MSc (Med), Faculty of Health Sciences, University of the Witwatersrand / The HER2 gene is amplified in 20-30% of breast cancers, a common cancer amongst South African women. HER2 amplification is associated with a poor prognosis and predicts response to treatments such as Herceptin. The gold standard for HER2 testing is Fluorescent in situ Hybridisation (FISH) with dual colour probes for the HER2 gene and chromosome 17 centromere (CEP17) internal control. According to international guidelines, a HER2/CEP17 ratio >2.2 is considered positive. The HER2 FISH test is complicated by the emergence of ambiguous cases with increased CEP17 signals that cannot be accounted for by chromosome 17 polysomy (> 6 copies of CEP17) and that may hide true HER2 gene amplification. The aims of this study were to characterise the HER2 amplicon, in particular the copy number of genes in the vicinity of the HER2 gene, and to design an alternative control probe that could clarify the HER2 gene status in ambiguous cases. In addition, results on 1558 breast cancer specimens sent for routine testing were analysed to determine the trends of HER2 amplification amongst South African women. The rate of HER2 gene amplification was significantly higher (p < 0.05) in African patients (52%) than in Caucasian patients (43%). In Caucasian women, the rate of HER2 amplification in the younger group (68%) was significantly higher (p < 0.05) than in the general Caucasian group (43%), while the same was not seen in the African cohort. Nineteen ambiguous cases with more than 9 copies of CEP17 were further investigated. FISH assays with four different probe kits (PathVysion HER-2: Poseidon Repeat free TOP2A, HER2, CEP17: and Vysis PML-RARA respectively) were performed to determine the copy number of the HER2, TOP2A, RARA genes and CEP17. An in-house dual colour probe kit was designed using the ACTG1 gene as a control for HER2. Of the 19 ambiguous cases, 16 had centromeric amplification, showing that CEP17 is no longer an adequate internal control in FISH HER2 testing. The TOP2A gene was only amplified in HER2 positive cases and the RARA gene was only amplified when the TOP2A gene was also amplified. FISH with ACTG1 as v a control clearly revealed HER2 amplification in ambiguous cases on image analysis and gave HER2/ACTG1 ratios significantly higher than HER2/CEP17 ratios. However, screening of an additional 40 unambiguous cases showed an increased copy number, although limited ( 8), of the ACTG1 gene in four patients; this warrants further testing to assess the value of this gene as a control. Interestingly, a trend was observed for ACTG1 increased copy number in HER2 negative cases, this may point to the presence of a driver gene whose amplification tends to be mutually exclusive from HER2 amplification.

<p>Breast cancer mortality in Sweden is now in decline, thanks to early detection and the wide use of adjuvant endocrine therapy and chemotherapy. </p><p>While hormone receptor status is predictive of response to endocrine treatment, there is no clinically useful predictive marker of a patient’s response to chemotherapy. Consequently, patients receive chemotherapy with considerable toxicity but minimal benefit. The aim of this thesis was to investigate a number of methods with the potential to predict response to chemotherapy and thus enhance treatment efficacy in breast cancer patients.</p><p>We found that topo IIα, the key target enzyme of topo II inhibitors, is significantly expressed in nonproliferating breast cancer cells. This finding may explain why topo II inhibitors are effective in patients with slow growing tumors and a low proliferation rate.</p><p>Topo IIα gene amplification was suggestive of increased response to anthracyclines in advanced breast cancer, whereas the oncogene HER2 had no predictive value by itself. These findings are in accordance with current knowledge.</p><p>Cyclin A, a marker of cell proliferation, showed good prognostic value but did not predict response to chemotherapy in advanced breast cancer.</p><p>In vitro chemosensitivity testing with FMCA predicted tumor response in patients with advanced breast cancer with a sensitivity of 89% and a specificity of 53%. Our results are consistent with the results from similar assays, which predict drug resistance with good accuracy while clinical drug sensitivity is less reliably predicted. The use of FMCA and similar assays is not yet recommended outside clinical trials; their main utility is in preclinical testing of new anti-cancer drugs, including targeted therapies.</p><p>The combination of epirubicin, capecitabine, and cisplatin (EXC) demonstrated high clinical response rate (74%) and pathological complete response rate (22%) in locally advanced breast cancer, but with cumbersome toxicity. The fluoropyrimidine biomarkers TS, TP, and DPD did not predict response to the EXC regimen.</p>

