Search

Purpose: Inhibition of monocarboxylate transporter (MCT) 1–mediated lactate transport may have cytostatic and/or cytotoxic effects on tumor cells. We report results from the dose-escalation part of a first-in-human trial of AZD3965, a first-in-class MCT1 inhibitor, in advanced cancer. Patients and Methods: This multicentre, phase I, dose-escalation and dose-expansion trial enrolled patients with advanced solid tumors or lymphoma and no standard therapy options. Exclusion criteria included history of retinal and/or cardiac disease, due to MCT1 expression in the eye and heart. Patients received daily oral AZD3965 according to a 3+3 then rolling six design. Primary objectives were to assess safety and determine the MTD and/or recommended phase II dose (RP2D). Secondary objectives for dose escalation included measurement of pharmacokinetic and pharmacodynamic activity. Exploratory biomarkers included tumor expression of MCT1 and MCT4, functional imaging of biological impact, and metabolomics. Results: During dose escalation, 40 patients received AZD3965 at 5–30 mg once daily or 10 or 15 mg twice daily. Treatment-emergent adverse events were primarily grade 1 and/or 2, most commonly electroretinogram changes (retinopathy), fatigue, anorexia, and constipation. Seven patients receiving ≥20 mg daily experienced dose-limiting toxicities (DLT): grade 3 cardiac troponin rise (n = 1), asymptomatic ocular DLTs (n = 5), and grade 3 acidosis (n = 1). Plasma pharmacokinetics demonstrated attainment of target concentrations; pharmacodynamic measurements indicated on-target activity. Conclusions: AZD3965 is tolerated at doses that produce target engagement. DLTs were on-target and primarily dose-dependent, asymptomatic, reversible ocular changes. An RP2D of 10 mg twice daily was established for use in dose expansion in cancers that generally express high MCT1/low MCT4).

Purpose:Inhibition of monocarboxylate transporter (MCT) 1–mediated lactate transport may have cytostatic and/or cytotoxic effects on tumor cells. We report results from the dose-escalation part of a first-in-human trial of AZD3965, a first-in-class MCT1 inhibitor, in advanced cancer.Patients and Methods:This multicentre, phase I, dose-escalation and dose-expansion trial enrolled patients with advanced solid tumors or lymphoma and no standard therapy options. Exclusion criteria included history of retinal and/or cardiac disease, due to MCT1 expression in the eye and heart. Patients received daily oral AZD3965 according to a 3+3 then rolling six design. Primary objectives were to assess safety and determine the MTD and/or recommended phase II dose (RP2D). Secondary objectives for dose escalation included measurement of pharmacokinetic and pharmacodynamic activity. Exploratory biomarkers included tumor expression of MCT1 and MCT4, functional imaging of biological impact, and metabolomics.Results:During dose escalation, 40 patients received AZD3965 at 5–30 mg once daily or 10 or 15 mg twice daily. Treatment-emergent adverse events were primarily grade 1 and/or 2, most commonly electroretinogram changes (retinopathy), fatigue, anorexia, and constipation. Seven patients receiving ≥20 mg daily experienced dose-limiting toxicities (DLT): grade 3 cardiac troponin rise (n = 1), asymptomatic ocular DLTs (n = 5), and grade 3 acidosis (n = 1). Plasma pharmacokinetics demonstrated attainment of target concentrations; pharmacodynamic measurements indicated on-target activity.Conclusions:AZD3965 is tolerated at doses that produce target engagement. DLTs were on-target and primarily dose-dependent, asymptomatic, reversible ocular changes. An RP2D of 10 mg twice daily was established for use in dose expansion in cancers that generally express high MCT1/low MCT4).

supplementary data: Methods, tables, figure.

Background: Oral squamous cell carcinoma (OSCC) is a global healthcare problem associated with poor clinical outcomes. Early detection is key to improving patient survival. OSCC may be preceded by clinically recognizable lesions, termed oral potentially malignant disorders (OPMD). As histologic assessment of OPMD does not accurately predict their clinical behavior, biomarkers are required to detect cases at risk of malignant transformation. Epidermal growth factor receptor gene copy number (EGFR GCN) is a validated biomarker in lung non–small cell carcinoma. We examined EGFR GCN in OPMD and OSCC to determine its potential as a biomarker in oral carcinogenesis.Methods: EGFR GCN was examined by in situ hybridization (ISH) in biopsies from 78 patients with OPMD and 92 patients with early-stage (stages I and II) OSCC. EGFR ISH signals were scored by two pathologists and a category assigned by consensus. The data were correlated with patient demographics and clinical outcomes.Results: OPMD with abnormal EGFR GCN were more likely to undergo malignant transformation than diploid cases. EGFR genomic gain was detected in a quarter of early-stage OSCC, but did not correlate with clinical outcomes.Conclusion: These data suggest that abnormal EGFR GCN has clinical utility as a biomarker for the detection of OPMD destined to undergo malignant transformation. Prospective studies are required to verify this finding. It remains to be determined if EGFR GCN could be used to select patients for EGFR-targeted therapies.Impact: Abnormal EGFR GCN is a potential biomarker for identifying OPMD that are at risk of malignant transformation. Cancer Epidemiol Biomarkers Prev; 25(6); 927–35. ©2016 AACR.

Supplementary Table 1 - Characteristics of the two groups of patients, including their clinical outcomes.

Supplementary Tables 1-4, Flowchart 1 from Relation of a Hypoxia Metagene Derived from Head and Neck Cancer to Prognosis of Multiple Cancers

Affymetrix U133plus2 GeneChips were used to profile 59 head and neck squamous cell cancers. A hypoxia metagene was obtained by analysis of genes whose in vivo expression clustered with the expression of 10 well-known hypoxia-regulated genes (e.g., CA9, GLUT1, and VEGF). To minimize random aggregation, strongly correlated up-regulated genes appearing in >50% of clusters defined a signature comprising 99 genes, of which 27% were previously known to be hypoxia associated. The median RNA expression of the 99 genes in the signature was an independent prognostic factor for recurrence-free survival in a publicly available head and neck cancer data set, outdoing the original intrinsic classifier. In a published breast cancer series, the hypoxia signature was a significant prognostic factor for overall survival independent of clinicopathologic risk factors and a trained profile. The work highlights the validity and potential of using data from analysis of in vitro stress pathways for deriving a biological metagene/gene signature in vivo. [Cancer Res 2007;67(7):3441–9]

Cross talk between cancer cells and their microenvironment can lead to molecular changes in adjacent tissues that can be utilised as biomarkers. One such example stems from the discovery that malignant melanomas with metastatic potential can downregulate autophagy in the overlying epidermis. Autophagy is essential for normal keratinocyte maturation. AMBRA1 is a key autophagy regulatory molecule and its expression in the epidermis is reduced in the epidermis overlying some early stage melanomas. Expression of AMBRA1 is maintained in the overlying epidermis in a subset of low risk melanomas. This finding has been translated into a novel biomarker for clinical use. Preliminary data suggest that AMBRA1 expression in neoplastic cells may be a useful biomarker for prediction of risk of progression in oropharyngeal and cutaneous squamous cell carcinoma. It is likely that it will be necessary to combination biomarker expression with Artificial Intelligence analysis and convention staging to provide accurate individualised prognostic information for clinical use.

