0.

Abtract

The means of using heat in order to raise benign cancer living cells and tissue has been practiced
for a significant period of time. Advanced research with means of optimizing this practice has
been done and clinical results have proven the fact that hyperthermia by itself, or in combination
with other treatments, can enhance the opportunity to overcome benign illnesses. However,
further research needs to be done in order to prescribe accurate dosages and to gain a better
understanding of the organism’s response to them.

This report focuses on the main subjects surrounding the employment of Hyperthermia for
treatments of various forms of benign diseases. Differences between traditional and
contemporary methods are discussed as well as the main difference between them. Moreover,
important mathematical models and units such as the Henrique’s model and the Thermal
Issoeffective Dosimetry (TID) unit are reviewed. Its efficacy, advantages, and disadvantages are
mentioned along with further explanation of the current problem of modeling tissue injury as a
function of temperature and time. Emphasis in the non-isothermal portion of the heating
procedure is given due to its high importance and impact on the accuracy of mostly all of the
mathematical models. A brief explanation of the vascular and cellular injury response is given
and a deeper explanation of the methods of cell heating along with in-vitro, in-vivo, and ex-vivo
experimentation is analyzed.

1. Introduction

Cancer development in urologic systems is a significant concern that needs to be further studied.
Benign Prostatic Hyperplasia (BPH) is one of the most commo n illnesses experience by men
over the ages of 50 years. About 50% of men between the years of 51-60 years and nearly 90%
of men over 80 years have experienced BPH in their lifetime [ CITATION Bho04 \t \l 1033 ].It is
estimated by the American Cancer Society that nearly 218 000 new cases will be documented in
2010, and about 32 000 of these cases are going to result in death [ CITATION Ame \l 1033 ]. The
number of cases estimated each year is very similar to past years [ CITATION HeX05 \t \l 1033 ].
Other malignant diseases such as renal cell carcinoma are very significant with estimations of
nearly 36 000 cases estimated for 2010. Several treatments such as chemotherapy, embolization,
chemoembolization, radiotherapy, and hyperthermia have been used to treat these diseases.

Hyperthermia treatments have been studied for the past 30 to 40 years. The main objective of
several studies of this nature is to have a better understanding of the biological response due to
the treatment [ CITATION HeX09 \t \l 1033 ], and therefore optimize the existing variables
surrounding it (temperature, duration of heating, number of heating sessions, and heating process
[ CITATION Rob74 \l 1033 ]) in order to design an adequate thermal therapy protocol. The heating
processes in the clinical field usually involve a wide variety of energy sources such as
microwave [ CITATION Lin87 \l 1033 ], radio frequency [ CITATION Cha10 \l 1033 ], the use of
magnetic nanoparticles [ CITATION Joh05 \l 1033 ],laser [ CITATION Pea92 \l 1033 ],[ CITATION Sei08 \l
1033 ],[ CITATION Sei081 \l 1033 ], and ultrasound [ CITATION Tro03 \l 1033 ].

2. Long Mild Hyperthermia Treatment

The use of long mild-temperature hyperthermia can be defined as the process of raising the
tissue’s temperature to ranges from approximately 40 to 44 °C for long periods of time. This
method has been successfully combined with other treatments such as chemotherapy,
brachytherapy, and radiotherapy [ CITATION Arm04 \l 1033 ],[ CITATION Rot07 \l 1033 ], [ CITATION
Lim05 \l 1033 ], [ CITATION Iss08 \l 1033 ],[ CITATION Fen08 \l 1033 ]. One of these studies involving
simultaneous treatments of mild temperature hyperthermia and brachytherapy concluded that the
combination of chemotherapy and brachytherapy with mild-temperature hyperthermia can
increase its effectiveness, practicality, and it can increase the overall comfort of the patient
[ CITATION Arm04 \l 1033 ].