Breast cancer mortality in Sweden is now in decline, thanks to early detection and the wide use of adjuvant endocrine therapy and chemotherapy. While hormone receptor status is predictive of response to endocrine treatment, there is no clinically useful predictive marker of a patient’s response to chemotherapy. Consequently, patients receive chemotherapy with considerable toxicity but minimal benefit. The aim of this thesis was to investigate a number of methods with the potential to predict response to chemotherapy and thus enhance treatment efficacy in breast cancer patients. We found that topo IIα, the key target enzyme of topo II inhibitors, is significantly expressed in nonproliferating breast cancer cells. This finding may explain why topo II inhibitors are effective in patients with slow growing tumors and a low proliferation rate. Topo IIα gene amplification was suggestive of increased response to anthracyclines in advanced breast cancer, whereas the oncogene HER2 had no predictive value by itself. These findings are in accordance with current knowledge. Cyclin A, a marker of cell proliferation, showed good prognostic value but did not predict response to chemotherapy in advanced breast cancer. In vitro chemosensitivity testing with FMCA predicted tumor response in patients with advanced breast cancer with a sensitivity of 89% and a specificity of 53%. Our results are consistent with the results from similar assays, which predict drug resistance with good accuracy while clinical drug sensitivity is less reliably predicted. The use of FMCA and similar assays is not yet recommended outside clinical trials; their main utility is in preclinical testing of new anti-cancer drugs, including targeted therapies. The combination of epirubicin, capecitabine, and cisplatin (EXC) demonstrated high clinical response rate (74%) and pathological complete response rate (22%) in locally advanced breast cancer, but with cumbersome toxicity. The fluoropyrimidine biomarkers TS, TP, and DPD did not predict response to the EXC regimen.

Urinary bladder cancer is a heterogeneous disease appearing in different forms, e.g. non-muscle invasive and muscle invasive. For all variants, the expression of proteins is interesting to analyze for diagnostic, predictive, prognostic and drug targeting purposes, since it reflects the altered gene expression causing the cancer. Since urothelial cells of the bladder are in direct contact with urine it is likely that this body fluid contains cancer-related proteins. In Paper I, unbiased analysis of proteins in urine from urinary bladder cancer patients and controls, using label-free quantification by mass spectrometry, was applied and four interesting proteins APOE, FGB, LRG and SERPINA1 were selected and further analyzed with western and dot blot. In Paper II, two more proteins, POLR1E and TOP2A, were validated as relevant proteins in bladder cancer urine. In Paper III and IV, the proteins GAL1 and STMN1 were investigated for their prognostic and therapeutic target potential in bladder cancer. In Paper II, III and IV, the expression of seven of the proteins were analyzed on tissue microarrays representing tumour tissue from 360 patients with different tumour stages. For the proteins identified by the urine screening approach, their protein expressions were confirmed in bladder cancer tissue. The expression level in tissue of five of the proteins, APOE, FGB, POLR1E (Paper II), GAL1 (Paper III) and STMN1 (Paper IV), increased with tumour stage, showing diagnostic relevance and three of the proteins, SERPINA1 (Paper II), STMN1 (Paper IV) and GAL1 (Paper III) had prognostic potential in urinary bladder cancer. In addition, GAL1 and STMN1 were demonstrated to be highly expressed in metastatic disease and inhibition of STMN1 reduced cell growth (Paper III and IV), indicating that these proteins are promising drug targets in urinary bladder cancer. In conclusion, the approach of this thesis has generated several candidate protein biomarkers in urine and tissue, validated with independent methods, which have the potential to improve the care for bladder cancer patients.