International Conference on
Technology in Cancer Research and
Treatment in the New Millennium
Adenine Press
2066 Central Avenue
Schenectady, NY 12304
Phone: 518 456-0784
Fax: 518 452-4955
stone@adeninepress.com
http://www.cancerwatch.org
2066 Central Avenue
Schenectady, NY 12304
Phone: 518 456-0784
Fax: 518 452-4955
stone@adeninepress.com
http://www.cancerwatch.org
Richard Mendelsohn, Ph. D.
Department of Chemistry, Rutgers University
73 Warren Street, Newark, NJ, 07102
The IR spectrum of a molecule provides a unique fingerprint that historically has been used for two primary purposes. The spectrum qualitatively identifies and quantitatively determines concentrations of components in mixtures. Equally important for the extension of this technology to the study of tissues is the observation that vibrational frequencies are sensitive to subtle changes in molecular conformations and environments.
The application of traditional IR microscopy to study tissues is limited by the following consideration: Although the technology provides excellent quality spectra, for an IR-based method to provide biomedically significant conclusions thousands of locations in many samples must be examined to compensate for statistical variations in tissue properties within and among individuals. Data collection, even from a single sample, becomes a tedious process with traditional point-by-point sampling techniques.
The efficient , routine use of IR microscopy for biomedical applications has become feasible recently thanks to the availability of focal plane array detectors in the mid-IR spectral region. The declassification by the military of these devices has resulted in the development of a novel technology termed infrared microscopic imaging. In this experiment, the single point detector on standard IR instruments is replaced by a detector consisting of a 64 x 64 array of elements imaged to the focal plane of an IR microscope. During each scan (~4 minutes), a complete IR spectrum is acquired from each detector element. Thus 4096 mid-IR spectra are acquired in a time only slightly longer than previously needed to acquire a spectrum from a single element detector. Data are generated ~1000x faster compared with conventional FT-IR microscopes and without much loss in spectral quality. The variation of any IR spectral parameter within the set of 4096 spectra that characterize a given spatial region may be quickly computed and presented as an image.
A joint project was established in 1989 between Rutgers University and the Hospital for Special Surgery in New York to apply IR microscopy to study normal and pathological states of mineralized tissue. As an example of the spectral information available from bone, a series of IR spectra taken at 20 µm intervals across an osteon (human iliac crest biopsy) are shown in Figure 1. Spectra acquired from the osteonal center show strong contributions from the PMMA (in which the section is embedded) as noted from the C=O stretching mode near 1720 cm-1. For spectra acquired at increasing radial distances from the center, the increasing presence of mineral deduced from the appearance of the hydroxyapatite <1,<3 contour (950-1200 cm-1 ), and protein, monitored from the appearance of the collagen Amide I contour (1610-1680 cm-1 ), are evident.
Typical information from IR microscopic imaging is given in Figure 2. Images are presented of the spatial distribution of the intensity of the HA peak ( Figure 2A) and collagen amide I mode (Figure 2B) in the vicinity of a single osteon. The 4096 intensities ( one per spectrum) of each parameter are color coded (blue<green<yellow<orange). Thus the spatial distribution of the two main chemical constituents of bone may be easily monitored.
In addition to quantitative determination of the tissue components, we have taken advantage of the sensitivity of FT-IR to subtle changes in the structure of bone to examine spatial alterations in hydroxyapatite structure. An index of mineral crystallinity/maturity has been developed. The image of this index presented in Figure 2C shows that the crystallinity/ maturation of the mineral increases from the center of the osteon to the periphery. Similarly, we have developed an index of the extent of collagen crosslinking in bone. An image of this parameter is shown in Figure 2D. Such a structural characterization of the organic phase of bone is not readily available from other physical approaches.
As time permits, the application of IR microscopic imaging to normal and pathological states of bone and cartilage will be described. These include determinations of molecular structure changes in osteoporotic bone, alterations in the spatial distribution of and orientation of collagen in osteoarthritic cartilage, changes in the mineral structure of bone in TGF-beta knockout mice, and the effect of estrogen therapy on fracture healing in a rat model.
IR imaging provides two major advantages for the study of mineralizing tissue:
1. The full power of IR spectroscopy for determination of molecular structure and interactions is obtained with a spatial resolution approaching the diffraction limit.
2. The speed of data collection permits examination of a statistically robust number of samples in normal and pathological states. Both inter- and intra-sample comparisons may be made.
