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We conducted next-generation DNA sequencing on 335 biliary tract cancers and characterized the genomic landscape by anatomic site within the biliary tree. In addition to frequent FGFR2 fusions among patients with intrahepatic cholangiocarcinoma (IHCC), we identified FGFR2 extracellular domain in-frame deletions (EID) in 5 of 178 (2.8%) patients with IHCC, including two patients with FGFR2 p.H167_N173del. Expression of this FGFR2 EID in NIH3T3 cells resulted in constitutive FGFR2 activation, oncogenic transformation, and sensitivity to FGFR inhibitors. Three patients with FGFR2 EIDs were treated with Debio 1347, an oral FGFR1/2/3 inhibitor, and all showed partial responses. One patient developed an acquired L618F FGFR2 kinase domain mutation at disease progression and experienced a further partial response for 17 months to an irreversible FGFR2 inhibitor, futibatinib. Together, these findings reveal FGFR2 EIDs as an alternative mechanism of FGFR2 activation in IHCC that predicts sensitivity to FGFR inhibitors in the clinic.Significance:FGFR2 EIDs are transforming genomic alterations that occur predominantly in patients with IHCC. These FGFR2 EIDs are sensitive to FGFR inhibition in vitro, and patients with these alterations benefited from treatment with FGFR inhibitors in the clinic.This article is highlighted in the In This Issue feature, p. 2355

Purpose:Receptor tyrosine kinase fusions in colorectal cancers are rare, but potentially therapeutically relevant. We describe clinical, molecular, and pathologic attributes of RTK fusion–associated colorectal cancer.Experimental Design:We identified all cases with RTK fusions in patients with colorectal cancer seen at Dana-Farber Cancer Institute (Boston, MA) who underwent OncoPanel testing between 2013 and 2018. Clinical, histologic, and molecular features were extracted from the patient charts and molecular testing results.Results:We identified 12 driver oncogenic fusions in various RTKs. These fusions occurred exclusively in BRAF and RAS wild-type tumors and were enriched in right-sided and mismatch repair–deficient (MMR-D) colorectal cancers. All of the MMR-D colorectal cancers with RTK fusions were found in tumors with acquired MMR-D due to MLH1 promoter hypermethylation and one was associated with a sessile serrated polyp. Molecular profiles of MMR-D colorectal cancer with RTK fusions largely resembled BRAF V600E–mutated MMR-D colorectal cancer, rather than those secondary to Lynch syndrome. We describe two patients with fusion-associated microsatellite stable (MSS) colorectal cancer who derived clinical benefit from therapeutic targeting of their translocation. The first harbored an ALK-CAD fusion and received sequential crizotinib and alectinib therapy for a total of 7.5 months until developing an ALK L1196Q gatekeeper mutation. The second patient, whose tumor contained an ROS1-GOPC fusion, continues to benefit from entrectinib after 9 months of therapy.Conclusions:RTK fusions in colorectal cancer are a rare, but important disease subgroup that occurs in RAS and BRAF wild-type tumors. Despite enrichment in acquired MMR-D tumors, RTK fusions also occur in MSS colorectal cancer and provide an important therapeutic target.

Supplementary Figure 1. Pan-TRK IHC in four cases identified as having NTRK fusions by OncoPanel analysis shows strong cytoplasmic staining confirming the presence of functional TRK fusion protein (40Ã-). Top left image demonstrates the specificity of TRK staining, which is present only in neoplastic cells, whereas adjacent normal crypts are negative. Supplementary Figure 2A. Two cases of ALK fusion in our cohort shared clinical and molecular features but differed in their microsatellite status. Both tumors were APC wild-type (wt) and contained biallelic RNF43 frameshift (fs) mutations. B. Copy number variation by chromosome is color-coded for two ALK fusion-associated colon cancers in our cohort. ALK+MMR-D cancer (top) displays a diploid genome, whereas ALK+MSS cancer (bottom) displays frequent copy number gains and losses. ALK fusion breakpoints are shown in both profiles (arrow). The vertical axis is the ratio of the number of reads for each specimen and a panel of normals in log base 2 scale. A value of 0 denotes no difference from normal (diploid). Supplementary Figure 3A. MMR-D RTK fusion CRC cases display a diploid genome similar to those in other MMR-D cases: BRAF V600E+mutated and Lynch syndrome. Copy number profile plots of RTK fusions or Wnt fusions with MSS CRC show extensive gains and losses and are shown for comparison. 3B. All MMR-D subgroups: BRAF V600E+, Lynch syndrome and RTK fusion show a significantly higher homopolymer indel rate (top) and TMB (bottom) than do MSS tumors with either RTK fusions or Wnt fusions. Supplementary Figure. 4. Histologic analysis with hematoxylin and eosin stained slides for two patients with NTRK1-LMNA fusions. Morphological analysis shows: A. mucinous and B. medullary histology.