While amoebic infection is widely known as a cause of gastroenteritis, keratitis, and meningoencephalitis, amoebae are challenging to recognize at unexpected sites. Despite multiple case reports of sinonasal amoebiasis, amoebic infection is not regularly considered in the differential diagnosis of sinonasal necroinflammatory disease. Here, we aim to characterize the pathologic features of sinonasal amoebiasis to facilitate better recognition. We identified sinonasal amoebiasis in 4 men, median age of 67 years (range: 37 to 71 y). All were immunocompromised, including 2 with chronic lymphocytic leukemia, 1 with human immunodeficiency virus, and 1 with human immunodeficiency virus and kidney transplant. Patients presented with nasal mucosal necrosis or polypoid masses, with facial ulceration in 1 patient and distant dermal nodules in another. Biopsies displayed extensive necrotic debris and inflammation. Although amoebic cysts were abundant in 3 cases, they were mistaken for yeast at frozen section in 1 case; 1 case showed only rare trophozoites that were not recognized on initial biopsy. Periodic acid Schiff and Grocott Methenamine Silver stains highlighted the organisms, and polymerase chain reaction confirmed Acanthamoeba species in 3 cases tested. 2 patients responded well to antiprotozoal medications, but 2 died of disease. Overall, sinonasal amoebiasis presents as a necroinflammatory process in patients immunocompromised for various reasons. Amoebae can mimic other organisms or be incredibly scarce, requiring active consideration to recognize amoebiasis and differentiate it from overlapping conditions like invasive fungal sinusitis, granulomatosis with polyangiitis, and natural killer/T-cell lymphoma. Because sinonasal amoebiasis is highly treatable when diagnosed promptly, pathologists play a critical role in the recognition of this rare necroinflammatory disease.

Oral potentially malignant disorders (OPMDs) are associated with an increased risk of occurrence of cancers of the lip or oral cavity. This paper presents an updated report on the nomenclature and the classification of OPMDs, based predominantly on their clinical features, following discussions by an expert group at a workshop held by the World Health Organization (WHO) Collaborating Centre for Oral Cancer in the UK. The first workshop held in London in 2005 considered a wide spectrum of disorders under the term "potentially malignant disorders of the oral mucosa" (PMD) (now referred to as oral potentially malignant disorders: OPMD) including leukoplakia, erythroplakia, proliferative verrucous leukoplakia, oral lichen planus, oral submucous fibrosis, palatal lesions in reverse smokers, lupus erythematosus, epidermolysis bullosa, and dyskeratosis congenita. Any new evidence published in the intervening period was considered to make essential changes to the 2007 classification. In the current update, most entities were retained with minor changes to their definition. There is sufficient evidence for an increased risk of oral cancer among patients diagnosed with "oral lichenoid lesions" and among those diagnosed with oral manifestations of 'chronic graft-versus-host disease'. These have now been added to the list of OPMDs. There is, to date, insufficient evidence concerning the malignant potential of chronic hyperplastic candidosis and of oral exophytic verrucous hyperplasia to consider these conditions as OPMDs. Furthermore, due to lack of clear evidence of an OPMD in epidermolysis bullosa this was moved to the category with limited evidence. We recommend the establishment of a global research consortium to further study the natural history of OPMDs based on the classification and nomenclature proposed here. This will require multi-center longitudinal studies with uniform diagnostic criteria to improve the identification and cancer risk stratification of patients with OPMDs, link them to evidence-based interventions, with a goal to facilitate the prevention and management of lip and oral cavity cancer.

Background: A large number of oral squamous cell carcinomas (OSCCs) are believed to be preceded by oral potentially malignant disorders (OPMD) that have an increased likelihood of malignant transformation compared to clinically normal mucosa. This study was performed to identify differentially expressed genes between OPMDs that underwent malignant transformation (MT) and those that did not, termed “non-transforming” (NT) cases. Methods: Total RNA was extracted from formalin-fixed paraffin-embedded tissue biopsies of 20 OPMD cases with known clinical outcomes (10 MT vs. 10 NT). Samples were assessed for quantity, quality and integrity of RNA prior to sequencing. Analysis for differential gene expression between MT and NT was performed using statistical packages in R. Genes were considered to be significantly differentially expressed if the False Discovery Rate corrected P-value was 1.90). Analysis of RNA-Sequencing outputs revealed 41 genes (34 protein-coding; 7 non-coding) that were significantly differentially expressed between MT and NT cases. The log2 fold change for the statistically significant differentially expressed genes ranged from −2.63 to 2.48, with 23 protein-coding genes being downregulated and 11 protein-coding genes being upregulated in MT cases compared to NT cases. Conclusion: Several candidate genes that may play a role in malignant transformation of OPMD have been identified. Experiments to validate these candidates are underway. It is anticipated that this work will contribute to better understanding of the etiopathogenesis of OPMD and development of novel biomarkers.

The basal-like molecular subtype of pancreatic ductal adenocarcinoma (PDAC) is associated with poor prognosis and upregulation in TP63ΔN (p40) network. Adenosquamous histology can be observed. This study assessed immunohistochemical p40 expression in fine needle biopsy (FNB) samples with PDAC and association with cytomorphological features of squamous differentiation and clinical data. 106 EUS FNBs with PDAC were assessed for eight cytomorphological features of squamous differentiation. P40 H-score (intensity 0–3 × percentage positive nuclei) was analysed for association with morphological features, patient age, gender, operability, chemotherapy and survival. P40 H-score in 14 paired FNBs and resections was compared. P40 h-score was 1–3 in 31%, 4–30 in 16% and > 30 in 13% of FNBs. It was significantly associated with intercellular bridges, elongated cell shape, sharp cell borders, angular nuclei with homogenous chromatin (p 30 (n = 9, 1.8 months) than for h-score ≤ 30 (n = 66, 6.7 months) not quite reaching statistical significance (p = 0.08). P40 was significantly associated with squamous morphology in FNBs with PDAC. P40 H-score > 30 showed a trend towards shorter survival in inoperable patients. Squamous differentiation may be a treatment target in PDAC.

