Insights of Clinical Significance From 109 695 Solid Tumor Tissue-Based Comprehensive Genomic Profiles.

BACKGROUND: FoundationOneCDx is approved in the US and Japan as a companion diagnostic test to identify patients with cancer who may benefit from treatment with 30 drug therapies in the US and 23 in Japan. Tumor profiling with FoundationOneCDx also detects genomic findings with evidence of clinical significance that may inform clinical care decisions beyond companion diagnostic claims. This observational study reports the breadth and impact of clinical decision insights from FoundationOneCDx solid tumor profiles. MATERIALS AND METHODS: Consecutive test result reports for patients with solid tumor diagnoses (n = 109 695) were retrospectively analyzed for clinically significant predictive, prognostic, and diagnostic genomic alterations and signatures, determined in accordance with professional guidelines. Interventional clinical trials with targeted therapies or immune checkpoint inhibitors were matched to tumor profiles based on evidence that the genomic finding may be an actionable, investigational, or hypothetical target in the patient's tumor type. RESULTS: In 14 predefined cancer types (80.7% of analyzed solid tumors), predictive, prognostic, and diagnostic markers were reported in 47.6%, 13.2%, and 4.5% of samples, respectively, accounting for a total of 51.2% of tumor profiles. Pan-cancer predictive markers of tumor mutational burden (TMB) of 10 or more mutations per megabase, high microsatellite instability (MSI), or NTRK1/2/3 fusions were observed in 15.6%, 2.0%, and 0.1% of solid tumors, respectively. Most solid tumor profiles (89.2%) had genomic results that could theoretically inform decisions on the selection of immunotherapy and targeted therapy clinical trials. CONCLUSION: For this real-world population of patients with FoundationOneCDx solid tumor profiles in the routine course of clinical care, clinically significant findings were reported for approximately half of patients with genomic results.

Genomic profiling of solid tumors harboring BRD4-NUT and response to immune checkpoint inhibitors.

BACKGROUND: The translocation t(15:19) produces the oncogenic BRD4-NUT fusion which is pathognomonic for NUT carcinoma (NC), which is a rare, but extremely aggressive solid tumor. Comprehensive genomic profiling (CGP) by hybrid-capture based next generation sequencing of 186+ genes of a cohort of advanced cancer cases with a variety of initial diagnoses harboring BRD4-NUT may shed further insight into the biology of these tumors and possible options for targeted treatment. CASE PRESENTATION: Thirty-one solid tumor cases harboring a BRD4-NUT translocation are described, with only 16% initially diagnosed as NC and the remainder carrying other diagnoses, most commonly NSCLCNOS (22%) and lung squamous cell carcinoma (NSCLC-SCC) (16%). The cohort was all microsatellite stable and harbored a low Tumor Mutational Burden (TMB, mean 1.7 mut/mb, range 0-4). In two index cases, patients treated with immune checkpoint inhibitors (ICPI) had unexpected partial or better responses of varying duration. Notably, four cases - including the two index cases - were negative for PD-L1 expression. Neo-antigen prediction for BRD4-NUT and then affinity modeling of the peptide-MHC (pMHC) complex for an assessable index case predicted very high affinity binding, both on a ranked (99.9%) and absolute (33 nM) basis. CONCLUSIONS: CGP identifies BRD4-NUT fusions in advanced solid tumors which carry a broad range of initial diagnoses and which should be re-diagnosed as NC per guidelines. A hypothesized mechanism underlying responses to ICPI in the low TMB, PD-L1 negative index cases is the predicted high affinity of the BRD4-NUT fusion peptide to MHC complexes. Further study of pMHC affinity and response to immune checkpoint inhibitors in patients with NC harboring BRD4-NUT is needed to validate this therapeutic hypothesis.

Exceptional Response to Everolimus in a Patient with Metastatic Castrate-Resistant Prostate Cancer Harboring a PTEN Inactivating Mutation.

