Human breast cancer in vitro: matching histo-pathology with small-angle x-ray scattering and diffraction enhanced x-ray imaging

Twenty-eight human breast tumour specimens were studied with small-angle x-ray scattering (SAXS), and 10 of those were imaged by the diffraction enhanced x-ray imaging (DEI) technique. The sample diameter was 20 mm and the thickness 1 mm. Two examples of ductal carcinoma are illustrated by histology images, DEI, and maps of the collagen d-spacing and scattered intensity in the Porod regime, which characterize the SAXS patterns from collagen-rich regions of the samples. Histo-pathology reveals the cancer-invaded regions, and the maps of the SAXS parameters show that in these regions the scattering signal differs significantly from scattering by the surrounding tissue, indicating a degradation of the collagen structure in the invaded regions. The DEI images show the borders between collagen and adipose tissue and provide a co-ordinate system for tissue mapping by SAXS. In addition, degradation of the collagen structure in an invaded region is revealed by fading contrast of the DEI refraction image. The 28 samples include fresh, defrosted tissue and formalin-fixed tissue. The d-values with their standard deviations are given. In the fresh samples there is a systematic 0.76% increase of the d-value in the invaded regions, averaged over 11 samples. Only intra-sample comparisons are made for the formalin-fixed samples, and with a long fixation time, the difference in the d-value stabilizes at about 0.7%. The correspondence between the DEI images, the SAXS maps and the histo-pathology suggests that definitive information on tumour growth and malignancy is obtained by combining these x-ray methods.     

A preliminary study of breast cancer diagnosis using laboratory based small angle x-ray scattering

Breast tissue collected from tumour samples and normal tissue from bi-lateral mastectomy procedures were examined using small angle x-ray scattering. Previous work has indicated that breast tissue disease diagnosis could be performed using small angle x-ray scattering (SAXS) from a synchrotron radiation source. The technique would be more useful to health services if it could be made to work using a conventional x-ray source. Consistent and reliable differences in x-ray scatter distributions were observed between samples from normal and tumour tissue samples using the laboratory based 'SAXSess' system. Albeit from a small number of samples, a sensitivity of 100% was obtained. This result encourages us to pursue the implementation of SAXS as a laboratory based diagnosis technique.     

Monte Carlo studies of x-ray scattering in transmission diagnostic radiology

Monte Carlo methods have been used to simulate the scattering of x rays in polystyrene and water phantoms. In particular, the ratio of the scattered to total x-ray fluence (scatter fraction) has been calculated for monoenergetic x-ray beams in the energy region relevant to diagnostic radiology and nuclear medicine (30-660 keV). Simulations have been made for representative values of the pertinent geometrical factors; phantom thickness from 5 to 21 cm, x-ray beam diameters of 10 and 25 cm, and scatterer-to-image-plane separations from 0 to 20 cm. As a function of x-ray energy, the scatter fraction was found to vary slowly between 30 and 100 keV, and to decrease between 100 and 660 keV. The present results were generated with a special transport code which included the effects of special geometries and the response of the x-ray detector. With the inclusion of these effects, the results resolved inconsistencies and showed good agreement with previous measured and calculated data.     

Small-angle x-ray scattering studies of human breast tissue samples

Small-angle x-ray scattering (SAXS) patterns are recorded from thin breast tissue samples containing healthy and cancerous regions. The SAXS patterns are compared with histo-pathological observations. The information available from SAXS is reviewed, and a model for scattering from collagen is presented. Scattering patterns of collagen at regions far from the tumours are essentially different from those at tumours. The axial period of collagen fibrils is 65.0 +/- 0.1 nm in healthy regions, and 0.3 nm larger in cancer-invaded regions. The average intensity of scattering from cancerous regions is an order of magnitude higher than the intensity from healthy regions. This is interpreted to arise from an increase of the specific surface area of the scatterers, which is due to a disruption of the molecular and supra-molecular structures in cancerous regions and invasion of new types of cells. The differences of the SAXS patterns are large and distinctive enough to suggest that these phenomena may be utilized in mammography.     

