Molecular pathology and thyroid FNA.

This review summarises molecular pathological techniques applicable to thyroid FNA. The molecular pathology of thyroid tumours is now fairly well understood. Molecular methods may be used as a rule-in test for diagnosis of malignancy in thyroid nodules, eg BRAF V600E point mutation, use of a seven-gene mutational panel (BRAF V600E, RAS genes, RET/PTC or PAX8/PPARG rearrangement), or as a comprehensive multigene next-generation sequencing panel, eg ThyroSeq v2. Molecular methods can also be applied as rule-out tests for malignancy in thyroid nodules, eg Afirma or ThyroSeq v2 or as markers of prognosis, eg TERT promoter mutation or other gene mutations including BRAF V600E, TP53 and AKT1, and as tests for newly defined tumour entities such as non-invasive follicular thyroid neoplasm with papillary like nuclei, or as a molecular marker(s) for targeted therapies. This review describes practical examples of molecular techniques as applied to thyroid FNA in routine clinical practice and the value of molecular diagnostics in thyroid FNA. It describes the range of molecular abnormalities identified in thyroid nodules and thyroid cancers with some practical applications of molecular methods to diagnosis and prognosis of thyroid nodules and thyroid cancer.

BRAF V600 co-testing is technically feasible in conventional thyroid fine needle aspiration (FNA) cytology smears and can reduce the need for completion thyroidectomy.

INTRODUCTION: While fine needle aspiration cytology (FNAC) is the mainstay of diagnosis in thyroid nodules, molecular markers of thyroid cancer have recently been shown to be of value in improving the diagnosis and reducing the rates of unnecessary surgery. METHOD: A technical method is presented for the assessment of the BRAF V600 gene mutation in thyroid cancer using a simple adaptation of a commercially available kit. After standard preparation and reporting of conventionally stained alcohol-fixed Papanicolaou or air-dried Giemsa-stained slides the coverslip is removed from one slide, the DNA is extracted and submitted for PCR analysis. RESULTS: Assessment of the BRAF V600 mutational status is feasible in very small quantities of DNA, requiring just greater than 5 ng per case from a single pre-stained FNA slide using this method. From the 14 cases examined thus far, one Thy4/Bethesda Class V case (suspicious of malignancy) has been identified with a BRAF V600 mutation and this patient, after multidisciplinary discussion, received a total thyroidectomy. CONCLUSION: Based on this methodology and other published results for the BRAF mutation, we believe that it is now feasible and cost effective for the UK NHS to BRAF co-test all Thy4/Bethesda Class V thyroid FNAs, as the additional cost of BRAF testing will still be much less than the cost of submitting all Thy4 (Bethesda Class V) patients to a partial and then a later completion thyroidectomy.

Targeting EGFR and PI3K pathways in ovarian cancer.

BACKGROUND: The epidermal growth factor receptor (EGFR) is expressed in ovarian cancer, but agents targeting this pathway have shown little effect as single agents. This may be due to the presence of alternative pathways, particularly activation of the PI3K/Akt/MTOR pathway. METHODS: We have therefore examined the effect of inhibitors of this pathway (ZSTK474 and sirolimus) in combination with the EGFR inhibitors erlotinib and gefitinib in ovarian cancer primary cell cultures. RESULTS: The single-agent EGFR inhibitors showed little activity, although some activity was seen with the single-agent PI3K inhibitor, ZSTK474. Combinations of ZSTK474 with EGFR inhibitors showed enhanced activity with some evidence of synergy, whereas sirolimus combinations were less active. The results were not explicable on the basis of PIK3CA mutation or amplification, or PTEN loss, although one tumour with a KRAS mutation showed resistance to EGFR inhibitors. However, there was correlation of the EGFR expression with sensitivity to EGFR and resistance to PI3K active agents, and inverse correlation in the sensitivity of individual tumours to agents active against these pathways, suggesting a mechanism of action for the combination. CONCLUSION: Phase I/II clinical trials with these agents should include further pharmacodynamic endpoints and molecular characterisation to identify patients most likely to benefit from this strategy.

Management of a large outbreak of COVID-19 at a British Army training centre: lessons for the future.

