LIFESCI 2A03 Final Notes

Module 1 - Introduction to Science, the Scientific Method, and Research Studies

Lecture 1

  • Science = Pursuit and application of knowledge and understanding of the natural + social world following a systematic methodology based on evidence

  • Characteristics of science (REMEMBER):

    1. Based on empirical knowledge

    2. Provides rational/natural explanations

    3. Testable

    4. Repeatable + reproducible + reliable

    5. Involves observation and experimentation

    6. Generality of principles

  • Empirical knowledge = Knowledge based on evidence

    • Evidence: data collection from observation/experimentation

  • Observation - common starting point of scientific investigations

  • Natural explanations = Explains what we observe in the natural world

    • Cannot be explained by “miraculous phenomenon”

  • Testable = Scientific explanations must generate specific expectations + can be tested through observation/experimentation

    • Can generate a hypothesis and a prediction

    • “If (hypothesis), then (prediction)”

  • Repeatable + reproducible + reliable = Scientific concepts can be confirmed by repeated/reproduced tests

    • More reproduction/repeats = More reliable findings

  • Experiment = Controlled setting in which the researcher manipulates/intervenes a natural process to observe its effects

  • Observation = When a researcher observes the focal individual/population without manipulating any processes/behaviours

    • In a natural setting

  • Generality of principles = Scientific explanations can be applied universally to explain common patterns

  • Research = Systematic, planned, multi-step process that uses discoveries to advance knowledge

  • Scientific research = Research with the purpose of scientific contribution

  • Importance of scientific knowledge:

    • Identifying new phenomena, materials, organisms

    • Solves scientific problems

    • Develops scientific inventions

    • Provides basis for government and industry policies

  • Research process is an interplay of inductive and deductive reasoning

  • Inductive reasoning = Process of using specific observations to make generalizations

    • Start with specific observation

    • Goal is to find general theory

  • Deductive reasoning = Process of determining a generalization to explain specific observations

    • Start with general theory

    • Goal is to observe and explain specific observation

  • Steps of the scientific method (REMEMBER):

    1. Determine the problem/question

    2. Formulate a hypothesis

    3. Formulate predictions based on hypothesis

    4. Test predictions

    5. Analyze data

    6. Ask “does the data support the hypothesis”

    7. Draw conclusions

    8. Communicate results

  • Scientific method is an iterative process, the steps can be repeated multiple times if needed

Lecture 2

  • Research hypothesis = Tentative explanation to a research question

  • Characteristics of a research hypothesis (REMEMBER):

    • Stated before experimental/observational studies

    • Based on prior knowledge

    • Testable

    • Can be falsified but NOT proven

    • Leads to predictions

    • Implies a relationships between independent (predictor) and dependent (outcome) variables

  • Types of relationships between variables (REMEMBER):

    • Correlation = Association between 2 variables, they change together in a predictable way

    • Causation = One event is the result of the occurrence of another event

  • Patterns correlations (REMEMBER):

    • Positive/direct linear = When x increases, y increase

    • Negative/inverse linear = When x increases, y decreases or vice versa

    • Non-linear relationship = Can be visualized as a non linear function (e.g. parabola)

    • No relationship

  • Directional vs. directional RH:

    • Directional = The hypothesis states either a positive or negative direction of the association

    • Directional hypotheses can ONLY be made if there is evidence of a directional relationship

    • If the RH is directional, the predictions must be directional

    • Directional RH uses a one tailed t-test

    • Nondirectional RH uses a two tailed t-test

  • Steps on testing a hypothesis (REMEMBER):

    1. Generate predictions using DEDUCTIVE reasoning

    2. Collect data

    3. Statistically analyze and interpret data (includes finding significance level)

    4. Generate a conclusion - either you fail or REJECT TO FAIL hypothesis

  • Null and alternate hypotheses = More specific hypotheses generated from the RH that can be tested

  • Null hypothesis (H0) = Implies no relationship between variables

  • Alternate/alternative hypothesis (Ha) = Implies relationship between variables

    • Corresponds with research hypothesis

  • 2 types of statistical analysis:

    • Hypothesis tests

    • Estimation statistics

  • Hypothesis tests:

    1. Researcher sets their a (significance level = likelihood of type 1 error) value

    2. Researcher chooses the statistical test to run (i.e.. t-test, chi squared test)

    3. Test statistic and p value are calculated

  • Estimation statistics:

    • More common in clinical studies

    1. Researcher sets their a value

    2. Researcher calculates parameter estimates (i.e. mean differences, risk ratio, correlation coefficient) - which quantifies the strength of associations

  • Types of statistical errors (REMEMBER):

    • Type 1 (alpha) error = Rejecting a true H0

    • Type 2 (beta) error = Failing to reject a false H0

  • Statistical power (a value) = 1 - beta value

    • Quantifies the ability to identify a real relationship when it exists

  • When to reject or fail to reject a hypothesis:

    • Case 1: if P < a

      • The confidence interval does not include the H0 value

      • Reject H0

      • Fail to reject Ha

      • Biological conclusion = There is significant evidence to support the hypothesized association

    • Case 2: if P > a

      • The confidence interval does include the H0 value

      • Fail to reject H0

      • Reject Ha

      • Biological conclusion = There is not enough evidence to support the hypothesized association

Lecture 3

  • Primary research = Researchers conduct their own studies

    • Includes collection and analysis of raw data

    • Types: observational research, experimental research

  • Secondary research = Analysis of studies that have already been performed

    • Types: narrative/traditional literature reviews, systematic reviews, meta-analyses

  • Primary and secondary research can be either:

    • Basic = Investigates basic principles and reasons for a particular event/process/phenomenon

