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.

Disparate Use of Chemoradiation in Elderly Patients With Localized Anal Cancer.

BACKGROUND: The incidence of squamous cell carcinoma of the anus (SCCA) is increasing, particularly among the elderly (age ≥65 years). We sought to compare patterns of care for the treatment of SCCA in elderly versus nonelderly patients. METHODS: Data for patients with stages I-III SCCA diagnosed from 2004 through 2015 were obtained from the National Cancer Database. Patients were categorized as having received standard-of-care (SOC) chemoradiation (CRT) with multiagent chemotherapy, non-SOC therapy, palliative therapy, or no treatment. Differences in treatment groups were tested using the chi-square test. We used logistic regression to identify predictors of SOC CRT and multiagent versus single-agent chemotherapy in patients receiving CRT. Propensity score matching was used to compare overall survival (OS) in elderly patients receiving multiagent versus single-agent chemotherapy for those receiving CRT. RESULTS: We identified 9,156 elderly and 17,640 nonelderly patients. A lower proportion of elderly versus nonelderly patients (54.5% vs 65.0%; P<.0001) received SOC CRT than other treatments or no treatment. In multivariate analysis, elderly patients were 38% less likely than nonelderly patients to receive SOC CRT (odds ratio, 0.62; 95% CI, 0.58-0.65; P<.0001). A higher proportion of the elderly were treated with single-agent versus multiagent chemotherapy (16.9% vs 11.8%; P<.0001), which resulted in a >1.5-fold increase in the likelihood of elderly patients receiving single-agent chemotherapy (odds ratio, 1.52; 95% CI, 1.39-1.66) in multivariate analysis. After propensity score matching, 3-year OS was higher in elderly patients who received CRT with multiagent versus single-agent chemotherapy (77.1% vs 67.5%; hazard ratio, 0.78; 95% CI, 0.68-0.89; P=.0002). CONCLUSIONS: In this comprehensive study of patients with stages I-III SCCA, elderly patients were less likely than nonelderly patients to receive SOC CRT. The low proportion of elderly patients receiving SOC CRT with multiagent chemotherapy for localized anal cancer suggests that the optimal treatment approach for this vulnerable population remains undefined.

Implementing precision cancer medicine in the genomic era.

The utilization of genomic data to direct treatment for cancer patients represents the central tenet in precision oncology, in which a patient is matched to a specific drug or therapy based on the genetic drivers detected in his or her tumor rather than the tumor's histologic classification. The expected but not always realized outcomes of molecularly matched therapies include increased response rates, more durable responses, deeper responses, and decreased number of therapy-related side effects. In this review, we will discuss different facets of utilizing genomic data to direct the increasingly complex care of cancer patients. We discuss the enlarging compendium of actionable genomic alterations and the development of novel molecular diagnostic assays for clinical application. Finally, we present an overview of the growing number of genomics-driven clinical trials and conclude with a discussion of future challenges in the implementation of precision oncology.

E2f1-3 switch from activators in progenitor cells to repressors in differentiating cells.

In the established model of mammalian cell cycle control, the retinoblastoma protein (Rb) functions to restrict cells from entering S phase by binding and sequestering E2f activators (E2f1, E2f2 and E2f3), which are invariably portrayed as the ultimate effectors of a transcriptional program that commit cells to enter and progress through S phase. Using a panel of tissue-specific cre-transgenic mice and conditional E2f alleles we examined the effects of E2f1, E2f2 and E2f3 triple deficiency in murine embryonic stem cells, embryos and small intestines. We show that in normal dividing progenitor cells E2f1-3 function as transcriptional activators, but contrary to the current view, are dispensable for cell division and instead are necessary for cell survival. In differentiating cells E2f1-3 function in a complex with Rb as repressors to silence E2f targets and facilitate exit from the cell cycle. The inactivation of Rb in differentiating cells resulted in a switch of E2f1-3 from repressors to activators, leading to the superactivation of E2f responsive targets and ectopic cell divisions. Loss of E2f1-3 completely suppressed these phenotypes caused by Rb deficiency. This work contextualizes the activator versus repressor functions of E2f1-3 in vivo, revealing distinct roles in dividing versus differentiating cells and in normal versus cancer-like cell cycles.

Manzamine A reduces androgen receptor transcription and synthesis by blocking E2F8-DNA interactions and effectively inhibits prostate tumor growth in mice.

