Proteomica Clinica

Esercitazioni in patologia clinica

CdL in medicina e chirurgia

La proteomica clinica: nuove frontiere per la medicina personalizzata

Massimo Papale, PhD
Dipartimento dell’emergenza e dei trapianti d’organo
Università di Bari
Centro di ricerca “BioAgroMed”
Università di Foggia
Foggia, 19 Aprile 2012

Biomarker definition

Any parameter that can be used to measure an interaction between a biological system and an environment agent, which may be chemical, physical or biological (WHO, 1993).

How many types of biomarkers?

• Proteins
• Electric signals
• Neuro-imaging
• Troponin I
• ECG
• TAC
• Gene expression
• Proteomic pattern
• Microarray
• Urine proteome
• SELDI-TOF

Why we need new biomarkers of kidney diseases

Although several clinical risk factors for the progression of CKD have been identified, there is still a lack of a consensus definition for CKD progression. Several definitions have been used in published studies, depending on the clinical setting and the duration of the study. This lack of uniformity in the definition adds to the difficulties in interpreting the published data, and will likely continue to be a limitation for future observational and interventional studies. P. Devarajan ACKD, Vol 17, No 6 (November), 2010: pp 469-479.

Improved biomarkers are clearly required to stratify subjects who are at greatest risk for CKD progression, and who might maximally benefit from increased surveillance, early prevention, and specific interventions. P. Devarajan ACKD, Vol 17, No 6 (November), 2010: pp 469-479.

How to monitor the natural history of renal disease?

Biomarkers for personalized care:

• Typical
• Earliest initiating
• Earliest baseline
• Current burden
• Clinical events
• Molecular risk
• Intervention detection cost

Disease time

Disease initiation and progression:

• Preclinical progression
• Therapeutic decision support
• Stable sources of dynamic genomics

New Genomics: Proteomics Biomarkers:

• Single nucleotide polymorphisms (SNPs)
• Haplotype mapping
• Molecular imaging
• Gene sequencing

How can we improve our diagnostic tools in renal diseases?

Renal Disease (Clinical Phase) and Renal Disease (Pre-clinical Phase):

The “Omics” Era:

• Transcriptomics
• Genomics
• Metabolomics
• Proteomics

For some time, the alpha-to-omega relationship of gene-to-protein was reversed and all biological buzzwords began with “gene or genomic.” However, in “Revolution Postponed,” Stephen S. Hall notes that “the Human Genome Project had not yet attained those miracles of medicine, so desperately awaited during the frenzied period marked by the sequencing of human DNA.” Notwithstanding the numerous advances brought forth by the substantial efforts of many researchers during this epochal phase of fundamental biology, another field increasingly emerged and with less fanfare: proteomics, the study of the proteome. Sci Am 303:60-67, 2010.

The word "proteome" is a blend of “protein and genome and was coined by Marc Wilkins in 1994 while working on the concept as a PhD student. He defined the Proteome as “The 'hierarchical' analysis for the mass screening of proteins and the analysis of genomes via their protein complement” (Wilkins et Al., Electrophoresis. 1995).

From proteins to proteomes

Large scale protein identification by two-dimensional electrophoresis and amino acid analysis. Marc R. Wilkins et al. Nature Biotechnology 14, 61 - 65 (1996). Publications dealing with proteomics: 31,485.

Clinical proteomics

Clinical proteomics (Nephrology) Fino a settembre 2010 Base metodologica dell’indagine proteomica. Comparison of techniques Genomics Proteomics Adapted from Negm et al. 2002.

Why proteomics?

Genes behavior

Cover Illustration, The Economist, Dec 13, 2002.

Why proteomics?

Different urine protein Different histological lesions in a glomerulus profiling. Klein JB. et al. – Am. J. Nephrology - 2004.

Applicazione della proteomica alla ricerca di biomarcatori di malattia: la proteomica clinica

La speranza Le insidie.

Key points:

• Appropriate sample collection
• Definition of the gold standard protocol for the chosen biological samples
• Definition of the clinical question and identification of the patients to be enrolled
• Identification and validation of the candidate biomarkers
• Appropriate sample collection: BIOBANKING of Biological Samples

Definition

Biobank is ‘a collection of biological material and the associated data and information stored in an organized system for a population or a large subset of a population’ (Organisation for Economic Cooperation and Development- OECD).

Collezione di materiale biologico in Italia

• Presenza di numerose collezioni “private” curate da singoli ricercatori il cui uso è limitato al gruppo di ricerca che ne dispone
• Carenza di personale dedicato
• Carenza di fondi per adeguare gli impianti alle normative vigenti (stoccaggio dei campioni in sicurezza; gestione informatizzata dei dati)

Impatto sociale e clinico limitato.

