGFR altered BTC is tolerable with very modest clinical activity. This is the first report of prospective assessment of FGFR inhibition in BTC using ponatinib, and the first study to report its effect on HRQoL. Further development of ponatinib will involve correlative studies to better refine patient selection, focus on combinations with other molecular targeted agents, conventional cytotoxic chemotherapy, and studies to better understand mechanisms of treatment resistance.

PURPOSECholangiocarcinomas (CCA) are a group of heterogeneous tumors arising from the biliary epithelia. Significant sequencing efforts have provided further insights into the molecular mechanisms of this disease including fibroblast growth factor receptor ( FGFR) alterations, which occurs in approximately 15%-20% of intrahepatic CCAs. Herein, we describe the FGFR inhibitor (FGFRi)-associated treatment toxicity and cancer-specific outcomes from a multicenter single-institution cohort.METHODSThis is a retrospective study of patients with CCA and known FGFR alterations treated with FGFRi. We describe the toxicity and efficacy in patients treated at Mayo Clinic between January 2010 and December 2020.RESULTSOur group identified 61 patients with advanced or metastatic CCA, 19 males (31%) and 42 females (69%), harboring FGFR alterations who received FGFRi. The most common grade 1 or higher adverse events for all patients included fatigue (92%), AST elevations (78%), anemia (80%), decreased platelet count (63%), and hyperphosphatemia (74%). Median progression-free survival on FGFRi was 5.8 months for all patients (95% CI, 4.9 to 9.0). Females had significantly longer progression-free survival at 6.9 months (95% CI, 5.2 to 11.8) on FGFRi compared with males at 4.9 months (95% CI, 2.8 to not estimable; P = .038).CONCLUSIONFGFRi are well tolerated with clinical efficacy. With the recent approval of FGFRi by the US Food and Drug Administration and ongoing clinical trials for new FGFRi, understanding outcomes and toxicity associated with these medications is important for precision oncology.

Abstract Background and Objectives Pancreatic neuroendocrine tumors (pNETs) in patients with hereditary cancer syndromes are typically multifocal. In contrast, sporadic pNETs are usually unifocal and the incidence of multifocal sporadic pNETs is unknown. The primary aim of this study was to investigate the incidence of multifocality in sporadic pNETs and any associated effect on recurrence risk and survival. Methods Patients who underwent resection of pNETs at Mayo Clinic from 2000 to 2019 were identified and clinical data were obtained from medical records. Syndromic disease was defined as pNETs arising in the setting of a hereditary cancer syndrome. Statistical comparisons were made using χ 2 , Fisher's exact, and Kruskal–Wallis tests and survival was assessed using the Kaplan–Meier method. Results Six hundred and sixty‐one patients with sporadic pNETs and fifty‐nine with syndromic pNETs were identified. Multifocal disease was present in 4.8% of sporadic patients and 84.7% of syndromic patients ( p < .001). Within patients with sporadic pNETs, clinicopathologic features and recurrence‐free and overall survival were similar between patients with unifocal and multifocal disease. Conclusions Multifocal sporadic pNETs are rare and multifocality is not associated with worse survival or increased recurrence risk. Patients with multifocal sporadic pNETs can likely be safely managed with a combination of resection and observation as indicated for each tumor.

AbstractIntroductionAlthough surgical resection is necessary, it is not sufficient for long‐term survival in pancreatic ductal adenocarcinoma (PDAC). We sought to evaluate survival after up‐front surgery (UFS) in anatomically resectable PDAC in the context of three critical factors: (A) margin status; (B) CA19‐9; and (C) receipt of adjuvant chemotherapy.MethodsThe National Cancer Data Base (2010–2015) was reviewed for clinically resectable (stage 0/I/II) PDAC patients. Surgical margins, pre‐operative CA19‐9, and receipt of adjuvant chemotherapy were evaluated. Patient overall survival was stratified based on these factors and their respective combinations. Outcomes after UFS were compared to equivalently staged patients after neoadjuvant chemotherapy on an intention‐to‐treat (ITT) basis.ResultsTwelve thousand and eighty‐nine patients were included (n = 9197 UFS, n = 2892 ITT neoadjuvant). In the UFS cohort, only 20.4% had all three factors (median OS = 31.2 months). Nearly 1/3rd (32.7%) of UFS patients had none or only one factor with concomitant worst survival (median OS = 14.7 months). Survival after UFS decreased with each failing factor (two factors: 23 months, one factor: 15.5 months, no factors: 7.9 months) and this persisted after adjustment. Overall survival was superior in the ITT‐neoadjuvant cohort (27.9 vs. 22 months) to UFS.ConclusionDespite the perceived benefit of UFS, only 1‐in‐5 UFS patients actually realize maximal survival when known factors highly associated with outcomes are assessed. Patients are proportionally more likely to do worst, rather than best after UFS treatment. Similarly staged patients undergoing ITT‐neoadjuvant therapy achieve survival superior to the majority of UFS patients. Patients and providers should be aware of the false perception of ‘optimal’ survival benefit with UFS in anatomically resectable PDAC.