Tumour Infiltrating Lymphocytes (TILs) and immune composition in breast cancer patients from Kenya: Spatial distributions and associations with risk factors and tumour characteristics.

BACKGROUND: The immune landscape of breast cancer (BC) in patients from Sub Saharan Africa is understudied. Our aims were to describe the distribution of Tumour Infiltrating Lymphocytes (TILs) within the intratumoural stroma (sTILs) and the leading/invasive edge stroma (LE-TILs), and to evaluate TILs across BC subtypes with established risk factors and clinical characteristics in Kenyan women. METHODS: Visual quantification of sTILs and LE-TILs were performed on Haematoxylin and eosin -stained pathologically confirmed BC cases based on the International TIL working group guidelines. Tissue Microarrays were constructed and stained with immunohistochemistry (IHC) for CD3, CD4, CD8, CD68, CD20, and FOXP3. Linear and logistic regression models were used to assess associations between risk factors and tumour features with IHC markers and total TILs, after adjusting for other covariates. RESULTS: A total of 226 invasive BC cases were included. Overall, LE-TIL (mean = 27.9, SD = 24.5) proportions were significantly higher than sTIL (mean = 13.5, SD = 15.8). Both sTILs and LE- TILs were predominantly composed of CD3, CD8, and CD68. We found higher TILs to be associated with high KI67/high grade and aggressive tumour subtypes, although these associations varied by TIL locations. Older age at menarche (≥ 15 vs. < 15 years) was associated with higher CD3 (OR: 2.06, 95%CI:1.26-3.37), but only for the intra-tumour stroma. CONCLUSION: The TIL enrichment in more aggressive BCs is similar to previously published data in other populations. The distinct associations of sTIL/LE-TIL measures with most examined factors highlight the importance of spatial TIL evaluations in future studies.

Breast cancer risk factors in relation to molecular subtypes in breast cancer patients from Kenya.

BACKGROUND: Few studies have investigated risk factor heterogeneity by molecular subtypes in indigenous African populations where prevalence of traditional breast cancer (BC) risk factors, genetic background, and environmental exposures show marked differences compared to European ancestry populations. METHODS: We conducted a case-only analysis of 838 pathologically confirmed BC cases recruited from 5 groups of public, faith-based, and private institutions across Kenya between March 2012 to May 2015. Centralized pathology review and immunohistochemistry (IHC) for key markers (ER, PR, HER2, EGFR, CK5-6, and Ki67) was performed to define subtypes. Risk factor data was collected at time of diagnosis through a questionnaire. Multivariable polytomous logistic regression models were used to determine associations between BC risk factors and tumor molecular subtypes, adjusted for clinical characteristics and risk factors. RESULTS: The median age at menarche and first pregnancy were 14 and 21 years, median number of children was 3, and breastfeeding duration was 62 months per child. Distribution of molecular subtypes for luminal A, luminal B, HER2-enriched, and triple negative (TN) breast cancers was 34.8%, 35.8%, 10.7%, and 18.6%, respectively. After adjusting for covariates, compared to patients with ER-positive tumors, ER-negative patients were more likely to have higher parity (OR = 2.03, 95% CI = (1.11, 3.72), p = 0.021, comparing ≥ 5 to ≤ 2 children). Compared to patients with luminal A tumors, luminal B patients were more likely to have lower parity (OR = 0.45, 95% CI = 0.23, 0.87, p = 0.018, comparing ≥ 5 to ≤ 2 children); HER2-enriched patients were less likely to be obese (OR = 0.36, 95% CI = 0.16, 0.81, p = 0.013) or older age at menopause (OR = 0.38, 95% CI = 0.15, 0.997, p = 0.049). Body mass index (BMI), either overall or by menopausal status, did not vary significantly by ER status. Overall, cumulative or average breastfeeding duration did not vary significantly across subtypes. CONCLUSIONS: In Kenya, we found associations between parity-related risk factors and ER status consistent with observations in European ancestry populations, but differing associations with BMI and breastfeeding. Inclusion of diverse populations in cancer etiology studies is needed to develop population and subtype-specific risk prediction/prevention strategies.

