Study Design

1
Study Design

• A study design is a careful advance plan of the
  analytic approach needed to answer the research
  question under investigation in a scientific way.
• The basics of study design
• A carefully formed research question and a
  clearly stated outcome measure
• Assessing the feasibility of study objectives and
  considering alternative research designs
• Defining the study population and key concepts in
  operational terms
• Selecting methods of sampling, data collection,
  and analysis appropriate to the study's
  objectives
• Developing realistic budgets and time schedules
  for each stage of the research.

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Types of Study

• Experimental/ Interventional Investigator
  controls the assignment of the exposure or of the
  treatment e.g. randomized controlled trial.
• Non-experimental/Observational The allocation or
  assignment of factors is not under control of
  investigator. For example, in a study to see the
  effect of smoking, it is impossible for an
  investigator to assign smoking to the subjects.
  Instead, investigator can study the effect by
  choosing a control group and find the cause and
  relation effect. Some examples are-
• Cross-sectional study
• Cohort study
• Case-control study

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Study Design

• Randomized controlled Trial Random allocation of
  different interventions (or treatments) to
  subjects in which one treatment group is for the
  purpose of determining the efficacy of the other
  treatment (s). E.g. placebo or standard
  medication can be used as a controlled to compare
  the efficacy of the other (s) treatment (s)
• Types of control groups
• Placebo control group Receive treatment
• Active control group For example a cancer
  patient cant be given placebo. Need to use a
  standard medication in the market.
• Types of randomized controlled trials
• Open trial Investigator and subject know the
  full details of the treatment.
• Single-blind trial Investigator knows about the
  treatment but subject does not.
• Double-blind Both investigator and subject do
  not know about the treatment

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Study Design

• Cross-sectional study A descriptive study of the
  relationship between diseases and other factors
  at one point of time (usually) in a defined
  population. This is also known as prevalence
  study or survey study.
• Cohort study Subjects who presently have a
  certain condition and/or receive a particular
  treatment are followed over time and compared
  with another group who are not affected by the
  condition under investigation. Cohort analysis
  attempts to identify cohorts effects. E.g.
  recruit a group of smokers and a group of
  non-smokers and follow them for a set period of
  time and note differences in the incidence of
  lung cancer between the groups at the end of this
  time.
• Case-control study A study that compares two
  groups of people those with the disease or
  condition under study (cases) and a very similar
  group of people who do not have the disease or
  condition (controls) and look back to see if they
  had the exposure of interest. E.g. two groups of
  people (lung cancer group and non-lung cancer
  group) are selected and compare for an exposure
  (smoke).

5
Study Design

• A Protocol is a document that describes the
  background, objective(s), design, methodology,
  data collection and management, variable
  assessment, statistical considerations, and
  organization of the study.
• Basically a protocol is a manuscript that
  describes every step from proposal to completion
  of the research study.

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Basic concepts of clinical trials

• A clinical trial is a research study to answer
  specific questions about vaccines or new
  therapies or new ways of using known treatments.
  
• Institutional Review Board (IRB) A committee of
  physicians, statisticians, researchers, and
  others that ensures that a clinical trial is
  ethical and that the rights of study participants
  are protected.
• Efficacy is the maximum ability of a drug or
  treatment to produce a result
• Baseline measurement is the measurement taken
  just before a participant starts to receive the
  experimental treatment which is being tested
• Change from baseline measurement is the
  difference between baseline and post-baseline
  measurements.
• Percent change from baseline (Change from
  baseline / baseline measurement) x 100
• Pharmacokinetics (PK) analysis explores what the
  body does to the drug. That is, the processes (in
  a living organism) of absorption, distribution,
  metabolism, and excretion of a drug or vaccine.
  It helps to decide the duration of doses.
• Pharmacodynamic (PD) analysis detects the effect
  of drug on the body or microorganisms of the
  body.

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Basic concepts of clinical trials

• Classification of clinical trials by their
  purposes
• Treatment trials Test experimental treatments,
  new combinations of drugs, or new approaches to
  surgery or radiation therapy.
• Prevention trials Look for better ways to
  prevent disease in people who have never had the
  disease or to prevent a disease from returning.
  These approaches may include medicines, vitamins,
  vaccines, minerals, or lifestyle changes.
• Diagnostic trials Conducted to find better tests
  or procedures for diagnosing a particular disease
  or condition.
• Screening trials Test the best way to detect
  certain diseases or health conditions.
• Quality of Life Trials (or Supportive Care
  trials) explore ways to improve comfort and the
  quality of life for individuals with a chronic
  illness.

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Study design of a Clinical Trial

• Title Reflects the main research interest.
• Background/Rational of study Importance of the
  study and previous study results will be
  explained.
• Study Objectives
• Primary objective (s) Focuses on the core
  research question (s)
• Secondary Objectives Focuses on the secondary/
  optional research questions
• Investigational Plan
• Variable (parameter) selections to achieve the
  research objectives. Avoid selection of
  unnecessary variables
• Overall study design and plan description Brief
  description of design and assessments.

