Effect of 2-methoxyestradiol on mammary tumor initiation and progression.

BACKGROUND: The anti-cancer agent 2-methoxyestradiol (2-ME) has been shown to have anti-proliferative and anti-angiogenic properties. Previously, the effect of 2-ME on early- and late-stage breast cancer (BC) was investigated in vivo using a transgenic mouse model (FVB/N-Tg(MMTV-PyVT)) of spontaneous mammary carcinoma. Anti-tumor effects were observed in late-stage BC with no effect on early-stage BC. Given the contrasting results obtained from the different BC stages, we have now investigated the effect of 2-ME when administered before the appearance of palpable tumors. METHODS: Each mouse received 100 mg/kg 2-ME on day 30 after birth, twice per week for 28 days, while control mice received vehicle only. Animals were terminated on day 59. Lung and mammary tissue were obtained for immunohistochemical analysis of CD163 and CD3 expression, and histological examination was performed to analyze tumor necrosis. Additionally, blood samples were collected to measure plasma cytokine levels. RESULTS: 2-ME increased tumor mass when compared to the untreated animals (p = .0139). The pro-tumorigenic activity of 2-ME was accompanied by lower CD3+ T-cell numbers in the tumor microenvironment (TME) and high levels of the pro-inflammatory cytokine interleukin (IL)-1ß. Conversely, 2-ME-treatment resulted in fewer CD163+ cells detectable in the TME, increased levels of tumor necrosis, increased IL-10 plasma levels, and low IL-6 and IL-27 plasma levels. CONCLUSION: Taken together, these findings suggest that 2-ME promotes early-stage BC development.

Effect of 2-methoxyestradiol treatment on early- and late-stage breast cancer progression in a mouse model.

The prevalence of breast cancer (BC) continues to increase and is the leading cause of cancer deaths in many countries. Numerous in vitro and in vivo studies have demonstrated that 2-methoxyestradiol (2-ME) has antiproliferative and antiangiogenic effects in BC, thereby inhibiting tumour growth and metastasis. We compared the effect of 2-ME in early- and late-stage BC using a transgenic mouse model-FVB/N-Tg(MMTV-PyVT)-of spontaneously development of aggressive mammary carcinoma with lung metastasis. Mice received 100 mg/kg 2-ME treatment immediately when palpable mammary tumours were identified (early-stage BC; Experimental group 1) and 28 days after palpable mammary tumours were detected (late-stage BC; Experimental group 2). 2-ME was administered via oral gavage three times a week for 28 days after initiation of treatment, whereas control mice received the vehicle containing 10% dimethyl sulfoxide and 90% sunflower oil for the same duration as the treatment group. Mammary tumours were measured weekly over the 28 days and at termination, blood, mammary and lung tissue were collected for analysis. Mice with a tumour volume threshold of 4000 mm3 were killed before the treatment regime was completed. 2-ME treatment of early-stage BC led to lower levels of mammary tumour necrosis, whereas tumour mass and volume were increased. Additionally, necrotic lesions and anti-inflammatory CD163-expressing cells were more frequent in pulmonary metastatic tumours in this group. In contrast, 2-ME treatment of late-stage BC inhibited tumour growth over the 28-day period and resulted in increased CD3+ cell number and tumour necrosis. Furthermore, 2-ME treatment slowed down pulmonary metastasis but did not increase survival of late-stage BC mice. Besides late-stage tumour necrosis, none of the other results were statistically significant. This study demonstrates that 2-ME treatment has an antitumour effect on late-stage BC, however, with no increase in survival rate, whereas the treatment failed to demonstrate any benefit in early-stage BC.

Comparative Molecular Genetics of Odontogenic Keratocysts in Sporadic and Syndromic Patients.