A Novel Spectroscopic Imaging and Chemometric Approach for Automatically Partitioning and Classifying Biological Tissue
E. Neil Lewis, PhD(1), Linda H. Kidder, PhD(1), Abigail S. Haka, BS(2), Steven Vogel, MS(3) and Steven Lowry, PhD(4)
Spectral Dimensions, Inc.(1), 3403 Olandwood Ct. Suite 102, Olney MD 20832, USA
Massachusetts Institute of Technology(2), 77 Massachusetts Avenue, George Harrison Spectroscopy Laboratory Rm. 6-014, Cambridge, MA 02139-4307
Thermo Spectra-Tech(3), 2 Research Dr., Shelton CT 06484
Thermo Nicolet(4), 5225 Verona Rd., Madison, WI 53711
There has recently been significant interest in developing optical spectroscopy as a tool to augment the current protocols for cancer diagnosis1-5. Vibrational spectroscopy is particularly appealing since it has the capability to probe changes in the biochemical composition of tissue that accompany disease progression. The incorporation of this type of quantitative parameter provides an ideal adjunct to conventional cancer diagnosis methodologies which rely heavily on the visual examination of stained tissue sections. Numerous vibrational spectroscopic studies have been performed in an attempt to elucidate differences between normal, benign, and malignant tissues. Examination of cell lines, homogenized tissue, as well as microscopy and bulk measurements of tissue biopsies have provided notable insight into the biochemical changes associated with malignancy. Although these approaches are well accepted within the spectroscopic community, applications in the medical sciences have been limited due in large part to a lack of cross discipline understanding. The substantial language and terminology barriers associated with such specialized fields as medicine and spectroscopy lead to inefficient communication amongst communities and hinder the progress of such multidisciplinary lines of study. Overcoming these barriers will not only facilitate superior research but may also result in improved patient care through modernized diagnostic methods. In an effort to surmount these barriers, we present a method based on infrared spectroscopic imaging microscopy which may have great benefits as an adjunct to current cancer diagnostic methodologies. The incorporation of an imaging approach is significant as it allows for the rapid pathological assessment of tissue biopsies, through image visualization, while simultaneously incorporating quantitative parameters that reflect changes in the molecular composition of the tissue. This hybrid approach is particularly attractive from a clinical standpoint since it embraces and enhances standard pathological protocols and does not attempt to supplant them. It can be easily integrated into the histopathological diagnosis routine, in which tissue is fixed, stained, visually examined and evaluated for abnormalities. These visible images of the stained tissue biopsy can be simultaneously compared and correlated with the corresponding IR images either by the pathologist or by other automated computational means.
To this end we have developed and applied a novel Fourier transform infrared (FTIR) spectroscopic imaging system6,7 which couples a mercury cadmium telluride (MCT) focal plane array detector (FPA) and a Michelson step-scan interferometer, and used it to investigate various types of human tissue. The MCT FPA used consists of 64 x 64 pixels, each 61 mm2, and has a spectral range of 2-10.5 microns. Imaging data set are typically collected at 16 cm-1 resolution requiring only a few minutes of data collection time. The resulting image cubes consist of 512 image planes and a total of 4096 infrared spectra each. We will demonstrate how we can utilize this novel infrared imaging microscope for highlighting biochemical variance within the epithelium of cancerous and pre cancerous conditions. We will also present images, generated by a variety of multivariate clustering algorithms, which demonstrate the successful automated partitioning of distinct tissue type domains while the analysis of the differences in their spectra provide insight into the chemical makeup of each tissue type. The implications of the use of this infrared imaging approach as an automated technique for the separation and examination of tissue domains in pathology will be discussed.
1. L. Chiriboga, P. Xie, H. Yee, D. Zarou, D. Zakim, and M. Diem, "Infrared spectroscopy of human tissue: IV. Detection of dysplastic and neoplastic changes of human cervical tissue via infrared microscopy", Cell. Mol. Biol., 44, 219-229, 1998.
2. E. Benedetti, E. Bramanti, F. Papineschi, I. Rossi, and E. Benedetti, "Determination of the Relative amount of Nucleic Acids and Proteins in Leukemic and Normal Lymphocytes by Means of Fourier Transform Infrared Microspectroscopy", Appl. Spectrosc., 51, 792-797, 1997.
3. P.T. Wong, R.K. Wong, T.A. Caputo, T.A. Godwin, B. Rigas, "Infrared spectroscopy of exfoliated human cervical cells: Evidence of extensive structural changes during carcinogenesis", Proc. Natl. Acad. Sci. USA, 88, 10988-10992, 1991.
4. K. Yano, S. Ohoshima, Y. Shimizu, T. Moriguchi, H. Katayama, "Evaluation of glycogen level in human lung carcinoma tissues by an infrared spectroscopic method", Cancer Letters, 110, 29-34, (1996).
5. R. Manoharan, K. Shafer, L. Perelman, J. Wu, K. Chem, G. Deinum, M. Fitzmaurice, J. Myles, J. Crowe, R.R. Dasari, M.S. Feld, "Raman Spectroscopy and Fluorescence Photon Migration for Breast Cancer Diagnosis and Imaging", Photochem. Photobiol., 67, 15-22, 1998.
