The clonal relationships between pre-cancer and cancer revealed by ultra-deep sequencing.

The study of the relationships between pre-cancer and cancer and identification of early driver mutations is becoming increasingly important as the value of molecular markers of early disease and personalised drug targets is recognized, especially now the extent of clonal heterogeneity in fully invasive disease is being realized. It has been assumed that pre-cancerous lesions exhibit a fairly passive progression to invasive disease; the degree to which they, too, are heterogeneous is unknown. We performed ultra-deep sequencing of thousands of selected mutations, together with copy number analysis, from multiple, matched pre-invasive lesions, primary tumours and metastases from five patients with oral cancer, some with multiple primary tumours presenting either synchronously or metachronously, totalling 75 samples. This allowed the clonal relationships between the samples to be observed for each patient. We expose for the first time the unexpected variety and complexity of the relationships between this group of oral dysplasias and their associated carcinomas and, ultimately, the diversity of processes by which tumours are initiated, spread and metastasize. Instead of a series of genomic precursors of their adjacent invasive disease, we have shown dysplasia to be a distinct dynamic entity, refuting the belief that pre-cancer and invasive tumours with a close spatial relationship always have linearly related genomes. We show that oral pre-cancer exhibits considerable subclonal heterogeneity in its own right, that mutational changes in pre-cancer do not predict the onset of invasion, and that the genomic pathway to invasion is neither unified nor predictable. Sequence data from this study have been deposited in the European Nucleotide Archive, Accession No. PRJEB6588.

A novel genomic signature reclassifies an oral cancer subtype.

Verrucous carcinoma of the oral cavity (OVC) is considered a subtype of classical oral squamous cell carcinoma (OSCC). Diagnosis is problematic, and additional biomarkers are needed to better stratify patients. To investigate their molecular signature, we performed low-coverage copy number (CN) sequencing on 57 OVC and exome and RNA sequencing on a subset of these and compared the data to the same OSCC parameters. CN results showed that OVC lacked any of the classical OSCC patterns such as gain of 3q and loss of 3p and demonstrated considerably fewer genomic rearrangements compared to the OSCC cohort. OVC and OSCC samples could be clearly differentiated. Exome sequencing showed that OVC samples lacked mutations in genes commonly associated with OSCC (TP53, NOTCH1, NOTCH2, CDKN2A and FAT1). RNA sequencing identified genes that were differentially expressed between the groups. In silico functional analysis showed that the mutated and differentially expressed genes in OVC samples were involved in cell adhesion and keratinocyte proliferation, while those in the OSCC cohort were enriched for cell death and apoptosis pathways. This is the largest and most detailed genomic and transcriptomic analysis yet performed on this tumour type, which, as an example of non-metastatic cancer, may shed light on the nature of metastases. These three independent investigations consistently show substantial differences between the cohorts. Taken together, they lead to the conclusion that OVC is not a subtype of OSCC, but should be classified as a distinct entity.

Investigation of p16(INK4a) as a prognostic biomarker in oral epithelial dysplasia.

BACKGROUND: Human papilloma virus is a risk factor for oropharyngeal cancer. Evidence for a similar aetiological role in the development of oral dysplasia or its transformation to oral cancer is not as clear. Meta-analyses estimate the prevalence of high-risk human papilloma virus (HPV) serotypes to be three times higher in pre-malignant lesions and cancer than in normal oral mucosa. However, this does not imply a causal relationship. Conflicting results are reported from the few studies examining the prognostic significance of HPV positivity in the development of oral cancer. We aimed to examine the ability of p16(INK4a) protein expression, a surrogate marker of HPV infection, to predict malignant progression in a large cohort of oral dysplasia patients. METHODS: One hundred forty eight oral dysplasia cases underwent immunohistochemical analysis using a monoclonal antibody against p16(INK4a) . Clinical factors were also collated on each case. Slides were double scored independently by two trained observers. Univariate analyses using both logistic and Cox regression models were performed. RESULTS: Thirty nine of 148 cases progressed to cancer. Ten of 148 cases (7%) were p16(INK4a) positive. High grade of dysplasia (P = 0.0002) and lesion morphology (P = 0.03) were found to be prognostic of malignant progression. p16(INK4a) score was not prognostic in this cohort (P = 0.29). This did not change with a time to event analysis (P = 0.24). CONCLUSION: Few studies have assessed the aetiological role of HPV in cancer development from dysplastic lesions. Our study, using one of the largest cohorts of oral dysplasia, demonstrated a low rate of p16(INK4a) positivity and was unable to confirm a prognostic ability for this biomarker.

