The future of fibroblast growth factor receptor inhibitors and mechanisms of resistance for cholangiocarcinoma.

INTRODUCTION: Cholangiocarcinoma (CCA) is a rare cancer that arises from the biliary tract. Despite advances in multimodal treatment, patients with CCA have a poor prognosis. Molecular profiling of CCA has identified unique genetic aberrations (GA) that may serve as therapeutic targets. A common GA in CCA is in the fibroblast growth factor receptors (FGFR). FGFRs are a group of transmembrane receptors that stimulate downstream pathways for cell proliferation and survival. AREAS COVERED: We herein review recent clinical trial data related to different FGFR inhibitors and the challenges within the field. An extensive literature search was performed to identify preclinical studies, clinical research, and clinical trials that evaluated the effectiveness of FGFR inhibitor therapy in patients with CCA. EXPERT OPINION: FGFR inhibitors have demonstrated effectiveness in pre-clinical studies and some clinical trials. Infigratinib, futibatinib, and pemigatinib are being evaluated in an open phase III trial versus gemcitabine/cisplatin as first-line treatment for locally advanced or metastatic CCA with FGFR GA (PROOF-301 NCT03773302, FOENIX-CCA3 NCT04093362, FIGHT-302 NCT03656536). Unfortunately, the effectiveness of FGFR therapy is often limited by acquired resistance mechanisms, and continued work is needed to understand and overcome these mechanisms of resistance.

Pan-cancer Landscape of Programmed Death Ligand-1 and Programmed Death Ligand-2 Structural Variations.

PURPOSE: Programmed cell death protein-1 (PD-1) receptor and ligand interactions are the target of immunotherapies for more than 20 cancer types. Biomarkers that predict response to immunotherapy are microsatellite instability, tumor mutational burden, and programmed death ligand-1 (PD-L1) immunohistochemistry. Structural variations (SVs) in PD-L1 (CD274) and PD-L2 (PDCD1LG2) have been observed in cancer, but the comprehensive landscape is unknown. Here, we describe the genomic landscape of PD-L1 and PD-L2 SVs, their potential impact on the tumor microenvironment, and evidence that patients with these alterations can benefit from immunotherapy. METHODS: We analyzed sequencing data from cancer cases with PD-L1 and PD-L2 SVs across 22 publications and four data sets, including Foundation Medicine Inc, The Cancer Genome Atlas, International Cancer Genome Consortium, and the Oncology Research Information Exchange Network. We leveraged RNA sequencing to evaluate immune signatures. We curated literature reporting clinical outcomes of patients harboring PD-L1 or PD-L2 SVs. RESULTS: Using data sets encompassing 300,000 tumors, we curated 486 cases with SVs in PD-L1 and PD-L2 and observed consistent breakpoint patterns, or hotspots. Leveraging The Cancer Genome Atlas, we observed significant upregulation in PD-L1 expression and signatures for interferon signaling, macrophages, T cells, and immune cell proliferation in samples harboring PD-L1 or PD-L2 SVs. Retrospective review of 12 studies that identified patients with SVs in PD-L1 or PD-L2 revealed > 50% (52/71) response rate to PD-1 immunotherapy with durable responses. CONCLUSION: Our findings show that the 3'-UTR is frequently affected, and that SVs are associated with increased expression of ligands and immune signatures. Retrospective evidence from curated studies suggests this genomic alteration could help identify candidates for PD-1/PD-L1 immunotherapy. We expect these findings will better define PD-L1 and PD-L2 SVs in cancer and lend support for prospective clinical trials to target these alterations.

Mainstreaming germline genetic testing for patients with pancreatic cancer increases uptake.