PURPOSE With the growing number of available targeted therapeutics and molecular biomarkers, the optimal care of patients with cancer now depends on a comprehensive understanding of the rapidly evolving landscape of precision oncology, which can be challenging for oncologists to navigate alone. METHODS We developed and implemented a precision oncology decision support system, GI TARGET, (Gastrointestinal Treatment Assistance Regarding Genomic Evaluation of Tumors) within the Gastrointestinal Cancer Center at the Dana-Farber Cancer Institute. With a multidisciplinary team, we systematically reviewed tumor molecular profiling for GI tumors and provided molecularly informed clinical recommendations, which included identifying appropriate clinical trials aided by the computational matching platform MatchMiner, suggesting targeted therapy options on or off the US Food and Drug Administration–approved label, and consideration of additional or orthogonal molecular testing. RESULTS We reviewed genomic data and provided clinical recommendations for 506 patients with GI cancer who underwent tumor molecular profiling between January and June 2019 and determined follow-up using the electronic health record. Summary reports were provided to 19 medical oncologists for patients with colorectal (n = 198, 39%), pancreatic (n = 124, 24%), esophagogastric (n = 67, 13%), biliary (n = 40, 8%), and other GI cancers. We recommended ≥ 1 precision medicine clinical trial for 80% (406 of 506) of patients, leading to 24 enrollments. We recommended on-label and off-label targeted therapies for 6% (28 of 506) and 25% (125 of 506) of patients, respectively. Recommendations for additional or orthogonal testing were made for 42% (211 of 506) of patients. CONCLUSION The integration of precision medicine in routine cancer care through a dedicated multidisciplinary molecular tumor board is scalable and sustainable, and implementation of precision oncology recommendations has clinical utility for patients with cancer.

BACKGROUND: Pathogenic POLE exonuclease domain mutations (pPOLE) undermine mismatch error correction during DNA replication, causing somatic ultramutation and response to immunotherapy. We examined the pan-cancer landscape of POLE mutations and applied a POLE-specific variant classification model. METHODS: Comprehensive genomic profiling was performed during clinical care. Mutational signature calling was performed via decomposition using the 96-feature single-base substitution COSMIC reference signatures. A POLE-specific classification model encompassing mutation position in the exonuclease domain, TMB, presence of POLE signature, absence of other signatures, germline frequency, and other features was applied to identify pPOLE mutations causative of ultramutation. RESULTS: POLE mutation status was evaluated in 458,437 samples (425,520 tissue biopsies (TB) and 32,917 liquid biopsies (LB)). One or more POLE alterations, including pathogenic alterations and variants of unknown significance (VUS), were detected in 3.8% of samples. 19,470 total alterations were identified, 84.8% of which were missense substitutions. Application of the POLE-specific classification model identified 35 unique pathogenic variants, many of which were VUS prior to this study. 749 samples harbored a pPOLE, more than half (56.6%) of which were either p.P286R (n=245) or p.V411L (n=179). pPOLE were found in 1.4% (199/13,688) of endometrial cancers (EC) and 0.5% (270/55,981) of colorectal cancers (CRC) and were rarer in a long tail of other malignancies. The overall pPOLE rate was significantly lower in LB than TB (0.02% vs 0.17%, P CONCLUSIONS: pPOLE were seen in both TB and LB across cancer types. The high rate of passenger mutations underscores the utility of this POLE-specific variant classification model. Because TMB can be underestimated when tumor purity is near the limit of detection for the assay, accurate detection and classification of pPOLE is critical for identifying patients who may benefit from immunotherapy. Citation Format: Rachel B. Keller, James Haberberger, Tyler Janovitz, Alexa B. Schrock, Hanna Tukachinsky, Lei Zhong, Douglas A. Mata, Lyle V. Lopez, Zoe Fleischmann, Radwa Sharaf, Ethan S. Sokol, Garrett M. Frampton, Nimesh R. Patel, Douglas I. Lin, Geoff R. Oxnard, Erik A. Williams, Julia A. Elvin, Brennan Decker. POLE-specific variant classification strategy is critical for identifying patients who may benefit from immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 305.

