Next-Generation Sequencing and Result Interpretation in Clinical Oncology: Challenges of Personalized Cancer Therapy.

The tools of next-generation sequencing (NGS) technology, such as targeted sequencing of candidate cancer genes and whole-exome and -genome sequencing, coupled with encouraging clinical results based on the use of targeted therapeutics and biomarker-guided clinical trials, are fueling further technological advancements of NGS technology. However, NGS data interpretation is associated with challenges that must be overcome to promote the techniques' effective integration into clinical oncology practice. Specifically, sequencing of a patient's tumor often yields 30-65 somatic variants, but most of these variants are "passenger" mutations that are phenotypically neutral and thus not targetable. Therefore, NGS data must be interpreted by multidisciplinary decision-support teams to determine mutation actionability and identify potential "drivers," so that the treating physician can prioritize what clinical decisions can be pursued in order to provide cancer therapy that is personalized to the patient and his or her unique genome.

EGFR modulates microRNA maturation in response to hypoxia through phosphorylation of AGO2.

MicroRNAs (miRNAs) are generated by two-step processing to yield small RNAs that negatively regulate target gene expression at the post-transcriptional level. Deregulation of miRNAs has been linked to diverse pathological processes, including cancer. Recent studies have also implicated miRNAs in the regulation of cellular response to a spectrum of stresses, such as hypoxia, which is frequently encountered in the poorly angiogenic core of a solid tumour. However, the upstream regulators of miRNA biogenesis machineries remain obscure, raising the question of how tumour cells efficiently coordinate and impose specificity on miRNA expression and function in response to stresses. Here we show that epidermal growth factor receptor (EGFR), which is the product of a well-characterized oncogene in human cancers, suppresses the maturation of specific tumour-suppressor-like miRNAs in response to hypoxic stress through phosphorylation of argonaute 2 (AGO2) at Tyr 393. The association between EGFR and AGO2 is enhanced by hypoxia, leading to elevated AGO2-Y393 phosphorylation, which in turn reduces the binding of Dicer to AGO2 and inhibits miRNA processing from precursor miRNAs to mature miRNAs. We also identify a long-loop structure in precursor miRNAs as a critical regulatory element in phospho-Y393-AGO2-mediated miRNA maturation. Furthermore, AGO2-Y393 phosphorylation mediates EGFR-enhanced cell survival and invasiveness under hypoxia, and correlates with poorer overall survival in breast cancer patients. Our study reveals a previously unrecognized function of EGFR in miRNA maturation and demonstrates how EGFR is likely to function as a regulator of AGO2 through novel post-translational modification. These findings suggest that modulation of miRNA biogenesis is important for stress response in tumour cells and has potential clinical implications.

Physician interpretation of genomic test results and treatment selection.

BACKGROUND: Genomic testing is increasingly performed in oncology, but concerns remain regarding the clinician's ability to interpret results. In the current study, the authors sought to determine the agreement between physicians and genomic annotators from the Precision Oncology Decision Support (PODS) team at The University of Texas MD Anderson Cancer Center in Houston regarding actionability and the clinical use of test results. METHODS: On a prospective protocol, patients underwent clinical genomic testing for hotspot mutations in 46 or 50 genes. Six months after sequencing, physicians received questionnaires for patients who demonstrated a variant in an actionable gene, investigating their perceptions regarding the actionability of alterations and clinical use of these findings. Genomic annotators independently classified these variants as actionable, potentially actionable, unknown, or not actionable. RESULTS: Physicians completed 250 of 288 questionnaires (87% response rate). Physicians considered 168 of 250 patients (67%) as having an actionable alteration; of these, 165 patients (98%) were considered to have an actionable alteration by the PODS team and 3 were of unknown significance. Physicians were aware of genotype-matched therapy available for 119 patients (71%) and 48 of these 119 patients (40%) received matched therapy. Approximately 46% of patients in whom physicians regarded alterations as not actionable (36 of 79 patients) were classified as having an actionable/potentially actionable mutation by the PODS team. However, many of these were only theoretically actionable due to limited trials and/or therapies (eg, KRAS). CONCLUSIONS: Physicians are aware of recurrent mutations in actionable genes on "hotspot" panels. As larger genomic panels are used, there may be a growing need for annotation of actionability. Decision support to increase awareness of genomically relevant trials and novel treatment options for recurrent mutations (eg, KRAS) also are needed. Cancer 2018;124:966-72. © 2017 American Cancer Society.

