1. Termin

Question 1

Tip cell formation

Answer 1

Tip cell = Endothelial cells that spearhead the vascular sprouts
- characterized by position, long and dynamic filopodia and migratory behavior
- VEGF activates tip cell migration
- respond to attractive and repulsive directional signals presented by the environment and thereby define the route in which the new sprout grows
- reuquired to create new connections between different sprouts to generate an interconnected and functional vascular network
- Tip cells express VEGFR2/3 together with several co-receptors (neuropilin-1 (NRP1))
- Tip cell attraction by VEGF gradients
- Upon activation: Tip cells express DLL4, that binds to Notch receptors on follower stalk cell

Question 2

Receiver operating characteristics

Answer 2

• Graphical plot that illustrates the diagnostic ability of a binary classier system as its discrimination threshold is varied
• to draw a ROC curve, only the true positive rate (TP) and false positive rate (FP) are needed
• Best possible prediction method would yield a point in the upper left corner or a coordinate (0,1) of the ROC space, representing 100% sensitivity (no false negatives) and 100% specificity (no false positives)
• points above the line of no discrimination represent good classification results (better than random); points below the line represent bad results (worse than random)

Question 3

Validity

Answer 3

Analytical validity is a measure of how well the test measures what it’s supposed to measure (a test designed to detect a mutation associated with melanoma should not give a positive result for an unrelated mutation associated with diabetes)

Question 4

Specificity

Answer 4

• ability to correctly identify patients without cancer
• a specific test gives a positive result only the the cancer is present
  -> no false positives

Question 5

Sensitivity

Answer 5

• ability to correctly identify patients with cancer
• a sensitive test correctly identifies everyone who has the cancer
  -> no false negatives

Question 6

Staging of cancer -> TNM staging system

Answer 6

Tumor, Nodes, Metastasis
- Description of the size of the primary tumor and the extension of spreading (tissue infiltration)
- involvement of lymph nodes
- spreading to distant organs (metastasis)

Question 7

Staging of cancer -> Grading

Answer 7

• Determination of cells anaplasia (loss of differentiation) in tumor tissue in comparison to the normal tissue
• degree of tumor aggressiveness
• Pathology: Variability in size, shape and staining of cells and their nuclei
• nucleus (high) and cytoplasm (low) ration
• Mytotic activity (high)
• Atypical mitoses (high)

Other parameters:
- measurement of serum tumor markers (LDH, alpha-fetoprotein, PSA)
- Completeness of the operation (resection, boundaries free of cancer cells or not)

Aims: Help to plan treatment and give an indication of prognosis

There are four grading categories with an additional one where the grade cannot be assessed

Tumors with highly abnormal cell appearance and large numbers of dividing cells
- tend to grow more quickly
- spread to other organs more frequently (metastasis)
- less responsive to therapy than cancers whose cells have a more normal appearance
-> poor prognosis

Question 8

Stem cell niche

Answer 8

• is a specialized microenvironment that protects stem cells and regulates their behavior
  Example: C. elegans germline stem cell niche: one cell, the mesenchymal distal tip cell, provides a niche for germline stem cells
• the stem cell niche can act on a stem cell by various mechanisms:
  -> Direct contact: direct contact between the stem cell and the niche cells
  -> Soluble factors: releasing of soluble factors by the niche
  -> Intermediate cell: Intermediate cells “communicate” between the niche and the stem cell

Question 9

Pre-metastatic niche

Answer 9

• Hypoxia
• ROS level increased
  -> both up regulate CSC stress signaling pathways
• in the case of hijacking of a normal stem cell niche: CSCs up regulate EMT pathway in the surrounding non tumorigenic cells and transform them into CSCs to further support the CSCs to colonize the new niche
• secreted soluble factors are key players in bone marrow cell mobilization during metastasis
• membrane vesicles derived from both tumor and host cells

Question 10

Pre-metastatic niche formation

Answer 10

Tumors metastasize to particular organs due to the migration of hematopoietic bone marrow cells expressing VEGFR1 and stromab cells to these particular sites poor to the formation of clinically relevant metastasis.
- influenced by many different bodily processes, including the suppression of the immune system and an increase in the presence of cytokines and other growth factors as well as extracellular matrix deposition and remodeling
- Hypoxia in the primary tumor and the movement of exosomes from the primary tumor to the secondary organ are additional phenomena that are partially responsible for the formation of pre-metastatic niches

Question 11

What is cancer?