    • Applied = Attempts to solve practical problems of interest to humans

  • Biological systems used in research:

    • Pre-clinical = non-human subjects

      • In vitro = Isolated cells in a dish/tube

      • Ex vivo = Isolated tissue/organ

      • In vivo = Model organism

    • Clinical = human subjects

  • Research questions can be either:

    • Descriptive (who, what, when, where)

    • Analytic (why, how)

  • Data collection can be either:

    • Longitudinal = Subjects are followed over time and data is collected at set intervals/events

      • Prospective = Data is collected over time as characteristics/circumstances change

      • Retrospective = Information is collected from an individual’s past (e.g. medical records, criminal history)

    • Cross-sectional = Data is collected at one point in time

Lecture 4

  • Sources of bias:

    • Limitations in study design

    • Technical limitations

    • Limited abilities of scientists to discern differences

    • Wishful thinking

  • When can bias occur:

    • In the design stage

    • During data collection/analysis

    • During publication

    • Types of biases (REMEMBER):

      • In the design stage

        • Sampling/recruitment/selection bias = Unrepresentative sampling of subjects

        • Allocation Bias = Unequal allocation of subjects into groups

      • During data collection/analysis

        • Performance bias = Differences in care provided to groups other than the manipulated variable

        • Recall/attrition bias = Incomplete/inaccurate/lost data

        • Assessment/measurement/detection bias = Errors in assessing/recording/analyzing data + data manipulation

      • During publication

        • Reporting bias = Selective reporting of data

        • Citation bias = Only citing studies that support own findings

        • Publication bias = Preference of journals to publish studies with significant results/studies that cite the journal

  • Example of limitations in science - Painter’s Human Chromosome Count

    • Painter reported 48 chromosomes in a human cell

      • This was accepted for 3 decades

    • Issues:

      • Sampling error = Low sample size

        • Source of tissue (testicular) had a low # of spermatogonia

      • Measurement error = Damaged chromosomes since tissue was not fixed

        • Chromosomes were clumped/curled

        • DNA was not adequately condensed to visualize

      • From researchers that reproduced the study:

        • Sampling bias

        • Assessment/detection bias

        • Skeptical to challenge previous count

    • Improvements:

      • Fixation of tissue

      • Hypotonic solution to prevent chromosome clumping

      • Use of colchicine for further DNA condensation

  • How researchers can minimize bias (REMEMBER):

    • Be aware of all potential sources of bias

    • Include:

      • Control groups

      • Matching

      • Randomization

      • Blinding

    • Declare study limitations

  • How editors and reviewers can minimize bias (REMMEBER):

    • Create and enforce guidelines for publication

    • Detect bias during review process

    • Avoid publication bias

Lecture 5

  • Study design for hypothesis driven studies require:

    • Variables of interest

    • Methods of sampling + assigning subjects

    • Determining the sample size

    • Determining how data is collected and analyzed

  • Types of errors in study design:

    • Random = Due to chance/natural variation in population

    • Systematic = Bias

    • Statistical = Type 1 and 2 errors

  • Confounding variables = Variables not of interest to the experimenter

  • How to control confounders:

    • Including control groups

    • Matching groups for known confounding factors

    • Subject randomization

    • Large sample sizes

    • Inclusion of confounder in statistical analysis

  • Types of control groups:

    • Negative control = When there is no effect expected, they produce no effect

      • Sham = Given the procedure without the actual treatment (surgery)

      • Vehicle = Administering the vehicle for the treatment without the treatment itself (saline, buffer)

      • Placebo = Administering inactive substance (sugar pill)

      • No treatment

    • Positive control = When there is treatment with a known expected effect, they produce the expected effect when they are given the treatment

      • Shows that the experimental setup works

  • Randomization can be applied at sampling and/or allocation to groups

  • Types of randomization:

    • Simple

    • Stratified

    • Block

  • Randomization can’t always, like in observational studies

  • Sample sizes must be large enough to represent the population

  • Sample size = # of biological replicates

  • However, we can’t always use the largest samples possible as it can be too expensive and time consuming

  • Blinding minimizes performance bias and detection/assessment bias

  • Types of blinding:

    • Single blind

    • Double blind

    • Full blind

    • Partial blind

  • How to study causation:

    • Control unknown and known confounders

    • Use interventional studies (longitudinal) that manipulate the predictor

    • Include dose-response analysis in design

  • Drawbacks of experimental studies:

    • Sometimes difficult to apply/generalize results

    • Some variable cannot be manipulated

    • Ethical considerations

Module 2 - Introduction to Stem Cells (SCs)

Lecture 1

  • Stem cells (SCs) = Cells capable of self-renewal and differentiation

  • Characteristics of SCs (REMEMBER):

    • Spherical shape

    • Self renewal through mitosis

    • Unspecialized cells that later differentiate

  • Types of SC divisions (REMEMBER):

    • Symmetric self-renewal = One SC divides into 2 SCs

    • Asymmetric division = One SC divides into 1 SC and 1 progenitor (which becomes a differentiated cell)

    • Symmetric differentiation (AKA symmetric division) = 1 SC becomes 2 progenitors (which become 2 differentiated cells)

  • Stem cells have either unlimited or limited proliferation potential

  • Hayflick limit = Term that applies to the limited proliferation of cells in culture

    • Depends on

      • Species

      • Tissue type

      • Age of donor

  • Limited proliferation potential is due to telomeres

    • Shorten over time

    • Telomerase becomes less active

  • Types of differentiation potential (REMEMBER):