New strategies for the study of complex diseases

Most complex diseases are elusive as they do not root in single defects, but are caused by a large number of small, often additive effects from genetic predisposition, lifestyle and the environment. Discovery of adequate biomarkers will depend critically on the study of large collections of well documented, up-to-date epidemiological, clinical and biological information and accompanying material from large numbers of patients and healthy persons. Large sample collections are needed to maximize the “omics” potentiality.

• Genome
• Proteome
• Transcriptome
• Metabonome
• Environment
• Diet

Modalità di conservazione dei campioni

• Secure storage upon the principle of having mirror sites, with samples being split and banked in separate locations (freezers or geographic).
• Robust data tracking system (secure data storage and effective interrogation) with a well-maintained record of every step in the banking process.
• Strictly access-controlled system with automatic data logging.
• Barcoding schemes to allow unique identification of each sample and aliquot while displaying no personally identifying information.

D.H. Jackson and R.E. Banks, Proteomics Clin. Appl. 2010, 4, 250–270.

Tipologia del materiale conservato

• Cellule
• Colture cellulari sia primarie che derivate
• Tessuti adulti e fetali normali e controlli pre-analitici patologici
• Acidi nucleici
• Proteine
• Liquidi biologici

Necessità di standardizzare le variabili pre-analitiche

D.H. Jackson and R.E. Banks, Proteomics Clin. Appl. 2010, 4, 250–270.

Aspetti etici e giuridici

Tutela della riservatezza

La tutela dei diritti della persona e in particolare della riservatezza di ogni individuo è uno degli aspetti più delicati nella gestione di una biobanca. Normativa vigente: Autorizzazione n. 2/2004 del Garante per la protezione dei dati personali “il trattamento dei dati genetici può essere effettuato solo da coloro che svolgono attività sanitarie in senso stretto o attività di ricerca”.

Informativa e consenso

L’informativa deve contenere:

• Le finalità perseguite
• I risultati conseguibili
• Il diritto dell’interessato di opporsi al trattamento per motivi legittimi
• La possibilità dell’interessato di limitare l’accesso ai propri dati e ai campioni
• La disponibilità ad usare questi ultimi per ulteriori scopi
• Il periodo di conservazione dei dati e dei campioni biologici

Consenso informato: autorizzazione alla raccolta e al trattamento dei dati derivanti dal materiale depositato.

Oltre i localismi: la politica europea per le infrastrutture di ricerca

“A European Research Infrastructure Consortium (hereinafter referred to as an ‘ERIC’) involves facilities, resources and related services that are used by the scientific community to conduct top-level research in their respective fields and covers major scientific equipment or sets of instruments” (art. 2).

European strategy forum on research infrastructures – ESFRI

“The ESFRI roadmap aims at building a coordinating, large scale European research infrastructure of already existing and de novo collections of biomedically relevant, quality-assessed samples (with the possibility to link to related clinical and epidemiological information), to enhance therapy and prevention of common and rare diseases, including cancer.”

Le attuali infrastrutture di ricerca europee

“BBMRI is a pan-European and internationally broadly accessible research infrastructure that includes samples from patients and healthy persons, representing different European populations (with links to epidemiological and health care information), molecular genomic resources and biocomputational tools to optimally exploit this resource for global biomedical research.”

I.N.M.I. "L. Spallanzani" I.R.C.C.S. Istituto Superiore di Sanità (HUB Italiano). Coordina le attività di 23 biobanche presenti sul territorio nazionale tra cui:

• Definition of the gold standard protocol for the chosen biological samples: STANDARDIZATION OF URINE ANALYSIS

Cancer biomarker discovery sample sources

Dilution of markers with distance from tumor:

• Neoplastic body
• Sample type cytology
• Tissue fluids
• Biomarker concentration high medium low seminal plasma serum biopsy sample(s) suitable for nipple aspirates plasma LCM fine needle
• Aspirates biologial samples and kidney biomarkers sample type biomarkers amount biopsy specimens high intermediate urine low serum/plasma
• Urine advantages less complex than serum and plasma un-invasive recruitment in large amount higher presence of kidney derived molecules

Standardization of urine analysis

• Fresh sample or frozen one?
• Time controlled collection or not?
• UrineStorage at RmT: how long can it stay? Between-subject differences?
• Can protease inhibitors efficiently prevent protein degradation?

Concluding remarks

The timing of the collection can affect the proteomic profile.