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TECHNICAL FIELD
This invention relates to a non-surgical method for treating tumors and
suppressing tumor growth. More specifically, this invention relates to a
method for the treatment of tumor-bearing animals, including humans,
involving exposure of a tumor to high energy shock waves ("HESW") to alter
the tumorigenic potential of tumor cells and suppress tumor growth. The
method of the invention provides a safe and effective alternative to
surgical techniques for the treatment of tumors.
BACKGROUND OF THE INVENTION
Surgical removal of tumors has been one of the conventional treatments for
cancer. Such treatment is attended by the high degree of pain and
disability, and increased susceptibility to infection which accompanies
any surgical procedure. In addition, scarring of tissue inevitably occurs.
Due to the substantial disadvantages of surgery, there continues to exist
a need for the non-invasive treatment of tumors.
Non-invasive techniques hae been employed in various types of medical
protocols. For example, U.S. Pat. Nos. 3,237,623, 3,735,755, and 4,441,486
refer to the use of ultrasound waves to destroy various tissues or cells,
and U.S. Pat. No. 4,315,514 refers to the use of selected frequencies of
ultrasound to destroy tumor cells. However, because long exposure to
ultrasound results in cellular (thermal) degradation, the use of
ultrasound waves for the suppression of tumor cells is not favored.
Shock wave technology is currently being used for the non-invasive
destruction of human renal and ureteral calcified deposits
("extracorporeal shock wave lithotripsy"). C. Chaussy et al., Lancet,
1:1265 (1980); C. Chaussy et al., J. Urology, 127:417 (1982); E. Schmiedt,
C. Chaussy, Urol Int 39:193 (1984). In this technique, HESW are focused by
a brass semi-ellipse of high acoustical impedance to a second focal point
(F.sub.2).
In contrast to an ultrasonic wave, which consists of sinusoidal wave form,
a shock wave consists of a single positive pressure spike with very steep
onset and gradual relaxation. While ultrasound can generate pressure waves
of approximately 0.1 bar, shock waves can generate pressure amplitudes of
up to 1000 bar or more.
Ultrasound is capable of creating a mechanical shock, which is felt at a
distance of a few microns. If the ultrasonic wave traveling through a
liquid is high enough in amplitude, a microscopic bubble or cavity is
produed. This phenomenon, known as cavitation, can produce bubbles of a
resonant size which collapse violently to produce high local pressure
charges of up to 20,000 atmospheres. For example, at 20 KHz, the resonant
bubble size is about 150 microns. H. Alliger, "Ultrasonic Disruption,"
American Laboratory, (1975).
Shock waves have a greater depth of penetration than ultrasonic waves.
Because of this feature, focused shock waves have been used to break up
urinary concrements, as discussed briefly above. In those procedures,
patients are submergeed in a water-bath and the urinary stone is
visualized by the use of two dimensional fluoroscopy. The generated shock
wave is propagated through both water and tissue at nearly identical
velocities as a result of similar acoustical imedance. However, the target
stone, with its high acoustical impedance, is said to absorb and reflect a
significant portion of the shock wave. By repeated shock wave exposures in
the range of 1000 to 2000 shocks, non-invasive stone disintegration is
said to be achieved. The multiple small fragments produced are then said
to pass through the intact urinary tract and be excreted in the urine.
Similary, U.S. Pat. No. 3,942,531 refers to the use of shock waves to
destroy calcified deposits in the urinary tract. The '531 patent refers to
an elliptical container which is applied directly on the skin of the body
in an airtight manner. This technique relies on the shock waves' selective
attack on the calcified stone, which is said to leave the surrounding
tissue intact.
To date, however, there has been no disclosure or investigation of the use
of shock waves to destroy or eliminate tissue growth abnormalities such as
tumors. On the contrary, clinical use of shock waves, to destory urinary
concrements, has been said to be based on the premise that shock waves,
meeting with the substantially greater acoustical impedance of the stone,
pulverize the stone while "other parts of the body are not affected
thereby" ('531 patent, Col. 1 , lines 61-63).
SUMMARY OF THE INVENTION
The present invention provides a nonsurgical method of treating tumors by
altering the tumorigenic potential of tumor cells and suppressing tumor
growth. As will be appreciated from the disclosure to follow, this
invention advantageously provides a method which has none of the
above-stated disadvantages of ultrasound techniques. The method of this
invention comprises a process for the treatment of tumor-bearing animals
including humans, by exposue of a tumor to HESW, which results in a
significant delay in tumor growth. More particularly, the method of this
invention comprises the steps of locating the tumor in the body of the
patient and generating a high energy shock wave towards a point that
coincides with the position of the tumor.
