Immunohistochemical expression of secreted frizzled receptor protein 1 in the invasive front of tongue squamous cell carcinoma.

The Wnt signalling pathway is involved in the development of oral cancer. When inactivated, secreted frizzled receptor protein 1 (SFRP1), a cellular Wnt-inhibitor protein, may enhance cancer development. The aim of this study was to assess the expression of SFRP1 in the invasive front of tongue squamous cell carcinoma (SCC). Immunohistochemical expression of SFRP1 in the cohesive and discohesive invasive front of 36 resection specimens of tongue SCC was investigated. Immunostaining was compared with tumour size, tumour thickness, and neural invasion. Immunoreactivity increased in depth from the surface of the tumour to the invasive front. There was a statistically significant association between type of invasive front and pattern of SFRP1 expression. A discohesive invasive front showed more intense immunoreactivity. No statistically significant associations were found between the intensity of staining and relevant prognostic factors. Tongue SSC has variable SFRP1 expression in different parts of the tumour and the greatest expression is in the invasive front. The more intense staining in the discohesive invasive front compared with the cohesive invasive front might contribute to the worse prognosis of cancers with a discohesive invasive front.

Combining viral genomics and clinical data to assess risk factors for severe COVID-19 (mortality, ICU admission, or intubation) amongst hospital patients in a large acute UK NHS hospital Trust.

Throughout the COVID-19 pandemic, valuable datasets have been collected on the effects of the virus SARS-CoV-2. In this study, we combined whole genome sequencing data with clinical data (including clinical outcomes, demographics, comorbidity, treatment information) for 929 patient cases seen at a large UK hospital Trust between March 2020 and May 2021. We identified associations between acute physiological status and three measures of disease severity; admission to the intensive care unit (ICU), requirement for intubation, and mortality. Whilst the maximum National Early Warning Score (NEWS2) was moderately associated with severe COVID-19 (A = 0.48), the admission NEWS2 was only weakly associated (A = 0.17), suggesting it is ineffective as an early predictor of severity. Patient outcome was weakly associated with myriad factors linked to acute physiological status and human genetics, including age, sex and pre-existing conditions. Overall, we found no significant links between viral genomics and severe outcomes, but saw evidence that variant subtype may impact relative risk for certain sub-populations. Specific mutations of SARS-CoV-2 appear to have little impact on overall severity risk in these data, suggesting that emerging SARS-CoV-2 variants do not result in more severe patient outcomes. However, our results show that determining a causal relationship between mutations and severe COVID-19 in the viral genome is challenging. Whilst improved understanding of the evolution of SARS-CoV-2 has been achieved through genomics, few studies on how these evolutionary changes impact on clinical outcomes have been seen due to complexities associated with data linkage. By combining viral genomics with patient records in a large acute UK hospital, this study represents a significant resource for understanding risk factors associated with COVID-19 severity. However, further understanding will likely arise from studies of the role of host genetics on disease progression.

The effect of tricyclic antidepressants on cutaneous melanoma cell lines and primary cell cultures.

The tricyclic antidepressants have previously been shown to exert activity against glioma cells in vitro. Initial studies in cell lines suggested that this might extend to melanoma cells. We have therefore conducted a study in primary cell cultures from metastatic cutaneous melanoma deposits using a well established ATP-based tumour chemosensitivity assay to confirm and extend these findings. Two cell lines and eight primary cell cultures from metastatic melanoma deposits were exposed to three tricyclic drugs, amitriptyline, nortriptyline and clomipramine, at concentrations ranging from 200 to 6.25 µmol/l in the ATP-based tumour chemosensitivity assay. All three drugs showed activity, although nortriptyline was more active than clomipramine or amitriptyline in both cell lines and primary cell cultures, with an IC50 of 9, 27 and 33 µmol/l, respectively. Tricyclic agents show activity against melanoma in vitro. This could be related to the lysosomal effects based on their cationic amphiphilic properties, or effects at the mitochondrial membrane.

Multiple pathways of SARS-CoV-2 nosocomial transmission uncovered by integrated genomic and epidemiological analyses during the second wave of the COVID-19 pandemic in the UK.