6. E.N. Lewis, P.J. Treado, R.C. Reeder, G.M. Story, A.E. Dowrey, C. Marcott, I.W. Levin, "Fourier transform spectroscopic imaging using an infrared focal-plane array detector", Anal Chem., 67, 3377-3381 1995.
7. E.N. Lewis, I.W. Levin, P.J. Treado, "Spectroscopic imaging devices employing imaging quality spectral filters", U.S. Patent No. 5,377,003, 1994.
Novel Prodrug Approach for Targeting Radionuclides to Tumors
A.I. Kassis, Ph.D., R.S. Harapanhalli, Ph.D., B.A. Dahman, M.S., N. Ho, Ph.D.,
S.J. Adelstein, M.D., Ph.D., Harvard Medical School
Antibody-directed enzyme–prodrug therapy (ADEPT) is an effective two-step approach which has been shown to deliver potent cytotoxic agents specifically to solid tumors. We have synthesized a water-soluble radioiodinated prodrug (ammonium 2-(2'-phosphoryloxyphenyl)-6-iodo-4-(3H)-quinazolinone) which is converted to a water-insoluble radioiodinated product (2-(2'-hydroxyphenyl)-6-iodo-4-(3H)-quinazolinone) by the action of alkaline phosphatase (ALP). Owing to its negatively-charged prosthetic groups, the radioiodoprodrug (*IPD) is highly hydrophilic and should not be internalized by mammalian cells. *IPD should also be rapidly cleared from the circulation at a rate compatible with its physical characteristics (e.g. low MW, high negative charge). Being a substrate for the pretargeted enzyme, this water-soluble molecule loses its prosthetic group and the resulting radioiodinated compound (*ID) should precipitate out because of its highly water-insoluble nature. The precipitated molecule is thus expected to be "indefinitely trapped" within the targeted solid tumor. When *IPD is radiolabeled with the beta-particle-emitting radionuclide iodine-131, the trapped, precipitated, radioactive molecule will maintain the radionuclide within the targeted tumor, thereby enhancing its residence time and delivering a high radiation dose specifically to the tumor and a low dose to the rest of the body.
Our preliminary results support our expectations and demonstrate that (i) alkaline phosphatase specifically converts IPD to ID both in vitro and in vivo, (ii) *IPD is rapidly cleared from circulation, (iii) *IPD has no affinity for any normal tissues in mice, and (iv), once formed in vivo, *ID remains "indefinitely" within the tissue where it was produced.
The Potential Role of Fourier Transform Infrared Spectroscopy and Imaging in Cancer Diagnosis Incorporating Complex Mathematical Methods
Christian P. Schultz, Ph. D.
BRUKER Optics Inc, 19 Fortune Drive, Billerica, Massachusetts 01821
Technology and application of infrared spectroscopy and imaging are currently going through a phase of rapid development opening up new diagnostic fields in biological sciences and many medical disciplines. Since infrared spectroscopic information of isolated cell material or tissues can be very specific, different cellular structures and cell biochemistry can successfully be identified and classified based on reference material. The application of this new technology can provide biomedical researchers and physicians with new diagnostic tools for the determination of diseases states and possibly with information on the success of therapeutic agents used in disease treatment. Although spectroscopy and hyper-spectral imaging carry the same level of information, imaging will always have the advantage of being easier to comprehend, since spectroscopic information can directly be correlated with structural information as in case of tissue pathology. However, in order to make the transition in to the medical field, non-spectroscopists must be able to perform those new infrared spectroscopy and imaging techniques to transform them in to diagnostic tools.
A necessary step in this process is the design of analytical tools that may automate the spectroscopy and imaging evaluation in biomedical diagnostics (often found in the process control environment). The current situation still requires specialized spectroscopists with an expertise in imaging applications or remote sensing technology. One purpose of this presentation is to demonstrate the potential of mutivariate statistics and artificial neural networks for the development of easier evaluation procedures of chemical information contained in infrared spectra and hyper-spectral images of tissue. Key elements of this approach are feature selection, data reduction, rapid data analysis and the definition of effective diagnostic procedures. In order to achieve this goal many reference spectra have to be collected, classified and converted into reduced data formats for each type of tissue. It is also recommended to analyze multiple samples from many subjects to cover statistical variations between individual subjects and those typical for the disease. Libraries of different tissue types can then be used to rapidly classify and identify tissue based on the extracted information provided by principle component analysis (PCA) and artificial neural networks (ANN). To confirm this approach infrared spectra of unknown samples from randomly picked subjects should also be classified by unsupervised procedures for completion. The success of this technique will depend on the response time, which has to be very fast to allow online diagnosis of tissue in the clinical environment. The aim beyond this task is to avoid the still very slow FPA imaging process for standard applications by replacing it with single micro-spot measurements followed by immediate identification of the sample thereby quickly supporting the assessment of the cytologist or pathologist.
