A study of FT-IR spectroscopy for the identification and classifcation of haematological malignancies

The aim of the work presented in this thesis was to explore the use of FT-IR spectroscopy, as a complementary clinical tool for haematological laboratory analysis. FT-IR spectra were measured from air-dried and frozen cell lines derived from lymphoma, lymphoid, myeloid leukaemia and normal and chronic lymphocytic leukaemia blood samples. Multivariate statistical analysis was used to extract important spectral information with the greatest discriminative power. Principal component fed linear discriminant spectral models have been tested with leave one out cross validation procedures. A preliminary unfiltered classification model using 50 frozen and air-dried samples correctly classified 54% of 18556 spectra. The performance improved with the three cell line group datasets, with 71% of 19903 spectra correctly classified. Furthermore, the use of the frozen spectra improved the performance of the three cell line group classification model considerably. Findings showed that 73.3% of 9920 spectra were correctly classified in the frozen datasets, whereas in the air-dried only 41.5% of 9983 spectra are correctly classified. Optimisation of the spectral models by selection of principal components, application of Savitsky-Golay filters and selecting spectra using standard deviation and absorption filter tool was investigated. Using the first 25 significant PCs, a 0 th derivative Savitsky-Golay filter and the absorbance filter tool on the frozen five cell line spectral dataset were shown to be the optimal parameters for constructing a classification model. When tested with leave one batch out cross validation 90% of the spectra were correctly classified for the five cell line model. Blood component classification models tested with leave one batch out cross validation performed well. The whole blood model correctly classified 70% of 1736 spectra, measured on 22 samples. The plasma model correctly classified 80.6% of 331 spectra and the buffy coat model correctly classified 99.5% of 1438 spectra. This demonstrated that the buffy coat (containing white blood cells) holds the key biochemical information for discrimination between the pathology of the blood samples. Partial least squares analysis has been demonstrated as a method to support whole blood count tests for real time prediction of cellular constituents. These findings demonstrate the potential of FT- IR spectroscopy as a clinical tool although more work is needed if it is to be applied in clinical practice.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Probing the action of a novel anti-leukaemic drug therapy at the single cell level using modern vibrational spectroscopy techniques

Acute myeloid leukaemia (AML) is a life threatening cancer for which there is an urgent clinical need for novel therapeutic approaches. A redeployed drug combination of bezafibrate and medroxyprogesterone acetate (BaP) has shown anti-leukaemic activity in vitro and in vivo. Elucidation of the BaP mechanism of action is required in order to understand how to maximise the clinical benefit. Attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, Synchrotron radiation FTIR (S-FTIR) and Raman microspectroscopy are powerful complementary techniques which were employed to probe the biochemical composition of two AML cell lines in the presence and absence of BaP. Analysis was performed on single living cells along with dehydrated and fixed cells to provide a large and detailed data set. A consideration of the main spectral differences in conjunction with multivariate statistical analysis reveals a significant change to the cellular lipid composition with drug treatment; furthermore, this response is not caused by cell apoptosis. No change to the DNA of either cell line was observed suggesting this combination therapy primarily targets lipid biosynthesis or effects bioactive lipids that activate specific signalling pathways

Determinants of insulin concentrations in healthy 1-week-old babies in the community: applications of a bloodspot assay