Introduction: Throughout the global COVID-19 pandemic, nosocomial transmission has represented a major concern for healthcare settings and has accounted for many infections diagnosed within hospitals. As restrictions ease and novel variants continue to spread, it is important to uncover the specific pathways by which nosocomial outbreaks occur to understand the most suitable transmission control strategies for the future. Methods: In this investigation, SARS-CoV-2 genome sequences obtained from 694 healthcare workers and 1,181 patients were analyzed at a large acute NHS hospital in the UK between September 2020 and May 2021. These viral genomic data were combined with epidemiological data to uncover transmission routes within the hospital. We also investigated the effects of the introduction of the highly transmissible variant of concern (VOC), Alpha, over this period, as well as the effects of the national vaccination program on SARS-CoV-2 infection in the hospital. Results: Our results show that infections of all variants within the hospital increased as community prevalence of Alpha increased, resulting in several outbreaks and super-spreader events. Nosocomial infections were enriched amongst older and more vulnerable patients more likely to be in hospital for longer periods but had no impact on disease severity. Infections appeared to be transmitted most regularly from patient to patient and from patients to HCWs. In contrast, infections from HCWs to patients appeared rare, highlighting the benefits of PPE in infection control. The introduction of the vaccine at this time also reduced infections amongst HCWs by over four-times. Discussion: These analyses have highlighted the importance of control measures such as regular testing, rapid lateral flow testing alongside polymerase chain reaction (PCR) testing, isolation of positive patients in the emergency department (where possible), and physical distancing of patient beds on hospital wards to minimize nosocomial transmission of infectious diseases such as COVID-19.

Predicting survival in malignant pleural mesothelioma using routine clinical and laboratory characteristics.

INTRODUCTION: The prognosis of malignant pleural mesothelioma (MPM) is poor, with a median survival of 8-12 months. The ability to predict prognosis in MPM would help clinicians to make informed decisions regarding treatment and identify appropriate research opportunities for patients. The aims of this study were to examine associations between clinical and pathological information gathered during routine care, and prognosis of patients with MPM, and to develop a 6-month mortality risk prediction model. METHODS: A retrospective cohort study of patients diagnosed with MPM at Queen Alexandra Hospital, Portsmouth, UK between December 2009 and September 2013. Multivariate analysis was performed on routinely available histological, clinical and laboratory data to assess the association between different factors and 6-month survival, with significant associations used to create a model to predict the risk of death within 6 months of diagnosis with MPM. RESULTS: 100 patients were included in the analysis. Variables significantly associated with patient survival in multivariate analysis were age (HR 1.31, 95% CI 1.09 to 1.56), smoking status (current smoker HR 3.42, 95% CI 1.11 to 4.20), chest pain (HR 2.14, 95% CI 1.23 to 3.72), weight loss (HR 2.13, 95% CI 1.18 to 3.72), platelet count (HR 1.05, 95% CI 1.00 to 1.10), urea (HR 2.73, 95% CI 1.31 to 5.69) and adjusted calcium (HR 1.47, 95% CI 1.10 to 1.94). The resulting risk model had a c-statistic value of 0.76. A Hosmer-Lemeshow test confirmed good calibration of the model against the original dataset. CONCLUSION: Risk of death at 6 months in patients with a confirmed diagnosis of MPM can be predicted using variables readily available in clinical practice. The risk prediction model we have developed may be used to influence treatment decisions in patients with MPM. Further validation of the model requires evaluation of its performance on a separate dataset.

The Alpha variant was not associated with excess nosocomial SARS-CoV-2 infection in a multi-centre UK hospital study.