A similar but slightly different situation arises when sampling of tissues is not an option. In cases where only fine needle aspirates or bodyfluids can be obtained, infrared spectroscopy can offer the same power of discrimination as in hyper-spectral imaging of tissue sections. The key for success here is simply the principle that these infrared techniques generally apply. Spectra of reference material of known origin are used for calibrating the classification and identification approach. In the next step pattern recognition techniques can be applied to sort out the different cell structures based on different spectral features caused by the different cell biochemistry of normal and cancer (abnormal) cells. This for example, allows the detection and staging of Chronic Lymphocytic Leukemia (CLL) and of different cancers of the thyroid gland as well as others. The same approach can be utilized to detect drug sensitivity in cancer cells and allows the determination and distinction of apoptotic from necrotic cell decay. Since cells can be harvested by aspiration, a direct comparison with tissue spectra of excised tissue samples would allow the detection of cell abnormalities based on their biochemistry though cytology may still be unaltered. The great potential of the new infrared methods can be seen in the additional information provided that may help a physician to improve diagnosis in critical cases where differential diagnosis is inconclusive.
Choosing a different spectral range may even support minimally invasive procedures in the future of new medical procedures in surgery. Near-Infrared spectroscopy and imaging are already on their way to enter the surgical area due to their unique properties they provide. First infrared fiberscopes have been built and are currently tested in the field with moderate success, since the technology will have to be adapted to the different environment. Again the challenge here is the evaluation of infrared spectral information, which has to be preprocessed and adapted to accommodate the needs of non-spectroscopists. In vitro models already suggest today that infrared assisted fiber optics technology will be able to distinguish disease states in vivo such as the detection vulnerable plaques in blood vessels. Some or many of the features presented will become more and more important in medical disease diagnosis and will hopefully be established within the next 5 years supporting physicians on a completely new level of information.
Fig 1: Infrared imaging evaluation of oral tissue thin sections based on principle component analysis (PCA). First loading factors are shown to illustrate the information depth that can be obtained from infrared hyper-spectral images.
Fig 2: Data evaluation of infrared hyper-spectral images based on principle component analysis (PCA) and artificial neural networks (ANN). Multiple clusters are shown in the ANN evaluation allowing the determination of non-classified regions in other areas of the tissue samples.
Phyllanthus urinaria - A potent Anti-Cancer Molecule Proven by the Modern tools of Biotechnology (Poster presentation)
Karthikeyen N.P., Velmurugan R., Balakrishnan A.
Center for Biotechnology, Anna University, Chennai
Natural products continue to provide the most productive source of leads for new medicines; they provide greater structural diversity than any combinatorial or other practicable synthetic approach. This vast resource has remained untapped for drug discovery. Our research target was identification of new therapeutic molecules from the traditional Indian system, which are more effective and less toxic. This is a fusion of traditional Indian system of medicine and modern tools of biotechnology to the benefit of humanity at large.
A rare variety of Phyllanthus - a plant product - used by traditional Siddha healers for centuries as an anticancer medicine was analyzed. The active molecule is identified and its molecular structure elucidated and patented. This is still an ongoing study with active collaboration between The Center for Biotechnology, Anna University, Chennai, India and National Institute of Immunology, New Delhi, India. We have isolated and identified an antiproliferative Lignin with vast therapeutic potential as a Radiation sensitiser as well as curative and palliative metastasis scavenger.
The crude extract of the plant [Phyllanthus] was screened using an in vitro bioactivity-based screen for anticancer, antibacterial, lymphocyte proliferation activities.
The anticancer activity was studied by their ability to inhibit the proliferation of Hep2 laryngeal cancer cell lines. It was compared to untreated controls. The same plant extract showed a stimulatory effect on lymphocyte proliferation
The extract was tested in vitro, on Hep2 epithelial cell line and on peripheral blood mononuclear cells, for its ability to modulate the proliferative index of these cells. A dose response assay with the extracts was carried out to determine the maximum effective dilution. Growth kinetics was monitored by 3H thymidine incorporation studies. Repetitive studies confirmed that Phyllanthus had marked antiproliferative action on Hep2 cell line.
The dried plant powder was extracted with different polar and non-polar solvents and each fraction was tested for antiproliferative effect. The ethyl acetate fraction that showed the maximum cytotoxicity was subjected to silica column fractionation. Each fraction was examined by TLC and similar profiles pooled together. The fractions that showed cytotoxicity were further purified by HPLC. Five fractions were obtained, and fraction 3 showed antiproliferative effect and on TLC showed only one spot. This was obtained in large concentrations and was then used for the analysis of structure by Mass spec and IR studies.