Answer 11

• cancer is the general name for a group of diseases characterized by uncontrolled cellular growth
• Neoplasia: the growth of new tissue
• Neoplasm/tumor: an abnormal growth of tissue forming a mass
• Transformation: process of converting a normal cell into a cell having attributes of a cancer cell

Question 12

Transformed cell

Answer 12

• Cell proliferation: uncontrolled
• number of cell divisions: unlimited (immortal)
• contact inhibition: no, multilayer cell culture
• dependent on growth factors: no
• tumor formation: yes, tumorigenic in laboratory animals

Immortalization: process whereby a cell normally having limited replicative potential acquires the ability to multiply indefinitely

Question 13

Cancer stem cell characteristics

Answer 13

Fraction with a small number of cells
- behave like stem cells
- self-renewal
- unlimited proliferative potential
- production of a large number of progeny which are in different state of differentiation (different surface markers) and with limited proliferative potential

Question 14

Therapeutic challenge of cancer stem cells

Answer 14

Radio- and chemotherapy resistance due to “stemless” inductions:
- Drug export: elevated expression and high activity of ABC transporters
- ROS decrease: High ALDH activity
- High survival: Anti-apoptotic molecules, high telomerase activity, DNA damage
- Quiescence

Question 15

Six hallmarks of (malignant) cancer

Answer 15

1) Self-suffiency in growth signals -> Activate H-Ras oncogene
2) Insensitivty to anti-growth signals -> Lose retinoblastoma suppressor
3) Evading apoptosis -> produce IGF survival factors
4) Limitless replicative potential -> turn on telomerase
5) Sustained angiogenesis -> produce VEGF inducer
6) Tissue invasion & metastasis -> inactivate E-cadherin

-> The hallmarks of cancer are thought to be necessarily acquired during the multistep pathogenesis pathways leading to most forms of human cancer
-> certain forms of cancer may be less dependent on one hallmark or another

7) Emerging Hallmarks
-> Avoiding immune destruction
-> Deregulation cellular energetics
8) Enabling characteristics
-> genome instability and mutation
-> Tumor-promoting inflammation

Question 16

Loss of heterozygosity

Answer 16

• Typically, cells possess two functional alleles of a given tumor suppressor gene
• the first hit may be an inactivating mutation or the silencing of the gene by promoter hypermethylation
• the second hit may involve the loss of the second allele (LOH) or a process similar to the first hit
• if the first hit happened in the germ line or during early development, the organism will have an increased tumor risk

-> bezeichnet den Verlust der normalen Funktion des Alles eines Gens in einer Zelle, in der das andere Allel bereits inaktiviert war
-> Beispiel: Retinoblastom (Tumor in der Netzhaut des Auges): Hier wird ein Defekt an einem Chromosom vererbt, wobei der Defekt erst dann zu einer Tumorbildung führen kann, wenn das zweite Gen durch externe Faktoren (wie chemische Karzinogene oder auch Strahlung) ebenfalls mutiert

Question 17

Natural endogenous DNA damages -> DNA alkylation

Answer 17

• endogenous and chemical
• O6-MG is an alkylation of the oxygen atom of guanine and highly mutagenic
• 1 of 8 unprepared O6-MG damages is leading to a mutation > high cancer risk
• Repaired by O6-methylguanine-DNA methyltransferase (MGMT)
• MGMT is a suicide enzyme (1 MGMT protein can repair 1 damage)

DNA damage -> Synthesis -> MGMT -> Transfer of Methyl group -> Degradation

Question 18

Oncogenes vs Tumor suppressor genes

Answer 18

Proto-oncogenes: code for proteins that stimulate the cell cycle and promote cell growth and proliferation

Tumor suppressor genes: code for proteins that repress cell cycle progression and promote apoptosis

-> oncogenes result from the activation (turning on) of photo-oncogenes
-> tumor suppressor genes cause cancer when the are inactivated (turned off)