INTRODUCTION: The COVID-19 pandemic has posed major challenges for infection control within training centres, both civilian and military. Here we present a narrative review of an outbreak that occurred at the Royal Military Academy Sandhurst (RMAS) in January-March 2021, in the context of the circulating, highly transmissible SARS-CoV-2 variant B.1.1.7. METHODS: Testing for SARS-CoV-2 was performed using a combination of reverse transcriptase PCR and Lateral Flow Devices (LFDs). Testing and isolation procedures were conducted in line with a pre-established symptom stratification system. Genomic sequencing was performed on 10 sample isolates. RESULTS: By the end of the outbreak, 185 cases (153 Officer Cadets, 32 permanent staff) had contracted confirmed COVID-19. This represented 15% of the total RMAS population. This resulted in 0 deaths and 0 hospitalisations, but due to necessary isolation procedures did represent an estimated 12 959 person-days of lost training. 9 of 10 (90%) of sequenced isolates had a reportable lineage. All of those reported were found to be the Alpha lineage B.1.1.7. CONCLUSIONS: We discuss the key lessons learnt from the after-action review by the Incident Management Team. These include the importance of multidisciplinary working, the utility of sync matrices to monitor outbreaks in real time, issues around Officer Cadets reporting symptoms, timing of high-risk training activities, infrastructure and use of LFDs. COVID-19 represents a vital learning opportunity to minimise the impact of potential future pandemics, which may produce considerably higher morbidity and mortality in military populations.

Nosocomial or not? A combined epidemiological and genomic investigation to understand hospital-acquired COVID-19 infection on an elderly care ward.

BACKGROUND: COVID-19 has the potential to cause outbreaks in hospitals. Given the comorbid and elderly cohort of patients hospitalized, hospital-acquired COVID-19 infection is often fatal. Pathogen genome sequencing is becoming increasingly important in infection prevention and control (IPC). AIM: To inform the understanding of in-hospital SARS-CoV-2 transmission in order to improve IPC practices and to inform the future development of virological testing for IPC. METHODS: Patients detected COVID-19 positive by polymerase chain reaction on Ward A in April and May 2020 were included with contact tracing to identify other potential cases. Genome sequencing was undertaken for a subgroup of cases. Epidemiological, genomic, and cluster analyses were performed to describe the epidemiology and to identify factors contributing to the outbreak. FINDINGS: Fourteen cases were identified on Ward A. Contact tracing identified 16 further patient cases; in addition, eight healthcare workers (HCWs) were identified as being COVID-19 positive through a round of asymptomatic testing. Genome sequencing of 16 of these cases identified viral genomes differing by two single nucleotide polymorphisms or fewer, with further cluster analysis identifying two groups of infection (a five-person group and a six-person group). CONCLUSION: Despite the temporal relationship of cases, genome sequencing identified that not all cases shared transmission events. However, 11 samples were found to be closely related and these likely represented in-hospital transmission. This included three HCWs, thereby confirming transmission between patients and HCWs.

Molecular basis of chemosensitivity of platinum pre-treated ovarian cancer to chemotherapy.

BACKGROUND: Ovarian cancer shows considerable heterogeneity in its sensitivity to chemotherapy both clinically and in vitro. This study tested the hypothesis that the molecular basis of this difference lies within the known resistance mechanisms inherent to these patients' tumours. METHODS: The chemosensitivity of a series of 31 ovarian tumours, all previously treated with platinum-based chemotherapy, was assessed using the ATP-based tumour chemosensitivity assay (ATP-TCA) and correlated with resistance gene expression measured by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) in a TaqMan Array following extraction of mRNA from formalin-fixed paraffin-embedded tissue. The results were standardised against a housekeeping gene (PBGD), and assessed by multiple linear regression. RESULTS: Predictive multiple linear regression models were derived for four single agents (cisplatin, gemcitabine, topotecan, and treosulfan), and for the combinations of cisplatin+gemcitabine and treosulfan+gemcitabine. Particularly strong correlations were obtained for cisplatin, gemcitabine, topotecan, and treosulfan+gemcitabine. No individual gene expression showed direct correlation with activity in the ATP-TCA. Genes involved in DNA repair and apoptosis were strongly represented, with some drug pumps also involved. CONCLUSION: The chemosensitivity of ovarian cancer to drugs is related to the expression of genes involved in sensitivity and resistance mechanisms.