- ItemCorrelating p16INK4a /Ki-67 co-expression and gene methylation with HIV infection and high-risk HPV in abnormal cervical squamous intraepithelial cells(Stellenbosch : Stellenbosch University, 2023-03) Louw, Meagan; Sanderson-November, Micheline; Neethling, Greta; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Anatomical Pathology.ENGLISH SUMMARY: Globally, cervical cancer is the fourth most common cancer amongst women. Persistent infection with high-risk human papillomavirus (HPV) is shown as the causal factor in cervical cancer development. Women living with HIV is six times more prone to cervical cancer development. The aim of this study was to identify the HR-HPV types, investigate the simultaneous expression of p16INK4a and Ki-67 and evaluate the methylation status of CADM1, MAL and miR124-2 genes in cytology samples from HIV-positive and HIV-negative women with LSIL, HSIL, ASC-US, and ASC-H Pap smear results. Study participants were women between the ages of 21 years to 60 years referred to the Colposcopy clinic at Tygerberg Academic Hospital. Exfoliated cervical intraepithelial cells were collected in Surepath medium and HR-HPV types were determined using the Hybrispot HPV direct flow chip kit, whereas the co- expression of p16INK4a/Ki-67 proteins was evaluated with the CINtec® Plus cytology immunocytochemistry kit. For methylation assays, DNA was isolated from the left-over exfoliated cells followed by the assessment of quantity and purity of isolated DNA using fluorometry and spectrophotometry, respectively. Isolated DNA was bisulfite converted and the methylation assays for the CADM 1, MAL and miR-124-2 genes were done using the respective EpiMelt assays. HPV DNA detection results were associated with cytological diagnosis as well as HIV status. In our study group 74 % (51/69; 95% CI: 62,1%-83%) of woman tested positive for HPV of which 70.6% (36/51) were of WLWH and 29.4% (15/51) of HIV negative women. Our results showed that p16INK4a /Ki-67 dual-staining was detected in 43.8% (25/57) of the samples with the HSIL cytology showing the highest p16INK4a /Ki-67 co-expression rate of 64% (16/25) compared to the other cytology groups. The proportion of the LSIL group with p16INK4a /Ki- 67 dual staining was 33,3% (4/12), whereas that of the ASC-US and ASC-H groups were 25% (2/8) and 23% (3/13) ASC-H, respectively. CADM1 methylation was detected in 12.3% (7/57) of samples, while MAL and the miR-124-2 genes showed methylation in 14% (8/57) and 12.3% (7/57)of the samples, respectively. HPV infection was detected in 28.1% (16/57) of the samples with methylated CADM1, MAL and miR-124-2 genes. A significant relationship was found between HR-HPV and miR-124-2 methylation. The logistic regression model analysis employing predictors such HR-HPV, p16INK4a and Ki- 67 co-expression, as well as the methylation status of the CADM1, MAL, and miR-124-2 genes, for LSIL cytology showed low sensitivity and high specificity, contrasting to that of the HSIL model with high sensitivity and low specificity. Therefore, we conclude that a larger study is warranted, with removing or adding predictors for model improvement.
- ItemTreatment of lentigo maligna of the head and neck with staged excision in South Africa : assessing surgical excision margins with Melan A, SOX10 and PRAME immunohistochemistry(Stellenbosch : Stellenbosch University, 2022-11) de Wet, Johann; Schneider, Johann Wilhelm; du Plessis, Pieter; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Anatomical Pathology.ENGLISH SUMMARY: Lentigo Maligna (LM) is a subtype of melanoma in situ that occurs on sun-damaged skin, typically on the head and neck area of older individuals. LM is associated with significant subclinical extension beyond the visible clinical margins and therefore recommended surgical excision margins may be inadequate for complete surgical clearance of the tumour. Staged Excision (SE) has emerged as the treatment of choice for LM of the head and neck. It allows for complete margin control, superior clearance, and lower recurrence rates compared to conventional wide local excision (WLE). Differentiating between actinic melanocyte hyperplasia (AMH) and LM at the peripheral margin complicates the assessment of completeness of excision when using this technique. Objectives: The study aimed to describe the patient demographics, tumour characteristics, and histological findings of LM cases on the head and neck treated with SE. Secondary objectives included: (1) To determine if standard recommended surgical excision margins for LM of the head and neck are adequate to achieve a 97% clearance rate and if any patient or tumour characteristics warranted wider margins, (2) To determine whether immunohistochemical (IHC) staining with SOX10 and PRAME aids in diagnosing LM on excision margins compared to conventional Hematoxylin and Eosin (H&E) and Melan A IHC staining. Methodology: The study involved a retrospective chart review of all patients diagnosed with LM of the head and neck and treated with SE at the Skinmatters Mohs Micrographic Surgery and Reconstructive Unit. Tissue sections of LM cases with LM reported to be present at margins were immunohistochemically stained with SOX10 and PRAME and reviewed by a Mohs surgeon and a pathologist with expertise in melanoma pathology. Results: The first component of the study showed that 6mm, 9mm, and 12mm surgical excision margins obtained complete excision in 60.94%, 71.88%, and 90.64% of the LM cases, respectively. A surgical excision margin of 18mm correlated with complete excision in 96.7% of tumors, while complete excision in 100% of LM cases required a 21mm margin. Recurrent tumors (p-value = 0.01) and tumour size larger than 20mm were associated with wider surgical excision margins (pvalue= 0.154). The second study component evaluated IHC stains and consisted of 35 sections. Based on H&E and Melan A IHC staining, 23 sections were diagnosed as LM by the initial pathologist. Further staining with SOX10 IHC showed only 8 cases consistent with a diagnosis of LM and 9 revealing actinic melanocyte hyperplasia (AMH). PRAME was positive in 5 of the 8 cases of LM and negative in all 9 cases of AMH (p=0.009). The presence of melanocyte nests (p=0.29) and pagetoid spread (p = 0.003) were the most reliable histological findings for distinguishing LM from its mimics. Conclusion: This study of LM in a South African population corroborates that the standard surgical excision margins recommended by international melanoma guidelines for LM are inadequate to achieve a 97% clearance rate. Recurrent LM cases and tumours larger than 20mm may require wider margins. The study further concluded that SOX10 is a more specific and sensitive marker for melanocytes when assessing for LM on excision margins compared to Melan A. The addition of PRAME can be useful to confirm or exclude the diagnosis in challenging cases.
- ItemDetermining the suitability of bio-specimens obtained by fine needle aspiration biopsy at a tertiary hospital in Malawi for immunocytochemical assessment(Stellenbosch : Stellenbosch University, 2022-04) Mulenga, Maurice; Schneider, Johann Werner; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology.ENGLISH SUMMARY: Fine-needle aspiration biopsy (FNAB) is a quick, economical, least invasive and easy to perform a minor surgical procedure. In resource-limited settings, FNAB is of utmost importance in providing a rapid diagnosis that facilitates timely and correct institution of treatment. The FNAB smear preparation provides an opportunity for either rapid on-site evaluation or routine diagnosis if ancillary tests are necessary to establish a specific diagnosis. Cell blocks (CB) prepared from FNAB specimens improve the diagnostic yield, increase the sensitivity and reduce false-positive interpretations of detecting a malignant neoplasm. In addition, CB allow for additional morphological evaluation with a better architectural pattern, enable the performance of numerous ancillary diagnostic studies, including immunocytochemistry and molecular studies and offer the storage of material that can be used for future research studies. Delays in fixing the cell block have been challenges in various cell block preparatory techniques. However, a special alcohol-based fixative, commercially available solution called CytoRich Red® (CRR) has been described to be comparative to liquid-based cytology due to its effectiveness in lysing red blood cells, reducing background material, and improving staining qualities of the nucleus and cytoplasm in routine preparations of non-gynaecological material in suspension or fluids. Despite this breakthrough, there is a paucity of data on the suitability of CRR cell blocks for immunocytochemical and DNA assessment from FNAB material obtained from solid tumours. This study aimed to establish and confirm the suitability of CytoRich Red® Cell Blocks and FNAB biospecimens obtained and prepared at Kamuzu Central Hospital, Lilongwe, Malawi, for cytomorphological and immunocytochemical assessment. This study analysed 144 cell blocks and 128 FNAB smears. It is one of the first within sub-Saharan Africa to describe diagnostic efficacy from FNAB specimens obtained from various superficial and deep masses fixed in CRR. It describes the advantage of using an alcohol-based fixative immediately to reduce pre-fixation time lag. This study showed that CRR-fixed cell blocks improve sensitivity and architectural preservation, and immunocytochemical staining characteristics of the aspirate compared to routine FNAB smears. It is envisioned that CRR-fixed cell blocks will be a source of extractable, stable and usable DNA that supports research in biorepositories and biobanks.