Introduction Clinical management of oral potentially malignant disorders relies on accurate histopathological assessment of the presence and grade of oral epithelial dysplasia. Whilst adjunctive laboratory tests have provided useful prognostic information, none are in widespread clinical use. This study was performed to assess the clinical utility of two contemporary oral epithelial dysplasia grading systems. Methods Patients were identified from a clinical database. Oral epithelial dysplasia grading was performed by three oral and maxillofacial pathologists blinded to clinical outcome using the WHO 2017 system and a binary classification. The primary outcome measure was the development of oral squamous cell carcinoma, termed 'malignant transformation'. Results 131 cases satisfied the inclusion criteria, of which 23 underwent malignant transformation. There was substantial inter-rater agreement between the study pathologists for both grading systems, measured using kappa statistics (κ = 0.753-0.784). However, there was only moderate agreement between the consensus WHO 2017 dysplasia grade for the study against the original grade assigned by a pool of six pathologists in the context of the clinical service (κ = 0.491). Higher grade categories correlated with an increased risk of developing cancer using both grading systems. Conclusion This study demonstrates that the WHO 2017 and binary grading systems are reproducible between calibrated pathologists and that consensus reporting is likely to improve the consistency of grading. The WHO and binary systems were prognostically comparable. We recommend that institutions implement consensus oral epithelial dysplasia grading and prospectively audit the effectiveness of risk stratifying their patients with oral potentially malignant disorders. (249 words).

Typically, HPV-related cancers are sexually transmitted, however, the natural history of HPV-related oropharyngeal squamous cell carcinoma (OPSCC) is unclear. HPV16 transmission has been reported previously between five couples with OPSCC. We report the clinico-pathological features of a further four couples with HPV-related OPSCC and compare them with the published cases.We identified four couples in long-term heterosexual relationships that all had HPV-related OPSCC. The couples were treated at three UK hospitals and presented between 2009 and 2015. HPV tests included p16 immunohistochemistry, high-risk HPV DNA in-situ hybridisation and Roche Cobas HPV test. DNA sequencing was used to determine the HPV variant.The four couples represented2% of patients with HPV-related OPSCC at the three contributing hospitals (8 of 457 consecutive patients). The couples' tumours all contained HPV16. The mean age was 63 years old (range 52-72 years). The interval between the index cancer and the partner's cancer was 16, 24, 26 and 64 months respectively. The majority of patients had Stage I disease (UICC TNM8). Six of eight patients are disease free, one patient is alive with disease and there was one death from loco-regional recurrence.This report highlights the occurrence of HPV-related OPSCC in heterosexual couples and raises the possibility of transmission of HPV16. Despite increasing prevalence of HPV-related OPSCC and increased awareness of the disease, there is a paucity of couples with the disease, suggesting either under-reporting or that the development of OPSCC following HPV transmission between couples is a rare event.

Background and PurposeAcute ischemic stroke caused by large vessel occlusions (LVO) is a major contributor to stroke deaths and disabilities; however, identification for emergency treatment is challenging.AimsTo evaluate the diagnostic accuracy of a panel of biomarkers for LVO prediction.Methods170 patients with suspected stroke were recruited retrospectively at one hospital. We analysed the plasma levels of D-dimer, OPN, OPG, GFAP, vWF, and ADAMTS13 in LVO vs non-LVO. Diagnostic performance was estimated by using blood biomarkers alone or in combination with NIHSS-derived stroke severity scales.ResultsOur patient cohort comprised 20% stroke mimics, 11% transient ischemic attack, 11% hemorrhagic stroke, 15% LVO ischemic stroke, 28% non-LVO ischemic stroke, and 15% ischemic stroke with unknown LVO status. Multivariable analysis found that the optimal set of blood biomarkers for LVO prediction was D-dimer (OR 15.4, 95% CI 4.9 to 57.6; p-valueConclusionsThe combination of D-dimer, GFAP, and stroke scales could provide a simple and highly accurate tool for identifying LVO patients.

Single molecule biomarkers are used extensively in head and neck pathology for diagnosis and increasingly for prognosis. Companion markers for therapy such as PDL-1 and NTRK are now finding applications in head and neck cancer care. Immunohistochemistry is an attractive option because of its rapid turnaround time and convenience but molecular testing is often necessary for validation. This chapter will focus on some selected biomarkers being developed for translational purposes. Adoptive T cell therapies are being trialled for head and neck cancer and have limited efficacy currently. Identification of biomarkers as targets is an attractive option for development, and the use of molecular sequencing to identify individual neo-antigens is a promising way forward for precision medicine approaches including adoptive T cell therapies.

BACKGROUND: This study was undertaken to develop and validate a gene expression signature that characterises oral potentially malignant disorders (OPMD) with a high risk of undergoing malignant transformation. METHODS: Patients with oral epithelial dysplasia at one hospital were selected as the ‘training set’ (n = 56) whilst those at another hospital were selected for the ‘test set’ (n = 66). RNA was extracted from formalin-fixed paraffin-embedded (FFPE) diagnostic biopsies and analysed using the NanoString nCounter platform. A targeted panel of 42 genes selected on their association with oral carcinogenesis was used to develop a prognostic gene signature. Following data normalisation, uni- and multivariable analysis, as well as prognostic modelling, were employed to develop and validate the gene signature. RESULTS: A prognostic classifier composed of 11 genes was developed using the training set. The multivariable prognostic model was used to predict patient risk scores in the test set. The prognostic gene signature was an independent predictor of malignant transformation when assessed in the test set, with the high-risk group showing worse prognosis [Hazard ratio = 12.65, p = 0.0003]. CONCLUSIONS: This study demonstrates proof of principle that RNA extracted from FFPE diagnostic biopsies of OPMD, when analysed on the NanoString nCounter platform, can be used to generate a molecular classifier that stratifies the risk of malignant transformation with promising clinical utility.