    • Totipotent = Can become any cell type + extraembryonic tissue

    • Pluripotent = Can become any cell type

    • Multipotent = Can become a few cell types

    • Unipotent = Can only become one cell type

  • Factors regulating SC function (quiescent or actively cycling)(REMEMBER):

    • Extrinsic = The environment where the SC is

    • Intrinsic = Proteins and miRNAs in the cell

  • SC niche = Cellular + non cellular components

Lecture 2

  • Types of SCs:

    • Embryonic stem cells (ESCs)

    • Adult tissue stem cells (ASCs)

    • Induced pluripotent stem cells (iPSCs)

  • ESCs = From the inner cell mass (ICM) of plastocyst

    • Unlimited proliferation

    • Pluripotent

    • Can form tumors

  • ASCs = From adult tissue

    • Somatic ASCs = Gives rise to somatic cells

      • Hayflick limit

      • Can be multi or unipotent

      • Replaces dead/damaged cells

      • Can be dormant for a long time

      • Hard to isolate and grow in lab

    • Germline = Gives rise to germline cells

  • Processes of ASCs in order:

    1. Symmetric self renewal

    2. Asymmetric self renewal

    3. Progenitor division

    4. Differentiation of progenitors

  • Intestinal epithelium = Monolayer of cells in intestine

    • Replaced every few days

  • SCs are found in intestinal epithelium and differentiate into

    • Absorptive cells

    • Goblet cells

    • Paneth cells

    • Enteroendocrine cells

  • iPSCs = Cells that are isolated from a patient and treated with “reprogramming factors”

    • Unlimited self-renewal

    • Pluripotent

  • Clinical applications of iPSPs (REMEMBER):

    • In vitro, clinical studies for human disease

    • Drug screening/toxicity test

    • Cell regenerative therapy

Module 3 - Non-clinical SC Research

Lecture 1

  • Non-clinical research = Research where the subjects of study are NOT humans

  • Types of non-clinical research:

    • Primary and secondary

    • Basic and applied

    • Observational and experimental

  • Model organisms = Non human species used by researchers to study conserved phenomena (processes that can only be observed in vivo)

  • Planaria = Small flatworms

  • Benefits of planaria as model organisms:

    • Easy to rear

    • Inexpensive

    • Similar molecular and cellular processes as higher organisms

    • Easy to manipulate

    • High regenerative ability throughout lifespan

    • “immortal under the edge of the knife”

  • Planarian SCs = neoblasts

    • Pluripotent

    • 20% of all cells

    • Involved in tissue homeostasis and regeneration

    • Only mitotically active cell population

  • BrdU (bromodeoxyuridine) = analogue of thymidine used to label neoblasts in planaria

  • Neoblasts cultured in different cell medium conditions (extrinsic factors) can give rise to all known types of differentiated cells in planaria

  • Single neoblasts grafted into irradiated planaria can restore the subject’s regeneration capacity

    • Process is formation of blastema (mass of cells)

Lecture 2

  • Research study #1 - Reddien et al

    • RQ = Are Smedwi genes required for neoblast function? If so, in which process (homeostasis/regeneration) and step (proliferation, migration, differentiation)?

    • Prior knowledge:

      • PIWI genes regulate SC function

      • 2 types of PIWI like genes in planaria - smwedi 1 and smwedi 2

  • Smwedi gene expression:

    • RH = Smwedi genes are expressed in neoblasts

    • Prediction = Patterns of smwedi 1 and 2 expression should resemble neoblast distribution

    • Ha = There is a relationship between the location of neoblasts and the location of smwedi gene expression

    • H0 = There is no relationship between the location of neoblasts and smwedi gene expression

    • Statistical conclusion = H0 is rejected, failed to reject Ha

    • Biological conclusion = Smwedi genes are expressed in neoblasts

  • Smwedi genes role in regeneration:

    • RH = Smwedi-2 is crucial for the proliferation of neoblasts during regeneration

    • Prediction = Reducing smwed-2 expression should block neoblast proliferation during regeneration

    • Ha = Smwedi-2 has a positive effect on the proliferation of neoblasts during regeneration

    • H0 = There is no effect of smwedi-2 expression on the proliferation of neoblasts

    • Statistical conclusion = Ha rejected, failed to reject H0

    • Biological conclusion = Smwedi-2 expression does not play a role in neoblast proliferation during regeneration

Lecture 3

  • Niche = Local tissue microenvironments that maintain and regulate SCs

    • Source of extrinsic stimuli

  • Example: Intestinal SC niche (Gut microbiota community)

    • Microorganisms in the intestinal tract

    • Association between an imbalance of gut microbiota community and GI related disorders

    • Extrinsic stimuli = Secreted/membrane bound bacterial components + by products and metabolites

  • Research study #2: Naito et al.

    • RQ: Does gut microbiota regulate intestinal epithelium homeostasis in the colon? If so, how?

  • Example: Hair follicles

    • Hair follicles - epithelial mini-organs that sustain cyclic hair growth

    • New bulge in follicle —> asymmetrical division of SC

    • Activity of hair follicles are time dependent

      • Leading to hair thinning and loss

  • Research study #3: Matsumura et al.

    • RQ: What causes hair follicle aging and age-dependent hair loss?

Lecture 4

Research Study #2 - Naito et al.

