When should we order a next generation sequencing test in a patient with cancer?

Technical advances in genome sequencing and the implementation of next-generation sequencing (NGS) in clinical oncology have paved the way for individualizing cancer patient therapy based on molecular profiles. When and how to use NGS testing in the clinic is at present an unsolved issue, although new research results provide evidence favoring this approach in some types of advanced cancer. Clinical research is evolving rapidly, from basket and umbrella trials to adaptative design precision oncology clinical studies, and genomic and molecular data often displace the classical clinical validation procedures of biomarkers. In this context, physicians must be aware of the clinical evidence behind these new biomarkers and NGS tests available, in order to use them in the right moment, and with a critical point of view. This review will present the status of currently available targeted drugs that can be effective based on actionable molecular alterations, and the NGS tests that are currently available, offering a practical guide for the application of Clinical Precision Oncology in the real world routine practice.probability of identifying a targetable mutation is low[62], the canceris in early stages with recognized and effective forms of standardtreatment, or the patient has an irreversible disease with very shortlife-expectancy. As with any other laboratory test, doctors andpatients must be sure before ordering an NGS test that its result willhave an impact of the therapeutic plan. In any case, standard single-gene molecular testing must always be performed when indicated,since important therapeutic targets might be potentially missed if nomolecular analyses were performed.Clinical trials are showing that NGS testing can have an impact inthe response rate and progression-free survival of patients, and cantherefore be a very useful strategy leading to new molecularly-tar-geted treatment indications. Key factors responsible for improvedresults in precision-oriented clinical research, include refining themolecular pathways studied, developing molecular testing that inte-grates standarised genomic tests with transcriptomic analysis andimmunohistochemistry, selecting more active targeted agents,designing combinations of targeted agents -also with other forms oftherapy, and providing early treatment recommendations with avail-able Molecular Multidisciplinary Boards. Interdisciplinary discussionare very important to help with the interpretation of unclear molecu-lar results that are oftentimes seen with NGS testing.Important unsolved issues that will need to be addressed in thefuture include deciding which is the best tissue to perform NGS (pri-mary tumor vs metastasis, tumor DNA vs circulating tumor DNA),when is the right moment to test (atfirst diagnosis of advanced dis-ease or when the disease is refractory), and whether there are NGSclinical trial designs that allow for the use of control groups. Finally,using a complete informed consent before NGS testing and communi-cating NGS reports to patients are two very important aspects of theprocedure that have raised ethical concerns, and that must be alwaysaddressed by the practicing oncologists when ordering a NGS test.Search strategy and selection criteriaWe identified references through PubMed with the search terms“cancer AND NGS,”“cancer AND next generation sequencing,”“can-cer AND genomics,”for articles published to March 30, 2020. Thefinalreference list was generated on the basis of originality and relevanceto the broad scope of this Review.FundingThis Review was funded in part by research funds from projectsPIE15/00068andPI17/01865(Instituto de Salud Carlos III) awardedto RC, projectsJR17/00007andPI17/008(Instituto de Salud CarlosIII), awarded to NR-L,PI15/01491andPI19/00549(Instituto de SaludCarlos III) awarded to AA, projectsSAF2017 82886-R(Ministerio deEconomía y Competitividad), INDISNET-S2011/BMD-2332(Fundaci on Ram on Areces), andHR17-00016("La Caixa" Foundation)awarded to FS-M, and projectsPI16/00354(Instituto de Salud CarlosIII) andB2017/BMD-3733from the Consejería de Educaci on, Juventudy Deporte, Comunidad de Madrid, awarded to MQ-F. The manuscriptis part of the activities of the endowed Chair of Personalised PrecisionOncology, Universidad Aut onoma de Madrid (UAM-Fundaci on Insti-tuto Roche)S

MEK inhibition enhances the response to tyrosine kinase inhibitors in acute myeloid leukemia