The present invention has none of the disadvantages associated with the
surgical treatment of tumors. HESW treatment would thus minimize the pain,
morbidity, mortality and convalescence expenses associated with
conventional surgical techniques.
The present invention also has none of the disadvantages of ultrasonic wave
medical treatment and its resultant thermal degradation of tissue. Rather,
the method herein disclosed recognizes and demonstrates that soft tumor
tissue can be affected by shock waves in order to suppress tumor
growth--which is unexpected in view of the afore-quoted belief that shock
waves leave all soft tissue "substantially intact". Thus, HESW therapy is
a desirable technique, which offers a safe and effective alternative to
surgical techniques for the removal of tumors.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A is a graphic representation of the in vitro tumorigenic potential
of R3327AT-3 rat prostatic cancer cells after exposure to HESW, plotting
mean tumor volume (cm.sup.3) against days.
FIG. 1B is a graphic representation of in vivo effect of one treatment of
HESW on R3327AT-3 tumor nodules, plotting tumor volume versus time.
FIG. 2 (comprising FIGS. 2A, 2B, 2C, and 2D) is a graphic representation of
flow cytometry for total DNA content, plotting the number of cells of
normal human lymphocytes, control R3327AT-3, and cells exposed to 800 and
1500 HESW, respectively, against relative DNA values.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
According to the method of this invention high energy shock waves are used
for the non-surgical treatment of tumors. This method advantageously
provides a non-invasive method for the treatment of tumor-bearing human
patients, which apparently disrupts tumor cells on a subcellular level. It
has been found that although tumors are composed of relatively "soft"
tissue, the high energy shock wave can selectively disrupt tumor cells,
and leave the normal cells of the surrounding tissue largely intact. The
term "tumor", as used herein, relates to tissue growth abnormalities which
may form a swelling or a group of malignant or benign, atypical or
abnormal cells which appear within or upon the body of the animal to be
treated. While not wishing to be bound by theory, it is believed that the
tumor cells are selectively affected by the shock wave because of their
relative density.
While tumors are actually composed of a heterogeneous population of cells,
tumor cells frequently possess a greater complement of DNA, i.e., they
have more DNA than the normal (2n) complement. Those tumor cells which
possess a greater complement of DNA have a greater density than normal
cells. FIG. 2, represents some tumor cells with the normal "2n" complement
of DNA (the first peak) and some cells with a larger than normal
complement (second peak). After treatment with HESW the second peak merges
with the first, indicating that normal cells are unaffected and only the
abnormal, denser cells are selectively killed.
According to one embodiment of this invention, the method of treatment
begins by first localizing the tumor tissue. This can be accomplished by
any conventional means, including ultrasound, visual means, fluoroscopy,
nuclear magnetic resonance, CT scanning, or any other method suitable for
localizing tumor growth.
The patient is anesthetized by any conventional means, and then placed in a
tub, preferably filled with de-gassed water to prevent bubble formation
which can interfere with the transmission of shock waves. If necessary,
the patient can be held in position with restraining straps.
Next, the high energy shock waves are generated outside the body. A shock
wave may be generated in water to enter into the patients biological soft
tissue in a largely unimpeded manner. Alternatively, the shock wave may be
generated at a point directly against the patient's body, so that the
shock wave would pass through the body to reach the tumor positioned at
the second focal point.
The HESW utilized in this invention may be generated by any suitable means.
For example, the HESW may be generated by underwater sparks whereby an
underwater electrode discharges stored electrical energy in approximately
0.5 microseconds. Once created, the spark causes vaporization of water,
which generates a spherically propagated pressure wave in the surrounding
liquid. Alternatively, the shock waves may be generated by laser, whereby,
the heat generated by laser light causes vaporization of water in an
explosive manner. In another embodiment, the shock waves may be created by
chemical explosion. Regardless of the means selected to generate the shock
waves, the frequency of the shock waves should be in the range of about
100,000 hz to 100 mhz, preferably between about 500,000 hz and 50 mhz.
According to one embodiment of the invention, shock waves are generated
from underwater sparks using the Dornier Lithotripter machine. This
machine has been used successfully in the treatment of urinary
concrements. FDA approval of its use in the treatment of such calcified
deposits was granted in December 1984, after the Lithotripter had been
widely used in the United States and Europe.