Question 19

Tumor suppressor genes

Answer 19

Functions:
1) Repression of genes that are essential for counting the progression of the cell cycle
2) Coupling the cell cycle to DNA damage
3) If damage cannot be repaired, the cell should initiate apoptosis
4) Proteins that function in DNA repair, preventing cells from replicating mutations
5) Some proteins involved in cell adhesion prevent tumor cells from dispersing, block loss of contact inhibition and inhibit metastasis

Some examples
pRb (retinoblastoma) -> proliferation
APC (colorectal cancer) -> proliferation
p53 (many tumors) -> Division, apoptosis
BRCA1 (breast cancer) -> DNA repair

Question 20

Tumor supressor genes -> Gatekeeper

Answer 20

Genes that directly hinder cell division or promote cell differentiation or cell death
- Mutations lead to the appearance of unregulated dividing cells (e.g. pRB in Retinoblastoma)

Question 21

Tumor suppressor genes -> Caretaker

Answer 21

Genes that encode proteins responsible for maintaining the integrity of the genome
-> Inactivation of a caretaker gene:
- does not promote tumor initiation directly
- but leads to genomic instability that increase the frequency of mutations (e.g. BRCA1 and 2 (repair of DNA breaks) in breast cancer)

Question 22

Tumor supressor genes -> Landscaper

Answer 22

Genes that encode products that help creative environments that control cell growth
- Regulation of extracellular metric proteins, cellular surface markers, cellular adhesion molecules and growth factors (e.g. PTEN)

Question 23

Loss of heterozygosity (LOH)

Answer 23

-> ONE mutant Rb allele
- Heterozygous configuration (Rb +/-): 1 wildtype and 1 defective gene copy
- appearance of wild-type phenotype
-> TWO mutant Rb gene copies
- Loss of heterozygosity (Rb -/-): 2 defective gene copies
- Appearance of retinoblastoma phenotype
-> Tumor suppressor genes are recessive!
-> Both Rb copies must be affected before an effect is manifested

mechanisms:
1) Loss of an entire chromosome by chromosome non-disjunction during mitosis
2) Loss of an entire chromosome by chromosome non-disjunction and recombination of the mutant allele
3) Chromosomal translocation
4) Deletion of the “wild type” locus (rare)

Question 24

Dominant-negative mutation of p53 gene

Answer 24

• p53 normally functions as a homotetrameric transcription factor
• missense mutation in the DNA binding domain results in the expression of altered proteins which results in the expression of altered proteins which can form tetrameters but cannot bind to DNA
• all tetramers with one or more mutant p53 subunits are defective in their function as transcription factor and are more stable than wild type p53

Question 25

Hepatitis B virus genome

Answer 25

- virus particles have a partially double-stranded genome - although HBV is a DNA virus, it replicates through a pre-genomic RNA intermediate - the virus encodes proteins from five genes on the minus strand of HBV DNA -> S gene: hepatitis B surface antigen (HBsAg) and glycosylated partner, transmembrane proteins in the virus envelope -> C gene: hepatitis B core antigen (HBcAg) which forms the nucleocapsid of the virus -> E protein: hepatitis B e antigen (HBeAg); variant of a c antigen, transferred into blood -> P region: viral reverse transcriptase; DNA-dependent DNA polymerase and RNase H activity required for virus replication -> X gene: hepatitis Bx (HBx), a small regulatory protein, transactivating protein and stimulates virus gene expression and replication, protects virus-infected cells against immune-mediated destruction

Question 26

Hepatitis C virus genome

Answer 26

- HCV is a positive, single-stranded RNA virus that encodes a large poly protein of about 3,000 amino acids from a single open reading frame (ORF) - Flanked by two untranslated regions (UTRs), which contain signals fro viral protein and RNA synthesis - Translation is initiated through an internal ribosomal entry site (IRES) in the 5' UTR - structural proteins: the core protein and the two envelope glycoproteins (E1 and E2) - p7 is an ion channel protein that promotes virus assembly - non-strucutral (NS) proteins are required for viral replication

