Discovery and technical validation of high-performance methylated DNA markers for the detection of cervical lesions at risk of malignant progression in low- and middle-income countries.

BACKGROUND: Cervical cancer remains a leading cause of death, particularly in developing countries. WHO screening guidelines recommend human papilloma virus (HPV) detection as a means to identify women at risk of developing cervical cancer. While HPV testing identifies those at risk, it does not specifically distinguish individuals with neoplasia. We investigated whether a quantitative molecular test that measures methylated DNA markers could identify high-risk lesions in the cervix with accuracy. RESULTS: Marker discovery was performed in TCGA-CESC Infinium Methylation 450 K Array database and verified in three other public datasets. The panel was technically validated using Quantitative Multiplex-Methylation-Specific PCR in tissue sections (N = 252) and cervical smears (N = 244) from the USA, South Africa, and Vietnam. The gene panel consisted of FMN2, EDNRB, ZNF671, TBXT, and MOS. Cervical tissue samples from all three countries showed highly significant differential methylation in squamous cell carcinoma (SCC) with a sensitivity of 100% [95% CI 74.12-100.00], and specificity of 91% [95% CI 62.26-99.53] to 96% [95% CI 79.01-99.78], and receiver operating characteristic area under the curve (ROC AUC) = 1.000 [95% CI 1.00-1.00] compared to benign cervical tissue, and cervical intraepithelial neoplasia 2/3 with sensitivity of 55% [95% CI 37.77-70.84] to 89% [95% CI 67.20-98.03], specificity of 93% [95% CI 84.07-97.38] to 96% [95% CI 79.01-99.78], and a ROC AUC ranging from 0.793 [95% CI 0.68-0.89] to 0.99 [95% CI 0.97-1.00] compared to CIN1. In cervical smears, the marker panel detected SCC with a sensitivity of 87% [95% CI 77.45-92.69], specificity 95% [95% CI 88.64-98.18], and ROC AUC = 0.925 [95% CI 0.878-0.974] compared to normal, and high-grade squamous intraepithelial lesion (HSIL) at a sensitivity of 70% (95% CI 58.11-80.44), specificity of 94% (95% CI 88.30-97.40), and ROC AUC = 0.884 (95% CI 0.822-0.945) compared to low-grade intraepithelial lesion (LSIL)/normal in an analysis of pooled data from the three countries. Similar to HPV-positive, HPV-negative cervical carcinomas were frequently hypermethylated for these markers. CONCLUSIONS: This 5-marker panel detected SCC and HSIL in cervical smears with a high level of sensitivity and specificity. Molecular tests with the ability to rapidly detect high-risk HSIL will lead to timely treatment for those in need and prevent unnecessary procedures in women with low-risk lesions throughout the world. Validation of these markers in prospectively collected cervical smear cells followed by the development of a hypermethylated marker-based cervical cancer detection test is warranted.

2-Hydroxypropyl-ß-cyclodextrin (HPßCD) as a Potential Therapeutic Agent for Breast Cancer.

Cholesterol accumulation is documented in various malignancies including breast cancer. Consequently, depleting cholesterol in cancer cells can serve as a viable treatment strategy. We identified the potency of 2-hydroxypropyl-ß-cyclodextrin (HPßCD), a cholesterol-depletor in vitro against two breast cancer cell lines: MCF-7 (Oestrogen-receptor positive, ER+) and MDA-MB-231 (Triple negative breast cancer (TNBC)). The results were then compared against two non-cancerous cell lines using cytotoxic-, apoptosis-, and cholesterol-based assays. Treatment with HPßCD showed preferential and significant cytotoxic potential in cancer cells, inducing apoptosis in both cancer cell lines (p < 0.001). This was mediated due to significant depletion of cholesterol (p < 0.001). We further tested HPßCD in a MF-1 mice (n = 14) xenograft model and obtained 73.9%, 94% and 100% reduction in tumour size for late-, intermediate-, and early-stage TNBC, respectively. We also detected molecular-level perturbations in the expression patterns of several genes linked to breast cancer and cholesterol signalling pathways using RT2-PCR arrays and have identified SFRP1 as a direct binding partner to HPßCD through SPR drug interaction analysis. This work unravels mechanistic insights into HPßCD-induced cholesterol depletion, which leads to intrinsic apoptosis induction. Results from this study potentiate employing cholesterol depletion as a promising unconventional anticancer therapeutic strategy, which warrants future clinical investigations.