FMS-like tyrosine kinase 3 (FLT3) is a key driver of acute myeloid leukemia (AML). Several tyrosine kinase inhibitors (TKIs) targeting FLT3 have been evaluated clinically, but their effects are limited when used in monotherapy due to the emergence of drug-resistance. Thus, a better understanding of drug-resistance pathways could be a good strategy to explore and evaluate new combinational therapies for AML. Here, we used phosphoproteomics to identify differentially-phosphorylated proteins in patients with AML and TKI resistance. We then studied resistance mechanisms in vitro and evaluated the efficacy and safety of rational combinational therapy in vitro, ex vivo and in vivo in mice. Proteomic and immunohistochemical studies showed the sustained activation of ERK1/2 in bone marrow samples of patients with AML after developing resistance to FLT3 inhibitors, which was identified as a common resistance pathway. We examined the concomitant inhibition of MEK-ERK1/2 and FLT3 as a strategy to overcome drug-resistance, finding that the MEK inhibitor trametinib remained potent in TKI-resistant cells and exerted strong synergy when combined with the TKI midostaurin in cells with mutated and wild-type FLT3. Importantly, this combination was not toxic to CD34+ cells from healthy donors, but produced survival improvements in vivo when compared with single therapy groups. Thus, our data point to trametinib plus midostaurin as a potentially beneficial therapy in patients with AML.We are particularly indebted to all the patients who participated in the study. This work was supported by the Instituto de Salud Carlos III (PI13/02378 and PI16/01530) and the CRIS foundation. M.L. had a postdoctoral fellowship from the Spanish Ministry of Economy and Competitiveness (FPDI-2013-016409) and holds a grant from the Spanish Society of Hematology and Hemotherapy.S

Monitoring vascular normalization induced by antiangiogenic treatment with (18)F-fluoromisonidazole-PET

This work was supported by the following sources: Fondo de Investigacion Sanitaria (Ministry of Health, Spain; numbers FIS PI10/0288, FIS PI13/00430, FIS PI 11/00616, CPII14/00005 and FIS PI14/00860; the first two awarded to MQF and the last three to MD), and "Fondo Europeo de Desarrollo Regional (FEDER) - Una manera de hacer Europa". MQF is a recipient of a 2010 Beca-Retorno from the AECC Scientific Foundation. Rosae Foundation and AVON Espana S.A.U. contributed to this work with unrestricted donations. Dovitinib was kindly provided by Novartis.BACKGROUND: Rationalization of antiangiogenics requires biomarkers. Vascular re-normalization is one widely accepted mechanism of action for this drug class. The interstitium of tumors with abnormal vasculature is hypoxic. We sought to track vascular normalization with (18)F-misonidazole ([18F]-FMISO, a probe that detects hypoxia) PET, in response to window-of-opportunity (WoO) treatment with the antiangiogenic dovitinib. METHODS: Two patient-derived pancreas xenografts (PDXs; Panc215 and Panc286) and the spontaneous breast cancer model MMTV-PyMT were used. Animals were treated during 1 week of WoO treatment with vehicle or dovitinib, preceded and followed by [18F]-FMISO-PET, [18F]-FDG-PET, and histologic assessment (dextran extravasation, hypoxia and microvessel staining, and necrosis, cleaved caspase-3 and Ki67 measurements). After WoO treatment, gemcitabine (pancreas)/adriamycin (breast) or vehicle was added and animals were treated until the humane endpoint. Tumor growth inhibition (TGI) and survival were the parameters studied. RESULTS: [18F]-FMISO SUV did not change after dovitinib-WoO treatment compared to vehicle-WoO (0.54 vs. 0.6) treatment in Panc215, but it decreased significantly in Panc286 (0.58 vs. 1.18; P < 0.05). In parallel, 10-KDa perivascular dextran extravasation was not reduced with dovitinib or vehicle-WoO treatment in Panc215, but it was reduced in Panc286. Whereas the addition of dovitinib to gemcitabine was indifferent in Panc215, it increased TGI in Panc286 (TGI switched from -59% to +49%). [18F]-FMISO SUV changes were accompanied by an almost 100% increase in interstitial gemcitabine delivery (665-1260 ng/mL). The results were validated in the PyMT model. CONCLUSIONS: [18F]-FMISO accurately monitored vascular re-normalization and improved interstitial chemotherapy delivery.This work was supported by the following sources: Fondo de Investigacion Sanitaria (Ministry of Health, Spain; numbers FIS PI10/0288, FIS PI13/00430, FIS PI 11/00616, CPII14/00005 and FIS PI14/00860; the first two awarded to MQF and the last three to MD), and "Fondo Europeo de Desarrollo Regional (FEDER) - Una manera de hacer Europa". MQF is a recipient of a 2010 Beca-Retorno from the AECC Scientific Foundation. Rosae Foundation and AVON Espana S.A.U. contributed to this work with unrestricted donations. Dovitinib was kindly provided by Novartis.S

