Radboud· me

r. cademy

Radboud· ^me

Examination Date

Start

B2RS3-5 Research semester 3 January 18th, 2019

13:00 h

After finishing the exam, you can take this examination set along with you.

Please hand in the OTHER part (the answering form) to the supervisor.

You are allowed to use a calculator of the type Casio FX-82MS.

The questions must be answered in English. lf you cannot remember a specific English term, you are allowed to use the Dutch term.

During the exam you have access on a computer to these books:

• Baynes & Dominiczak: Medical Biochemistry

• Campbell: Statistics at square one

• Donders: Literature Measurement errors

• Fletcher: Clinical Epidemiology

• van Oosterom en Oostendorp: Medische Fysica

• Petrie and Sabin: Medical Statistics at a Glance

• Turnpenny: Emery's Elements of Medical Genetics

• Form with statistica! formulas

GENERAL INSTRUCTIONS:

• This exam consists of 5 open questions.

• The available time is 2 hours.

• You are allowed to use scrap paper that will be handed out. Do not use the scrap paper for your answers and do not hand it over to the supervisor.

• Check if your examination set is complete.

• Please write vour name and student number on each page of the answering form.

• Write your answers on the answering form in the open space below the questions.

Read the questions carefully before phrasing your answers.

• Be concise and complete in your answers.

• lf necessary you can also use the backside of the pages.

• Refrain from using abbreviations in your answers, and write legibly (illegible answers are considered incorrect).

• Please do not use a pencil.

• The use of audiovisual and technica! devices is not allowed, unless it is mentioned explicitly elsewhere on this page. Any inappropriate use of such equipment is regarded as fraud.

• Except for the exam farms, some loose writing material, your student and registration card your table should be empty. No boxes or cases are allowed.

• After finishing the exam, please hand the answering form to the supervisor. lf you have

comments about the questions we refer you to the hyperlink of the digital comment form that is included in your "studenten webdossier" below "toetsen".

SUCCESS!

ATTENTION !!

FIRST PUT YOUR NAME AND STUDENT NUMBER AT THE BEGINNING OF EACH QUESTION.

vrb/810S1BT1516.docx/18-12-2018

Take-home set Research Exam Semester 3 - 2018-2019 January 18, 2019

Question 1

Q5: Cancer etiology and prognosis - prof. dr. B. Kiemeney (20 points)

Molecular Markers lncrease Precision of the European Association of Urology Non­

Muscle-lnvasive Bladder Cancer Progression Risk Groups Abstract, Journal of clinical research 20 18

Purpose: The European Association of Urology (EAU) guidelines for non-muscle-invasive bladder cancer (NMIBC) recommend risk stratification based on clinicopathologic

parameters. Our aim was to investigate the added value of biomarkers to improve risk stratification of NMIBC.

Experimental Design: We prospectively included 1 ,239 patients in follow-up for NMIBC in six European countries between the years 2008 and 201 3. Fresh-frozen tumor samples were analyzed for GATA2, TBX2, TBX3, and ZIC4 methylation (Note: methylation of promoters of genes usually repress gene transcription) and FGFR3, TERT, PIK3CA, and RAS mutation status (Note: gene mutations may alter gene activity). A regression analysis identified markers that were significantly associated with progression to muscle-invasive disease.

Results: In our cohort, 276 patients had a low, 273 an intermediate, and 555 a high risk of tumor progression based on the EAU NMIBC guideline. Fifty-seven patients (4.6%)

progressed to muscle-invasive disease. Overall, wild-type FGFR3 and methylation of GATA2 and TBX3 were significantly associated with progression (Hazard Ratio (HR)=

0.34, 2.53, and 2.64, respectively).

Conclusions: We conclude that the addition of selected biomarkers to the EAU risk stratification increases its accuracy and identifies a subset of NMIBC patients with a very high risk of progression.