Human Homolog of Drosophila Ariadne (HHARI) is a marker of cellular proliferation associated with nuclear bodies.

HHARI (also known as ARIH1) is an ubiquitin-protein ligase and is the cognate of the E2, UbcH7 (UBE2L3). To establish a functional role for HHARI in cellular proliferation processes, we performed a reverse genetics screen that identified n=86/522 (16.5%) ubiquitin conjugation components that have a statistically significant effect on cell proliferation, which included HHARI as a strong hit. We then produced and validated a panel of specific antibodies that establish HHARI as both a nuclear and cytoplasmic protein that is expressed in all cell types studied. HHARI was expressed at higher levels in nuclei, and co-localized with nuclear bodies including Cajal bodies (p80 coilin, NOPP140), PML and SC35 bodies. We confirmed reduced cellular proliferation after ARIH1 knockdown with individual siRNA duplexes, in addition to significantly increased levels of apoptosis, an increased proportion of cells in G2 phase of the cell cycle, and significant reductions in total cellular RNA levels. In head and neck squamous cell carcinoma biopsies, there are higher levels of HHARI expression associated with increased levels of proliferation, compared to healthy control tissues. We demonstrate that HHARI is associated with cellular proliferation, which may be mediated through its interaction with UbcH7 and modification of proteins in nuclear bodies.

Current concepts in hypophosphatasia: case report and literature review.

BACKGROUND. Hypophosphatasia (HP) is characterized by defective mineralization of bone and teeth because of deficient alkaline phosphatase activity. There are generally six recognized clinical forms, of which the most severe is often lethal prenatally or early in life. In milder forms, such as odontohypophosphatasia (OHP), premature exfoliation of primary teeth may be the only clinical manifestation. CASE REPORT. A 20-month-old girl was referred to the Specialist Paediatric Salaried Dental Service within the Harrogate and District NHS Foundation Trust with mobility of tooth numbers 71 and 81. Clinical examination revealed grade III mobile 71 and 81, with minimal gingival inflammation and plaque deposits. There were no other dental findings and no significant medical history. Tooth numbers 71 and 81 exfoliated prematurely with no evidence of root resorption, shortly after presentation. Haematological and urinary investigations showed no abnormalities. Histological examination showed a complete absence of cementum. A diagnosis of OHP was made. After 10 months of dental follow-up, no further teeth have increased mobility. CONCLUSION. Odontohypophosphatasia should be included as a differential diagnosis in children presenting with early loss of primary teeth. The dentist may be the first health care professional to whom the patient presents.

Periodontal healing following avulsion and replantation of teeth: a multi-centre randomized controlled trial to compare two root canal medicaments.

BACKGROUND: Non-setting calcium hydroxide (Ultracal XS(®) ) is recommended by the International Association of Dental Traumatology as the initial medicament following avulsion and replantation for mature teeth. There is experimental evidence to suggest Ledermix(®) , placed as an alternative inter-visit dressing may improve periodontal healing. AIM: This study investigated, using a multi-centre randomized controlled trial, the effect of two root canal medicaments, Ledermix(®) and Ultracal XS(®) , on periodontal healing of avulsed and replanted teeth. MATERIAL AND METHODS: Children were recruited if they fulfilled all inclusion criteria. Treatment followed a standardized protocol. Assessment of periodontal healing or ankylosis was made clinically and radiographically by an experienced, 'blinded', clinician at 12months. RESULTS: Over 200 patients were assessed for eligibility at five centres. Twenty-nine patients were eligible for inclusion. Final analysis involved 22 patients with 27 teeth. Ankylosis was detected in four of the 12 teeth in the Ledermix(®) group and nine of 15 in the Ultracal XS(®) group. No significant difference between medicaments was found in the proportion of teeth or patients showing periodontal healing. DISCUSSION: There was no significant difference in periodontal healing between the two medicaments at either a tooth or patient level. The numbers recruited fell short of an estimated power calculation. For patients meeting the inclusion criteria and completing the trial, periodontal healing was seen in 52% of teeth at the 12-month assessment between both groups. The only factor found to significantly influence the periodontal outcome was dry time.

Human LPLUNC1 is a secreted product of goblet cells and minor glands of the respiratory and upper aerodigestive tracts.

