OpenUCT :: Browsing by Subject "Anatomical Pathology"

Browsing by Subject "Anatomical Pathology"

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Open Access
Biomarker identification in HIV and non-HIV related lymphomas
(2016) Magangane, Pumza Samantha; Naidoo, Richard; Govender, Dhiren
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. Initially differences in the clinicopathological features between HIV negative and HIV positive DLBCL patients were determined by conducting a retrospective study of patients treated at GSH. Subsequent to this, potential protein biomarkers for DLBCL were determined using MALDI imaging mass spectrometry (IMS) and characterised using LCMS. The expression of one of the biomarkers, heat shock protein (Hsp) 70, was confirmed on a separate cohort of samples using immunohistochemistry. Our results indicate that the clinicopathological features for HIV negative and HIV positive DLBCL are similar except for median age, and frequency of elevated LDH levels. Several clinicopathological factors were prognostic for all DLBCL cases including age, gender, stage and bone marrow involvement. In addition, tumour extranodal site was also a prognostic indicator for the HIV negative cohort. 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 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. Expression of Hsp70 correlated with poor outcome in the HIV negative cohort. In conclusion, this study identified potential biomarkers for HIV negative and HIV positive DLBCL from both clinical and molecular sources. These may be used as diagnostic and prognostic markers complementary to current clinical management for DLBCL.
Open Access
Cyclooxygenase-2 in cervical neoplasia and the relationship with specific human papillomavirus types
(2000) Hofmeyr, Michael Devitt; Hall, Pauline de la Motte
Includes bibliographical references.
Open Access
Cytohistologic correlation of suspected Cervicofacial Head & Neck Extra-Pulmonary Tuberculosis in children: A retrospective case series
(2023) Jackson, Christopher; Pillay, Komala; Peer Shazia
Background Tuberculosis (TB), especially extrapulmonary TB, is a difficult diagnosis to make in children due to the paucibacillary nature of paediatric disease and difficulty in obtaining sputum and tissue samples for microbiology confirmation. Lymphadenopathy in children with suspected cervicofacial TB are amenable to FNA or surgery for further cytological and histological assessment. It is therefore important to understand how well the morphologic features (from cytology and histology) correlate with defined reference standards (TB culture and molecular evidence of MTb) for the diagnosis of TB and the reliability of these features. Aim The aim of the study is to determine how well the cytology and histology-made TB diagnoses in children with suspected cervicofacial EPTB correlates with TB culture and MTb PCR results. Materials and methods This is a descriptive retrospective study that involved a re-appraisal of all patients with suspected cervicofacial EPTB who had histology and cytology performed at Red Cross Children's Hospital identified from the National Health Laboratory Service (NHLS) Trakcare system over a 5 year period (2012-2017). Following identification of histopathology accession numbers, histopathology reports and slides were retrieved from the archive of the Division of Anatomical Pathology/ National Health Laboratory Service, Red Cross Children's Hospital, Cape Town for evaluation. In addition, results for Genexpert testing and TB culture were identified using the National Health Laboratory Service (NHLS) Trakcare system. In patients that did not have either of the above, MTb PCR testing was performed. Results Data from the reports of 76 children with suspected cervicofacial TB were included in this study. More biopsies were submitted for histology (48) than for cytology (22). Six children had biopsies for both cytology and histology done. Most children had suspected and confirmed TB involvement of the cervical lymph nodes. On histology, the feature that correlated the best with proven TB was necrotising granulomatous inflammation (79.5% of cases had confirmed TB). On cytology, necrotising inflammation, necrotising granulomatous and non-necrotising granulomatous inflammation correlated well with proven TB. The sensitivity of cytology was 77.3% against TB culture and 81.8% against GXP for TB diagnosis. Whilst for histology the sensitivity was 82.5% against TB culture and 90.3% against GXP as reference standards for TB diagnosis. Conclusion FNA for cytology is a safer procedure with less complications than biopsy for histology. Also, the use of cytology together with a GXP renders a rapid and accurate diagnosis of TB and our findings are supportive for the combined use of these modalities as first line investigations. However, every attempt should still be made to obtain a sample for TB culture (as the WHO recommended gold standard for TB confirmation).
Open Access
The db mouse as a model for steatohepatitis
(2006) Sutherland, Jason Robert; Hall, Pauline de la Motte; Marais, David
Fatty liver disease is a collective phrase for a spectrum of diseases characterised by increased liver fat content. It ranges from fatty infiltration of the liver to an inflammatory condition, steatohepatitis, which may lead onto cirrhosis. Although not associated with alcohol consumption, non-alcoholic steatohepatitis (NASH) has strong associations with obesity, diabetes and dyslipidaemia. Overlapping pathological mechanisms may be involved. The course of the disease will remain unpredictable, and specific treatment will only be able to be instituted once the pathogenesis is fully understood. This thesis reviews current understanding of the pathogenesis and explores the suitability of a recently defined obese diabetic mouse model for its value as a model in the heterozygous and homozygous states. Observations revealed that the db/wt phenotype has a larger mass than the wt/wt and responds with hyperglycaemia. Lipid accumulation occurs in this model when alcohol is administered and lipid peroxidation occurs but histological changes of steatosis and steatohepatitis do not occur. The db/db model is phenotypically distinguished by a large amount of fat storage, diabetes and macrovesicular steatosis that has more lipid peroxidation but no steatohepatitis even when alcohol further increases lipid peroxidation. The model, as explored, did not reveal steatohepatitis either alone, or with alcohol as a single additional stressor, but both the db/wt and db/db mouse model could be further investigated to explore whether additional stressors could induce steaotohepatitis in this model.
