An immunohistochemical and microsatellite analysis of nephroblastomas.

Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2008.The aims of this study were: (i) to determine the association between p53, bcl-2, pRb, p21, cyclin A and p-glycoprotein immunoexpression and prognosis, and (ii) to determine the frequency of loss of heterozygosity and microsatellite instability at 11 p, 16q and mismatch repair gene loci and their association with prognosis, in nephroblastomas in South African children. There were 138 cases (111 of whom received preoperative chemotherapy) in the immunohistochemical study and, 70 cases (48 with preoperative chemotherapy) in the microsatellite study. The following monoclonal antibodies were used after heat induced epitope retrieval; p53, bcl-2, pRb, p21, cyclin A and p-glycoprotein. Six polymorphic microsatellite markers were selected from the 11p region, 5 from the 16q region and 6 from the loci of known mismatch repair genes. Automated fluorescent DNA technology was used in the analysis. The results of the immunohistochemical and microsatellite studies were correlated with patient age, gender, preoperative chemotherapy, SlOP histological classification, SlOP histological risk group, clinicopathological stage, patient outcome and survival using X2 , Fisher's exact test, Cox regression model and Kaplan-Meier estimates. The majority of patients presented with advanced disease. Anaplastic tumours and high-risk histology were associated with high disease stage. Mortality was directly related to increasing stage and histological risk group. Multivariate analysis showed that clinicopathological stage was the only factor significantly associated with survival (p<0.001) (hr=5.6, 95%CI: 2.1-14.9). High expression of p53 was more frequent in anaplastic tumours suggesting that p53 mutations are common events in this tumour type (p<0.001). Despite the strong association with tumour histology, there was no association with stage. Although p53 expression was found to be a predictor of survival in the univariate analysis this was not retained in the multivariate analysis. Tumours treated with preoperative chemotherapy showed higher bcl-2 immunoreactivity (p=0.027 but lower levels of pRb (p=0.040) and cyclin A expression (p<0.001). All anaplastic tumours showed high expression of pRb compared to the other histological types (p=0.003). Expression of xxii pRb was significantly associated with survival in the univariate analysis but not in the multivariate analysis. High cyclin A expression was associated with high risk histology (p<0.001). Cyclin A expression was found to be a significant predictor of survival in both the univariate (hr=1.7; 95%CI 1.2-2.4; p=0.002) and multivariate analyses (hr=1.7; 95%CI1.1-2.7; p=0.032). Although tumours with high risk histology were more likely to express high levels of p-glycoprotein, this did not reach significance. LOH at 11 p was seen in 64.7% of 68 informative cases. LOH at 11 p13 was more frequent than LOH at 11p15. LOH for both 11p13 and 11p15 was found in 39.7% of all tumours. MSI at 11 p was seen in 22.1 % of informative cases. The majority showed MSI for one marker only. LOH 16q was seen in 66.7% of 66 informative cases. MSI at 16q was seen in 16.7% of cases. LOH for 016S496 and 016S520 appear to be related to tumour histology and risk group. The most frequent locus for LOH was 16q21-22, which is known to harbour important genes, such as, E2F4 and E-cadherin. LOH for MMR markers was seen in 43.5% of 69 informative cases. MSI was seen in 11.6% of tumours. In the multivariate analysis there was no significant correlation between LOH at any of the loci studied and survival. There were no tumours with high frequency MSI. Low frequency MSI was of no clinicopathological significance. The following conclusions are made: (i) p53 mutations determined by high p53 expression is a frequent finding in anaplastic tumours, (ii) Bcl-2 may play a role in the chemoresistance of nephroblastomas, (iii) Rb gene alterations are not important in the development of nephroblastoma and anaplasia, (iv) Cyclin A expression is an independent predictor of survival, (v) p-glycoprotein may be responsible for the chemoresistance in a proportion of nephroblastomas, (vi) MSI is a rare occurrence in nephroblastoma and does not play a role in the development of nephroblastoma, (vii) LOH at 11 p and 16q are frequent findings in nephroblastomas, (viii) LOH for the specific 16q markers (016S496 and 016S520) may have an important prognostic role in nephroblastoma

Immunohistochemical and Biochemical Characterization of Mucin in Pseudomyxoma Peritonei: A Case Study

