JOURNAL

PRESENTATION
BY Moderated by
Dr. Anjali Suresh, PhD
RUTH N
Professor
MPT 2nd SEM
Department of physiotherapy
23MP3002
Title
“The Effects of Exercise Duration and Intensity on
Breast Cancer-Related DNA Methylation:
A Randomized Controlled Trial”
Arielle S. Gillman,Timothy Helmuth et.al,

Cancers 2021 article published by MDPI

 BACKGROUND
• Physical activity lowers cancer risk, including breast cancer, but the exact
mechanism isn’t fully understood. One theory is that exercise may reduce
risk by influencing gene methylation.

• DNA methylation involves adding a small chemical group to DNA,

which can turn genes on or off. This process regulates gene activity and is
crucial for normal cell function and stability.

• New findings suggest that methylation patterns can predict cancer before
diagnosis, highlighting the importance of understanding and influencing
DNA methylation.
 Factors Association with Methylation

Body Mass Elevated Poorer Physical Cardiorespirat APC Tumor Physical Aerobic
Index Weight Aerobic Activity ory Fitness Suppressor Activity and Exercise
(BMI) Status Fitness Levels (VO2 max) Gene Global DNA vs. Control
Methylation

Associated Linked to Associated Related to Associated Inversely Positive No

with hypomethyl with differenti with related to association significant
Methylatio ation of hypometh al differential promoter found, but the differences
n of cancer- inflammato ylation of methylati methylation of hypermethyl effect was in DNA
related ry genes inflammat on of breast cancer- ation in attenuated methylation
genes ory genes genes related genes women after adjusting of breast
specific without for other cancer-
to breast breast cancer factors related
cancer genes
 BRCA1 Breast Cancer 1, Early Onset

RUNX3 Runt-Related Transcription Factor 3

PAX6 Paired Box 6

TUMOUR
DECREASED
SUPRESSOR
Polypeptide N- METHYLATION
GENES GALNT9
Acetylgalactosaminyltransferase 9
SIM1 Single-Minded 1

FBLN2 Fibulin 2

AURKA Aurora Kinase A

BCAR1 Breast Cancer Anti-Estrogen Resistance 1 DECREASED
ONCOGENES
BPI Fold Containing Family A, Member 4 / METHYLATION
BPIF4AP/BASE
Bactericidal Permeability Increasing Protein, BASE

TLR4 Toll-Like Receptor 4

INCREASED
INFLAMMATION-
Aim and Objective
Examine changes in DNA methylation from baseline to post-exercise
intervention.

Explore whether the positive effects of exercise on DNA methylation

might revert if exercise was not continued after the program.
 Methodology
• Type of study: Experimental study - Randomized controlled trial (RCT) with a
factorial design.

• Study Design: Factorial design, with participants evenly allocated across four
conditions based on intensity and duration.

• Study sampling : Randomization- PI used online random number generator to generate

the random allocation sequence

• Study subject: Pre-menopausal women recruited from the Denver metro area.

• Study setting: Conducted at a research facility in the Denver metro area, including
laboratory-based tests and supervised exercise sessions.
• Sample size: 276 women (Mean age = 37.25, SD = 4.64) from the Denver metro
area. where 135 completing the trial and 81 having viable post-exercise
methylation data.
• Study time period: Recruitment began on 1/30/2014 and the six-month follow-up
was completed by 8/17/2017.
• Independent Variables (Intervention Factors)

- Exercise Intensity: 55–65% of VO2max Vs. 75–85% of VO2max.

- Exercise Duration: 20 minutes Vs. 40 minutes per session.
• Trial Registration: The study was registered as a randomized controlled trial on
ClinicalTrials.gov (Registration number: NCT02032628).
Table 1. Study eligibility criteria.

