Berretta, M.

Catapano, O.

De Lucia, D.

Del Pup, L.

Di Francia, R.

Lus, G.

Pezone, A.

Tommaselli, A.

Tommaselli, P. A.

English

Archivio della ricerca - Università degli studi di Napoli Federico II

INTRODUCTIONOver the past 30 years, the clinical laboratory hasevolved into a complex, technology-driven enter-prise whose main purposes are diagnosing andscreening for disease, monitoring health and ther-apeutic response, and gauging deviations from nor-mal physiology in humans.Advances in diagnostic medicine, have beenachieved through the application of science and tech-nology as a result of a synergic effort among Univer-sities, industries, governments, and privateinstitutions. We are now entering the era of Molecu-lar Diagnostics, which is bringing forth the newestand most powerful science and technology availablefor the modern-day practice of diagnostic laboratorymedicine. Among the numerous important areas toconsider with molecular diagnostics there are theemerging issues concerning the development of ge-netic assays and their use for testing individual pa-tient responses for personalized pharmaceuticaltherapy. Here, we highlight the most relevant discus-sion presented by speakers in the conference “Mole-cular Diagnostics in the clinical practice” held inCaserta at “Research Centre CETAC on May 23-242014 (Figure 1). At the above faculty have been pre-sented the latest reports from literature in the fieldsof newest genomic test suitable in clinical practice.Knowledge-Base sessionDr. Oriana Catapano introduced the knowledge-base of “Old and new techniques in molecular bi-ology.”In the last seventy years, many improvementshave been made onto DNA techniques.Karyotype, FISH, PCR, Real-Time PCR,MLPA, Sanger sequencing are actually used to di-agnose genetics syndromes, tumours, viral or bac-terial infections, to predict or to follow uppharmacological therapies and for many others ap-plications. Many technologies like Array technolo-gies (aCGH, Gene Expression Profiling,SNP-array, etc.) and Next Generation Sequencingplatforms, developed into research laboratories, arenow largely used in clinical diagnostics. They havethe advantage to give more information in less timethan traditional techniques, giving a higher detec-tion rate and resolution power, allowing to identifyunknown genetic alterations and improving knowl-edge on molecular pathological mechanisms1.However, the reagents high costs and data man-agement are the main limitations to the diffusion ofthese techniques in all diagnostics laboratories2.Dr.Antonio Pezone clarified the Knowledge-baseof “DNA methylation is a "scar" induced by damagedrepaired DNA and remodelled by transcription.”1MOLECULAR DIAGNOSTICS INTHE CLINICAL PRACTICE –“RESEARCH CENTRE CETAC” CASERTA (ITALY)THE 23/24 MAY 2014R. DI FRANCIA1, O. CATAPANO2, A. PEZONE3, G. LUS4, A. TOMMASELLI1,M. BERRETTA5, L. DEL PUP6, P.A. TOMMASELLI7, D. DE LUCIA81National Cancer Institute “Fondazione G. Pascale”, Naples, Italy2MeriGen Molecular Biology Laboratory, Naples, Italy3Department of Molecular Medicine & Biotechnology, University “Federico II”, Naples, Italy4Centro per la Sclerosi Multipla, II Clinica Neurologica, Second University of Naples, Italy5Department of Medical Oncology, National Cancer Institute, Aviano (PN), Italy6Department of Surgical Oncology, National Cancer Institute, Aviano (PN) Italy7Department of Radiology, National Cancer Institute “Fondazione G. Pascale”, Naples, Italy8Azienda Policlinico at Second University of Naples, ItalyCorresponding Author: Raffaele Di Francia, MD; e-mail: raffaele.difrancia@istitutotumori.na.itWCRJ 2014; 1 (2): e220DNA is modified by methylation, which is lay-ered on the primary genetic information and altersgene expression. Somatic DNA methylation is un-stable or metastable and varies with age and dis-eases.Using a defined genetic system, we find thatfaithful repair of a DNA lesion in a reference geneleaves methylation marks, as “scars” on one strandof the repaired segment, which are transmitted tohalf of the daughter cells. These DNA methylatedsites are stable and inherited. If the scars occur ingenes, which inhibit growth, the silencing of thesegenes by methylation will “foster” the growth ofthe cells and favor cancer. In cancer, cells with thesame scars accumulate and evolve with malig-nancy3.We are deciphering the code of these epigeneticsignatures by deep sequencing methylated alleles(epialleles) in cancer4. For example, by analyzingthe growth suppressor, p16 CDKN2A methylationtraits in myeloid leukemia, we have monitored theprogression of the disease from the onset to theend. We are currently able, to detect patient-spe-cific signatures (corresponding to DNA damageevents experienced