  • Prior knowledge:

    • Gut microbiota community called CSCM (niche) is found close to intestinal epithelia

    • Mice deficient in TLRs (receptors that mediate immune responses) and LPS (lipopolysaccharides) show increased cell proliferation in these niches

  • RH = CSCM regulates intestinal epithelial homeostasis

  • Prediction = Mice colonized with germs will show different #s of proliferative cells compared to germ free (GF) mice

  • Methods:

    • Ex vivo - analyzed organoids from mice after they were/weren’t mono-colonized

  • Ha = CSCM has an effect on intestinal epithelial homeostasis

  • H0 = CSCM has no effect on intestinal epithelial homeostasis

  • Statistical conclusion = Rejected H0, failed to reject Ha

  • Biological conclusion = CSCM regulates intestinal epithelial homeostasis by regulating proliferation through LPS

Lecture 5

Research study #3 - Matsumura et al.

  • Prior knwoledge:

    • Increase in hair thinning and loss with age

    • Patients with COL17A1 deficiency show alopecia

    • Miniaturization of HFs = Underlying cause of male pattern baldness (androgenic alopecia)

  • RH = Age-dependent hair loss is driven by HF miniaturization as a loss of collagen XVII and SC depletion

  • RH1 = Age dependent hair loss has a positive correlation with miniaturization of HFs

  • Ha = Age dependent hair loss is positively associated with miniaturization of HFs

  • H0 = There is no relationship between HF miniaturization and hair loss

  • RH2 = Aging related hair loss and HF miniaturization is linked to loss of collagen XVII and HFSCs

  • Prediction = HFs of old mice should have lower levels of collagen XVII and of S100A6 (SC marker)

  • H0 = Aging related hair loss and HF miniaturization are not related to loss of collagen XVII and HFSCs

  • RH3 = Age dependent loss of COL17A1 triggers HF miniaturization and hair loss

  • Prediction = Young mice with mutant COL17A1 + knockout gene for expression of bulge will have more miniaturized HFs compared to control mice

  • Ha = Age dependent loss of COL17A1 will have a positive effect on HF miniaturization and hair loss

  • H0 = No relationship between the 2 variables

Tutorial 5

1. Why is the null hypothesis a valuable tool in experimental design? What is the null hypothesis that will be tested in this experiment?

  • Value of the Null Hypothesis:
    The null hypothesis (H₀) is essential in experimental design because it provides a baseline for comparison, allowing researchers to measure the strength of evidence against it. By assuming that no real effect or difference exists, the null hypothesis helps determine if the observed results are due to chance.

  • Null Hypothesis in Fisher’s Experiment:
    In the "Lady Tasting Tea" experiment, the null hypothesis states that the lady cannot distinguish between tea with milk added first and tea with milk added second.
    Mathematically:

H0:The lady’s guesses are purely random.

  • Comparison with Initial Assumption:
    If your original answer was that the null hypothesis suggested the lady could distinguish the order of milk and tea, then it differs from Fisher’s null hypothesis, which assumes no ability to differentiate. Fisher’s null hypothesis forms the foundation for calculating the probability of her success by random guessing.

2. Why is it important to offer the model more than just two cups? Was your answer the same as Fisher’s answer? Describe Fisher’s reasoning.

  • Importance of More Than Two Cups:
    Offering only two cups (one milk-first and one tea-first) is inadequate because it creates a 50% chance of guessing correctly, making it impossible to distinguish genuine skill from random guessing.

  • Fisher’s Reasoning:
    Fisher advocated for offering 8 cups (4 with milk-first, 4 with tea-first) because it reduces the likelihood of success by chance alone. With 8 cups, the probability of guessing all correctly by chance is:

1/70 = 0.014

This design ensures that success is unlikely unless the subject genuinely possesses the ability to discriminate.

3. How many cups did you say the lady should taste? How many cups did Fisher say she should taste? Describe Fisher’s answer, including mathematical considerations.

  • Your Initial Estimate vs. Fisher’s Suggestion:
    If you initially suggested fewer than 8 cups, your design would not have been as statistically rigorous as Fisher’s. Fisher specified 8 cups because:

    • 4 with milk-first and 4 with tea-first.

    • This number balances a manageable experimental size while minimizing the probability of guessing correctly by chance.

  • Mathematical Considerations:
    Fisher calculated the probability of perfect success by chance using the hypergeometric distribution. With 8 cups, the chance of randomly identifying all correctly is 1/70 =

This design provides a stringent test for detecting real ability.

  • Factors Impacting Significance and Constraints:

    • Sample size: Small sample sizes reduce power and precision.

    • Bias and systematic errors: Can lead to misleading results.

    • Variability in responses: Uncontrollable variability can affect outcomes.

4. How should the cups be prepared? Does every cup need to be identical in every way except for the order of milk and tea? What does Fisher suggest about variation?

  • Cup Preparation:
    Ideally, the cups should be identical except for the order of adding milk and tea to eliminate confounding variables.

  • Can Cups Be Identical?
    In practice, it is nearly impossible to make every cup truly identical. Small variations in temperature, mixing, or cup quality can occur.

  • Fisher’s View on Uncontrollable Variation:
    Fisher acknowledged that slight, uncontrollable variations are inevitable. However, randomization minimizes the impact of these variables, ensuring that differences between cups are equally likely to affect all experimental conditions.

5. Why is randomization important in experimental design? In what order should the cups be presented? How did Fisher recommend choosing the order?

  • Importance of Randomization:
    Randomization prevents systematic bias by ensuring that any uncontrolled variables are equally distributed across experimental conditions.

  • Order of Presentation:
    Fisher recommended randomizing the order of presentation of the cups to ensure that the sequence of tasting does not inadvertently influence the results. Without randomization, subconscious cues or fatigue could bias the experiment.

  • Fisher’s Method for Choosing Order:
    Fisher’s approach involved randomly assigning the order of the cups, using principles of combinatorial probability to calculate the likelihood of success by chance.

6. Would it be acceptable to add two more cups to the original 8 cups? Why or why not? Why is it important not to change the design mid-experiment?