Long PLUNC1 (LPLUNC1, C20orf114) is a member of a family of poorly described proteins (PLUNCS) expressed in the upper respiratory tract and oral cavity, which may function in host defence. Although it is one of the most highly expressed genes in the upper airways and has been identified in sputum and nasal secretions by proteomic studies, localisation of LPLUNC1 protein has not yet been described. We developed affinity purified antibodies and localised the protein in tissues of the human respiratory tract, oro- and nasopharynx. We have complemented these studies with analysis of LPLUNC1 expression in primary human lung cell cultures and used Western blotting to study the protein in cell culture secretions and in BAL. LPLUNC1 is a product of a population of goblet cells in the airway epithelium and nasal passages and is also present in airway submucosal glands and minor glands of the oral and nasal cavities. The protein is not expressed in peripheral lung epithelial cells. LPLUNC1 is present in bronchoalveolar lavage fluid as two glycosylated isoforms and primary airway epithelial cells produce identical proteins as they undergo mucociliary differentiation. Our results suggest that LPLUNC1 is an abundant, secreted product of goblet cells and minor mucosal glands of the respiratory tract and oral cavity and suggest that the protein functions in the complex milieu that protects the mucosal surfaces in these locations.

Characterisation and expression of SPLUNC2, the human orthologue of rodent parotid secretory protein.

We recently described the Palate Lung Nasal Clone (PLUNC) family of proteins as an extended group of proteins expressed in the upper airways, nose and mouth. Little is known about these proteins, but they are secreted into the airway and nasal lining fluids and saliva where, due to their structural similarity with lipopolysaccharide-binding protein and bactericidal/permeability-increasing protein, they may play a role in the innate immune defence. We now describe the generation and characterisation of novel affinity-purified antibodies to SPLUNC2, and use them to determine the expression of this, the major salivary gland PLUNC. Western blotting showed that the antibodies identified a number of distinct protein bands in saliva, whilst immunohistochemical analysis demonstrated protein expression in serous cells of the major salivary glands and in the ductal lumens as well as in cells of minor mucosal glands. Antibodies directed against distinct epitopes of the protein yielded different staining patterns in both minor and major salivary glands. Using RT-PCR of tissues from the oral cavity, coupled with EST analysis, we showed that the gene undergoes alternative splicing using two 5' non-coding exons, suggesting that the gene is regulated by alternative promoters. Comprehensive RACE analysis using salivary gland RNA as template failed to identify any additional exons. Analysis of saliva showed that SPLUNC2 is subject to N-glycosylation. Thus, our study shows that multiple SPLUNC2 isoforms are found in the oral cavity and suggest that these proteins may be differentially regulated in distinct tissues where they may function in the innate immune response.

Influence of sodium chloride content in electrolyte solution on electrochemical impedance measurements of human dentin.

BACKGROUND: The aim of this study was to investigate the influence of sodium chloride (NaCl) content in electrolyte solution on electrochemical impedance measurements of human dentin by employing electrochemical impedance spectroscopy. MATERIALS AND METHODS: Dentin samples were prepared from extracted molars. Electrochemical impedance measurements were carried out over a wide frequency range (0.01Hz-10MHz). After measurements, samples were characterized using scanning electron microscopy. RESULTS: Electrochemical impedance measurements showed that the mean values of dentin electrical resistance were 4284, 2062, 1336, 53 and 48kΩ at different NaCl contents in electrolyte solution. One-way ANOVA test of mean values of dentin electrical resistance revealed a significant difference (P < 0.0001) as a function of NaCl content in electrolyte solution. Comparing electrical resistance values of dentin samples at 0.05% w/v and 0.9% w/v concentrations were found to be significantly different (P < 0.05 at 95% confidence level). Scanning electron microscopy revealed structure of dentin sample with intertubular dentin matrix and distribution of patent dentinal tubules. CONCLUSION: This in vitro study indicated, through electrochemical impedance spectroscopy measurements, that electrical resistance of dentin was affected by the concentration of NaCl in electrolyte solution. It is clear from the current study that NaCl concentration in electrolyte solution has a marked influence on dentin electrical resistance. Therefore, this baseline data need to be considered in any future study on dental samples.

WFDC2 (HE4): a potential role in the innate immunity of the oral cavity and respiratory tract and the development of adenocarcinomas of the lung.

