Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=ljii20 Journal of Immunoassay and Immunochemistry ISSN: 1532-1819 (Print) 1532-4230 (Online) Journal homepage: https://www.tandfonline.com/loi/ljii20 Multiplex assay for multiomics advances in personalized-precision medicine Maria-Linda Popa, Radu Albulescu, Monica Neagu, Mihail Eugen Hinescu & Cristiana Tanase To cite this article: Maria-Linda Popa, Radu Albulescu, Monica Neagu, Mihail Eugen Hinescu & Cristiana Tanase (2019) Multiplex assay for multiomics advances in personalized- precision medicine, Journal of Immunoassay and Immunochemistry, 40:1, 3-25, DOI: 10.1080/15321819.2018.1562940 To link to this article: https://doi.org/10.1080/15321819.2018.1562940 Published online: 11 Jan 2019. Submit your article to this journal Article views: 22 View Crossmark data
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Full Terms & Conditions of access and use can be found athttps://www.tandfonline.com/action/journalInformation?journalCode=ljii20
Multiplex assay for multiomics advances inpersonalized-precision medicine
Maria-Linda Popa, Radu Albulescu, Monica Neagu, Mihail Eugen Hinescu &Cristiana Tanase
To cite this article: Maria-Linda Popa, Radu Albulescu, Monica Neagu, Mihail EugenHinescu & Cristiana Tanase (2019) Multiplex assay for multiomics advances in personalized-precision medicine, Journal of Immunoassay and Immunochemistry, 40:1, 3-25, DOI:10.1080/15321819.2018.1562940
To link to this article: https://doi.org/10.1080/15321819.2018.1562940
Multiplex assay for multiomics advances inpersonalized-precision medicineMaria-Linda Popaa,b, Radu Albulescua,c, Monica Neagua,d, Mihail Eugen Hinescua,b,and Cristiana Tanasea,e
aBiochemistry-Proteomics Department, Victor Babes National Institute of Pathology, Bucharest,Romania; bCellular and Molecular Biology and Histology Department, “Carol Davila” University ofMedicine and Pharmacy, Bucharest, Romania; cPharmaceutical Biotechnology Department, NationalInstitute for Chemical-Pharmaceutical R&D, Bucharest, Romania; dFaculty of Biology, University ofBucharest, Bucharest, Romania; eCajal Institute, Titu Maiorescu University, Bucharest, Romania
ABSTRACTBuilding the future of precision medicine is the main focus incancer domain. Clinical trials are moving toward an array ofstudies that are more adapted to precision medicine. In thisdomain, there is an enhanced need for biomarkers, monitoringdevices, and data-analysis methods. Omics profiling usingwhole genome, epigenome, transcriptome, proteome, andmetabolome can offer detailed information of the humanbody in an integrative manner. Omes profiles reflect moreaccurately real-time physiological status.
Personalized omics analyses both disease as a whole andthe main disease processes, for a better understanding of theindividualized health. Through this, multi-omic approaches forhealth monitoring, preventative medicine, and personalizedtreatment can be targeted simultaneously and can lead clin-icians to have a comprehensive view on the diseasome.
Precision medicine improves the disease treatment and prevention by ana-lyzing the interaction of gene variability, environment, and individual life-style. In 2015, the President of the USA, Barack Obama, firmly requested theimplementation of precision medicine, a new model of patient-poweredresearch, in everyday clinical practice. In this regard, for 2016, the USAGovernment assigned 215 million dollars to accelerate biomedical discoveriesthat will provide new tools, knowledge, and patient-targeted therapies toclinicians.
Precision medicine represents an important approach of healthcare aimingto improve patient-specific and individualized diagnoses, medical decisions,medication, therapies, and prognoses and to increase life quality. It is an
CONTACT Cristiana Tanase [email protected] Department of Biochemistry-Proteomics, Victor BabesNational Institute of Pathology, no. 99-101 Splaiul Independentei, 050096 sect. 5, Bucharest, RomaniaColor versions of one or more of the figures in the article can be found online at www.tandfonline.com/LJII.
JOURNAL OF IMMUNOASSAY AND IMMUNOCHEMISTRY2019, VOL. 40, NO. 1, 3–25https://doi.org/10.1080/15321819.2018.1562940
advanced step in understanding medicine, serve patients, and healthcareimprovement. The implementation of precision medicine will facilitate theimplementation of new therapeutic strategies, drug discovery, and develop-ment of the gene-oriented treatment (Figure 1).
Precision medicine for the future
Precision medicine is based on advanced omic technologies, such as next-generation sequencing, protein and gene microarray, laser capture microdis-section, implying the correlation of genomics, epigenomics, proteomics,metabolomics with the clinical phenotypes of the individual patient. Thedevelopment of multiplex genotyping technologies and high-throughputgenomic profiling allow the analysis of individual patient genome fromperipheral blood or small biopsy material.