  • Adding More Cups:
    No, it is not acceptable to add two more cups after establishing the design. Changing the experimental setup midway introduces bias and undermines the validity of the results.

  • Value of Predefined Experimental Design:
    Predefining the design ensures:

    • Consistency and reproducibility.

    • Maintenance of the original probability calculations.

    • Protection against cherry-picking favorable outcomes.

7. Can you conclude from the lady’s success that most people can tell the difference between milk-first and tea-first? Design an experiment to test this broader question.

  • Conclusion from Her Success:
    No, success in one individual does not imply that most people possess the same ability. Fisher’s test evaluated the skill of one individual, not the general population.

  • Designing a Broader Experiment:
    To test this question more broadly:

    • Sample Size: Recruit a large and diverse sample of participants.

    • Experimental Setup:

      • Randomly assign 8 cups (4 milk-first, 4 tea-first) to each participant.

      • Randomize the order of cup presentation.

    • Control Group: Include a control group with individuals who self-report no ability to differentiate.

    • Data Collection: Measure the number of correct guesses per participant.

    • Statistical Analysis: Use a chi-square test or binomial test to determine if the overall proportion of correct responses differs significantly from chance.

Module 4 - Clinical SC Research

Lecture 1 - Overview of Clinical Studies

Learning Outcomes

  • Identify different types of clinical research studies and their characteristics

  • Explain advantages and disadvantages of each type

  • Explain the application and purpose of blinding and randomization in clinical research

Pre-Clinical vs. Clinical Research

  • Pre-clinical = Involves isolated cells, tissues, or organs

    • Less complex

    • Fewer ethical issues

  • Clinical = Involves human subjects

    • More complex

    • More ethical considerations

Why Conduct Clinical Studies?

  • To gain medical knowledge for:

    • prevention of disease development or recurrence

    • diagnosis

    • treatment

    • palliative/support care symptom management

Types of Clinical Studies

  • Divided into 2 main categories:

    1. Observational = Researchers observe and analyze what happens to people in different situations based on exposure

      • No interventions

    2. Experimental (interventional) = Researchers manipulate/assign a specific treatment/factor to subjects

Study Type

Design

Group Assignment

Measures/Reports

Key Use

Cohort

Longitudinal

By exposure

Risk Ratio (RR)

Measure incidence of disease

Case-Control

Longitudinal

By outcome (disease status)

Odds Ratio (OR)

Study rare diseases

Cross-Sectional

Single time pt.

By exposure & outcome (same time)

Risk Ratio (RR, from prevalence)

Measure prevalence of disease

Clinical Trials (CTs)

Prospective

By intervention (assigned)

Risk Ratio (RR)

Test safety/efficacy of interventions; strongest evidence of causality

RR and OR

  • Risk ratio (RR) and odds ratio (OR) are measures of association used to quantify the relationship between an exposure/intervention, and an outcome

RR

  • RR (AKA relative risk) = Compares the risk of an event in one group to the risk of the same event in another group

  • Interpretation:

    • RR = 1 - There is no association, as the risk of the event is the same in both groups

    • RR >1 - There is a positive association, as the risk of the event is higher in the exposed/intervention group compared to the control

    • RR <1 - There is a negative association (AKA protective effect), as the risk of the event is lower in the exposed/intervention group compared to the control

  • Calculation:


  • Example:

    Imagine a clinical trial testing a new drug to prevent heart attacks. After 5 years, 5% of the participants taking the drug had a heart attack, while 10% of the participants taking a placebo had a heart attack.

    • Risk in the drug group = 5% = 0.05

    • Risk in the placebo group = 10% = 0.10

    The Risk Ratio is:

    RR=0.05/0.10 = 0.5

    This means that the risk of having a heart attack in the drug group is 0.5 times (or 50%) of the risk in the placebo group. In other words, the drug reduces the risk of heart attack by 50%.

OR

  • OR = Compares the odds of the exposure in the group with the disease to the odds of exposure in the group without the disease

  • Interpretation:

    • OR = 1 - No association, as the odds of exposure are the same in both groups

    • OR >1 - Positive association, as the odds of exposure are higher in the cases compared to controls

    • OR < 1 - Negative association (AKA protective effect), as the odds of exposure are lower in the cases compared to the controls

  • Calculation


  • Example: In a case-control study looking at the association between smoking and lung cancer, researchers found that 80% of lung cancer patients (cases) were smokers, while 20% of a control group without lung cancer were smokers.


    First, calculate the odds of exposure (smoking) in each group:

    Odds of smoking in cases = 0.8/(1-0.8) = 4

    Odds of smoking in controls = 0.2/(1-0.2) = 0.25


    Then, calculate the Odds Ratio:

    OR=40.25=16

    This means that the odds of smoking are 16 times higher in people with lung cancer compared to people without lung cancer.