Germline genetic testing is recommended for all patients with pancreatic cancer (PC) but uptake rates are low. We implemented a mainstreaming program in oncology clinics to increase testing for PC patients. Genetic counselors trained oncology providers to offer a standardized multigene panel and obtain informed consent using an educational video. Pre-test genetic counseling was available upon request. Otherwise, patients with identified pathogenic variants, strong family history, or questions regarding their results were referred for post-test genetic counseling. We measured rates of testing and genetic counseling visits. From September 2019 to April 2021, 245 patients with PC underwent genetic testing. This represents a 6.5-fold increase in germline testing volume (95% confidence interval 5.2-8.1) compared to previous years. At least one pathogenic or likely pathogenic variant (PV/LPV) was found in 34 (13.9%) patients, including 17 (6.9%) PV/LPVs in high or moderate risk genes and 18 (7.3%) in low risk or recessive genes. Five (2.0%) PVs had implications on treatment selection. 22 of the positive patients (64.7%) and an additional 8 PC patients (1 negative, 3 VUS, and 4 pre-test) underwent genetic counseling during the study period. Genetic counselors saw 2.0 PC patients/month prior to this project, 1.6 PC patients/month during this project, and would have seen 2.2 PC patients/month if all patients with pathogenic variants attended post-test counseling. Conclusions Mainstreaming genetic testing expands access for PC patients without overwhelming genetic counseling resources.

Individualized, heterologous chimpanzee adenovirus and self-amplifying mRNA neoantigen vaccine for advanced metastatic solid tumors: phase 1 trial interim results.

Checkpoint inhibitor (CPI) therapies provide limited benefit to patients with tumors of low immune reactivity. T cell-inducing vaccines hold promise to exert long-lasting disease control in combination with CPI therapy. Safety, tolerability and recommended phase 2 dose (RP2D) of an individualized, heterologous chimpanzee adenovirus (ChAd68) and self-amplifying mRNA (samRNA)-based neoantigen vaccine in combination with nivolumab and ipilimumab were assessed as primary endpoints in an ongoing phase 1/2 study in patients with advanced metastatic solid tumors (NCT03639714). The individualized vaccine regimen was safe and well tolerated, with no dose-limiting toxicities. Treatment-related adverse events (TRAEs) >10% included pyrexia, fatigue, musculoskeletal and injection site pain and diarrhea. Serious TRAEs included one count each of pyrexia, duodenitis, increased transaminases and hyperthyroidism. The RP2D was 1012 viral particles (VP) ChAd68 and 30 µg samRNA. Secondary endpoints included immunogenicity, feasibility of manufacturing and overall survival (OS). Vaccine manufacturing was feasible, with vaccination inducing long-lasting neoantigen-specific CD8 T cell responses. Several patients with microsatellite-stable colorectal cancer (MSS-CRC) had improved OS. Exploratory biomarker analyses showed decreased circulating tumor DNA (ctDNA) in patients with prolonged OS. Although small study size limits statistical and translational analyses, the increased OS observed in MSS-CRC warrants further exploration in larger randomized studies.

Association of CD274 (PD-L1) Copy Number Changes with Immune Checkpoint Inhibitor Clinical Benefit in Non-Squamous Non-Small Cell Lung Cancer.

BACKGROUND: We sought to characterize response to immune checkpoint inhibitor (ICI) in non-squamous non-small cell lung cancer (NSCLC) across various CD274 copy number gain and loss thresholds and identify an optimal cutoff. MATERIALS AND METHODS: A de-identified nationwide (US) real-world clinico-genomic database was leveraged to study 621 non-squamous NSCLC patients treated with ICI. All patients received second-line ICI monotherapy and underwent comprehensive genomic profiling as part of routine clinical care. Overall survival (OS) from start of ICI, for CD274 copy number gain and loss cohorts across varying copy number thresholds, were assessed. RESULTS: Among the 621 patients, patients with a CD274 CN greater than or equal to specimen ploidy +2 (N = 29) had a significantly higher median (m) OS when compared with the rest of the cohort (N = 592; 16.1 [8.9-37.3] vs 8.6 [7.1-10.9] months, hazard ratio (HR) = 0.6 [0.4-1.0], P-value = .05). Patients with a CD274 copy number less than specimen ploidy (N = 299) trended toward a lower mOS when compared to the rest of the cohort (N = 322; 7.5 [5.9-11.3] vs 9.6 [7.9-12.8] months, HR = 0.9 [0.7-1.1], P-value = .3). CONCLUSION: This work shows that CD274 copy number gains at varying thresholds predict different response to ICI blockade in non-squamous NSCLC. Considering these data, prospective clinical trials should further validate these findings, specifically in the context of PD-L1 IHC test results.