Odontogenic keratocysts (OKCs) are common cysts of odontogenic origin that usually occur as a single nonsyndromic cyst in isolation (sporadic) or as syndromic multiple cysts as a manifestation of naevoid basal cell carcinoma syndrome. Alterations involving the PTCH gene are the most commonly identified factor associated with up to 85% and 84% of naevoid basal cell carcinoma syndrome and sporadic cases, respectively. Other Hedgehog pathway and non-Hedgehog pathway-associated genes have been implicated in the pathogenesis of OKCs. This pilot study used the Affymetrix OncoScan molecular assay to perform a comparative genomic analysis between 4 sporadic and 3 syndromic cases of OKC to identify molecular drivers that may be common and/or distinct in these 2 groups. The majority of alterations detected in both groups were copy number neutral loss of heterozygosity. Despite distinct molecular signatures observed in both groups, copy number neutral loss of heterozygosity alterations involving chromosome 9q affecting not only PTCH but also the NOTCH1 gene were detected in all syndromic and 3 sporadic cases. Loss of heterozygosity alterations involving 16p11.2 affecting genes not previously described in OKCs were also detected in all syndromic and 3 sporadic cases. Furthermore, alterations on 22q11.23 and 10q22.1 were also detected in both groups. Of note, alterations on 1p13.3, 2q22.1, and 6p21.33 detected in sporadic cases were absent in all syndromic cases. This study demonstrates that a more common group of genes may be affected in both groups of OKCs, whereas other alterations may be useful in distinguishing sporadic from syndromic cysts. These findings should be validated in larger OKC cohorts to improve molecular diagnosis and subsequent patient management.

Slc7a8 Deletion Is Protective against Diet-Induced Obesity and Attenuates Lipid Accumulation in Multiple Organs.

Adipogenesis, through adipocyte hyperplasia and/or hypertrophy, leads to increased adiposity, giving rise to obesity. A genome-wide transcriptome analysis of in vitro adipogenesis in human adipose-derived stromal/stem cells identified SLC7A8 (Solute Carrier Family 7 Member 8) as a potential novel mediator. The current study has investigated the role of SLC7A8 in adipose tissue biology using a mouse model of diet-induced obesity. slc7a8 knockout (KO) and wildtype (WT) C57BL/6J mice were fed either a control diet (CD) or a high-fat diet (HFD) for 14 weeks. On the HFD, both WT and KO mice (WTHFD and KOHFD) gained significantly more weight than their CD counterparts. However, KOHFD gained significantly less weight than WTHFD. KOHFD had significantly reduced levels of glucose intolerance compared with those observed in WTHFD. KOHFD also had significantly reduced adipocyte mass and hypertrophy in inguinal, mesenteric, perigonadal, and brown adipose depots, with a corresponding decrease in macrophage infiltration. Additionally, KOHFD had decreased lipid accumulation in the liver, heart, gastrocnemius muscle, lung, and kidney. This study demonstrates that targeting slc7a8 protects against diet-induced obesity by reducing lipid accumulation in multiple organs and suggests that if targeted, has the potential to mitigate the development of obesity-associated comorbidities.

Sexual Dimorphism in Changes That Occur in Tissues, Organs and Plasma during the Early Stages of Obesity Development.

Despite obesity being a major health concern, information on the early clinical changes that occur in plasma and tissues during obesity development and the influence of sexual dimorphism is lacking. This study investigated changes in tissue and organ histology, macrophage infiltration, plasma hormones, lipid, and chemokine and cytokine levels in mice fed on a high fat diet for 11-weeks. An increase in adiposity, accompanied by adipocyte hypertrophy and macrophage infiltration, was observed to be significantly greater in males than females. Important changes in cell morphology and histology were noted in the lungs, liver, kidney, spleen, and heart, which may indicate early signs for developing obesity associated comorbidities. Leptin, but not adiponectin, was significantly altered during weight gain. Additionally, leptin, but not adiposity, correlated with insulin levels. Interestingly, GM-CSF, TNFα, and IL-12 (p70) were not produced in the early stages of obesity development. Meanwhile, the production of MCP-1, IP-10, RANTES, IL-10, IL-6, KC, and IL-9 were greatly influenced by sexual dimorphism. Importantly, IL-6/IL-10 axis of anti-inflammatory cytokine regulation was observed only in females and may account for their significantly lower weight gain compared to males. This study provides new knowledge on how sexual dimorphism may influence the development of obesity and associated comorbidities.

Oral plasmablastic lymphoma: A clinicopathological study of 113 cases.