Next-generation sequencing (NGS) technologies and all the gathered dataobtained by implementing these methods have changed cancer investigationand provided support for clinicians in treatment decision-making. NGStechnologies have permitted an “omics” approach to cancer, allowinga genomic, transcriptomic, and epigenomic characterization of the diseaseof individual patients.
The main target of personalized medicine is to understand the molecularmechanism of the disease and to integrate it with individual pharmacoge-nomics profile that defines the response to drugs.
The personalized treatment implies the characterization of predictive(diagnostic), prognostic, treatment and prevention biomarkers. These can
Figure 1. Workflow to develop precision medicine (Adapted from Chen et al.[2]).
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be evaluated by omics techniques and some of them are already used in themanagement of several malignancies, such as chronic myeloid leukemia,cutaneous melanoma, colon, breast, and lung cancer. Diagnostic biomarkersare represented by key mutations and modification in signaling pathwaysinvolved in disease development. They offer information on drug responseand play a major role in therapy decisions. Prognostic biomarkers includesomatic germline mutations, epigenetic modifications, changes of microRNAlevels and circulating tumor cells, and are used to determine the outcome ofthe disease. Treatment and prevention biomarkers are necessary for indivi-dual therapy guidance and patient monitoring with different outcome risks.
The interaction between the genome-transcriptome-proteome profile ofthe patient and the environmental perturbations influences processes such asinflammation, thrombosis, fibrosis, immune response, cell proliferation,apoptosis, necrosis, and often generates distinct patho-phenotypes, clinicalsyndromes, and diseases. In spite of the great advances in science andtechnology in the twenty-first century, cancer still presents high morbidityand mortality, being a heavy social and economic global burden. For Europeare estimated almost 4 million new cases of cancer (excluding non-melanomaskin cancer) and almost 2 million deaths from cancer in 2018.[1] Moreover,according to latest world cancer estimations of WHO, cancer incidence andmortality could achieve pandemic proportions by 2025.
Implementation of precision medicine will allow aiming individual-specific targets, minimizing the harmful impact on normal tissue, and alsoreducing the costs of managing diseases on global level, along with mortalityand morbidity.
The molecular profiling of each individual patient involves advanced techni-ques such as germline DNA-NGS, tumor immunohistochemistry (IHC), tumorsequencing (NGS – next-generation sequencing/WES – whole-exome sequen-cing/WGS – whole-genome sequencing), tumor transcriptomics/proteomics,circulating cell-free DNA and circulating tumor cells.
Precision medicine: personal omics profile for understanding andmanaging health and disease
New therapies are mandatory for a targeted population of patients thatpresent abnormal gene fusions and mutations, methylation and acetylation,aberrations and variants; all these alterations can lead to protein over-expression. Precision medicine is based on human genetics and genomics,signaling pathways, individual gene interactions or most likely gene interac-tion networks, molecular regulations and controls, or functional mechan-isms. Omics profiling of transcriptomes, proteomes, cytokines, metabolomes,and autoantibodies has revealed a wide variation of the molecular compo-nents during the progression of the disease. In this regard, an integrated
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analysis of multi-omic data can lead to enhanced prediction of disease riskand evolution.
As stated in the Introduction section, precision medicine is based onadvanced omics technologies. Patients should receive a particular therapytailored to their own molecular characteristics. The panel of omes that shouldcharacterize an individual comprises genome, epigenome, transcriptome,proteome, metabolome, antibodyome, and all other omes that can lead topathology monitoring, that could offer preventative measures, and, in theend, crown precision medicine as an integrated medical approach.
All the technology arrays that omics analyze have the purpose to trans-form the traditional symptom-oriented diagnosis medicine toward preven-tion and early diagnostics.[3,4] Conventional medicine diagnosis andtreatment is limited by the concept of few universal targets for the samedisease, simplifying the complexity of an individual that develops thedisease.[5] These limitations can be overridden by large-scale genetic andmolecular profiling; thus, medicine is focusing on the individual particu-larities developing a certain disease. Omics technologies, like high-throughput DNA sequencing and mass spectrometry, can now simulta-neously identify tens of thousands of new molecules that generate a hugeamount of information for a certain biological system. This data can leadto complex molecular signatures, intra- and inter-cellular networks thatcan characterize the system in a particular time frame describing accu-rately certain pathology. This complex and combined information pin-points the genetic susceptibility of an individual in a real-timephysiology, terms that were published as Personal Omics Profile (iPOP).[6] Recently published, the results of iPOP analysis, including transcrip-tome, proteome, and metabolome data obtained in 20 time points during14 months, have shown detailed molecular differences between differentphysiological states, health, or disease (viral infections). These results haveimportant ground for precision medicine, as they can proactively foreseethe onset of the disease. The iPOP profile can be moreover customized byintroducing omes data relevant for a specific pathology, includingepigenome,[7] gut microbiome,[8] microRNA profiles,[9] and immunereceptor repertoire.[10] Macro-data such as behavioral parameters (nutri-tion, exercise, stress control, sleep) may also be added to the profile[11,12]
in the attempt to meticulously characterize the individual profile.