The compound was identified as Lignin. A patent corporation treaty [PCT] for the antiproliferative activity of the compound and the bioactivity-based screening that led to the identification has been filed.
Use of this compound in patients with Head and neck cancers showed a marked radiation sensitiser activity, which was very effective in primary curative radiotherapy.
The micro metastasis inhibitor activity of the molecule was quite useful for follow up drug therapy and also in palliation, in case of secondaries.
Even on long term administration there was no inhibition on the haemopoietic activity.
This Anticancer compound - used for centuries by traditional healers of siddha system of medicine - with ability to assist lymphocyte proliferation, favors further multicentric interactive clinical trials.
Metabolic and Functional Imaging in the 21st Century.
Abass Alavi, M.D.
University of Pennsylvania, Philadelphia, PA
In recent years, FDG PET imaging has become a major tool for diagnosing, staging, restaging and monitoring various malignant conditions. FDG PET is an imaging technique that allows a unique approach for the assessment of the degree and the extent of cancer which is complementary to other techniques. Alterations in glucose metabolism, which are commonly noted in most cancers as revealed as functional images, FDG PET provides information that may be distinct from that obtained by conventional imaging methods. As a result of considerable technological advances made in data acquisition and processing, high quality whole-body images can be obtained in a time frame which is comparable to other diagnostic techniques. Although spatial resolution of PET is inferior to that of CT or MR, it reflects metabolic phenomena that take place at the molecular level. PET provides images which reveal cellular or intracellular biochemical processes that are dependent on the presence of specific enzymes or membrane receptors. FDG is by far the most widely used radiotracer for clinical purposes, but other radiopharmaceuticals are being actively investigated, including tracers for amino acid uptake, cell proliferation and tumor hypoxia. The ability to evaluate metabolic alterations in tumor cells prior to any detectable anatomical or structural changes results in a significant impact in patient management and substantially improves our understanding of the evaluation of the disease processes over time.
Basic Physics and Quality Assurance for Linac Radiosurgery
Tao-Seng Chen, Ph.D.
Medical Physics, Albany Medical College, Albany, NY
Linac radiosurgery is a single application procedure, utilizing a linear accelerator to deliver a high dose of radiation to a stereotactically defined intracranial lesion. The single fraction procedure consists of: (1) rigidly attaching a stereotactic head ring to the skull of the patient; (2) acquiring the scanned CT and MRI or angiogrphy images of the intracranial lesion and critical structures; (3) accurately defining the shape and location of the lesion in the head frame system; (4) developing a three-dimensional computer plan with isodose distributions for evaluation and prescribing radiation dose for treatment; (5) performing the quality assurance (QA) of treatment machine; and (6) setting up treatment parameters from the appoved plan for radiation dose delivery. The goal of radiosurgery is to deliver a highly concentrated dose in the lesion with steep dose gradients outside the target volume. The rapid dose falloff from the edge of the treatment volume significantly spares normal brain tissues. Linac radiosurgery relies on extreme accuracy of radiation delivery.
The basic physics applied to Linac radiosurgery includes investigating various factors contributing to the net uncertainty of the procedure and the accuracy of dose measurements of small radiation beams. Mechanical precision and accuracy of the treatment machine is an essential element in radiosurgery. In this presentation, the uncertainty of Linac coordinate system from gantry, collimator and treatment couch movement will be addressed. The limitation of spatial resolution of images obtained from the CT, MRI, and angiography will also be discussed. Several different dosimeters may be used for dose measurements in radiosurgery. Small cylindrical or parallel plane ionization chambers, silicon diodes, scintillation detectors, thermoluminescent dosimeters (TLD), and films have been used for the dose measurements of tissue maximum ratios, beam profiles and scatter correction factors. The accuracy of measured doses in the small radiation beams with various dosimeters will be evaluated.
It is extremely important to develop a comprehensive QA program for the Linac radiosurgery procedure. The QA in Linac radiosurgery is a multidisciplinary program not only for medical physics but also for radiation oncology, radiology and neurosurgery as well. The medical physics QA program for radiosurgery developed at Albany Medical Center consists of several steps which can be summarized as follows: (1) treatment machine preparation and isocenter accuracy check, (2) laser beam precision adjustment and film test, (3) verification of the computer treatment plan, and (4) patient’s setup and treatment. The details of each QA step will be illustrated in this presentation.