Observational Studies

Cohort Studies

  • Cohort study = A study that follows groups of people over time

    • Prospective or retrospective

  • To see how certain exposures affects the likelihood of developing a disease/outcome

  • Participants are grouped based on exposure to a risk factor

  • Main advantage - Can measure the incidence (new cases) of disease, good for studying common outcomes

  • Primary statistical measure: Risk ratio (AKA RR)

Case Control Studies

  • Case control study = Researchers group subjects based on the outcome (whether they have a particular disease or not), then they look in time to determine exposures or risk factors they might have had

    • Retrospective

  • Main advantage - Useful for studying rare diseases because researchers already have subjects with the disease (rather than wasting their time with a cohort study to see if any participants get the disease)

  • Main disadvantage - Relies on participants’ memory and/or past records

    • Subject to bias

  • Main statistical measure: Odds ratio (AKA ORR) = Odds of exposure in cases vs. controls

Cross-Sectional Studies

  • Cross-sectional study = Researchers collect data from a population @ a single point in time

  • “Snapshot” of the population = Both the exposure and the outcome are measured simultaneously

  • Main advantage - Useful for determining the prevalence of a disease/condition in a population

  • Main disadvantage - Cannot study causation or changes over time

Experimental/Interventional Studies

Clinical Experimental Studies

  • AKA clinical trials (CT)

  • Involves assigning interventions/treatments to participants

  • Main advantage - Strongest evidence for cause and effect relationships

  • Main statistical measure: RR

  • Types of CTs:

    1. Randomized control trials (RCTs) = Participants are randomly assigned to groups to minimize bias

    2. Non-randomized trials = Assignment is not random, which can introduce bias

    3. Community/cluster trials = Groups (e.g. schools, towns) are assigned to interventions, rather than individuals

    4. Field trials = Preventative interventions are tested in healthy populations

Key Terminology

  • Exposure (AKA predictor/risk factor) = Independent variable that is naturally determined, it is not something that can be manipulated by the researcher

    • e.g. Age, sex, smoking

  • Intervention (AKA treatment) = Independent variable assigned and manipulated by the researcher

    • e.g. drug administration, medical procedure

Special Study Designs

  • Pilot and feasibility studies = Small-scale versions of larger studies

    • Used to test whether a full-scale study is practical

    • Also used to refine study procedures

  • Multi-Arm Multi-Stage trials (AKA MAMS) = Trials that test multiple treatments @ once, allowing researchers to add/drop treatment groups as results become available

  • Nested case-control and case-cohort studies = Variations that combine features of cohort and case-control studies

    • Often used to improve efficiency or reduce bias

Lecture 2 - Function and Cancer

Learning Outcomes

  • Understand the characteristics of heterogeneous tumore

  • Explain what cancer stem sells (CSCs) are and their origin

  • Identify potential tissue/organ sources of CSCs that drive tumor relapse

What is Cancer?

  • Involves the formation and growth of abnormal cell populations

    • Interferes with normal vital functions

  • Cause - Uncontrolled cell proliferation

Tumor Heterogeneity

  • Tumor heterogeneity = The structural and functional diversity observed within many tumors

  • Question - Is cancer driven by a specific population of cancer cells?

    • Is a subset of cells within the tumor primarily responsible for driving the tumor’s growth and spread?

Cancer, SC Number, and Function

  • Cancer SCs = Cancer cells with high self-renewal capacity

    • Thought to drive tumor growth

  • Characteristics of cancer stem cells include:

    • their ability to self-renew,

    • differentiate into various cell types, and

    • resist conventional therapies

SC Division Theory of Cancer

  • CSC division is the driving force of carcinogenesis (initiation of cancer formation)

  • Carcinogens → accumulation of cell divisions in SCs ←→ accumulation of DNA alterations → cancer

Hierarchal Model of Tumor Growth

  • There is a structured organization of cells within tumors

    • Each cell type has a distinct role

  • Hierarchal model of tumor growth:

    1. Cancer stem cells (CSCs) = High self-renewal capacity which sustains the tumor

    2. Transit amplifying (TA) cells = They divide faster than CSCs but have a finite # of divisions before they stop

    3. Non-proliferative cancer cells = Cells that do not actively proliferate

Example of Cancer - Glioblastoma Multiforme (GBM)

  • GBM = Type of brain tumor originating from astrocytes

  • The most common and aggressive form of malignant brain tumors

    • Rapid growth

    • Treatment resistant

  • Mainly observed in adults from 45-75 years old

  • Poor prognosis = typically given 1 year to live after diagnosis

  • Treatment (often not curative):

    • Surgery

    • Radiation

    • Chemo using temozolomide

Neural SC Niches and GBM

  • Neural SC niches (AKA NSC niches) = Microenvironment where neural stem calls reside

    • Subventricular zone (SVZ) of lateral ventricles

    • Sub granular zone (SGZ) of dentate gyrus within the hippocampus

  • Issues within the NSC niches can give rise to brain tumor propagating cells (AKA BTPCs), which can initiate and help sustain tumor growth

Lecture 3 - Research Study 1

Learning Outcomes

  • Identify key features, advantages, and limitations of this type of observational studies

  • Explain what progression-free survival is and how it can be studied

  • Explain what prognostic factors are and how their effect can be studied

  • Identify variables, formulate RH, predictions, and conclusions

Research Study #1

  • RQ - Are brain SC niches a reservoir for glioma CSCs driving relapse? If so, can irradiation of the SC niches improve patient survival?

  • RH - Irradiation of NSC niche decreases GBM recurrence and improves patient prognosis

  • Prediction 1 (P1) - Patients that receive ‘high’ dose PV (SVZ) irradiation should survive longer than those treated with ‘low’ doses

  • P2 - Patients that receive ‘high’ dose PV (SVZ) irradiation should have a lower risk of progression than those that received ‘low’ doses

Previous Observations + Findings

  • GBMs are hierarchally organized - They contain distinct cell populations with varying self-renewal capacities

  • GBMs often recur even after chemo + radiotherapy

  • NSCs can undergo malignant transformation which leads to tumor development

    • This was observed in mice models

Study Design

  • Population was composed of individuals with a grade 3 or grade 4 tumor

  • N = 55

  • Divided the population based on radiation dose to the PV zone (AKA PV volume):

