Chapter 14: Technology and drugs and diagnostics development (Secondary Details)

Question 1

When looking at the phase I results of imatinib in GIST, what would recommend as the maximum tolerated dose?

Answer 1

400mg (500mg has too many side-effects)

Question 2

When looking at the efficacy of the Phase III results of imatinib in GIST, what would you recommend as effective dose?

Answer 2

The difference between once and twice a day is limited, and as we saw earlier that a high dose can give lots of side-effects, 400 mg is the tolerated dose. Moreover, the side-effects were also measured comparing the one- and two-dose, as you can see in this figure, and the high amount of side effects reflects the earlier found results

Question 3

What did the phase I study show when Vemurafenib was investigated in melanoma?

Answer 3

The tumor cells disappeared

Question 4

A ‘3+3’ study design was used in the phase I trial of Vemurafenib and the results are seen in the table. What would you predict to be the maximum tolerated dose (MTD)?

Answer 4

1120mg was considered too toxic, so therefore they chose 960mg to be the MTD

Question 5

What does this PET-scan of a metastatic melanoma patient treated with BRAF-inhibition (Vemurafenib) show?

Answer 5

That there is a very good response. The black spots are high metabolic places and you see an immense decrease in day 15 compared to the baseline

Question 6

We know that the combined treatment of BRAF- and MEK-inhibitors is superior to BRAF-inhibitors alone, but did it also increase the survival rate?

Answer 6

Yes it did, it increased from ~18 months to ~25 months

Question 7

What does this waterfall plot show?

(the change in growth of the tumor is shown on the y-axis and the x-axis shows the patients)

Answer 7

That the KRAS mutations do not show any response to the treatment, and the wild type KRAS show about 60-70% response to the treatment

Question 8

Can you explain this diagram?

Answer 8

The left diagram shows how when the EGFR is mutated this normally results in proliferation and angiogenesis, when there is no mutation in one of the downstream proteins, when antibodies are administered this pathway this will result in sensitivity and the pathway is blocked. The right diagram shows how when, even when the EGFR is blocked, the downstream pathways are still activated and result in resistance, because the antibody does not work

Question 9

This is a graphic representation of how molecular diagnostics can be used to detect if patients are sensitive to the treatment or not

Answer 9

Very interesting! :)

Question 10

This is for illustration!!! What diseases can be treated with imatinib?

Answer 10

Chromic myeloid leukemia, acute lymphoblastic leukemia, meylodysplastic and myeloproliferative disease (with PDGR gene re-arrangements)

Question 11

This is for illustration!!! What diseases can be treated with Vemurafenib?

Answer 11

Melanoma, Erdheim-Chester disease

Question 12

Explain the typical protocol of microarrays (in great detail), which use of this figure

Answer 12

Thousands of gene-specific hybridization probes are applied to a glass slide or silicon chip (a). The DNA probes are usually bound to defined locations on the grid by robotic or laser technology. RNA is isolated from a biological sample, such as a tumor, and copied to incorporate fluorescent nucleotides or a fluorescent tag (b). The chip is then incubated with labeled RNA or complementary DNA (cDNA) from the tumor sample (c). Unhybridized RNA is washed off, and the microarray is then scanned under a laser and analyzed by computer (d). A sample microarray image is shown in e.

Question 13

What are the two types of microarrays that are common? What is the difference between the two?

Answer 13

cDNA microarrays and oligonucleotide microarrays. The difference between the two is due to the nature of the probes. In cDNA microarrays, each probe has its own ideal hybridization temperature (based on factors such as CG content), and thus intensities of a test sample must always be compared with a control sample processed at the same time. In oligonucleotide microarrays, the synthetic probes are disgned such that all the probes have identical hybridization temperatures,, allowing absolute values of expression to be measured within one sample

Question 14

In what way can the results of microarrays be visualized?

Answer 14

• Heat map that uses color to represent levels of gene expression
• Genes may be arranged in rows, and time points may be arranged in columns
• A red box may be used to indicate an increase in expression relative to a control
• A green box may be used to indicate a decrease in expression relative to the control
• A black box represents no change
• Similarities in the patterns of gene expression can be depicted in clusters in a cluster analysis diagram

Question 15

Can you give an example of how the use of microarrays lead to the identification of a molecular subtype of cancer

For illustration

Answer 15

The application of a lymphochip, a microarray that screens for genes important in cancer, immunology and lymphoid cells, helped to define two molecularly distinct forms of a particular lymphoma. Results from the lymphochip showed to distinct patterns of gene expression that were differentiation stage-specific and also corresponded to different lcinical outcomes (76% of one subgroup was alive after 5 years, compared with only 16% of the other).

Question 16

Whole-genome expression profiling has been used for classification and progneosis of lymphoma. What is tested in trials?

Answer 16

Different protocols for identifying patients based on their molecular subtype for personalized treatment. DNA arrays have also been used to refine molecular calssifications to other cancers, including breast cancer

Question 17

What were the aims and results of two studies that studied the gene expression for breast cancer?