Preventing disease through precision medicine
Easier to prevent than to manage, risk assessment and early detection ofa disease gets new insights through precision medicine. Disease susceptibility,disease evolution, thoroughly linked to drug response are important issuesunraveled by a person’s genomic information.[11,12] Hence complex projects
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and databases (e.g. Personal Genome Project[13] are starting to increase theirknowledge in this domain. Combining genomic information as predictors ofdisease susceptibility with factors such as environment would enlarge thedata for disease prevention.[14,15] Collaborative international efforts devel-oped by International Cancer Genome Consortium (http://www.icgc.org/)and the Cancer Genome Atlas (http://cancergenome.nih.gov/) have shownDNA sequencing for a large array of cancers, e.g. breast cancer,[16,17] chroniclymphocytic leukemia,[18] hepatocellular carcinoma,[19] pediatricglioblastoma,[20] melanoma,[21] ovarian cancer,[22] small cell lung cancer,[23]
Sonic-Hedgehog medulloblastoma,[24] clear cell renal cell carcinoma,[25] andJAK2-negative myeloproliferative neoplasm.[26] Cancer genome sequencingcan indicate potential targets and reveal specific drugs in precision therapyfor specific patients. Whole genome and exome sequencing can identifyhereditary genetic diseases or diseases that did not have any obvious geneticlink up to now. In line with this sequencing, pharmacogenomics is anotherexpanding field in precision medicine. Hence, it is known from extendedclinical experience that the same drug can display different effects on differ-ent individuals, effects triggered by both genomic background and environ-mental factors.[11,27]
As important the genomic profiling is (e.g. whole transcriptomesequencing),[28] proteome analysis encompassing the actual players of thebiological functions is gaining its place in precision medicine domain.Gaining momentum in the last years, through mass spectrometryanalysis,[29,30] proteomics is important because it detects expressed muta-tions, influencing the biological event.[6,31,32]
MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight)imaging technology (MALDI-MSI) can identify spatial proteome profiling intissue samples.[33,34]
Another expanding field is metabolomics, where metabolite profiling canidentify the biological outcome of a specific drug in particular samples.[6,35]
Metabolic analogs can perform as drugs for specific proteins and/or cellularpathways. The metabolome profiling can be associated with differentdiseases[36] in specific individuals, hence another tool for precisionmedicine.[37,38]
A glimpse into the problem: precision medicine is addressing all the areasin medical sciences, i.e. we can speak of this approach in terms of cancer,chronic obstructive pulmonary disease, cardiovascular diseases, brain dis-eases, and any other disease or disorder that threats or affects human lifeand/or health. For the present review, the selection is narrowed to only onemajor class of diseases, namely cancer.
Precision medicine uses many “drivers”, and one of these drivers isobviously the intensive use of various technologies for analyzing the cohortsof molecular components of life. Different tools were setup for the in-depth
analysis of entire classes of molecules, and we now speak of several “omics”that seem to have freshened the hope that mankind will be the winner in thebattle with cancer.
Genomics represents, historically, the first “omics” discipline, and, prob-ably, it is also the most mature when we analyze the achievements in the fieldof biomarker discovery, especially when addressing cancer.
While Sanger or “first generation” sequencing is still largely applied, weincreasingly find out the achievements of “Next-generation Sequencing”based on either “Whole Genome Sequencing” or on targeted sequencing“Whole Exome Sequencing”. While Next-gen technology just started a fewyears ago, we already speak of an emerging technology, Single nucleotidesequencing.
A remarkable feature of NGS, highly used in genomics, transcriptomics,and epigenomics, offers insights for each molecular class investigated.[39]
An example of multiple data provided by NGS is published by Parkeret al.,[40] who analyzed at transcriptional and epigenetic level the glioblas-toma heterogeneity. According to their findings, promoter methylation statusof DNA repair enzyme O(6)-methylguanine DNA methyltransferase(MGMT) is the most important clinical biomarker in glioblastoma, predict-ing the therapeutic response, but other aberrations in DNA repair mechan-isms may also contribute. Genome sequencing (based on NGS) permitted thecharacterization of chromosome impairment, such as gene deletions, ampli-fications, translocations and sequence inversions. The same “machinery” canbe also employed for the evaluation of epigenetic modifications.
By whole exome sequencing, a higher sequence depth is achieved, witha lower cost compared to the whole genome sequencing. This fact is due tothe exome representing about 1% of the genome size, and the analysis effectivelyfocusing on parts of the genome that will be translated in a functional protein.[41]
The most significant impact of NGS on cancer genomics has been theability to re-sequence, analyze and compare the tumor tissue andmatched normal genome in an individual patient. With the significantlyreduced cost of sequencing, it is now possible to sequence multiplepatient samples of a given cancer type. NGS is useful in understandingthe affected pathways behind cancer development. This requiresa preliminary investigation to map genes that potentially lead to tumordevelopment (oncogenes) since many mutations may occur without car-cinogenic consequences.