Processing and Analysis of Tissue Samples from Esophageal Cancer Patients in an African Setting.

Although infectious diseases continue to present a major health care problem in Africa, the incidence of cancer is increasing rapidly on the African continent and this merits an increased investment in cancer research in low to medium resource settings. Esophageal squamous cell carcinoma (ESCC) has a high incidence in Eastern and Southern Africa, with late clinical presentation and a very poor prognosis. There is limited research on the molecular pathology of this cancer in Africa, partly as a result of a lack of infrastructure for biobanking and sample processing in many African countries. The aim of this study was to establish a practical and robust workflow to collect, store, and process esophageal cancer samples such that both the tissue architecture and quality of the samples would be preserved and suitable for future genomic research. We developed a workflow that allows storage of fresh biopsy tissue in sterile Eppendorf tubes containing RNAlater, an efficient RNAse inhibitor. We collected 142 ESCC biopsy samples and showed that storage in RNAlater for up to 18 months did not alter tissue morphology, thus allowing histologic assessment by experienced pathologists and determination of tumor content in each biopsied sample. DNA and RNA extracted from tissue samples was assessed for purity, molecular size, and yield. The quantity and quality of nucleic acids obtained were suitable for genomic applications, and whole-exome sequencing of DNA from tumor tissues produced sequence data with a high proportion of both usable reads and correct base calling. We conclude that this workflow may be applicable to a wide range of malignancies for future genomic research in low-resource settings.

In Vivo Modelling of Hepatitis B Virus Subgenotype A1 Replication Using Adeno-Associated Viral Vectors.

The paucity of animal models that simulate the replication of the hepatitis B virus (HBV) is an impediment to advancing new anti-viral treatments. The work reported here employed recombinant adeno-associated viruses (AAVs) to model HBV subgenotype A1 and subgenotype D3 replication in vitro and in vivo. Infection with subgenotype A1 is endemic to parts of sub-Saharan Africa, and it is associated with a high risk of hepatocellular carcinoma. Recombinant AAV serotype 2 (AAV2) and 8 (AAV8) vectors bearing greater-than-genome-length sequences of HBV DNA from subgenotype A1 and D3, were produced. Transduced liver-derived cultured cells produced HBV surface antigen and core antigen. Administration of AAV8 carrying HBV subgenotype A1 genome (AAV8-A1) to mice resulted in the sustained production of HBV replication markers over a six-month period, without elevated inflammatory cytokines, expression of interferon response genes or alanine transaminase activity. Markers of replication were generally higher in animals treated with subgenotype D3 genome-bearing AAVs than in those receiving the subgenotype A1-genome-bearing vectors. To validate the use of the AAV8-A1 murine model for anti-HBV drug development, the efficacy of anti-HBV artificial primary-microRNAs was assessed. Significant silencing of HBV markers was observed over a 6-month period after administering AAVs. These data indicate that AAVs conveniently and safely recapitulate the replication of different HBV subgenotypes, and the vectors may be used to assess antivirals' potency.

The Pathology of Lymphocytes, Histiocytes, and Immune Mechanisms in Mycobacterium tuberculosis Granulomas.