Approximately 8-10% of pancreatic cancers do not harbor mutations in KRAS. Understanding the unique molecular and clinical features of this subset of pancreatic cancer (PC) is important to guide patient stratification for clinical trials of molecularly targeted agents. To this end we investigated a cohort of 795 PC patients from Dana-Farber Cancer Institute who had undergone somatic genomic characterization with OncoPanel, a targeted next-generation sequencing panel with coverage of more than 400 cancer-associated genes. A total of 9.2% (73/795) of cases in our cohort were KRAS WT. The KRAS WT cohort was statistically enriched for MSI-H PC and acinar cell carcinomas (p = 0.0035, p < 0.0001 respectively). Actionable alterations in alternative MAPK drivers were identified in 44% (32/73) of KRAS WT cases. BRAF alterations accounted for 56% (18/32) of detected alternative MAPK drivers, the majority of which (72%) were Class II which exhibit dimer-dependent constitutive activity. Receptor Tyrosine Kinase (RTK) fusion events in BRAF, NTRK1, NRG1, NTRK3, ROS1, and FGFR2 accounted for 25% (8/32) of detected alternative MAPK drivers in KRAS WT tumors. BRAF in-frame deletions showed increased sensitivity to dual pan-RAF and MEK inhibition in organoid models and one patient with a ROS1 fusion received prolonged clinical benefit from targeted therapy. In addition to alternative MAPK drivers, mutations in GNAS (p = 0.0014) and ARID2 (p = 0.045) were significantly enriched in KRAS WT PC, whereas TP53 mutations were significantly less frequent in KRAS WT cases. Interestingly, although not statistically significant, rates of mutation in the other canonical tumor suppressor genes (CDKN2A, SMAD4) were also lower in KRAS WT PC. Clinically, KRAS Mutant (MUT) PC were associated with a decreased overall survival (OS) compared to the KRAS WT cohort [median OS 17.5 vs 24.0 months, HR 1.38, p = 0.036], however this relationship was no longer significant after accounting for other clinical factors. For patients with KRAS WT PC, those with SMAD4 alterations had a significantly decreased OS (HR 6.24, p < 0.001), whereas presence of TP53 or CDKN2A mutations had no significant impact. Lastly, we found that KRAS WT PC was associated with a younger age of onset. Interestingly, we noted that KRAS WT PC patients with a younger age of onset had tumors with few oncogenic alterations whereas no such association was seen in KRAS MUT patients. Validation of this finding in a separate dataset is required and is currently ongoing. In summary, our clinical and genomic characterization of KRAS WT PC identifies a high prevalence of alternative MAPK drivers that are amenable to targeted therapies. Our cohort also recapitulates the previously reported clinical characteristics of KRAS WT PC and identifies the presence of SMAD4 alterations as significantly associated with decreased overall survival in KRAS WT PC. Additional analysis from multiple sources will be critical to risk stratify these patients further and to validate age-related findings. Citation Format: Harshabad Singh, Rachel B. Keller, Kevin S. Kapner, Julien Dilly, Srivatsan Raghavan, Chen Yuan, Eizabeth Cohen, Michael Tolstorukov, Emma Hill, Elizabeth Andrews, Lauren K. Brais, Annacarolina Da Silva, Kimberly Perez, Douglas A. Rubinson, Benjamin L. Schlechter, Michael H. Rosenthal, Jason L. Hornick, Valentina Nardi, Yvonne Li, Hersh Gupta, Andrew Cherniack, Mathew L. Meyerson, James M. Cleary, Jonathan A. Nowak, Brian M. Wolpin, Andrew A. Aguirre. Clinical-genomic analysis of KRAS wild-type pancreatic cancer confirms alternative targetable drivers and provides insight for age and risk related clinical stratification [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A001.