Background: Oral potentially malignant disorders are a clinical conundrum as there are no reliable methods to predict their behaviour. We combine conventional oral epithelial dysplasia grading with DNA ploidy analysis to examine the validity of this approach to risk assessment in a cohort of patients with known clinical outcomes. Methods: Sections from diagnostic biopsies were assessed for oral epithelial dysplasia using the WHO grading system, and DNA ploidy analysis was performed using established methods. Patients reviewed for a minimum of 5 years who did not develop oral squamous cell carcinoma were classified as “non-transforming” cases. Patients that developed oral squamous cell carcinoma ≥ 6 months after the initial diagnostic biopsy were classified as having “malignant transformation.” Results: Ninety cases were included in the study. Seventy cases yielded informative DNA ploidy results. Of these 70 cases, 31 progressed to cancer. Oral epithelial dysplasia grading and DNA ploidy status were both significantly associated with clinical outcome (P < 0.05). Severe dysplasia had a hazard ratio of 3.50 (CI: 1.46, 8.45; P = 0.005) compared to cases with mild dysplasia. Aneuploidy had a hazard ratio of 2.09 (CI: 1.01, 4.32; P = 0.046) compared to cases with a diploid/tetraploid status. Receiver operating characteristic analysis gave an area under the curve of 0.617 for DNA ploidy status and 0.688 when DNA ploidy status was combined with dysplasia grading. Conclusion: Our findings suggest that combining dysplasia grading with DNA ploidy status has clinical utility which could be used to develop novel management algorithms.

Acute ischemic stroke caused by large vessel occlusions (LVOs) is a major contributor to stroke deaths and disabilities, however, identification for emergency treatment is challenging. We recruited two separate cohorts of suspected stroke patients and screened a panel of blood-derived protein biomarkers for LVO detection. Diagnostic performance was estimated by using blood biomarkers in combination with NIHSS-derived stroke severity scales. Multivariable analysis demonstrated that D-dimer (OR 16, 95% CI 5–60, p-value &lt, 0.001) and GFAP (OR 0.002, 95% CI 0–0.68, 0.05) comprised the optimal panel for LVO detection. Combinations of D-dimer and GFAP with a number of stroke severity scales increased the number of true positives, while reducing false positives due to hemorrhage, as compared to stroke scales alone (p-value &lt, 0.001). A combination of the biomarkers with FAST-ED resulted in the highest accuracy at 95% (95% CI: 87–99%), with sensitivity of 91% (95% CI: 72–99%), and specificity of 96% (95% CI: 90–99%). Diagnostic accuracy was confirmed in an independent cohort, in which accuracy was again shown to be 95% (95% CI: 87–99%), with a sensitivity of 82% (95% CI: 57–96%), and specificity of 98% (95% CI: 92–100%). Accordingly, the combination of D-dimer and GFAP with stroke scales may provide a simple and highly accurate tool for identifying LVO patients, with a potential impact on time to treatment.

AimsUnderstanding the molecular mechanisms of underlying disease has led to a movement away from the one-drug-fits-all paradigm towards treatment tailored to the genetic profile of the patient. The Biocartis Idylla platform is a novel fully automated, real-time PCR–based in vitro diagnostic system. The Idylla NRAS-BRAF mutation test has been developed for the qualitative detection of mutations in NRAS and BRAF oncogenes, facilitating genetic profiling of patients with cancer. The aim of this study was to carry out a formal clinical performance evaluation.MethodsTwo-hundred and forty-two formalin-fixed paraffin-embedded (FFPE) human malignant colorectal cancer (CRC) tissue samples were identified in departmental archives and tested with both the Idylla NRAS-BRAF mutation test and the Agena Bioscience MassARRAY test.ResultsThe overall concordance between the Idylla NRAS-BRAF mutation test and the MassARRAY comparator reference test result was 241/242 (99.59%, lower bound of one-sided 95% CI=98.1%) for NRAS and 242/242 (lower bound of 95% one-sided 95% CI=98.89%) for BRAF. The Idylla NRAS-BRAF test detected one NRAS mutation that had not been reported by the MassARRAY comparator reference test. Reanalysis of this sample by droplet digital PCR confirmed that the mutation was present, but at an allelic frequency below the stated sensitivity level of the MassARRAY system.ConclusionThese results confirm that the Idylla NRAS-BRAF mutation test has high concordance with a widely used NRAS-BRAF test, and is therefore suitable for use as an in vitro diagnostic device for this application.

Standardized, synoptic pathologic reporting for tumors greatly improves communication among clinicians, patients, and researchers, supporting prognostication and comparison about patient outcomes across institutions and countries. The International Collaboration on Cancer Reporting is a nonprofit organization whose mission is to develop evidence-based, universally available surgical pathology reporting data sets. Within the head and neck region, lymph node excisions and neck dissections are frequently performed as part of the management of head and neck cancers arising from the mucosal sites (sinonasal tract, nasopharynx, oropharynx, hypopharynx, oral cavity, and larynx) along with bone tumors, skin cancers, melanomas, and other tumor categories. The type of specimen, exact location (lymph node level), laterality, and orientation (by suture or diagram) are essential to accurate classification. There are significant staging differences for each anatomic site within the head and neck when lymph node sampling is considered, most importantly related to human papillomavirus–associated oropharyngeal carcinomas and mucosal melanomas. Number, size, and site of affected lymph nodes, including guidelines on determining the size of tumor deposits and the presence of extranodal extension and soft tissue metastasis, are presented in the context of prognostication. This review elaborates on each of the elements included in the data set for Nodal Excisions and Neck Dissection Specimens for Head & Neck Tumours.