Prostate cancer is among the most common types of cancer in men. Early detection and proper medical intervention is crucial to ensuring successful treatment. Here we describe a patient clinically presenting with castrate-resistant prostate carcinoma. Comprehensive genomic profiling identified a PTEN inactivating mutation in the patient's tumor. After being heavily pretreated, the patient showed stable disease on everolimus, a PI3K-Akt-mTOR pathway inhibitor.

MET Genomic Alterations in Head and Neck Squamous Cell Carcinoma (HNSCC): Rapid Response to Crizotinib in a Patient with HNSCC with a Novel MET R1004G Mutation.

Identification of effective targeted therapies for recurrent/metastatic head and neck squamous cell carcinoma (HNSCC) remains an unmet medical need. A patient with platinum-refractory recurrent oral cavity HNSCC underwent comprehensive genomic profiling (CGP) that identified an activating MET mutation (R1004). The patient was treated with the oral MET tyrosine kinase inhibitor crizotinib with rapid response to treatment.Based on this index case, we determined the frequency of MET alterations in 1,637 HNSCC samples, which had been analyzed with hybrid capture-based CGP performed in the routine course of clinical care. The specimens were sequenced to a median depth of >500× for all coding exons from 182 (version 1, n = 24), 236 (version 2, n = 326), or 315 (version 3, n = 1,287) cancer-related genes, plus select introns from 14 (version 1), 19 (version 2), or 28 (version 3) genes frequently rearranged in cancer. We identified 13 HNSCC cases (0.79%) with MET alterations (4 point mutation events and 9 focal amplification events). MET-mutant or amplified tumors represent a small but potentially actionable molecular subset of HNSCC. KEY POINTS: This case report is believed to be the first reported pan-cancer case of a patient harboring a MET mutation at R1004 demonstrating a clinical response to crizotinib, in addition to the first documented case of head and neck squamous cell carcinoma (HNSCC) with any MET alteration responding to crizotinib.The positive response to MET inhibition in this patient highlights the significance of comprehensive genomic profiling in advanced metastatic HNSCC to identify actionable targetable molecular alterations as current treatment options are limited.

Unique mutation patterns in anaplastic thyroid cancer identified by comprehensive genomic profiling.

INTRODUCTION: Anaplastic thyroid cancer (ATC) is a highly aggressive thyroid cancer. Those ATC with genomic alterations (GAs) in TSC2, ALK, and BRAF may respond to targeted therapies. METHODS: Comprehensive genomic profiling on 90 ATC specimens identified base substitutions, short insertions and deletions, amplifications, copy number alterations, and genomic rearrangements in up to 315 cancer-related genes and 28 genes commonly rearranged in cancer. RESULTS: Median patient age was 65 (range, 33-86) years, 50 patients were male. There was a mean of 4.2 GA per case, range 1-11. The most common GA were TP53 (66%), BRAF (34%), TERT (32%), CDKN2A (32%), and NRAS (26%). BRAF V600E and NRAS/HRAS/KRAS alteration were mutually exclusive. BRAF, CDKN2A, PIK3CA, and JAK2 were more frequent in patients >70 years of age; while myc, PTEN, and NRAS were more common in those ≤50 years. CONCLUSION: ATC shows many GA with potential therapeutic significance and suggesting different molecular pathways can lead to ATC.

Pediatric, Adolescent, and Young Adult Thyroid Carcinoma Harbors Frequent and Diverse Targetable Genomic Alterations, Including Kinase Fusions.