Open Access
The development of a "new" stain and its comparison with currently available stains for the evaluation of mycobacteria in processed tissue
(2006) Jamieson, Craig; Hall, Pauline de la Motte
Includes bibliographical references.
Open Access
Differentiating follicular thyroid carcinoma from the follicular variant of papillary thyroid carcinoma
(2022) Rikhotso, Tshikani Norman; Govender, Dhirendra
Introduction: Differentiating follicular adenoma (FA), follicular thyroid carcinoma (FTC) and follicular variant of papillary thyroid carcinoma (FVPTC) remain a diagnostic challenge in some cases. This is as a result of the assessment of nuclear features being highly subjective, and the threshold for confirming cytomorphological features for papillary thyroid carcinoma varying greatly among pathologists which results in poor interobserver agreement. Diagnostic challenges may be encountered with some encapsulated follicular-patterned neoplasms of the thyroid due to uncertainty about the presence of capsular or vascular invasion. Immunohistochemistry may provide a better alternative to distinguishing these entities. Aims and objectives: To study the expression of biomarkers HBME-1, CD15, CK-19 and BRAF V600E in follicular adenoma, follicular carcinoma and follicular variant of papillary thyroid carcinoma. To ascertain the usefulness of these markers in differentiating these follicular-patterned thyroid neoplasms. Materials and methods: This is a ten-year retrospective study in which seventy-nine cases consisting of follicular adenoma (n=26), follicular thyroid carcinoma (n=25) and follicular variant of papillary thyroid carcinoma (n=28) were retrieved and reviewed. Four immunohistochemical stains (CD15, CK-19, HBME-1 and BRAF V600E) were performed and scored in tumour tissue. Data were analysed to determine if there was any correlation between the expression of the immunomarkers and the three follicular patterned thyroid neoplasms. Results: The patients' ages ranged from 13 to 74 years. There was a female bias with a female-to-male ratio of 4:1. HBME-1 expression showing varying intensity and proportion was seen in 7 (28%) FA, 15 (65%) FTC and 22 (79%) FVPTC. CK-19 expression showing varying intensity and proportion was seen in 5 (19%) FA, 3 (13%) FTC and 18 (72%) FVPTC. BRAF V600E expression showing varying intensities and proportions was seen in 3 (11%) of FVPTC. FA and FTC cases were all negative for BRAF V600E. CD15 expression showing varying intensity and proportion was seen in 2 (8%) FA, 6 (24%) FTC and 9 (33%) FVPTC. Statistical analysis detected a significant association between group and HBME-1 and CK-19 (H-score, proportion and intensity). The post-hoc comparison revealed that follicular adenoma was significantly more likely to have negative HBME-1 staining and a lower H-score when compared to follicular thyroid carcinoma and follicular variant of papillary thyroid carcinoma. The follicular variant of papillary thyroid carcinoma was significantly more likely to have an HBME-1 intensity of three and a higher H-score compared to follicular adenoma. Post-hoc comparison revealed that follicular adenoma was significantly more likely to have a negative CK-19 and a lower H-score when compared to follicular thyroid carcinoma and follicular variant of papillary thyroid carcinoma. Follicular variant of papillary thyroid carcinoma was significantly more likely to have a CK-19 intensity of three and a higher H-score compared to follicular thyroid carcinoma. HBME-1 had an overall specificity and sensitivity of 72% and 74% for distinguishing FA from FTC and FVPTC (benign from malignant). CK-19 had an overall specificity and sensitivity of 80.8% and 43.8% for distinguishing FA from FTC and FVPTC (benign from malignant). CK-19 had specificity of 87% and sensitivity of 72% for distinguishing FTC from FVPTC. Combining HBME-1 and CK-19 did not significantly increase the sensitivity and specificity of these markers. There was statistically no significant association between group and BRAF V600E and CD15 biomarkers. Conclusion: The study, within the small sample size power limitations, has shown that CK-19 may have a role in distinguishing follicular thyroid carcinoma from follicular variant of papillary thyroid carcinoma. In addition, HBME-1 and CK-19 may be used in differentiating benign follicular-patterned thyroid lesions from malignant follicular patterned thyroid lesions.