Inclusion Criteria Exclusion Criteria

1. BMI >39 kg/m2

2. Diabetic or on a restricted diet
1. Between ages of 30–45
3. Controlled hypertension (resting systolic
2. <60min of moderate intensity exercise
BP > 150 mmHg or diastolic BP
per week
> 90 mmHg)
3. Pre-menopausal
4. Cardiovascular or respiratory disease
4. Non-smoker
5. Serious arrythmias at rest during the
5. Willing to accept random assignment
VO2Max test
6. Willing to provide blood samples
6. Reported history of breast neoplasia
7. Willing and physically capable of
7. Currently receiving treatment for any
engaging in moderate exercise activity
type of cancer
(i.e., no injuries, physical impairments, or
8. Currently taking psychotropic
pre-existing contraindications) as
medications except for depressionand
assessed by a study physician
anxiety
8. Ability to successfully complete a
9. Currently under treatment for any
VO2 Max test without evidence of cardiac
psychiatric disorder
or other abnormalities
10. Currently under treatment for alcohol or
9. Planningto remain in the Denver metro
drug abuse
area for the next 10 months
11. Currently pregnant or attempting to
become pregnant
Demographics Data collected via an online survey administered using REDCap.
VO2max Measurement: VO2max (mL/kg/min) assessed with spirometry.
Confirmed by a respiratory quotient ≥1.1 or plateau in VO2.
Timing: Measured at baseline and after the intervention.
Exercise Volume Issue: Actual number of exercise sessions varied (25-64 sessions).
Calculation Calculation: Total exercise volume in kcal/kg/week based on
attendance, condition assignment, and VO2max, following ACSM
guidelines
BMI Height and weight measured in kg/m².
Gene Methylation Assessment: DNA methylation at CpG sites analyzed using
pyrosequencing at EpigenDx.
Data: Percent methylation reported, averaged for each gene to reduce
statistical errors.
Follow-Up Exercise Method: Self-reported moderate-intensity exercise behavior measured
Behavior using the Stanford 7-Day Physical Activity Recall (PAR) six months
after the intervention.
 Procedure
1. Study Approval - Colorado Multiple Institutional Review Board ,Colorado Clinical &
Translational Sciences Institute.
2. Participant Recruitment- Advertisements (local publications, flyers, online) , Description of
exercise program opportunity
3. Eligibility Assessment - Initial contact and assessment
First appointment
Enrollment and informed consent
Medical screening by study physician
Self-report and PAR completion
4.Baseline Assessments - Online assessments via REDCap
Blood sample collection: 2 samples (2.5 mL each) , BD Vacutainer® CPT (for
PBMC),PAXgene Blood RNA tube (for transcriptome).
Processing and storage: Centrifuge and store PBMCs at −70°C, DNA extraction and
quantification
5. Exercise Intervention-VO2max testing to determine parameters, Random
allocation to exercise conditions, Exercise sessions: Treadmill, elliptical, Heart
rate monitoring.
6. Post-Intervention Assessments
End of 16 weeks: Second VO2max test
Follow-up blood draw
Six-month follow-up: Complete PAR
Blood samples for methylation analysis
 Recruited
Consented
Participants Retention ateach phase:
Participants
(number of Phone Screen  Consent 250/1363=19.7%
N =276
callers) Consent  Baseline 269/276 =97.4%

n =1363 Baseline  VO2 234/269 =87.0%

VO2  Started Exercising 219/234 =93.6%

Completed Exercise  VO 2 135/137 =98.5%

Top reasons for no consent:
CompletedBaseline
Final Follow-u p 116/136 =85.3%
Unable to contact: 21.0%
Assessment
Started Exercising then dropped 82/219 =37.4%
Did not meet exercise criteria: 13.2%
N =269

BMI too high: 9.3% Withdrawn =120

Not menstruating regularly: 6.4% Excluded=21
Smokingcriteria: 5.4% Withdrawn- No Contact/ Unreachable: 41
CompletedBaseline
Unwilling to Participate in 16 week Withdrawn- Time commitment: 33
VO2max
study: 5.3% Withdrawn- No longer interested: 16
N =234
A ge: 5.0% Withdrawn- Changed jobs/schedule change: 16
Refused: 4.5% Withdrawn- health concerns: 9
Planningto get pregnant: 3.7% Excluded- High BP: 11
Diabetes: 2.8% Began supervised exercise Excluded- Heart Cond: 3
Psychotropic medication: 2.3% N =219 Other: 12
Unwilling to commute: 2.2% Viable methylation data Total =141
N =188

40Min High 20Min High 40Min Moderate 20Min Moderate

Intensity Intensity Intensity Intensity
n =62 started n =55 started n =55 started n =47 started
n =33 completed n =31 completed n =41 completed n =32 completed

Completedfollow-up &
Dropped after Finishedexercise
second VO2max
startingexercise sessions
n =135
n =82 n =137
Viable methylation dataN =81
 Recruited
Consented
Participants Retention ateach phase:
Participants
(number of Phone Screen  Consent 250/1363=19.7%
N =276
callers) Consent  Baseline 269/276 =97.4%

n =1363 Baseline  VO2 234/269 =87.0%

VO2  Started Exercising 219/234 =93.6%

Completed Exercise  VO 2 135/137 =98.5%
Top reasons for no consent:
CompletedBaseline
Final Follow-u p 116/136 =85.3%
Unable to contact: 21.0% Assessment
Started Exercising then dropped 82/219 =37.4%
Did not meet exercise criteria: 13.2% N =269