OBJECTIVES: Recently emerging SARS-CoV-2 variants have been associated with an increased rate of transmission within the community. We sought to determine whether this also resulted in increased transmission within hospitals. METHODS: We collected viral sequences and epidemiological data of patients with community and healthcare associated SARS-CoV-2 infections, sampled from 16th November 2020 to 10th January 2021, from nine hospitals participating in the COG-UK HOCI study. Outbreaks were identified using ward information, lineage and pairwise genetic differences between viral sequences. RESULTS: Mixed effects logistic regression analysis of 4184 sequences showed healthcare-acquired infections were no more likely to be identified as the Alpha variant than community acquired infections. Nosocomial outbreaks were investigated based on overlapping ward stay and SARS-CoV-2 genome sequence similarity. There was no significant difference in the number of patients involved in outbreaks caused by the Alpha variant compared to outbreaks caused by other lineages. CONCLUSIONS: We find no evidence to support it causing more nosocomial transmission than previous lineages. This suggests that the stringent infection prevention measures already in place in UK hospitals contained the spread of the Alpha variant as effectively as other less transmissible lineages, providing reassurance of their efficacy against emerging variants of concern.

SARS-CoV-2 lineage B.1.1.7 is associated with greater disease severity among hospitalised women but not men: multicentre cohort study.

BACKGROUND: SARS-CoV-2 lineage B.1.1.7 has been associated with an increased rate of transmission and disease severity among subjects testing positive in the community. Its impact on hospitalised patients is less well documented. METHODS: We collected viral sequences and clinical data of patients admitted with SARS-CoV-2 and hospital-onset COVID-19 infections (HOCIs), sampled 16 November 2020 to 10 January 2021, from eight hospitals participating in the COG-UK-HOCI study. Associations between the variant and the outcomes of all-cause mortality and intensive therapy unit (ITU) admission were evaluated using mixed effects Cox models adjusted by age, sex, comorbidities, care home residence, pregnancy and ethnicity. FINDINGS: Sequences were obtained from 2341 inpatients (HOCI cases=786) and analysis of clinical outcomes was carried out in 2147 inpatients with all data available. The HR for mortality of B.1.1.7 compared with other lineages was 1.01 (95% CI 0.79 to 1.28, p=0.94) and for ITU admission was 1.01 (95% CI 0.75 to 1.37, p=0.96). Analysis of sex-specific effects of B.1.1.7 identified increased risk of mortality (HR 1.30, 95% CI 0.95 to 1.78, p=0.096) and ITU admission (HR 1.82, 95% CI 1.15 to 2.90, p=0.011) in females infected with the variant but not males (mortality HR 0.82, 95% CI 0.61 to 1.10, p=0.177; ITU HR 0.74, 95% CI 0.52 to 1.04, p=0.086). INTERPRETATION: In common with smaller studies of patients hospitalised with SARS-CoV-2, we did not find an overall increase in mortality or ITU admission associated with B.1.1.7 compared with other lineages. However, women with B.1.1.7 may be at an increased risk of admission to intensive care and at modestly increased risk of mortality.

The effect of pentamidine on melanoma ex vivo.

Pentamidine is a small molecule inhibitor of the Ca(+)-binding protein S100B and disrupts the S100B-p53 protein-protein interaction; this is thought to restore wild-type p53 tumour suppressor function in melanoma. Additional anticancer effects may be the result of inhibition of regenerating liver family phosphatases. In this study, we have used a standardized ATP-tumour chemosensitivity assay to investigate the effect of pentamidine on cells derived from 18 skin melanoma samples and one uveal melanoma sample. The cells were tested at six concentrations from which the IC(50) and IC(90) were calculated. To allow comparison between samples, an index(sum) was calculated based on the percentage of tumour growth inhibition at each concentration. Of the skin melanoma samples tested, 78% exhibited an index(sum) less than 300 indicating strong inhibition. The median index(sum) of 237 also indicates considerable activity against these samples. The median IC(90) (30.2 micromol/l) may be clinically achievable in a proportion of patients. The uveal melanoma sample exhibited an index(sum) of 333 indicating moderate inhibition, and 86% inhibition at test drug concentration (37.96 micromol/l). These results show that pentamidine has activity against melanoma, and support the prospect of its development for therapeutic use.

Activity of mevalonate pathway inhibitors against breast and ovarian cancers in the ATP-based tumour chemosensitivity assay.