In this embodiment of the invention, the shock waves are focused to reach
the target tumor tissue. A reflector, which has the shape of an
upward-opened semiellipsoid, is incorporated into the floor of the Dornier
tub. Electrodes protrude horizontally into the ellipsoid reflector in such
a way that the discharge occurs at the lower focal point of the ellipsoid.
The waves are focused by placing the electrode at one focal point of the
symmetrical ellipsoidal cavity (F1). The shock waves are reflected by the
walls of the ellipsoid reflector and concentrated in the second focal
point (F2), where the tumor is located.
Energy for the discharge is stored in the pulse current generator. The
electrode is connected mechanically and electrically to the pulse current
generator. Ignition of the underwater spark is controlled by an
electrocardiographic triggering unit that monitors the patient's heart
rate.
The tumor is localized via the focusing system of the Lithotripter. This
system consists of a two-axis X-ray system which is permanently secured to
the Lithotripter mounting and radiates horizontally at an angle of
55.degree. through the windows in the floor of the tub, so that the X-ray
axes intersect at the focal point F2 of the reflector; the corresponding
image intensifiers are mounted on a movable pivoting arm. When the patient
has been lowered into the tub, the image intensifiers are manually placed
into the radiation path of the X-ray tubes and then are moved by motor
along the radiation path as close as possible to the patient. An automatic
cut-off prevents further movement of the image intensifiers when contact
is made with the patient. The fluoroscopic (X-ray) picture is transferred
to two television display units on which, with the aid of cross hairs, the
exact position of the tumor in relation to the focal point (F2) may be
determined.
In the preferred method of the present invention, the patient is positiond
so that the tumor is in line with the second focal point of the reflector.
The patient may be placed on an overhead support, which permits the
optimum application of the shock waves. The movement of the patient
support in three coordinates is achieved by a positioning unit, which is
guided from a crane runway installed on the ceiling of the room. The
initial movement of the patient support to the position above the bath is
carried out manually. The positioning procedures--the vertical,
longitudinal, and lateral movements--are controlled from a control cabinet
by an hydraulic drive unit.
The patient is thus placed in the bathtub of stainless steel, constructed
so that a tumor in any position on the body can be localized and
positioned in the upper focal point of the reflector. The shock wave
electrode, the elliptical reflector and the windows required by the
passage of X-rays should be located in the floor of the tub. The water
should be temperature controlled and de-gassed to remove dissolved oxygen,
carbon dioxide and nitrogen.
Abdominal organs can tolerate pressure waves in the 2.5-3 kilobar range.
Lungs, however, must be protected from the shock wave by styrofoam or a
similar material to avoid significant damage. The characteristics of
styrofoam which make it a desirable material to block shock waves are
described in C. Chaussy, et al. Extracorporeal Shock Wave Lithotripsy,
page 35. For the treatment of a tumor located in the lung, a hole may be
cut in a protective vest, positioned over the portion of the lung
containing the tumor.
The patient should be scheduled to receive a series of fractionated HESW
treatments. Each session should involve administration of between 500 and
6000 shocks, preferably 800 to 3000 shocks per treatment. The method of
this invention may be used in conjunction with conventional cancer
treatment such as chemotherapy, immunotherapy or radiation. It is believed
that one of the benefical effects of HESW treatments in conjunction with
such conventional cancer treatment is the possible reduction in the amount
of alternative treatment needed.
EXAMPLES
The following examples demonstrate the method of HESW treatment of tumor
cells according to the invention. These examples are set forth for the
purpose of illustration only and are not to be construed as limiting the
scope of the invention in any manner.
Example 1
This example demonstrates the effects of HESW on Dunning R3327AT-3 rat
prostatic carcinoma cells in vitro. R3327AT-3 cells were provided to me by
Dr. J. T. Issacs of the Brady Urological Institute, Johns Hopkins
University School of Medicine, Baltimore, Md. R3327AT-3 rat prostatic
carcinoma is a hormone insensitive, anaplastic prostate cancer. J. T.
Issacs et al., Cancer Res. 38:4353 (1978). Histologically, it appears as
sheets of anaplastic cells without acini or glandular elements. There is
no evidence of secretory activity. The doubling time is between 1 day in
vitro and 1.7 days in vivo. When 100,000 viable cells are injected into
rats, the time to achieve a tumor volume of 1 cm.sup.3 is approximately 12
days with eventual large local growth and lymphatic and pulmonary
metastases.