Investigating toxic metal levels in popular edible fishes from the South Durban basin: implications for public health and food security.

Contamination of the ocean by heavy metals may have ecosystem-wide implications because they are toxic even if present in trace levels, and the relative ease of their bioaccumulation by marine organisms may affect human health, primarily through consumption of contaminated fish. We evaluated metal concentrations in six different popular edible fish species and estimated the potential health risks from consumption of contaminated fish. There was no correlation between fish length and average metal accumulation although the fish species tended to accumulate significantly more Al and Zn (P < 0.05) than any of the other metals. Significantly higher Mn concentrations were found in fish gills compared to other body parts in all fish species. Bronze seabream, Catface rockcod, and Slinger seabream had significantly higher mean Cr concentration in the liver than in either the tissues or gills. The highest concentration of Zn in fleshy tissue was in Horse mackerel (56.71 µg g-1) followed by Bronze seabream (31.07 µg g-1). Al levels ranged from 5.6 µg g-1 in Atlantic mackerel to 35.04 µg g-1 in Horse mackerel tissue while Cu and Cr concentrations were highest in the tissues of Horse mackerel (6.83 and 1.81 µg g-1, respectively) followed by Santer seabream (3.15; 1.09 µg g-1) and Bronze seabream (3.09; 1.30 µg g-1), respectively. The highest tissue concentration of Mn was detected in Bronze seabream (8.23 µg g-1) followed by Catface rockcod (6.05 µg g-1) and Slinger seabream (5.21 µg g-1) while Pb concentrations ranged from a high of 8.44 µg g-1 in Horse mackerel to 1.09 µg g-1 in Catface rockcod. However, the estimated potential health risks from fish consumption as determined by the target hazard quotient (THQ) and hazard index (HI) were significantly lower than 1, implying that metals were not present in sufficiently high quantities to be of any health and/or food and security concern in the studied fishes.

The role of miRNA-21 and epithelial mesenchymal transition (EMT) process in colorectal cancer.

AIMS: The study was conducted to assess the expression levels of epithelial mesenchymal transition (EMT) proteins (E-cadherin, N-cadherin, snail-1 and vimentin) and miRNA-21. In addition, we correlated these data with clinicopathological features in Colorectal cancer. METHODS: H&E slides from a total of 59 formalin fixed paraffin embedded tissue blocks were examined by a pathologist to demarcate normal and tumour regions. Immunohistochemical analysis of mismatch repair proteins (MLH1, MSH2 and MSH6) and EMT markers (E-cadherin, N-cadherin, snail-1 and vimentin) was performed. The miRNA-21 expression levels were determined using qRT-PCR and the data was analysed using the relative quantification method. The Fisher's exact and Pearson's χ2 tests were used to correlate snail-1, E-cadherin, miRNA-21 and clinicopathological data. RESULTS: Our results showed a statistically significant correlation between high miRNA-21 expression levels and E-cadherin positive cases. There was also an association between high miRNA-21 expression levels and negative snail-1 expression. No significant correlation was seen between miRNA-21 expression levels and clinicopathological features. Moreover, high expression levels of miRNA-21 were significantly associated with the sporadic cases. CONCLUSIONS: Our data suggest that miRNA-21 in association with E-cadherin and snail-1 does not play a significant role in the development and progression of this disease.

Determining protein biomarkers for DLBCL using FFPE tissues from HIV negative and HIV positive patients.