BMI too high: 9.3% Withdrawn =120

Not menstruating regularly: 6.4% Excluded=21
Smoking criteria: 5.4% Withdrawn- No Contact/ Unreachable: 41
CompletedBaseline
Unwilling to Participate in 16 week Withdrawn- Time commitment: 33
VO2max
study: 5.3% Withdrawn- No longer interested: 16
N =234
A ge: 5.0% Withdrawn- Changed jobs/schedule change: 16
Refused: 4.5% Withdrawn- health concerns: 9
Planning to get pregnant: 3.7% Excluded- High BP: 11
Diabetes: 2.8% Began supervised exercise Excluded- Heart Cond: 3
Psychotropic medication: 2.3% N =219 Other: 12
Unwilling to commute: 2.2% Viable methylation data Total =141
N =188

40Min High 20Min High 40Min Moderate 20Min Moderate

Intensity Intensity Intensity Intensity
n =62 started n =55 started n =55 started n =47 started
n =33 completed n =31 completed n =41 completed n =32 completed

Completedfollow -up &

Dropped after Finishedexercise
second VO2max
startingexercise sessions
n =135
n =82 n =137
Viable methylation dataN =81

Completed final follow-up

n =116
Viable methylation data
N =88

Figure 1. CONSORT diagram of participant flow throughout study.

 Table represents baseline characteristics for the 81 participants with viable post
test methylation data. Baseline characteristics were not significantly different
across conditions (all p’s > 0.38) .
Overall Sample Low Intensity + 20 Low Intensity + 40 High Intensity + 20 High Intensity + 40
Characteristic min min min
(n= 21) (n= 23) (n= 17) (n= 20)
Age 37.14(4.71) 37.00(4.86) 37.26 (4.60) 37.82(5.45) 33.55(4.29)
Race (% White) 65.4% 61.9% 65.2% 70.6% 65.0%
BMI (kg/m2 ) 29.60(5.69) 28.22(6.02) 29.36 (4.95) 29.59(5.69) 31.32(6.08)
VO2 max
27.37(5.29) 28.18(5.73) 26.78 (5.38) 27.70(5.41) 26.89(4.85)
Self-reported exercise
18.27 (31.94) 12.86 (15.13) 22.85(48.14) 17.65(22.23) 19.25 (29.92)
 Table 4 : Systematic Differences (Completion vs. Dropout)

Completed Trial (n= 135) Enrolled, Not Completed (n= 141) Test StatisticforGroup
Characteristic
M (SD) Differences
Age 37.43(4.71) 37.08 (4.59) t(267)=0.60), p= 0.545
Race (% white) 59.7% 51.5% χ 2(6) = 6.69, p= 0.035
Education (% college
71.6% 54.1% χ2(7) = 14.13, p= 0.05
BMI (kg/m2) 28.54(5.62) 29.38 (5.15) t(261) = −1.25, p= 0.211
BaselineVO2max
27.95(5.80) 26.46 (4.80) t(229)= 2.08, p= 0.04
Self-reported exercise 17.13(32.61) 15.56 (25.02) t(266)= 0.441, p=0.66
 Statistical analysis
1. Sample Size determination:
• To detect the effect of exercise intensity and duration on DNA methylation power analysis was
done using G*Power 3.0.372 with a two-tailed alpha of 0.05 and a power level of at least 0.80.
The initial recruitment target was 300 women Ultimately, 276 women were recruited due to
resource limitations, leading to the study being exploratory
• Given the final sample size was smaller than required, analyses were considered exploratory
and aimed at generating hypotheses for future research.

2. Statistical Analyses:
Repeated Measures ANCOVA: Methylation changes were assessed at three time points: pre-
intervention, post-intervention, and six months post-intervention.
Exercise quantity was operationalized in three ways: Exercise volume (kcal/kg/week), Change
in VO2max, Duration/intensity condition assignment.
Three models were run for each gene.
 Results
1. Change in Methylation: Baseline, Post-Intervention, and Follow-Up
Gene Methylation Change Linear Effect Quadratic Effect Interpretation

AURKA Increased b = 0.33, p = 0.04 Not significant .Healthy increase in methylation over time

BCAR1 Increased b = 0.36, p = 0.006 Not significant Healthy increase in methylation over time.