Native Fluorescence Imaging of Head and Neck Tumors
A. Katz and R. R. Alfano
New York State Center for Advanced technology in Ultrafast Photonics
Institute for Ultrafast Spectroscopy and Lasers,
The City College of New York, New York, NY 10031
Howard E. Savage and Steven A. McCormick
Dept. of Pathology, New York Eye and Ear Infirmary,
New York, NY 10003
Carcinogenesis is accompanied by cellular, molecular and structural changes which modify the optical properties of tissue. Native fluorescence from tissues can monitor these changes and thereby provide a real-time, non-invasive method to detect, in vivo, malignancy and pre!malignancy before there is visual indication. One does not have to remove tissue to obtain information about the state of the tissue. Fluorescence was first applied to the detection of cancer in human tissue by Alfano et al (1) and has since been extended to tissues of many different origins. The use of excitation and emission spectroscopy in the UV has produced diagnostic accuracies greater than 90% using algorithms based on ratios of emission intensities at key wavelengths (2, 3). Algorithms based on intensity ratios have the advantage of being independent of fluctuations in excitation power and collection geometry. Some of the key tissue fluorophores with diagnostic significance are tryptophan, collagen, elastin, flavins and nicotinamide adenine dinucleotide (NADH). Glasgold et al,(4) using a rat esophagus model, demonstrated that changes in basal cell layer thickness can be observed in excitation spectra. In there work, it was shown that the thickening of the basal cell layer resulting from tumor growth, produces a corresponding reduction of collagen emission (lex = 340 nm; lem = 380 nm) from the underlying stroma. Studies performed at the CCNY group on ex vivo tissues of multiple organs has demonstrated that the ratio of tryptophan emission (lex =300 nm; lem = 340 nm) to NADH emission (lex = 300 nm; lem = 440 nm) was consistently higher for malignant tissue than for non-malignant tissue. This is due, in part, to the fact that NADH fluoresces while NAD does not.
The effectiveness of fluorescence as a real-time, less invasive, diagnostic aide can be further increased by the use of fluorescence imaging. Fluorescence imaging systems can allow a physician to examine larger areas of tissue as part of a routine screening examination. The higher spatial resolution offered by optical imaging systems permits identification of small foci of abnormal cells. In addition to detecting cancer or pre-cancer, the fluorescence imaging system developed at CCNY can identify and image different structures found in head and neck tissues which may provide useful information to the physician.
In this present work, fluorescence images of ex vivo head and neck tissues, including tongue, parotid gland, thyroid and floor of mouth were acquired at multiple combinations of emission and excitation wavelengths for the purposes of identifying features which may be indicative of malignancy. Fluorescence maps were generated by ratioing fluorescence image intensities. These fluorescence ratio maps enhanced the ability to recognize regions of tumor and other features in tissues. Histopathological analysis was performed on the tissue samples. Location and shape of features observed in the fluorescence images were correlated with structures observed in histopathology.
All the samples examined in this research exhibited some similar spectral characteristics in their respective fluorescence images. The tumor regions of the samples consistently demonstrated weaker emission at lem = 440 nm than the non-tumor regions. The regions of normal tissue exhibited higher emission than the cancer samples at lem = 380 nm for lex = 340 nm excitation, while for 300 nm excitation no clear differences were observed in the 380 nm emission intensity.
The ability of fluorescence imaging to locate and identify tumor regions as well as other structures in tissues is evidenced in the fluorescence image of a thyroid specimen shown in Figure 1 for lex = 340 nm and lem = 380 nm. These wavelengths correspond to the collagen absorption and emission peaks, respectively. Pathological analysis identified a collagen capsule. The center of the capsule is a papillary carcinoma. The carcinoma has broken out of the capsule and spread to other areas of the thyroid as identified in Figure 1. Pathology also identified regions of normal thyroid, connective tissue, and fibrous tissue in the sample. The image has been inverted, i.e. darker regions represent higher emission intensities. It is observed that the collagen capsule, as expected, has very strong emission at 380 nm. The fibrous and connective tissue with high collagen content also exhibit strong 380 nm emission, while the cancer regions exhibit weak collagen emission. The location of the break in the capsule where the tumor has broken out can be identified by the low intensity area towards the bottom of the collagen ring. Below, and on either side of the capsule are regions of weak collagen signal surrounded by stronger signal areas. The weak signal areas have been identified by pathology as pockets of tumor surrounded by higher collagen content fibrous tissue. The normal tissue region towards the top of the sample has also has higher 380 nm emission than the tumor areas. The normal connective tissue on the upper right, with its higher collagen content is seen to have higher emission intensity than the other regions, with the exception of the capsule.
Identification of locations of tumor regions can be further enhanced by forming intensity ratio images. Figure 2 shows a pseudo color image of the specimen shown in Figure 1. The image was formed by the ratio, R = I(lex 340; lem 300) /I(lex 340; lem 380). The high ratio regions shown as bright red and yellow areas correspond to the regions of tumor.