BACKGROUND: Epidemiological research into insulin secretion and insulin action would be helped by improved ability to measure insulin concentrations in large groups of healthy babies in the neonatal period. Such research is often restricted by the invasive nature of blood sampling. AIMS: We assessed the use of an assay that can measure insulin from bloodspots taken during routine Guthrie testing 7 days after delivery. STUDY DESIGN AND SUBJECTS: Insulin and glucose were measured in 366 seven-day-old infants from heel-prick bloodspots. Time since last feed and type of feed were recorded. RESULTS: Bloodspot insulin concentrations in normal 7-day-old infants were much lower (median (IQR): 15.4 pmol/l (<10-28.5)) than fasting insulin concentrations in adult males (44.3 pmol/l (30.6-72.6)) (p<0.001). Insulin and glucose concentrations were correlated (r=0.33, p<0.001). Insulin and glucose fell significantly with time from feed. Bottle fed infants had higher insulin concentrations but similar glucose concentrations compared to breast fed infants. Detailed analysis to account for confounders was limited due to the skewed distribution of time since feed and the lower limit of the assay leading to non-continuous insulin data. CONCLUSIONS: In the largest study of normal 7-day-old children to date we have shown insulin concentrations are low compared to adults and vary with glucose, time from feed, and type of feed. This validates the use of the bloodspot insulin assay as a potential research tool for large-scale epidemiological studies. However, careful study design would be required in future use to reduce the variation caused by timing and type of feeding and the problem of one third of values being at or below the lower limit of this assay.Not heldPublished version, accepted version (12 month embargo

NEO-EXCEL phase III neoadjuvant trial of pre-operative exemestane or letrozole plus /- celecoxib in the treatment of ER positive postmenopausal early breast cancer

COX2 has been implicated in breast tumorigenesis, tumour proliferation & invasion. The role of COX2 in carcinogenesis is thought to be related to its abilities to increase production of prostaglandins, convert pro-carcinogens to carcinogens, inhibit apoptosis, promote angiogenesis, modulate inflammation & immune function & increase tumour cell invasiveness. COX2 inhibition may synergise with aromatase inhibition in controlling endocrine responsive breast cancer. The COX2 product prostaglandin E2 (PGE2) & cytokines such as interleukin-6 (IL6) can up regulate aromatase expression suggesting that aromatase inhibition may be more effective in combination with a COX2 inhibitor. There may be additional COX2 mediated anticancer activity. The hypothesis addressed is that activity of aromatase inhibitors(AI) as neoadjuvant endocrine therapy for early breast cancer may be enhanced by the addition of a COX2 inhibitor. TRIAL OBJECTIVES To determine whether the activity of AIs as neo-adjuvant endocrine therapy for ER positive breast cancer in postmenopausal women may be enhanced by the addition of the selective COX2 inhibitor celecoxib. TRIAL DESIGN Prospective phase III multicentre randomised trial. Patients were randomised to receive 16 weeks of exemestane 25 mg daily or letrozole 2.5 mg daily (open label) and celecoxib 400 mg twice daily or matched placebo (double blinded). Translational research tumour samples were collected before, during & after therapy. KEY ELIGIBILITY CRITERIA Post menopausal, ER positive, invasive cancer, 2cms or greater with calipers & visible on USS. PRIMARY OUTCOME MEASURE Objective clinical response to neoadjuvant treatment by RECIST criteria. RESULTS Primary Outcome; Response to treatment has been calculated for 266 patients (Table 1). Response rate was 73% in the celecoxib arm & 55% in the placebo arm (p=0.0022). The response rates 4 arm comparison are shown in Table 2. After adjustment for AI effect the significant difference in response rates remained (p=0.0023); the difference in response rates was greater in the exemestane treated group (29%) compared to the letrozole group (7%) although heterogeneity between AI arms was statistically non-significant (p=0.06). Secondary outcome; There was an USS response rate of 42% v 37% for celecoxib & placebo arms respectively (p=0.2513) CONCLUSION The addition of the COX2 inhibitor celecoxib to an AI significantly & substantially increased the clinical response from 55% to 73%. Effect on tumour size assessed with USS is less marked with a non-significant increase in responses from 37% to 42%. This work was supported by CRUK: CRUK/06/005 and Pfizer. Citation Format: Rea D, Francis A, Poole C, Brookes C, Stein R, Bartlett J, Dunn J, Canney P, Sutton R, Daoud R, Hallissey M, Achuthan R, Grant M, Babrah J, Smith S, Fraser J, Desai A, Al Dubaisi M, Patel A, Bristol J, Chandrasekharan S, Prest C, Jewkes A. NEO-EXCEL phase III neoadjuvant trial of pre-operative exemestane or letrozole +/- celecoxib in the treatment of ER positive postmenopausal early breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD2-02