BACKGROUND: Thyroid carcinoma, which is rare in pediatric patients (age 0-18 years) but more common in adolescent and young adult (AYA) patients (age 15-39 years), carries the potential for morbidity and mortality. METHODS: Hybrid-capture-based comprehensive genomic profiling (CGP) was performed prospectively on 512 consecutively submitted thyroid carcinomas, including 58 from pediatric and AYA (PAYA) patients, to identify genomic alterations (GAs), including base substitutions, insertions/deletions, copy number alterations, and rearrangements. This PAYA data series includes 41 patients with papillary thyroid carcinoma (PTC), 3 with anaplastic thyroid carcinoma (ATC), and 14 with medullary thyroid carcinoma (MTC). RESULTS: GAs were detected in 93% (54/58) of PAYA cases, with a mean of 1.4 GAs per case. In addition to BRAF V600E mutations, detected in 46% (19/41) of PAYA PTC cases and in 1 of 3 AYA ATC cases, oncogenic fusions involving RET, NTRK1, NTRK3, and ALK were detected in 37% (15/41) of PAYA PTC and 33% (1/3) of AYA ATC cases. Ninety-three percent (13/14) of MTC patients harbored RET alterations, including 3 novel insertions/deletions in exons 6 and 11. Two of these MTC patients with novel alterations in RET experienced clinical benefit from vandetanib treatment. CONCLUSION: CGP identified diverse clinically relevant GAs in PAYA patients with thyroid carcinoma, including 83% (34/41) of PTC cases harboring activating kinase mutations or activating kinase rearrangements. These genomic observations and index cases exhibiting clinical benefit from targeted therapy suggest that young patients with advanced thyroid carcinoma can benefit from CGP and rationally matched targeted therapy. The Oncologist 2017;22:255-263 IMPLICATIONS FOR PRACTICE: The detection of diverse clinically relevant genomic alterations in the majority of pediatric, adolescent, and young adult patients with thyroid carcinoma in this study suggests that comprehensive genomic profiling may be beneficial for young patients with papillary, anaplastic, or medullary thyroid carcinoma, particularly for advanced or refractory cases for which clinical trials involving molecularly targeted therapies may be appropriate.

TMPRSS2-ERG fusions unexpectedly identified in men initially diagnosed with nonprostatic malignancies.

Background: TMPRSS2-ERG gene fusions are frequently found in prostate cancer and are pathognomomic for prostatic origin. In a series of cancer cases assayed with comprehensive genomic profiling (CGP) in the course of clinical care, we reviewed the frequency of TMPRSS2-ERG fusions in patient tumors of various histologic subtypes. Methods: Frequency of TMPRSS2-ERG fusions was determined in comprehensive genomic profiles from 64,263 cancer cases submitted to Foundation Medicine to assess genomic alterations suggesting benefit from targeted therapy. Genomic results from an index case of prostate cancer that underwent evolution from adenocarcinoma to pure squamous cell carcinoma are presented. Results: TMPRSS2-ERG fusions were identified for 0.86% (250/29030) of male patients and not found for female patients (0/35233). TMPRSS2-ERG fusions were detected in six tumors that were classified as squamous carcinoma, five of which were of unknown primary site. The index case is a patient with a large left retrovesical mass diagnosed as squamous carcinoma by morphologic examination and a history of Gleason 9 prostate cancer with prior prostatectomy and salvage radiation therapy. TMPRSS2-ERG was detected by genomic profiling in the squamous cell tumor, the primary adenocarcinoma of the prostate, and in a metachronous prostatic adenocarcinoma metastasis. Based on these results, the patient received androgen deprivation therapy. A phylogenetic tree demonstrating clonal and histopathologic evolution of prostate cancer in the index patient was constructed. Conclusions: In this large CGP dataset, TMPRSS2-ERG fusion was seen in ~30% of prostate cancers regardless of histologic type; the fusion was on occasion detected in advanced cancers not initially carrying a diagnosis of prostate carcinoma. CGP of advanced cancers in men may reveal prostatic origin by detection of the pathognomomic TMPRSS2-ERG fusion gene.

Genomic alterations in human epidermal growth factor receptor 2 (HER2/ERBB2) in head and neck squamous cell carcinoma.