Open Access
The diversity of malignant rhabdoid tumours : a morphological, immunohistochemical and ultrastructural review of cases from the Red Cross Children's Hospital and Groote Schuur Hospitals
(1997) Mostert, Colin; Kaschula, R O C
Malignant rhabdoid tumours of the kidney are rare childhood neoplasms. Extra-renal rhabdoid tumours are known to have a distinctive biological behaviour and do not always occur in the paediatric age group. As the histogenesis of rhabdoid tumours, and their apparent relationship to nephroblastoma is still unclear, careful assessment of new cases is required. This investigation illustrates diverse ultrastructural, light microscopic and immunohistochemical findings. These features are related to each other and to the biological behaviour of renal rhabdoid tumours, and six extra-renal lesions with rhabdoid features obtained from the Pathology Archives of the Red Cross Children's Hospital and Groote Schuur Hospital. In this series primitive epithelial elements are a dominant feature, but ultrastructural features of one renal rumour suggest diverse differentiation. The extra-renal lesions investigated include three undifferentiated rhabdoid lesions, a primitive neuro-ectodermal tumour, a malignant epithelioid Schwannoma and a possible undifferentiated hepatocellular carcinoma; all showing areas of extensive rhabdoid differentiation. Pseudo-rhabdoid cells in an additional two cases were also examined. These particular tumours were a nephroblastoma and a fibro-lamellar carcinoma of the liver. These rhabdoid tumour mimics were ultrastructurally different from true rhabdoid cells. Strong immunohistochemical co-expression of Vimentin and cytokeratin in rhabdoid tumour cell inclusions has been noted by previous investigators. (Vogel, 1984) (Gansler, 1991), (Berry, 1992). We speculate that the predominant line of differentiation in renal rhabdoid tumours is epithelial although, as in nephroblastoma multiple lines of differentiation may occur. The extra-renal lesions appear to represent more than one entity, but once again epithelial or neuro-epithelial differentiation appears to be present. Ultrastructural examination is a more useful investigation than immunohistochemistry because of inherent non-specific uptake of antibodies by the filamentous cytoplasmic inclusions.
Open Access
Evaluation of the P13K pathway and downstream effect in Her-2 positive and negative breast carcinomas
(2014) Rademan, Anina; Govender, Dhiren
Breast cancer management continues to be a challenge due to the heterogeneity of the disease and the fact that individuals with the same stage and pathological diagnosis may respond differently to treatment as a result of differences in gene expression. Several components of the Her-2/PI3K/Akt pathway were evaluated for their relevance as potential prognostic markers or indicators for treatment. A secondary objective was to evaluate the CISH technique for its suitability for analysis of Her-2 gene amplification on archived, Papanicolaou-stained fine needle aspiration (FNA) samples. Tissue blocks from a retrospective series of 93 primary breast carcinoma cases were selected, based on their Her-2 status. Twenty six of these cases received trastuzumab treatment while 67 did not. Expression of Her-2, ER, PI3K, PTEN, p-Akt, BCL2, NFÎºB, MDM2 and p53 were analysed and compared with various clinicopathological features. The CISH technique was evaluated for its suitability for analysis of Her-2 gene amplification on archived, Papanicolaou-stained FNA samples.
Open Access
Gastric remnant carcinoma : histochemical and immunohistochemical profile
(2004) Elazzabi, Tawfik; Mall, Anwar S
Gastric remnant carcinoma (GRC) is a gastric cancer that develops in gastric remnant more than five years after resection for benign disease. GRC comprises 1 %-9% of all gastric cancers. Partial gastrectomy for peptic ulcer is thought to be a risk factor for GRC. Pancreato-duodenal and bile reflux may play an important part in the aetiology of GRC. Primary gastric carcinoma (pGC) is a gastric cancer that arises in un-operated stomach and chronic gastritis is a well-known risk factor. Consequently there appear to be differences in the aetiology of GRC and PGC. According to many studies, surgical treatment of early GRC (Stage I or II) resulted in the same or better prognosis with similar stage PGC. However if diagnosed late, GRC has a worse prognosis than PGC at the same stage. In this study haematoxylin and eosin, alcian blue pH 2.5, periodic acid Schiff, high iron diamine and Giemsa stains as well as immunohistochemical methods (eight antibodies against MUCI to MUC6) were used to determine the type of mucin and the pattern of staining in twenty cases of GRC and twenty PGC (ten cases of intestinal type PGC, ten diffuse type PGC) and ten normal gastric mucosal biopsies. The aim of the study was to describe the morphology of GRC and the adjacent gastric mucosa, as well as to determine the histochemical and immunohistochemical mucin profile of GRC and to compare this with that of PGC and normal mucosa.
Open Access
Hereditary non-polyposis colorectal carcinoma (HNPCC) : morphological and immunohistochemical studies
(2005) Holm, Hannes; Hall, Pauline de la Motte
Families with hereditary non-polyposis colorectal carcinoma (HNPCC) are not uncommon along the West-Coast of South Africa. These patients present with early onset carcinomas mostly colorectal, predominantly in the right colon. They may develop tumours of other organs, including uterus, breast, stomach and skin. To evaluate and compare the microscopic characteristics of three groups of colorectal carcinomas (HNPCC, early onset colorectal carcinomas and sporadic colorectal carcinomas). 2. To determine the features most characteristic of the group.
