Molecular Technology: Lecture 2

biomarker

endogenous or injected molecules whose presence or metabolism correlates with important disease related physiological processes and disease outcomes

• the accuracy of biomarkers vary, so not all biomarkers are suitable for medical development

types of biomarkers

• molecular (blood glucose)

• histologic

• radiographic (tumor size)

• physiologic (blood pressure)

biological marker

measurable indicator that can tell us something about a person’s health or disease state: disease process, biological process, a response to treatment or medicine, or a psychological condition

biomarker characteristics

• objectively measured & evaluated

• indicator of normal biological or pathological processes, or response to therapeutic intervention

• biological molecule found in blood, other body fluids, or tissues

• sign of normal or abnormal process, or condition of disease

• how well body responds to a treatment

• AKA molecular marker & signature molecule

ideal biomarker characteristics

• have great sensitivity, specificity, accuracy, high predictive value

• safe and easy to measure

• cost-efficient to follow up

• modifiable with treatment

• consistent across gender and ethnic groups

validation of biomarkers

• accuracy

• precision

• limit of detection

• interference, cross-reactivity

• sample preparation/conditions

• performance around the cut-off

• potential for carryover, cross-hybridization

genomics

• genome sequencing

• genome variation

• genome annotation

transcriptomics

• microarrays

• gene expression data

proteomics

• mass spectrometry

• protein chips

metabolomics

• NMR

• Mass spectrometry

other technologies for biomarker discorvery

• fluorescent indicators

• lab-on-chip

• nuclear magnetic resonance

• mass spectrometry / liquid chromatography

• nanobiotechnology

• imaging

phase I of biomarker evaluation

identified markers are prioritized based on their diagnostic/prognostic/theraputic value that could suggest their evolution into routine clinical use

phase II of biomarker evaluation

establishing an assay with clear intended clinical use. The assays need to be validated for reproducibility and shown to be portable among different laboratories

phase III of biomarker evaluation

an investigator evaluates the sensitivity and specificity of the test for the detection of diseases that have yet to be detected clinically

phase IV of biomarker evaluation

evaluates the sensitivity and specificity of the test on a prospective cohort. An investigator can estimate the false referral rate based on tested biomarkers and describe the extent and characteristics of the disease detected

phase V of biomarker evaluation

evaluates the overall benefits and risks of the new diagnostic test on the screened population

biomarkers in use

• diagnosis in symptomatic patients

• early detection (screening)

• monitoring of disease

biomarkers for predicting the future

• risk assessment

• prognosis

• prediction

ideal marker for diagnosis

• should have great sensitivity (>0.9), specificity (>0.9), and accuracy in reflecting total disease burden

• a tumor marker should also be prognostic of outcome and treatment

samples for biomarker detection

blood, urine, tissue or other body fluids samples

biomarker and screening

• real time indicators of the presence of cancer

• marker must be highly specific, minimize false positive and negatives

• must be able to clearly reflect the different stages of disease

• marker must be easily detected without complicated medical procedures

• the disease markers released to serum and urine are good targets for application of early screening

• should be cost effective

safety (monitoring) biomarkers

constantly monitored safety lab biomarkers can act as common vital organ function tests applied across different therapeutic areas or as specialized testing applied to detect unique toxicities

prognosis biomarkers

• may provide information about a patients expected outcome

• help determine which cancers may grow rapidly and/or metastasize

predicting biomarker

• may be used to predict a patient’s response to treatment

• or determine the optimal drug dose or type used for breast cancer tx

  • (since breast cancer is heterogenous disease, different cancers respond different to same treatments)

monitoring biomarker

used to predict and monitor a patients breast cancer recurrence of the disease AFTER treatment

efficacy biomarkers

demonstrate a change in all or at least a good proportion of treated subjects

• the more positive the biomarker, the higher the efficacy

efficacy biomarker classifications

• surrogate biomarkers/endpoints

• predictive biomarkers

• pharmacodynamic biomarkers

• prognostic biomarkers

surrogate endpoints

• laboratory or physical measurement used in clinical trials to indicate a drug’s response and can be used in place of a clinical endpoint

• used to assess the benefit or harm of a therapeutic drug agent

• can provide the concept for which a candidate drug is to be used

validated biomarkers as surrogate endpoints advantages

• they might be able to be measured earlier, more easily, or frequently, with high precision

• may be less affected by other treatments, reduce sample size required, and allow for faster decisions

• ethical advantages in diseases with poor prognosis

surrogate biomarker examples

• blood pressure (hypertension)

• cholesterol, LDL (dyslipidiemia)

• Glycosylated hemoglobin (diabetes)

• intraocular pressure (glaucoma)

• biomarkers tumor shrinkage, response rate (cancer)

predictive biomarkers

• stratify patient population into responders and non responders to treatment

• predict whether or not a drug will have the intended effect

• forecast the extent to which a drug can be effective and/or toxic in different patient populations

• predict the risk or outcome of a disease in a patient population without the involvement of therapy

limitation for biomarkers

• expensive

• storage

• laboratory errors

• normal range is difficult to establish

• lack of making different selections before initiating discovery phase

• lack in biomarker characterization/validation strategies

• robustness of analysis techniques used in clinical trials

challenges of using biomarkers in medicine development

• technical challenges

• regulatory challenges

• ethical challenges

technical challenges

• must be validated by scientific evidence to ensure that the biomarker is accurate, reliable, sensitive, and specific

• show a reasonable relationship to the disease being studied

• enough “predictive ability?”