BPIF4AP Increased then Decreased Not significant b = 0.41, p = 0.005 Healthy increase post-intervention, but
(p = 0.89) decrease at follow-up (unhealthy).

BRCA1 Increased b = 0.06, p = 0.04 Not significant Worsening methylation over time.
(p = 0.26)

SIM1 Increased b = 0.23, p < 0.001 b = −0.11, p = 0.004 Unhealthy increase from post-intervention
to follow-up.
• No significant linear or quadratic changes in methylation were found for FBLN2, GALNT9, PAX6, RUNX3, TLR4, or
TLR6.
• AURKA and BCAR1 showed healthy, sustained improvements in methylation due to exercise.
• BPIF4AP improved during exercise but worsened after it ended.
• BRCA1 and SIM1 showed negative trends in methylation, potentially indicating unhealthy changes.
Average percent methylation for each gene at each time point, on average across
condition. Y-axis for each panel represents % methylation. Bars are standard error bars.
Change in Methylation from Baseline to Post-Intervention Based on Quantity of Exercise
Completed
Exercise Volume
Exercise volume calculated in kcal/kg/week was not associated with post-intervention methylation for any of the
candidate genes.

Vo2 max
BRCA1 Methylation: Significant Negative Association: BRCA1 methylation decreased as VO2max increased.
• Effect size: b = −0.05 , 95% Confidence Interval: [−0.10, −0.01], t(66) = −2.52, p = 0.01.

FBLN2 Methylation:
• Trending Negative Association: There was a trend similar to BRCA1, but it wasn’t statistically significant.
• Effect size: b = −0.08, 95% Confidence Interval: [−0.117, 0.01], t(67) = −1.80, p = 0.08.
• The trend suggests that those who increased VO2max had less FBLN2 methylation, but this finding was marginal.
Other Genes:
• No significant associations were found between VO2max and methylation changes for the other genes studied.
Six-Month Follow-Up Methylation Change as a Function of Level of
Continued Exercise
Researchers looked at how this self-reported exercise affected gene methylation, while
considering previous methylation levels, BMI, and age.

They found that more exercise was linked to lower methylation levels of the tumor
suppressor gene GALNT9. This suggests a potential beneficial effect of exercise on this
gene. The change was statistically significant (p = 0.02).

No significant effects were found for other genes.

 Discussion
• This study explored whether higher volumes of exercise (both in duration
and intensity) were linked to healthy changes in the methylation of genes
associated with breast cancer. The results were mixed:
• For some genes, such as AURKA and BCAR1, there were positive changes
in methylation due to exercise, or at least prevention of negative changes in
genes like BPIF4AP and SIM1.
• Changes in BRCA1 methylation were significantly linked to improvements
in VO2max, a measure of cardiovascular fitness, which was related to the
volume of exercise completed.
• At the six-month follow-up, participants who reported more minutes of
exercise showed lower methylation of GALNT9, a tumor suppressor gene,
suggesting potential long-term benefits of exercise.
 Strengths and Limitations
Strengths:
• The experimental design with random assignment allows for stronger conclusions about the effects of
exercise on DNA methylation.
• Exercise and fitness improvements were objectively measured, unlike previous studies that relied on self-
reported data.
Limitations:
• Small Sample Size: The final sample size was smaller than planned, possibly limiting the ability to detect
significant effects.
• Differences in Groups: There were differences in education and baseline fitness between those who
completed and did not complete the study.
• Methylation Data: Data came from mixed blood cell types, which can vary in methylation patterns.
• No Control Group: Without a no-exercise control group, it’s unclear if changes were solely due to
exercise or just time.
• Additional Exercise: Participants might have done extra exercise outside the study, affecting results.
• Age and Gender: Findings may not apply to men or people outside the 30-45 age range .
 Conclusion
This study is important for understanding breast cancer prevention. It provides early
insights into how more exercise might lower breast cancer risk by affecting DNA
changes.
The results suggest that exercise could influence factors related to cancer risk and set
the stage for more research on how to use exercise to improve health.
but don’t know the exact amount of exercise needed to make a difference, this study
is a good first step and highlights the need for further investigation.