Granuloma formation is the pathologic hallmark of tuberculosis (TB). Few studies have detailed the exact production of cytokines in human granulomatous inflammation and little is known about accessory molecule expressions in tuberculous granulomas. We aimed to identify some of the components of the immune response in granulomas in HIV-positive and -negative lymph nodes. We investigated the immunohistochemical profiles of CD4+, CD8+, CD68+, Th-17, Forkhead box P3 (FOXP3) cells, accessory molecule expression (human leukocyte antigen [HLA] classes I and II), and selected cytokines (interleukins 2, 4, and 6 and interferon-γ) of various cells, in granulomas within lymph nodes from 10 HIV-negative (-) and 10 HIV-positive (+) cases. CD4+ lymphocyte numbers were retained in HIV- granulomas, whereas CD4+:CD8 + cell were reversed in HIV+ TB granulomas. CD68 stained all histiocytes. Granulomas from the HIV+ group demonstrated a significant increase in FOXP3 cells. Interleukin-2 cytoplasmic expression was similar in both groups. Interferon-gamma (IFN-γ) expression was moderately increased, IL-6 was statistically increased and IL-4 expression was marginally lower in cells from HIV- than HIV+ TB granulomas. Greater numbers of cells expressed IFN-γ and IL-6 than IL-2 and IL-4 in HIV- TB granulomas. This study highlights the varied cytokine production in HIV-positive and -negative TB granulomas and indicates the need to identify localized tissue factors that play a role in mounting an adequate immune response required to halt infection. Although TB mono-infection causes variation in cell marker expression and cytokines in granulomas, alterations in TB and HIV coinfection are greater, pointing toward evolution of microorganism synergism.

Using Systematized Nomenclature of Medicine clinical term codes to assign histological findings for prostate biopsies in the Gauteng province, South Africa: Lessons learnt.

BACKGROUND: Prostate cancer (PCa) is a leading male neoplasm in South Africa. OBJECTIVE: The aim of our study was to describe PCa using Systemized Nomenclature of Medicine (SNOMED) clinical terms codes, which have the potential to generate more timely data. METHODS: The retrospective study design was used to analyse prostate biopsy data from our laboratories using SNOMED morphology (M) and topography (T) codes where the term 'prostate' was captured in the narrative report. Using M code descriptions, the diagnosis, sub-diagnosis, sub-result and International Classification of Diseases for Oncology (ICD-O-3) codes were assigned using a lookup table. Topography code descriptions identified biopsies of prostatic origin. Lookup tables were prepared using Microsoft Excel and combined with the data extracts using Access. Contingency tables reported M and T codes, diagnosis and sub-diagnosis frequencies. RESULTS: An M and T code was reported for 88% (n = 22 009) of biopsies. Of these, 20 551 (93.37%) were of prostatic origin. A benign diagnosis (ICD-O-3:8000/0) was reported for 10 441 biopsies (50.81%) and 45.26% had a malignant diagnosis (n = 9302). An adenocarcinoma (8140/3) sub-diagnosis was reported for 88.16% of malignant biopsies (n = 8201). An atypia diagnosis was reported for 760 biopsies (3.7%). Inflammation (39.03%) and hyperplasia (20.82%) were the predominant benign sub-diagnoses. CONCLUSION: Our study demonstrated the feasibility of generating PCa data using SNOMED codes from national laboratory data. This highlights the need for extending the results of our study to a national level to deliver timeous monitoring of PCa trends.

Rhabdomyosarcoma Arising in a Giant Congenital Melanocytic Naevus: Case Report and Literature Review.

Giant congenital melanocytic naevi (GCN) are rare, disfiguring lesions which carry a significant risk of malignant transformation. Melanoma is the most common malignancy documented in association with these lesions. Although exceedingly rare, other malignant neoplasms, including mesenchymal tumours such as rhabdomyosarcoma (RMS), may complicate GCN. This report documents a fatal embryonal RMS arising in a GCN on the distal left lower limb of a 4-month-old female infant, who had ipsilateral inguinal lymph node metastases at the time of presentation. To date there have been only 7 prior reports in the English literature of RMS complicating GCN. Differential diagnoses include small cell melanoma, rhabdoid melanoma, and melanoma with divergent RMS differentiation. A distinction between the latter and de novo RMS arising in GCN may have potential prognostic and therapeutic implications.

A potpourri of pathogenetic pathways in endometrial carcinoma with a focus on Lynch Syndrome.

