A new medical instrument, which combines Raman spectroscopy and resonance sensor technology, for detecting prostate cancer.
Prostate cancer has the highest incidence of all cancers in Europe and USA, and is the third leading cause of cancer-related death after lung and colorectal cancer in European men. Since 1995 an elevation of 16% of the number of deaths due to prostate cancer has been experienced in Europe, and in our aging population the prevalence of the disease is expected to increase. The most common method to detect prostate cancer is to measure the concentration of prostate specific antigen (PSA) in the blood. However, the PSA test has many limitations. A high concentration of PSA indicates prostate cancer, but the PSA level may also be elevated in men with a normal prostate. Furthermore, many men that harbor prostate cancer have a low PSA level.
Still today the most common method for diagnosing cancer is histopathology, which means that the disease is proven in vitro (outside the living organism) using a microscope. If the PSA level is elevated, a biopsy (obtaining a tissue specimen for microscopic analysis) is performed to establish if the patient harbors cancer. In a biopsy a needle is passed into the prostate and a tissue sample is withdrawn as the needle is removed. Figure 1 shows histopathologic sections of cancerous and healthy prostate tissue. Histopathology is a time-consuming method that requires skilled staff able to perform the biopsy and interpret the results correctly. Furthermore, it can be problematic to use in some cases, especially for prostate cancer. Tumors in the prostate can usually not be localized either by palpation or by any commercially available imaging method, such as ultrasound or magnetic resonance imaging. Palpation of the prostate means that the physician examines the hardness of the prostate tissue via the rectum using the fingers. The physician looks for tissue areas that are harder than the surrounding tissue; tumors are generally harder than healthy tissue. Since the position of a possibly present tumor rarely can be determined it is unfortunately necessary to take biopsies from several areas of the prostate. It is rather common that a present cancer is overlooked, it has been estimated that this occurs in 3 out of 10 biopsy examinations. The biopsies are very small, less than 1/1000 of the volume of the whole prostate, which explains why many tumors are missed. European guidelines recommend that 10-12 biopsies are taken. To increase the number of biopsies does not seem to increase the detection rate. The pain experienced by patients, the risk of complications, such as infections, and the costs increase with the number of biopsies. A major problem with prostate cancer management is that current methods cannot distinguish between aggressive and indolent tumors. To remove the prostate is a risky intervention, e.g. impotence is a common adverse effect. For men with indolent tumors it may be unnecessary to remove the prostate. More men die with prostate cancer than from prostate cancer. On the other hand it is very important that aggressive, life-threatening tumors are identified and removed at an early stage. Unfortunately, many men are treated too late today.
To address these problems we are currently developing a novel device for detecting and diagnosing tumors in various tissue types in vivo (in the living organism), in the first place prostate cancer. It is envisioned as a handheld, easy-to-use device that with a high reliability can discover cancer and other lesions. The instrument will combine two detection techniques in order to increase the diagnostic accuracy and also receive complementary information about the molecular cellular changes. Our idea is to utilize a resonance sensor, which can measure the hardness of the tissue, in combination with a Raman spectrometer, which supplies information about the molecular cellular changes. The combination of these two methods into one instrument poses a promising alternative to the multiple-biopsy procedure presently in use.
Raman spectroscopy is a light-based method where the sample is illuminated with laser light. The light interacts with the sample and undergoes color shifts. The spectrum of colors that is reflected from the sample contains information about the molecular composition of the sample. The Raman technique is useful for distinguishing cancerous and healthy tissue; this has been shown in many studies. Cancerous prostate tissue can be identified due to elevated levels of DNA, cholesterol and choline and a reduced concentration of oleic acid. Raman spectroscopy is very promising for distinguishing aggressive and indolent tumors. Recently it was seen that a Raman sensor was able to disclose a tumor, which was invisible to the physicians, in the chest of a patient that was operated. Drawbacks of Raman are that the laser light can cause heating, dehydration and photo-induced changes of the tissue; the degree to which the tissue is affected depends on the wavelength and the effect of the laser.
Due to a generous grant from the Kempe foundation we have recently been able to purchase one of the thinnest fiberoptic Raman probes in the world, see Figure 2, and a high quality Raman spectroscope. The Raman probe is merely 0.8 mm thin and delivers high quality spectra.
An example of a spectrum of porcine prostate tissue, collected using this probe, is shown in Figure 3. The relative intensity of the labeled peaks at 1448 cm-1 and 1661 cm-1 is an indicator of many types of cancers, such as breast and brain cancer. We also have access to a 1.2 mm Raman probe of the same model, and a Raman micro spectrometer with which we can investigate the microscopic properties of tissue samples. The principle of a Raman micro spectrometer is shown in Figure 4.
Supported by the Kempe Foundation, we have also had the opportunity to purchase a micro-resonance sensor. This resonance sensor allows us to measure tissue stiffness at the micro level. The sensor tip is a very thin glass needle, the tip of a glass sphere with a diameter of only 50 microns, see Figure 5. To this resonance sensor system, we have added a Raman probe, see Figure 6, which makes this system unique in the world.