Validation and Characterization of FGFR2 Rearrangements in Cholangiocarcinoma with Comprehensive Genomic Profiling.

Cholangiocarcinoma (CCA) is a heterogeneous biliary tract cancer with a poor prognosis. Approximately 30% to 50% of patients harbor actionable alterations, including FGFR2 rearrangements. Pemigatinib, a potent, selective fibroblast growth factor receptor (FGFR) FGFR1-3 inhibitor, is approved for previously treated, unresectable, locally advanced or metastatic CCA harboring FGFR2 fusions/rearrangements, as detected by a US Food and Drug Administration-approved test. The next-generation sequencing (NGS)-based FoundationOneCDx (F1CDx) was US Food and Drug Administration approved for detecting FGFR2 fusions or rearrangements. The precision and reproducibility of F1CDx in detecting FGFR2 rearrangements in CCA were examined. Analytical concordance between F1CDx and an externally validated RNA-based NGS (evNGS) test was performed. Identification of FGFR2 rearrangements in the screening population from the pivotal FIGHT-202 study (NCT02924376) was compared with F1CDx. The reproducibility and repeatability of F1CDx were 90% to 100%. Adjusted positive, negative, and overall percentage agreements were 87.1%, 99.6%, and 98.3%, respectively, between F1CDx and evNGS. Compared with evNGS, F1CDx had a positive predictive value of 96.2% and a negative predictive value of 98.5%. The positive percentage agreement, negative percentage agreement, overall percentage agreement, positive predictive value, and negative predictive value were 100% for F1CDx versus the FIbroblast Growth factor receptor inhibitor in oncology and Hematology Trial-202 (FIGHT-202) clinical trial assay. Of 6802 CCA samples interrogated, 9.2% had FGFR2 rearrangements. Cell lines expressing diverse FGFR2 fusions were sensitive to pemigatinib. F1CDx demonstrated sensitivity, reproducibility, and high concordance with clinical utility in identifying patients with FGFR2 rearrangements who may benefit from pemigatinib treatment.

Genomic and Transcriptomic Characterization of Relapsed SCLC Through Rapid Research Autopsy.

INTRODUCTION: Relapsed SCLC is characterized by therapeutic resistance and high mortality rate. Despite decades of research, mechanisms responsible for therapeutic resistance have remained elusive owing to limited tissues available for molecular studies. Thus, an unmet need remains for molecular characterization of relapsed SCLC to facilitate development of effective therapies. METHODS: We performed whole-exome and transcriptome sequencing of metastatic tumor samples procured from research autopsies of five patients with relapsed SCLC. We implemented bioinformatics tools to infer subclonal phylogeny and identify recurrent genomic alterations. We implemented immune cell signature and single-sample gene set enrichment analyses on tumor and normal transcriptome data from autopsy and additional primary and relapsed SCLC data sets. Furthermore, we evaluated T cell-inflamed gene expression profiles in neuroendocrine (ASCL1, NEUROD1) and non-neuroendocrine (YAP1, POU2F3) SCLC subtypes. RESULTS: Exome sequencing revealed clonal heterogeneity (intertumor and intratumor) arising from branched evolution and identified resistance-associated truncal and subclonal alterations in relapsed SCLC. Transcriptome analyses further revealed a noninflamed phenotype in neuroendocrine SCLC subtypes (ASCL1, NEUROD1) associated with decreased expression of genes involved in adaptive antitumor immunity whereas non-neuroendocrine subtypes (YAP1, POU2F3) revealed a more inflamed phenotype. CONCLUSIONS: Our results reveal substantial tumor heterogeneity and complex clonal evolution in relapsed SCLC. Furthermore, we report that neuroendocrine SCLC subtypes are immunologically cold, thus explaining decreased responsiveness to immune checkpoint blockade. These results suggest that the mechanisms of innate and acquired therapeutic resistances are subtype-specific in SCLC and highlight the need for continued investigation to bolster therapy selection and development for this cancer.