Table 1. 1 - Results of the regression analysis that was performed on the progression to non-muscle-invasive bladder cancer

Factor Univaria ble Multivaria ble

HR (Cl 95%) p-Value HR (Cl 95%) p-Value Age (years) 1 .04 (1 .01 -1 .07) 0.004

Biomarkers

FGFR3 mutation 0.34 (0.71 - 0.68) 0.002 0.43 (0.21 - 0.87) 0.01 9 GATA2 methylation 2.53 (1 .36 - 4.71 ) 0.003 2.23 (1 .1 6 - 4.31 ) 0.01 6 TBX3 methylation 2.64 (1 .41 - 4.92) 0.002 1 .85 (0.94 - 3.65) 0.076

a. Which regression analysis was performed in this study (1 pt)?

Provide 1 reason why this specific regression analysis is used (2pt)

b. One of the reported limitations in this study is that they found a very low number of people who had progression to muscle-invasive disease when compared to (much) older studies. Provide an explanation for the lower number found in this study (3pts).

c. Please take a look at Table 1 .1 and explain in words the results for the factor 'Age (Years)'. (4 pts)

The authors of this article also looked at the classification groups and progression-free survival in a Kaplan-Meier Curve using the EAU-classification model combined with the FGFR3 and GATA2 status. They obtained the following figure 1 .1 .

1.0 ···-�·-····� •••••••••••••••••••••••••••••••••

0.8

0.6

0.4

0.2

0.0 0

... _ -- ...

•·• G d

." - 00

_."Moderate

...n

Poor

12 24

-

....

" ^___

,

--- ---

p

^<

0.01

36 48 60

Figure 1. 1: Progression Free Survival curve tor patients with Non-Muscle-lnvasive Bladder Cancer. Dotted line, good health status; dashed line, moderate health status; solid line, poor health status. P-value is based on Jog-rank test

d. In figure 1 .1 , what is the 3-year progression free survival risk for the poor health risk group?

(2 pts)

e. The log-rank test tells you that p ^< 0.01 . Please provide one reason why only a log­

rank test is not very informative. (3 pts)

f. Take a look at the figure 1 . Is this risk classification useful in the clinic? Please explain your answer. (5 pts)

Research exam Semester 3 (2018-2019 )- January 18, 2019 - Take away version 2

Question 2

Q5, Q6: Modelling physiological systems - dr. T. Oostendorp (15 points)

Eye rotation

When studying the motion of the eye lid, the eye lid can be modelled as a mass-spring system. The agonist and antagonist muscle are modelled together as a single spring with spring constant

k.

and resting position y0. When the muscle moved the eye lid trom open position _Yb to closed position

Ya (see figure 2.1 ), the resting position of the muscle pair changes value trom _Yb to _Ya.

Two farces are involved:

1 . The muscle force:

Fmuscle

⁼

k(yo

^-

y),

y

^b ----�----�

y

--- --�---�---,..-

Figure 2. 1 Eye lid geometry

2. The friction force

Ftriction•

which is proportional to the eye lid velocity.

a. Explain, using Newton's law

(F

^{= m · a)} that the differential equation tor the eye lid position

y(t)

is (4 pt):

d2 d

m

dt2 y(t)

⁼

k(y0

^-

y(t))

^{- f3}

dty(t)

b. Why is there a minus sign in front of f3

�y(t)

in the differential equation? (2 pt)

dt

We will use this model to study the motion the eye lid makes when it changes position trom 'closed' to 'open'.

c. Is

y0

a parameter or a variable in this study? Explain your answer. (2 pt)

d. The figure below is the incomplete Simulink diagram tor this study. Complete the diagram. (7 pts)

Yo

y(t) J

Question 3

Q5: Molecular Cancer Research - dr. P. Groenen (15 points)

1. Western blot: procedures and interpretation

Small cell lung cancer (SCLC), the most aggressive type of lung cancer, accounts for approximately 1 5% of lung cancer cases and is responsible for 25% of lung cancer-related deaths. Here, investigators studied AZD3965, an MCT1 specific inhibitor. They also

investigated the effects of hypoxia. Two cell lines were studied (COR-L 1 03 and NCl- H 1 048), tubulin was used as reference protein.