Question 27

Tip cell formation

Answer 27

- Endothelial cells spearheading the vascular sprouts - characterized by position, long and dynamic filopodia and migratory behavior - respond to attractive and repulsive directional cues presented by the environment and thereby define the route in which the new sprout grows - required to create new connections between different sprouts to generate an interconnected and functional vascular network

Question 28

Epithelial to mesenchymal transition (EMT)

Answer 28

- loss of polarity - loss of cell-to-cell and cell-to-basal lamina junctions - acquisition of a motile and migratory phenotype - enabling the invasion of the basal lamina - epithelial cells loose their cell polarity and cell-cell adhesion and gain migratory and invasive phenotype of mesenchymal cells - EMT is involved on physiological embryonic development, but also recapitulated under pathological conditions, prominently in fibrosis and in metastasis of carcinomas -> EMT program is a spectrum of transitional stages between the epithelial and mesenchymal phenotypes

Question 29

EMT in context of metastasis

Answer 29

- epithelial cell marker: E-cadherin, cytokeratin - mesenchymal cell marker: N-cadherin, vimentin - signaling pathways induce EMT-transcription factors (EMT-TFs) such as Twist

Question 30

Epithelial vs mesenchymal

Answer 30

EPITHELIAL - cell polarity - cell adhesion (to each other and to extracellular matrix) - stationary - high level of E-cadherin - low level of N-cadherin MESENCHYMAL - no cell polarity - loss of cell adhesion - ability to migrate and invade - low level of E-cadherin - high level of N-cadherin

Question 31

Pre-metastatic niche -> Establishment of the pre-metastatic niche (PMN)

Answer 31

- vascular leakiness: Clot formation and increased micro vessels permeability -> vascular endothelial growth factor (VEGFA) -> Angiopoietin-like 4 (ANGPTL4) -> Focal adhesion kinase (FAK) - local increase in cytokines like chemokine C-C motif ligand 2 (CCL2) correlates with the recruitment of various bone marrow-derived cell populations - clotting by platelets promote initiation of coagulation and clot formation by tissue factors (TF)

Question 32

Pre-metastatic niche -> Evolution of the pre-metastatic niche

Answer 32

Extracellular matrix (ECM) is actively deposited and remodeled - accumulation f fibronectin (FN) and cross linking of collagen I (via lysyl oxidase (LOX)) - secretion of matrix metalloproteinases (MMPs) Inflammatory cell recruitment - transforming growth factor-beta (TGFbeta)-dependent production of proteins such as periostin - up regulation of S100 proteins generates inflammation and recruitment of hematopoietic progenitor cells and differentiated immune cells

Question 33

Cancer therapy types

Answer 33

1) Hormone therapy 2) Surgery 3) Bone marrow transplantation 4) Chemotherapy 5) Targeted therapy 6) Radiation therapy 7) Immunotherapy

Question 34

Graft versus host disease

Answer 34

- occurs during allogeneic stem cell therapy - inflammatory mechanisms mediated by donor lymphocytes infused into the recipient - tissues produce molecules such as proinflammatory cytokines and chemokines - increase in expression of key receptors on antigen-presenting cells - enhancing cross penetration of polypeptide proteins to the donor immune cells Skin: maculopapular skin rash Upper gastrointestinal tract: nausea, anorexia Lower gastrointestinal tract: watery diarrheo, severe abdominal pain, bloody diarrhea or ileum

Question 35

Graft versus host disease -> Mechanism

Answer 35

1) Host APC activation 2) Donor T-cell activation 3) Cellular and inflammatory effectors

Question 36

Design of personalized clinical trials

Answer 36

Modern clinical trial strategies (Umbrella or basket strategy) include gene expression profiling into the descision of treatment type UMBRELLA CLINICAL TRIALS Multiple drugs tested against multiple genetic mutations within a single cancer type -> plasmaMATCH for advanced breast cancer BASKET CLINICAL TRIALS A handful (typically one or two) of drugs tested against a handful of genetic mutations across multiple cancer types -> vermurfenib is a treatment for a rare blood cancer Erdheim Chester Disease (EVD) -> patients have BRAF V600 genetic mutation