Infigratinib (BGJ398) in previously treated patients with advanced or metastatic cholangiocarcinoma with FGFR2 fusions or rearrangements: mature results from a multicentre, open-label, single-arm, phase 2 study.

BACKGROUND: Treatment options are sparse for patients with advanced cholangiocarcinoma after progression on first-line gemcitabine-based therapy. FGFR2 fusions or rearrangements occur in 10-16% of patients with intrahepatic cholangiocarcinoma. Infigratinib is a selective, ATP-competitive inhibitor of fibroblast growth factor receptors. We aimed to evaluate the antitumour activity of infigratinib in patients with locally advanced or metastatic cholangiocarcinoma, FGFR2 alterations, and previous gemcitabine-based treatment. METHODS: This multicentre, open-label, single-arm, phase 2 study recruited patients from 18 academic centres and hospitals in the USA, Belgium, Spain, Germany, Singapore, Taiwan, and Thailand. Eligible participants were aged 18 years or older, had histologically or cytologically confirmed, locally advanced or metastatic cholangiocarcinoma and FGFR2 fusions or rearrangements, and were previously treated with at least one gemcitabine-containing regimen. Patients received 125 mg of oral infigratinib once daily for 21 days of 28-day cycles until disease progression, intolerance, withdrawal of consent, or death. Radiological tumour evaluation was done at baseline and every 8 weeks until disease progression via CT or MRI of the chest, abdomen, and pelvis. The primary endpoint was objective response rate, defined as the proportion of patients with a best overall response of a confirmed complete or partial response, as assessed by blinded independent central review (BICR) according to Response Evaluation Criteria in Solid Tumors, version 1.1. The primary outcome and safety were analysed in the full analysis set, which comprised all patients who received at least one dose of infigratinib. This trial is registered with ClinicalTrials.gov, NCT02150967, and is ongoing. FINDINGS: Between June 23, 2014, and March 31, 2020, 122 patients were enrolled into our study, of whom 108 with FGFR2 fusions or rearrangements received at least one dose of infigratinib and comprised the full analysis set. After a median follow-up of 10·6 months (IQR 6·2-15·6), the BICR-assessed objective response rate was 23·1% (95% CI 15·6-32·2; 25 of 108 patients), with one confirmed complete response in a patient who only had non-target lesions identified at baseline and 24 partial responses. The most common treatment-emergent adverse events of any grade were hyperphosphataemia (n=83), stomatitis (n=59), fatigue (n=43), and alopecia (n=41). The most common ocular toxicity was dry eyes (n=37). Central serous retinopathy-like and retinal pigment epithelial detachment-like events occurred in 18 (17%) patients, of which ten (9%) were grade 1, seven (6%) were grade 2, and one (1%) was grade 3. There were no treatment-related deaths. INTERPRETATION: Infigratinib has promising clinical activity and a manageable adverse event profile in previously treated patients with locally advanced or metastatic cholangiocarcinoma harbouring FGFR2 gene fusions or rearrangements, and so represents a potential new therapeutic option in this setting. FUNDING: QED Therapeutics and Novartis.

Fibroblast growth factor receptors in cancer: genetic alterations, diagnostics, therapeutic targets and mechanisms of resistance.