This calibration step typically involves: (i) comparison with othersequenced genomes (via dbSNP) and to other resources for variant discoverysuch as the 1000 Genomes Project[42] (www.1000genomes.org), followed by(ii) comparison of remaining variant sites between the tumor and the normalgenome. One caveat of this approach is the decision whether a mutationdiagnosis is a false positive, which gives an incorrect interpretation. Another
caveat is a false negative, which is harder to evaluate, the result appearing asa lack of sequencing coverage or the result is actually correct (true positive).Information about the prevalence of any mutation in a cell population allowsone to infer how early in the path toward cancer development that particularmutation occurred.[43] Recent progress of comprehensive genome-wide ana-lysis has revealed information regarding genomic mutations and rearrange-ments in each individual tumor[44]; however, during the process of tumorinvasion, metastasis, and treatment exposure, cancer genomes are dynami-cally changing and evolving. At this moment, no established technique existsfor tracking this genomic tumor evolution in the living tumor of a patient,although liquid biopsy to detect circulating tumor cells and circulating cell-free DNA could be a promising approach.[45,46] ctDNA represents a mixtureof tumor DNA derived from various tumors existing in patient, so ctDNAwill include mutations appearing in all existing tumors. Compared to otherpotential biomarkers, it is considered that mutations in ctDNA have a lowfalse-positive rate since such somatic mutations occur only in cancerous andpre-cancerous lesions.
While NGS is the “front line” of biomarker discovery, array and PCR-based methods are considered the solution for the clinical large-scaleapplication.[46–49]
Predictive biomarkers help matching targeted therapies with patients andpreventing toxicity of standard therapies. Such prognostic biomarkers aresomatic germline mutations, alterations in DNA methylation patterns, mod-ified levels of microRNA – from the “genomic/transcriptomics” group, ordifferent protein and protein signatures, as well as blood circulating tumorcells (CTC). Such biomarkers, based on different molecular diagnosticstechniques are already in use in clinical practice for lung, breast, colon,lung cancer, melanoma, and chronic myeloid leukemia. Examples of mole-cularly targeted therapies based on such biomarkers are: tyrosine kinaseinhibitors (for gastrointestinal tumors and chronic myeloid leukemia); agentsblocking HER2/neu (HER2/neu-positive breast cancer), ALK inhibitors (lungcancer showing the EML4-ALk fusion), EGFR inhibitors (for EGFR-mutatedlung cancer).[50] Proteomics represents the second (as frequency of use)technology in biomarkers research. Compared to genomics and transcrip-tomics has a major disadvantage in the lack of the “amplificability” feature;however, proteomics creates a picture of the effective players in the field ofa certain disease, and not only the “programming” molecules. Therefore, it isworth all the effort to obtain proteomic signatures from patients, for diag-nostic, prognostic, and monitoring purposes.[51–53]
The most frequently used group of platforms used in proteomic discoveryis centered on mass-spectrometry (MS) but usually coupled to an “in line”tool for separation of integral or fragmented proteins. The separation step iseither based on liquid chromatographic (LC) devices or, more recently, on
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capillary electrophoresis devices. There is also an increasing use of theBiacore systems in the discovery chain.[44] These technologies involve differ-ent “dialects”, like LC/MS, nano-spray LC-MS/MS, MALDI-ToF-MS/MS,CE-MS (capillary electrophoresis-MS) or CE-MS/MS. The oldest proteomicapproach that is still in use is the combination of 2DE (bidirectional gelelectrophoresis) and LC/MS (or tandem MS). The power of the newest MSdevices enables the use of “fully MS/MS motorized” platforms, that use oneof the MS stages for further sequencing the peptide fragments.[54] These LC-MS techniques are based on the analysis of peptide fragments of digestedproteins, which serve as “surrogates” for the integral proteins.