The resonance sensor is based on a piezoelectric ceramic element that can be set into vibration, much like a vibrating guitar string, by an electrical circuit, as shown in Figure 5. It vibrates at a particular frequency, the so called resonance frequency, which changes when the sensor tip comes in contact with e.g. tissue. The frequency shift is proportional to the stiffness of the tissue. Tumors can be detected since they normally are harder than soft healthy tissue. Studies performed by our colleagues at Umeå University show that the resonance sensor can distinguish tumors and healthy epithelial prostate tissue, which is a relatively soft type of tissue. Currently, the method has limitations in distinguishing tumors and naturally occurring hardenings, such as prostate stones (see Figure 1). However, tumors usually develop in the anterior part of the prostate gland, which mainly consists of soft epithelial tissue. Thus, a hardening in this area indicates cancer. This is promising for detecting prostate cancer in vivo using the resonance sensor.
The combination of the two techniques can minimize the drawbacks that are associated with each method. The proposed sensor can be made relatively small; it can be pictured like a kind of pen with a wire in one end. The instrument can be of great use in e.g. operations of cancer, to enable the surgeons to remove the whole tumor but a minimum of healthy tissue. The applications for an instrument that combines resonance and Raman techniques are countless. From a medical point of view many different areas of the human body can potentially be interrogated. Imagine e.g. how powerful a gastroscope (a long flexible instrument used to look inside the stomach) equipped with this technique could be. The versatility of the instrument could certainly be used for many other purposes also outside the medical sector.
Hand in hand with the experiments we will also develop mathematical algorithms to analyze the obtained data and to aid in making reliable diagnoses. The algorithms should be able to automatically detect cancerous tissue. This project is unique, and we have patented the idea to combine resonance sensors with Raman spectroscopy.
Measurements on porcine prostate tissue
We have studied porcine tissue recovered from slaughtered non-castrated pigs in our preliminary studies. The obtained results are very similar to published data on human prostate tissue, and show good spectral resolution and reproducibility. It is clear that the prostate tissue is inhomogeneous; spectra differ depending on which tissue type that is measured. The samples should be immersed in physiological saline when refrigerated and during measurements, to provide a natural milieu for the tissue. This prevents the tissue from dehydrating, which may cause spectral changes.
Investigation of how near-infrared laser light affects tissue
To ensure that reliable results are obtained using Raman spectroscopy it is important to assure that the laser irradiation does not harm the tissue. We have investigated how the near-infrared laser light (830 nm) that we use affects porcine prostate tissue. The Raman micro spectrometer was used for this purpose. We showed that no changes of the Raman signal could be seen during a 5 minutes laser illumination at maximum power. However, a reduction of the spectral background with increased illumination time was observed, which likely is due to fluorescent species being bleached, i.e. they lose their ability to fluoresce after being illuminated for a while. That does not mean that the tissue is harmed, our conclusion is that the laser illumination from the laser we use is gentle to the tissue.
Is snap-freezing of tissue in liquid nitrogen a good preservation method?
Many researchers use freezing in liquid nitrogen and subsequent storage in -80 degrees as a preservation method. It is then assumed that tissue that has been thawed is unaffected by the process and comparable to fresh tissue. However, there are not many studies that confirm this, and none that have examined prostate tissue. Therefore, we have investigated if Raman measurements on fresh prostate tissue differ from measurements on tissue that has been preserved using this method. The results proved that prostate tissue that has been frozen and then is thawed, show small but statistically significant differences as compared to fresh tissue. Mainly, it seems that the protein structure is altered. This is important information for researchers that use freezing in liquid nitrogen as a preservation method.
Combined measurements using fiberoptic Raman spectroscopy and resonance sensor technology
We have conducted our first measurements where both fiberoptic Raman spectroscopy and resonance sensor technology have been used for measurements at the same points of a tissue sample. Our fiberoptic Raman probe was used in conjunction with a commercially available resonance sensor system, the Venustron®. The instruments were set up side by side and a computerized translation stage (XYZ-stage) was used to ensure that the measurements were taken at precisely the same spots. Abdominal pork tissue (pork belly) was used as a model system. The resonance sensor measurements showed that one type of fat was harder than the other tissue types. The Raman data revealed that this kind of fat consisted of a relatively high degree of saturated fat. In summary, the results showed that for tissue classification the Raman probe provided additional information to the resonance sensor measurements, which is promising for the development of a combined instrument.
We have performed measurements on porcine prostate with our combination of micro-resonance sensor, and Raman spectroscopy. The combined measurement results were analyzed by a statistical classifier, a support vector machine (SVM). Three histologically identified tissue types could be distinguished. We have also made our first measurements on human prostate tissue using this method. Cancerous tissue could then be distinguished from healthy tissue.
Molding of a Raman probe into a resonance sensor element
A feasibility study of molding a fiberoptic Raman probe into a cylindrical resonance sensor element has been conducted. Rubber latex has successfully been used before in the construction of functional medical instruments, therefore it seems like an appropriate material for this application also. The Raman probe was substituted by a steel pipe of the same diameter. It was concluded that changes of the frequency properties was minimized when small amounts of molding material and a thin steel pipe were used.
We will continue to study how the two methods can best be combined. A resonant sensor with an integrated Raman probe will be constructed. The new prototype, and the mathematical methods for the classification of tissue will be evaluated by more measurements.