Expression of metastasis suppressor BRMS1 in breast cancer cells results in a marked delay in cellular adhesion to matrix.

Metastatic dissemination is a multi-step process that depends on cancer cells' ability to respond to microenvironmental cues by adapting adhesion abilities and undergoing cytoskeletal rearrangement. Breast Cancer Metastasis Suppressor 1 (BRMS1) affects several steps of the metastatic cascade: it decreases survival in circulation, increases susceptibility to anoikis, and reduces capacity to colonize secondary organs. In this report, BRMS1 expression is shown to not significantly alter expression levels of integrin monomers, while time-lapse and confocal microscopy revealed that BRMS1-expressing cells exhibited reduced activation of both ß1 integrin and focal adhesion kinase, and decreased localization of these molecules to sites of focal adhesions. Short-term plating of BRMS1-expressing cells onto collagen or fibronectin markedly decreased cytoskeletal reorganization and formation of cellular adhesion projections. Under 3D culture conditions, BRMS1-expressing cells remained rounded and failed to reorganize their cytoskeleton and form invasive colonies. Taken together, BRMS1-expressing breast cancer cells are greatly attenuated in their ability to respond to microenvironment changes. © 2013 Wiley Periodicals, Inc.

Syndecan-1 is required for robust growth, vascularization, and metastasis of myeloma tumors in vivo.

Myeloma tumors are characterized by high expression of syndecan-1 (CD138), a heparan sulfate proteoglycan present on the myeloma cell surface and shed into the tumor microenvironment. High levels of shed syndecan-1 in the serum of patients are an indicator of poor prognosis, and numerous studies have implicated syndecan-1 in promoting the growth and progression of this cancer. In the present study we directly addressed the role of syndecan-1 in myeloma by stable knockdown of its expression using RNA interference. Knockdown cells that were negative for syndecan-1 expression became apoptotic and failed to grow in vitro. Knockdown cells expressing syndecan-1 at approximately 28% or approximately 14% of normal levels survived and grew well in vitro but formed fewer and much smaller subcutaneous tumors in mice compared with tumors formed by cells expressing normal levels of syndecan-1. When injected intravenously into mice (experimental metastasis model), knockdown cells formed very few metastases as compared with controls. This indicates that syndecan-1 may be required for the establishment of multi-focal metastasis, a hallmark of this cancer. One mechanism of syndecan-1 action occurs via stimulation of tumor angiogenesis because tumors formed by knockdown cells exhibited diminished levels of vascular endothelial growth factor and impaired development of blood vessels. Together, these data indicate that the effects of syndecan-1 on myeloma survival, growth, and dissemination are due, at least in part, to its positive regulation of tumor-host interactions that generate an environment capable of sustaining robust tumor growth.

Correction: A feasibility study of returning clinically actionable somatic genomic alterations identified in a research laboratory.

[This corrects the article DOI: 10.18632/oncotarget.16018.].

Precision Oncology Decision Support: Current Approaches and Strategies for the Future.

With the increasing availability of genomics, routine analysis of advanced cancers is now feasible. Treatment selection is frequently guided by the molecular characteristics of a patient's tumor, and an increasing number of trials are genomically selected. Furthermore, multiple studies have demonstrated the benefit of therapies that are chosen based upon the molecular profile of a tumor. However, the rapid evolution of genomic testing platforms and emergence of new technologies make interpreting molecular testing reports more challenging. More sophisticated precision oncology decision support services are essential. This review outlines existing tools available for health care providers and precision oncology teams and highlights strategies for optimizing decision support. Specific attention is given to the assays currently available for molecular testing, as well as considerations for interpreting alteration information. This article also discusses strategies for identifying and matching patients to clinical trials, current challenges, and proposals for future development of precision oncology decision support. Clin Cancer Res; 24(12); 2719-31. ©2018 AACR.

Targeting TRK family proteins in cancer.

