SUMMARY Defective Hippo/YAP signaling in the liver results in tissue overgrowth and development of hepatocellular carcinoma (HCC). Here, we uncover mechanisms of YAP-mediated hepatocyte reprogramming and HCC pathogenesis. YAP functions as a rheostat maintaining metabolic specialization, differentiation and quiescence within the hepatocyte compartment. Increased or decreased YAP activity reprograms subsets of hepatocytes to different fates associated with deregulation of the HNF4A, CTNNB1, and E2F transcriptional programs controlling hepatocyte quiescence and differentiation. Importantly, treatment with siRNA-lipid nanoparticles (siRNA-LNPs) targeting YAP restores hepatocyte differentiation and causes pronounced tumor regression in a genetically engineered mouse HCC model. Furthermore, YAP targets are enriched in an aggressive human HCC subtype characterized by a proliferative signature and absence of CTNNB1 mutations. Thus, our work reveals Hippo signaling as a key regulator of positional identity of hepatocytes, supports targeting YAP using siRNA-LNPs as a paradigm of differentiation-based therapy, and identifies an HCC subtype potentially responsive to this approach.

ALK, ROS1, and NTRK rearrangements define a new rare subtype of mCRC with extremely poor prognosis. Primary tumor site, MSI-high, and RAS and BRAF wild-type status may help to identify patients bearing these alterations. While sensitivity to available treatments is limited, targeted strategies inhibiting ALK, ROS, and TrkA-B-C provided encouraging results.

589 Background: ALK, ROS1, and NTRKs fusions occur in 0.2-2.4% of CRCs, and represent therapeutic targets, as indicated by promising reports of individual patients treated with tyrosine kinase inhibitors. Clinical and molecular features of mCRCs harboring these fusions have not been elucidated. Methods: mCRC harboring ALK, ROS1 and NTRKs fusions were identified taking advantage of screening programs worldwide (Ignyta STARTRK trials; Foundation Medicine Database; Memorial Sloan Kettering-IMPACT; Australian MAX trial post-hoc analysis). Clinicopathological and molecular characteristics of ALK, ROS1, NTRKs rearranged cases were compared with those of non-rearranged cases, included in Ignyta’s STARTRK trials program. Results: 27 ALK, ROS1 or NTRKs rearranged (including a novel SCYL3-NTRK1 fusion) and 319 not rearranged mCRCs patients were included. Rearrangements were more frequent in older patients (p = 0.024), and tumors that were right-sided (80% vs 30%; p < 0.001), RAS wild-type (93% vs 52%; p < 0.001), MSI-high (48% vs 8%; p < 0.001) and spread more frequently to lymph nodes (46% vs 25%; p = 0.03) and less frequently to the liver (42% vs 66%; p = 0.026). At a median follow-up of 28.5 months, patients bearing rearranged tumors had a shorter overall survival (OS) when compared to non-rearranged (15.6 vs 33.7 mos; HR: 2.17, 95% CI 1.03-4.57; p < 0.001). In the multivariable model including significant prognostic variables (primary site, primary resection, BRAF and MMR status), rearrangements were still associated with shorter OS (HR: 2.78, 95% CI 1.27-6.07; p = 0.011). All of the 4 patients with rearranged tumors evaluable for benefit from anti-EGFRs experienced disease progression as best response. One patient with an MSI-high EML4-ALK rearranged tumor experienced durable response to PD-1 blockade. Conclusions: ALK, ROS1 and NTRKs rearrangements define a new molecular subtype of mCRCs associated with unfavorable prognosis, and specific clinicopathological and molecular features. Since sensitivity to available treatment options, including anti-EGFRs, is very limited, targeted approaches with or without immunotherapy should be investigated in these patients.

Introduction: Molecular profiling of tumor samples is becoming routine practice in the clinic, particularly for therapy selection. However, in some cases, tumor tissue is not available for testing. Liquid biopsy, which enables the analysis of circulating tumor DNA (ctDNA) shed from the tumor into the blood, can be used as a surrogate for conventional tissue based testing to detect somatic mutations. Furthermore, ctDNA profiling can have potential applications in tumor burden monitoring. Some technical challenges must be overcome to accurately and sensitively detect the often low amount of ctDNA present in plasma. The AVENIO ctDNA Analysis Kits (Targeted, Expanded and Surveillance Kits; Research Use Only) were developed to sensitively detect all 4 major mutation classes: single nucleotide variants, insertions/deletions, fusions, and copy number variations, using a hybrid capture target enrichment workflow, molecular barcoding, and next generation sequencing. We previously presented a comprehensive study of 370+ samples demonstrating analytical performance of the assays on the Illumina NextSeq 500. As ctDNA based analysis becomes more prevalent in the clinical research community, there is great desire for high performing and cost-effective assays that are easy to implement and available across multiple sequencing platforms. Methods: We used the commercially available AVENIO ctDNA Analysis Kits (Expanded, Target and Surveillance Kits; Research Use Only) pre-sequencing workflow to process a panel of cell line blends and plasma-derived ctDNA samples in order to compare the performance of the assays on multiple Illumina sequencing platforms: NextSeq 500, HiSeq 4000 and HiSeq 2500. Ninety samples were tested across all platforms. The samples contain SNVs at 0.5% minor allele frequency (MAF), deletions at 1% MAF, selected fusions at 1% MAF, and MET amplification at 2.3 copies. Analysis was done using the AVENIO ctDNA analysis pipeline for all sequencing runs across the sequencing platforms. Results: On the NextSeq 500, the AVENIO ctDNA Analysis Kits achieve sensitivities of &gt;99% for 0.5% SNVs, &gt;99% for 1% deletions, &gt;96% for 1% fusions, and &gt;99% for 2.3 copies of MET amplification, with 96% to &gt;99% specificity for all mutations. Similar performance is observed across additional Illumina platforms, HighSeq 4000 and HiSeq 2500. Across all platforms, key sequencing metrics are consistent to achieve required sequencing depths, on-target rates, and uniformity. Also, it is noted that in order to achieve required depths, each platform requires different levels of sample multiplexing. We demonstrated that the AVENIO ctDNA Analysis Kits achieve high sensitivity and specificity across multiple high throughput sequencing platforms. We also will highlight the key performance differences and considerations when performing the assay across these sequencing platforms. Citation Format: Jonathan Choi, Richard Dannebaum, Ashla Singh, Rob Foley, Jorge Dinis, Cindy Choi, Bosun Min, Jingchuan Li, Liang Feng, Fergal Casey, Janet Jin. Performance of the AVENIO ctDNA assays across multiple high-throughput next-generation sequencing platforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3648.