COR-L 103 NCf"H1048

Hypoxia

^{+ + + +}

AZD3965

^{+ + + +}

MCT1

^•.

! ..._

^{•_· _. ___ ._. _•_• __}

^{44 kDa}

MCT4. :48 kDa

Figure 3. 1. Protein expression analyses of MCT 1, MCT ₄ and tubulin in COR-L 103 and NCl-H1048 cel/ lines treated with the MCT 1 inhibitor AZD3965 in either a normoxic or a hypoxic environment.

a. Based on the results in Figure 3.1 , what is the correct interpretation about the protein loading and what does this mean for the interpretation of the MCT1 and MCT 4 protein differences observed in the cell lines COR-L 1 03 and NCl-H1 048? (4pts)

b. Explain the effects of hypoxia on the AZD3965 inhibitor sensitivity for the two cell lines as shown in Figure 3.1 . (4 pts)

ll. Tumor heterogeneity

Targeted therapies have been developed to counteract processes of cancer proliferation by inhibition of phosphorylated kinases. However, in tumors several different cell

populations may arise, leading to tumor heterogeneity, as schematically illustrated in Figure 3.2.

In order to determine the diagnosis and treatment options for a patient with this tumor, tumor cells are obtained by collecting a tumor biopsy. In this biopsy, all five subclones depicted in Figure 3.2 are included (red, green, yellow, light blue and dark blue).

By DNA extraction and genetic analysis, five mutations are identified in this biopsy:

- an activating mutation in the P/3K oncogene - an activating mutation in the BRAF oncogene - an activating mutation in GNAQ/ 1 1 oncogene

- an activating mutation in the receptor tyrosine kinase (RTK) CKIT - an inactivating mutation in the tumor suppressor PTEN

Research exam Semester 3 (2018-2019)- January 18, 2019 - Take away version 4

Primary _tumor wi"th m.ultiple

subclones

?' 0 o···. .

O� ··

^{. .}

•· -+-C

�

Figure 3. 2 Schema tic illustration of the development of tumor heterogeneity. The different tumor populations are indicated with different colors.

G;;m>tem <>.oopled

reeeptor

�

_{RAS ---}

/

/ T

NF1

MEK ERK

l

PTEN

· · · ·· ··"··"' ' -·

Figure 3. 3 Schema tic overview of oncogenic intracellular pathways.

c. Based on the presence of these mutations and on the pathway scheme (Figure 3.3), what is the best combination of two targeted therapies from the list below that will effectively inhibit oncogenic signaling in all five subclones. Explain why this is the best combination therapy. (4 pts)

- MEK inhibition - BRAF inhibition - CKIT inhibition - Pl3K inhibition - PTEN inhibition - GNAQ/1 1 inhibition

d. From a patient with melanoma, the melanoma tissue is obtained to identify the oncogenic pathway that is activated. By genetic analysis, an activating BRAF V600E mutation is identified in the melanoma. Next, a student in the lab performs a Western blot to determine the amount of phosphorylated ERK (pERK). The following antibodies are available: an antibody directed against phosporylated ERK and an antibody against tubulin. In addition, a tissue that has no activating BRAF mutation is available.

Please draw the Western blot that shows the activation status of the pathway in the melanoma and in the tissue without the activating BRAF mutation. Please mind the incorporation of the necessary controls in the experiment (3 pts ).

Question ₄

Q6: Statistics - dr. _J. in't Hout (15 points)

Background The benefits of blood pressure lowering treatment for prevention of

cardiovascular disease are well established. However, the extent to which these effects differ by drug class or other factors is less clear. Ettehad et al. (Lancet, 201 6) performed a systematic review and meta-analysis to clarify these differences.