Background: Oral squamous cell carcinoma (OSCC) is a global healthcare problem associated with poor clinical outcomes. Early detection is key to improving patient survival. OSCC may be preceded by clinically recognizable lesions, termed oral potentially malignant disorders (OPMD). As histologic assessment of OPMD does not accurately predict their clinical behavior, biomarkers are required to detect cases at risk of malignant transformation. Epidermal growth factor receptor gene copy number (EGFR GCN) is a validated biomarker in lung non–small cell carcinoma. We examined EGFR GCN in OPMD and OSCC to determine its potential as a biomarker in oral carcinogenesis. Methods: EGFR GCN was examined by in situ hybridization (ISH) in biopsies from 78 patients with OPMD and 92 patients with early-stage (stages I and II) OSCC. EGFR ISH signals were scored by two pathologists and a category assigned by consensus. The data were correlated with patient demographics and clinical outcomes. Results: OPMD with abnormal EGFR GCN were more likely to undergo malignant transformation than diploid cases. EGFR genomic gain was detected in a quarter of early-stage OSCC, but did not correlate with clinical outcomes. Conclusion: These data suggest that abnormal EGFR GCN has clinical utility as a biomarker for the detection of OPMD destined to undergo malignant transformation. Prospective studies are required to verify this finding. It remains to be determined if EGFR GCN could be used to select patients for EGFR-targeted therapies. Impact: Abnormal EGFR GCN is a potential biomarker for identifying OPMD that are at risk of malignant transformation. Cancer Epidemiol Biomarkers Prev; 25(6); 927–35. ©2016 AACR.

Oral potentially malignant disorders (PMD) are recognisable mucosal conditions that have an unpredictable risk of transformation to squamous cell carcinoma (SCC), a lethal and deforming disease of rising incidence. Contemporary management is based on clinical recognition of suspicious lesions and incisional biopsy to enable histopathological assessment and grading of dysplasia, together with excision of high-risk lesions and long-term surveillance. However, it is impossible to predict clinical outcome or risk of malignant transformation. Our aim was to evaluate the relevance of previously identified oral precursor lesions for the development of SCC and staging of disease. We therefore retrospectively reviewed 1248 cases of SCC diagnosed in oral and maxillofacial surgery units at Newcastle upon Tyne and Sunderland hospitals between 1996 and 2009. Of them, 58 identifiable precursor lesions became malignant but only 25 had been dysplastic on initial biopsy; 19 of 33 non-dysplastic lesions exhibited lichenoid inflammation only. SCC arose most often on the ventrolateral tongue and floor of the mouth, with a mean transformation time of 29.2 months. Transformation time was significantly shorter in men (p=0.018) and those over 70 years of age (p=0.010). Patients who consumed more than 21 units of alcohol/week and those who had had interventional laser surgery to treat precursor lesions, had higher-staged tumours (p=0.048). Although retrospective, this study shows that the results of incisional biopsy and grading of dysplasia have limited use as predictive tools, and supports the view that cancer may arise in the absence of recognisable epithelial dysplasia. Our findings confirm the importance of clinical vigilance and active surveillance in the management of all patients with clinically suspicious oral lesions, irrespective of the histological findings.

Introduction: BT1718 is a targeted Bicycle peptide-conjugate designed to deliver the anti-tubulin agent, DM1 to tumors expressing membrane type 1-matrix metalloprotease (MT1-MMP; MMP14; MT1). In vivo preclinical studies demonstrated that anti-tumor activity of BT1718 is dependent on the level of tumor MT1-MMP expression. In patient tumors MT1-MMP expression has been reported in tumor and stromal cells, both of which may contribute to the potential for anti-tumor effects following BT1718 dosing. BT1718 is currently being investigated in a Phase 1/2 clinical trial, which includes both dose escalation (ongoing) and dose expansion cohorts enrolling patients with advanced solid tumors that have exhausted standard therapeutic options. The dose expansion cohorts will enroll patients with tumors expressing high levels of MT1-MMP following establishment of the recommended Phase 2 dose. Here we describe the analysis of TMAs stained using a clinical grade MT1-MMP IHC assay to guide which patient populations to include in BT1718 dose expansion cohorts. Methods: A clinical grade MT1-MMP IHC assay was developed on the Ventana platform using a Millipore MT1-MMP primary antibody (MAB3328) at 1:6000 and detected using Optiview chemistry. Cancer indications reported in the literature with high MT1-MMP expression including ovarian, bladder, triple negative breast, esophageal, and NSCLC were stained and MT1-MMP expression levels estimated by consensus review of two pathologists using an H-score scale (staining intensity*percent positivity). H-scores (0-300) were derived separately for tumor membrane (TM), cytoplasm (TC), and stroma (TS) for each case. Results: MT1-MMP expression in TM/TS, but not TC is likely to yield the greatest potential for BT1718 binding and subsequent anti-tumor activity. Therefore, analyses of TM/TS H-scores were used to identify BT1718 dose expansion cohorts. Histograms were generated separately for TM and TS H-scores with a bin-width of 50. The distribution of MT1-MMP staining in TM and TS was different (Table 1) with H-score between 0-49 being the most frequently populated TM bin, regardless of indication. In contrast, TS scores were typically higher (e.g. H-score=100-149), with the clearest example being ovarian cancer (89% of cases TM 0-49 & only 20% of cases TS 0-49). In addition, within NSCLC, tumor subtype analysis demonstrated that cases of squamous histology appeared enriched for higher TM H-scores (TM≥150=36%) compared to adenocarcinoma cases (TM≥150=2%). Moreover, various TM and/or TS H-score boundaries were modelled with the aim of delivering a proposed cut-off for recruiting patients with high MT1-MMP expression to the expansion cohorts, the results of this modelling will be presented. Table 1: Distribution of MT1-MMP TM and TS H-scores across multiple indications p> Citation Format: Tara Gelb, Chris Bacon, Philip Sloan, Mike Rigby, Stephanie Guerrera, Fiona Kelly, Stefan Symeonides, Stephen J Blakemore. MT1-MMP Immunohistochemistry (IHC) analysis of tumor microarrays (TMAs) using a novel scoring system guides patient selection for BT1718 expansion cohorts [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A047. doi:10.1158/1535-7163.TARG-19-A047

Whilst dental healthcare professionals naturally focus on assessment of the teeth and the supporting tissues, they also have an important role in assessing the whole oro-facial complex and the neck. Assessment of the neck is particularly important, not least, because it contains the regional lymph nodes that are involved in immune surveillance of the head and neck region. The neck also contains the major salivary glands: the sub­mandibular gland and the tail of the parotid gland. Mid-line structures include the hyoid bone, larynx, and trachea, along with the thyroid gland and parathyroid glands. The assessment of these anatomical structures should form part of the routine clinical examination. The dis­covery of an abnormality in the neck, which may not have been noticed by the patient, may expedite the diagnosis of significant disease and facilitate a timely intervention. A through understanding of the anatomy of the neck is essential and informs the clinical examination. It is also important to understand the concept of the anatomical levels that map out the lymph node groups of the neck (Chapter 1; Fig. 1.2). Accurate assessment of the neck is usually best achieved by a combination of visual inspection and palpation, with the patient in a slightly reclined position, the clinician standing behind the patient. Any lumps, e.g. enlarged lymph nodes, are described by anatomical site, size, consistency (cystic, soft, rubbery, hard), whether the lump is mobile or fixed to the underlying tissue, and if palpation elic­its pain or discomfort. The combination of these parameters will help to formulate the differential diagnosis; for example, an isolated hard lump that is fixed to underlying structures is likely to represent meta­static cancer, whereas, bilateral soft lumps that are mobile and painful to palpation are likely to represent lymphadenitis as a consequence of systemic infection. Ultrasound examination can be used to ascertain important informa­tion about a neck lump such as the site (precise anatomical location, superficial or deep), size, consistency (solid or cystic), and multi-focality. Doppler settings can help to establish the vascularity of a lesion and its proximity to major vessels.