Open Access
The histopathology and immunohistochemical expression of cell cycle regulators and mismatch repair gene proteins in colorectal carcinoma : a comparative study
(2015) Sookhayi, Raveendra; Govender, Dhiren
Introduction: It has been reported that HNPCC colorectal carcinomas demonstrate a better prognosis compared to sporadic carcinoma, however the exact mechanism for this is still uncertain. It is possible that tumour morphology, location and cell cycle markers may be indicators of the underlying molecular mechanism. In a resource limited setting these factors may help to stratify which cases need further molecular testing and genetic counselling. Aims and objectives: To characterise the macroscopic and microscopic pathology in three cohorts of patients. The cohorts include (1) patients with CRCs that are < 50 years and mutation negative, (2) < 50 years and DNA mismatch repair gene mutation positive and (3) more than 50 years (sporadic). To investigate the immunoexpression of the cell cycle regulators (p21, p27, p53, c-myc, cyclin D1 and cyclin E) and MMP-7 in each cohort. To compare the immunoexpression of each marker between cohort s. To correlate the immunoexpression of each marker with tumour type, stage and grade. Materials and methods: In total, 17 mutation negative, 15 mutation positive and 28 sporadic adenocarcinoma resection cases were available for study. The histopathological features of all cases were reviewed. The cases were stained with antibodies against p21, p27, cyclin D1, cyclin E, p53, c- myc MMP -7, MLH1, MSH2 and MSH6. Results were considered statistically significant if P < 0.05, and P <0.017 if 3 pairs of medians were compared. Results: The mutation positive tumours were more frequently right sided tumours and showed mucinous differentiation, tumour infiltrating lymphocytes and an expanding border. The sporadic and mutation negative cohort s showed similar morphology. In the sporadic cohort, the five tumours that were MLH1 negative demonstrated morphological features of MSI-H tumours. MLH1 mutations were the commonest. MLH1 immuno expression was lost in the mutation positive tumours and was statistically significant when compared to the other two cohorts. There was no statistical significance among the three cohorts for MSH2 and MSH6 immunoexpression. There was no statistically significant difference in immunoexpression for p21, p27, p53 and MMP-7 among the three cohorts. Furthermore, there was no association with tumour type and stage. Cyclin D1 expression was increased in the mutation positive cohort and was statistically significant when compared to the mutation negative cohort only. Cyclin E expression was also increased in the mutation positive cohort and was statistically significant when compared to the sporadic cohort only. Conclusion: The morphological features of colorectal carcinomas can be helpful in identifying MS I-H tumours and cases requiring further molecular studies. The cell cycle marker expression s did not explain the expected differences in patient outcome and prognosis. The mutation negative cohort in our population continues to remain enigmatic and further testing at the molecular level is required, that may reveal another novel pathway of colorectal carcinogenesis or other novel mutations in mismatch repair genes.
Open Access
Human immunodeficiency virus (HIV) and Human papillomavirus (HPV) infection and cell cycle regulators in preinvasive lesions and invasive carcinomas of the anus
(2017) De Jager, Louis Johann; Govender, Dhirendra
Introduction: Anal cancer is a rare disease which accounts for 1.5% of gastrointestinal tract malignancies. The majority of these carcinomas are squamous cell carcinomas and are associated with high risk-HPV infection. HIV infection appears to interact synergistically with high risk-HPV in the development of squamous cell carcinoma at this site. Aims and objectives: To review the pathology of anal carcinomas and anal intraepithelial neoplasia (AIN) diagnosed between 2003 and 2012. To investigate the frequency of high risk-HPV infection and HIV infection in premalignant and malignant epithelial anal lesions using immunohistochemistry and to investigate the effect of these infections on Langerhans cell density. To investigate the role of cell cycle and WNT signalling pathway markers in the pathogenesis of these lesions. Materials and methods: This was a retrospective study and 51 cases of anal carcinoma and precursor lesions were identified during the study period. Where possible, blocks which contained normal and dysplastic tissue and invasive carcinoma were selected. Ten immunohistochemical stains (p24, p16, pRb, E-cadherin, CD1a, Langerin, Bcl-2, Ki-67, HPV L1 capsid protein and β-catenin) were performed and scored in normal, dysplastic and carcinomatous tissue. Data were analysed to determine if there were statistically significant differences in the expression of markers in different subtypes of carcinomas, grades of differentiation of carcinomas and in the range from normal to carcinoma. Results: The patients' ages ranged from 24 to 81 years. There were 26 females and 24 males; one patient did not have age or sex information available. Twenty-one cases did not have information available on HIV status. Eleven cases demonstrated squamous cell dysplasia only and 40 cases demonstrated invasive carcinoma, 36 of these being squamous cell carcinomas. p24 was positive in only two known HIVpositive cases. p16 demonstrated block positive staining in 35 out of 36 squamous cell carcinomas and 14 out of 18 high grade squamous intraepithelial lesions. There was a significant decrease in the proportion of pRb-positive cells from well to poorly differentiated squamous cell carcinomas (p=0.03). HIV status did not influence the expression of markers. The subtype of carcinoma did not have a significant effect on the proportion of pRb-positive cells. Differentiation of squamous cell carcinoma had a significant effect on the E-cadherin expression score (the more well differentiated a carcinoma, the higher the E-cadherin score; p=0.04). There was a significant difference in E-cadherin expression between normal tissue and squamous cell carcinoma, and dysplastic tissue and squamous cell carcinoma (p=0.002 and p=0.004, respectively). Differentiation, subtype of squamous cell carcinoma and HIV status did not influence the density of CD1a/Langerin-positive Langerhans cells. No significant difference in the density of CD1a/Langerin-positive cells was demonstrated amongst normal, dysplastic and squamous cell carcinoma tissue, regardless of HIV status. The differentiation, subtype of squamous cell carcinoma and HIV status, did not have a significant effect on the Bcl-2 expression. There was a significant difference in Bcl-2 expression among normal, dysplastic and cancerous tissue (p=0.02). There was no significant difference in the Ki-67 proliferation index amongst the different subtypes of squamous cell carcinoma and the degrees of differentiation. HPV L1 capsid IHC only stained two squamous cell carcinomas and nine cases with dysplastic squamous epithelium (AIN I and AIN II). There was no case which showed abnormal localisation of β-catenin. Conclusion: Less than 20% of HIV-positive cases showed positive p24 staining. p24 does not appear to be a useful stain to determine HIV status in non-lymphoid tissues. p16 is known to be a surrogate marker for high risk-HPV infection, and the fact that 35 out of 36 squamous cell carcinomas showed block positive staining suggests that the majority of squamous cell carcinomas in this study were associated with high risk-HPV infection. The mean density of CD1a- and Langerin-positive cells was increased in HIVpositive patients. HPV L1 capsid IHC showed a low sensitivity of detecting AIN and invasive SCC of the anus. Including vaccinations against high risk-HPV in the South African Expanded Programme on Immunisation may reduce the burden of anal dysplastic lesions and invasive squamous cell carcinoma in future.