Fibroblast growth factor receptors (FGFRs) are aberrantly activated through single-nucleotide variants, gene fusions and copy number amplifications in 5-10% of all human cancers, although this frequency increases to 10-30% in urothelial carcinoma and intrahepatic cholangiocarcinoma. We begin this review by highlighting the diversity of FGFR genomic alterations identified in human cancers and the current challenges associated with the development of clinical-grade molecular diagnostic tests to accurately detect these alterations in the tissue and blood of patients. The past decade has seen significant advancements in the development of FGFR-targeted therapies, which include selective, non-selective and covalent small-molecule inhibitors, as well as monoclonal antibodies against the receptors. We describe the expanding landscape of anti-FGFR therapies that are being assessed in early phase and randomised controlled clinical trials, such as erdafitinib and pemigatinib, which are approved by the Food and Drug Administration for the treatment of FGFR3-mutated urothelial carcinoma and FGFR2-fusion cholangiocarcinoma, respectively. However, despite initial sensitivity to FGFR inhibition, acquired drug resistance leading to cancer progression develops in most patients. This phenomenon underscores the need to clearly delineate tumour-intrinsic and tumour-extrinsic mechanisms of resistance to facilitate the development of second-generation FGFR inhibitors and novel treatment strategies beyond progression on targeted therapy.

Characterization of Clonal Evolution in Microsatellite Unstable Metastatic Cancers through Multiregional Tumor Sequencing.

Microsatellites are short, repetitive segments of DNA, which are dysregulated in mismatch repair-deficient (MMRd) tumors resulting in microsatellite instability (MSI). MSI has been identified in many human cancer types with varying incidence, and microsatellite instability-high (MSI-H) tumors often exhibit increased sensitivity to immune-enhancing therapies such as PD-1/PD-L1 inhibition. Next-generation sequencing (NGS) has permitted advancements in MSI detection, and recent computational advances have enabled characterization of tumor heterogeneity via NGS. However, the evolution and heterogeneity of microsatellite changes in MSI-positive tumors remains poorly described. We determined MSI status in 6 patients using our previously published algorithm, MANTIS, and inferred subclonal composition and phylogeny with Canopy and SuperFreq. We developed a simulated annealing-based method to characterize microsatellite length distributions in specific subclones and assessed the evolution of MSI in the context of tumor heterogeneity. We identified three to eight tumor subclones per patient, and each subclone exhibited MMRd-associated base substitution signatures. We noted that microsatellites tend to shorten over time, and that MMRd fosters heterogeneity by introducing novel mutations throughout the disease course. Some microsatellites are altered among all subclones in a patient, whereas other loci are only altered in particular subclones corresponding to subclonal phylogenetic relationships. Overall, our results indicate that MMRd is a substantial driver of heterogeneity, leading to both MSI and subclonal divergence. IMPLICATIONS: We leveraged subclonal inference to assess clonal evolution based on somatic mutations and microsatellites, which provides insight into MMRd as a dynamic mutagenic process in MSI-H malignancies.

Infigratinib in patients with advanced cholangiocarcinoma with FGFR2 gene fusions/translocations: the PROOF 301 trial.

Cholangiocarcinoma is an aggressive malignancy with poor overall survival. Approximately 15% of intrahepatic cholangiocarcinomas contain FGFR alterations. Infigratinib is an oral FGFR 1-3 kinase inhibitor. Favorable results from a Phase II trial of infigratinib in advanced/metastatic FGFR-altered cholangiocarcinomas has led to its further investigation in the front-line setting. In this article we describe the design, objectives and rationale for PROOF 301, a Phase III multicenter, open label, randomized trial of infigratinib in comparison to standard of care gemcitabine and cisplatin in advanced/metastatic cholangiocarcinoma with FGFR2 translocations. The results of this study have the potential to define a new role for a chemotherapy-free, targeted therapy option in the front-line setting for these patients. Clinical Trial Registration: NCT03773302 (ClincalTrials.gov).

Phase I Trial of Trametinib with Neoadjuvant Chemoradiation in Patients with Locally Advanced Rectal Cancer.