Previous data suggest that lipophilic statins such as fluvastatin and N-bisphosphonates such as zoledronic acid, both inhibitors of the mevalonate metabolic pathway, have anti-cancer effects in vitro and in patients. We have examined the effect of fluvastatin alone and in combination with zoledronic acid in the ATP-based tumour chemosensitivity assay (ATP-TCA) for effects on breast and ovarian cancer tumour-derived cells. Both zoledronic acid and fluvastatin showed activity in the ATP-TCA against breast and ovarian cancer, though fluvastatin alone was less active, particularly against breast cancer. The combination of zoledronic acid and fluvastatin was more active than either single agent in the ATP-TCA with some synergy against breast and ovarian cancer tumour-derived cells. Sequential drug experiments showed that pre-treatment of ovarian tumour cells with fluvastatin resulted in decreased sensitivity to zoledronic acid. Addition of mevalonate pathway components with zoledronic acid with or without fluvastatin showed little effect, while mevalonate did reduced inhibition due to fluvastatin. These data suggest that the combination of zoledronic acid and fluvastatin may have activity against breast and ovarian cancer based on direct anti-cancer cell effects. A clinical trial to test this is in preparation.

Resistance gene expression determines the in vitro chemosensitivity of non-small cell lung cancer (NSCLC).

BACKGROUND: NSCLC exhibits considerable heterogeneity in its sensitivity to chemotherapy and similar heterogeneity is noted in vitro in a variety of model systems. This study has tested the hypothesis that the molecular basis of the observed in vitro chemosensitivity of NSCLC lies within the known resistance mechanisms inherent to these patients' tumors. METHODS: The chemosensitivity of a series of 49 NSCLC tumors was assessed using the ATP-based tumor chemosensitivity assay (ATP-TCA) and compared with quantitative expression of resistance genes measured by RT-PCR in a Taqman Array following extraction of RNA from formalin-fixed paraffin-embedded (FFPE) tissue. RESULTS: There was considerable heterogeneity between tumors within the ATP-TCA, and while this showed no direct correlation with individual gene expression, there was strong correlation of multi-gene signatures for many of the single agents and combinations tested. For instance, docetaxel activity showed some dependence on the expression of drug pumps, while cisplatin activity showed some dependence on DNA repair enzyme expression. Activity of both drugs was influenced more strongly still by the expression of anti- and pro-apoptotic genes by the tumor for both docetaxel and cisplatin. The doublet combinations of cisplatin with gemcitabine and cisplatin with docetaxel showed gene expression signatures incorporating resistance mechanisms for both agents. CONCLUSION: Genes predicted to be involved in known mechanisms drug sensitivity and resistance correlate well with in vitro chemosensitivity and may allow the definition of predictive signatures to guide individualized chemotherapy in lung cancer.

The SENSOR Study: Protocol for a Mixed-Methods Study of Self-Management Checks to Predict Exacerbations of Pseudomonas Aeruginosa in Patients with Long-Term Respiratory Conditions.

BACKGROUND: There are an estimated three million people in the United Kingdom with chronic obstructive pulmonary disease (COPD), and the incidence of bronchiectasis is estimated at around 0.1% but is more common in COPD and severe asthma. Both COPD and bronchiectasis are characterized by exacerbations in which bacteria play a central role. Pseudomonas aeruginosa is isolated from sputum samples from 4% to 15% of adults with COPD and is more likely to be isolated from patients with severe disease. Earlier detection of exacerbations may improve morbidity and mortality by expediting treatment. Aseptika Ltd has developed a system for patients to self-monitor important physiological measurements including levels of physical activity, peak flow, forced expiratory volume (FEV1), and biomarkers for P aeruginosa in sputum. OBJECTIVE: We aim to test this system in 20 participants with P aeruginosa colonization and 10 controls with Haemophilus influenzae. METHODS: We plan to recruit 30 adult participants with COPD or non-CF bronchiectasis who have cultured P aeruginosa or H influenzae during an exacerbation in the last 6 months. They must produce sputum on most days and should have been stable for 4 weeks prior to entry. Daily data collected will include symptoms, health care usage, medication, weight, FEV1, physical activity level, blood pressure, oxygen saturation, and temperature. Sputum and urine samples will be provided daily. These data will be analyzed to assess predictive value in detecting upcoming exacerbations. Qualitative data will be gathered through self-administered questionnaires and semistructured interviews to gather information on participant coping and their use of the technology involved. RESULTS: Recruitment has been completed and results from the study should be available at the end of 2017. CONCLUSIONS: The SENSOR study aims to test a home-monitoring system in people with chronic airway infection and is currently underway.