The tropomyosin receptor kinase (TRK) family includes TRKA, TRKB, and TRKC proteins, which are encoded by NTRK1, NTRK2 and NTRK3 genes, respectively. Binding of neurotrophins to TRK proteins induces receptor dimerization, phosphorylation, and activation of the downstream signaling cascades via PI3K, RAS/MAPK/ERK, and PLC-gamma. TRK pathway aberrations, including gene fusions, protein overexpression, and single nucleotide alterations, have been implicated in the pathogenesis of many cancer types, with NTRK gene fusions being the most well validated oncogenic events to date. Although the NTRK gene fusions are infrequent in most cancer types, certain rare tumor types are predominately driven by these events. Conversely, in more common histologies, such as lung and colorectal cancers, prevalence of the NTRK fusions is well below 5%. Selective inhibition of TRK signaling may therefore be beneficial among patients whose tumors vary in histologies, but share underlying oncogenic NTRK gene alterations. Currently, several TRK-targeting compounds are in clinical development. The ongoing Phase 2 trials with entrectinib and LOXO-101, two of the leading TRK inhibitors, are designed as 'basket trials', inclusive of patients whose tumors harbor NTRK gene fusions, independent of histology. Additional Phase 1 studies of other TRK inhibitors, including MGCD516, PLX7486, DS-6051b, and TSR-011, are underway. Interim data examining NTRK-rearranged tumors treated with entrectinib or LOXO-101 demonstrate encouraging activity, with patients achieving rapid and durable responses. Consequently, both drugs have achieved orphan designation from regulatory agencies, and efforts are underway to further expedite their development.

"Personalized Cancer Therapy": A Publicly Available Precision Oncology Resource.

High-throughput genomic and molecular profiling of tumors is emerging as an important clinical approach. Molecular profiling is increasingly being used to guide cancer patient care, especially in advanced and incurable cancers. However, navigating the scientific literature to make evidence-based clinical decisions based on molecular profiling results is overwhelming for many oncology clinicians and researchers. The Personalized Cancer Therapy website (www.personalizedcancertherapy.org) was created to provide an online resource for clinicians and researchers to facilitate navigation of available data. Specifically, this resource can be used to help identify potential therapy options for patients harboring oncogenic genomic alterations. Herein, we describe how content on www.personalizedcancertherapy.org is generated and maintained. We end with case scenarios to illustrate the clinical utility of the website. The goal of this publicly available resource is to provide easily accessible information to a broad oncology audience, as this may help ease the information retrieval burden facing participants in the precision oncology field. Cancer Res; 77(21); e123-6. ©2017 AACR.

Clinical Use of Precision Oncology Decision Support.

PURPOSE: Precision oncology is hindered by the lack of decision support for determining the functional and therapeutic significance of genomic alterations in tumors and relevant clinically available options. To bridge this knowledge gap, we established a Precision Oncology Decision Support (PODS) team that provides annotations at the alteration-level and subsequently determined if clinical decision-making was influenced. METHODS: Genomic alterations were annotated to determine actionability based on a variant's known or potential functional and/or therapeutic significance. The medical records of a subset of patients annotated in 2015 were manually reviewed to assess trial enrollment. A web-based survey was implemented to capture the reasons why genotype-matched therapies were not pursued. RESULTS: PODS processed 1,669 requests for annotation of 4,084 alterations (2,254 unique) across 49 tumor types for 1,197 patients. 2,444 annotations for 669 patients included an actionable variant call: 32.5% actionable, 9.4% potentially, 29.7% unknown, 28.4% non-actionable. 66% of patients had at least one actionable/potentially actionable alteration. 20.6% (110/535) patients annotated enrolled on a genotype-matched trial. Trial enrolment was significantly higher for patients with actionable/potentially actionable alterations (92/333, 27.6%) than those with unknown (16/136, 11.8%) and non-actionable (2/66, 3%) alterations (p=0.00004). Actionable alterations in PTEN, PIK3CA, and ERBB2 most frequently led to enrollment on genotype-matched trials. Clinicians cited a variety of reasons why patients with actionable alterations did not enroll on trials. CONCLUSION: Over half of alterations annotated were of unknown significance or non-actionable. Physicians were more likely to enroll a patient on a genotype-matched trial when an annotation supported actionability. Future studies are needed to demonstrate the impact of decision support on trial enrollment and oncologic outcomes.

ALK: a tyrosine kinase target for cancer therapy.

The anaplastic lymphoma kinase (ALK) gene plays an important physiologic role in the development of the brain and can be oncogenically altered in several malignancies, including non-small-cell lung cancer (NSCLC) and anaplastic large cell lymphomas (ALCL). Most prevalent ALK alterations are chromosomal rearrangements resulting in fusion genes, as seen in ALCL and NSCLC. In other tumors, ALK copy-number gains and activating ALK mutations have been described. Dramatic and often prolonged responses are seen in patients with ALK alterations when treated with ALK inhibitors. Three of these-crizotinib, ceritinib, and alectinib-are now FDA approved for the treatment of metastatic NSCLC positive for ALK fusions. However, the emergence of resistance is universal. Newer ALK inhibitors and other targeting strategies are being developed to counteract the newly emergent mechanism(s) of ALK inhibitor resistance. This review outlines the recent developments in our understanding and treatment of tumors with ALK alterations.