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Study design of a Clinical Trial

• Selection of Study Population
• Inclusion criteria A set of conditions to
  include a subject in the study. E.g. a adult
  study will include subjects only of age 18 or
  more.
• Exclusion criteria A set of conditions under
  which a subject (met inclusion criteria) will be
  excluded from the study. E.g. protocol
  violations, Non-compliance of the treatment etc.
• Sample size calculation Based on the effect size
  and the statistical power needed to test of main
  research question. Here are some useful websites
  for power and sample size calculation-
• compare means, compare proportions, population
  survey
• http//department.obg.cuhk.edu.hk/researchsupport/
  Sample_size_EstMean.asp
• Experimental Design, survival analysis
• http//hedwig.mgh.harvard.edu/sample_size/size.htm
  l
• Regression/multiple regression
• http//www.danielsoper.com/statcalc/calc01.aspx

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Study design of a Clinical Trial

• Description of the treatment groups/ treatment
  administration/ treatment period
• Randomization of the treatment to the subjects
• Detailed descriptions of assessment/collection of
  all parameters/variables including sign and
  symptoms (adverse events)
• Statistical Methods Detailed descriptions of the
  statistical analyses of all variables in the
  study. A typical clinical trial may include-
• Hypothesis and Decision rules
• Rules for handling missing values
• Interim/Final analysis
• Subjects Disposition/summaries including the
  summary of the reasons of early termination

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Study design of a Clinical Trial

• Statistical Methods (continued)
• Disease Diagnosis/History Usually descriptive
  statistics is enough
• Summary of Medical/Surgical history
• Demographics (age, sex, race, BMI, height,
  weight, etc.) and baseline characteristics
  Usually descriptive statistics are enough but
  these variables are often used as covariates in
  efficacy analysis.
• Efficacy analysis Needs some reasonable
  statistical analysis to justify the research
  goal. Researchers sometimes perform analysis on
  the change from baseline and percent change from
  baseline values instead of the observed values.

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Study design of a Clinical Trial

• Statistical Methods (continued)
• Safety analysis (if subjects receives
  medication) Vital signs (temperature, blood
  pressures, respiration, pulse etc.), ECG/MRI
  results, Laboratory parameters, Physical exams,
  Adverse Events, pregnancy tests etc.)- Usually
  summary of the observed values and change from
  baseline values are provided.
• Prior/concomitant medications Summary of the all
  medications taken during the study or just
  immediate prior to study ( usually not more than
  one month) are provided.
• Quality of life measurements Both summary
  statistics and reasonable statistical analysis
  are required.
• Pharmacokinetic (PK) pharmacodynamic (PD)
  parameters Summary statistics is enough for most
  cases.

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Study design of a Clinical Trial

• Ethics
• Independent Ethics committee (IEC) or
  Institutional Review Board (IRB)
• Ethical conduct of the study Guidelines of Food
  and Drug Administration (FDA) and International
  Conference on Harmonization (ICH) for good
  clinical practices and maintaining the quality of
  research.
• Patient information and consent A document that
  describes the rights and risks of the study
  participants, and includes details about the
  study, such as its purpose, duration, required
  procedures, and key contacts. The participant
  then decides whether or not to sign the document.
  
• Data collection and management
• Storage security
• Protection from data loss
• Checking inconsistency of the data

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Experimental Design for Microarray Experiments

• Suzanne McCahan, Ph.D.
• Molecular Biologist

15
Microarray Research

• Should be Hypothesis Driven
• Test a specific statement
• Ask a specific question
• Involves data mining
• Often generates new hypotheses

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Microarrays can measure

• Gene Expression
• Chromatin Structure
• Methylation of Cytosine
• Histone Binding
• Array Comparative Genomic Hybridization (aCGH)
• Amplification of Chromosomal Regions
• Deletion of Chromosomal Regions

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General Background

• The application and type of array to be used
  determine what should be considered when
  designing microarray experiments.
• A general understanding of microarrays is needed.

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General Characteristics of Microarrays

• Microarrays are small.
• This is an picture of an Affymetrix GeneChip.

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Microarrays are comprised of DNA probes

• Probes are attached to (or synthesized on) a
  surface.
• Oligos - 25 bp
• Oligos 50-70 bp
• Cloned or Amplified DNA
• PCR 500 bp
• BAC (Bacterial Artificial Chromosome) 300kb

Image courtesy of Affymetrix.
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Hybridization

• Strands represent
• A probe on an array
• Labeled DNA or RNA from a sample (This is also
  referred to as the target.)

C T A A G A G C
G A T T C T C G
Image courtesy of Affymetrix.
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Hybridization
Fluorescence where labeled DNA (or RNA)
hybridizes to probe.
No fluorescence where labeled DNA (or RNA) does
NOT hybridize to probe.
Images courtesy of Affymetrix.
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DNA Microarrays

• There are many probes on a single microarray.
• Amount of target is relative to the intensity of
  fluorescent signal.

