The Localisation and Micro-mapping of Trace Elements in Breast Tumours

An active area of research for over 30 years has been the study of concentrations of trace elements in relation to breast disease in order to help understand the disease process. Studies by our group have shown statistically significant changes in levels of Cu, Fe, Zn and K in breast tissue, these changes being associated with cancer (1),(2). These studies were carried out using synchrotron radiation to excite an XRF response from elements of interest and utilising calibration samples for the quantification of the elemental concentrations. There are several reasons for investigating elemental concentrations in cancers depending on the roles the elements play. Three trace elements of interest in this work were iron, copper and zinc. Copper and zinc are known to act as catalysts for antioxidant enzymes (superoxide dismutase 1, SOD). These enzymes have a role to play in the defense against disease. However, copper can also act as a catalyst for the production of hydroxyl radicals that are linked to tissue destruction and has an important role in angiogenesis. Iron is necessary for the growth of cancer and transporters for its uptake are often upregulated in cancer. Zinc is a co-factor for a group of enzymes that protects tumours from acidosis (carbonic anhydrases), which are also potential therapy targets. Our previous studies have shown that typical concentrations of these elements in breast tumours are approximately 1pmm, 7ppm and 15ppm for cu, zn and fe respectively. We have continued with this work by examining the spatial distribution of a number of trace elements within breast cancer. In this report we show a method of using a synchrotron based micro probe to determine the localisation of trace elements in breast tumours at approximately cellular level. Measurements were made on individual samples approximately 10µm thick (mounted on 4µm thick ultralene film) of a formalin fixed paraffin embedded tissue microarray of human primary invasive breast carcinomas. The samples were obtained from the Cancer Research UK Tumour Pathology Group, University of Oxford, Each section on the array was a section of 1.0mm diameter. The data was collected using the synchrotron X-Ray Fluorescence Microprobe (SY-XRF) at Hasylab, beamline L (HYMO). The white beam is monochromatised by a double multilayer monochromator with a bandpass ∆E/E ~ 2%. The beam is focussed by a polycapillary halflens (X- Ray Optical Systems, Inc.) which provides a beam of 10-25 µm diameter, depending on energy. The excitation energy was set to 12 keV. By using this energy, data is collected for Cl, Ca, K, P, S, Ti, Fe, Cu and Zn. Recent improvements in spectrometer sensitivity and detector count rate capability have improved the limits of detection for XRF. By reducing the measurement time per point, larger areas of sample can be scanned without loss of spatial resolution which is important as physiological relevant areas are often several square millimetres. At 20µm spatial resolution this results in many thousands of points per scan, and in order to keep such scans to an acceptable time (several hours), a continuous scanning mode for collecting data has been developed. In this mode the sample is moved continuously across the beam, not stepwise as in conventional mode. The multi channel analyser (MCA) opens for a pre defined time interval and subsequently the data is read out and written to memory. In addition the signals needed for normalisation e.g. ionisation chamber signals, detector deadtime, ring current, are saved. The sampling time for each data point is 5 seconds which will allow samples to be scanned at the rate of approximately 6 hours each. The samples are supported on an XYZ table with a reproducible positioning of about 0.5 µm. The fluorescence signal is recorded using a Peltier cooled energy dispersive Si drift detector (Radiant