PURPOSE: The RAS/RAF/MEK/ERK signaling pathway is critical to the development of colorectal cancers, and KRAS, NRAS, and BRAF mutations foster resistance to radiation. We performed a phase I trial to determine the safety of trametinib, a potent MEK1/2 inhibitor, with 5-fluorouracil (5-FU) chemoradiation therapy (CRT) in patients with locally advanced rectal cancer (LARC). PATIENTS AND METHODS: Patients with stage II/III rectal cancer were enrolled on a phase I study with 3+3 study design, with an expansion cohort of 9 patients at the MTD. Following a 5-day trametinib lead-in, with pre- and posttreatment tumor biopsies, patients received trametinib and CRT, surgery, and adjuvant chemotherapy. Trametinib was given orally daily at 3 dose levels: 0.5 mg, 1 mg, and 2 mg. CRT consisted of infusional 5-FU 225 mg/m2/day and radiation dose of 28 daily fractions of 1.8 Gy (total 50.4 Gy). The primary endpoint was to identify the MTD and recommended phase II dose. IHC staining for phosphorylated ERK (pERK) and genomic profiling was performed on the tumor samples. RESULTS: Patients were enrolled to all dose levels, and 18 patients were evaluable for toxicities and responses. Treatment was well tolerated, and there was one dose-limiting toxicity of diarrhea, which was attributed to CRT rather than trametinib. At the 2 mg dose level, 25% had pathologic complete response. IHC staining confirmed dose-dependent decrease in pERK with increasing trametinib doses. CONCLUSIONS: The combination of trametinib with 5-FU CRT is safe and well tolerated, and may warrant additional study in a phase II trial, perhaps in a RAS/RAF-mutant selected population.

Co-occurrence of multiple endocrine neoplasia type 4 and spinal neurofibromatosis: a case report.

Multiple Endocrine Neoplasia (MEN) type 4 is a rare genetic condition that results from variants of the CDKN1B gene and predisposes individuals to develop endocrine tumors. Spinal neurofibromatosis (SNF) is an uncommon subtype of neurofibromatosis type 1 (NF1) characterized by bilateral neurofibromas of all spinal roots. Here we report a case of the co-occurrence of these syndromes, which has not yet been described in the literature. A male in his 60s presented with Gleason 5 + 4 localized prostate adenocarcinoma treated with radical prostatectomy. Two years later, he developed liver and bone metastasis consistent with trans-differentiation into small cell carcinoma. He developed hypercalcemia due to primary hyperparathyroidism from a parathyroid adenoma treated surgically. His family history was significant for a first-degree relative with a clinical diagnosis of NF1 and several second-degree relatives with multiple café-au-lait macules. Spine MRI showed multiple bilateral neurofibromas. Germline genetic testing showed a pathogenic variant in the CDKN1B gene, a variant in the NF1 gene, and a normal MEN1 gene. In this rare case of MEN4 and SNF, the patient was asymptomatic for much of his life. In addition to parathyroid adenoma and spinal neurofibromas, he had prostate adenocarcinoma with trans-differentiation into metastatic small cell cancer. Whether this diagnosis was coincidental or related to an emerging phenotype remains to be elucidated.

Efficacy of FGFR Inhibitors and Combination Therapies for Acquired Resistance in FGFR2-Fusion Cholangiocarcinoma.

The fibroblast growth factor receptor (FGFR) signaling pathway is aberrantly activated in approximately 15% to 20% of patients with intrahepatic cholangiocarcinoma. Currently, several FGFR kinase inhibitors are being assessed in clinical trials for patients with FGFR-altered cholangiocarcinoma. Despite evidence of initial responses and disease control, virtually all patients eventually develop acquired resistance. Thus, there is a critical need for the development of innovative therapeutic strategies to overcome acquired drug resistance. Here, we present findings from a patient with FGFR2-altered metastatic cholangiocarcinoma who enrolled in a phase II clinical trial of the FGFR inhibitor, infigratinib (BGJ398). Treatment was initially effective as demonstrated by imaging and tumor marker response; however, after 8 months on trial, the patient exhibited tumor regrowth and disease progression. Targeted sequencing of tumor DNA after disease progression revealed the FGFR2 kinase domain p.E565A and p.L617M single-nucleotide variants (SNV) hypothesized to drive acquired resistance to infigratinib. The sensitivities of these FGFR2 SNVs, which were detected post-infigratinib therapy, were extended to include clinically relevant FGFR inhibitors, including AZD4547, erdafitinib (JNJ-42756493), dovitinib, ponatinib, and TAS120, and were evaluated in vitro Through a proteomics approach, we identified upregulation of the PI3K/AKT/mTOR signaling pathway in cells harboring the FGFR2 p.E565A mutation and demonstrated that combination therapy strategies with FGFR and mTOR inhibitors may be used to overcome resistance to FGFR inhibition, specific to infigratinib. Collectively, these studies support the development of novel combination therapeutic strategies in addition to the next generation of FGFR inhibitors to overcome acquired resistance in patients.