Cancer cell adaptation to chemotherapy.

BACKGROUND: Tumor resistance to chemotherapy may be present at the beginning of treatment, develop during treatment, or become apparent on re-treatment of the patient. The mechanisms involved are usually inferred from experiments with cell lines, as studies in tumor-derived cells are difficult. Studies of human tumors show that cells adapt to chemotherapy, but it has been largely assumed that clonal selection leads to the resistance of recurrent tumors. METHODS: Cells derived from 47 tumors of breast, ovarian, esophageal, and colorectal origin and 16 paired esophageal biopsies were exposed to anticancer agents (cisplatin; 5-fluorouracil; epirubicin; doxorubicin; paclitaxel; irinotecan and topotecan) in short-term cell culture (6 days). Real-time quantitative PCR was used to measure up- or down-regulation of 16 different resistance/target genes, and when tissue was available, immunohistochemistry was used to assess the protein levels. RESULTS: In 8/16 paired esophageal biopsies, there was an increase in the expression of multi-drug resistance gene 1 (MDR1) following epirubicin + cisplatin + 5-fluorouracil (ECF) chemotherapy and this was accompanied by increased expression of the MDR-1 encoded protein, P-gp. Following exposure to doxorubicin in vitro, 13/14 breast carcinomas and 9/12 ovarian carcinomas showed >2-fold down-regulation of topoisomerase IIalpha (TOPOIIalpha). Exposure to topotecan in vitro, resulted in >4-fold down-regulation of TOPOIIalpha in 6/7 colorectal tumors and 8/10 ovarian tumors. CONCLUSION: This study suggests that up-regulation of resistance genes or down-regulation in target genes may occur rapidly in human solid tumors, within days of the start of treatment, and that similar changes are present in pre- and post-chemotherapy biopsy material. The molecular processes used by each tumor appear to be linked to the drug used, but there is also heterogeneity between individual tumors, even those with the same histological type, in the pattern and magnitude of response to the same drugs. Adaptation to chemotherapy may explain why prediction of resistance mechanisms is difficult on the basis of tumor type alone or individual markers, and suggests that more complex predictive methods are required to improve the response rates to chemotherapy.

The in vitro effect of gefitinib ('Iressa') alone and in combination with cytotoxic chemotherapy on human solid tumours.

BACKGROUND: Activation of the epidermal growth factor receptor (EGFR) triggers downstream signaling pathways that regulate many cellular processes involved in tumour survival and growth. Gefitinib ('Iressa') is an orally active tyrosine kinase inhibitor (TKI) targeted to the ATP-binding domain of EGFR (HER1; erbB1). METHODS: In this study we have used a standardised ATP-based tumour chemosensitivity assay (ATP-TCA) to measure the activity of gefitinib alone or in combination with different cytotoxic drugs (cisplatin, gemcitabine, oxaliplatin and treosulfan) against a variety of solid tumours (n = 86), including breast, colorectal, oesophageal and ovarian cancer, carcinoma of unknown primary site, cutaneous and uveal melanoma, non-small cell lung cancer (NSCLC) and sarcoma. The IC50 and IC90 were calculated for each single agent or combination. To allow comparison between samples the IndexSUM was calculated based on the percentage tumour growth inhibition (TGI) at each test drug concentration (TDC). Gefitinib was tested at concentrations ranging from 0.0625-2 microM (TDC = 0.446 microg/ml). This study represents the first use of a TKI in the assay. RESULTS: There was heterogeneity in the degree of TGI observed when tumours were tested against single agent gefitinib. 7% (6/86) of tumours exhibited considerable inhibition, but most showed a more modest response resulting in a low TGI. The median IC50 value for single agent gefitinib in all tumours tested was 3.98 microM. Interestingly, gefitinib had both positive and negative effects when used in combination with different cytotoxics. In 59% (45/76) of tumours tested, the addition of gefitinib appeared to potentiate the effect of the cytotoxic agent or combination (of these, 11% (5/45) had a >50% decrease in their IndexSUM). In 38% of tumours (29/76), the TGI was decreased when the combination of gefitinib + cytotoxic was used in comparison to the cytotoxic alone. In the remaining 3% (2/76) there was no change observed. CONCLUSION: The in vitro model suggests that gefitinib may have differential effects in response to concomitant cytotoxic chemotherapy with the agents tested during this study. The mechanism involved may relate to the effect of TKIs on growth rate versus their effect on the ability of the cell to survive the stimulus to apoptosis produced by chemotherapy.