Open Access
Human Papillomavirus DNA extraction and genotype analysis by multiplex real time polymerase chain reaction from formalin fixed paraffin wax-embedded cervical carcinoma specimens
(2019) Price, Brendon; Govender, Dhirendra; Naidoo, Richard
Introduction: Cervical squamous cell carcinoma is most commonly caused by persistent infection by high risk human papillomavirus (hrHPV) genotypes. The exact type of hrHPV varies geographically and is the basis for HPV–based vaccination for cervical squamous cell carcinoma prevention. Little is known regarding local hrHPV genotypes within the Western Cape population of South Africa. Aims and objectives: This was a pilot study aiming to extract of high quality genomic DNA from archival FFPE cervical squamous cell carcinoma cases and identify hrHPV genotypes by multiplex real time PCR (RT-PCR). Materials and methods: A retrospective search identified a total of 57 cases of cervical squamous cell carcinoma for the period 2004-2014. This was reduced to a final number of 23 that exhibited sufficient tumour burden for DNA extraction. The most common age group was 40-49 years. HIV status was as follows: two HIV-positive, 14 HIV-negative and 7 HIV unknown. DNA was extracted from archival FFPE cervical squamous cell carcinoma samples using QIAGEN QIAamp® DNA FFPE Tissue kit. Housekeeping genes were detected by endpoint PCR using standard primers for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) sequences to determine the quality and integrity of extracted DNA for downstream PCR amplification experiments. HrHPV DNA amplification was optimised using a touchdown PCR technique with L1 consensus gene GP5+/GP6+ primers. HrHPV genotypes were detected using a four colour multiplex hrHPV genotyping kit. Samples showing positive results in overlapping probe filter detection spectra were subjected to DNA Sanger sequencing for final confirmation of specific hrHPV genotype. Results: Standard xylene DNA extraction methods using QIAamp® system yielded adequate amounts of DNA with average final concentration of 463.2 ng/l and A260/A280 ratio of 1.86. Housekeeping genes were successfully detected in all samples, confirming that no significant DNA degradation of target sequences occurred within the archival time range of 2004-2014. HPV L1 detection via GP5+/GP6+ primers with endpoint PCR was not achieved via standard cycling conditions and required the use of a touchdown technique with gradually decreasing annealing temperatures. This method successfully identified HPV L1 sequences in 22 out of 23 cases. Multiplex RT-PCR with four colour hydrolysis probes identified hrHPV genotypes in 22 of 23 cases with relative frequencies of HPV genotypes: 16>>18=39=45>33. Most cases showed infection with a single hrHPV genotype (HPV 16 and one case with HPV 33) with four cases demonstrating two genotypes (two with HPV 16&18, one with 16&33 and one with 39&45) and one case with three genotypes (HPV 16, 39, 45). Interestingly, none of the HIV-positive cases showed multiple hrHPV genotype infection. Four hrHPV cases with overlapping spectra for HPV 18/31 and 45/59 were subjected to Sanger sequencing for confirmation of genotype. Three of four cases showed 100% match for genotypes 18 and 45 with the final case demonstrating only co-infective HPV 16.Conclusion: Commercial DNA extraction kits yield adequate amounts of intact, amplifiable DNA in archival FFPE cervical carcinoma specimens. Touchdown PCR is necessary for HPV detection in extracted FFPE DNA cases using GP5+/GP6+ L1 primers. RT-PCR using multicolour hydrolysis probes is a rapid, sensitive technique for hrHPV genotype screening of cervical squamous cell carcinoma specimens. A three colour detection system rather than four colour kit is recommended for future studies in order to avoid extra cost in DNA sequencing cases with overlapping spectra. This pilot study demonstrates hrHPV genotype prevalence similar to that in other populations and suggests that vaccination with currently available formulations would provide a sufficiently wide coverage of HPV genotypes. Future studies will include application of the FFPE DNA extraction, endpoint PCR and RT-PCR techniques to the remainder of the cases in the original cohort.