Figure 1 Survival Curves for Asynchronous and G1 cells (CHO) [ CITATION Sap78 \l 1033 ]

However, this traditional hyperthermia method has not been very effective due to development
of thermotolerance [ CITATION Dew94 \t \l 1033 ], [ CITATION Sap78 \l 1033 ], [ CITATION Son95 \l
1033 ]. Figure 1 shows a plot of two types of CHO cell’s survival fraction vs. time of
temperatures from 41.5 °C to 46 °C. For temperatures in the range of 43 °C to 46°C a shoulder
followed by an exponential reduction can be seen. On the other hand, for temperatures in the
range of 42.5°C to 41.5 °C the same shoulder and exponential drop can be seen, but it is
followed by a plateau in cell killing, which can be seen as tolerance being developed over a
period of time [ CITATION Rot07 \l 1033 ], [ CITATION Sap78 \l 1033 ]. The fact that there is no
plateau on higher temperature plots shows that heating tissue to temperatures great than 43°C is a
process that does not experience thermotolerance.

3. High Temperature Hyperthermia Treatment

High-temperature hyperthermia (<50 °C) has been gaining much more attention throughout the
years in the community [ CITATION HeX09 \t \l 1033 ], [ CITATION Thr99 \l 1033 ], [ CITATION Rot07 \l
1033 ], [ CITATION Bho04 \l 1033 ], [ CITATION HeX05 \t \l 1033 ], [ CITATION Cha10 \l 1033 ],
[ CITATION Cor05 \l 1033 ], [ CITATION Fen08 \l 1033 ]. This is due to the fact that the process
requires much less time [ CITATION Bho04 \l 1033 ], apart from it being a minimally invasive and
real time monitored process compared to other treatments such as surgery and chemotherapy,
hence, more comfortable for the patient [ CITATION HeX09 \t \l 1033 ]. An accurate mathematical
model describing tissue injury at high temperatures is still needed in order to accurately prescribe
an adequate heat dosage that would give expected outcomes [ CITATION Dew94 \t \l 1033 ],
[ CITATION HeX09 \t \l 1033 ],[ CITATION HeX05 \t \l 1033 ] .

Experimentation with porcine skin [ CITATION Hen47 \l 1033 ] was used in order to create a model
based on the Arrhenius equation (usually referred as Arrhenius Model) [ CITATION Dew94 \t \l
1033 ],[ CITATION HeX09 \t \l 1033 ], [ CITATION Hen47 \l 1033 ], [ CITATION Yan \l 1033 ], [ CITATION
HeX05 \t \l 1033 ], which states that the injury rate (k) of the tissue depends on its activation
energy and frequency factor. The activation energy ∆ E is defined as the energy required for a
chemical reaction to occur. Moreover, the frequency factor A is defined as the number of
collisions that result in a reaction per second. The survival fraction (S) is dependent on the injury
rate and the time lapse of exposure to heat (t) [ CITATION HeX09 \t \l 1033 ].
t

−∫ kdt
(1)
S=e 0

−∆ E /(Rg T )
k=Ae (2)

This model is widely used to analyze experimental data. It is a practical tool to define important
rate process parameters for use in clinical protocols. However, it has some limitations and
disadvantages which are going to be further explained in this report.

Moreover, Thermal Isoeffective Dosimetry (TID) is a practical unit developed in the 1970s that
describes the time needed for a hyperthermic process at 43°C (t 43 ¿ to induce the same amount of
tissue damage as that induced at an arbitrary temperature (T) for an arbitrary period of time (Δt).

t 43=∆ t R(T −43) (3)

Where R is said to be 2 for temperatures greater than 43°C and can vary from 4 to 6 for
temperatures smaller than 43 °C due to thermotolerance [ CITATION Dew94 \t \l 1033 ]. The main
reason 43 °C was established as the reference temperature is due to the linear break that occurs in
R at this temperature [ CITATION Lep80 \t \l 1033 ]. This tool is used for clinical convenience and
it is initially based on data taken from the already mentioned experimentation with porcine skin
[ CITATION Hen47 \l 1033 ]. However, it is effective only for certain cell lines and for a range of
temperatures being from 43°C to approximately 57°C (depending on the cell line)[ CITATION
Dew94 \t \l 1033 ],[ CITATION Cor05 \l 1033 ]. It has been stated that there is no real break in R for
a wide variety of human cells [ CITATION Cor05 \l 1033 ]. The justification for such break at 43 °C
comes when generalizing the behavior of the majority of cell lines on the system.

