Answer Happy

Data Analysis and Interpretation (DAI). Answer ALL questions in this section. This section contributes two thirds of the marks for the examination paper. 1. BRCA2 is a tumour suppressor gene. CRISPR was used to knock-out one (BRCA2"') or both alleles (BRCA2) of BRCA2 in Hela cells. The parental line, which has 2 functional copies of BRCA2 (BRCA2*, was used as a control. Cells were treated with the PARP inhibitor Olaparib and the effect upon cell viability (Figure 1a) and cell death (Figure 1b) measured. a) b) Olaparib sensitivity (HeLa cells) BRCA2 + 23,918 1,082 BRCA2 23,663 1,337 DMSO 5.35% Cellular viability BRCA2+/+ BRCA21- + BRCA2- 0.5 23,536 1,464 19,679 5,321 PI Olaparib 0 2 4 8 uM olaparib AnnexinV-FITC Figure 1. CRISPR was used to knock-out the BRCA2 gene in Hela cells. a) Wild-

Figure 1. CRISPR was used to knock-out the BRCA2 gene in Hela cells. a) Wild- type (BRCA2"*), heterozygote (BRCA2*) and homozygote (BRCA2") cells were treated with different concentrations of Olaparib, a PARP inhibitor, and the number of cells surviving quantified. b) BRCA2** and BRCA2" cells were treated with Olaparib or DMSO (vehicle control) and Annexin V staining performed to quantify the number of cells alive/dead. A total of 25,000 cells were analysed per condition and the number of cells with low or high Annexin V are provided in the figure. (a) Evaluate the data in Figure 1a (25%). (b) Using the data provided in Figure 1b, calculate the percentage of cells alive and dead in response to Olaparib treatment in the BRCA2+/+ and BRCA2- 7- cells (25%). (c) Evaluate the percentages that you have calculated. What effect does Olaparib have on the cells and does BRCA2 status affect this? [25%). (d) With reference to the data in Figure 1, evaluate if Olaparib is an effective treatment for all cancer patients and summarise how this information could be used to identify which patients would benefit most from this therapy. [25%] 2. Programmed death-ligand 1 (PD-L1) expression has been found to be an important factor in cancer patient survival and hence therapies have been designed to target this molecule. PD-L1 expression was analysed in patients with non-small cell lung cancer (NSCLC) and survival monitored (Figure 2a). To assess the effect of PDL 1-targeting therapies, a NSCLC mouse model was treated with anti-PDL1 with and without the nhamothorannustin Cinnlatin T..macrolimalan mendiminnallinare

2. Programmed death-ligand 1 (PD-L1) expression has been found to be an important factor in cancer patient survival and hence therapies have been designed to target this molecule. PD-L1 expression was analysed in patients with non-small cell lung cancer (NSCLC) and survival monitored (Figure 2a). To assess the effect of PDL 1-targeting therapies, a NSCLC mouse model was treated with anti-PDL1 with and without the chemotherapeutic Cisplatin. Tumour volume was measured using callipers for up to 60 days (Figure 2b). a) b) 100- 2000 PD-L1 negative PD-L1 positive 80- 1500 Placebo Cisplatin Anti-PDL1 Anti-PDL1+Cisplatin 60- Survival (%) 1000 Tumor volume (mm) 40- 500 20- 0 0 80 100 120 20 40 60 Months 0 10 20 30 40 50 60 Day Figure 2. a) Cancer samples were taken from patients with non-small cell lung cancer (NSCLC) and Programmed death-ligand 1 (PD-L1) expression analysed. Patients were split into those whose tumours expressed PDL-1 (PD- L1 positive) and those that did not (PD-L1 negative), and survival was recorded

2. Programmed death-ligand 1 (PD-L1) expression has been found to be an important factor in cancer patient survival and hence therapies have been designed to target this molecule. PD-L1 expression was analysed in patients with non-small cell lung cancer (NSCLC) and survival monitored (Figure 2a). To assess the effect of PDL 1-targeting therapies, a NSCLC mouse model was treated with anti-PDL1 with and without the chemotherapeutic Cisplatin. Tumour volume was measured using callipers for up to 60 days (Figure 2b). a) b) 2000 100- PD-L1 negative PD-L1 positive 80- 1500 Placebo Cisplatin Anti-PDL1 Anti-PDL1+Cisplatin 60- Survival (%) 1000 Tumor volume (mm) 40- 500 20- 0 0 80 100 120 20 40 60 Months 0 10 20 30 40 50 60 Day Figure 2. a) Cancer samples were taken from patients with non-small cell lung cancer (NSCLC) and Programmed death-ligand 1 (PD-L1) expression analysed. Patients were split into those whose tumours expressed PDL-1 (PD- L1 positive) and those that did not (PD-L1 negative), and survival was recorded

10-10 Wed 18 May 9 100% faser essex.ac.uk 0+ 0 20 40 80 100 120 10 20 30 40 50 . 60 60 Months Day Figure 2. a) Cancer samples were taken from patients with non-small cell lung cancer (NSCLC) and Programmed death-ligand 1 (PD-L1) expression analysed. Patients were split into those whose tumours expressed PDL-1 (PD- L1 positive) and those that did not (PD-L1 negative), and survival was recorded over 120 months. b) A mouse model of NSCLC was used to investigate the effects of targeting PD-L1. Mice were treated with placebo (control), Cisplatin, anti-PDL1, Cisplatin + anti-PDL1. Tumour volume was measured using callipers for up to 60 days. (a) Evaluate the data in Figure 2a [25%]. (b) With reference to Figure 2a, discuss why PD-L1 expression affects patient survival (25%). (c) Evaluate the data in Figure 2b (25%). (d) Using the data in Figure 2a and b, discuss if the anti-PD-L1 therapy is an effective treatment option for NSCLC (25%). END OF SECTION AD

10-10 Wed 18 May 9 100% faser essex.ac.uk 0+ 0 20 40 80 100 120 10 20 30 40 50 . 60 60 Months Day Figure 2. a) Cancer samples were taken from patients with non-small cell lung cancer (NSCLC) and Programmed death-ligand 1 (PD-L1) expression analysed. Patients were split into those whose tumours expressed PDL-1 (PD- L1 positive) and those that did not (PD-L1 negative), and survival was recorded over 120 months. b) A mouse model of NSCLC was used to investigate the effects of targeting PD-L1. Mice were treated with placebo (control), Cisplatin, anti-PDL1, Cisplatin + anti-PDL1. Tumour volume was measured using callipers for up to 60 days. (a) Evaluate the data in Figure 2a [25%]. (b) With reference to Figure 2a, discuss why PD-L1 expression affects patient survival (25%). (c) Evaluate the data in Figure 2b (25%). (d) Using the data in Figure 2a and b, discuss if the anti-PD-L1 therapy is an effective treatment option for NSCLC (25%). END OF SECTION AD