BACKGROUND: Despite recent advances, survival outcomes for those with metastatic or recurrent head and neck squamous cell carcinoma (HNSCC) have remained poor. Novel approaches should be investigated to improve outcomes. METHODS: A retrospective chart review was performed of a patient who presented with a TNM classification III HNSCC of the oropharynx, positive for human papillomavirus (HPV) who had a complete response to a human epidermal growth factor receptor 2 (HER2)-targeted therapy. Amplification rates of HER2 in the HNSCC Cancer Genome Atlas Network (TCGA) dataset and the FoundationOne genomic profiling dataset were evaluated. RESULTS: Comprehensive genomic profiling of the tumor obtained from the dermal metastasis identified amplification of HER2. Data from TCGA and FoundationOne showed that the frequency of HER2 alteration was not observed to vary significantly with HPV tumor status. CONCLUSION: This case demonstrates the application of genomic profiling to guide treatments in a patient with HNSCC with advanced metastatic disease refractory to standard of care therapies. © 2016 Wiley Periodicals, Inc. Head Neck 39: E15-E19, 2017.

Comprehensive Genomic Profiling of Clinically Advanced Medullary Thyroid Carcinoma.

OBJECTIVE: The aim of this study was to determine the genomic alterations of cancer-related genes in advanced medullary thyroid carcinoma during the course of clinical care. METHODS: Hybrid-capture-based comprehensive genomic profiling was performed on 34 consecutive medullary thyroid carcinoma cases to identify all four classes of genomic alterations, and outcome for an index patient was collected. RESULTS: RET was mutated in 88% (30/34) of cases, with RET M918T being responsible for 70% (21/30) of the RET alterations. The other RET alterations were RET E632_L633del, C634R, C620R, C618G/R/S, V804M, and RET amplification. Two of the four RET wild-type patients harbored mutations in KRAS or HRAS (1/34 each). The next most frequent genomic alterations were amplifications of CCND1, FGF3, and FGF19 and alterations in CDKN2A (3/34 each). One case with a RET M918T mutation developed acquired resistance to progressively dose-escalated vandetanib. When the mTOR inhibitor everolimus was added to continued vandetanib treatment, the patient achieved a second 25% reduction of tumor volume (RECIST 1.1) for 8 months. CONCLUSIONS: Comprehensive genomic profiling identified the full breadth of RET alterations in metastatic medullary thyroid carcinoma and possible cooperating oncogenic driver alterations. This approach may refine the use of targeted therapy for these patients.

CDK4/6 and IGF1 receptor inhibitors synergize to suppress the growth of p16INK4A-deficient pancreatic cancers.

Loss-of-function mutations in p16(INK4A) (CDKN2A) occur in approximately 80% of sporadic pancreatic ductal adenocarcinoma (PDAC), contributing to its early progression. Although this loss activates the cell-cycle-dependent kinases CDK4/6, which have been considered as drug targets for many years, p16(INK4A)-deficient PDAC cells are inherently resistant to CDK4/6 inhibitors. This study searched for targeted therapies that might synergize with CDK4/6 inhibition in this setting. We report that the IGF1R/IR inhibitor BMS-754807 cooperated with the CDK4/6 inhibitor PD-0332991 to strongly block proliferation of p16(INK4A)-deficient PDAC cells in vitro and in vivo. Sensitivity to this drug combination correlated with reduced activity of the master cell growth regulator mTORC1. Accordingly, replacing the IGF1R/IR inhibitor with the rapalog inhibitor temsirolimus broadened the sensitivity of PDAC cells to CDK4/6 inhibition. Our results establish targeted therapy combinations with robust cytostatic activity in p16(INK4A)-deficient PDAC cells and possible implications for improving treatment of a broad spectrum of human cancers characterized by p16(INK4A) loss.

Loss of RBF1 changes glutamine catabolism.

Inactivation of the retinoblastoma tumor suppressor (pRB) alters the expression of a myriad of genes. To understand the altered cellular environment that these changes create, we took advantage of the Drosophila model system and used targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) to profile the metabolic changes that occur when RBF1, the fly ortholog of pRB, is removed. We show that RBF1-depleted tissues and larvae are sensitive to fasting. Depletion of RBF1 causes major changes in nucleotide synthesis and glutathione metabolism. Under fasting conditions, these changes interconnect, and the increased replication demand of RBF1-depleted larvae is associated with the depletion of glutathione pools. In vivo (13)C isotopic tracer analysis shows that RBF1-depleted larvae increase the flux of glutamine toward glutathione synthesis, presumably to minimize oxidative stress. Concordantly, H(2)O(2) preferentially promoted apoptosis in RBF1-depleted tissues, and the sensitivity of RBF1-depleted animals to fasting was specifically suppressed by either a glutamine supplement or the antioxidant N-acetyl-cysteine. Effects of pRB activation/inactivation on glutamine catabolism were also detected in human cell lines. These results show that the inactivation of RB proteins causes metabolic reprogramming and that these consequences of RBF/RB function are present in both flies and human cell lines.