BACKGROUND: The Whey Acidic Protein domain is an evolutionarily conserved motif found in a number of proteins, the best studied of which are antiproteinases involved in the innate immune defence of multiple epithelia. We recently characterised the WFDC2 gene which encodes a two WAP domain-containing protein, initially suggested as a marker for epididymis, and showed that it is highly expressed in the lung and salivary gland. The precise location of WFDC2 protein in these sites has not been described. METHODS: We used immunohistochemistry to localise WFDC2 in normal tissues of the respiratory tract, naso- and oropharynx, as well as in chronically inflamed lung from Cystic Fibrosis and a range of pulmonary carcinomas. We have complemented these studies with molecular analysis of WFDC2 gene expression in primary human lung cell cultures. RESULTS: WFDC2 is expressed in some epithelial cells of the upper airways as well as in mucous cells and ducts of submucosal glands. No staining was seen in peripheral lung. Intense staining is found in major salivary glands and in minor glands of the nose, sinuses, posterior tongue and tonsil. Studies with the related protein Secretory Leukocyte Protease Inhibitor (SLPI) show that although both proteins are expressed in similar tissues, the precise cellular localisation differs. Significant increases in expression and localisation of WFDC2 are seen in patients with Cystic Fibrosis. SLPI expression was greatly reduced in the same samples. In cultures of tracheobronchial epithelial cells, expression of WFDC2 and SLPI are differentially regulated during differentiation yet WFDC2 is not induced by pro-inflammatory mediators. The majority of adenocarcinomas stain with WFDC2 whilst a significant minority of squamous, small cell and large cell carcinomas exhibit focal staining. There is no clear association with tumour grade. CONCLUSION: We believe that these studies support the hypothesis that WFDC2 may be a component of the innate immune defences of the lung, nasal and oral cavities and suggest that WFDC2 functions in concert with related WAP domain containing proteins in epithelial host defence. We also suggest that WFDC2 re-expression in lung carcinomas may prove to be associated with tumour type and should be studied in further detail.

Are plasma cell-rich inflammatory conditions of the oral mucosa manifestations of IgG4-related disease?

AIM: The aim of this study was to characterise plasma cell infiltrates, in terms of IgG4 positivity, in specific and non-specific plasma cell-rich chronic inflammatory conditions of the oral mucosa. Exploring the possibility that specific plasma cell-rich oral inflammatory conditions have association with or represent an oral manifestation of immunoglobulin G4-related disease (IgG4-RD). METHODS: Ten patients with plasma cell-rich chronic inflammatory conditions of the oral mucosa were identified (seven--plasma cell mucositis and three--non-specific diffuse oral mucosal inflammation with ulceration). For each patient, the clinical record and H&E-stained sections were reviewed. Immunohistochemistry for IgG and IgG4 antibodies was performed on sections from the corresponding paraffin block, permitting calculation of the mean number of IgG4+ plasma cells per high-power field (HPF) and the IgG4+/IgG+ plasma cell ratio. RESULTS: In all the cases, only one histological hallmark of IgG4-RD--a dense lymphoplasmacytic infiltrate--was seen. Review of the medical histories did not reveal any features representing other manifestations of IgG4-RD. The number of IgG4+ plasma cells exceeded 100 per HPF in half of the cases. Only two cases had an IgG4+/IgG+ plasma cell ratio of >40%; both of which were in the non-specific oral inflammatory group. CONCLUSIONS: Our study suggests that plasma cell mucositis does not meet microscopic criteria for IgG4-RD. It importantly reinforces the opinion that IgG4+ plasma cells are major components of chronic inflammation in the oral cavity and the pertinence of correct contextual interpretation of histopathological features with clinical findings.

Novel approaches to the diagnosis of basal cell nevous syndrome.

In 1960, Robert James Gorlin and William Goltz, both American physicians, defined a new syndrome. Little did they realize that 40 years later, the pathways involved in its development would be provoking serious and sustained interest amongst a plethora of specialists. Fruit-fly biologists, oncologists, geneticists, dermatologists, indeed, hardly a medical or dental specialist gets excluded. To date, there have been some major breakthroughs in identifying abnormal gene sequences. Much has been discovered about this syndrome and its pivotal role in a number of cancer pathways but much more waits to be done or explained. This article sets out to discuss the current position and aims to stimulate further work on this intriguing and puzzling disorder.

In vitro analysis of 'smear layer' on human dentine using ac-impedance spectroscopy.

OBJECTIVE: To analyse smear layers on human dentine using ac-impedance spectroscopy. METHODS: Dentine samples were prepared from extracted, sound, third molars. Impedance measurements were carried out on dentine samples before and after etching. After measuring, samples were examined under scanning electron microscope (SEM) to correlate electrical measurements with structure. RESULTS: Marked differences in impedance before and after etching were demonstrated. SEM investigation showed that a smear layer overlies dentine surfaces before etching, but completely disappeared after etching, leaving open dentinal tubules. CONCLUSIONS: The clinical removal of smear layers is still subjective. This objective method, based on combined ac-impedance and admittance measurement in vitro, has the potential to allow development of standardised techniques and if developed further may be of use in vivo.