A major help in biomarker discovery is provided by isotopic labelingtechniques: iTRAQ (isobaric tag for relative and absolute quantitation),SILAC (Stable Isotope Labeling with Amino Acids in Cell Culture).A relative quantitation of proteins is deduced from the intensity of peptidepeaks derived from that protein or from the number of MS/MS eventstriggered during LC/MS/MS analysis. iTRAQ associated with tandem MSwas used to discover potential biomarker proteins in cerebrospinal fluid fromschwannoma patients,[55] or for non-small-cell lung cancer.[56] However,even if they motorize some of the most powerful instruments, SILAC andiTRAQ technologies rely most often on patient-derived cell cultures and noton direct patient samples.[57,58] Full LC-MS techniques for the relative quan-titation of proteins are increasingly applied for the purpose of biomarkerdiscovery.[54,59–61]
Some of our studies were based on the application of proteomicstechnologies in precision medicine. In glioblastomas, we have found sig-nificant dysregulation in serum level of cytokines and angiogenic factors,with over threefold up-regulation of IL-6, IL-1beta, TNF-alpha, and IL-10and up to twofold up-regulation of VEGF, FGF-2, IL-8, IL-2, and GM-CSF.[62] Simultaneous identification of proteomic signatures could providepotential biomarkers panels for diagnostic and personalized treatment ofdifferent subsets of glioblastoma, as we already showed in other.[63–66]
Proteomic technologies – SELDI-ToF MS and LC-MS/MS have beenused to investigate the protein profile of glioblastoma and establishedseveral novel candidate diagnostic biomarkers, e.g. S100A8, S100A9 andCXCL4. Our data proposed an alternative and efficient approach by usinga novel combination of multiple technologies.[67] Also, we have studiedprotein biomarkers in cancers of the digestive tract to improve tumorscharacterization and possible personalized therapies. The novel biomarkersare expected to provide, hopefully, less invasive or non-invasive diagnostictools to make possible earlier detection of disease, and provide morereliable selection in the drug discovery process, and provide guidance forpersonalized medicine.[62]
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MALDI-imaging emerged more recently as a combined technology, link-ing resources from MS platforms with microscopy, thus offering a powerfultool for tissue proteomics. Basically, this combination provides a preciseoverlapping of protein spectra to the biological material, making possiblean accurate allocation of the proteins throughout the tissue sample, forexample, pin-pointing proteomic differences between tumoral and non-tumoral areas and even detecting proteins that append to special cell popula-tions like cancer stem cells (CSCs).[4,68–70]
Precision medicine: an approach for delivering improved healthcareto patients
Precision medicine is a further step to understanding medicine, improvepatients’ treatment, and aid healthcare. The diagnostics and therapyapproaches that are developed through Precision Medicine will facilitatethe practice of Personalized Medicine. The implementation of precisionmedicine will also facilitate the implementation of new therapeuticstrategies, drug discovery, and development of gene-oriented treatment.
In the panel of therapeutical approaches, precision medicine and immune-oncology have spoken in a prominent voice driven by the latest advances ingenomics with its genome-wide sequencing, big-data analytics, proteomics-based new technologies, and blood-based sampling.
Patient’s population groups are units of dissimilarities. Recently developedresearch has discovered that while a therapy works perfectly in one certaingroup, in a supposed similar group the same intervention seems useless.
Significant advancement in precision medicine is due to the developmentof advanced technologies such as genome-wide sequencing, and also to thecomprehension of the tumor immune microenvironment, that facilitated thestudy of the pre-malignancy biology.
Several clinical reports focused on the identification of driver mutations incirculating cell-free DNA of patients with various premalignant conditions(premalignant lung lesions, clonal hematopoiesis as premalignant state) sug-gest that we have entered a transformative period in cancer prevention andearly detection.[71] The rapid advance of high-throughput technologies allowsthe analysis of biological phenomena at omics levels leading to significantadvances in personalized and precision medicine. The therapy can be selectedbased on patient molecular characteristics. The health monitoring and pre-ventive measures rely also on individual omes as well as the integrated profilesof multiple omes, such as the genome, the epigenome, the transcriptome, theproteome, the metabolome, the antibodyome, and other omics information.These advances can transform medicine from disease diagnosis and treatmentto disease prevention and early diagnostic. Advances in the achieving of large-scale genetic and molecular profiling sustain the personalized and precision
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medicines as the paradigm of future healthcare. Conventional disease diagnosisand treatment are based on patient symptoms and have major limits: theycenter on late symptoms and usually ignore preclinical patho-phenotypes, riskfactors, and biological mechanisms of the disease.
Advances in the ability to perform large-scale genetic and molecularprofiling are expected to overcome these limitations by addressing indivi-dualized differences in diagnosis and treatment in unprecedenteddetail.[50]
Goh et al., using network analysis methods, reported that genes related tosimilar disorders present a greater probability of association between theirproducts and a greater connection among their transcription profiles com-pared with those that are not associated with similar disorders. Similarly,proteins that are associated with the same disease present an increasedaffinity to interact with each other. These observations sustain the conceptof disease-specific functional modules, which include an ample network ofknown human genetic diseases and disease-related gene network[72,73]
(Figures 2 and 3).
Figure 2. Human disease network. Every node represents a distinct disorder, colored by type ofdisease or affected organ. The size of each node is proportional to the number of genesassociated with the disorder. The lines between the same disorder classes are colored with thesame color; the lines connecting different disorder classes are colored gray; the height of lines isproportional to the number of genes associated with respective disorders. The name of disordersassociated with more than 10 genes is mentioned in the figure. (Reproduced from Goh et al.[73]).