by the cells) and partially, dis-ease-specific marks. More samples should be ana-lyzed to validate epigenetic traits specific of thedisease and its stage. In both cases, this representsa viable method to personalize cancer history andtreatment5.Molecular Neurology sessionProf. Giacomo Lus proceeded to examine “Themolecular markers of neurodegeneration in multi-ple sclerosis as predictors of the clinical evolu-tion.”2The complex etiopathogenesis and the consid-erable heterogeneity of multiple sclerosis (MS)disease has led to identify it as a complex disorderthat can be further subdivided into different sub-types, each possibly characterized by a commonpathophysiological molecular mechanisms andprobably by a similar prognosis of the disease andtreatments response. The study of biomarkers canbe considered as the most promising indicator ofnumerous pathological disorders; in MS identifi-cation of these parameters, especially if used inclinical practice, it is still in the early stages6. Theidentification of anti-aquaporin-4 as a specificother form of demyelinating disease of the centralnervous system, such as the neuromyelitis optica(NMO), is by far the most successful example ofdiagnostic biomarkers. Ultimately, in the next fewyears the research should be focused on the iden-tification and validation of biomolecular parame-ters that, combined with those clinical andneuroimaging already present, may provide greaterpredictability to the prognosis of MS7.Prof Domenico De Lucia highlighted the suit-able “Laboratory Markers in the Diagnosis of Ve-nous Thromboembolis.”The purpose of this brief review is to discussthe value of selected blood tests that may be use-ful in the diagnosis of venous thromboembolism(VTE), or in the identification of a congenital oracquired defect associated with the developmentof VTE. It is extremely important in today’s cost-conscious world to make sure that each test that isordered will be clinically useful in the managementof the patient. This approach is emphasized in thisreview.Selected blood tests may be useful in the diag-nosis of venous thromboembolism (VTE), or in theidentification of a congenital or acquired defect as-Figure 1. Conference scene.sociated with the development of VTE. Severalstudies have shown the D-dimer assay to have ahigh negative predictive value but poor specificitywhen used in the detection of VTE.The presence of such genetic thrombophiliamarkers as factor V Leiden, prothrombin 20210Amutation, and antiphospholipid antibodies signifi-cantly increases patient’s risk of a thromboticevent. Other markers such as hyperhomocysteine-mia and deficiencies of antithrombin, protein C, orprotein S, when combined with the previous mu-tations, also significantly increase patient’s risk ofa thrombotic event.We feel that it is important to identify theseultra-high-risk patients to provide adequate coun-seling about the risk of thrombosis before electivesurgical procedures. Often, lifelong anticoagula-tion may be needed as these patients and familymembers may need testing before taking birth con-trol pills or hormonal replacement.Pharmacogenomics and molecularoncology sessionDr Raffaele Di Francia reported a pilot study on“Cost-effectiveness of pharmacogenomics testingin Fluoropyrmidine/oxaliplatin-based therapy.”In general, there are three main types of eco-nomic analysis for genotyping that differ primarilyin the evaluation of health outcome: cost-effec-tiveness, cost-utility and cost-benefit parameters.The primary aim of a cost-effectiveness analysis isto provide sufficiently robust information for de-cision-makers to allocate resources to healthcareinterventions. Overviews of cost-effectivenessstudies on PGx technologies are now available; allthe reviews until 2012, used different inclusion cri-teria and assessed the quality of analyzed studiesusing different approaches. Sutton et al8 reportsthat different meta-analysis methods used to eval-uate the accuracy of diagnostic tests can affect andinterfere with the economic evaluation. Recently,several methods to assess the quality of cost-ef-fectiveness, cost-utility and cost-benefit of PGxtests have become available. A relevant example isthe National Institute for Health and Clinical Ex-cellence (NICE). NICE forms a Diagnostic Advi-sory committee, which is willing to stimulatePharma and Academic communities to produce arobust set of data, including the design and datasource in economic models of healthcare9. It iswell known that PGx tests, performed before drugtreatment, lower overall medical costs and providehigher quality and longer life expectancy10.The methods and cost of genotyping specificgermline polymorphisms in four drug-metaboliz-ing genes, associated