I maintained the Dunning R3327AT-3 cells in standard tissue culture at
37.degree. C. in RPMI 1640 supplemented with 10% fetal calf serum, 1%
L-glutamine, 1% non essential amino acids, 1 ng/ml dexamethasone, 100
.mu./ml of penicillin and 100 mcg/ml of streptomycin. I grew the cells to
subconfluence and created a single cell suspension by mechanical
dispersion. I then transferred 1.5 ml aliquots with concentrations of
1.times.10.sup.6 cells/ml to 5 ml polypropylene test tubes (Falcon,
Cockeysville MD). I then prepared a separate group of experimental cells
and a separate control group of cells, which were treated identically
except for the HESW treatment.
I next placed my experimental cell suspension of R3327AT-3 cells in a test
tube holder designed to fit a base portion over the brass ellipse in the
Dornier Lithotripter and hold a test tube in the second focal point of the
Dornier Lithotripter (supplied by Urotech, Ltd., Houston, Tex.).
I then subjected the cells to HESW. I set the operating voltage at 18 kilo
volts so as to deliver 100 shocks/min to the cells which were to receive a
total of 800 and 1500 HESW. The waterbath temperature was kept constant at
37.degree. C. and the water level was always above the cell suspension
within the test tube. I placed the unshocked control cells peripherally in
the water bath, but out of the focus of the HESW.
I determined the percentage of viable cells immediately after exposure to
HESW and again at 24 hours in cells returned to tissue culture. Table 1
indicates my observations: immediately after HESW, there was a moderate
decrease in viability of the cells, as determined by trypan blue
exclusion.
I next performed a clonogenic assay by plating five thousand viable cells
in triplicate and incubating them at 37.degree. C. for six days, according
to the method of B. R. Rao et al., Cancer Res., 38:4431 (1978). After
methanol fixation and hematoxylin staining, I counted the colonies which
consisted of greater than 50 cells and expressed my results in colony
forming units/5000 cells plated.
I observed that after 24 hours of incubation following HESW treatment, the
viability of the treated cells returned to the level of the control cells.
This represents the death or repair of sublethally damaged cells. I also
observed a marked decrease in the ability of the treated cells to form
colonies after HESW exposure (Table 1). The percentage decrease of cells
capable of colony formation varied with the number of HESW delivered.
These results indicate that HESW profoundly influences colony formation at
shock levels which are not necessarily capable of causing immediate cell
death.
TABLE 1
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HIGH ENERGY SHOCK WAVES:
EFFECT ON VIABILITY PERCENTAGE AND
CLONOGENIC POTENTIAL OF R-3327AT-3
PROSTATIC CARCINOMA IN VITRO
% cells viable
Number of
0 24 clonogenic
% of
Time: hours hours survivors control
______________________________________
CONTROL 97% 95% 245 .+-. 11
(100%)
800 SHOCKS
88% 96% 72 .+-. 17
(29%)
1500 SHOCKS
75% 100% 12 .+-. 02
(5%)
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I subjected samples of R3327AT-3 cells to flow cytometric determination on
DNA content following HESW exposure (FIG. 2). The fluorescent dye used was
acridine orange, according to the method of F. Traganos et al., J.
Histochem Cytochem 25:46 (1977). The results demonstrated a selective loss
of the cells containing twice the normal complement of DNA (G2M polulation
of cells) which corresponded to an increase in shocks administered. Cells
maintained in tissue culture for 48 hours showed a reversion to control
DNA distribution.
Next, I exposed 100,000 R3327AT-3 cells in 0.5 cc of media to HESW, and
then injected them into the right anterior thigh of male Copenhagen rats
weighing 150-200 grams. When the tumors became palpable, I measured them
and calculated mean tumor volumes using the formula
L.times.W.times.H.times.0.523, according to the method of J. K. Smolev et
al., Cancer Treat Rep. 61:273 (1977).
In those rats receiving cells exposed to 800 and 1500 HESW, there was a
pronounced delay in the time for the mean tumor volume to achieve 1
cm.sup.3 (FIG. 1a). The time for the tumor to reach a mean volume of 1
cm.sup.3 was 12 days for control rats receiving control cells and 15 and
17 days for rats receiving 800 and 1500 shocked cells, respectively. At 17
days the mean tumor volume of the rats receiving cells exposed to 1500
shocks was 1.00 cm.sup.3, whereas the control tumor volume was 7.92
cm.sup.3 (p=0.002). Therefore, a growth delay equal to a decrease tumor
volume of 87% was achieved. This correlates with the 95% decrease (compare
Table 1) which I observed with the in vitro clonogenic assay.