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Clinical trials for therapeutic development in precision medicine
The development of therapeutic agents that target molecular mechanisms isdriving innovation in clinical-trial strategies. Although progress has beenmade, modifications to existing core paradigms in drug development willbe required to obtain the promise of precision medicine. One of the maintargets of precision medicine is the oncology domain and many trials havebeen developed in the last years. For example, crizotinib, an ALK inhibitorfor ALK fusion proteins, is used in lung cancer while vemurafinib, a BRAFinhibitor is used for melanoma patients with the BRAF V600E mutation.Trastuzumab is a HER2-targeted antibody for breast cancer, targeting proteinover-expression.[74]
These clinical trial results drove the ad hoc post-trial analyses, identifyingthe factors that induce these dissimilarities. For example, Gleevec (imatinib)doubled the survival rates of leukemia patients, but only for those presentingPhiladelphia translocation.[75] The same case was found in colorectal cancerfor Erbitux (cetuximab), that improved patient’s survival, but only for thosethat had a mutated EGFR.[76] Acknowledging these individualities, severalnew types of clinical trials were designed. Basket trials have been recentlydeveloped in the framework of precision medicine. This type of trial, actuallytests the effectiveness of a drug on the basis of its mode of action, not takinginto account the type of disease it was designed for. For example, one of the
Figure 3. Disease gene network. Each node represents a single gene, the connection between twogenes shows that these genes are involved in the same disease. The size of each node is proportional tothe number of specific disorders in which the gene is involved. Gene related to more than five diseasesare indicated with the gene symbol in the figure. (Reproduced from Goh et al.[73]).
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first precision clinical trials is NCI-Molecular Analysis for Therapy Choice(NCI-MATCH) launched in August 2015.[77]
This large trial (over 3,000 patients enrolled) analyzes patients’ tumors fortheir genomic alterations toward which there is already a drug (“actionablemutations”). The goal is to allocate therapies based on their individualabnormality and analyze if this type of precision medicine will show itseffectiveness. The uniqueness of the trial is that new treatments can beadded or seized anytime during the trial. Ten trial arms were opened in2015 for advanced solid tumors and for non-responsive lymphomas and bythe middle of 2016 additional arms were opened. In NCI-MATCH, tumorsamples from 3,000 patients will be subjected to DNA sequencing for identi-fying genomic abnormalities that can be targets for known drugs, eitheralready approved or during approval in other clinical trials. The investigatorsare foreseeing over 20 drugs/therapy approaches to be tested.[78]
In differently designed trials, umbrella trials, multiple drugs are tested forone disease. This approach is being used in melanoma where genomicallyguided therapy is given to late-stage patients, after choosing from over 40drugs.[77,79]
Nevertheless, there are still gaps for a proper precision therapy, just tounderline that, although the patient is carrying a particular mutation, theindividual’s responsiveness appends to many unknown factors. For example,vemurafenib, approved for melanoma carrying the BRAF (V600E) mutationcan be not as efficient in some groups because some tumor cells developother mutations, or their metastasis gain another profile, or with aging thereis a different mutation profile.[77]
Another type of trials are N-of-1 trials (clinical trial in which a singlepatient is enrolled, a single case study) where relevant data are collected forone person on a day-to-day basis or periodically over months or years. In thiscase, statistical analysis is based on a person’s response to a therapy.[77] Froma macropopulational point of view, it looks useless and costly, but thisapproach was used in an Australian study, for pain levels, swelling, andother symptoms reporting for the drugs tested in osteoarthritis and chronicpain. Before and after data for different therapies have given the investiga-tors, a more-effective individualized prescription.[80] In cancer trials, N-of-1type could guide clinicians for detecting an early disease onset. For example,when serum CA125 increases to 35 U/mL, this indicates an onset of ovariancancer, but when assessed regularly, even a level of 25 U/mL is an indicatorof onset when the patient had a previous 15 U/mL level of serum CA125.[81]
In a clinical trial designated to individual levels, the goal is to establish anindividual threshold for identifying with precision the disease onset.[6]
In the precision medicine portrait, there are several milestones that wereachieved. In 2015, the first clinical reports that used mutations detected incirculating cell-free DNA evaluated patients with premalignant lesions of the
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lung. For oral cancer, there was performed a molecular selection in chemo-prevention randomized controlled trial (RCT). In duodenum cancer in RCThigh efficacy was obtained when targeting signaling pathway alterations thatwere linked to germline mutation. Identification of HPV as a major cause ofcancer burden was also an important step in precision medicine, as HPV16could induce specific tumors, such as oropharyngeal cancer. In addition todeveloping new epigenetic drugs, new repurposed classic drugs such as aspirin,metformin, and tamoxifen were identified as immune-modulators.[71]