regulatory challenges

• developers of novel biomarkers are being encouraged to engage with regulators at an early stage

• validating biomarkers to meet regulatory standards can be complex and expensive

• if intended to be used as a surrogate endpoint

  • a dedicated clinical trial is required to test the link between the biomarker and clinical endpoint

ethical challenges

• linked to storage and use of tissue samples and the associated handling of personal medical data

• targeted medicine

• medicine is developed for people who fall outside of the population treated

tumor marker may be present in

• blood circulation

• body cavity fluids

• cell membranes

• cell cytoplasm

• DNA

biochemical properties of good tumor marker

1. should be present in or produced by the tumor itself

2. not be present in healthy tissues

3. plasma level should be at a minimum in healthy subjects

4. specific for a tissue, and different immunological properties when it is synthesized on other tissues

5. plasma level should be in proportion to the size of tumor and activity of tumor

6. half life should not be long

7. present at a detectable level even if the tumor size is small

8. useful for prediction and the recurrence of the tumor

uses of tumor markers

• screening general population

• differential diagnosis of symptomatic patients

• clinical staging of cancer

• estimating tumor volume

• prognostic indicator for disease progression

• evaluating success of tx

• detect recurrence of cancer

• monitoring response to therapy

• radioimmunolocalaization of tumor masses

serum level of marker reflects tumor burden

• level of a marker may be used as a prognostic indicator for disease progression and patient survival

  • after tx the marker should decrease

• the magnitude of marker reduction may reflect the degree of tx success

• in recurrence, the marker increases again

• tumor marker values correlate with the effectiveness of tx

biomarkers in periodontal disease

GCF, saliva, serum

saliva

• contains mix of enzymes, proteins, water, antibodies, ions, and mucins

• produced by parotid, submandibular, and sublingual glands

• unique biochemical composition

salivary biomarkers

• enzymes (-ase)

• immunoglobulins (IgA, IgG, IgM)

• protein

• phenotypic marker (keratin)

• host cell (leukocytes)

• ion (calcium)

• hormones (cortisol)

• bacteria

• volatile compounds (sulphide, marcaptan, picolines, pyridines)

IL 1b

• proinflammatory cytokine

• function: osteoclastic activity in periodontitis

• influence: immune cell recruitment, cell proliferation, tissue destruction, vascular smooth muscle cell contraction

alkaline phosphatase (ALP)

• mixed saliva of adult periodontitis patients revealed the highest enzyme activities

• associated with alveolar bone loss (key feature of PD disease)

C-reactive protein (CRP)

• synthesized in liver

• systemic marker and indicator of acute phase of an inflammatory response

• circulating CRP reaches saliva via GCF or salivary glands

• high levels associated with chronic and aggressive periodontal disease

Gingival Crevicular Fluid

• thought to be an inflammatory exudate or to cleanse material from the crevice

• have antimicrobial properties & antibody activity

GCF biomarkers

1. host-derived enzymes and their inhibitors

2. tissue breakdown products

3. inflammatory mediators and host response modifiers

Matrix Metalloproteinases

• key enzyme in extracellular collagen matrix degradation

• significantly increases he risk of periodontal disease

• MMP-8 indicates disease severity but also disease activity

• MMP-1 (interstitial collagenase) also appeared to be activated in periodontitis

beta glucuronidase

• degradation of the connective tissue ground substance: glycoproteins, proteoglycans

• may be obtained from both saliva and GCF

• positively associated with spirochetes, P. Gingivalis, P. intermedia

• negatively associated with cocci

aspartate amino transferase (AST)

cell death within the periodontal tissues

neutrophil elastase

marker of intracrevicular PMN activity, anti-bacterial activity

Cathepsin B

intracellular collagenolytic breakdown

osteocalcin

• most abundant non-collagenous protein of mineralized tissue, it is a small calcium-binding protein of bone

• source: osteoblasts

• function: promotes hydroxyapatite binding and accumulation of bone, chemotactically attracts osteoclast progenitor cells and blood monocytes

GCF osteocalcin

• has a positive correlation with clinical parameters in a cross-sectional study of patients with periodontitis

• higher in both periodontitis and gingivitis patients

• contradicting results has potential as a bone specific marker but not as a predictive indicator for periodontal disease

Osteopontin

• found in bone matrix

• highly concentrated at sites where osteoclasts are attached to the underlying mineral surface

• source: osteoblasts and osteoclasts

• function: holds a dual function in bone maturation

pro-inflammatory cytokines

• monocytes, macrophages, fibroblasts, and endothelial cells respond to plaque microorganisms by secreting chemokines and inflammatory cytokines (TNF-a, PGE2, IL-1b, IL6)

• can be obtained from GCF and saliva (IL1, TNF-a)

TNF-a

• origin: mononuclear phagocyte

• the main stimulus for release is the LPS of bacterial cell walls

• functions: bone resorption, inhibit bone collagen synthesis, induce collagenases, stimulate osteoclast differentiation in the presence of M-CSF

• act synergistically with cytokines and induces release of IL-1

IL-6

• produced by various cells such as activated monocytes or macrophages, endothelial cells, and fibroblasts

• Functions: B cells promote growth and facilitate maturation of the b cells causing immunoglobulin secretion, osteoclast formation and activity

• higher in areas of gingival inflammation, plays a role in bone resorption

IL-17

• proinflammatory cytokine produced by T-helper 17 cells

• stimulate carious cell types to produce other inflammatory cytokines and chemokines

• supports immune responses resulting in osteoclastic bone resorption