Tumor P70S6K hyperactivation is inversely associated with tumor-infiltrating lymphocytes in triple-negative breast cancer

Purpose: Triple-negative breast cancer (TNBC) is characterized by large heterogeneity and relative lack of available targeted therapies. To find therapeutic strategies for distinct patients with TNBC, several approaches have been used for TNBC clustering, including recently immune and phosphoproteomic patterns. Based on 70-kDa ribosomal protein S6 kinase (P70S6K)-TNBC clustering, the current study explores the immune profiling in TNBC tumors. Methods: Stromal tumor-infiltrating lymphocytes (sTILs) were evaluated in human TNBC tumor samples. Furthermore, immunohistochemistry staining for CD8, CD4, Foxp3, and CD20 was performed in tissue microarrays (TMA) sections. Results: Histological analysis showed decreased sTILs, CD20+ cells, and CD8+/CD4+ ratio in high phosphorylated P70S6K (p-P70S6K) tumors. Moreover, p-P70S6K score was directly correlated with CD4+ and Foxp3+ T cells, while it was inversely correlated with CD8+/CD4+ and CD8+/Foxp3+ ratios. Conclusion: sTIL infiltration and lymphocyte profiling vary in the context of hyperactivation of P70S6K in TNBC tumorsThe project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 893597. RC is a recipient of the ISCIII grants: PI17/01865 and PI20/01458. MQF is a recipient of the following Grants: AES-PI19/00454 funded by the ISCIII and co-funded by the European Regional Development Fund (ERDF) and B2017/BMD3733 (Immunothercan-CM)—Call for Coordinated Research Groups from Madrid Region—Madrid Regional Government—ERDF funds. The study was also funded by CRIS Contra el Cancer Foundatio

Essentiality of fatty acid synthase in the 2D to anchorage-independent growth transition in transforming cells

Upregulation of fatty acid synthase (FASN) is a common event in cancer, although its mechanistic and potential therapeutic roles are not completely understood. In this study, we establish a key role of FASN during transformation. FASN is required for eliciting the anaplerotic shift of the Krebs cycle observed in cancer cells. However, its main role is to consume acetyl-CoA, which unlocks isocitrate dehydrogenase (IDH)-dependent reductive carboxylation, producing the reductive power necessary to quench reactive oxygen species (ROS) originated during the switch from two-dimensional (2D) to three-dimensional (3D) growth (a necessary hallmark of cancer). Upregulation of FASN elicits the 2D-to-3D switch; however, FASN's synthetic product palmitate is dispensable for this process since cells satisfy their fatty acid requirements from the media. In vivo, genetic deletion or pharmacologic inhibition of FASN before oncogenic activation prevents tumor development and invasive growth. These results render FASN as a potential target for cancer prevention studies.M.Q.F. is a recipient of the following grants: FIS PI13/00430 and FIS PI16/00354 funded by the Instituto de Salud Carlos III (ISCIII) and co-funded by the European Regional Development Fund (ERDF) and AECC Scientific Foundation (Beca de Retorno 2010). R.C. is a recipient of the following grants: FIS PI11/00832 and FIS PI14/00726 funded by the Instituto de Salud Carlos III (ISCIII) and co-funded by the European Regional Development Fund (ERDF), II14/00009 and PIE15/00068 from the Ministerio de Sanidad, Spain. N.S.C. is a recipient of an NIH grant (5R35CA197532). O.Y.T. is a recipient of the grants BFU2014-57466 from the Ministerio de Economia y Competitividad (MINECO). J.P.B. is funded by MINECO (SAF2016-78114-R), Instituto de Salud Carlos III (RD12/0043/0021), Junta de Castilla y Leon (Escalera de Excelencia CLU-2017-03), Ayudas Equipos Investigacion Biomedicina 2017 Fundacion BBVA, and Fundacion Ramon Areces. This study was partially supported by the generous donations from Fundacion CRIS Contra el Cancer and AVON Spain. We thank Drs. Erwin Wagner and Nabil Djouder for their critical review of the paper.S