Next-generation sequencing analysis for detecting human papillomavirus in oral verrucous carcinoma.

OBJECTIVE: The etiology of oral verrucous carcinoma is unknown, and human papillomavirus 'involvement' remains contentious. The uncertainty can be attributed to varied detection procedures and difficulties in defining 'gold-standard' histologic criteria for diagnosing 'verrucous' lesions. Their paucity also hampers investigation. We aimed to analyze oral verrucous lesions for human papillomavirus (HPV) subtype genomes. STUDY DESIGN: We used next-generation sequencing for the detection of papillomavirus sequences, identifying subtypes and computing viral loads. We identified a total of 78 oral verrucous cases (62 carcinomas and 16 hyperplasias). DNA was extracted from all and sequenced at a coverage between 2.5% and 13%. RESULTS: An HPV-16 sequence was detected in 1 carcinoma and 1 hyperplasia, and an HPV-2 sequence was detected in 1 carcinoma out of the 78 cases, with viral loads of 2.24, 8.16, and 0.33 viral genomes per cell, respectively. CONCLUSIONS: Our results indicate no conclusive human papillomavirus involvement in oral verrucous carcinoma or hyperplasia.

Next-generation sequencing for simultaneous determination of human papillomavirus load, subtype, and associated genomic copy number changes in tumors.

Human papillomavirus (HPV) infection in cases of squamous cell carcinoma of the oropharynx is a powerful predictive and prognostic biomarker. We describe how the use of next-generation sequencing can provide a novel method for the detection of HPV in DNA isolated from formalin-fixed paraffin-embedded tissues. Using this methodology in a cohort of 44 head and neck tumors, we identified the samples that contained HPV sequences, the viral subtype involved, and a direct readout of viral load. Specificity of HPV detection by sequencing compared to traditional detection methods using either PCR or p16 immunohistochemistry was 100%. Sensitivity was 50% when either compared to PCR [confidence interval (CI) = 29% to 71%] or 75% when compared to p16 (CI = 47% to 91%). In addition, we demonstrate the ability of next-generation sequencing to detect other HPV subtypes that would not have been detected by traditional methods, and we demonstrated the ability to apply this method to any tumor and any virus in a panel of eight human cancer cell lines. This methodology also provides a tumor genomic copy number karyogram, and in the samples analyzed here, a lower level of chromosome instability was detected in HPV-positive tumors compared to HPV-negative tumors, as observed in previous studies. Thus, the use of next-generation sequencing for the detection of HPV provides a multiplicity of data with clinical significance in a single test.

Discoloration of teeth after avulsion and replantation: results from a multicenter randomized controlled trial.

INTRODUCTION: There is evidence to suggest that Ledermix, placed as an intervisit root canal dressing, might improve periodontal healing after replantation of avulsed teeth. As a part of a multicenter randomized controlled trial, we aimed to compare the effect of 2 root canal medicaments, Ledermix and Ultracal XS, on the discoloration of replanted teeth. METHODS: Discoloration was investigated by using 3 methods: patient satisfaction with the color of replanted teeth, clinical photographs taken at baseline and 12-month reviews, and estimation of color change by using CIELAB scores for baseline and 12-month photographs. RESULTS: Twenty-two patients (27 teeth) were recruited. Ten patients (12 teeth) were randomized to the Ledermix group and 12 patients (15 teeth) to the Ultracal XS group. At 12 months, 8 patients were concerned with the discoloration of their teeth. Seven came from the Ledermix group and 1 from the Ultracal XS group. This difference was significant (Fisher exact test, P = .009). Standardized photographs were taken for the patients recruited at one center only (17 patients). There was significant discoloration of teeth from baseline with Ledermix, causing a darkening and gray-brown discoloration (mean change from baseline to 12 months, L∗ = -5.1, a∗ = 0.3, b∗ = -1.2, and ΔE = 8.1) and Ultracal XS, causing a yellowing and lightening of teeth (L∗ = 1.9, a∗ = 0.3, b∗ = 3.3, and ΔE = 5.4). There was a significant difference for the L∗ and b∗ variables (independent t test) between the 2 groups. CONCLUSIONS: Both root canal medicaments cause discoloration, with Ledermix proving less acceptable to patients.