DLBCL is the most common lymphoma subtype occurring in older populations as well as in younger HIV infected patients. The current treatment options for DLBCL are effective for most patients yet the relapse rate is high. While many biomarkers for DLBCL exist, they are not in clinical use due to low sensitivity and specificity. In addition, these biomarkers have not been studied in the HIV context. Therefore, the identification of new biomarkers for HIV negative and HIV positive DLBCL, may lead to a better understanding of the disease pathology and better therapeutic design. Protein biomarkers for DLBCL were determined using MALDI imaging mass spectrometry (IMS) and characterised using LC-MS. The expression of one of the biomarkers, heat shock protein (Hsp) 70, was confirmed on a separate cohort of samples using immunohistochemistry. The biomarkers identified in the study consisted of four protein clusters including glycolytic enzymes, ribosomal proteins, histones and collagen. These proteins could differentiate between control and tumour tissue, and the DLBCL immunohistochemical subtypes in both cohorts. The majority (41/52) of samples in the confirmation cohort were negative for Hsp70 expression. The HIV positive DLBCL cases had a higher percentage of cases expressing Hsp70 than their HIV negative counterparts. The non-GC subtype also frequently overexpressed Hsp70, confirming MALDI IMS data. The expression of Hsp70 did not correlate with survival in both the HIV negative and HIV positive cohort. This study identified potential biomarkers for HIV negative and HIV positive DLBCL from FFPE tissue sections. These may be used as diagnostic and prognostic markers complementary to current clinical management programmes for DLBCL.

GSTM1, GSTP1, and NQO1 polymorphisms and susceptibility to atopy and airway hyperresponsiveness among South African schoolchildren.

Glutathione-S-transferases (GSTM1 and GSTP1) and nicotinamide quinone oxidoreductase (NQO1) genes play an important role in cellular protection against oxidative stress which has been linked to asthma pathogenesis. We investigated whether common, functional polymorphisms in GSTM1, GSTP1 and NQO1 influence airway hyperreactivity (AHR) and atopy among schoolchildren in South Africa. Genomic DNA was extracted from 317 primary schoolchildren, aged 9-11 years, from urban, low socioeconomic communities of Durban, South Africa. GSTM1 (null vs. present genotype), GSTP1 (Ile105Val; AA â†’ AG + GG), and NQO1 (Pro/187Ser; CC â†’ CT/TT) genotypes were determined using polymerase chain reaction (PCR) methods. Atopy was defined as a positive skin-prick test to any of several common allergens. Airway hyperreactivity (AHR) was evaluated by pulmonary function testing before and after methacholine challenge. Among the children, 30% were GSTM1 null, 65% carried the G allele for GSTP1, and 36% carried the C allele for NQO1. The frequency of GSTM1, GSTP1, and NQO1 variants among our South African sample was similar to frequencies found in similar ethnic groups worldwide. Marked airway reactivity (PC(20) ≤ 2 mg/ml) was found in 10.3% of children and approximately 40% of them were atopic. No significant associations for GSTM1 and NQO1 with either AHR or atopy were identified. A significant protective effect against atopy was found among children with one or two copies of the GSTP1 G allele.

Biotin-directed assembly of targeted modular lipoplexes and their transfection of human hepatoma cells in vitro.

The asialoglycoprotein receptor, which is abundantly and near exclusively expressed on hepatocytes, has received much attention in the design of non-viral hepatotropic DNA delivery systems. Thus, asialoglycoproteins and hexopyranosyl ligands have been coupled to DNA-binding cationic polymers and liposomes in the assembly of complexes intended for uptake by liver parenchymal cells. The aim of the study was to construct a hepatocyte-targeted multimodular liposome-based transfecting complex, in which the biotin-streptavidin interaction provides the cohesive force between the ligand asialorosomucoid and the liposome bilayer, and to evaluate its transfection capabilities in the hepatocyte-derived human transformed cell line HepG2. Dibiotinylated asialoorosomucoid was attached to cationic liposomes constructed from 3beta[N-(N',N'-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T):dioleoylphosphatidylethanolamine:biotinylcholesterylformylhydrazide (MSB1) (48:50:2 mole ratio) through streptavidin interposition. Liposome-pGL3 DNA interactions were studied by gel band shift and ethidium displacement assays. The cytotoxicity of assemblies was evaluated in the HepG2 cell line and transfection capabilities determined by measuring the activity of the transgene luciferase. Binding assays showed that all DNA was liposome associated at a DNA (negative):liposome (positive) charge ratio of 1:1. Accommodation of a streptavidin dibiotinylated asialoorosomucoid assembly was achieved at a DNA:liposome:streptavidin dibiotinylated asialoorosomucoid ratio of 1:4:9 (weight basis). Complexes showed optimal transfection activity at this ratio, which was reduced 10-fold by the presence of the competing ligand asialofetuin. The streptavidin-biotin interaction has been applied for the first time to the assembly of hepatocyte-targeted lipoplexes that display asialoorosomucoid and that are well tolerated by a human hepatoma cell line in which transfection is demonstrably achieved by receptor mediation. Favorable size and charge ratio characteristics suggest that this system may be suitable for in vivo application.