The androgen receptor (AR) is the main driver in the development of castration-resistant prostate cancer, where the emergence of AR splice variants leads to treatment-resistant disease. Through detailed molecular studies of the marine alkaloid manzamine A (MA), we identified transcription factor E2F8 as a previously unknown regulator of AR transcription that prevents AR synthesis in prostate cancer cells. MA significantly inhibited the growth of various prostate cancer cell lines and was highly effective in inhibiting xenograft tumor growth in mice without any pathophysiological perturbations in major organs. MA suppressed the full-length AR (AR-FL), its spliced variant AR-V7, and the AR-regulated prostate-specific antigen (PSA; also known as KLK3) and human kallikrein 2 (hK2; also known as KLK2) genes. RNA sequencing (RNA-seq) analysis and protein modeling studies revealed E2F8 interactions with DNA as a potential novel target of MA, suppressing AR transcription and its synthesis. This novel mechanism of blocking AR biogenesis via E2F8 may provide an opportunity to control therapy-resistant prostate cancer over the currently used AR antagonists designed to target different parts of the AR gene.

Synergistic function of E2F7 and E2F8 is essential for cell survival and embryonic development.

The E2f7 and E2f8 family members are thought to function as transcriptional repressors important for the control of cell proliferation. Here, we have analyzed the consequences of inactivating E2f7 and E2f8 in mice and show that their individual loss had no significant effect on development. Their combined ablation, however, resulted in massive apoptosis and dilation of blood vessels, culminating in lethality by embryonic day E11.5. A deficiency in E2f7 and E2f8 led to an increase in E2f1 and p53, as well as in many stress-related genes. Homo- and heterodimers of E2F7 and E2F8 were found on target promoters, including E2f1. Importantly, loss of either E2f1 or p53 suppressed the massive apoptosis in double-mutant embryos. These results identify E2F7 and E2F8 as a unique repressive arm of the E2F transcriptional network that is critical for embryonic development and control of the E2F1-p53 apoptotic axis.

Allele-specific tumor spectrum in pten knockin mice.

Germline mutations in the tumor suppressor gene PTEN (phosphatase and tensin homology deleted on chromosome 10) cause Cowden and Bannayan-Riley-Ruvalcaba (BRR) syndromes, two dominantly inherited disorders characterized by mental retardation, multiple hamartomas, and variable cancer risk. Here, we modeled three sentinel mutant alleles of PTEN identified in patients with Cowden syndrome and show that the nonsense Pten(4-5) and missense Pten(C124R) and Pten(G129E) alleles lacking lipid phosphatase activity cause similar developmental abnormalities but distinct tumor spectra with varying severity and age of onset. Allele-specific differences may be accounted for by loss of function for Pten(4-5), hypomorphic function for Pten(C124R), and gain of function for Pten(G129E). These data demonstrate that the variable tumor phenotypes observed in patients with Cowden and BRR syndromes can be attributed to specific mutations in PTEN that alter protein function through distinct mechanisms.

ALK fusions in the pan-cancer setting: another tumor-agnostic target?

Anaplastic lymphoma kinase (ALK) alterations (activating mutations, amplifications, and fusions/rearrangements) occur in ~3.3% of cancers. ALK fusions/rearrangements are discerned in >50% of inflammatory myofibroblastic tumors (IMTs) and anaplastic large cell lymphomas (ALCLs), but only in ~0.2% of other cancers outside of non-small cell lung cancer (NSCLC), a rate that may be below the viability threshold of even large-scale treatment trials. Five ALK inhibitors -alectinib, brigatinib, ceritinb, crizotinib, and lorlatinib-are FDA approved for ALK-aberrant NSCLCs, and crizotinib is also approved for ALK-aberrant IMTs and ALCL, including in children. Herein, we review the pharmacologic tractability of ALK alterations, focusing beyond NSCLC. Importantly, the hallmark of approved indications is the presence of ALK fusions/rearrangements, and response rates of ~50-85%. Moreover, there are numerous reports of ALK inhibitor activity in multiple solid and hematologic tumors (e.g., histiocytosis, leiomyosarcoma, lymphoma, myeloma, and colorectal, neuroendocrine, ovarian, pancreatic, renal, and thyroid cancer) bearing ALK fusions/rearrangements. Many reports used crizotinib or alectinib, but each of the approved ALK inhibitors have shown activity. ALK inhibitor activity is also seen in neuroblastoma, which bear ALK mutations (rather than fusions/rearrangements), but response rates are lower (~10-20%). Current data suggests that ALK inhibitors have tissue-agnostic activity in neoplasms bearing ALK fusions/rearrangements.

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.

Cell proliferation in the absence of E2F1-3.