Endometrial carcinoma is the most frequently occurring female genital tract malignancy in developed nations, with a rising annual incidence. Endometrioid endometrial carcinoma (EEC), the most common histological variant, differs in morphologic and molecular characteristics from serous carcinomas but morphological distinction of high-grade EECs from serous carcinomas may prove difficult. Thus, molecular categorization of tumors may allow for better tumor classification with greater insight into the underlying biology of endometrial carcinomas with new therapeutic options. Microsatellite instability (MSI) is a commonly occurring molecular aberration in EECs and has been identified in most Lynch Syndrome (LS) associated tumors. This tumor syndrome predisposes afflicted individuals to a myriad of tumors including endometrial carcinoma. Herein, the molecular signature of endometrial tumors as well as LS, and its clinical manifestations are reviewed. Understanding of the pathogenetic pathways allows for greater comprehension of occurrences at a molecular level which are then appreciated at a cellular and tissue level, by the histopathologist. The molecular classification of endometrial tumors allows for further targeted therapeutic options for affected patients. Screening tests for patients with suspected LS enables surveillance of other tumors in the affected patient and her family with the potential to decrease morbidity and mortality. It is envisioned that this overview will allow for enhanced comprehension of genetic pathways by practicing pathologists, oncologists, gynecologists and other members of the multidisciplinary team, all of whom are involved in the management of the patient with an endometrial malignancy.

A rare case of concomitant bilateral nipple and vulval condyloma accuminatum.

In this case study, we have presented a rare case of concomitant human papillomavirus condylomata lesions of both breast nipples and giant vulval warts in an HIV-positive woman on antiretroviral therapy. The woman underwent successful surgical excision and reconstruction for both breast nipple condylomata and vulval warts. Histology of the excised breast nipple and vulval tissue confirmed low-risk human papillomavirus infection. Concomitant vulval and bilateral breast nipple condylomata pose a management dilemma. It is possible that the pathogenesis of these lesions is through both direct contact spread as well as haematogenous dissemination of the human papillomavirus.

Trans-Urethral Snare of Bladder Tumor (TUSnBT) with Stone Basket Retrieval: A Novel Time-Saving Technique in the Endoscopic Management of Papillary Bladder Lesions.

BACKGROUND: To assess if trans-urethral snare of bladder tumor (TUSnBT) with subsequent stone basket retrieval can be used as an effective, time-saving adjunct resection technique for papillary bladder lesions. METHODS: Via standard cystoscopy, TUSnBT was performed using a standard endoscopic polypectomy snare with subsequent tumor extraction utilizing a standard stone retrieval basket, when lesions were more than 10 mm in diameter. Smaller lesions were removed with the polypectomy snare. Standard trans-urethral resection of bladder tumors (TURBT) of the tumor bed was performed post TUSnBT. Histological assessment was performed and assessed separately per session. RESULTS: In total, 18 papillary lesions, measuring between 9 and 26 mm, were resected via TUSnBT. Operative TUSnBT time, ranged between 10 and 60 seconds duration per lesion. No significant postoperative morbidity was experienced by patients within this cohort. Histo-pathological assessment revealed adequate muscle representation in 83.3 % of TUSnBT grouped sessions assessed. CONCLUSION: TUSnBT with stone retrieval basket retrieval is a feasible method in selected papillary bladder lesions, and may be coupled with standard TURBT resection techniques. This method is less time consuming and would prove beneficial in select lesions. It may also be beneficial to assist with reducing the resection time or inadvertent bladder perforation encountered during the conventional TURBT.

Disulfiram with or without metformin inhibits oesophageal squamous cell carcinoma in vivo.