We previously reported the presence of MUC2, MUC5AC and, for the first time, MUC5B in a 58-year-old male with pseudomyxoma peritonei (PMP). This is a report on the biochemical and immunohistochemical characterization of mucin in a 50-year-old female with the same rare illness. A right oophorectomy and appendicectomy and a resection of the involved omentum were performed. Approximately a litre of crude material in the sol and gel phases was obtained from the patient during laparotomy. This was briefly homogenized in 6 M guanidinium hydrochloride and proteolytic inhibitors and purified by density gradient centrifugation in caesium chloride. At laparotomy it was noted that the patient had appendiceal and ovarian masses as well as extensive mucinous deposits in the omentum and peritoneum. A mucinous adenocarcinoma of the appendix and ovary was confirmed on histology. The cells expressed both sulphated and non-sulphated acidic mucins. The presence of MUC2, MUC5AC, MUC5B and a-1-acid glycoprotein was shown by Western blotting and MUC4 by immunohistochemical staining. MUC1 and MUC6 were not detectable in the tissue. The study confirms that MUC2, MUC5AC and MUC5B are produced in the mucus of patients with PMP. The expression of MUC4 in this disease has not been previously reported

The C-Type Lectin Receptor CLECSF8/CLEC4D Is a Key Component of Anti-Mycobacterial Immunity

Open Access funded by Wellcome Trust: Under a Creative Commons license Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved. Acknowledgments We would like to thank S. Hardison, P. Redelinghuys, J. Taylor, C. Wallace, A. Richmond, S. Hadebe, A. Plato, F. Abbass, L. Fick, N. Allie, R. Wilkinson, K. Wilkinson, S. Cooper, D. Lang, and V. Kumar for reagents and assistance, and the animal facility staff for the care of our animals. This work was supported by the MRC (UK) and Wellcome Trust (G.D.B.); MRC (South Africa) and Sydney Brenner Fellowship (M.J.M.); Vici (M.G.N.), Vidi (R.v.C.), and Veni grants (T.S.P.) from the Netherlands Organization for Scientific Research; the Royal Netherlands Academy of Arts and Sciences (T.H.M.O.); EC FP7 projects (NEWTBVAC, ADITEC; T.H.M.O.); Carnegie Corporation and CIDRI (J.C.H.); and the University of Aberdeen (B.K.).Peer reviewedPublisher PD

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

TNF-dependent regulation and activation of innate immune cells are essential for host protection against cerebral tuberculosis

BACKGROUND: Tuberculosis (TB) affects one third of the global population, and TB of the central nervous system (CNS-TB) is the most severe form of tuberculosis which often associates with high mortality. The pro-inflammatory cytokine tumour necrosis factor (TNF) plays a critical role in the initial and long-term host immune protection against Mycobacterium tuberculosis (M. tuberculosis) which involves the activation of innate immune cells and structure maintenance of granulomas. However, the contribution of TNF, in particular neuron-derived TNF, in the control of cerebral M. tuberculosis infection and its protective immune responses in the CNS were not clear. METHODS: We generated neuron-specific TNF-deficient (NsTNF / ) mice and compared outcomes of disease against TNF f/f control and global TNF / mice. Mycobacterial burden in brains, lungs and spleens were compared, and cerebral pathology and cellular contributions analysed by microscopy and flow cytometry after M. tuberculosis infection. Activation of innate immune cells was measured by flow cytometry and cell function assessed by cytokine and chemokine quantification using enzyme-linked immunosorbent assay (ELISA). RESULTS: Intracerebral M. tuberculosis infection of TNF / mice rendered animals highly susceptible, accompanied by uncontrolled bacilli replication and eventual mortality. In contrast, NsTNF / mice were resistant to infection and presented with a phenotype similar to that in TNF f/f control mice. Impaired immunity in TNF / mice was associated with altered cytokine and chemokine synthesis in the brain and characterised by a reduced number of activated innate immune cells. Brain pathology reflected enhanced inflammation dominated by neutrophil influx. CONCLUSION: Our data show that neuron-derived TNF has a limited role in immune responses, but overall TNF production is necessary for protective immunity against CNS-TB

Tumour infiltrating lymphocytes (TILs) and immune composition in breast cancer patients from Kenya: Spatial distributions and associations with risk factors and tumour …