Funding: This work was funded by a grant from the National Cancer Institute, 1R01CA179963 to Angela Bryan.
NIH/NCRR Colorado CTSI Grant Number UL1 RR025780 supported the use of research electronic data capture
(REDCap). Publication of this article was funded by the University of Colorado Boulder Libraries Open Access
Fund
Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration
of Helsinki and approved by the Colorado Multiple Institutional review Board (COMIRB) (protocol number 13-
2314, approved on 19 May 2014).
 Critical appraisal
1) a. Was the assignment of patients to treatments randomised?
Yes. The participants were assigned to different treatment groups randomly. The
research team used a computer program to generate a random list, and each participant
was assigned to a group based on this list when they came for their first exercise
session. This process helped ensure fairness and reduced the chance of bias in the study.
b. Were the groups similar at the start of the trial?
Yes. the groups were similar at the start in terms of age, BMI, and fitness levels.
2) a. Aside from the allocated treatment, were groups treated equally?
Yes. In this study, apart from the different exercise programs given to each group, all
groups were treated the same.
• Everyone was randomly placed into a group.
• DNA changes were measured for everyone at the same time points
• Exercise and fitness levels were checked the same way for all participants.
• Follow-up was done the same way for all, using the same questionnaires and tests.
b. Were all patients who entered the trial accounted for? And were they analysed in the
groups to which they were randomised?
No. Patient Accounting: Not all patients who started the trial were included in the final
results due to dropouts and data issues.
Analysis by Randomization: The study aimed to analyze participants in their assigned
groups, but the final analysis might not perfectly reflect the original randomization due to
data challenges.
3.Were measures objective or were the patients and clinicians kept “blind” to which
treatment was being received?
Objective measures- Yes. The study used devices and tests (like VO2max and heart rate
monitors) to measure outcomes.
Blinding- No. Participants and researchers knew which exercise condition each participant
was assigned to.
Results:
1. How large was the treatment effect?
No. The treatment effect size was variable across different outcomes and Overall, the effects
were generally modest and varied by gene.
For some genes (AURKA, BCAR1): Modest positive changes in methylation were observed.
For others (BPIF4AP, SIM1): Changes were moderate, with some prevention of negative
effects.
VO2max: Significant relationship with BRCA1 methylation, but no other significant effects
were noted.
GALNT9: Lower levels of methylation were associated with more self-reported exercise at
follow-up.
2. How precise was the estimate of the treatment effect?
No. The estimate of the treatment effect was not very precise, as the study had a smaller
sample size than planned. This makes the results more preliminary and less certain.
My Remarks:
However, there are some aspects where the results and purpose of the study do not fully
align:
Group Comparisons: The study aimed to know whether dose response relationship exists
between quantity of exercise completed and the degree of change in DNA methylation
but detailed results on how different exercise groups specifically influenced methylation
were not provided.The focus was on overall changes in methylation over time, effects of
exercise volume, and the association between VO2max and methylation, rather than
detailed comparisons between the different exercise conditions.
This finding provides preliminary evidence that maintaining exercise behavior may have
long-term effects on methylation, at least for this particular gene.
Overall, these analyses provide some support for the idea that DNA methylation may be
one mechanism by which exercise behavior may reduce the risk of breast cancer
 PEDRO scoring
• Random Allocation: Yes (Study used random allocation for intervention assignment).
• Concealed Allocation: Yes (Allocation was concealed using a random number generator).
• Baseline Comparability: Yes (Baseline characteristics were reported and considered).
• Blinding of Subjects: No (Participants were not blinded to their intervention condition).
• Blinding of Therapists: No (Therapists delivering the interventions were not blinded).
• Incomplete Data: Yes (Study reported handling of incomplete data).
• Statistical Analysis: Yes (Appropriate statistical analyses were used).
• Outcome Measures: Yes (Valid and reliable measures were used for VO2max and
methylation).
• Follow-Up: Yes (Follow-up data was reported and analyzed).
• Sample Size: Yes (Sample size calculation was provided).
PEDro Score Calculation
Based on the provided information, the study could potentially score 8/11 on the PEDro
Clinical implementation and translating in
Indian clinical setup.
yes, The study suggests that exercise can influence DNA methylation, potentially
affecting breast cancer risk.

Aerobic exercise has the potential to reduce cancer risk by influencing DNA
methylation patterns related to cancer. The evidence is promising, but further research is
needed to fully understand the extent and mechanisms of this effect.

so, If we have the necessary resources, staff, and facilities, and if we can meet ethical,
regulatory, and financial requirements, then it is likely feasible to implement the study in
the setting.
THANK YOU