Frequent accumulation of nuclear E-cadherin and alterations in the Wnt signaling pathway in esophageal squamous cell carcinomas.

Esophageal squamous cell carcinoma is frequently associated with poor prognosis, as a result of high levels of lymph node metastasis. So far, very few genetic abnormalities have been associated with this disease, and its molecular etiology remains largely unknown. To assess whether the Wnt pathway contributes to esophageal squamous cell carcinoma, we characterized the expression and subcellular localization of the key Wnt signaling components in all 30 cases of esophageal squamous cell carcinomas analyzed. We found abnormal expression and/or localization in glycogen synthase kinase-3 alpha/beta (34%), Axin2 (48%), alpha-catenin (31%), MYC (73%) and cyclin D1 in 46% of cases. Only 13% of tumors showed nuclear accumulation of beta-catenin. By contrast, 60% showed nuclear expression of E-cadherin using an antibody that recognizes the cytoplasmic domain of E-cadherin. When the same tumors were stained with antibody raised against the extracellular domain of E-cadherin, the expression was lost. A direct correlation was found between nuclear E-cadherin and the increased nuclear cyclin D1, one of the AP-1 target genes in these tumors. By transfection experiments, the cytoplasmic portion of E-cadherin was found to activate the AP-1 transcription factor pathway and induced cyclin D1 promoter activity, but beta-catenin/Tcf transcription activity was unaffected. Nuclear expression of E-cadherin was also detected in tumors other than squamous cell carcinoma, including pancreatic and colon cancers, albeit at lower frequency. Nuclear accumulation of a portion of E-cadherin in esophageal squamous cell carcinoma and the other types of tumors indicates that, in addition to the previously implicated tumor suppressor activity of E-cadherin, modified forms of this glycoprotein might also play a role in growth promotion.

Abnormalities of chromosome 17 in oesophageal cancer.

BACKGROUND: Oesophageal cancer is the most common malignancy encountered in South African males, especially in the Eastern Cape and surrounding region of South Africa. There are a number of risk factors and predisposing conditions that have been implicated in the aetiology of the disease. The tylosis oesophageal cancer (TOC) gene, localised to a small region on chromosome 17q25, has been shown to be associated with oesophageal squamous cell carcinoma. AIM: To investigate loss of heterozygosity (LOH) and microsatellite instability (MSI) in the region of the TOC locus. METHODS: In 74 oesophagectomy specimens for squamous cell carcinoma, microsatellite PCR was performed using five fluorescently labelled TOC markers. The PCR products were analysed and the data correlated with clinicopathological findings. RESULTS: LOH ranged from 25% to 60%. LOH for the individual markers was as follows: D17S1839, 25%; D17S1864, 36%; D17S1817, 38%; D17S785, 47.8%; and D17S579, 60%. MSI ranged from 4.1% to 6.8% for the five loci in the 17q region. MSI was 4.1% for the markers D17S579, D17S785 and D17S1817. Marker D17S1864 showed MSI to occur in 4 cases (5.4%) and marker D17S1839 in 5 cases (6.8%). CONCLUSION: No significant relationship between genetic and clinical parameters was observed; however, aberrations in poorly differentiated tumours were high for markers D17S579 and D17S1864 (25% and 37%, respectively), indicating that these markers may have an underlying role in the molecular pathogenesis of oesophageal squamous cell carcinoma. In addition, 63% of patients who died showed LOH for the markers D17S579, D17S1864 and D17S1817.

Sanguinarine downregulates AT1a gene expression in a hypertensive rat model.