Open Access
Identifying Children with Constitutional Mismatch Repair Deficiency (CMMR-D) Syndrome in the Expanding Lynch Syndrome population in Cape Town
(2021) Tu, Sindy Jen-Yi; Pillay, Komala; Ramesar, R; Wessels, A
INTRODUCTION: Constitutional Mismatch Repair Deficiency (CMMR-D) syndrome is a rare tumour predisposition and polyposis syndrome that presents in childhood. It is caused by mutations in mismatch repair (MMR) genes that result in a tumour spectrum including colorectal cancers, high-grade gliomas, non-Hodgkin T-cell lymphomas and leukaemias. It is characterized by biallelic germline mutation of one of four possible MMR genes resulting in loss of protein expression that can be identified by applying immunohistochemistry to formalin fixed paraffin embedded tissue sections. Use of MMR immunohistochemistry is established in the setting of Lynch syndrome (LS); however, the pattern of loss of staining in the background, non-tumour tissue is unique to CMMR-D syndrome. CMMR-D syndrome is seen in LS families and occurs as a result of consanguinity or founder effect. The South African population has LS families concentrated in the Western Cape and Northern Cape Provinces and the mixed ancestry population shows a unique MLH1 c1528C>T mutation which may have implications on the incidence, penetrance and severity of CMMR-D syndrome seen in our population. The diagnosis of CMMR-D syndrome includes clinical findings outlined in the European Consortium's Care of CMMRD document and confirmation of the biallelic mutation in one of the MMR genes. MMR immunohistochemistry can be used in the diagnosis of CMMR-D syndrome by identifying cases for targeted molecular genetic tests. However, MMR immunohistochemical staining patterns are not usually described in detail, particularly the loss of staining of the affected gene in the background, non-tumour tissue, the key feature of CMMR-D syndrome. METHODS: We performed a retrospective analysis of archival formalin fixed paraffin embedded tissue of children attending Red Cross Children's Hospital with tumours that form part of the CMMR-D spectrum, outlined by the Care for CMMRD criteria. We used the criteria of high-grade gliomas (WHO Grade III or IV) occurring before 25 years of age, cutaneous lesions suggestive of CMMR-D syndrome and patients with a first or second degree relative diagnosed with LS. MMR immunohistochemistry was applied, and the staining pattern was documented in terms of proportion of tumour staining and intensity of staining using a modified Allred Scoring system. Specific attention was given to the characterization of the staining pattern of the background normal tissue. RESULTS: 21 samples taken from 18 patients were evaluated. 16 samples represented brain tumours, predominantly high-grade gliomas. Three samples were excluded due to suboptimal staining despite positive external controls. 12 samples showed intact staining of all four MMR stains. Two samples showed staining of unknown significance. Four samples from 3 different patients showed staining patterns compatible with MMR deficiency. This included two patients, each with a biopsy showing high-grade glioma and two samples of the same patient taken at a 1-year interval of a Burkitt lymphoma. Of these four samples, three samples showed loss of staining in background non-tumour tissue with positive external control, the unique staining pattern for CMMR-D syndrome. These cases will be referred for confirmatory testing by molecular genetic techniques. CONCLUSION: MMR immunohistochemistry can be used in the evaluation of CMMR-D syndrome, but care is needed in evaluating adequacy of staining, the pattern and scoring of staining of both the tumour and the background non-tumour tissue. Endothelial cells are easy to identify and evaluate as background tissue which is useful in extra-intestinal tumours. Neurons and choroid plexus can also be evaluated as background tissue in brain tumour samples. Selection bias in this study resulted in the underrepresentation of lymphomas and colorectal carcinomas. Improved characterization and search for Non-Hodgkin T-cell lymphomas and inclusion of samples of colorectal carcinomas of adolescents and adults would be needed to include these tumours. Use of MMR immunohistochemistry in postmortem tissue samples is not recommended because of suboptimal staining, even with a short post-mortem interval of 1 day. The diagnosis of CMMR-D syndrome depends on clinical application of Care for CMMRD criteria, MMR immunohistochemistry in conjunction with molecular genetic testing. It is important to identify cases of CMMR-D syndrome and offer cancer screening to prevent development of other cancers in the index patient. It also provides an opportunity for genetic counselling and testing of the parents and at-risk siblings.
Open Access
An immunohistochemical assessment of endomyocardial biopsy specimens from the South African arrhythmogenic right ventricular cardiomyopathy registry
(2014) Morse, Nicole; Wainwright, Helen; Mayosi, Bongani
Arrhythmogenic right ventricular cardiomyopathy / dysplasia (ARVC/D) is a genetic disease causing fibro-fatty replacement of the right ventricular myocardium, resulting in cardiac arrhythmias and sudden death. Part of the diagnostic work up for these patients includes a biopsy of the endocardium which has historically been difficult to interpret and of limited value in the early stages of disease. This study will focus on novel immunohistochemical stains of the cardiac desmosomes. These will be used to try to aid in the early diagnosis of ARVC.