- ItemComparison of Xpert® Breast cancer STRAT4 assay and immunohistochemistry for the evaluation of breast cancer biomarkers in South African patients(Stellenbosch : Stellenbosch University, 2020-12) Dube, Welile Vumile; De Jager, Louis; Kotze, Maritha J.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Anatomical Pathology.Background: Breast cancer is one of the most common cancers diagnosed in women and approximately 60% of breast cancer related deaths are reported in low-and middle-income countries. Breast cancer is a highly heterogeneousdisease, and molecular subtyping is paramount foreffective treatment of patients. Therefore, it is important to validate new molecular methods for assessing cancer biomarkers for cost-effective use in resource-poor settings.Aim:Aretrospective study was performed to determine the concordance between aQuantitativeReverse TranscriptionPolymerase Chain Reaction(RT-qPCR) CE-IVD assay (Xpert® Breast Cancer STRAT4*) and the current gold standard methods of immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) for determining estrogenreceptor (ER),progesterone receptor(PR), human epidermal growth factor receptor 2 (HER2)andproliferation index(KI-67)expression in breast carcinomas.Method: One hundred and one cases of breast carcinoma were retrieved from the archives of the Division of Anatomical Pathology, Tygerberg Academic Hospital. The original stained slides were reviewed and IHC expression of ER, PR, HER2 and KI-67 scored. Three-micron sections were cut from formalin-fixed paraffin embedded (FFPE) tissue blocks and processed according to the instructions of the manufacturer. The assay was run on the resultant lysates.Results:The overall percentageagreement between the Xpert® STRAT4 assay and IHC / FISH results were 85.15% for ESR, 89.90% for PGR,91.09% for ERBB2, 90.72% for MKI67 (when using a cut off of 10%) and 84.54% for MKI67 (when using a cut-off of 20%). The positive percentage agreement for ESR, PGR, ERBB2, MKI67 with 10% cut-off andMKI67 with 20% cut-offwere 82.76%, 94.64%, 68.97%, 91.30% and 96.05%, respectively, and the negative percentage agreement were 100%, 84.09%, 91.67%, 80.00% and 42.86%, respectively. Conclusion:The studyhas shownthattheXpert® BreastCancer STRAT4 assay shows good concordance with IHC and FISH in detecting breast cancer biomarkers, and may become a supplementary or alternativestandard of care aftervalidation studies are performed.
- ItemPrevalence of succinate dehydrogenase deficiency in paragangliomas and pheochromocytomas at Tygerberg Hospital: a retrospective review.(Stellenbosch : Stellenbosch University, 2020-12) Bruce-Brand, Cassandra; Van Wyk, A.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Anatomical Pathology.IntroductionPheochromocytomas (PC) and paragangliomas (PGL) are rare neural crest-derived tumours that occur at adrenal and extra-adrenal sites. These tumours may be sporadic but a significant proportion are caused by familial syndromes due to germline mutations. Mutations of the succinate dehydrogenase (SDH) complex make up the bulk of syndromic cases in the international literature. SDH mutated cases are now known to have higher rates of metastatic disease, a younger age of onset, an association with other SDH mutated tumours as well as implicationsforfirst degree relatives. An immunohistochemical stain for SDHB that has excellent correlation with SDH mutation status has been developed and is routinely used in many centres to infer SDH mutation status. Loss of staining is seen when there is a mutation of any of the SDH subunit complexes. The prevalence of SDHmutated tumoursis not known in the South African setting. MethodsA retrospective laboratory-based study was conducted at Tygerberg Hospital in South Africa to assess the prevalence of SDH deficiency in all PC and PGLs between 2005 and 2015. These tumours were further stratified by other characteristics:tumour site, patient age, sex and presence of metastatic disease. Fifty-two cases met the inclusion criteria and the SDHB immunohistochemical stain was performed on these cases. Germline testing or sequencing of these cases was not performed. ResultsThirty-six percent of cases showed loss of staining of SDHB by immunohistochemistry. Head and neck PGLs made up the bulk of cases (50%) and females were strongly represented, particularly at head and neck sites (73%). Loss of staining was significantly correlated with a younger age at presentation(z= -3.59, p< .001).There was no correlation betweenloss of staining and tumour site or patient sex. The inter-observer agreement in interpretation of the immunohistochemical stain was excellent (Cohen’s kappa= 0.917, p< .001). Conclusion The prevalence of SDH deficiency in our setting, as shown by loss of immunohistochemical staining for SDHB, is comparable to the literature and makes up a significant proportion of our PC/PGL cases. This highlights the need for performance of this stain in our setting in order to recognise these syndromic cases. Many patients in South Africa do not have access to genetic testing upon diagnosis of a PC or PGL as this is costly and not widely available. Many studies have shown excellent correlation of the immunohistochemical stain with underlying SDH mutation status. Immunohistochemistry is widely available in South African pathology laboratories and is relatively affordable. Although interpretation of this stain can be challenging, we report excellent inter-observer agreement in a generalist pathology practice when following published guidelines for interpretation. We therefore advocate for routine use of this stain in all PC/PGL cases diagnosed in our setting.