BACKGROUND: Plasmablastic lymphoma (PBL) is an aggressive neoplasm that commonly develops in HIV-positive patients, usually affecting the oral cavity. EBV is present in the majority of cases, therefore, playing an important role in the pathogenesis of this neoplasm. METHODS: PBL diagnosed from 2000 to 2020 were retrieved from the archives of the Department of Oral Pathology and Oral Biology at the University of Pretoria, South Africa. The patients' clinical information including gender, age, tumour location and HIV status was obtained from the original histopathology request forms. A morphological description was assessed using H&E-stained slides, with diagnoses confirmed by immunohistochemistry, and EBV detection performed via in situ hybridisation. RESULTS: During the 20 years period investigated, 113 PBL were found. Males outnumbered females (M:F ratio of 3:1), with a median age of 41 years (range 8-62). The gingiva (50 cases or 44.2%) and the palate (23 cases or 20.4%) were the most affected sites. All cases with available information were HIV positive. The tumours were composed of a diffuse proliferation of immunoblasts or plasmablasts in all cases. A starry-sky pattern, tissue necrosis, cellular pleomorphism and mitotic figures were common microscopic findings. IHC for CD3 and CD20 were negative in all cases, while positivity for CD38, CD138 and MUM1 was observed in 70.2%, 79.2% and 98.9%, respectively. EBV was present in 100% of the cases. CONCLUSION: PBL is a frequent diagnosis in South Africa, due to the country's HIV burden, where it usually affects the oral cavity and is always associated with EBV infection.

Expression of Mucins in Salivary Gland Mucoepidermoid Carcinoma.

Mucoepidermoid carcinoma (MEC) is the most common malignant salivary gland tumour in both adults and children. Histological grading of MEC is subjective, but plays an important role in predicting patient prognosis. Epithelial mucin (MUC) status may aid in establishing a more accurate grade. This study aimed to investigate the expression of various mucins (MUC1, MUC2, MUC4 and MUC5AC) in MECs to determine a possible correlation with tumour grade. Fifteen cases of each tumour grade (low-, intermediate-, and high-grade) were retrieved from the pathology archives of the Department of Oral Pathology and Oral Biology at the University of Pretoria. The patients included 23 men and 22 women, and ranged from 13 to 85 years (mean 49.8 years). Sections from formalin-fixed paraffin-embedded (FFPE) tissue were used for fluorescence in situ hybridization (FISH) for MAML2 rearrangements and MUC immunohistochemical analysis. The percentage immunohistochemical expression of the neoplastic mucous cells was evaluated first, followed by the overall percentage expression of all tumour cells. The results indicated that MUC1 overexpression may be a reliable marker of high-grade MECs, whereas MUC4 overexpression may be more indicative of low-grade tumours. MUC5AC expression was considered an unreliable marker in determining grade. MUC2 was only expressed in a single case of MEC and may be considered a useful marker to exclude MEC as a diagnostic possibility. This study demonstrates that MECs show an altered MUC expression pattern that can be used for diagnostic purposes and to aid in establishing a more accurate tumour grade.

Amplification of 3q26.2, 5q14.3, 8q24.3, 8q22.3, and 14q32.33 Are Possible Common Genetic Alterations in Oral Cancer Patients.

The lack of clinical biomarkers for head and neck cancer subtypes limits early diagnosis and monitoring of disease progression. This study investigates genetic alterations in clinically identical tumor, tumor-adjacent dysplastic epithelium (TADE) and normal epithelium (NE) in five oral cancer patients to identify differences and commonalities between oral cancer, TADE and NE. A VELscope®Vx device was used to identify TADE and NE surrounding a clinical tumor for analysis of genetic alterations using the OncoScan® assay. One of the tumor samples examined was an "M" class tumor with a high confidence BRAF:p.G469A:c.1406G>C somatic mutation, which is the first to be reported in oral cancer. Another tumor showed mosaicism in genetic alterations, indicating the presence of multiple clones. Overall, each patient's tumor, TADE and NE showed a distinct genetic profile which indicates intertumoral clonal/genetic diversity. Interestingly, four tumors showed gain of 3q26.2, 5q14.3, 8q24.3, 8q22.3, 14q32.33 and loss/LOH in 9p21.3 while all TADE had LOH on 22q11.23. In addition, some genetic alterations progressed from NE through TADE into tumor in individual patients. Furthermore, no molecular event was identified that is common to all NE and/or TADE that progressed into tumor. This pilot study demonstrates the presence of genetic heterogeneity in oral tumorigenesis, and suggests that there might exist some common genetic alterations between tumors and TADE. However, this observation would need to be further investigated and validated in a larger cohort of oral cancer patients for its potential role in oral tumorigenesis.