The in vivo effect of HESW was manifest by a 5-day delay in tumor growth
(see Table 2). These results demonstrate that HESW not only suppresses
tumor clonogenic capacity but also delays the rate of subsequent tumor
development in rats.
TABLE 2
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TUMOR PRODUClNG POTENTIAL OF
HESW TREATED R3327AT-3 CELLS
DAYS TO TUMOR VOLUME AT DAY 17
1 cm.sup.3
17 days in cm.sup.3
______________________________________
CONTROL 12 7.92 .+-. 1.77
800 SHOCKS
15 3.93 .+-. 0.83
1500 SHOCKS
17 1.00 .+-. 0.23
______________________________________
control vs. 800 (p + 0.079)
control vs. 1500 (p = 0.002)
800 vs. 1500 (p = 0.0092)
Example 2
This example demonstrates the effect of HESW on Dunning R3327AT-3 rat
prostatic carcinoma cells in vivo. Ten rats with small palpable tumors
(largest volume dimension 0.25 cm.sup.3) in the anterior thigh were
exposed to shocks targeted at the tumor using fluroscopic control. I
placed the rats under ketamine anesthesia (8.0 mg/kg) and then placed them
into a holder designed to tie the rats to a plastic platform.
With fluoroscopic guidance, using the Dornier machine, I maneuvered the
tumors into the second focus of the HESW. 1500 HESW were delivered with
operating voltage set at 18 kv and at a rate of 100 shocks/min (FIG. 1b).
Ten tumor bearing rats treated similarly to those receiving the HESW but
not subjected to HESW, served as my controls.
As a result of the HESW treatment, there was a retardation in growth rate
during the first week after treatment (p>0.05). No rats died or suffered
any ill-effects as a result of the HESW treatment and no control rats died
during this period. One treated rat died of anesthetic complication on day
1, a second of small bowel obstruction on day 3, and a third of apparent
cannibalism on day 4.
Tumors in the right anterior thigh of rats (largest volume dimension 1
cm.sup.3) were subjected directly to 600 HESW and samples were obtained
for light microscopy and transmission electron microscopy. After
hematoxylin and eosin staining, I examined sections by light microscopy.
There was no evidence of histologic damage after HESW, apart from
hemorrhage surrounding the tumor, which had no detrimental effect on the
animals.
However, transmission electron microscopy (1% glutaraldehyde fixation;
6,600 magnification ) demonstrated progressive ultrastructural evidence of
nuclear and cytoplasmic damage after HESW exposure. There was marked
nuclear disruption with nuclei present in the cytoplasm. In addition,
autolytic digestion granules were in the cytoplasm, and the mitochondria
were damaged. The damage was most marked two days after HESW exposure.
Similar ultrastructural damage was not observed in control rats with tumors
exposed to HESW. Rat prostatic carcinoma R3327AT-3 cells from control rats
which had not been exposed to 600 HESW, had intact tumor cells, and tumor
matrix, normal unclear morphology with the cytoplasmic organ cells intact.
Example 3
This example demonstrates the effect of HESW on a melanoma line. I followed
a process similar to that described in Example 1, using the human melanoma
line, SK-Mel-28. The human melanoma line was obtained through the courtesy
of Dr. Lloyd Old's laboratory of Memorial Sloan Kettering Cancer Center,
1275 York Avenue, N.Y.C., N.Y. 10021. My experiments revealed an increase
in sensitivity to HESW as compared to R3327AT-3, treated above in Examples
1 and 2.
I subjected the melanoma cells to 400 shocks, and observed that viability
was decreased to 50%. After administering 800 shocks, viability was
decreased to 5%. Colony formation was reduced to 5% of control by 400
shocks. This demonstrates a spectrum of susceptibility of cells to HESW.
While I have herein presented a number of embodiments of this invention, it
is apparent that my basic construction can be altered to provide other
embodiments which utilize the process of this invention. Therefore, it
will be appreciated that the scope of this invention is to be defined by
the claims appended hereto rather than by the specific embodiments which
have been presented hereinbefore by way of example.
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