Open Access
An immunohistochemical study of beta-catenin in HNPCC colon tumours
(2004) Watkins, Jennifer G; Hall, Pauline de la Motte
Beta-catenin is normally complexed with adenomatous polyposis coli (APC) protein and E-cadherin adhesion molecule, and localized on the cell membrane. If APC/beta-catenin is disrupted, beta-catenin transfers into the nucleus, where it functions as a transcriptional activator, causing unregulated cell proliferation. The localisation of beta-catenin in H NPCC adenomas has not been studied but a shift in beta-catenin to the nucleus has been previously demonstrated in a range of col 0 recta I cancers, including those in HNPCC. The aim of the first part of the study was to determine whether there is a beta-catenin shift occurring as an early event in HNPCC tumours. Coded sections of tumours were immunohistochemically stained with antibody against beta-catenin and counterstained in haematoxylin. 14 HNPCC adenomas, 13 HNPCC carcinomas, 10 FAP adenomas, 10 FAP carcinomas, 10 sporadic adenomas and carcinomas and 10 juvenile polyps -three with dysplasia and seven without- were studied. A score was given for loss of membrane staining (0-1), presence of cytoplasmic staining (0-2) or nuclear staining (0- 2) and a total out of five obtained. An shift in beta-catenin was demonstrated at the adenoma phase in HNPCC. HNPCC, tumours were compared with sporadic tumours and a statistically significant similarity in prevalence of beta-catenin shift found in adenomas and carcinomas. The early shift in beta-catenin in HNPCC led to the second part of the study evaluating the "down-stream" effects of this shift in HNPCC tumours. TheHNPCC sections were immunohistochemically stained with E-cadherin, cmyc and cyclin 01. The results showed a positive correlation between Ecadherin loss, increased cyclin 01 and a shift in beta-catenin. No significant change in c-myc or correlation between c-myc and a shift of beta-catenin was found. In conclusion the study indicates that disruption of the APC/beta-catenin pathway plays a similar role in HNPCC tumours to that in sporadic tumours. A notable exeption is the effect on c-myc and further study is needed in this regard.
Open Access
Interaction between DC-SIGN and DC-SIGNR with HHV-8 (LANA-1) and HIV-p24 in Castleman disease
(2018) Chetty, Dharshnee Rama; Govender, Dhiren
Background: Castleman disease (CD) is a lymphoproliferative disorder with four subtypes, some of which are aetiologically linked to Human Herpes virus 8 (HHV-8) which is known to cause diseases preferentially occurring in HIV-infected individuals. There has been a notable increase in the number of patients with HIV/HHV-8 associated CD diagnosed in the Groote Schuur hospital complex. Aims: The aim of the study was to determine the role of DC-SIGN, DC-SIGNR, p24 and HHV-8 (LANA-1) in Castleman disease. Our objectives were to identify the presence of DC-SIGN and DC-SIGNR in HHV-8 infected cells, determine whether HHV-8 and p24 (HIV) co-infection occurs in the same cells and to determine whether HHV-8 infects B and/or T cells. This study not only represents the largest and first immunophenotypic investigative evaluation of CD but also signifies the first double staining immunohistochemical analysis of CD diagnosed at Groote Schuur hospital. Methods: This was both a retrospective descriptive as well as an analytic cross-sectional immunohistochemistry study. Fifty cases of CD diagnosed at the Division of Anatomical Pathology, National Health Laboratory Service, Groote Schuur hospital over a ten and half year period were included in the study. Double immunohistochemistry was used to characterise HHV-8 infected cells using LANA-1 antibody, in conjunction with DC-SIGN, DC-SIGNR, p24, CD20 and CD3. Immunophenotypic analysis was then performed to assess 1) the number of infected HHV-8 cells and 2) number and distribution of cells co-expressing HHV-8 and DC-SIGN, DC-SIGNR, p24, CD20 and CD3. The immunophenotypic profiles were then compared to the CD morphologic subtypes. Results: The study cohort included 26 male and 24 female patients (M: F = 1.08:1), mean age 37.7 years. There were 16 hyaline vascular CD (HV-CD), 16 plasmablastic CD (Pb-CD). Nine plasma cell CD and 9 mixed-CD subtypes. There was a statistically significant association between HIV (n=45) and HHV-8 (n=40) positivity (p < 0.0002). CD4 counts and HAART enrolment were not predictive of CD development (p = 0.6120). Concurrent Kaposi sarcoma was seen in 16% (n=8) of the cohort. When comparing Pb-CD and HV-CD, there were statistically significant differences in density of LANA-1 infected cells (p<0.0002), LANA-1/DC-SIGN co-expressing cells (p <0.0072) and LANA-1/p24 co-expressing cells (p<0.0001). Conclusions: The findings of this study suggest that DC-SIGN may have a role in HHV-8 entry into cells. Furthermore, there is evidence that HIV and HHV-8 co-infection may function synergistically in CD. It is possible that DC-SIGN and DC-SIGNR facilitate dual viral entry into cells and influence viral replication and persistent infection.
Open Access
International Academy of Cytology Yokohama System for reporting Breast Fine Needle Aspiration Biopsy (FNAB) cytology: A Retrospective Study in a Single South African Tertiary Institution
(2021) Pamacheche, Patricia Nee Pariza; Chetty, Dharshnee Rama
Introduction: Breast carcinoma is the most common malignancy amongst women in South Africa. Triple assessment has been pivotal in the work up and management of breast carcinoma. Breast cytology has been used as a component of the triple assessment. Although core needle biopsy (CNB) is the gold standard and the preferred diagnostic modality, there is still a role for fine needle aspirate cytology (FNAC) in resource limited settings. The present study was conducted at Groote Schuur Hospital in Cape Town, South Africa. Aims: 1. To assess the utility of the International Academy of Cytology (IAC) Yokohama System for Reporting Breast FNAC five category stratifications in our institution. 2. To assess the respective risk of malignancy (ROM) for each category. 3. To assess the diagnostic yield of the breast FNAB at our institution by comparing it to the matched histopathology over a 12-month period. Methodology: A retrospective longitudinal descriptive study was done. A computerized search on TrakCare NHLS for the year 2019, identified 884 patients who had breast cytology and corresponding histology specimens. The cytology categories(C1-C5) were first reclassified according to the IAC Yokohama system. The new cytology category was then compared to the histological diagnosis for each patient. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and risk of malignancy (ROM) were calculated. Results: The sensitivity, specificity, PPV, and NPV were 83.10%, 93.01%, 88.86% and 89.13% respectively. The Cohen's kappa coefficient was 0.659 and percentage agreement was 80.85%. The ROM was calculated; insufficient (9.09%), benign (4.46%), atypia (45.28%), suspicious for malignancy (72.5%) and malignant (91,09%). Conclusion: Breast aspiration cytology performed at GSH has shown good correlation with histopathology as well a high sensitivity and specificity comparable to international standards. The ROM is comparable to previous similar studies. Overall, our results show that breast aspiration cytology is a rapid, accurate and cost-effective diagnostic procedure in our institution that is very useful in the diagnosis of benign and malignant breast lesions.