This work demonstrates that fluorescence imaging at multiple wavelengths in the ultraviolet coupled with intensity ratio maps generated from these images, can be a clinical tool to identify regions of tumor and other structures in tissue. The extension of this work to in vivo measurements may provide physicians with a valuable clinical tool to aide in the identification of tumor.
References and Footnotes
1. Alfano, R. R., Tang, G., Pradham, A., Lam, W., Choy, D. S. J., and Opher, E. Fluorescence spectra from cancerous and normal human breast and lung tissues, IEEE Journal of Quantum Electronics. QE-23: 1806-1811, 1987.
2. Alfano, R. R., Das, B. B., Cleary, J., Prudente, R., and Celmer, E. J. Light sheds light on cancer - distinguishing malignant tumors from benign tissues and tumors, Bulletin of the New York Academy of Medicine. 67: 143-150, 1991.
3. Das, B. B., Glassman, W. L., Alfano, R. R., Cleary, J., R.Prudente, E.Celmer, and Lubicz, S. UV-fluorescence spectroscopic technique in the diagnosis of breast, ovarian, uterus, and cervix cancer. In: Laser-Tissue Interaction II, Los Angeles, CA, 1991, pp. 368-373.
4. Glasgold, R., Glasgold, M., Savage, H., Pinto, J., Alfano, R., and Schantz, S. P. Tissue autofluorescence as an intermediate endpoint in NMBA-induced esophageal carcinogenesis., Cancer Letters. 85: 223-232, 1994.
Figure 1. Fluorescence image from thyroid specimen with papillary carcinoma, lex = 340 nm,
lem = 380 nm. Image is inverted, i.e. darker areas are higher intensity.
Figure 2. Pseudo color ratio map (I(lex 340; lem 300) /I(lex 340; lem 380) of papillary carcinoma specimen. High ratios, indicated by yellow and red regions were histopatholoically identified as being malignant. Blue regions are normal tissue and black area is a collagen capsule.
Computer-Aided Diagnosis of Breast Cancer: Evidence and Potentials
Yulei Jiang, Ph.D.
Department of Radiology, The University of Chicago, Chicago, IL 60637
Mammography is currently the most effective method for detecting breast cancer. However, interpretation of mammograms can be challenging. About 10-20% of breast cancers are missed at mammography, many of which are visible in retrospect. In addition, only 10-40% of lesions suspicious mammographically prove to be malignant by biopsy. Computer-aided diagnosis (CAD) techniques are computer-based image-analysis methods that are designed to aid radiologists by providing a "second opinion." CAD techniques are developed to detect subtle cancers that might be missed by radiologists and to differentiate malignant and benign lesions. The potential of computer-aided detection to help radiologists detect more cancers has been shown by laboratory observer studies and clinical evaluations. Commercial computer-aided detection systems are becoming available. The potential of computer-aided diagnosis of malignant and benign lesions is being demonstrated. We present a CAD technique that classifies clustered microcalcifications in mammograms as malignant or benign. This technique when used by radiologists in an observer study improved radiologists' diagnostic accuracy and biopsy recommendations. It helped radiologists correctly diagnose more malignant lesions while recommending fewer benign lesions to biopsy. In addition, it reduced the variability in radiologists' interpretation of mammograms and surpassed the performance gains possible from double readings by two radiologists. These results demonstrate the potential effectiveness of CAD to help radiologists reduce the number of biopsies performed on benign lesions while increasing the sensitivity of mammography.
Very High Energy Electrons (50 – 250 MeV) in Radiation Therapy
Colleen DesRosiers, M.S.,Vadim Moskvin, Ph.D., Lech Papiez, Ph.D.