All these steps were driven by the “omics-related domain that can pinpointeach individual molecular characteristic.[82]
Precision medicine in cutaneous melanoma
The number of new cases of cutaneous melanoma has been increasing inrecent decades[83] and an association with higher socioeconomic status hasbeen described.[84] Detected early, melanoma may be cured by surgicalexcision, but the advanced (metastatic) disease is commonly incurable, with5-year overall survival (OS) under 10% and a median survival under1 year.[85]
The first medical evidence of the existence of cutaneous melanoma andmetastasis of this skin cancer was found in pre-Columbian mummies, about2,400 years ago, therefore this being a disease that was associated to humanssince their early history. We came a long way from the mid-twentieth-centurywide resection surgery with sentinel lymph node removal and first cytostaticdacarbazine in this disease. Thus, in the last years, seven new medicines gotapproval for advanced melanoma, over half of them being immune-mediated.The breakthrough anti-CTLA-4 ipilimumab has been followed by anti-PD1inhibitors and very recently, the first oncolytic immunotherapy, talimogenelaherparepvec (T-VEC) was approved. New therapies are currently being eval-uated or considered. These drugs are directed to various targets and are in thepreliminary phases of clinical investigation showing already new benefits.[86] Incomparison to mucosae or ocular melanoma, the cutaneous form is character-ized by different features. Thus, among all kinds of cancers, cutaneous mela-noma has the highest rate of mutations. For example, cutaneous melanoma has100–120 mutations/megabase in comparison to prostate cancer displaying only10 to 12 mutations/megabase.[87] Studies on standard melanoma cell lines andon excised tumors have shown mainly mutations in the NRAS gene.Subsequent studies have revealed mutations in the MAP kinase. In 2002, forthe first time, mutations were reported in the BRAF gene, mutations that arepresent in over 50% of melanoma metastasis. In this mutation, the mostcommon mutation induces glutamic acid substitution with valine (V600E).As with other types of cancer, cutaneous melanoma’s personalized therapy isstill in the early stages of development. On one hand, current guidelines
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recommend testing tumors for relevant mutations (NRAS, BRAF, KIT, GNAQ/11, and/or BAP1) to determine prognosis and to guide/individualizetherapy.[88] On the other hand, the manipulation of the immune status of thepatient is another branch in the developing personalized therapy in melanoma.
In melanoma, immunotherapies have the advantage to induce durable anti-tumor responses and could be personalized. Adoptive cell therapy (ACT) is oneof these approaches. It is based on genetically engineered T-cells armed tospecific “neoantigens”. The fact that this is a feasible therapeutical protocolcreates new opportunities for other tumor types to be attached when displayingpotential antigens and that can be revealed by tumor genomic sequencing. AsACT has been used in leukemia and was further tested in experimental mela-noma models, ACT awaits its early clinical studies in melanoma.[89] In the samearea, chimeric antigen receptor (CAR) T cells are used in hematological malig-nancies, but rapidly expanding to immunogenic cancers like melanoma andrenal cell carcinoma. Ex vivo expanded tumor-infiltrating lymphocytes (TIL)that can be genetically engineered to harbor CAR were subject to other solidcancer clinical trials. The efficacy of this approach was not satisfying, probablybecause individualized particularities of the tumor microenvironment caninduce a clear immunosuppressive effect. Thus, besides CAR T cells the futurewill need to unveil specific tumor microenvironment targets.[90]
Autologous monocyte-derived dendritic cells (DCs) that are loaded withmRNA encoding several specific melanoma antigens were recently tested inmelanoma patients with a high risk of recurrence. The loading was done withmRNA encoding a chimeric protein spanning MAGE-A1, -A3, -C2,Tyrosinase, Melan-A/MART-1, or gp100, and the sequence that targetsHLA class II. After 6 years follow-up, the median relapse-free survival wasregistered as 22 months, while 2-year and 4-year survival rates are 93% and70%, respectively.[91] Another recent report showed that recombinantMAGE-A3 protein (recMAGE-A3) was inoculated in resected stage IIB-IVMAGE-A3(+) melanoma patients. All patients developed specific antibodiesand good CD4 + T cell response.[92]
Precision medicine in cutaneous melanoma has at least two paths, onerepresented by specific and selective targeting of individual oncoproteinsexpressed by tumors and second customizing immune response to antigensexpressed by tumor and hindering the immunosuppression developed bytumor microenvironment.
Precision medicine in lung cancer
Lung cancer has a death toll of over 158,000 deaths each year only in USA.With a very low 5-year survival rate (18%), this type of cancer is anotherintense subject for precision medicine. In non-small cell lung cancer (NSCL),several clinical trials are opened and there are ongoing evaluations of the
16 M.-L. POPA ET AL.
efficacy of new drug combinations, mainly those that stimulate the immunesystem.