Detection of Microsatellite Instability Biomarkers via Next-Generation Sequencing.

A high level of microsatellite instability (MSI-H+) is an emerging predictive and prognostic biomarker for immunotherapy response in cancer. Recently, MSI-H+ has been detected in a variety of cancer types, in addition to the classical cancers associated with Lynch Syndrome. Clinical testing for MSI-H+ is currently performed primarily through traditional polymerase chain reaction (PCR) or immunohistochemistry (IHC) assays. However, next-generation sequencing (NGS)-based approaches have been developed which have multiple advantages over traditional assays. For instance, NGS has the ability to interrogate thousands of microsatellite loci compared with just 5-7 loci that are detected by PCR. In this chapter, we detail the biochemical and computational steps to detect MSI-H+ from analysis of paired tumor and normal samples through NGS. We begin with DNA extraction, describe sequencing library preparation and quality control (QC), and outline the bioinformatics steps necessary for sequence alignment, preprocessing, and MSI-H+ detection using the software tool MANTIS. This workflow is intended to facilitate more widespread usage and adaptation of NGS-powered MSI detection, which can be eventually standardized for routine clinical testing.

Tumor heterogeneity and acquired drug resistance in FGFR2-fusion-positive cholangiocarcinoma through rapid research autopsy.

Cholangiocarcinoma is a highly aggressive and lethal malignancy, with limited treatment options available. Recently, FGFR inhibitors have been developed and utilized in FGFR-mutant cholangiocarcinoma; however, resistance often develops and the genomic determinants of resistance are not fully characterized. We completed whole-exome sequencing (WES) of 11 unique tumor samples obtained from a rapid research autopsy on a patient with FGFR-fusion-positive cholangiocarcinoma who initially responded to the pan-FGFR inhibitor, INCB054828. In vitro studies were carried out to characterize the novel FGFR alteration and secondary FGFR2 mutation identified. Multisite WES and analysis of tumor heterogeneity through subclonal inference identified four genetically distinct cancer cell populations, two of which were only observed after treatment. Additionally, WES revealed an FGFR2 N549H mutation hypothesized to confer resistance to the FGFR inhibitor INCB054828 in a single tumor sample. This hypothesis was corroborated with in vitro cell-based studies in which cells expressing FGFR2-CLIP1 fusion were sensitive to INCB054828 (IC50 value of 10.16 nM), whereas cells with the addition of the N549H mutation were resistant to INCB054828 (IC50 value of 1527.57 nM). Furthermore, the FGFR2 N549H secondary mutation displayed cross-resistance to other selective FGFR inhibitors, but remained sensitive to the nonselective inhibitor, ponatinib. Rapid research autopsy has the potential to provide unprecedented insights into the clonal evolution of cancer throughout the course of the disease. In this study, we demonstrate the emergence of a drug resistance mutation and characterize the evolution of tumor subclones within a cholangiocarcinoma disease course.

Homologous recombination and DNA repair mutations in patients treated with carboplatin and nab-paclitaxel for metastatic non-small cell lung cancer.