Different methods of cell quantification can lead to different results: a comparison of digital methods using a pilot study of dendritic cells in HIV-positive patients

BACKGROUND: Although new digital pathology tools have improved the positive cell quantification, there is a heterogeneity of the quantification methods in the literature. The aim of this study was to evaluate and propose a novel dendritic cells quantification method in squamous cell carcinoma comparing it with a conventional quantification method. MATERIAL AND METHODS: Twenty-six squamous cell carcinomas HIV-positive cases affecting the oropharynx, lips and oral cavity were selected. Immunohistochemistry for CD1a, CD83, and CD207 was performed. The immunohistochemical stains were evaluated by automated examination using a positive pixel count algorithm. A conventional quantification method (unspecific area method; UA) and a novel method (specific area method; SA) were performed obtaining the corresponding density of positive dendritic cells for the intratumoral and peritumoral regions. The Mann-Whitney U test was used to verify the influence of the quantification methods on the positive cell counting according to the evaluated regions. Data were subjected to the ANOVA and Student's t-test to verify the influence of the tumour location, stage, histological grade, and amount of inflammation on the dendritic cells density counting. RESULTS: The cell quantification method affected the dendritic cells counting independently of the evaluated region (P-value < 0.05). Significant differences between methods were also observed according to the tumour features evaluations. CONCLUSIONS: The positive cell quantification method influences the dendritic cells density results. Unlike the conventional method (UA method), the novel SA method avoids non-target areas included in the hotspots improving the reliability and reproducibility of the density cell quantification

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Molecular profile of tongue cancer in an 18-year-old female patient with no recognizable risk factor.

BACKGROUND: The occurrence of oral tongue squamous cell carcinoma (TSCC) in nonsmoking young adults, especially females, has increased. Yet, there is no clear evidence to support the existence of any single determinant. This case reports the presence of TSCC in an 18-year-old female with no recognizable risk factor for oral cancer development. METHODS: Histological examination and p16 immunohistochemistry were performed. Formalin-fixed paraffin-embedded sections were prepared from resected tissue and DNA was extracted for molecular OncoScan analysis. RESULTS: Histological and immunochemical analysis showed a p16-negative poorly differentiated keratinizing squamous cell carcinoma. OncoScan analysis of this tumor revealed a high confidence TP53:p.R213*:c.637C>T somatic mutation as well as copy number alterations of chromosomal regions including gains of 1p, 3q, 5p, 7p, 8p, 8q, 11q, 15q, 17q, and 20p, and losses on 1p, 3p, 18q, and 22q. CONCLUSION: The TP53:p.R213*:c.637C>T mutation detected is indicative of a genetic predisposition to cancer and it is the first to be reported in TSCC in a nonsmoking young adult. LEVEL OF EVIDENCE: Case report.

Heterozygosity of p16 expression in an oral squamous cell carcinoma with associated loss of heterozygosity and copy number alterations.

BACKGROUND: Oral field cancerization describes a multifocal development process involving many cells at once in response to prolong exposure to carcinogens. This case demonstrated differential p16 expression in different sections of the same HPV negative tumor on the floor of mouth in a patient with history of prolonged smoking. METHODS: Histological examination, presence of HPV infection and OncoScan analysis of DNA extracted from two well-defined areas with different p16 expression profiles were performed. RESULTS: Histological and immunochemical analysis revealed the presence of a dual architectural pattern squamous cell carcinoma with a p16 negative and a p16 positive component. OncoScan analysis showed genetic changes that define field cancerization of the p16 negative tumor as revealed by mosaicism in both loss of heterozygosity and copy number alterations in cancer-associated genes located on 3p, 7p, 9p 11q, and 17p. CONCLUSION: These changes were indicative of field cancerization in response to tobacco exposure.

Correlation between dysplasia and ploidy status in oral leukoplakia.

Oral leukoplakia and other potentially malignant disorders (PMD) may progress to oral squamous cell carcinoma (OSCC). The gold standard for assessing the potential for malignant transformation remains histologic examination with the aim of grading the dysplastic changes. However, not all lesions with dysplasia will progress to OSCC. DNA ploidy has been suggested as a method to predict the clinical behaviour of PMD. This study reports on the use of high-resolution flow cytometry to determine the ploidy status of formalin-fixed, paraffin-embedded material from PMD compared to their dysplasia grade on histology. Aneuploidy was found in 13 % of mild, 31 % of moderate, and 54 % of severe dysplasia cases. This difference was statistically significant (p = 0.011). The differences in ploidy status were more significant when grouping the dysplasia into low-risk and high-risk categories (p = 0.008). These findings indicate that the ploidy status of PMD as determined by high-resolution flow cytometry may be of value in predicting biological behaviour in PMD such as leukoplakia.

Dominant genetic aberrations and coexistent EBV infection in HIV-related oral plasmablastic lymphomas.