Radiation Oncology, Indiana University, Indianapolis IN 46202
The success of a radiation therapy treatment largely depends on the ability to deliver high radiation dose to the tumor or target while minimizing dose outside of this region. The design of radiation therapy equipment, and treatment techniques, has been continually evolving to meet the criteria of minimizing dose to healthy tissue. To this end the high energy linear accelerator, the cyclotron for heavy particle therapy and the Gamma Knife, which utilizes 201 beams in a hemispherical array aimed at a target minimizing overlap in normal tissue, have been developed. Theoretically, there is no limit on the dose to be delivered to the tumor, i.e. the greater the better. So typically, the factor limiting the radiation dose delivered to the tumor is the radiation dose that can be tolerated by the surrounding normal tissue structures. Since it is the tolerance dose of normal tissue that is a limiting factor, and this tolerance dose is volume dependent (Enami, et al.), reducing the high dose region to normal tissues is an aim of radiotherapy treatment planning. The toxicity produced as a result of the tolerance dose being reached is a deterministic effect. A deterministic effect has a threshold of dose below which, the probability of causing harm is minimal. Therefore, exposing even large volumes of normal tissues to doses below this threshold is expected to be inconsequential in terms of expressed damage. This is definitely a contributing factor to the success of the Gamma Knife. More recent designs of some therapy units (Nomos, tomotherapy) are conceptually similar to the Gamma Knife treatment. In these approaches small beams from a swept out arc are made to impinge on a target as a patient is translated through the plane perpendicular to beams axes. It is the apparent goal in these therapies to maximize the tumor dose and minimize the normal tissue volume receiving high doses (and consequently raising the volume of normal tissue receiving low dose). These approaches deliver intensity modulated radiotherapy (IMRT) by way of dynamic multileaf collimation (DMLC). This means that the beam shape, and beam intensity profile, are changing with the change of beam orientation relative to patient position. This is achieved by using software controlled mechanical motions of multileaf jaws that are correlated with positions of the gantry relative to patient body.
The mechanical shaping of beams’ intensities is an attempt to achieve a ‘scanning’ beam, i.e. a small ‘pencil’ beam which may be translated over a patient from all directions, ‘dwelling’ for longer times in positions that result in highest tumor/target dose ratio and ‘dwelling’ for shorter times (or not at all) in positions that result in unfavorable tumor/target dose ratio. Electromagnetic scanning can be achieved with charged particle beams but it cannot be applied to photon beams. This is why the cumbersome method of mechanical leaf positioning is utilized in the case of photon beams to achieve the conformal therapy. In contrast, electromagnetic scanning provides much more elegant method of achieving intensity modulation of beams. Currently this scanning technique is being investigated in the application heavy particle and proton therapies (Pedroni, et al., Jones, et al., Carlsson, et al.) However, high costs of cyclotron facilities would not allow heavy particle therapy with electromagnetic scanning to become a routine radiotherapy treatment in foreseeable future.
Electromagnetic scanning may also be applied for electron beams. Commonly available energies of electron beams in radiation therapy (4-30 MeV) limit their clinical usefulness as most of the dose is deposited in relatively superficial tissues and high angular spread of beams prohibits conformality. The use of higher energy electrons (30-50 MeV) has been explored recently and has shown some promise for treating deep-seated tumors and also has shown usefulness of electromagnetic scanning as a mechanism for achieving IMRT (Karlsson) with electron beams. Nevertheless, clinical usefulness of electron beams in this range of energies is still limited by the lack of their penetrating ability.
This paper explores the use of very high energy electrons (50-300 MeV) whose dose distributions from Monte Carlo simulations have been investigated recently (DesRosiers, et al.). These investigations have shown that clinically predictable and uniform dose distributions at the interface between inhomogeneites (lung tissue, bone, soft tissue, air) are achievable with very high energy electrons. Very high energy electron beams have the caveat of producing neutrons and inducing radioactivity within the tissues. However, with the use of several overlapping beams, the increased dose from neutron production and radioactivity enhances the target/normal tissue dose ratio and is not a disadvantage. The technology for producing these beams is available although there is no treatment unit currently designed for medical application.
The proposed design of this equipment incorporates an electromagnetic scanning device that controls the position of the pencil beam (< 0.5 cm diameter at the patient surface) and the angle of incidence with respect to the patient. With electromagnetic scanning, it is possible to achieve hundreds of orientations of this pencil beam in a fraction of the time required for tens of beams conventionally. By maximizing the number of directions from which the beam can impinge upon the target, the total deliverable dose to the target can be maximized while minimizing the volume of normal tissue receiving high doses, in a very similar fashion as the Gamma Knife. Controlling the ‘dwell’ time of each individual pencil beam further assures optimization of the tumor/normal tissue dose ratio.
References
Enami B, Lyman J, Brown A, et al. Tolerance of normal tissue to therapeutic irradiation. Int J Radiat Oncol Biol Phys 1990; 21:109-22
Pedroni E, Bohringer T, Coray A, et al. Initial experience of using an active beam delivery technique at PSI. Strahlentherapie und Onkologie. 1999; 175 Suppl 2:18-20
Jones DT, Schreuder AN, Symons JE, et al. Status report of the NAC particle therapy programme. Strahlentherapie und Onkologie 1999; 175 Suppl 2: 30-2
Karlsson M, Zackrisson A. Exploration of new treatment modalities offered by high energy (up to 50 MeV) electrons and photons. Radiother Oncol 1997; 43: 303-309
DesRosiers C, Moskvin V, Bielajew A, Papiez L. 150-250 MeV electron Beams in Radiation Therapy. Phys Med Biol 2000; 45:1781-1805