The parameter R is assumed to be 2 from 43°C to 57°C because several experiments done on
mammalian cell samples have shown a similar linear constant on the data. On the traditional
range of interest, an approximation of R being 2 gives an output error of less than 2% [ CITATION
Sap84 \t \l 1033 ], although this error gets bigger for higher temperatures. Even though this has
been known for a significant period of time, R has been incorrectly used as a parameter in the
TID model for a wider range of temperatures (>57°C) [ CITATION HeX05 \t \l 1033 ], [ CITATION
HeX09 \t \l 1033 ]. R is indeed extracted from experimental data, but is not even close to 2 for
several temperatures and cell lines [ CITATION Dew94 \t \l 1033 ], [ CITATION HeX05 \t \l 1033 ]. It
has been stated that it is dependent on the activation energy and temperature [ CITATION Sap84
\t \l 1033 ].
−∆ E

R=e (R g T ( T +1) ) (4)

Finding the activation energy with available R has been done in the past [ CITATION Dew94 \t \l
1033 ]. But different activation energies have been found when they were extracted from the use
of eq. 1 and 2 [ CITATION HeX09 \t \l 1033 ]. This states that erroneous generalizations have been
made regarding the R parameter and it is not always the well known value of 2.
 1

0.9

0.8

0.7
Fraction Cell Survival

0.6

0.5

0.4

0.3

0.2

0.1

0
35 40 45 50 55 60 65
Temperature (C)

Figure 2: Plot of Survival Fraction vs. Temperature for different temperatures. ‘o’ using Δt=1 min, while continuous plot
uses t43 for each temperature with Δt=1 min. (With assumption of constant activation energy and frequency factor)

If one assumes, for the sake of the argument, that the activation energy and frequency factor of a
cell line are constant with temperature, the same survival rates should be found for a certain time
at different temperatures and for their respective equivalent times at 43°C. As expected, the
Arrhenius model and the TID do not correlate with each other for temperatures greater than
approximately 53 °C. Figure 2 shows a plot of the survival fraction (S) vs. temperature (T). The
activation energy is 384928 J/(M*K) and the frequency factor is 3.8e57 (1/s). These are the rates
processes of albumen [ CITATION Yan \l 1033 ]. One plot is using ∆ t as 1 min for each temperature
T and the other one is using ∆ t as its respective t 43for each T. One can see that there is no
similitude between these plots at temperatures greater than 55 °C. Now that this assumption has
been proven to be erroneous, it can be seen that the activation energy plays an important role and
it depends on the overall time of heating as well.

Due to the fact that cell or tissue damage is dependent on the temperature and time, it seems
useful to multiply the temperature T with its heating time ∆ t (hence creating the unit °C min or
°C sec) in order to find some direct type of correlation between different temperatures and
therefore trying to find a better model for the thermal dose (within a great range of temperatures)
[ CITATION Dew94 \t \l 1033 ]. Studies state that such a unit is not useful; as stated before, the
basic relationship between time and temperature has to do with the tissue’s activation energy
[ CITATION Dew94 \t \l 1033 ], [ CITATION HeX09 \t \l 1033 ], [ CITATION HeX05 \t \l 1033 ].
Figure 3: Theoretically Generated temperature profiles. A,B and C profiles are all equivalent to the dashed profile using
equivalent minute calculations. [ CITATION Sap84 \t \l 1033 ]

The curves in Figure 3 are all said to produce the same damage. This was done with a calculation
referred to as cumulative equivalent minutes and it is going to be further explained later in the
report. It can be seen that a simple integration over time would not yield the same results.

It appears that the best approach to find R is to first find the activation energy using methods
further explained in section 5 and then make use of eq. 4. The main constraint is that not only the
TID is effective only at a limited range, but the Arrhenius model too. This is because both the
TID the Arrhenius model are limited to describe the Isothermal Phase of the procedure [ CITATION
Fen08 \l 1033 ].