A sound understanding of clinical oral pathology is essential if a dental clinician is to navigate successfully through clinical guidelines, make timely referrals to specialists, and provide good care for patients. This new edition of Soames' & Southam's Oral Pathology provides a clear and friendly guide for students, practitioners, and the whole dental team. Thoroughly updated for today's clinical practice, this textbook covers 'must-know' oral pathology and integrates key aspects of oral medicine. It begins by explaining the principles of clinical assessment, the synthesis of a differential diagnosis, and the selection of further investigations including laboratory tests. Ten chapters bring this theory to life by looking at the clinical and pathological features of a wide range of common oral diseases including oral cancer, salivary gland disorders, and diseases of the jaws. Two new chapters address skin diseases affecting the oro-facial region and neck lumps. A final chapter highlights the importance of clinical oral pathology in the context of systemic human disease. New radiology content includes examples of cross-sectional imaging. Photomicrographs have been replaced with carefully selected images to illustrate key pathological features. Each chapter includes key points boxes and tables to aid learning. Written by experts in both oral pathology and oral medicine, this new edition is a must-have for dentistry students, and those working in the field, providing current and trustworthy information.

The most common oral diseases are dental caries and periodontal dis­ease. The diagnosis and treatment of these diseases are the focus of the majority of dentists, dental therapists, and dental hygien­ists. Nevertheless, it is the responsibility of the dental healthcare pro­fessional to provide a holistic approach to management that ensures both good oral and general health for their patients. A broad knowledge of the range of diseases that affect the oral cavity is essential and also an appreciation that oral disease may be the first sign of an underlying systemic disease. Before this knowledge can be applied, the clinician must obtain an accurate patient history and undertake a systematic clinical examination. These key clinical skills underpin both medicine and dentistry, and are an absolute requirement to formulate a differ­ential diagnosis. The use of imaging modalities and laboratory tests are often required to reach a definitive diagnosis, and it is the justification and informed choice of these additional investigations that will facili­tate a timely and accurate diagnosis. Following diagnosis, appropriate treatment can be instituted, the ideal outcome being a return to health or control of the patient’s symptoms in recalcitrant chronic diseases. Obtaining a clear and precise clinical history is essential. The clinician must listen carefully to the information conveyed by the patient and then use direct questioning to collect additional data to construct an accurate picture of the patient’s problem. This can be a significant challenge and requires excellent communication skills to build a good patient–clinician relationship. The most common presenting problems relate to pain or the development of a lesion: swelling, lump, ulcer, or white/red patch. To establish a comprehensive pain history, the features listed in Table 1.1 should be addressed. Obtaining an accurate history for a lesion is dependent on the patient noticing the abnormality in the first instance and, as a consequence, the information may be rather vague; however, it is important to ascertain the key points listed in Table 1.2. A comprehensive medical history (Box 1.1) will identify any concur­rent disease that may be relevant to the presenting oral condition (Chapter 10) and highlight any issues relating to proposed medical or surgical interventions.

This chapter emphasizes the importance of oral disease in the context of systemic disease. Oral disease may be the first sign of an underlying systemic disorder, which should trigger further investigations or referral to a medical or specialist practitioner, expediting early diagnosis and appropriate clinical care. In those patients with known illnesses, dental healthcare professionals should have an appreciation of the effects of these diseases on the oro- facial complex; patients can then benefit from the supportive care of the dental team. Coeliac disease (gluten-induced enteropathy) is a genetically deter­mined inflammatory small bowel disease that is induced by gluten in the diet. Gluten is present in wheat, rye, and barley. The inflammatory reaction results in malabsorption due to morphological abnormalities in the small intestinal mucosa. It was once thought of as a rare disease of childhood, but now it is understood to be a common disease that can be diagnosed at any age. Studies undertaken since the advent of sero­logical screening indicate a prevalence of 0.5–1% in the population, with adult presentations now more frequent than childhood ones. This new appreciation has led to the concept of the ‘coeliac iceberg’. At the tip of the iceberg are those with overt disease, with the lower groups classified in order as: silent coeliac disease, latent coeliac disease, and healthy individuals with genetic susceptibility. Severe presentations in childhood classically were of diarrhoea, failure to thrive, and weakness. However, it is now understood that patients with coeliac disease may have minimal gastrointestinal symptoms of malabsorption at presenta­tion and experience other rather non-specific symptoms of the disease. The increasing recognition of the disease is attributed to several factors, including new serological assays, advances in flexible endoscopy allow­ing clinicians to take duodenal biopsies more easily, and an increased index of suspicion in looking for the disease. The disease is associated with an increased rate of osteoporosis, infertility, autoimmune diseases, and malignancy, especially lymphoma. Treatment should involve a gluten-free diet, which is a major undertaking and for which dietetic advice is essential.