with Fluropyrimidine/oxali-platin-based therapy, have been reported. Relativecosts of these PGx tests have been evaluated by“subjective” criteria due to lack of specific guide-lines.The aim of this study is to provide informationon the advantages and limitations, in terms of costsof the most common available methods for molec-ular detection of variations of DYPD, TYMSMTHFR and GSTP1 genes11.The retrospective and prospective trials evalu-ating the pharmacoeconomic impact of genotyp-ing testing inFluoropyrimidine/oxaliplatin-based-therapy willprovide answers on the possibility to incorporatePGx testing into routine clinical practice.Dr Antonio Pasquale Tommaselli clarified theimportant role of “Integrated Thyroid diagnostics–cytologic, immunohistochemical, biomolecular-of fine needle aspiration.”Thyroid cancer is the most common malignanttumor of the endocrine system. These tumors fre-quently have genetic alterations leading to the ac-tivation of the mitogen-activated protein kinase(MAPK) signaling pathway.Most common mutations in papillary carcino-mas are point mutations of the BRAF and KRASgenes and RET/PTC rearrangement. These geneticalterations are found in >70% of papillary carci-nomas and they rarely overlap in the same tumor12.In follicular carcinomas, the second most com-mon type of thyroid malignancy, include RAS mu-tations and PAX8-PPAR  rearrangement. RETpoint mutations are crucial for the development ofmedullary thyroid carcinomas.The traditional diagnostic approach to this clin-ical situation is ultrasound-guided fine-needle as-piration (FNA) of the thyroid nodule followed bycytologic examination, which together reliably es-tablish the diagnosis in 70% to 80% of cases. Inthe rest of nodules the presence of cancer cannot beruled out by FNA cytology. New approaches to di-agnosis of cancer in thyroid nodules are based ondetection of aforementioned mutational markers,which can be reliably detected in cells aspiratedduring the FNA procedure. These markers offersignificant improvement in the diagnostic accuracyof FNA cytology and are poised to make a pro-found effect on the management and therapy of pa-tients with thyroid nodules13.Dr. Massimiliano Berretta introduced theknown “Molecular markers of colorectal cancer.”Over the past 30 years, there has been a greatinterest in clinical and molecular prognostic fac-tors in metastatic colorectal cancer (CRC).MOLECULAR DIAGNOSTICS IN THE CLINICAL PRACTICE3This interest is even greater today with the ad-vent of molecularly targeted agents that havechanged dramatically the treatment algorithms andthe survival for patients with metastatic CRC.CRC is one of the most commonly diagnosedcancers in the world and remains the second lead-ing cause of cancer death in Western countries.Survival for patients with metastatic CRC hasimproved dramatically over the past decade. In themid 1990s, the median overall survival (OS) forpatients with metastatic CRC treated with a 5-flu-orouracil (5-FU)-based regimen was only about 12months, increased to approximately 18 monthswith the addition of irinotecan and oxaliplatin14.The introduction of biologic agents has led to asubstantial jump in OS, approaching 30 months insome studies and allowing significant advances inthe study of CRC prognosis and outcome.Despite the advances in dosing and schedulingof chemotherapy in both adjuvant and advancedsettings, and a greater emphasis on early detection,the outlook still remains poor for most patients.Molecular analyses have shown that not allCRCs have the same natural history15.Cancers belonging to a particular pathologicstage may display significant clinical heterogene-ity, which may reflect underlying molecular het-erogeneity.Individual patients with same stage tumoursmay have different long term prognosis and re-sponse to therapy. In addition, some prognosticfactors are likely to be more important than others.These findings led, over the last eight decades, toextensive research of other possible prognostic fac-tors, in attempt to improve the identification of pa-tients more likely to have a poorer clinical outcomeand therefore more likely to benefit from more ag-gressive treatment strategies.The selection of the most beneficial treatmentregimes in CRC remains a challenge and is hin-dered by a lack of well established prognosticmarkers that correlate with survival or disease-freesurvival (DFS) and predictive markers for re-sponse to a particular therapy.Therefore, information regarding which pa-rameters influence the prognosis would be valu-able in the interpretation and design of clinicaltrials, and could also have implications for the clin-ical management decisions in the palliative setting.CONCLUSIONS AND FUTURE