Optimization of proteinase K incubation protocol duration during DNA extraction from oral squamous cell carcinoma FFPE samples

Background: Formalin-fixed paraffin-embedded (FFPE) specimen archives are a valuable source of sample material for molecular biological analysis. However, the DNA isolated from FFPE samples is usually low in concentration and fragmented. Thus, it is necessary to optimize the FFPE DNA extraction protocol to obtain the best results. Proteinase K incubation is undoubtedly crucial in DNA extraction procedures, but this step is often not well explained in the manufacturer’s manual. Purpose: This study aimed to find the optimal duration for proteinase K incubation protocols to achieve the highest DNA yields. Methods: Fifteen paraffin blocks of Oral Squamous Cell Carcinoma (OSCC) specimens were obtained, and the cancerous areas were microdissected into smaller cuts for DNA extraction. The samples were randomly divided into three groups (n=5) and subjected to three different proteinase K incubation protocols: one-hour incubation at 56ºC as per the manufacturer’s instructions (Group I), 24-hour incubation at 56ºC (Group II), and 48 hours at room temperature with an additional four hours at 56ºC (Group III). The extracted DNA was then quantified using a Nanodrop spectrophotometer. The recorded data were analyzed using ANOVA-LSD. Results: The highest DNA concentration was found in Group III (107.74 ± 41.92), which was significantly higher compared to Group II (59.46 ± 30.32) and Group I (6.46 ± 1.97) (p<0.05). Conclusion: In conclusion, modifying the duration of proteinase K incubation protocols can lead to different DNA yield results. In this study, the most optimized protocol for proteinase K incubation, resulting in the highest DNA yields, was 48 hours at room temperature with an additional four hours at 56ºC

Diagnosis of oral potentially malignant disorders: Overview and experience in Oceania

The diagnosis and management of oral potentially malignant disorders (OPMD) should be the same the world over, but there are important nuances in incidence, aetiological factors, and management opportunities that may lead to differences based on ethnogeography. In this review, we update and discuss current international trends in the classification and diagnosis of OPMD with reference to our experience in various regions in Oceania. Oceania includes the islands of Australia, Melanesia (including Papua New Guinea, Fiji, Solomon Islands, Micronesia and Polynesia (including New Zealand, Samoa, Tonga) and hence has diverse populations with very different cultures and a range from well-resourced high-population density cities to remote villages

Revisiting the association between candidal infection and carcinoma, particularly oral squamous cell carcinoma

Background: Tobacco and alcohol are risk factors associated with cancer of the upper aerodigestive tract, but increasingly the role of infection and chronic inflammation is recognized as being significant in cancer development. Bacteria, particularly Helicobacter pylori, and viruses such as members of the human papilloma virus family and hepatitis B and C are strongly implicated as etiological factors in certain cancers. There is less evidence for an association between fungi and cancer, although it has been recognized for many years that white patches on the oral mucosa, which are infected with Candida, have a greater likelihood of undergoing malignant transformation than those that are not infected. Objective: This article reviews the association between the development of oral squamous cell carcinoma in potentially malignant oral lesions with chronic candidal infection and describes mechanisms that may be involved in Candida-associated malignant transformation

Immune Responses in Primary Oral Squamous Cell Carcinoma and Lymph Node Metastasis