Critically short telomeres and toxicity of chemotherapy in early breast cancer

Cumulative toxicity from weekly paclitaxel (myalgia, peripheral neuropathy, fatigue) compromises long-term administration. Preclinical data suggest that the burden of critically short telomeres ( 21.9% CSTs) had 2-fold higher number of neuropathy (P = 0.04) or fatigue (P = 0.019) episodes and >3-fold higher number of myalgia episodes (P = 0.005). The average telomere length was unrelated to the incidence of side effects.The percentage of CSTs, but not the average telomere size, is associated with weekly paclitaxel-derived toxicity.This work was supported by the Fondo de Investigación Sanitaria [FIS PI10/00288 and FIS PI13/00430]; AECC Scientific Foundation [Beca de Retorno-2010, to MQF]; Spanish Ministry of Economy and Competitiveness Projects [SAF2013-45111-R]; Madrid Regional Government Projects [S2010/BMD- 2303]; AXA Research Found; Fundación Botin; AVON Spain; and Boehringer-Ingelheim Spain.S

18F-fluoromisonidazole PET and Activity of Neoadjuvant Nintedanib in Early HER2-Negative Breast Cancer: A Window-of-Opportunity Randomized Trial.

Purpose: We previously detected promising efficacy of neoadjuvant nintedanib (a multityrosine kinase inhibitor, TKI) in early HER2-negative breast cancer. In a preclinical study, we monitored stromal hypoxia with 18F-fluoromisonidazole-positron emission tomography (18F-FMISO-PET); we found that reoxygenation of tumors (or lack of it) during a window-of-opportunity (WoO) treatment with TKIs correlated with the benefit (or lack of it) from TKI-plus-chemotherapy combinations. We studied the predictive role of 18F-FMISO-PET for the TKI nintedanib in the neoadjuvant setting in a phase II WoO randomized trial.Experimental Design: Patients were randomized to a 14-day WoO of nintedanib preceded and followed by an 18F-FMISO-PET, followed by nintedanib plus weekly paclitaxel (Arm A) or an 18F-FMISO-PET followed by weekly paclitaxel (Arm B) before surgery. The endpoint was residual cancer burden (RCB). The objective was to detect the patients with no response (RCB-III) on the basis of the baseline or evolutive 18F-FMISO-PET values/changes.Results: One-hundred and thirty HER2-negative patients were randomized. Seventeen (27.9%), 34 (55.7%), and 8 (13.1%) patients had an RCB of III, II, and I/0, respectively, in Arm A. In this arm, baseline hypoxic tumors had a 4.4-fold higher chance of experiencing RCB = 3 (P = 0.036) compared with baseline normoxic tumors. Nintedanib WoO induced tumor reoxygenation in 24.5% of the patients; those not reoxygenating showed a trend toward higher chance of experiencing RCB-III (6.4-fold; P = 0.09). In Arm B, 18F-FMISO-PET lacked predictive/prognostic value.Conclusions: Baseline hypoxic tumors (measured with 18F-FMISO-PET) do not benefit from neoadjuvant nintedanib. Clin Cancer Res; 23(6); 1432-41. ©2016 AACR

Stem cell-like transcriptional reprogramming mediates metastatic resistance to mTOR inhibition

Inhibitors of the mechanistic target of rapamycin (mTOR) are currently used to treat advanced metastatic breast cancer. However, whether an aggressive phenotype is sustained through adaptation or resistance to mTOR inhibition remains unknown. Here, complementary studies in human tumors, cancer models and cell lines reveal transcriptional reprogramming that supports metastasis in response to mTOR inhibition. This cancer feature is driven by EVI1 and SOX9. EVI1 functionally cooperates with and positively regulates SOX9, and promotes the transcriptional upregulation of key mTOR pathway components (REHB and RAPTOR) and of lung metastasis mediators (FSCN1 and SPARC). The expression of EVI1 and SOX9 is associated with stem cell-like and metastasis signatures, and their depletion impairs the metastatic potential of breast cancer cells. These results establish the mechanistic link between resistance to mTOR inhibition and cancer metastatic potential, thus enhancing our understanding of mTOR targeting failure