Background: The immune landscape of breast cancer in patients from Sub Saharan Africa is understudied and findings are mainly extrapolated from studies in Caucasians and African Americans. Given the prognostic value of Tumour Infiltrating Lymphocytes (TILs) in breast cancer subtypes, the paucity of specific TIL marker data among BC patients from Africa, and the limited data on TIL spatial heterogeneity, our aim was to describe the distribution of TILs within the intratumoral stroma and the leading/invasive edge stroma, and evaluate their association across BC subtypes with established risk factors in Kenyan women with BC. Methods: Visual quantification of overall TILs within the intratumoral stroma (sTILs) and the stroma at the leading edge/invasive edge of the tumour (LE-TILs) were performed on Haematoxylin and Eosin stained whole slide sections of pathologically confirmed breast cancer based on the guidelines of the International TIL working group. Tissue Microarrays (TMAs) of tumour were also were constructed and stained with immunohistochemistry for CD3, CD4, CD8, CD68, CD20 and Fox P3. The TMA slides were scanned on the Aperio Digital pathology slide scanner, imported into the Aperio, assigned a template, segmented and annotated before analysis using a nuclear algorithm. Linear and logistic regression models were used to assess the associations between risk factors and tumor features with IHC markers and total TILs, after adjusting for other covariates. Results: A total of 226 invasive breast cancer cases were included in the final analysis for TILs and immune cell markers. The mean age at diagnosis of these breast cancer patients was 48.3 years. The distribution of the BC molecular subtypes defined by IHC markers was 56.2%, 16.8%, 9.3%, and 17.7 % (luminal A, luminal B, HER2-enriched, and TN breast cancers, respectively). On the whole we found that LE TIL proportions were higher than sTIL (41.3% versus 15% for TILs ≥30 and 19.3% and 4.4% for TILs ≥50 respectively). After adjusting for other tumor characteristics, we found higher TILs associated with high KI67 and high grade (for both sTIL and LE-TIL), TNBC (for sTIL only), and HER2 status, luminal B subtype, and smaller tumor size (\u3c4 cm) (for LE-TIL only). Both sTILs and LE-TILs were predominantly composed of CD3, CD8 and CD68 with a much smaller contribution of CD4 and CD20. The percent positive cells for Fox3 were markedly lower compared to other IHC markers for both sTILs and LE-TILs. Conclusions: Our findings with regards to enrichment of TILs in more aggressive breast cancers are similar to what has been previously published. The distinct associations between intratumoral stroma and leading edge TIL measures for some factors highlight the importance of spatial TIL evaluations in future studies. Our findings add to the body of knowledge regarding TILs and TIL subtypes in breast cancer, which underscore the need to conduct studies specific to African populations

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

Abstract: Background: The immune landscape of breast cancer in patients from Sub Saharan Africa is understudied and findings are mainly extrapolated from studies in Caucasians and African Americans. Given the prognostic value of Tumour Infiltrating Lymphocytes (TILs) in breast cancer subtypes, the paucity of specific TIL marker data among BC patients from Africa, and the limited data on TIL spatial heterogeneity, our aim was to describe the distribution of TILs within the intratumoral stroma and the leading/invasive edge stroma, and evaluate their association across BC subtypes with established risk factors in Kenyan women with BC. Methods: Visual quantification of overall TILs within the intratumoral stroma (sTILs) and the stroma at the leading edge/invasive edge of the tumour (LE-TILs) were performed on Haematoxylin and Eosin stained whole slide sections of pathologically confirmed breast cancer based on the guidelines of the International TIL working group. Tissue Microarrays (TMAs) of tumour were also were constructed and stained with immunohistochemistry for CD3, CD4, CD8, CD68, CD20 and Fox P3. The TMA slides were scanned on the Aperio Digital pathology slide scanner, imported into the Aperio, assigned a template, segmented and annotated before analysis using a nuclear algorithm. Linear and logistic regression models were used to assess the associations between risk factors and tumor features with IHC markers and total TILs, after adjusting for other covariates. Results: A total of 226 invasive breast cancer cases were included in the final analysis for TILs and immune cell markers. The mean age at diagnosis of these breast cancer patients was 48.3 years. The distribution of the BC molecular subtypes defined by IHC markers was 56.2%, 16.8%, 9.3%, and 17.7 % (luminal A, luminal B, HER2-enriched, and TN breast cancers, respectively). On the whole we found that LE TIL proportions were higher than sTIL (41.3% versus 15% for TILs ≥30 and 19.3% and 4.4% for TILs ≥50 respectively). After adjusting for other tumor characteristics, we found higher TILs associated with high KI67 and high grade (for both sTIL and LE-TIL), TNBC (for sTIL only), and HER2 status, luminal B subtype, and smaller tumor size (\u3c4 \u3ecm) (for LE-TIL only). Both sTILs and LE-TILs were predominantly composed of CD3, CD8 and CD68 with a much smaller contribution of CD4 and CD20. The percent positive cells for Fox3 were markedly lower compared to other IHC markers for both sTILs and LE-TILs. Conclusions: Our findings with regards to enrichment of TILs in more aggressive breast cancers are similar to what has been previously published. The distinct associations between intratumoral stroma and leading edge TIL measures for some factors highlight the importance of spatial TIL evaluations in future studies. Our findings add to the body of knowledge regarding TILs and TIL subtypes in breast cancer, which underscore the need to conduct studies specific to African populations