Background: Oral squamous cell carcinoma (OSCC) develops in an immune cell-rich environment, where inflammatory cells in the tumour microenvironment establish an anti-tumour response by secreting pro-inflammatory cytokines. At the same time the cancer cells may induce various mechanisms suppressing the anti-tumour response such as regulating a network of suppressive cytokines and the recruitment of suppressive regulatory T cells (Tregs). These escape mechanisms are seen at the local tumour site and similar mechanisms may also occur in regional lymph nodes (LN). In this thesis it is postulated that the escape of malignant oral keratinocytes from the primary site and their metastasis to regional lymph nodes is orchestrated by Tregs and their associated immune repertoire. Objectives: 1. To determine the nature, composition and relationships of T cell subsets, including Tregs, and other associated immune repertoires that may be involved in creating a tolerogenic state within primary OSCC tissues. 2. To investigate the gene expression profile of immune cells with respect to cytokines involved in immune tolerance (including IL-17, TLR2, IL-10, TGFβ and IL-6) in primary OSCC, metastatic OSCC and in regional lymph nodes with OSCC metastases and to correlate the expression within the different tissues with Tumour, Nodes, Metastasis staging. Methods:Twenty five formalin fixed paraffin embedded (FFPE) archival cases of OSCC and control tissues were stained via single and/or double immunohistochemistry (IHC) and immunofluorescence (IF) with T cell receptor markers including CD3, CD4, CD8, CD56, CD68, CD25/FoxP3, CD4/FoxP3, CD8/FoxP3 antibodies and other associated immune cell markers including Fas and TLR2/FoxP3 antibodies. For investigation of human T cell anergy and immune tolerance genes expression a further 32 FFPE archival cases of OSCC were divided into the following groups:- 1) primary OSCC without metastasis 2) OSCC with associated metastatic LN and 3) the metastatic lymph nodes. Group 4 was non-specific inflammatory control tissues. The expression of human T cell anergy and immune tolerance genes was determined using focused array technology via RT-PCR. Results: A reduction in NK and CD8+ T cells with a smaller reduction in CD4+ T cells was observed in primary OSCC tissues. A higher frequency of CD25+FoxP3+ Tregs together with an increase in apoptosis was observed in the infiltrates associated with OSCC compared with controls. The reduction in CD8+ T cells and the presence of Tregs in primary OSCC tissues was confirmed with IF observation, although CD8+FoxP3+ cells were not detected. FoxP3+ Tregs was observed to be closely associated with naïve CD4+ cells. TLR2+ FoxP3+ Tregs were also observed in the OSCC tumour microenvironment. In addition, apparent co-localisation between TLR2+ and FoxP3+ cells was observed. The gene analysis studies demonstrated active regulation of T cell anergy and tolerance genes in primary OSCC and in metastatic lymph nodes. STAT3 gene was significantly down-regulated in all OSCC groups with the highest fold change in the metastatic lymph nodes. This may indicate a greater modulation the anti-tumour response in the nodes compared with the primary tissues. The immune suppression mechanisms were similar in lymph nodes with and without extracapsular (ECS) spread, though the suppression mechanism was stronger in lymph nodes with ECS. Treg expression was elevated in all stages of OSCC in comparison to controls. Conclusions:Immune suppression appears to occur in the OSCC microenvironment and this suppression becomes more profound as the cancer progresses. In the later stages of OSCC, Tregs appear to exert profound control of the tolerance processes. As the tumour metastasises to cervical lymph nodes suppression appears to be less dependent on Tregs and switches to suppression via the STAT3 pathway, in order to prevent pro-inflammatory revival. Finally, it is concluded that the anti-tumour immune response does not fail entirely. Immune cells were not entirely suppressed by OSCC, particularly in the early stages of the cancer process. This may be an indication that OSCC eradication could be achieved with selected immunotherapy

Toll-like receptors and cancer, particularly oral squamous cell carcinoma

It is becoming increasingly apparent that the tumour microenvironment plays an important role in the progression of cancer. The microenvironment may promote tumour cell survival and proliferation or, alternatively may induce tumour cell apoptosis. Toll-like receptors are transmembrane proteins, expressed on immune cells and epithelial cells, that recognise exogenous and endogenous macromolecules. Once activated they initiate signalling pathways leading to the release of cytokines and chemokines which recruit immune cells inducing further cytokine production, the production of angiogenic mediators and growth factors, all of which may influence tumour progression.This paper examines the actions of toll-like receptors in carcinogenesis with particular emphasis on their role in oral squamous cell carcinom

Stevens-Johnson Syndrome following Failure of Genetic Screening prior to Carbamazepine Prescription