A feasibility study of returning clinically actionable somatic genomic alterations identified in a research laboratory.

PURPOSE: Molecular profiling performed in the research setting usually does not benefit the patients that donate their tissues. Through a prospective protocol, we sought to determine the feasibility and utility of performing broad genomic testing in the research laboratory for discovery, and the utility of giving treating physicians access to research data, with the option of validating actionable alterations in the CLIA environment. EXPERIMENTAL DESIGN: 1200 patients with advanced cancer underwent characterization of their tumors with high depth hybrid capture sequencing of 201 genes in the research setting. Tumors were also tested in the CLIA laboratory, with a standardized hotspot mutation analysis on an 11, 46 or 50 gene platform. RESULTS: 527 patients (44%) had at least one likely somatic mutation detected in an actionable gene using hotspot testing. With the 201 gene panel, 945 patients (79%) had at least one alteration in a potentially actionable gene that was undetected with the more limited CLIA panel testing. Sixty-four genomic alterations identified on the research panel were subsequently tested using an orthogonal CLIA assay. Of 16 mutations tested in the CLIA environment, 12 (75%) were confirmed. Twenty-five (52%) of 48 copy number alterations were confirmed. Nine (26.5%) of 34 patients with confirmed results received genotype-matched therapy. Seven of these patients were enrolled onto genotype-matched targeted therapy trials. CONCLUSION: Expanded cancer gene sequencing identifies more actionable genomic alterations. The option of CLIA validating research results can provide alternative targets for personalized cancer therapy.

S6K1 promotes invasiveness of breast cancer cells in a model of metastasis of triple-negative breast cancer.

Breast cancer is the second-leading cause of oncology-related death in US women. Of all invasive breast cancers, patients with tumors lacking expression of the estrogen and progesterone hormone receptors and overexpression of human epidermal growth factor receptor 2 have the poorest clinical prognosis. These referred to as triple-negative breast cancer (TNBC) represent an aggressive form of disease that is marked by early-onset metastasis, high tumor recurrence rate, and low overall survival during the first three years post-diagnosis. In this report, we discuss a novel model of early-onset TNBC metastasis to bone and lungs, derived from MDA-MB-231 cells. Breast cancer cells injected intravenously produced rapid, osteolytic metastases in long bones and spines of athymic nude mice, with concurrent metastasis to lungs, liver, and soft tissues. From the bone metastases, we developed a highly metastatic luciferase-tagged cell line variant named MDA-231-LUC Met. In this report, we demonstrate that the Akt/mTOR/S6K1 axis is hyperactivated in these cells, leading to a dramatic increase in phosphorylation of S6 ribosomal protein at Ser235/236. Lastly, we provide evidence that inhibition of the furthest downstream kinase in the mTOR pathway, S6K1, with a highly specific inhibitor PF-4708671 inhibits cell migration, and thus may provide a potent anti-metastatic adjuvant therapy approach.

Inhibition of type I insulin-like growth factor receptor signaling attenuates the development of breast cancer brain metastasis.

Brain metastasis is a common cause of mortality in cancer patients, yet potential therapeutic targets remain largely unknown. The type I insulin-like growth factor receptor (IGF-IR) is known to play a role in the progression of breast cancer and is currently being investigated in the clinical setting for various types of cancer. The present study demonstrates that IGF-IR is constitutively autophosphorylated in brain-seeking breast cancer sublines. Knockdown of IGF-IR results in a decrease of phospho-AKT and phospho-p70s6k, as well as decreased migration and invasion of MDA-MB-231Br brain-seeking cells. In addition, transient ablation of IGFBP3, which is overexpressed in brain-seeking cells, blocks IGF-IR activation. Using an in vivo experimental brain metastasis model, we show that IGF-IR knockdown brain-seeking cells have reduced potential to establish brain metastases. Finally, we demonstrate that the malignancy of brain-seeking cells is attenuated by pharmacological inhibition with picropodophyllin, an IGF-IR-specific tyrosine kinase inhibitor. Together, our data suggest that the IGF-IR is an important mediator of brain metastasis and its ablation delays the onset of brain metastases in our model system.