OUTLOOKThe clinical diagnostic laboratory performs testingof patient samples, provides the guidelines forstandardizing test development and utilization, andis the site most likely to standardize Pharmacoge-nomics testing. As a result, the hospital-based clin-ical laboratory is the logical site to perform rou-tine molecular testing.Furthermore, the major issues to consider forthe clinical laboratories (who are responsible forproviding PG services), are: i) the availability ofFDA-cleared tests; ii) the current absence of pub-lic reimbursement; iii) the need for genotyping ac-curacy; and iv) the need to find clinical expertise tointerpret laboratory data results16.The rapidly growing area of molecular diag-nostics is ideally suited to clinical laboratories andsuitable testing is a necessary and critical step tomove personalized medicine into practice.Hopefully, the future implementation of themethods for genotyping, will result in personalizedtreatments and eventually, in shifting the balancefrom disease relapse towards disease eradication. Itis important and quite timely for the clinical diag-nostic industry to step up, assuming these new re-sponsibilities, and pharmaceutical andbiotechnology companies should join each other,in order to develop a commercial test suitable forroutine molecular diagnostics.Duality/conflict of interest:None declared/applicable.References1. Riegel M. Human molecular cytogenetics: From cells tonucleotides. Genet Mol Biol 2014; 37(1 Suppl): 194-220.2. Voelkerding KV, Dames SA, Durtschi JD. Next-genera-tion sequencing: from basic research to diagnostics. ClinChem 2009; 55: 641-645.3. Cuozzo C, Porcellini A, Angrisano T, Morano A, Lee B, DiPardo A, Messina S, Iuliano R, Fusco A, Santillo MR,Muller MT, Chiariotti L, Gottesman ME, Avvedimento EV.DNA damage, homology-directed repair, and DNAmethylation. PLoS Genet 2007; 3: e110.4. Perillo B, Ombra MN, Bertoni A, Cuozzo C, Sacchetti S,Sasso A, Chiariotti L, Malorni A, Abbondanza C, Avved-imento EV. DNA oxidation as triggered by H3K9me2demethylation drives estrogen-induced gene expression.Science 2008; 319: 202-206.5. Morano A, Angrisano T, Russo G, Landi R, Pezone A,Bartollino S, Zuchegna C, Babbio F, Bonapace IM, AllenB, Muller MT, Chiariotti L, Gottesman ME, Porcellini A,Avvedimento EV. Targeted DNA methylation by homol-ogy-directed repair in mammalian cells. Transcription re-shapes methylation on the repaired gene. Nucleic AcidsRes 2014; 42: 804-821.6. Comabella M, Montalban X. Body fluid biomarkers inmultiple sclerosis. Lancet Neurol 2014; 13: 113-126.7. Fernández O. Integrating the tools for an individualizedprognosis in multiple sclerosis. J Neurol Sci 2013; 331: 10-13.8. Sutton A, Ades AE, Cooper N, Abrams K. Use of indi-rect and mixed treatment comparisons for technologyassessment. Pharmacoeconomics 2008; 26: 753-767.49. Dhalla IA, Garner S, Chalkidou K, Littlejohns P. Perspec-tives on the National Institute for Health and Clinical Ex-cellence's recommendations to use health technologiesonly in research. Int J Technol Assess Health Care 2009;25: 272-280.10. Berretta M, Di Francia R, Tirelli U. Editorial – The newoncologic challenges in the 3RD millennium. WCRJ2014; 1: e133.11. Di Francia R, Siesto RS, Valente D, Del Buono A, PuglieseS, Cecere S, Cavaliere C, Nasti G, Facchini G, BerrettaM. Current strategies to minimize toxicity of oxaliplatin:selection of pharmacogenomic panel tests. Anti CancerDrugs 2013; 24: 1069-1078.12. Nikiforov YE, Yip L, Nikiforova MN. New strategies in di-agnosing cancer in thyroid nodules: impact of molecu-lar markers. Clin Cancer Res 2013; 19: 2283-2288.13. Nikiforov YE. Molecular diagnostics of thyroid tumors.Arch Pathol Lab Med 2011; 135: 569-577.14. Berretta M, Cappellani A, Fiorica F, Nasti G, Frustaci S,Fisichella R, Bearz A, Talamini R, Lleshi A, Tambaro R,Cocciolo A, Ristagno M, Bolognese A, Basile F,Meneguzzo N, Berretta S, Tirelli U. FOLFOX4 in the treat-ment of metastatic colorectal cancer in elderly patients:a prospective study. Arch Gerontol Geriatr 2011; 52: 89-93.15. Di Francia R, Valente D, Pugliese S, Del Buono A,Berretta M. What health professions in oncology needsto know about pharmacogenomics? WCRJ 2014; 1:e90.16. Di Francia R, Valente D, Pugliese S, Del Buono A,Berretta M. What health professions in oncology needsto know about pharmacogenomics? WCRJ 2014; 1:e90.MOLECULAR DIAGNOSTICS IN THE CLINICAL PRACTICE5

MOLECULAR DIAGNOSTICS IN THE CLINICAL PRACTICE" RESEARCH CENTRE CETAC" CASERTA (ITALY) THE 23/24 MAY 2014

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MOLECULAR DIAGNOSTICS IN THE CLINICAL PRACTICE" RESEARCH CENTRE CETAC" CASERTA (ITALY) THE 23/24 MAY 2014

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