    1. High bilaterial PV dose RT (>43 Gy) = Patients that received a radiation dose higher than 43 gray

    2. Low bilateral PV dose RT (<43 Gy) = Patients who received a radiation dose lower than 43 gray

  • Further divided the PV dose groups into progression or no progression after bilateral PV radiation

    • Progression = Relapse/recurrence of the tumor after initial treatment

  • Analyzed:

    • Ipsilateral hippocampal formation (HF)

    • Ipsilateral PV

    • Total radiation dose received by the tumor

Results

  • Median PFS for the high PV dose group = 15 months

  • Median PFS for the low PV dose group = 7.2 months (p < 0.03)

    • Statistically significant difference (p < 0.03) between the two groups

Analyzing the Effect of Prognostic Factors (AKA Hazard Ratio)

  • Prognostic factors = Characteristics of a disease/patient that can be used to predict the likely outcome of the disease

  • Prognostic factors for the risk of tumor progression:

    • Location of the tumor

    • Extent of surgical intervention

    • Total tumor does - amt of radiation delivered to the tumor

  • Hazard ratio = Measures how a specific prognostic factor affects the rate @ which an event occurs over time

  • HR interpretation:

    • < 1: Lower risk of event (progression) in high dose group, indicating a protective effect of high-dose irradiation.

    • = 1: No difference between groups, suggesting that the factor has no impact on the risk of progression.

    • > 1: Higher risk of event (progression) in high dose group, indicating that high-dose irradiation may be associated with increased risk of progression

Study Limitations

  • Was a retrospective analysis

    • They need prospective studies to confirm the correlation

  • Overall survival was not assessed

    • They focused only on progression-free survival

    • Individuals who did survive and had progression were not included in the findings

  • Researchers assumed that treatment and other factors were consistent during the follow-up period

  • Did not evaluate the potential long-term effects of radiation on NSC niches

    • Memory impairment, impaired tissue regeneration

  • Lacked MGMT (O6-methylguanine DNA-methyltransferase) promoter methylation data for most patients

    • This is an important predictor of response to temozolomide chemotherapy

MGMT and Temozolomide (TMZ)

  • TMZ is an alkylating agent that methylates DNA, leading to cell death.

  • MGMT = Enzyme that removes methyl groups from DNA, repairing the damage caused by TMZ

  • High levels of MGMT expression can make cancer cells resistant to TMZ by repairing the DNA damage it causes

Summary of Research Study #1

  • Prescribed high dose (>43 Gy) to the periventricular region may improve GBM patient outcome

  • RCTs (randomized controlled trials) are needed to confirm the benefits of high-dose periventricular irradiation and to assess its potential side effects

    • Impact on OS: RCTs should evaluate the impact of high-dose periventricular irradiation on overall survival (OS)

    • Impact on adult neurogenesis, memory, and tissue repair: RCTs should also assess the potential effects of high-dose periventricular irradiation on adult neurogenesis, memory, and tissue repair

Lecture 4 - Research Study #2

Learning outcomes

  • Identify the key features, advantages, and limitations of this type of study (historical control study)

  • Explain what a historical control is and how it differs from a concurrent control

  • Identify variables and formulate RH, predictions, and conclusions

Research Study #2

  • RQ - Can NPC-sparing strategy be efficient @ controlling tumor relapse and survival while mitigating treatment-induces neurocognitive decline

  • Previous observations/findings:

    • Higher radiation doses → improved survival, BUT ALSO negative effects on cognitive function

    • Lower radiation doses → Reduced negative cognitive effects, but less effective against the tumor

  • RH - Low (sparing) irradiation to NPC niches does not impair tumor contro lor patient survival

  • P1 - Patients treated with NPC sparing RT and those treated with standard RT will have similar overall survival rates

  • P2 - Patients treated with NPC sparing RT will have similar progression free survival compared to those with standard RT

Neural Progenitor Cells (NPCs)

  • Neural progenitor cells (NPCs, AKA neural stem cells/NSCs) = Stem cells that reside in the SC niches of the brain

    • Helps maintain homeostasis

    • May be the source of cancer stem cells that drive tumor relapse after treatment

Study Design

  • Population - Patients with GBM receiving NPC niche sparing radiation therapy

    • Sample size, n = 30 (only 17 completed neurocognitive assessment)

  • NPC niche sparing RT = Radiation that is delivered in a way that avoids as much of the NSC niches as possible

    • Depends on tumor location and geometry

  • Outcomes measured:

    • Progression free survival (PFS) = Time from treatment until the tumor worsens/returns

    • Overall survival (OS) = Time from treatment until death or up to 24 months after RT

    • Neurocognition = Cognitive function after treatment, with a focus on verbal memory

  • Historical controls were used - patients in the past who received standard (non NPC sparing) RT

    • Historical control = A group of subjects from previous studies used for comparison instead of a concurrent control group

      • Concurrent control = Control group enrolled and treated @ the same time as the experimental group

  • Variables:

    • IV = Type of radiation therapy (NPC-sparing RT vs. standard RT)

    • DVs:

      • Overall survival (OS)

      • Progression free survival (PFS)

      • Neurocognitive outcomes

Results

  • OS and PFS after NPC-sparing RT were similar to the historical controls

  • Patients who received NPC niche-sparing RT showed better verbal memory performance than the historical controls

Study Limitations

  • Larger and more comprehensive studies are needed (only 17 participants did a neurocognitive assessment)

  • Using historical controls may introduce bias if patient populations or treatments differ over time

  • MGMT methylation was not uniformly measured

  • Does not address whether higher radiation doses may improve survival compared to standard RT

    • Only addressed if the survival rates were similar


Lecture 5 - Intro to CTs

Learning Outcomes

  • Identify key features of clinical trials (CTs) and differentiate them from observational studies