BRAF V600 co-testing in thyroid FNA cytology: short-term experience in a large cancer centre in the UK.

AIMS: To ascertain whether BRAF V600 mutational analysis is useful for diagnosis of thyroid cancer in thyroid fine needle aspirate (FNA). METHODS: Over 8 months thyroid FNAs reported as Thy 3F (neoplasm possible/suggestive of follicular neoplasm), Thy4 (suspicious of malignancy) and Thy 5 (malignant) were tested for BRAF V600 mutation and managed as malignant if mutations were present. RESULTS: Of 207 FNAs from 176 patients, 5 were Thy 5, 19 Thy 4, 36 Thy 3f, 13 Thy 3a, 84 Thy 2 and 50 Thy 1. 11 Thy 3f, 15 Thy 4 and 3 Thy 5 FNAs were tested for BRAF V600 mutation. 0 Thy 3F cases, 6 Thy 4 and 1 Thy 5 (24% of the total tested) showed evidence of mutation. Four patients with BRAF V600 mutation underwent surgery to remove all thyroid tissue, two patients received a lobectomy and one patient is awaiting thyroidectomy. All patients with BRAF V600 mutation were found to have malignancy on final histology, with a diagnostic sensitivity for malignancy excluding coincidental microcarcinoma of 43% and specificity of 100%. CONCLUSIONS: BRAF V600 mutational analysis can enable single-stage total thyroidectomy for carcinoma if gene mutation is present in preoperative FNA. BRAF V600 co-testing may reduce the need for completion thyroidectomy with implied cost savings and lower patient morbidity associated with completion thyroidectomy when the cytology is inconclusive but where BRAF V600 mutation is identified in preoperative thyroid FNA.

Activity of EGFR, mTOR and PI3K inhibitors in an isogenic breast cell line model.

BACKGROUND: The epidermal growth factor receptor family is expressed in breast cancer, and agents targeting this pathway have single agent effects (e.g. traztuzumab). Development of resistance may be due to the presence of alternative pathways, particularly activation of the PI3K/Akt/MTOR pathway. We have therefore examined the effect of inhibitors of this pathway (ZSTK474 and sirolimus) in combination with the epidermal growth factor (EGFR) inhibitors erlotinib and gefitinib in breast MCF10a isogenic cell lines with EGFR, BRAF, AKT, and PI3K mutations. RESULTS: PI3K mutation conferred increased activity of EGFR inhibitors against MCF10a cells in comparison with the parental cell line and other mutations studied. Combination of EGFR inhibitors with either the PI3K inhibitor ZSTK474 or the MTOR inhibitor sirolimus showed increased activity. CONCLUSIONS: These results are encouraging for the use of combinations targeting the PI3K and EGFR pathway simultaneously.

Isolation and culture of colon cancer cells and cell lines.

The preparation of cells from heavily contaminated tissue is challenging. It is usually best to avoid such specimens if possible, but for the study of colorectal and some other tumours, it is inevitable that this must be overcome. The best methods seem to use a combination of (1) debridement of necrotic or infected areas of the tumour, (2) enzymatic dissociation in the presence of antibiotics, (3) density centrifugation to remove debris, and (4) extensive washing of the tumour-derived cells prior to their use in cell culture methods. It is possible to obtain cells from up to 80-90% tumours with careful technique and cooperation from the clinical team.