We studied the in vivo effects of sanguinarine in a hypertensive rat model and its effects on AT1a mRNA expression in kidney tissues. Rats received daily for 14 d sanguinarine 0.1 mg/kg (SangL) and 0.3 mg/kg (SangH), losartan 1 mg/kg by weight (Los), or DMSO (Con). Blood pressures were monitored regularly and urine volume and sodium concentration was measured on days 0, 7, and 14. On day 15, animals were anesthetized (sodium thiopentane, 50 mg/kg), blood samples for aldosterone levels were taken, and kidneys were removed for AT1a mRNA expression. Los and SangH groups showed reduced AT1a mRNA expressions by 4.22- and 5.9-fold, respectively. In the SangL group it was reduced by 2.7-fold. Decreases in systolic blood pressures mirrored decreases in AT1a mRNA expressions in all groups. Los and SangH groups showed reductions in systolic blood pressure of 12.3% and 19.3%, respectively, whereas in the SangL group, it was reduced by 8.07%. Urine output in the Los group increased (228% mean increase from days 0-14), whereas sodium excretion decreased by 69.6% (mean decrease from days 0-14). In the SangL and SangH groups, urine volumes increased significantly by 108.3% and 115% (mean increase from days 0-14), respectively. Urinary sodium excretion increased significantly by 60.9% in the SangH group. We concluded that sanguinarine reduces blood pressure in the Dahl rat because of decreased AT1 receptor expression and reduced aldosterone levels. The action of losartan on increased urinary volume and decreased sodium excretion may be attributed to reduced vasopressin secretion.

Microsatellite analysis of the APC gene and immunoexpression of E-cadherin, catenin, and tubulin in esophageal squamous cell carcinoma.

The histological features that accompany the development and progression of solid tumors are known to be controlled by a distinct cascade of molecular events. One such event is the inactivation of tumor suppressor genes, such as the adenomatous polyposis coli (APC) gene. Disruption of the cadherin-catenin cell adhesion complex also plays a role in the initial steps of cancer invasion and metastasis whereas alterations in cell structural molecules, such as tubulin, may contribute to the cancer phenotype. The understanding of the status of these molecules in ESSC may provide novel markers that could impact on management of the disease. The present study examined alterations in the microsatellite sequence of the APC gene via fluorescent-based polymerase chain reaction in 100 cases of primary esophageal squamous cell carcinoma. In addition, the expression of E-cadherin, alpha- and beta-catenin, and alpha- and beta-tubulin was analyzed using immunohistochemistry. These data were then statistically compared with each other as well as the relevant clinicopathologic data. Although the APC markers (D5S210, D5S346, D5S299, and D5S82) tested did show an overall high frequency of allelic imbalance/loss of heterozygosity (62.48%) and microsatellite instability (41.27%), they did not show prognostic significance in the study cohort and were not correlated with the immunohistochemical data. The tubulin proteins showed no significant change in expression in the tumor tissue The decreased immunoreactivity of E-cadherin was statistically correlated with the presence of lymph node metastases (P = .0180). Although alpha- and beta-catenin as well as E-cadherin showed no direct prognostic value, E-cadherin may warrant further investigation as an indirect prognostic indicator by allowing more accurate prediction of lymph node metastases.

Microsatellite analysis of the DCC gene in nephroblastomas: pathologic correlations and prognostic implications.