Open Access
Investigating the relationship between miRNA expression and epithelial mesenchymal transition in colorectal cancer
(2016) Jaca, Anelisa; Naidoo, Richard; Locketz, Michael L
Introduction: Epithelial-mesenchymal transition (EMT) is characterized by the loss of an epithelial phenotype and gain of a mesenchymal phenotype, i.e., migratory and metastatic properties. The EMT process is therefore characterized by a low expression of E-cadherin and high expression of mesenchymal markers (e.g., N-cadherin, snail and vimentin). It is stated that cells which have undergone EMT also gain stem cell features. Therefore, both EMT and stem cell phenotypes have been implicated in carcinogenesis and metastasis of tumour cells. Furthermore, EMT is regulated by small non-coding molecules (miRNAs) that either function as tumour suppressors or oncogenes (oncomirs). Tumour suppressor miRNAs reverse EMT while oncomirs activate it. Therefore, investigating the relationship between miRNAs and EMT is important in addressing metastasis of colorectal cancers (CRC). Aims and Objectives: The aim of the study was to determine the association between miRNA (miRNA-21 and miRNA-34a) expression levels and EMT in CRC. In addition, this investigation aimed to correlate miRNA and EMT data with clinicopathologic features of the study cohort. Methodology: A total of 100 CRC (including 8 known HNPCC cases) Formalin Fixed Paraffin Embedded (FFPE) tissue blocks and their corresponding H&E slides were collected from the archives of the Division of Anatomical Pathology at the University of Cape Town. Subsequently, the FFPE tissue blocks were sectioned at 3μm and IHC analysis of 4 EMT markers (E-cadherin, N-cadherin, snail-1 and vimentin) and 1 stem cell marker (CD44V6) was performed. The stains were then evaluated and scored by a pathologist. The IHC data were then correlated with clinicopathologic features. Furthermore, 59 cases (FFPE tissues and corresponding H&E slides) which included the 8 HNPCCs were randomly selected for miRNA analysis. The H&Es were examined by a pathologist to demarcate normal and tumour regions. RNA was then extracted from 59 tumours and 12 normal tissues using a High Pure FFPET Isolation Kit (Roche). Subsequently, cDNA was synthesized and qRT-PCR was performed to determine the expression levels of miRNA-21 and miRNA-34a. MiRNA-21 and miRNA-34a expression levels were ascertained using the relative quantification method. Moreover, the clinical significance of the two miRNAs was evaluated in relation to MSI status. Therefore, IHC analysis of MLH1, MSH2 and MSH6 mismatch repair proteins was performed on the Ventana platform. Statistical analysis was performed using Fisher's and Pearson's Chi Square tests in Stata 12 to correlate EMT and clinicopathologic data. Additionally, the Mann-Whitney non-parametric test in GraphPad prism 6 was used to determine miRNA-21 and miRNA-34a expression in relation to EMT and MSI data. Results: Our results showed low expression of E-cadherin in 77% of cases. In addition, there was decreased expression of N-cadherin and vimentin in 98% whilst snail-1 expression was decreased in 65% of the cases. Low expression of CD44v6 was also seen in 78% of the cases. There was no correlation between EMT/stem cell markers and clinicopathologic data. Furthermore, increased miRNA-21 expression was significantly associated with grade, lymph node metastasis and age of patients. There was a significant correlation between high miRNA- 21 expression and down-regulated snail-1 and N-cadherin expression. MiRNA-34a expression was not associated with any of the clinicopathologic features. In addition, high miRNA-34a expression was linked with low expression of snail-1 and CD44v6. Increased miRNA-21 expression was related with MSS tumours, whereas there was no relationship between miRNA- 34a and MSI status. Conclusion: Our investigation shows that there is an inverse association between miRNA (miRNA-21 and miRNA-34a) expression and two EMT (N-cadherin and snail-1) markers in our colorectal cancer cohort. Our data also show that both miRNA-21 and miRNA-34a cannot be used as biomarkers to determine progression of the cancer. Contrary to previous studies, our findings indicate that miRNA-21 does not activate EMT in this CRC cohort. However, similar to other studies our results confirm that miRNA-34a may be repressing snail-1 expression, thereby inhibiting EMT in the cancer.
Open Access
An investigation of criteria for the morphologic diagnosis of infection by Chlamydia trachomatis in the uterine cervix
(2004) Knight, Bryan Kenyon
Includes bibliographical references (leaves 180-211).