Oesophageal squamous cell carcinoma (OSCC) is highly prevalent in developing countries but there has been little recent progress into efficacious yet affordable treatment strategies. Drug repurposing is one attractive approach for cancer therapy. Disulfiram (DSF), used to treat alcoholism, inhibits cancer growth and we previously found that DSF perturbs protein degradation/turnover pathways in vitro. This was enhanced by combining DSF with the anti-diabetic drug metformin (Met). Here, we investigated DSF with/without Met, against OSCC in vivo. Nude mice injected subcutaneously with the human OSCC cell line WHCO1, were treated with 30 mg/kg or 50 mg/kg DSF three times per week and with/without Met, for 21 days. DSF and DSF/Met-treated animals had significantly smaller tumours compared to untreated, vehicle and positive control cisplatin-treated groups. This effect for DSF was independent of copper, with no significant accumulation of copper in tumours, together with maintained proteasome activity. However, increases in total ubiquitinated proteins, LC3B-II, LAMP1 and p62 in DSF and DSF/Met groups, indicate that autophagy is inhibited. These findings show that DSF and DSF/Met significantly impede OSCC tumour growth in vivo and offer prospective alternative chemotherapy approaches for OSCC.

Conjunctival leiomyosarcoma: A fairly common tumour in an uncommon site.

BACKGROUND: Leiomyosarcomas of soft tissue are malignant tumours that are not infrequently encountered in clinical medicine and histopathology. Such sarcomas of the eye are, however, a rare occurrence. PURPOSE: Herein, the histopathological features of a case of conjunctival leiomyosarcoma are described. A 38-year-old HIV-positive male, who was otherwise fit and healthy, presented with redness of his right eye and a mass of the conjunctiva. Clinically, he did not have any soft-tissue masses elsewhere in the body. METHOD: He underwent monoblock excision of the conjunctival mass which was confirmed histologically to be a leiomyosarcoma. Unfortunately, the patient had not returned for follow-up examination. CONCLUSION: The differential diagnosis of a conjunctival spindle cell neoplasm is broad. While a spindle cell carcinoma is the most likely tumour, other tumours must be borne in mind so as not to misdiagnose primary sarcomas in this unusual location.

The First Report of Urothelial (Clear Cell Variant) Bladder Cancer in a Child.

Urothelial bladder tumours in childhood are extremely rare, with most cases being low grade, superficial disease of the papillary subtype with favourable outcomes. Urothelial (clear cell variant) carcinoma of bladder (UCCVCB) is an exceptional histological finding in both the adult and paediatric population. Herein we describe the first case of UCCVCB diagnosed in childhood. With this case being only the fourth report of confirmed muscle invasion in childhood, we discuss the entity of UCCVCB, and furthermore review all previously reported confirmed cases of childhood muscle invasive urothelial bladder cancer, in general.

Using Systematized Nomenclature of Medicine clinical term codes to assign histological findings for prostate biopsies in the Gauteng province, South Africa: Lessons learnt

Background: Prostate cancer (PCa) is a leading male neoplasm in South Africa.Objective: The aim of our study was to describe PCa using Systemized Nomenclature of Medicine (SNOMED) clinical terms codes, which have the potential to generate more timely data.Methods: The retrospective study design was used to analyse prostate biopsy data from our laboratories using SNOMED morphology (M) and topography (T) codes where the term 'prostate' was captured in the narrative report. Using M code descriptions, the diagnosis, sub-diagnosis, sub-result and International Classification of Diseases for Oncology (ICD-O-3) codes were assigned using a lookup table. Topography code descriptions identified biopsies of prostatic origin. Lookup tables were prepared using Microsoft Excel and combined with the data extracts using Access. Contingency tables reported M and T codes, diagnosis and sub-diagnosis frequencies.Results: An M and T code was reported for 88% (n = 22 009) of biopsies. Of these, 20 551 (93.37%) were of prostatic origin. A benign diagnosis (ICD-O-3:8000/0) was reported for 10 441 biopsies (50.81%) and 45.26% had a malignant diagnosis (n = 9302). An adenocarcinoma (8140/3) sub-diagnosis was reported for 88.16% of malignant biopsies (n = 8201). An atypia diagnosis was reported for 760 biopsies (3.7%). Inflammation (39.03%) and hyperplasia (20.82%) were the predominant benign sub-diagnoses.Conclusion: Our study demonstrated the feasibility of generating PCa data using SNOMED codes from national laboratory data. This highlights the need for extending the results of our study to a national level to deliver timeous monitoring of PCa trends