3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Targeting LRP/LR for the treatment of metastatic lung and colorectal cancer through impediment of telomerase activity(2018) Baichan, PavanThe 37 kDa/67 kDa laminin receptor (LRP/LR) plays a vital role in the malignancy of various cancer types contributing to invasion, adhesion, apoptosis evasion, proliferation and tumour angiogenesis. In addition, LRP/LR interacts with the catalytic reverse transcriptase subunit, TERT, of the ribonucleoprotein telomerase. Both LRP/LR and telomerase are implicated in cancer progression and knockdown of LRP/LR causes a decrease in telomerase activity in breast cancer cells. In the current study, LRP/LR was downregulated in lung adenocarcinoma (A549) and late-stage colorectal carcinoma (DLD-1) cells in an attempt to impede telomerase activity and ultimately impede cancer progression. Western blotting analysis showed a significant decrease in LRP/LR levels in HEK293 (Human Embryonic Kidney Cells) and A549 cells after siRNA mediated LRP/LR knockdown. To confirm LRP/LR knockdown confocal microscopy was performed; a reduction in LRP/LR protein levels was observed which also resulted in a subsequent decrease in hTERT mRNA levels with a corresponding decrease in hTERT levels in HEK293, A549, and DLD-1 cell lines. Furthermore, siRNA mediated knockdown of LRP/LR significantly decreased telomerase activity in HEK293, A549, and DLD-1 cells. The effect of LRP/LR downregulation on cellular viability was investigated via the MTT assay and a significant decrease in cell viability in A549 and DLD-1 cells was observed. Since downregulation of LRP/LR impedes telomerase activity and decreases cell viability, siRNAs directed against LRP mRNA acts as potential alternative therapeutic tools for treatment of lung adenocarcinoma and late-stage colorectal carcinoma.Item Epidermal growth factor receptor and anaplastic lymphoma kinase mutations detected by immunohistochemistry in lung adenocarcinoma in patients from Johannesburg(2017) Vorajee, NaseemaLung cancer is the leading cause of cancer related death worldwide. Despite the availability of conventional cytotoxic chemotherapy, prognosis even with treatment is poor. Treatment targeted to specific molecular alterations called Tyrosine kinase inhibitors (TKI) has been found to be effective in certain subtypes of non small cell carcinoma (NSCC) that have the epidermal growth factor receptor (EGFR) gene mutation and the anaplastic lymphoma kinase (ALK) translocation. EGFR mutations are seen in 18-25% of lung adenocarcinomas (AC)s and are represented in more than 90% of cases by the E746_A750 deletion on exon 19 and the L858R point mutation on exon 21. The ALK translocation is seen in 2-7% of lung cancers, and involves the EML4-ALK fusion gene product. Immunohistochemistry (IHC) has the potential of being used as an initial screening tool that can facilitate a shorter diagnostic time and fast track treatment options. Little is known about the mutational status of patients with these mutations in South Africa. This study examines the use of IHC as a means of detecting the most common EGFR mutations and the ALK translocation in lung cancer. Method: Biopsies of patients from the Charlotte Maxeke Johannesburg Academic Hospital (CMJAH) and Helen Joseph Hospital (HJH) sent to the National Institute for Occupational Health (NIOH) from 1st January 2008 to 30th June 2014 were reviewed. A total of 3901 histology reports were accessed. There were 297 lung carcinomas comprising 117 (39%) ACs and 128 (46%) squamous carcinomas (SCC)s. A total of 119 biopsies comprising 107 ACs (90%), 4 (3%) adenosquamous carcinomas (ADSCC)s, 1 (1%) large cell carcinoma (LC) and 7 (6%) NSCC (nos.) comprised the cohort. One hundred and eleven biopsies were available for mutational analysis, as 8 biopsies were excluded due to insufficient tissue availability. The mutation specific antibodies, EGFR SP111 and EGFR SP125 were used to detect the E746_A750 deletion and the L858R point mutations respectively. The high affinity ALK D5F3 antibody was used for the detection of the EML4-ALK translocation. Results: The majority of patients in the study were Black males (61%). There were no Asians. The mean age was 58 years with a SD of 11.5. Most patients (76%) were younger than 65 years. The EGFR IHC stain was positive in 10/111 (9%) biopsies, of v which 8 were from Blacks, 6 were from males, 4 were from smokers and 2 were from non-smokers. There was however no significant difference between the proportions of Black or White and male or female among those who tested positive to EGFR and those who tested negative (p>0.05) and no significant association was found between the variables age, sex and smoking history (p>0.05). The ALK IHC was positive in 8 (7%) patients. All 8 patients were Black, six were male, four had a smoking history and two patients were non-smokers. A significant association was found between race and positive ALK IHC (p=0.03). There was no significant association with age (p=0.081). The acinar growth pattern was found in 80% of the EGFR IHC positive biopsies, with 10% of biopsies showing either lepidic, solid or micropapillary patterns. Although several growth patterns were seen in ALK IHC positive biopsies, there was a slight predominance of the acinar and solid growth patterns. Discussion: The EGFR IHC was positive in 9% of patients, which is half the number of cases described in literature from Western and Eastern countries, where the prevalence is usually more than 18%. There may be several reasons for the lower rate of EGFR IHC positivity. Antigen degradation, intratumoral heterogeneity and a low sensitivity of the EGFR IHC antibody may have contributed. The demographic profile of patients with positive result following EGFR IHC differs from the literature although the results were not found to be statistically significant. The sensitivity of the EGFR IHC test as described in the literature ranges from as low as 61% to 100%. A good correlation between EGFR IHC and EGFR polymerase chain reaction, in confirming the presence of the EGFR mutation, is described with biopsies that show strong positive cytoplasmic staining with IHC. Strong positive cytoplasmic staining with EGFR IHC was found in these 10 biopsies. The ALK IHC result was positive in 7% of patients, which is on the upper limit of the 2-7% rate recorded in most literature. A significant association was found with Blacks. Although an association with younger patients and the ALK mutation was found, this was not statistically significant. As ALK IHC is associated with an almost 100% sensitivity and specificity, there is a possibility that the ALK mutational rate in South African Black patients may be higher than the rate in other international populations. SCC is the more common subtype of lung cancer (46%) compared to AC vi (39%) in this group of patients from Johannesburg. This differs from studies in Cape Town and international studies, where a shift in trend from SCC to AC is observed. Conclusion: This study confirms the presence of the EGFR mutation and ALK translocation in patients with adenocarcinoma from Johannesburg using immunohistochemistry. We have proposed a diagnostic algorithm for patients with lung cancer in South Africa where EGFR IHC and ALK IHC can be used as rapid initial screening tests to identify patients with the EGFR mutation and ALK translocation respectively, provided established guidelines for IHC interpretation are followed. This approach allows patients with lung cancer who have the EGFR and ALK mutations to be fast tracked towards receiving targeted therapy.