Medline was searched for large-scale blood pressure lowering trials. Randomised controlled trials of blood pressure lowering treatment were eligible for inclusion if they included a minimum of 1 000 patient-years of follow-up in each study arm.

In total, the authors included 1 23 studies with 61 3,81 5 participants.

a. How realistic is the danger of publication bias in this meta-analysis? (1 p) Explain. (2p)

b. The authors performed a fixed-effect meta-analysis. How are the weights calculated that are assigned to the studies, using a fixed-effect model? (2p)

c. Consider a small study, taken from a meta-analysis with quite some between-study heterogeneity. Is this study relatively more important (for the pooled result) in a fixed­

effect or in a random-effects meta-analysis? ( 1 p) Explain your answer (2p ).

d. To evaluate the relation between reduction in major cardiovascular disease events in relation to the achieved blood pressure reduction, the authors performed meta­

regression. This resulted in an estimated risk ratio (RR) of 0.80 per 1 0 mmHg of reduction.

Assume a cardiovascular disease event rate of 1 0% in the control group. Explain which event rate you expect in the experimental group if the difference in SBP between the control and experimental group is 1 5 mm Hg? (4p)

Research exam Semester 3 (2018-2019 )- January 18, 2019 - Take away version 6

0

0

0

0

₀

0

⁰

0 0

0

0 5 10 15 20

Reduction in SBP

(mm Hg)

Figure 4. 1. Meta-regression plot of the percentage risk reduction in all-cause mortality (y-axis) regressed against the difference in achieved systolic blood pressure (SBP) between treatment arms (x-axis).

e. Figure 4.1 shows the fixed-effect meta-regression results for all-cause mortality in relation to systolic blood pressure reduction. Studies are indicated with small and large circles (bubbles). Relate the bubble size to the importance of the studies (1 p) and the weights of the studies (2p).

Question 5

Q6: Measuring and modelling reflexes - dr E. Tanck & dr. T. Oostendorp (20 points) Statie exercise

A young man (80 kg) lifts his right leg from a vertical position to horizontal to train the quadriceps muscle and holds his leg in a position of 45° (see figure below). A physical therapist is interested to know how high the forces are in the hip joint and the m.

quadriceps when the leg is at a position of 45° during this statie exercise. You are assisting him with the calculations.

Known data: the young man weights 80 kg, his leg weights 8 kg, use g = 1 0 m/s2 tor the gravitational acceleration. You can assume that all distances, masses, angles and attachment points for ligaments and muscles are known.

Figure 5. 1

a. Create on the next page a free body diagram (FBO) to calculate the forces in the hip joint and the m. quadriceps. Make your diagram large enough so everything can be labeled clearly. Create a legend to specify all components of the FBO. (7 points) b. Give the Equilibrium equations that belong to your FBO (4 points). You do not have to

calculate the forces.

c. What happens to the force in the m. quadriceps when the man lifts his leg to 90°

degrees, so parallel to the floor? Choose from: higher than, lower than or equal to the force when lifting to 45°, and explain your answer using an equilibrium equation. (4 points)

The quadriceps is innervated by the femoral nerve. A researcher wants to record the activity of the femoral nerve. He finds that the amplitude of the ElectroNeuroGram (ENG) trom the femoral nerve is about 40 µV, while the amplitude of ElectroMyoGram (EMG) trom the quadriceps muscle is about 700 µV.

d. Explain why the amplitude from the quadriceps is so much larger than that from the femoral nerve. (2 points)

Because the ENG from the femoral nerve is so small, the researcher plans to use signal averaging in order in improve the signal-to-noise ratio.

e. Explain why it does not make sense to use signal averaging when recording voluntary activity of the femoral nerve. (3 points)

Research exam Semester 3 (2018-2019 ) - January 18, 2019 - Take away version 8