4. Non-Isothermal Phase

The reason why the TID and the Arrhenius Model only work for a certain range of temperatures
is because at this range, the non-isothermal (ramping) phase of the process is neglected. At
temperatures higher than 57°C, the non-isothermal phase of the process accounts for a great
percentage of the total injury of the tissue [ CITATION HeX09 \t \l 1033 ], [ CITATION HeX05 \t \l
1033 ]. There has been experimentation done that its main objective is raising the temperature of
the tissue up to a peak temperature and lowering it back to the initial temperature with no real
exposure time at the peak (in order to account only for the damage done by the non-isothermal
process).
Figure 4: Measured and predicted cell viability vs. hold time for SN12 cells. Solid line is the predicted cell viability using
the non-isothermal method with all available data points while dashed line is the predicted cell viability using the same
method but using only data points with 0 hold time. [ CITATION HeX05 \t \l 1033 ]

Figure 4 shows one of these experiments with SN12 cells [ CITATION HeX05 \t \l 1033 ]. It can be
seen that there is a big difference in cell viability drop with increasing hold time for low and high
temperatures for SN12 cells. Also, the drop at high temperatures is so sudden that it can be said it
might not be too influenced in the hold time but more in the accumulation of heat while the
temperature was raised to its desired peak (non- isothermal phase). In this study, different
activation energy and frequency factor constants were extracted by using all available data points
and by using only the data from thermal histories with zero hold time (non-isothermal portion).
It was concluded that kinetic parameters obtained by experimental data are substantially
dependant on both isothermal and non-isothermal portions of the heating process. It was also
concluded that the maximum temperature below which the cell injury due to the non-isothermal
heating is less than 10% would be in the vicinity of 60 °C at heating and cooling rates of 130
°C/min and 65 °C/min respectively [ CITATION HeX05 \t \l 1033 ].

Non-Isothermal heating becomes a major disadvantage when applying the Arrhenius Model as
well. Usually, the value of k eq. 2 is assumed constant throughout the process for simplicity
purposes [ CITATION HeX09 \t \l 1033 ], [ CITATION Jen09 \l 1033 ], [ CITATION Yan \l 1033 ].

S=e−kt (5)

By doing this, the extraction of k from data results fairly easy (due to its linearity when it is
analyzed in a semi-log scaled plot). In other words, equations 2 and 3 can be easily used for
constant temperature, therefore, constant activation energy, frequency factor, and injury rate. In
in-vivo and even in-vitro experimentation, this is not true. Every heating process has its non-
isothermal portion and temperature is not always constant. This portion can be neglected if the
isothermal portion is much greater, but at high temperatures, where heating durations account for
approximately 1 to 2 min, such assumptions result to be erroneous. This model has the limitation
of recording all the cellular damage over the entire heating process period and throughout a wide
temperature range. Also, it is extremely sensible and small changes in the parameters would
show significant changes in the sample’s survival. This is due to its double exponential form
[ CITATION Fen08 \l 1033 ].

It can be concluded that the TID and the Arrhenius model are both affected by the Non-
Isothermal portion of the heating process and it does not yield accurate results for high
temperatures were ramping becomes significant. Studies from this past decade and late 90s focus
mainly on the non-isothermal phase in order to present adequate dosage at higher temperatures
on the clinic [ CITATION Thr99 \l 1033 ], [ CITATION HeX09 \t \l 1033 ], [ CITATION HeX05 \t \l 1033 ].
What has been done in order to solve this problem is to divide eq. (1) for every T and sum each
time in order to get the cumulative time, often referred as CEM 43 (cumulative equivalent minutes
at T=43°C) [ CITATION Dew94 \t \l 1033 ], [ CITATION HeX09 \t \l 1033 ], [ CITATION HeX05 \t \l
1033 ], [ CITATION Thr99 \l 1033 ], [ CITATION Bho04 \l 1033 ], [ CITATION Cha10 \l 1033 ].

5. Other Models

An interesting model based on the Arrhenius model uses classical arguments of statistical
thermodynamics in deriving the Boltzmann distribution law to logically distribute the two states
a cell can be in (dead or alive) [ CITATION Fen08 \l 1033 ].
γ
−( +αt +β )
T
e
S= γ
(6)
−( +αt + β)
T
1+e

The parameters γ , α and βcan be found by using the least-squares regression when the
experiment is performed (with adequate procedure and data recollection). In other words,
compute the natural logarithm of the ratio of the cell death with respect to the cell survival for
every temperature with its respective time, and then use bilinear regression methods to find the
means for each parameter.
 Figure 5: Plot of two state model (solid line) with traditional Arrhenius model (dashed line) according to kinetic
parameters extracted from experimentation with PC3 Cells. [ CITATION Fen08 \l 1033 ]

These parameters are also much less sensitive and give a better approximation for a wider range
of temperatures [22]. It can be seen on Figure 5 that a more detailed approximation is given if
more kinetic parameters are used. The traditional kinetic parameters (activation energy and
frequency factor) show a larger error margin of 15 to 20%, while the two state model parameters
show one of 3 to 5% [ CITATION Fen08 \l 1033 ].