Monte carlo simulation of the compton scattering technique applied to characterize diagnostic x-ray spectra

The quality control of x-ray tubes for medical radiodiagnostic services is very important for such devices. Therefore, the development of new procedures to characterize the x-ray primary beam is highly interesting in order to obtain an accurate assessment of the actual photon spectrum. The Compton scattering technique is very useful to determine x-ray spectra (in the 10-150 kVp range), avoiding a pile-up effect in the detector since a large room is not usually available to apply other techniques. In this work, this process has been simulated using a Monte Carlo code, MCNP 4C. Some geometrical models have been developed and different techniques have been studied in order to improve statistics and accuracy in the acquisition of Pulse Height Distribution (PHD). The effect of both the collimation of the primary beam and the scattering angle of the spectrometer has been analyzed. Results obtained using simulation models have been compared with experimental measurements.     

Structural characterization of the human cerebral myelin sheath by small angle x-ray scattering

Myelin is a multi-lamellar membrane surrounding neuronal axons and increasing their conduction velocity. When investigated by small-angle x-ray scattering (SAXS), the lamellar quasi-periodical arrangement of the myelin sheath gives rise to distinct peaks, which allow the determination of its molecular organization and the dimensions of its substructures. In this study we report on the myelin sheath structural determination carried out on a set of human brain tissue samples coming from surgical biopsies of two patients: a man around 60 and a woman nearly 90 years old. The samples were extracted either from white or grey cerebral matter and did not undergo any manipulation or chemical-physical treatment, which could possibly have altered their structure, except dipping them into a formalin solution for their conservation. Analysis of the scattered intensity from white matter of intact human cerebral tissue allowed the evaluation not only of the myelin sheath periodicity but also of its electronic charge density profile. In particular, the thicknesses of the cytoplasm and extracellular regions were established, as well as those of the hydrophilic polar heads and hydrophobic tails of the lipid bilayer. SAXS patterns were measured at several locations on each sample in order to establish the statistical variations of the structural parameters within a single sample and among different samples. This work demonstrates that a detailed structural analysis of the myelin sheath can also be carried out in randomly oriented samples of intact human white matter, which is of importance for studying the aetiology and evolution of the central nervous system pathologies inducing myelin degeneration.     

Classification of breast tissue using a laboratory system for small-angle x-ray scattering (SAXS)

Structural changes in breast tissue at the nanometre scale have been shown to differentiate between tissue types using synchrotron SAXS techniques. Classification of breast tissues using information acquired from a laboratory SAXS camera source could possibly provide a means of adopting SAXS as a viable diagnostic procedure. Tissue samples were obtained from surgical waste from 66 patients and structural components of the tissues were examined between q = 0.25 and 2.3 nm(-1). Principal component analysis showed that the amplitude of the fifth-order axial Bragg peak, the magnitude of the integrated intensity and the full-width at half-maximum of the fat peak were significantly different between tissue types. A discriminant analysis showed that excellent classification can be achieved; however, only 30% of the tissue samples provided the 16 variables required for classification. This suggests that the presence of disease is represented by a combination of factors, rather than one specific trait. A closer examination of the amorphous scattering intensity showed not only a trend of increased scattering intensity with disease severity, but also a corresponding decrease in the size of the scatterers contributing to this intensity.     

Apparent exchange rate for breast cancer characterization

Although diffusion MRI has shown promise for the characterization of breast cancer, it has low specificity to malignant subtypes. Higher specificity might be achieved if the effects of cell morphology and molecular exchange across cell membranes could be disentangled. The quantification of exchange might thus allow the differentiation of different types of breast cancer cells. Based on differences in diffusion rates between the intra‐ and extracellular compartments, filter exchange spectroscopy/imaging (FEXSY/FEXI) provides non‐invasive quantification of the apparent exchange rate (AXR) of water between the two compartments. To test the feasibility of FEXSY for the differentiation of different breast cancer cells, we performed experiments on several breast epithelial cell lines in vitro. Furthermore, we performed the first in vivo FEXI measurement of water exchange in human breast. In cell suspensions, pulsed gradient spin‐echo experiments with large b values and variable pulse duration allow the characterization of the intracellular compartment, whereas FEXSY provides a quantification of AXR. These experiments are very sensitive to the physiological state of cells and can be used to establish reliable protocols for the culture and harvesting of cells. Our results suggest that different breast cancer subtypes can be distinguished on the basis of their AXR values in cell suspensions. Time‐resolved measurements allow the monitoring of the physiological state of cells in suspensions over the time‐scale of hours, and reveal an abrupt disintegration of the intracellular compartment. In vivo, exchange can be detected in a tumor, whereas, in normal tissue, the exchange rate is outside the range experimentally accessible for FEXI. At present, low signal‐to‐noise ratio and limited scan time allows the quantification of AXR only in a region of interest of relatively large tumors. © 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.     