Question 37

Targeted therapy

Answer 37

1) Sustaining proliferative signaling -> EGFR inhibitors 2) Evading growth suppressors -> Cyclin-dependent kinase inhibitors 3) Avoiding immun destruction -> Immune activating anti-CTLA4 mAb 4) Enabling replicative immortality -> Telomerase inhibitors 5) Tumor promoting inflammation -> Selective anti-inflammatory drugs 6) Activating invasion and metastasis -> Inhibitors of HGF/c-Met 7) Inducing angiogenesis -> Inhibitors of VEGF signaling 8) Genome instability and mutation -> PARP inhibitors 9) Resisting cell death -> Proapoptotic BH3 mimetics 10) Deregulating cellular energetics -> Aerobic glycolysis inhibitors

Question 38

Biomarker used in cancer

Answer 38

Biomarkers are measured as an indicator of normal biological processes, pathogenic processes or responses to an exposure or intervention, including therapeutic interventions Human papilloma virus (HPV) -> Cervical cancer BRCA1 and BRCA2 gene mutations -> Breast ovarian, cervical cancer CA125 protein -> ovarian cancer alpha-fetoprotein -> Hepatocellular carcinoma Prostate specific Antigen (PSA) -> Prostate cancer HER2/neu gene mutations -> Breast cancer Calprotectin -> Colorectal inflammations and cancer KIT gene -> Acute myelogenous leukemia and gastrointestinal stromatumors EGFR, ALK and KRAS gene mutations -> lung cancer

Question 39

What are microRNAS?

Answer 39

Feature: Single stranded (ss) non-coding RNAs (20-25 nucleotides) Occurence: Almost all eukaryotes encode miRNAs Function: - RNA silencing: Suppress gene expression in a sequence specific manner at post-transcriptional level - RNA interference (RNAi) - posttranscriptional gene silencing (PTGS) - components of ribonucleoprotein complexes: RNA induced silencing complex (RISC) - miRNAs bind to complementary target mRNAs - complementary in part or in whole to target mRNAs

Question 40

Formation of the RNA-induced silencing complexes (RISC)

Answer 40

Loading of the miRNA duplex - Compose of one of the four Human Argonaute proteins (AGO1-4), Dicer and a dsRNA binding protein (e.g. TRBP or PACT) - Loading of miRNA duplex onto Argonaute Selection of the mature miRNA strand - Pre-RISC consist of a Argonaute and a miRNA duplex - Removal of the passenger strand leads to the formation of an activated RISC - the active RISC (miRISC) contains a single stranded miRNA (guide strand) which directs the complex to the target mRNA for posttranscriptional gene silencing

Question 41

Model of microRNA (miRNA)-mediated gene silencing

Answer 41

- miRISC binds to the 3' UTR of the target mRNA - AGO recruits the scaffold protein GW182 - GW182 interacts with poly(A)-binding protein and de-adenylase complexes 1) Repression of cap-dependent translation via CCR4-NOT (de-adenylation independent) 2) Deadenylation by de-adenylase complexes 3) Removement of 5'cap by decapping proteins 4) mRNA degradation by 5'-3' exonuclease

Question 42

Apoptosis occurs throughout the whole life of an organism

Answer 42

Removal of individual cells during embryogenesis and in adult organisms 1) Morphogenesis 2) Maintain of homeostasis of tissues, organs and organ systems 3) Elimination of cells with damaging potential

Question 43

Essential functions during embryo genesis

Answer 43

Morphogenesis - Shaping many developing structures - Tissues and organ development Examples Formation of - interdigital spaces of fingers and toes - middle ear spaces - nostrils - vaginal opening Elimination of about 50% of all neurons Important function during sexual differentiation -> In the male, the Müllerian ducts are lost -> In the female, the wolffian ducts are lost

Question 44

Essential functions in adult organism: life balance

Answer 44

Maintaining tissue architecture and homeostasis - Regeneration of tissues, organs and organ systems - Maintenance of cell count - About 10x10^9 cells are generated by mitosis each day and the same number of cells die through apoptosis Deregulated apoptosis 1) Too little cell death - Autoimmune diseases - cancer 2) Too much cell death - neurodegenerative diseases - AIDS

