T.K. Ong

Using next-generation sequencing for high resolution multiplex analysis of copy number variation from nanogram quantities of DNA from formalin-fixed paraffin-embedded specimens

The use of next-generation sequencing technologies to produce genomic copy number data has recently been described. Most approaches, however, reply on optimal starting DNA, and are therefore unsuitable for the analysis of formalin-fixed paraffin-embedded (FFPE) samples, which largely precludes the analysis of many tumour series. We have sought to challenge the limits of this technique with regards to quality and quantity of starting material and the depth of sequencing required. We confirm that the technique can be used to interrogate DNA from cell lines, fresh frozen material and FFPE samples to assess copy number variation. We show that as little as 5 ng of DNA is needed to generate a copy number karyogram, and follow this up with data from a series of FFPE biopsies and surgical samples. We have used various levels of sample multiplexing to demonstrate the adjustable resolution of the methodology, depending on the number of samples and available resources. We also demonstrate reproducibility by use of replicate samples and comparison with microarray-based comparative genomic hybridization (aCGH) and digital PCR. This technique can be valuable in both the analysis of routine diagnostic samples and in examining large repositories of fixed archival material.

Duplex in the assessment of the free radial forearm flaps: Is it time to change practice?

Radial forearm free flaps (RFFFs) are safe, but critical ischaemia of the hand has been described and is catastrophic. Every effort should therefore be made to improve the safety margin even further. Colour flow duplex ultrasound (US) is a simple, non-invasive and effective assessment tool. We compared it with Allens test to identify serious vascular anomalies. We studied 121 patients who were listed to have a RFFF harvested, all of whom had both duplex US assessment and Allens testing of the selected arm. The significance of differences in proportions was assessed using McNemars test. Five of the 121 patients had an alternative flap selected as a consequence of the duplex assessment. A single flap failed. There were no ischaemic vascular complications that affected the hand.

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. Copyright

The configuration of clinics and the use of biopsy and photography in oral premalignancy: a survey of consultants of the British Association of Oral and Maxillofacial Surgeons

Oral dysplastic lesions may have an increased chance of becoming oral squamous cell carcinoma, but to date their management remains controversial. The aim of this survey was to explore the current practical aspects of the management of patients with dysplasia by oral and maxillofacial consultants in the UK. In the survey we asked consultants about the numbers of patients they see with oral premalignant lesions, the frequency and specialty of designated hospital clinics, their use of photographs and biopsy, factors that influence their decision whether to biopsy a lesion at the first appointment, the procedure for treatment and follow-up and their use (if any) of chemopreventive agents. We found a wide variation in the practical aspects of managing patients with dysplasia, and the lack of consistency among clinicians supports the idea of an initiative to establish more robust national guidelines to use as a gold standard in the future.

Biopsy examination of squamous cell carcinoma of the tongue: Source of significant prognostic information?

Histological analysis of tumour resection for squamous cell carcinoma (SCC) of the tongue yields prognostic information. We analysed histological slides of biopsy and tumour resection specimens using an adapted malignancy grading score and analysed variables of neck dissections. There was moderate correlation between biopsy and tumour resection using malignancy grading scores (correlation coefficient 0.45); good agreement of tumour grade (79%), tumour depth (76%), and type of invasive front (80%), but correlation was only fair to moderate (κ=0.38, κ=0.51, and κ=0.41, respectively). Correlation of the biopsy grading score and invaded nodes in the neck, extra capsular spread, and soft tissue disease was not significant.

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.

The genomic road to invasion—examining the similarities and differences in the genomes of associated oral pre-cancer and cancer samples

BackgroundIt is frequently assumed that pre-invasive lesions are simpler precursors of cancer and will contain a limited subset of the genomic changes seen in their associated invasive disease. Driver mutations are thought to occur early, but it is not known how many of these are present in pre-invasive lesions. These assumptions need to be tested with the increasing focus on both personalised cancer treatments and early detection methodologies.MethodsWe examined genomic copy number changes in 256 pre-invasive and invasive samples from 69 oral cancer patients. Forty-eight samples from 16 patients were further examined using exome sequencing.ResultsEvidence of a shared ancestor of both dysplasia and carcinoma was seen in all but one patient. One-third of dysplasias showed independent copy number events. The remainder had a copy number pattern that was similar to or simpler than that of the carcinoma. All dysplasias examined contained somatic mutations absent in the related carcinoma.Previously observed copy number changes and TP53 mutations were very frequently observed, and almost always shared between dysplasia and carcinoma. Other gene changes were more sporadic. Pathway analysis confirmed that each patient’s disease developed in a different way.Examining the numbers of shared mutations and the rate of accumulation of mutations showed evidence that all samples contain a population of sub-clones, with little evidence of selective advantage of a subset of these.ConclusionsThese findings suggest that most of the genomic changes driving oral cancer occur in the pre-cancerous state by way of gradual random accumulation rather than a dramatic single event.