Prognostic parameters in breast cancer

Carcinoma of the breast is the most common cancer in Australian women. Current methods of treatment and refinements in therapeutic regimens are based on our understanding of the biological behaviour of the disease. Several prognostic parameters have been identified which predict survival and allow the selection of patients who may benefit from adjuvant therapies. The more important of these parameters include tumour size, histologic type, histologic grade, axillary lymph node status, estrogen receptor status and tumour growth fraction. Diagnostic pathologists who are responsible for the evaluation of these parameters should be cognizant of their relative prognostic values and also of other factors such as cellular antigens, lectin binding and oncogenes which may have potential roles in predicting survival and therapeutic responses. This review provides an update of prognostic parameters which are assessed through examination of the excised specimen.     

Fourier transform infrared imaging and small angle x-ray scattering as a combined biomolecular approach to diagnosis of breast cancer

Fourier transform infrared (FTIR) microspectroscopic imaging and small angle x-ray scattering (SAXS) were combined to investigate the supramolecular structure of collagen from 27 tissue sections from patients undergoing mastectomy, excisional biopsy, or mammoplasty. Both techniques were correlated by matching the scattering profile from the SAXS data with the integrated area of the infrared collagen region (1300-1180 cm(-1)). The FTIR spectral profiles and multivariate analysis of various tissue components showed consistent differences between all major tissue components, particularly between cancer and normal tissue cells. Analysis of the SAXS data revealed broad differences between cancer and normal tissue, but were inconclusive due to the small sample size. Parameters were extracted from each technique in relation to their characterization of collagen to reveal a good correlation between the two techniques, which diagnostically parallels with gold-standard Hematoxylin and Eosin (H&E) stained sections. The results show that the integrated area of collagen region in the FTIR spectrum for cancerous samples is greater than that for noncancerous samples indicating collagen disorder. This supports the notion that collagen is structurally disrupted in cancer tissue consistent with the interpretation of the SAXS data. Overall, both these techniques successfully distinguished cancer from normal breast tissue. Integration of these two techniques was able to better segregate cancer as well as provide a more complete understanding of the differences in collagen on all structural levels during breast cancer development.     

The influence of diagnostic radiography on the incidence of breast cancer and leukemia

Little accurate information is available on the influence of diagnostic radiography on the incidence of cancer in the general population, largely because of a lack of knowledge of the numbers and the age distributions of patients undergoing radiographic examinations. We used such information from a closed population in Maine that was followed for a year, along with data from the literature on the absorbed dose from typical examinations and a mathematical model linking dose to the incidence rate of cancer, to estimate the numbers of cases of radiologically induced cancer of the bone marrow and breast. Our data indicate that about 1 percent of all cases of leukemia and less than 1 percent of all cases of breast cancer result from diagnostic radiography. In addition, such tumors generally occur late in life; the incidence of radiation-induced leukemia was highest at 69 years, and the incidence of radiation-induced breast cancer was highest at 76 years. Thus, diagnostic radiography has only a small influence on the occurrence of leukemia and breast cancer.     

Evaluation of x-ray diffraction enhanced imaging in the diagnosis of breast cancer

The significance of the x-ray diffraction enhanced imaging (DEI) technique in the diagnosis of breast cancer and its feasibility in clinical medical imaging are evaluated. Different massive specimens including normal breast tissues, benign breast tumour tissues and malignant breast tumour tissues are imaged with the DEI method. The images are recorded respectively by CCD or x-ray film at different positions of the rocking curve and processed with a pixel-by-pixel algorithm. The characteristics of the DEI images about the normal and diseased tissues are compared. The rocking curves of a double-crystal diffractometer with various tissues are also studied. The differences in DEI images and their rocking curves are evaluated for early diagnosis of breast cancers.     

The use of cadmium telluride detectors for the qualitative analysis of diagnostic x-ray spectra

A method is introduced for the evaluation of x-ray spectra from x-ray machines operating in the range 50-100 kVp using a cadmium telluride (CdTe) detector with low detection efficiency. The pulse height distribution obtained with this kind of detector does not represent the true photon spectra owing to the presence of K-escape, Compton scattering, etc.; these effects were evaluated using a Monte Carlo method. A stripping procedure is described for implementation on a Univac 1100/82 computer. The validity of our method was finally tested by comparison with experimental results obtained with a Ge detector and with data from the literature; the results are in good agreement with published data.