Question 45

Exon skipping in Duchenne Muscular Atrophy

Answer 45

DMD patient: out of frame -> Premature stop codon -> no dystrophin produced Eteplirsen treated DMD patient: Reading frame restored -> Shortened, functional dystrophin produced

Question 46

Spinal Muscular Atrophy -> Nusinersen

Answer 46

-> Antisense treatment of patient with SMA -> no SMAmRNA produced - gehört zu den Antisense-Oligonukleotiden - Nusinersen beeinflusst das alternative Spleißen des SMN2-Gens, sodass es funktionell in ein SMN1-Gen umgewandelt wird. Dadurch steigen die SMN-Spiegel im ZNS und im peripheren Nervengewebe an

Question 47

Spinal Muscular Atrophy -> Zolgensma

Answer 47

-setzt einen rekombinanten AAV9-Vektor ein, um eine gesunde Kopie des SMN1-Gens als doppelsträngige DNA in die betroffene Nervenzellen einzuschleusen. Die DNA wird jedoch nicht in die chromosomale DNA des Patienten integriert, sondern getrennt von ihr als Episom im Zellkern abgelegt. Ein im Arzneimittel enthaltener konstitutiver Promoter bewirkt die Expression des therapeutischen Gens, sodass eine anhaltende, kontinuierliche Biosynthese des SMN-proteins erfolgt.

Question 48

Spinal Muscular Atrophy -> Risdiplam

Answer 48

- Wirkung beruht auf einer erhöhten Produktion von stabilem und funktionellem "survival motor neuron protein" (SMN-Protein) - Die spinale Muskelatrophie wird durch eine Loss-of-function-Mutation des SMN1-Gens auf dem langen Arm von Chromosom 5 (5q) ausgelöst. Das SMN1-Gen kodierte das SMN-Protein, das eine zentrale Bedeutung für die Funktion von Motorneuronen hat. Das homologe SMN2-Gen kodiert ebenfalls für das SMN-Protein. Beim Spleißen der SMN2-mRNA wird jedoch Exon7 entfernt, sie resultiert daher in einer instabilen verkürzten Variante des Proteins, das seine Funktion nicht erfüllen kann. - Risdiplam beeinflusst das alternative Spleißen und bewirkt die Inklusive von Exon 7. Somit kann das SMN-Protein wieder in voller Länge produziert werden. Je nachdem, wie viele Kopien vom SMN2-Gen vorliegen, kann somit der Verlust von SMN1 teilweise kompensiert werden.

Question 49

Duchenne muscular dystrophy -> different therapies

Answer 49

Eteplirsen -> Antisense oligonucleotide Victolarsen -> Antisense oligonucleotide Golgodirsen -> Antisense oligonucleotide Casimersen -> Antisense oligonucleotide

Question 50

EMT - transcription factors

Answer 50

- Extracellular signaling pathways including TGF-beta, Wnt and Notch activate EMT-inducing transcription factors (EMT-TFs) such as snail, slug, Twist and ZEB-1/2. - EMT-TFs recruit various co-repressors which cooperatively repress CDH1 transcription - ZEB-1/2 is regulated through negative feedback loop involving microRNAs - CDH1 promoter is flanked by repressive histone marks like H3K9me2, H3K27me3, H4K20me1; loss of histone variant: H2A.Z - DNA methyltransferases can be recruited to the CDH1 promoter to repress gene transcription

Question 51

Tip and stalk cell communication

Answer 51

How to select which cell is a tip and which is a stalk cell? - tip cells express VEGFR2/3 together with several co-receptors (neuropilin-1 (NRP1)) - tip cell attraction by VEGF gradients - upon activation: tip cells express DLL4, that binds to Notch receptors on follower stalk cells - in stalk cells: Notch is cleaved by gamma-secretase releasing Notch intracellular domain (NICD) - NICD dampens VEGFR2/3 expression and increases VEGFR1 expression - VEGFR1 is released by stalk cells and binds to VEGF molecules to block local VEGF signaling - Enhancement: Jagged1 (Jag1) is expressed by stalk cells and negatively regulates Notch activity in tip cells