Cell sensitivity assays: the ATP-based tumor chemosensitivity assay.

The ATP-based tumor chemosensitivity assay (ATP-TCA) is a standardised system which can be adapted to a variety of uses with both cell lines and primary cell cultures. It has a strong track record in drug development, mechanistic studies of chemoresistance and as an aid to clinical decision-making. The method starts with the extraction of cells in suspension from continuous cell culture (for cell lines), malignant effusions or biopsy material. Enzymatic digestion is used to obtain cells from tumor tissue. The assay uses a serum-free medium and polypropylene plates to prevent the growth of non-neoplastic cells over a 6-day incubation period followed by detergent-based extraction of cellular ATP for measurement by luciferin-luciferase assay in a luminometer. The assay results are usually shown as percentage inhibition at each concentration tested, and can be used with suitable software to examine synergy between different anticancer agents.

Measuring gene expression from cell cultures by quantitative reverse-transcriptase polymerase chain reaction.

Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) offers a robust method for the measurement of RNA levels for any gene within cells harvested at any point before or during cell culture. The key elements of RNA extraction followed by a two-step qRT-PCR method (reverse transcription and PCR) are described, followed by a brief section on analysis of the results. There are a number of excellent kits available commercially for much of this work, but it is essential to ensure that the quality and quantity of cDNA produced is adequate for the standard PCR or array to be used.

Efficacy of anti-cancer agents in cell lines versus human primary tumour tissue.

The discovery of anti-cancer drugs has become dependent on cell lines, which are used to screen potential compounds for activity as well as to explore cancer biology. Cell lines produce rapid results, but their relevance to patient outcomes is questionable as they undergo selection over many passages to a point where they are no longer representative of their originating tumour. This has led to the increasing use of primary cell cultures, primary tumour cell explants, early passage cell lines, and xenografts to improve the accuracy of results during drug development. Over the last few years, there has been an increasing interest in these methods and they are now firmly established, with a plethora of different techniques available. For instance, explant and three-dimensional models allow cell:cell interactions to be examined in live cells, and endpoints can include the measurement of gene expression and image analysis. In the future, anti-cancer drug development is likely to use a combination of molecular, cell line, primary or early passage cell culture, and xenograft methods for lead optimisation before clinical trials are contemplated.

The molecular basis of the chemosensitivity of metastatic cutaneous melanoma to chemotherapy.

BACKGROUND: Chemotherapy benefits relatively few patients with cutaneous melanoma. The assessment of tumour chemosensitivity by the ATP-based tumour chemosensitivity assay (ATP-TCA) has shown strong correlation with outcome in cutaneous melanoma, but requires fresh tissue and dedicated laboratory facilities. AIM: To examine whether the results of the ATP-TCA correlate with the expression of genes known to be involved in resistance to chemotherapy, based on the hypothesis that the molecular basis of chemosensitivity lies within known drug resistance mechanisms. METHOD: The chemosensitivity of 47 cutaneous melanomas was assessed using the ATP-TCA and correlated with quantitative expression of 93 resistance genes measured by quantitative reverse transcriptase PCR (qRT-PCR) in a Taqman Array after extraction of total RNA from formalin-fixed paraffin-embedded tissue. RESULTS: Drugs susceptible to particular resistance mechanisms showed good correlation with genes linked to these mechanisms using signatures of up to 17 genes. Comparison of these signatures for DTIC, treosulfan and cisplatin showed several genes in common. HSP70, at least one human epidermal growth factor receptor, genes involved in apoptosis (IAP2, PTEN) and DNA repair (ERCC1, XPA, XRCC1, XRCC6) were present for these agents, as well as genes involved in the regulation of proliferation (Ki67, p21, p27). The combinations tested included genes represented in the single agent signatures. CONCLUSIONS: These data suggest that melanoma chemosensitivity is influenced by known resistance mechanisms, including susceptibility to apoptosis. Use of a candidate gene approach may increase understanding of the mechanisms underlying chemosensitivity to drugs active against melanoma and provide signatures with predictive value.