We present common cytogenetic features in the largest cohort of plasmablastic lymphoma (PBL) of the oral cavity published to date. This cohort included 45 patients, 32 of whom had a known HIV status, of which 31 were HIV positive. Ninety eight per cent of all PBL cases were known to be EBV positive. In line with previous studies, we found that rearrangements of the MYC gene was the most common genetic abnormality seen in 60% of cases with the immunoglobulin heavy chain (IGH) locus as a partner in 51% of cases. Additional complex genetic aberrations were frequent, in particular, an increased copy number of the CCND1 gene was seen in 41% of cases with true amplification of CCND1 in 15% of cases. Aneuploidy was also observed for the BCL6 gene in 28% of cases. Interestingly, rearrangements of both IGH genes were detected in 16% of cases with t(14;18) and t(11;14) respectively involved in conjunction with a t(8;14) in two cases. These bi-allelic IGH rearrangements have not been described before in oral PBL. Our results reinforce the notion that EBV infection and MYC rearrangements are important events in the pathogenesis of oral PBL. The genetic diversity and complexity observed in these cases, underlines the importance to genetically characterise PBL patients at presentation as this may inform the choice of more effective treatment modalities.

Plasmablastic lymphomas with light chain restriction - plasmablastic extramedullary plasmacytomas?

BACKGROUND: It is diagnostically difficult to differentiate plasmablastic lymphomas (PBLs) from plasma cell neoplasms with plasmablastic differentiation. Plasmablastic lymphomas are currently classified as 'PBL of the oral mucosa' and 'PBL with plasmacytic differentiation'. METHODS: Forty-five cases of PBL were retrieved from the Departments of Oral Pathology of the Universities of Pretoria and Limpopo, South Africa. Clinical features and HIV status were recorded and each case was classified as 'PBL of the oral mucosa type' or as 'PBL with plasmacytic differentiation'. Immunohistochemistry included: CD45, CD3, CD20, CD79a, CD38, CD138, MUM1, Ki-67 and kappa and lambda light chains. Positivity was recorded based on the percentage of positive staining cells as focal (5-20%); intermediate (20-70%) or diffuse (>70%). In situ hybridization was performed for Epstein-Barr virus (EBV) and HHV-8. Results were recorded as positive or negative. RESULTS: All cases showed some degree of plasmacytic differentiation. All were negative for CD20 with reactive T cells detected with CD3. Diffuse and strong positive staining was found with Ki-67 and MUM1, but variable immunoreactivity was found with CD79a, CD45, CD38 and CD138. Twenty cases (47%) showed light chain restriction. Epstein-Barr virus was detected in 44/45 cases and HHV-8 in none. CONCLUSIONS: The morphological classification of PBLs is not valid as all cases showed some degree of plasmacytic differentiation. We propose that PBLs with light chain restriction be reclassified as 'plasmablastic extramedullary plasmacytomas' and managed accordingly. The rest represents true PBLs. The true nature of these neoplasms as an entity should be further investigated with molecular and genetic studies.

An investigation of the role of oral epithelial cells and Langerhans cells as possible HIV viral reservoirs.

BACKGROUND: The role of the oral mucosa as a target of human immunodeficiency virus (HIV-1) infection and persistence is unclear. HIV-1 has been reported in oral epithelial cells, but this has not been confirmed. Cellular reservoirs may impede antiretroviral therapies and should be identified. This study was performed to determine the presence of HIV-1 in oral epithelial and Langerhans cells (LCs) of HIV-1-positive antiretroviral naïve patients. Non-invasive brush biopsy technique for future in vivo HIV research was also evaluated. METHODS: Oral mucosal cells were harvested from the buccal mucosae, dorsal tongue and the gingiva of the mandibular teeth of 35 HIV-1-positive patients using a Cytobrush Plus cell collector. Epithelial cells were purified from the samples by flow cytometric cell sorting using cytokeratin stains after which the epithelial cell samples were further purified and divided into superficial and deep epithelial cells by laser microdissection on Pap stained cytospin smears. LCs were picked up individually by laser microdissection from CD1a stained cytospin smears. Purified epithelial and LC samples were tested for the presence of HIV-1 DNA by polymerase chain reaction analysis. RESULTS: Ten of the patients had HIV-1 DNA in one or more of the sampled anatomical locations. No HIV-1 DNA could be demonstrated in any of the purified superficial or deep epithelial or LC samples. CONCLUSIONS: HIV-DNA can be found using non-invasive oral brush biopsies and should be investigated further as an experimental model for in vivo oral HIV research. Better ways to purify the different cell types should be investigated.