As in melanoma, there is a panel of frequently mutated genes. Top 10mutated genes include AKT1, BRAF, CTNNB1/beta-catenin, EGFR, ERBB2,HRAS, KRAS, NRAS, PIK3CA, and P53. These mutations and other mutatedgenes can encode for neo-antigens that influence the overall clinical responseof the patients. Early-stage NSCL has CD8 + TILs that can react to neoanti-gens expressing high levels of programmed cell death protein-1 (PD-1). InNSCL patients, the sensitivity to PD-1 and CTLA-4 blockade increased weretumors were rich in clonal neoantigens. In groups with clinical clear benefitT cells recognizing clonal neoantigens could be detected. Neoantigen hetero-geneity and tumor particularities can definitely influence immunesurveillance.[93] Durvalumab and tremelimumab have their clinical trials inlung cancer and results show that over 20% of patients have either a completeor a partial response. Cancer clinical trials have also an ongoing issue; in theera of precision and personalized medicine, this is once more delicate,namely the cost/value equilibrium in this expensive targeted therapy.[94]
Analog to melanoma, in lung cancer, one of the main arms for precisionmedicine is the immune-therapy. Immune checkpoint inhibitors, CTLA-4and PD-1/PD-L1 (PD ligand-1) were tested in lung cancer as well. Thesuccess of the drugs led to the approval both in squamous and non-squamous NSCL. Moreover, the treatment is prescribed after minute mole-cular detection in tumors of PD-L1.[95] Ongoing studies on NSCLC wouldpinpoint a new key factor modulating the immune response to immunecheckpoint inhibitors.[96] Patients in late stages of lung cancer with thepoorest prognosis being brain metastasis need to benefit from precisionmedicine. In this case, once more targeted therapies and immunotherapyrepresent the solution for late stages NSCLC.[97]
Challenges and promises – perspectives on precision medicine
Precision medicine, a key component of Horizon 2020 (the EU FrameworkProgramme for Research and Innovation) is being rapidly embraced bybiomedical researchers, pioneering clinicians and scientific funding programsin both the European Union and USA.
One of the main challenges is represented by heterogeneous informationwhich must be integrated into personalized predictive models.
In precision medicine, patient’s literacy in this domain gains increasingimportance. Hence, two separate multinational surveys of oncologists andpatients gained data that reflect self-reported and physician-assessed levels ofpatient cancer literacy in treatment decisions making. The results have shownthat patients and physicians are aware that a tumor has individual particula-rities and that personalized treatment plan is more efficacious.[98]
JOURNAL OF IMMUNOASSAY AND IMMUNOCHEMISTRY 17
Building the future of precision medicine it seems that there is an impor-tant particularity in cancer domain. There is a paired molecular target withits molecular test that gives an oncogenic mutation-driven cancer therapy.Another emerging point is that classical clinical trials are moving toward anarray of trials that are more adapted to precision medicine. In this domain,there is an enhanced need for biomarkers, monitoring devices, and data-analysis methods. Entering a new era of precision medicine it seems that thetools developed by immune-oncology are revolutionizing cancer therapy.
Through personalized omics, health monitoring, preventative medicine,and personalized treatment can be targeted simultaneously. Complex diseasessuch as autism[99] or Alzheimer’s disease,[100] can have thus an array offactors revealed leading to the onset of the disease.
Several health-related fields, such as nutritional systems biology, persona-lized diet and its relationship to health can take advantage of the complexomics analysis.[101]
One of the important issues in precision medicine application are thetechnologies costs, but as miniaturization and lab-on-a-chip analysis willdevelop, precision medicine will come one step closer to the patients.[102]
As stated by us in previous sections, individuals are a dissimilarities unit, butdiseases can be a similarities unit. Hence, data-driven disease similarity strategycan lead clinicians to have a comprehensive view on the diseasome. This iscomprised of a network of regulation mechanisms that become dysfunctional,and further generate a pathological condition. These networks provide informa-tion on pathogenesis mechanisms, as well as specific targets for drug development.
Conclusion
In the last years, significant changes have been made regarding cancer pre-vention, early detection and therapy and Precision medicine are revolutioniz-ing cancer therapy and prevention. The standard treatment of advanced-stagecancers evolved from classical empirical treatment strategy based on theclinical profile to one based on the molecular profile of the tumor.
Omics profiling using whole genome, epigenome, transcriptome, pro-teome, and metabolome, can offer detailed information of the human bodyin an integrative manner. Omes profiling reflects more accurately real-timephysiological status. Diseasome can provide information on pathogenesismechanisms, as well as specific targets for drug development.
Personalized omics analyses both disease as a whole and disease processeswithin for a better understanding of the individualized health. Through thisapproach health monitoring, preventative medicine and personalized treat-ment can be targeted simultaneously.
18 M.-L. POPA ET AL.
Acknowledgments
All authors had equal contribution in preparing this paper.
Funding
The work has been partially supported by the Executive Agency for Higher Education, Research,Development and Innovation Funding, under grant PN-III-P1.1-PD-2016-2093, contract no.121/2018, Bucharest, Romania.
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