Examination of the face and hands can identify significant skin diseases and also provide clues to the presence of underlying systemic disease. Many patients ignore even malignant skin tumours because they are often painless, subtle in appearance, and may be slow-growing. Dental healthcare professionals should be aware of how to recognize malig­nant skin tumours. If suspicious, but unsure of the nature of the lesion, the patient should be referred to their general medical practitioner for further evaluation. If malignancy is obvious, then an urgent referral to an appropriate specialist (dermatologist, plastic surgeon, or oral and max­illofacial surgeon) should be made using the ‘2-week wait’ (2WW) path­way (Chapter 1). Benign lesions and inflammatory diseases are more common and are important considerations in the differential diagnosis of head and neck skin abnormalities. It is important that the dental healthcare professional should be able to recognize common skin infections involving the oro-facial region. Some infections, such as erysipelas, can mimic cellulitis associated with a dental infection. When infection is diagnosed, it is vital to consider the underlying or predisposing factors, as these may be not only important diagnoses, but also may require treatment to achieve an effective clin­ical outcome. The adage ‘infection is the disease of the diseased’ is a useful reminder when dealing with patients presenting with infection. Direct inoculation of Streptococcus into skin through minor trauma is the most common initiating factor for erysipelas, which occurs in iso­lated cases. Infection involves the upper dermis and, characteristically, spreads to involve the dermal lymphatic vessels. Clinically, the disease starts as a red patch that extends to become a fiery red, tense, and indurated plaque. Erysipelas can be distinguished from cellulitis by its advancing, sharply defined borders and skin streaking due to lymphatic involvement. The infection is most common in children and the elderly, and whilst classically a disease affecting the face, in recent years it has more frequently involved the leg skin of elderly patients. Although a clinical diagnosis can be made without laboratory testing, and treat­ment is antibiotic therapy, when the diagnosis is suspected in dental practice, referral to a medical practitioner is recommended.

A wide variety of processes can affect the formation of teeth during development. The number, size, shape, and quality of dental hard tis­sue may be abnormal and teeth may erupt early or be prematurely shed or resorbed. When a child presents with a tooth abnormality, the clin­ical and radiographic features are often distinctive and management depends on diagnosis (Box 5.1). Broadly, developmental abnormal­ities of the teeth can be either genetically determined or acquired as a result of injurious processes affecting the developing teeth. It can be problematic to make a diagnosis, particularly when teeth initially erupt. Sometimes pathological examination of a shed or extracted tooth by ground sectioning (for enamel) or conventional sectioning of a decalci­fied tooth can provide a diagnosis. Research has provided insights into the genetic and structural basis of dental anomalies, and has resulted in a complex and extensive classification of subtypes. Minor abnormal­ities, such as failure of development of a few teeth or enamel erosion in adult life, may be dealt with in general dental practice, but it is advisable to refer younger patients with more complex or extensive dental abnor­malities to a specialist in child dental health, with links to expert diag­nostic facilities and input from orthodontic and restorative colleagues. The publically available Online Mendelian Inheritance in Man (OMIM) database provides an invaluable resource for genetic disorders, including dental abnormalities. Supernumerary teeth are common and may be rudimentary in form or of normal morphology, when they are referred to as supplemental teeth. The most common supernumerary tooth occurs in the mid- line of the maxillary alveolus and is referred to as a mesiodens, which usually has a conical shape. Eruption of adjacent normal successor teeth may be impeded by a mesiodens, which is an indication for its removal. Most supernumerary teeth occur as a sporadic event in devel­opment, but multiple extra teeth can be found in certain developmen­tal disorders. Failure of development of tooth germs results in teeth missing from the dental arch and is referred to as hypodontia. Most often the missing teeth are third molars, second premolars, and upper lateral incisors. Hypodontia is more common in the permanent dentition than in the primary teeth.

The oral mucosa lines the oral cavity and comprises a surface squamous epithelium with underlying lamina propria. Below the mucosa is the submucosa, which is composed of fibrous tissue and adipose tissue, and contains lobules of minor salivary glands and neurovascular bundles. In places, there is no submucosa and the lamina propria is continuous with periosteum, forming the resilient mucoperiosteum that covers the maxilla and mandible. The squamous epithelium is composed of keratinocytes arranged in lay­ers: there is a basal cell layer that rests on the basement membrane, a prickle cell layer, and usually a keratinized layer. The keratino­cytes are attached to each other by desmosomes and the basal keratino­cytes are attached to the basement membrane by hemi- desmosomes. The basement membrane is important in maintaining the integrity of the mucosa by sticking the squamous epithelium to the underlying lamina propria. There are two patterns of keratinization: par­akeratosis and orthokeratosis. In parakeratinized epithelium the surface keratinocytes become flattened and the nucleus becomes dark and shrunken (pyknotic). These terminally differentiated squamous cells are eventually lost at the surface by desquamation. In orthokeratiniza­tion, there is a granular cell layer (containing numerous keratohyaline granules) between the prickle cell layer and the keratinized layer. The surface squames become flattened and do not contain any discernible nuclear material. Whilst the majority of cells in squamous epithelium are keratinocytes, there are also accessory cells such as melanocytes, Langerhans cells, and neurosensory cells (Merkel cells and taste buds). The lamina propria is the connective tissue that lies immediately below the epithelium. It is divided into the superficial papil­lary layer (sometimes referred to as the corium) and the deeper reticu­lar layer. The lamina propria is composed of fibrous tissue with a rich neurovascular supply and contains fibroblasts that elaborate collagen and elastin fibres along with other extracellular matrix proteins. The lamina propria also contains components of the mucosal immune defence system such as Langerhans cells, macrophages, mast cells, and lymphocytes. The clinical appearance of the oral mucosa is dependent on the thick­ness of the epithelium, the amount of surface keratinization, melanin (and other) pigmentation, and the vascularity of the lamina propria.

The term ‘oral cancer’ encompasses all malignant neoplasms affecting the oral cavity. The majority, greater than 90%, are squamous cell car¬cinomas. The remainder are uncommon and comprise minor salivary gland adenocarcinomas, malignant melanoma, sarcomas, haemato-logical malignancies, and metastases to the oral cavity from cancers at other sites. Oral squamous cell carcinoma is a malignant epithelial neoplasm that arises from the lining mucosa of the oral cavity. The tumour shows vary¬ing degrees of squamous differentiation and is characterized by invasion of local structures and metastasis to regional lymph nodes, followed by metastasis to other organ systems (e.g. lungs and bones) later in the course of the disease. Epidemiological data pertaining to oral cancer can be difficult to evalu¬ate because of variations in the methods of data collection (Box 3.1). Notwithstanding these confounding variables, a database produced by the International Agency for Research on Cancer (GLOBOCAN), esti-mated there were over 400,000 new cases of lip, oral, and pharyngeal cancer worldwide in 2012, placing the disease in ninth position with breast, prostate, lung, colorectal, cervical, stomach, liver, and uterine cancer being more common. These data suggest that oral cancer is uncommon, but there are enormous variations worldwide. Whereas oral cancer is relatively uncommon in the UK, accounting for 2% of all cancers, in India and parts of South-East Asia it is the most common malignant neoplasm and accounts for around a third of all cancers. Furthermore, the incidence rates for large countries, such as India and the USA, conceal regional and ethnic variations. For example, incidence rates tend to be higher in urban as opposed to rural communities, and in the USA are higher for blacks than whites. In the United Kingdom, inci¬dence rates are slightly higher in Scotland than in England and Wales. In the United Kingdom the incidence of oral cancer is 9 per 100,000 of the population, which represents around 6,800 new cases per annum. The disease is more common in men than in women; the male:female ratio is currently 2:1. Oral cancer incidence increases with age, and the majority of cases (greater than two-thirds) are diagnosed after the age of 50 years old; less than 5% occur in individuals below the age of 40 years old.