OBJECTIVES: Chemotherapy remains a cornerstone treatment in non-small cell lung cancer either in combination with checkpoint inhibitors or as subsequent therapy. Identifying molecular predictors of response allows for optimal treatment selection. We performed genomic analysis on tumor samples of patients treated with carboplatin and nab-paclitaxel as part of a phase II trial to evaluate the prognostic and predictive value of mutations in DNA repair pathway in patients treated with this regimen. MATERIALS AND METHODS: Next-generation sequencing libraries were produced using a capture-based targeted panel covering the coding exons of 278 genes on patients treated on clinical trial NCT00729612. Overall survival (OS) and progression-free survival (PFS) were assessed as part of the clinical outcomes and correlated with mutation analysis. RESULTS: Of 63 patients enrolled, 25 patients had sufficient and acceptable DNA isolated from archival tumor samples for targeted sequencing. The most commonly altered pathways included DNA repair (DR) including Fanconi anemia and homologous recombination, JAK-STAT signaling, IGF-1, mTOR, and MAPK-ERK. Four patients with mutations in homologous recombination mutations had a shorter PFS (hazard ratio [HR] = 4.54, 95% CI 1.2, 17.1, p = 0.026) and OS (HR = 6.3, 95% CI 1.8, 21.3, p = 0.003). CONCLUSION: In this analysis of patients with predominantly squamous cell non-small cell lung cancer treated with carboplatin and nab-paclitaxel in a phase II trial, patients with mutations in homologous recombination pathways had shorter overall and progression-free survival. Validation on additional datasets of patients treated with platinum-based chemotherapy and immunotherapy combinations is warranted.

Characterization of a KLK2-FGFR2 fusion gene in two cases of metastatic prostate cancer.

BACKGROUND: The fibroblast growth factor receptor (FGFR) signaling pathway is activated in multiple tumor types through gene amplifications, single base substitutions, or gene fusions. Multiple small molecule kinase inhibitors targeting FGFR are currently being evaluated in clinical trials for patients with FGFR chromosomal translocations. Patients with novel gene fusions involving FGFR may represent candidates for kinase inhibitors. METHODS: A targeted RNA-sequencing assay identified a KLK2-FGFR2 fusion gene in two patients with metastatic prostate cancer. NIH3T3 cells were transduced to express the KLK2-FGFR2 fusion. Migration assays, Western blots, and drug sensitivity assays were performed to functionally characterize the fusion. RESULTS: Expression of the KLK2-FGFR2 fusion protein in NIH3T3 cells induced a profound morphological change promoting enhanced migration and activation of downstream proteins in FGFR signaling pathways. The KLK2-FGFR2 fusion protein was determined to be highly sensitive to the selective FGFR inhibitors AZD-4547, BGJ398, JNJ-42756943, the irreversible inhibitor TAS-120, and the non-selective inhibitor Ponatinib. The KLK2-FGFR2 fusion did not exhibit sensitivity to the non-selective inhibitor Dovitinib. CONCLUSIONS: Importantly, the KLK2-FGFR2 fusion represents a novel target for precision therapies and should be screened for in men with prostate cancer.

Genomic characterization of metastatic ultra-hypermutated interdigitating dendritic cell sarcoma through rapid research autopsy.

Interdigitating dendritic cell sarcoma (IDCS) is an extremely rare cancer of dendritic cell origin that lacks a standardized treatment approach. Here, we performed genomic characterization of metastatic IDCS through whole exome sequencing (WES) of tumor tissues procured from a patient who underwent research autopsy. WES was also performed on a treatment-naïve tumor biopsy sample obtained from prior surgical resection. Our analyses revealed ultra-hypermutation, defined as >100 mutations per megabase, in this patient's cancer, which was further characterized by the presence of three distinct mutational signatures including UV radiation and APOBEC signatures. To characterize clonal heterogeneity, we used the bioinformatics tool Canopy to leverage single nucleotide and copy number variants to catalog six subclones across various metastatic tumors. Truncal alterations, defined as being present in all clonal tumor cell populations, in this patient's cancer include point mutations in TP53 and CDKN2A and amplifications of c-KIT and APOBEC3A-H, which are likely driver mutations. In summary, we have performed genomic characterization evaluating tumor mutational burden (TMB) and heterogeneity in a patient with metastatic IDCS. Despite ultra-hypermutation, this patient's cancer was not responsive to treatment with PD-1 inhibition. Our results underscore the importance of characterizing clonal heterogeneity in TMB-high cancers.