Microsatellite instability has been reported in a wide variety of cancer types. Inactivation or loss of tumour suppressor genes has been shown to result in cell cycle deregulation and neoplastic growth. We conducted a microsatellite study using fluorescent-based DNA technology to determine whether mutations in the microsatellite sequences of the deleted in colorectal cancer (DCC) gene, a tumour suppressor at 18q21.1, have any pathologic correlation or prognostic significance in nephroblastomas. Normal and tumour DNA was isolated from 106 cases of nephroblastoma using the standard proteinase K digestion and phenol-chloroform extraction method from paraffin wax-embedded tissue. Polymerase chain reaction using three microsatellite markers; D18S21, D18S34 and D18S58, for the DCC gene were performed. The polymerase chain reaction products were analysed on the ALF Express Automated DNA sequencer. The results were correlated with age at diagnosis, preoperative chemotherapy, clinicopathological stage, histological classification and patient outcome using chi(2) test. Allelic imbalance/loss of heterozygosity appeared to be a more frequent genetic aberration than microsatellite instability with 20% of cases showing allelic imbalance/loss of heterozygosity and only 9% of cases showing microsatellite instability. Genetic aberrations were more frequent in unfavourable histology tumours compared to favourable histology tumours (P=0.012). All patients with genetic aberrations for more than one DCC marker died independent of histological classification and stage (P=0.016). There was no statistically significant difference when DCC aberrations were compared with age at diagnosis, preoperative chemotherapy and clinicopathological stage. In conclusion, this study has found that multiple aberrations involving the DCC locus may play a role in the progression of nephroblastomas, and hence confer a poorer prognosis.

Apolipoprotein E polymorphism and outcome after closed traumatic brain injury: influence of ethnic and regional differences.

OBJECT: The presence of the apolipoprotein E-epsilon4 (APOE-epsilon4) allele is reported to be associated with poor outcome after traumatic brain injury (TBI). This study was performed to determine if the presence of the APOE-epsilon4 allele influenced outcome in a cohort of black patients with TBI who had homogeneous neuropathological findings. METHODS: Venous blood was collected at the time of admission to determine the APOE genotype in black Zulu-speaking patients who presented with traumatic cerebral contusions. The frequency of the APOE-epsilon4 allele's appearance was correlated with outcome at a minimum of 6 months of follow up. Univariate and multivariate analyses were performed to determine independent risk factors and to control for confounding factors. In 110 black Zulu-speaking patients with traumatic cerebral contusions, genotypes for APOE were analyzed. Eleven of 45 (24.4%) with the APOE-epsilon4 allele experienced a poor outcome, compared with 10 (15.4%) of 65 without this allele (p = 0.34). Both patients with homozygous APOE-epsilon4 alleles experienced a good outcome (Glasgow Outcome Score 5). Univariate and multivariate analysis revealed no significant relationship in patients with the APOE-epsilon4 allele with regard to age, admission Glasgow Comas Scale score, contusion volume, type of neurosurgical management, and outcome. The risk of a poor outcome was, however, greater in patients with the APOE-epsilon4 allele (relative risk 1.59; 95% confidence interval 0.74-3.42). CONCLUSIONS: The authors recorded no relationship between APOE-epsilon4 allele status and outcome after TBI in black patients. Given the high regional susceptibility to the APOE gene, further studies, possibly even community-based investigations and studies conducted in other geographic areas, are probably warranted.

Chromosome 2p, 3p, 5q and 18q status in sporadic gastric cancer.

AIM: The genetic make-up of gastric cancers in low-risk population groups from South Africa is largely unknown. The purpose of this study was to ascertain the incidence of microsatellite instability and loss of heterozygosity in this population. METHODS: Thirty-seven gastrectomy specimens for sporadic gastric cancer were analysed for the following clinicopathological parameters: age, gender, race, histopathological type, size of tumour, lymph node status and the presence/absence of Helicobacter pylori. DNA was then extracted from paraffin-embedded tissue and seven microsatellite markers in 2p, 3p, 5q and 18q loci were examined using automated DNA fluorescent technology. RESULTS: Only eight cases showed microsatellite instability (MSI) for one marker and were thus categorised as MSI-low. In the 3p region, loss of heterozygosity (LOH) was detected in 21.7-38.3% of informative cases, whilst in the 18q region CLOH ranged from 25 to 38.4%. LOH was not seen in the 2p locus and only one case showed LOH in the 5q region. When the molecular changes were compared with clinicopathological parameters, a statistically significant relationship did not emerge with any single parameter. CONCLUSIONS: This study shows that sporadic gastric cancer from a low-risk population in South Africa is MSI-low or MSI-stable, and that LOH in the 3p and 18q regions is similar to that seen in other low-risk populations from different geographical regions.