The salivary glands consist of three paired major glands—parotid, sub­mandibular, and sublingual—and the countless minor salivary glands found in almost every part of the oral cavity, except the gingiva and anterior regions of the hard palate. The secretion of saliva is essential for the normal function and health of the mouth, and disorders of salivary gland function predispose to oral disease. Functional disorders in salivary secretion may be associated with primary salivary gland disease but in other cases are a consequence of systemic factors, such as medi­cations, endocrine disturbances, and neurological disease, which are discussed in Chapter 10. Developmental anomalies of the salivary glands are rare. Aplasia of one or more major glands and atresia of one or more major salivary gland ducts have been reported. Congenital aplasia of the parotid gland may be associated with other facial abnormalities, e.g. ectodermal dysplasia, mandibulofacial dysostosis, and hemifacial microsomia. Heterotopic salivary tissue has been reported from a variety of sites in the head and neck region, the most frequent being its inclusion at the angle, or within the body, of the mandible, called a Stafne bone cavity. It is usually an incidental radiological finding and appears as a round or oval, well-demarcated radiolucency between the premolar region and angle of the jaw, and is typically located beneath the inferior dental canal. The radiographic appearances are due to a saucer-shaped depression or concavity of varying depth on the lingual aspect of the mandible, which contains salivary tissue in continuity with the submandibular gland. Accessory parotid tissue within the cheek or masseter muscle is rela­tively common and is subject to the same diseases that may affect the main gland. Age changes can be detected in both major and minor salivary glands. Reduction in the weights of submandibular and parotid glands has been reported with increasing age, associated in the submandibular gland with an age-dependent reduction in flow rates. By contrast, sev­eral studies have demonstrated that there is no significant reduction in parotid flow rates in the elderly.

Odontogenic cysts and tumours arise from inclusion of tooth-forming epithelium and mesenchyme in the jaw bones during development. Cysts also arise from non-odontogenic epithelium trapped during fusions or from vestigial structures. In addition, bone cysts that can arise at other skeletal sites may also occur in the jaws. Odontogenic cysts and tumours may be classified according to their putative developmental origins and biology. The classification of jaw cysts is shown in Fig. 6.1. Odontomes are hamartomatous develop­mental lesions of the tooth-forming tissues. Odontogenic tumours are uncommon and are usually benign. Ameloblastoma is the most com­mon odontogenic tumour and is described in detail. The other odon­togenic tumours are rare and only the principal features are presented. Very rare congenital lesions of possible odontogenic origin are men­tioned in the final section. A cyst may be defined as pathological cavity lined by epithelium with fluid or semi-fluid contents. However, clinically, the term encompasses a broader range of benign fluid-filled lesions, some of which do not possess an epithelial lining. The preferred definition is, therefore, ‘a pathological cavity having fluid or semi-fluid contents that has not been created by the accumulation of pus’. Cysts are commonly encountered in clinical dentistry and are generally detected on radiographs or as expansions of the jaws. Most cysts have a radiolucent appearance and are well circumscribed, often with a corticated outline. At least 90% of jaw cysts are of odontogenic origin. The clinico-pathological features of jaw cysts are summarized in Table 6.1. The incidence of the four most common jaw cysts are provided in Table 6.2. The epithelial lining of odontogenic cysts originates from residues of the tooth-forming organ. • Epithelial rests of Serres are remnants of the dental lamina and are thought to give rise to the odontogenic keratocyst, lateral periodon­tal, and gingival cysts. • Reduced enamel epithelium is derived from the enamel organ and covers the fully formed crown of the unerupted tooth. The dentiger­ous (follicular) and eruption cysts originate from this tissue, as do the mandibular buccal and paradental cysts. • Epithelial rests of Malassez form by fragmentation of Hertwig’s epi­thelial root sheath that maps out the developing tooth root. Radicular cysts originate from these residues.

Most invasive dental procedures involving removal of teeth or bone are followed by uneventful healing. However, dentists should be aware that generalized abnormalities of bone, such as osteoporosis and Paget’s dis­ease of bone, may complicate these procedures and, rarely, can lead to ongoing clinical problems. The effects of radiotherapy to the jaws and bisphosphonate treatment are well-described causes of osteonecrosis and delayed healing. Diagnosis of bone disorders often depends on integrating the results of clinical, imaging, pathological, genetic, and biochemical investigations. Although the bony skeleton is often thought of as forming just a rigid framework, it should be remembered that bone is a living, responsive tissue that plays an important role in metabolism. During development, some bones develop from a cartilaginous template and others, such as most of the craniofacial bones, form in fibrous membranes. Bone matrix is laid down by osteoblasts that are derived from the extensive meshwork of bone-lining cells that cover the bone surfaces. The bone matrix contains osteocytes that are responsive to mechanical stresses. Bone matrix is removed by osteoclasts that move over the bone sur­face, resulting in scalloped pits termed Howship’s lacunae. Bone mat­rix can be woven or lamellar in pattern. Pathologists often examine sections of bony lesions in polarized light to determine whether the pattern of the collagenous matrix is woven or lamellar, because it can be pivotal for diagnosis. It is also important for clinicians to be aware that, in order to produce a histological section of bone, the tissue must first be fixed and then demineralized to soften the matrix. When a bone biopsy is performed, the patient should be made aware that additional time will be needed to process the biopsy. Following extraction of a tooth, the socket rapidly fills with blood, which then clots. Granulation tissue, which consists of proliferating endothelial cells and fibroblasts derived from remnants of the periodontal ligament and surrounding alveolar bone, grows into the clot and organization commences. Osteoclasts begin to remodel the crestal bone and remove any small spicules of bone detached during the extraction.