Phosphorylation puts the pRb tumor suppressor into shape.

In this issue of Genes & Development, Burke and colleagues (pp. 1156-1166) describe how the structure of retinoblastoma protein (pRb) is altered by phosphorylation at T373 or S608. These modifications cause specific conformational changes and alter pRb's interaction with E2F via two distinct mechanisms. The structures suggest that the panel of phosphorylation sites represents a versatile set of tools that are used to sculpt pRb in precise, but very different, ways.

Genome-wide analysis of Epstein-Barr virus Rta DNA binding.

The Epstein-Barr virus (EBV) lytic transactivator Rta activates promoters through direct binding to cognate DNA sites termed Rta response elements (RREs). Rta also activates promoters that apparently lack Rta binding sites, notably Zp and Rp. Chromatin immunoprecipitation (ChIP) of endogenous Rta expressed during early replication in B95-8 cells was performed to identify Rta binding sites in the EBV genome. Quantitative PCR (qPCR) analysis showed strong enrichment for known RREs but little or no enrichment for Rp or Zp, suggesting that the Rta ChIP approach enriches for direct Rta binding sites. Rta ChIP combined with deep sequencing (ChIP-seq) identified most known RREs and several novel Rta binding sites. Rta ChIP-seq peaks were frequently upstream of Rta-responsive genes, indicating that these Rta binding sites are likely functioning as RREs. Unexpectedly, the BALF5 promoter contained an Rta binding peak. To assess whether BALF5 might be activated by an RRE-dependent mechanism, an Rta mutant (Rta K156A), deficient for DNA binding and RRE activation but competent for Zp/Rp activation, was used. Rta K156A failed to activate BALF5p, suggesting this promoter can be activated by an RRE-dependent mechanism. Rta binding to late gene promoters was not seen at early time points but was specifically detected at later times within the Rta-responsive BLRF2 and BFRF3 promoters, even when DNA replication was inhibited. Our results represent the first characterization of Rta binding to the EBV genome during replication, identify previously unknown RREs, such as one in BALF5p, and highlight the complexity of EBV late gene promoter activation by Rta.

Epstein-Barr virus LF2 protein regulates viral replication by altering Rta subcellular localization.

The switch from Epstein-Barr virus (EBV) latent infection to lytic replication is governed by two viral transactivators, Zta and Rta. We previously reported that the EBV protein LF2 binds Rta, inhibits Rta promoter activation, and blocks EBV replication in cells. In addition, LF2 induces SUMO2/3 modification of Rta. We now show that this modification occurs at four lysines within the Rta activation domain (426, 446, 517, and 530) and that sumoylation of Rta is not essential for its repression. Coexpression studies demonstrated that Rta is sequestered to the extranuclear cytoskeleton in the presence of LF2. We mapped the LF2 binding site to Rta amino acids (aa) 476 to 519 and showed that LF2 binding is critical for Rta relocalization and repression. The core of this binding site, Rta aa 500 to 526, confers LF2-mediated relocalization and repression onto the artificial transcription factor GAL4-VP16. Mutational analysis of LF2 provided further evidence that Rta redistribution is essential for repression. Rta localization changes during replication of the LF2-positive P3HR1 genome, but not during replication of the LF2-negative B95-8 genome. BLRF2 protein expression was decreased and delayed in P3HR1 cells compared with B95-8 cells, consistent with reduced Rta activity. By contrast, BMRF1 expression, regulated primarily by Zta, did not differ significantly between the two cell lines. Our results support a model in which LF2 regulates EBV replication by binding to Rta and redistributing it out of the nucleus.