Answer 17

The main purpose of analzying the selected genes was to identify tumors that are unlikely to metastasize in order to spare patients the trauma of chemotherapy. Only a small proportion of patients whose breast cancer has nto spread to the lymph nodes develop metastases (20%) and really require chemotherapy. Most node-negative breast cancer patients are cured by surgery and radiotherapy alone. Scores of less that 17 (low recurrance score) or scores of 31 and higher (high recurrence score) for the test are being used to predict the benefit of chemotherapy for breast cancer patients. Such gene profiling tests may spare patients from receiving unneccessary chemotherapy

Question 18

How does spectrometry analysis work?

(= allows for the simultaneous examination of thousands of proteins in a biological sample)

Answer 18

Proteins are treated with enzymes to create peptides. These peptides can be fractionated prior to separation and ionization in the spectrometer. Afterwards, the ionized petpides are fragmented, ant eh mass-to-charge ratios of the ionized products provide amino acid sequence information thorugh the spectrum produced and bioinformatics

Question 19

Where was the biomarker prostate-specefic antigen (PSA) used for?

Answer 19

Elevated levels of PSA detceted in the blood has been the conentional prostate tumor for screening (in the past)

Question 20

There are 70-80% negative biopsy rates. Why is that?

Answer 20

PSA is not specific for prostate cancer. Elevated levels are also detected in benign prostate conditions

Question 21

The prostate cancer antigen 3 (PCA3) gene has been identified as the most prostate cancer-specific gene described thus far.

Answer 21

Nice

Question 22

What is a futuristic idea to detect CTCs and miRNAs in the clinic?

Really don’t think you have to know this, but I bet you can’t imagine what’s on the other side of this card ;)

Answer 22

To implant a gene chip under the skin to monitor changes in such biomarkers, thus speeding up diagnosis and facilitating early treatment

Question 23

How does CRISPR-Cas9 work?

Answer 23

The CRISPR-Cas9 system is derived from the prokaryotic adaptive immune system. There are two components of the system: a DNA endonuclease (Cas9) and a chimeric single guide RNA (sgRNA) that guides Cas9. The sgRNA contains two parts: a CRISPR RNA part that binds to a 20-nucleotide genomic DNA target site by Watson-Crick base pairing and a trans-activating CRISPR RNA ocmponent that binds to the Cas9 endonuclease. the target sequence must be near an NGG or NAG trinucleotide (protospacer adjacent motifs PAMs). These molecules generate double-strand breaks 3 bp 5’ of the PAM in the targetsite that is repaired by either of two endogenous DNA repair pathways: non-homologous end joining or homology directed repair. Repair leads to either small insertions or deletions., creasting a loss of funciton mutation or pecise modification for creating gain-of-function muations

Question 24

How can CRIPR be used?

Answer 24

To engineer embryonic stem cells rapidly for “traditional” mouse models but has the advantage of being able to target mutliple loci at a time, and has been used to engineer tumor-associated chromosomal translocations. It can also be used to study engineer cells ex vivo to study lelukemias. It has even been used to make somatic mutations in in vivo in mice to indcue tumors of the liver. The potential for applying this to patients is also exciting and inclused ex vivo engineering of immune cells for immunotherapy

Question 25

What are some molecular features that may be examined by MFR?

Answer 25

overexpression of receptors, gene expression or cellular location.

Question 26

What are some functional features examined by MFI?

Answer 26

aspects of angiogenesis (e.g. vascular volume, vascular permeability, hypoxia) and metabolism (glycolytic activity)

Question 27

What are the advantages of MFI?

Answer 27

MFI promises to have a major impact on eaerly diagnosis, as well as on the monitoring of disease progression upon treatment. Early dection is associated with a 5-year survival rate of more than 90%. Refined imagin may also decrease the number of biopsies taken in the clinic. MFI is another approach that will halp match tumor to therapy. Such imaging may also serve as a guide to the administration of future therapeutics such as nano– and microdevices

Question 28

We are going to look at the development of imatinib, in relation to the stages of drug development. Roughly explain these steps

Answer 28

1. Chromosomal translocation that is characteristic of CML
2. BCR-ABL was identified as molecular target, as the translocation results in elevated tyrosine kinase activity
3. A lead compound (=kinase inhibitor) was identified in a chemical screen
4. Optimization of the coumpound by addition of small chemical groups iwth good bioavailability
5. Pre=clinical testing: selective inhibition of BCR-ABL kinase activity and induction of apoptosis in cell culture and leukemic cells from patients and in animal models
6. Clinical trials (phase I) documented safety and efficacy
7. Phase II and III showed positive results
8. Approval of FDA

Question 29

How does imitinib resistance occur?

Answer 29

By point mutations in the BCR-ABL gene that interfere with the binding of imatinib, but maintain a functional tyrosine kinase domain. Thus a second-generation inhibitor is made that only binds to the active conformation of the ABL-kinase

Question 30

What therapy can be given to patients who got both imatinib and second-generation tyrosine kinase inhibitor, and still were resistant?

Answer 30

Third-generation tyrosine kinase inhibitos, and include allosteric inhibitors that do not compete for ATP binding

Question 31

A novel trial design is randomized discontinuation trial design. How does this work?

Answer 31

Patients receive one or two cycles of a cytostatic drug, and upon completion, those who have stable disease are then randomized to placebo or to continue the same treatment. The aim of this design is to enrich the patient population with slowly progressive cancer and eliminate those with rapidly progressive cancer