Survival after surgery for oral cancer: a 30-year experience.

Oral squamous cell carcinoma is the most common intraoral malignancy, for which we advocate radical primary resection with adjuvant treatment where indicated. The main aims of this paper are to identify the overall survival of a consecutive series of patients and to relate survival to clinical and pathological factors. Kaplan-Meier curves were produced for site, sex, TNM status, and use of postoperative radiotherapy. The data were analysed using IBM SPSS Statistics for Windows and probabilities of less than 0.05 were accepted as significant. A total of 921 patients were recorded in the database with a diagnosis of oral squamous cell carcinoma out of a total of 1958 with salivary gland conditions or other cancers of the head and neck (43.1%). The earliest date of diagnosis was 1973, and the data were censored at 31 March 2016. The database comprised 340 women (36.9%) and 581 men (63.1%). A total of 339 patients died (34.5%): 117 women (33.7%) and 222 men (65.5%). The mean (range) age at death was 73.4 (31.4-97.5) years for women and 68.7 (33.3-95.5) years for men (t (337)=3.28, p=0.001). Our overall survival was somewhat better than the 56% five-year survival reported for oral cancer in England in 2010, which may be a reflection of the treatment. This work supports the view that aggressive management may improve overall survival.

Margins and survival in oral cancer

In the surgical management of oral squamous cell carcinoma (SCC) we aim to resect the tumour with clear margins in all planes. The aim of this study was to identify and compare overall survival in a group of 591 patients who had resections, and to relate this to the clearance of margins at the tumour bed. We used life tables to calculate survival at one, two, three, five, and 10 years after diagnosis by margin (clear=5mm or more; close=2-5mm; and involved=less than 2mm). Kaplan-Meier curves were produced for the margins alone, which were defined as clear in 480 patients (81%), close in 63 (11%), and involved in 48 (8%). Five-year survival was 81%, 75%, and 54% for clear, close, and involved margins, respectively, which highlights the importance of clear margins for survival. There is a significant prognostic implication associated with close, and particularly with involved, margins.

Delays in head and neck surgery—managers, theatre usage and suboptimal efficiency

Achieving maximum efficiency means that there are daily ressures from managers, towards optimal theatre utilisaion. In an attempt to improve team efficiency 41 consecutive econstructions with radial free forearm flaps – a flap comonly used in head and neck surgery1 – were recorded and nalysed prospectively. The mean average procedure time (on the table to finish urgery) was 7.85 h (range 5.67–11.17 h). The time for all the teps of each operation was recorded in minutes (Table 1). he mean average procedure time was 7.85 h which is comarable to a study by Chalian et al.2 If we look at unilateral elective neck dissection and peri-oral resection with simulaneous harvesting of RFFF, our mean time at 5.93 h was a ot faster than if we added a tracheostomy, another neck disection and lip–jaw split, which came in at 8.93 h. We next ooked at why we finished late. This was analysed as follows. irstly, the sample was arbitrarily divided at 8 h (procedure ime) into 2 groups, roughly the halfway mark for the group nd compared each of the stages of the procedure between he two groups. For completion, the first seven finishes were lso studied in close detail and were compared with the last even finishes. The early finish group (less than 8 h) had fewer rocedures such as tracheostomies, lip–jaw splits and neck issections. The late finishes (more than 8 h) had very late tarts (3/7 or 43%), longer mean times for several stages (e.g. racheostomies 5 min, neck 9 min and resections 14 min) and our patients (57%) had bilateral neck dissections. However, here were still times where no surgery was being carried out, or example when setting the patient up in theatre, cathetersing or re-draping after tracheostomy. There was a trend for he mean non-surgical time to be longer for the seven late nishes; one possible explanation may be due to the total umber of procedures being carried out with change-over of tages/staff. Therefore, inefficiency is not a major factor for he late finishes. We have found this exercise using organisational analysis seful. The benchmark times can be added together to predict ith a reasonable accuracy how long we are likely to over-run.