Raman spectroscopy in head and neck cancer.

In recent years there has been much interest in the use of optical diagnostics in cancer detection. Early diagnosis of cancer affords early intervention and greatest chance of cure. Raman spectroscopy is based on the interaction of photons with the target material producing a highly detailed biochemical 'fingerprint' of the sample. It can be appreciated that such a sensitive biochemical detection system could confer diagnostic benefit in a clinical setting. Raman has been used successfully in key health areas such as cardiovascular diseases, and dental care but there is a paucity of literature on Raman spectroscopy in Head and Neck cancer. Following the introduction of health care targets for cancer, and with an ever-aging population the need for rapid cancer detection has never been greater. Raman spectroscopy could confer great patient benefit with early, rapid and accurate diagnosis. This technique is almost labour free without the need for sample preparation. It could reduce the need for whole pathological specimen examination, in theatre it could help to determine margin status, and finally peripheral blood diagnosis may be an achievable target.

Actinomycosis mimicking a tonsillar neoplasm in an elderly diabetic patient.

Actinomycosis is a slowly progressive infection that can occur anywhere in the body. Three distinct clinical entities are described: cervico-facial, abdomino-pelvic and thoracopulmonary. Actinomyces are anaerobic, gram positive, non-acid-fast, branched filamentous bacteria that form part of the normal oral, colonic and vaginal flora of humans. The cervico-facial form of the disease is commonest and results from direct invasion of commensal oral actinomyces into local tissues. The most frequently isolated species is A. israelii. We describe a case where Actinomycosis caused massive unilateral hypertrophy of the tonsil, mimicking neoplasia. This is an unusual presentation of Actinomycosis.

Raman spectroscopy and advanced mathematical modelling in the discrimination of human thyroid cell lines.

Raman spectroscopy could offer non-invasive, rapid and an objective nature to cancer diagnostics. However, much work in this field has focused on resolving differences between cancerous and non-cancerous tissues, and lacks the reproducibility and interpretation to be put into clinical practice. Much work is needed on basic cellular differences between malignancy and normal. This would allow the establishment of a clinically relevant cellular based model to translate to tissue classification. Raman spectroscopy provides a very detailed biochemical analysis of the target material and to 'unlock' this potential requires sophisticated mathematical modelling such as neural networks as an adjunct to data interpretation. Commercially obtained cancerous and non-cancerous cells, cultured in the laboratory were used in Raman spectral measurements. Data trends were visualised through PCA and then subjected to neural network analysis based on self-organising maps; consisting of m maps, where m is the number of classes to be recognised. Each map approximates the statistical distribution of a given class. The neural network analysis provided a 95% accuracy for identification of the cancerous cell line and 92% accuracy for normal cell line. In this preliminary study we have demonstrated th ability to distinguish between "normal" and cancerous commercial cell lines. This encourages future work to establish the reasons underpinning these spectral differences and to move forward to more complex systems involving tissues. We have also shown that the use of sophisticated mathematical modelling allows a high degree of discrimination of 'raw' spectral data.

Potential for Raman spectroscopy to provide cancer screening using a peripheral blood sample.

Cancer poses a massive health burden with incidence rates expected to double globally over the next decade. In the United Kingdom screening programmes exists for cervical, breast, and colorectal cancer. The ability to screen individuals for solid malignant tumours using only a peripheral blood sample would revolutionise cancer services and permit early diagnosis and intervention. Raman spectroscopy interrogates native biochemistry through the interaction of light with matter, producing a high definition biochemical 'fingerprint' of the target material. This paper explores the possibility of using Raman spectroscopy to discriminate between cancer and non-cancer patients through a peripheral blood sample. Forty blood samples were obtained from patients with Head and Neck cancer and patients with respiratory illnesses to act as a positive control. Raman spectroscopy was carried out on all samples with the resulting spectra being used to build a classifier in order to distinguish between the cancer and respiratory patients' spectra; firstly using principal component analysis (PCA)/linear discriminant analysis (LDA), and secondly with a genetic evolutionary algorithm. The PCA/LDA classifier gave a 65% sensitivity and specificity for discrimination between the cancer and respiratory groups. A sensitivity score of 75% with a specificity of 75% was achieved with a 'trained' evolutionary algorithm. In conclusion this preliminary study has demonstrated the feasibility of using Raman spectroscopy in cancer screening and diagnostics of solid tumours through a peripheral blood sample. Further work needs to be carried out for this technique to be implemented in the clinical setting.