Image courtesy of Affymetrix.
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Numbers of Probes on Microarrays

• Gene Expression
• Affymentrix Rat GeneChip has 300,00 probes
  representing 15,000 genes
• Chromatin Structure
• Agilent Mouse CpG Island Array has 100,000
  probes
• aCGH (Amplification/Deletion)
• NimbleGen Human X Chromosome Tiling Array
  385,000 probes

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Keep comparisons simple

• Two well defined groups is best, although more
  can be used.
• Normal vs control
• Untreated vs treated (one drug)
• Less complex samples are better than complex
  ones.
• Cell lines are the least complex
• Blood
• RBC should be removed, hemoglobin mRNA and
  protein can interfere
• Mononuclear cells are better than total white
  cells
• Solid tissue
• Tumor only, no contaminating normal tissue
• Muscle only, no contaminating fat

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Decrease Variability

• Samples should be as much the same as possible
• If from patients
• Exact same tissue
• Strict criteria for diagnosis
• Only meds to be studied
• Same pubertal stage
• Handled in a similar manner (immediately on ice)
• Same quality of starting material (RNA or DNA)
• Hybridization, washing and scanning should be
  done by a single person at a single location.

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How many samples should be included in a study?

• Many tests are done on a single sample.
• Each hybridization is expensive. This usually
  limits the number of samples that can be included
  in an experiment.
• With the usual budget, it is not feasible to use
  standard statistical tools to determine the
  number of samples to be included in a study and
  analyze the data.

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How many samples should be included in a study?

• The best way to determine sample size is to do a
  pilot study to obtain data from a particular
  experimental system and do a power analysis
  taking the challenges of microarrays into
  consideration.
• A few publications assess sample size with public
  gene expression microarray data sets. The
  results vary with dataset. One estimation for
  sample size was 10-12 per group.
• When a pilot study is not feasible, a general
  rule of thumb is 5 10 samples per group.

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1-Color Hybridizations

• Common format for gene expression arrays
• RNA or DNA from each sample is hybridized to a
  single array
• If there are two groups (control and treated)
  with 10 samples each, then
• A total of 20 samples will be used
• A total of 20 arrays will be used

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2-Color Hybridizations

• Format for some gene expression, some aCGH, and
  all chromatin structure arrays
• RNA or DNA from two samples simultaneously
  hybridized to a single array
• One sample is experimental
• Other sample is control
• Each of the two samples is labeled with a
  different fluorochrome.
• One array is needed for each pair of samples

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aCGH Arrays Detection of Amplification/Deletion

• Most platforms for aCGH require 2-color
  hybridizations
• Tumor Studies
• Hybridize labeled DNA from tumor and normal
  tissue from a single subject (patient/animal)
  together.
• Genetic Studies
• Hybridize labeled DNA from control subject with
  DNA from diseased subject
• The same control should be used with all diseased
  subjects.
• The control could be DNA from a single individual
  or from a single pool of individuals

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Confirmation of results is necessary

• Since there is such disparity in the number of
  samples examined and the number of tests
  performed (e.g. level of transcripts measured)
  microarray experiments results must be confirmed.

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Methods for confirmation of results

• Additional samples that were not examined on the
  microarray should be used.
• Quantitative PCR methods are often performed.
• Confirmation usually involves a small number of
  genes (or chromosomal regions, depending on the
  type of array that was used).
• The combination of a larger sample size and small
  number of tests allows standard statistical
  methods to be used.

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Example of a microarry experiment

• Modeled after
• Insight into Pathogenesis of
  Antibiotic-Resistant Lyme Arthritis through Gene
  Expression Profiling
• AnneMare Brescia, MD
• Principal Investigator

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Hypothesis

• There are differences in gene expression of
  synovial fibroblasts from individuals with acute
  Lyme synovitis and chronic Lyme synovitis and
  these differences allow for the perpetuation of
  inflammation in chronic Lyme synovitis.

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Biological material Cell lines

• Collect synovial fluid
• Site of disease activity
• Prospectively dont know whether the case is
  acute or chronic Lyme disease at time of
  collection
• Culture cells from fluid
• Primary cells
• Adherent cells are selected
• Consistent phenotype - no monocytes
• Harvest while cells are dividing
• At same passage
• Before reaching confluence

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Synovial fluid from 3 groups

• Control
• Injured joints
• Acute Lyme Disease
• Lyme synovitis resolved within 2 months of
  initiation of antibiotic therapy
• Chronic Lyme Disease
• Lyme synovitis persisted for six or more months
  despite antibiotic therapy

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Experimental Details

• Uniform samples
• Cell line probably representing single cell type
• Affymetrix GeneChip
• Single color hybridization
• Human gene expression arrays 15 chips
• Biological replicates
• 5 samples per group

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Overview of Analysis

• Determine differential expression
• Confirm results with real-time RT-PCR
• Determine functional categories that are
  represented by differentially expressed genes
• Replication?
• Recruitment and/or activation of immune system?
• Unexpected results can generate additional
  hypotheses

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The Future

• New technologies may replace microarrays
• High through-put sequencing is on the horizon
• Less expensive
• Faster
• Short sequences (25 nt 400 nt)
• Presents new computing challenges
• Experimental design will need to be adjusted