One can state that the integral of the injury rate (k) over the total time (constant temperature) is
equal to the natural log of the ratio of the undamaged material at the beginning of the procedure (
C t=0) to the undamaged material concentration at the end of the procedure (C t=t ) [ CITATION f

Pea92 \l 1033 ]. This relationship is based on equation 2 and 3 (Arrhenius Model).

tf −∆ E
C t =0
Ω=ln
( )
C t=tf
=∫ A e R T dt
0
g
(7)

This is the main approach used to find the kinetic parameters of different cell lines at different
temperatures [ CITATION Yan \l 1033 ], [ CITATION HeX09 \t \l 1033 ], [ CITATION HeX05 \t \l 1033 ].
By itself, this approach has the exact same limitations as the Arrhenius model due to the fact that
it is actually just a derivation of it. However, special optimization techniques have been used to
yield more accurate results with it. In order to predict the transient temperature of tissue and to
isolate thermal damage to predetermined targeted areas for treatment in a system, a minimization
algorithm was created in order to optimize the hyperthermia treatment protocol. This algorithm
was based on eq. 4 and the bio-heat transfer equation. It was experimentally proven, with
acceptable error, that the thermal damage calculated agreed with the results of the experiment
[ CITATION Gay06 \l 1033 ].
 6. Cellular Response to heat

Several studies have been done in order to gain a better understanding of the cell response to
heat. It has been stated that cancerous cells are more sensitive to heat than healthy ones
[ CITATION Ver08 \t \l 1033 ]. The main objective is to define which event causes definite cell
death and ends the cell reproduction process.

An important cell’s response to heat is the irreversible one of protein aggregation and unfolding [
CITATION Lep82 \t \l 1033 ]. Hyperthermic effects on the cell include the exposition of
hydrophobic groups and aggregation of proteins due to this exposure. The nucleus will
eventually get packed with aggregated proteins and significant DNA changes occur due to it
(disruptions of the proteins inside the nucleus cause changes in DNA’s proper functionality in
terms of replication and repair procedures) [ CITATION Rot07 \l 1033 ].

Moreover, the application of heat to a cell induces metabolic changes such as the increase of
lactate rates and decrease in pH. These alter the microenvironment of the tumor and it is yet
another cause of death [ CITATION Ver08 \t \l 1033 ].

Other systems and organelles experience injury during the heating process, these including the
cytoskeleton, the ribosome, Golgi apparatus, etc. [ CITATION HeX03 \t \l 1033 ]. Even though
there are many more studies that describe other parts of the cell that show an injury response
after heating, the heating process is thought to have a general injury effect on cells by the
community [ CITATION HeX03 \t \l 1033 ].However, the thermal insult effect on membranes of the
cells is the one considered as the general one due to its effects and its easiness to identify.

A study done on Chinese Hamster Ovary (CHO) cells had the particular objective of defining at
which physical state a cell would stop proliferating. At a state were a large degree of blebbing
was occurring (mentioned as state 4 on this study), the cell was not able to divide and it
eventually died. Therefore, it was concluded that the survival of a cell can be calculated by the
cells degree of blebbing observed after it was heated. It was suggested that blebbing occurred
with the rupture of the plasma membrane. Therefore, the critical objective that has to be achieved
in order to kill and stop cell’s division is to damage the plasma membrane [ CITATION Bor86 \l
1033 ]. A known cause of cell death is the cell’s lipid bilayer transition from an ordered state to a
chaotic liquid state when it is subjected to heat. This process interrupts the cell’s membrane
functionality and it eventually leads to its death[ CITATION Mel76 \l 1033 ].