Failure to screen susceptible individuals for human leucocyte allele B∗1502 leads to the onset of Stevens-Johnson syndrome (SJS). We report a case of a 27-year-old Malay female who was treated with carbamazepine following the diagnosis of trigeminal neuralgia without a genetic screening. She was prescribed 150 mg of carbamazepine initially and the dose was increased to 300 mg following the initial dose. A sudden development of skin and mucous membrane ulcers was observed and this warranted immediate hospitalization. A diagnosis of SJS was made and she was treated immediately with intravenous corticosteroids. Genetic screening prior to carbamazepine prescription is essential especially in susceptible populations

Stevens-Johnson Syndrome following Failure of Genetic Screening prior to Carbamazepine Prescription

Failure to screen susceptible individuals for human leucocyte allele B∗1502 leads to the onset of Stevens-Johnson syndrome (SJS). We report a case of a 27-year-old Malay female who was treated with carbamazepine following the diagnosis of trigeminal neuralgia without a genetic screening. She was prescribed 150 mg of carbamazepine initially and the dose was increased to 300 mg following the initial dose. A sudden development of skin and mucous membrane ulcers was observed and this warranted immediate hospitalization. A diagnosis of SJS was made and she was treated immediately with intravenous corticosteroids. Genetic screening prior to carbamazepine prescription is essential especially in susceptible populations

Lyophilised Platelet-Rich Fibrin: Physical and Biological Characterisation

Background: Platelet-rich fibrin (PRF) has gained popularity in craniofacial surgery, as it provides an excellent reservoir of autologous growth factors (GFs) that are essential for bone regeneration. However, the low elastic modulus, short-term clinical application, poor storage potential and limitations in emergency therapy use restrict its more widespread clinical application. This study fabricates lyophilised PRF (Ly-PRF), evaluates its physical and biological properties, and explores its application for craniofacial tissue engineering purposes. Material and methods: A lyophilisation method was applied, and the outcome was evaluated and compared with traditionally prepared PRF. We investigated how lyophilisation affected PRF&rsquo;s physical characteristics and biological properties by determining: (1) the physical and morphological architecture of Ly-PRF using SEM, and (2) the kinetic release of PDGF-AB using ELISA. Results: Ly-PRF exhibited a dense and homogeneous interconnected 3D fibrin network. Moreover, clusters of morphologically consistent cells of platelets and leukocytes were apparent within Ly-PRF, along with evidence of PDGF-AB release in accordance with previously reports. Conclusions: The protocol established in this study for Ly-PRF preparation demonstrated versatility, and provides a biomaterial with growth factor release for potential use as a craniofacial bioscaffold

Potential of Lyophilized Platelet Concentrates for Craniofacial Tissue Regenerative Therapies

Objective: The use of platelet concentrates (PCs) in oral and maxillofacial surgery, periodontology, and craniofacial surgery has been reported. While PCs provide a rich reservoir of autologous bioactive growth factors for tissue regeneration, their drawbacks include lack of utility for long-term application, low elastic modulus and strength, and limited storage capability. These issues restrict their broader application. This review focuses on the lyophilization of PCs (LPCs) and how this processing approach affects their biological and mechanical properties for application as a bioactive scaffold for craniofacial tissue regeneration. Materials and Methods: A comprehensive search of five electronic databases, including Medline, PubMed, EMBASE, Web of Science, and Scopus, was conducted from 1946 until 2019 using a combination of search terms relating to this topic. Results: Ten manuscripts were identified as being relevant. The use of LPCs was mostly studied in in vitro and in vivo craniofacial bone regeneration models. Notably, one clinical study reported the utility of LPCs for guided bone regeneration prior to dental implant placement. Conclusions: Lyophilization can enhance the inherent characteristics of PCs and extends shelf-life, enable their use in emergency surgery, and improve storage and transportation capabilities. In light of this, further preclinical studies and clinical trials are required, as LPCs offer a potential approach for clinical application in craniofacial tissue regeneration