  • Recognize different CT designs and phases

  • Explain the advantages and limitations of different CT designs

Clinical Trials - Review

  • Participants receive interventions based on the researcher’s protocol

  • Designed to assess the safety and therapeutic effect of interventions

    • Therapeutic effects being assessed:

      • Efficacy - Performance of the intervention under ideal and controlled circumstances

      • Effectiveness - Performance of an intervention under real world settings

  • Prospective + analytical (evaluates the relationship between an intervention and an outcome)

Types of Controls in CTs

  • Standard treatment (active) = The current standard of care

    • The intervention is compared to this standard of care

  • Placebo = An inactive substance or treatment

  • Dose-response = Comparing different dosages of the same intervention

    • the lower/higher dose is used as a control for the new dose researchers are implementing

  • Historical = Using data from previous studies as a control group

Different Designs of CTs

  • Studies can be either:

    • Randomized or non randomized

    • Parallel or crossover

  • Randomized = Participants are randomly assigned to different treatment groups

    • Randomization can occur @ different stages of the CT

    • 3 types of randomization: simple, block, stratified

      • Simple randomization = Each individual is randomly assigned

      • Block randomization - Participants are divided into subgroups (blocks) then randomized within each block

      • Stratified randomization = Dividing patients into subgroups (strata) according to prognostic factors (ie. smokers, non smokers, drinkers) then randomly assigning treatments within each stratum

    • There can be equal (balanced) or unequal (unbalanced) #s of participants in each group

  • Non randomized = Participants are not randomly assigned

    • Potential for bias - Selection bias, confounding variables

  • Parallel = Participants remain in their assigned group throughout the study

  • Crossover = Participants switch treatment groups during the study

    • Allows each participant to serve as their own control

Blinding/Masking in CTs

  • Ideally included when its feasible to reduce/prevent performance, assessment, or attrition bias

  • Types of blinding:

    • Single-blind = Participants are unaware of their treatment assignment

    • Double-blind = Both participants and researchers are unaware of treatment assignments

    • Triple-blind = Participants, researchers, and data analysts are unaware of treatment assignments

Phases of CTs

  • Determined by the U.S. FDA

  • Each phase differs from the others in:

    • Goal

    • # and type of participants

    • Length

    • Presence of comparison groups, types of comparison groups present, and # of comparison groups present

  • 5 phases overall:

    1. Early phase 1 (AKA “0”)

    2. Phase I

    3. Phase II

    4. Phase III

    5. Phase IV

Phase I - Safety and Dosage

  • Phase I trial = Trial focused on assessing the safety and dosage of a new treatment in a small group of people

  • Usually done on healthy volunteers

  • n = 20 - 100

  • Done in specialized centers

  • Open (not blind)

Phase II - Efficacy and Side Effects

  • Phase II trial =Trial to verify the therapeutic effect of a treatment (efficacy), the possible side effects, and its feasibility

  • Treatment is given to patients with the disease

  • n = 100 - 500

  • Done in medical institutions and private practices

  • Multiple doses are used

  • Blinded

  • Comparative

Phase III - Comparison to Existing Treatments

  • Phase III trial = Larger trial comparing the new treatment to the current standard treatment to determine its effectiveness and monitor side effects

  • n = 1000 - 5000

  • Done in medical institutions and private practices

  • Blinded

  • Comparative

Phase IV - Post-Marketing Surveillance (Safety Monitoring)

  • Phase IV trial = A post-marketing surveillance study to monitor the long-term safety and effectiveness of a treatment after is has been approved and is available to the public

  • n = very large

  • Leads to new areas of application of the treatment

Lecture 6 - Research Study 3

Learning Outcomes

  • Identify key features, advantages, and limitations of this type of study (randomized phase II CT)

  • Explain the difference between primary and secondary outcome measures

  • Identify dependent, independent, and confounding variables

Research Study #3

  • RQ - Do high doses of radiation in the SVZ (subventricular zone - a NPC/NSC niche) improve the survival of GBM patients

  • RH - A higher than routine radiation dose to the SVZ decreases the chance of relapse, improving the survival of GBM patients

Previous Observations/Findings

  • NSCs in the SVZ might contribute to the recurrence of GBM

    • From preclinical data

  • Patients receiving higher radiation doses to the SVZ might have improved survival

    • From retrospective observational data

Study Design - A Phase II Therapeutic Trial

  • Gathers preliminary data on a drug’s effectiveness

  • Randomized + parallel

  • No blinding (AKA open)

  • ¾ patients will be treated with the higher dose of RT

  • ¼ patients will be given the standard radiation treatment plan

    • Control = Standard treatment

  • Inclusion/exclusion criteria:

    • Both sexes

    • Adults (18+)

    • Patients newly diagnosed and histologically confirmed with GBM

    • Must have undergone gross total resection or subtotal resection

    • Radiation must be done within 12 weeks of surgery

      • But CANNOT be before surgery

    • Patients must be scheduled to receive temozolomide as well as RT (during and after RT)

    • Cannot have prior malignancy (cannot be diagnosed with a recurrent GBM before the study)

    • Cannot be pregnant/breastfeeding

    • Cannot be using Avastin or another VEG-F inhibitor

Outcome Measures

  • Primary Outcome: The most important outcome for evaluating the effect of an intervention/treatment.

    • One or more primary outcomes.

    • Example: Progression-Free Survival (PFS)

  • Secondary Outcome: Is not as important as the primary outcome but still of interest. May become a primary outcome in future studies.

    • More than one secondary outcome.

    • Examples: Overall Survival (OS), location of tumor progression, neurocognitive changes