Fortunately, possibly or nearly all of the membranes of the cell suffer some type of irreversible
damage in the range of 41-45 °C [ CITATION Bor86 \l 1033 ]. This means that higher temperatures
are guaranteed to lead damage to the membrane. Currently, a good method to measure cell
survival is to use special dyes such as Trypan blue dye that stains the cells which membrane is
damaged [ CITATION HeX05 \t \l 1033 ][ CITATION Jen09 \l 1033 ][ CITATION Bho041 \l 1033 ].

7. In-Vivo, Ex-Vivo and In-Vitro Experimentation

There has been an extensive variety of methods used for in vitro experimentation. The most
common one is heating a cell sample or tissue using a water bath. However, this method is not
very useful when the temperatures tested are as high as <50°C due to the short period of time it
takes to completely injure the specimen at these temperatures [ CITATION HeX09 \t \l 1033 ],
[ CITATION HeX05 \t \l 1033 ]. Other method such as attaching cell samples in between coverglass
slips or suspending them on small volume flasks are more useful because these require much less
time to heat up and cool down [ CITATION HeX05 \t \l 1033 ]. The best method to induce heat on
the cell is with a heating stage due to its high accuracy and high heating rates. Experimentation
with attached and suspended cells was carried [ CITATION HeX05 \t \l 1033 ] and it was concluded
that there is a difference in the kinetic parameters obtained by data from attached and suspended
cells. Further research has to be done in order to state which experimentation method, cell
medium, and cell environment resembles the biological phenomena in a better manner.

In vivo experimentation has not been as practiced as in vitro due to the fact that is it more
challenging to measure data. In-vivo experimentation usually measures prostate size, peak urine
flow , quality of life, among others are used as variables to measure the efficacy of the treatment
[ CITATION Tro03 \l 1033 ], [ CITATION Lin87 \l 1033 ]. Moreover, ex-vivo experimentation uses units
of depth, area and volume to describe the tissue’s ablation and coagulation after the heating
procedure.

Figure 6: Cross Section of Prostate lobe after in-vivo laser treatment and perineal incision. Top bar is in cm Scale
[ CITATION Sei08 \l 1033 ]

Figure 6 shows the results of a laser treatment done on Beagle dogs [ CITATION Sei08 \l 1033 ].
This method is more common for ex-vivo experimentation and it can be seen that the results can
be calculated with a high level of accuracy. Moreover, secondary effects are easier to identify
with the use of this method
Even though in-vivo measurement method might seem ambiguous, it does work if one uses it to
statistically compare procedures such as ultrasound and nanoparticles heating. A study involving
transurethral microwave thermotherapy done on 541 men was done and data was acquired
through time after the procedure. It was concluded that 45% to 50% of the patient improved their
quality of life and 35% to 40% increase their peak urinary flow rate 48 months after treatment
[ CITATION Tro03 \l 1033 ] and laser induced heat procedures have proved to be effective for
relieving bladder obstruction [ CITATION Sei081 \l 1033 ].

The main limitation in vivo and ex-vivo experimentation has is the inability to deliver an exact
heat dosage due to the lack of an accurate mathematical model, especially because vascular and
cellular level damages differ in many ways. Further study has to be done in order to combine
these two types of cell injuries in order to provide optimum heating protocols for effective
treatment.

8. Conclusion

The main purpose of this report is to briefly summarize different creations of mathematical
models, heating methods and studies that have contributed to the understanding of the
complicated procedure of hyperthermia therapy.

It was reviewed that long mild temperature hyperthermia has its advantages such as the overall
comfort of the patient, however, its efficacy is limited by the natural response of the body and it
is not useful over time.

Several some mathematical models such as the TID and the Arrhenius model were reviewed and
their flaws were shown in order to create a target for upcoming studies. The non-isothermal
phase of the heating procedure was emphasized due to its high importance. New mathematical
models have to take this phase into account in order to be useful at high temperatures, where the
procedure has been proved to be more efficient.

In order to gain a better understanding of the cell or tissue’s survival and temperature/time
relationship, it is important to study vascular and cell injury response at a greater depth. It would
mainly improve in-vitro experimentation methods and data interpretation.

In-vivo and ex-vivo experimentation has shown that this treatment has potential; however, it has
not been yet been optimized due to the lack of understanding of the heat-damage relationship.
Once again, emphasis on this relationship is the primary target that has to be studied in order to
overcome the numerous obstacles to the final objective of optimizing Hyperthermia treatments
for clinical use.
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