E2F transcription factors regulate the progression of the cell cycle by repression or transactivation of genes that encode cyclins, cyclin dependent kinases, checkpoint regulators, and replication proteins. Although some E2F functions are independent of the Retinoblastoma tumor suppressor (Rb) and related family members, p107 and p130, much of E2F-mediated repression of S phase entry is dependent upon Rb. We previously showed in cultured mouse embryonic fibroblasts that concomitant loss of three E2F activators with overlapping functions (E2F1, E2F2, and E2F3) triggered the p53-p21(Cip1) response and caused cell cycle arrest. Here we report on a dramatic difference in the requirement for E2F during development and in cultured cells by showing that cell cycle entry occurs normally in E2f1-3 triply-deficient epithelial stem cells and progenitors of the developing lens. Sixteen days after birth, however, massive apoptosis in differentiating epithelium leads to a collapse of the entire eye. Prior to this collapse, we find that expression of cell cycle-regulated genes in E2F-deficient lenses is aberrantly high. In a second set of experiments, we demonstrate that E2F3 ablation alone does not cause abnormalities in lens development but rescues phenotypic defects caused by loss of Rb, a binding partner of E2F known to recruit histone deacetylases, SWI/SNF and CtBP-polycomb complexes, methyltransferases, and other co-repressors to gene promoters. Together, these data implicate E2F1-3 in mediating transcriptional repression by Rb during cell cycle exit and point to a critical role for their repressive functions in cell survival.

Effectiveness of a novel 1% glucose isotonic electrolyte solution for intraoperative fluid therapy in children: a randomized controlled trial.

BACKGROUND: An appropriate electrolyte solution is important for safe intraoperative anesthesia management in children. This trial assessed the effectiveness of a novel 1% glucose isotonic electrolyte solution in intraoperative fluid therapy in children. METHODS: This trial analyzed data from 100 patients aged older than 1 month with an ASA score of I to II who received general anesthesia. Patients were randomly assigned to receive either the novel electrolyte solution (containing glucose, sodium, potassium, chloride, and bicarbonate) or lactated Ringer's solution intraoperatively as a maintenance fluid. Patient demographics and the results of blood gas analysis at 1, 2, and 3 hours were documented, and changes in glucose and electrolyte concentrations and the acid-base status were analyzed. RESULTS: During infusion of the novel solution, the glucose and potassium concentrations were stable. Conversely, the solution was linked to increased sodium levels but decreased bicarbonate levels, although both changes were within the physiological ranges. In addition, pH remained stable during the intraoperative period. Hypoglycemia, hyperglycemia, hyponatremia, or hypernatremia was not detected. CONCLUSIONS: The novel 1% glucose isotonic electrolyte solution helped to maintain glucose and electrolyte concentrations and acid-base stability, and it may therefore improve children's safety during the intraoperative period.

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.

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.

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.

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.

Rapid Research Autopsy: Piecing the Puzzle of Tumor Heterogeneity.

Tumor heterogeneity decreases the effectiveness of anticancer therapies and is an important topic in translational cancer research, given its relevance in clinical oncology. Here, we discuss how rapid research autopsy of cancer patients can elucidate heterogeneity-associated processes including cancer evolution and acquired therapeutic resistance. In practice, rapid research autopsy is performed shortly after a patient's passing to procure multiple metastatic tumor samples for genomic studies through next-generation sequencing and development of patient-derived xenografts or organoids. Mechanistic insights gained from research autopsy studies of cancer patients can help identify new targets for therapeutic intervention. Finally, the success of research autopsy programs is bolstered by collaboration across different medical and scientific disciplines in addition to support from patients and families.

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.

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.

Landscape of Microsatellite Instability Across 39 Cancer Types.

PURPOSE: Microsatellite instability (MSI) is a pattern of hypermutation that occurs at genomic microsatellites and is caused by defects in the mismatch repair system. Mismatch repair deficiency that leads to MSI has been well described in several types of human cancer, most frequently in colorectal, endometrial, and gastric adenocarcinomas. MSI is known to be both predictive and prognostic, especially in colorectal cancer; however, current clinical guidelines only recommend MSI testing for colorectal and endometrial cancers. Therefore, less is known about the prevalence and extent of MSI among other types of cancer. METHODS: Using our recently published MSI-calling software, MANTIS, we analyzed whole-exome data from 11,139 tumor-normal pairs from The Cancer Genome Atlas and Therapeutically Applicable Research to Generate Effective Treatments projects and external data sources across 39 cancer types. Within a subset of these cancer types, we assessed mutation burden, mutational signatures, and somatic variants associated with MSI. RESULTS: We identified MSI in 3.8% of all cancers assessed-present in 27 of tumor types-most notably adrenocortical carcinoma (ACC), cervical cancer (CESC), and mesothelioma, in which MSI has not yet been well described. In addition, MSI-high ACC and CESC tumors were observed to have a higher average mutational burden than microsatellite-stable ACC and CESC tumors. CONCLUSION: We provide evidence of as-yet-unappreciated MSI in several types of cancer. These findings support an expanded role for clinical MSI testing across multiple cancer types as patients with MSI-positive tumors are predicted to benefit from novel immunotherapies in clinical trials.