Adjunctive Intraoperative Photodynamic Therapy in an animal model

M.G. Dilkes MS FRCS FRCS(ORL)

ENT Department, St Bartholomew’s Hospital, West Smithfield,

London EC1Y 0DT, U.K.

Keywords:

Photodynamic Therapy, Adjunctive, Surgery, Foscan

Summary

This experiment was performed by delivering intraoperative

photodynamic therapy to the surgical bed after excision of a

locally invasive, malignant tumour. The aim of the study was to

find out if this led to a reduction in the local recurrence rate

of the tumour. This was a blind, controlled experiment. The

treatment arm involved surgical removal and intraoperative

photodynamic therapy. The control groups were surgery alone,

surgery and photosensitiser diluent, and surgery and laser light.

The results show a statistically significant reduction in the

local tumour recurrence rate up to 4 weeks after surgery (the

experiment end point) in the photodynamic therapy (PDT) group

when compared to the control groups.

Introduction

The local recurrence of malignant disease after surgical removal

is a major cause of morbidity and death. The cause of local

recurrence is usually due to the incomplete removal of tumour, or

burst and spill of malignant cells onto the operative bed, during

surgery. The latter scenario is thought to occur when a viable

cell population is spilt. This cell population re-establishes a

local blood supply, and can continue to grow. Local recurrence is

a particular problem in the treatment of head and neck cancer,

where there is a high rate of positive (tumour involved) margins

due to the proximity of vital structures, and a confined

operative space, which, when faced with large tumours and

necrotic centers, can lead to tumour rupture and spillage.

Photodynamic therapy works by the intracytoplasmic activation of

a previously inert drug (a photosensitiser). Activation is by

light of a specific wavelength. This process causes singlet

oxygen to be released, with consequent intracellular failure and

cell death. This effect mimicks apoptosis, and leaves surrounding

tissue generally intact, although some local damage does occur

when treating a tumour in a bed of normal tissue. The relative

lack of local tissue damage is due to the nature of the healing

that occurs after PDT, and because tumours tend to contain more

photosensitiser than normal tissue does.

The potential value of adjunctive intraoperative photodynamic

therapy (AIOPDT) lies in the fact that by using powerful new

second generation photosensitising drugs and new lasers,

efficient adjunctive treatment can be delivered to the operative

bed following malignant tumour excision, in around 30 minutes.

This means that there is little additional to the overall

operating time, with the potential benefit of reducing the local

recurrence rate.

Material and Methods

This experiment was designed to test the hypothesis that AIOPDT

is effective in reducing the local malignant tumour recurrence

rate in an animal model. The aim of this study was to determine

whether the tumour model HSN fibrosarcoma, which bears a close

resemblance to our target clinical tumour, mucosal squamous cell

carcinoma of the Head and Neck , shows a statistically

significant reduction in local recurrence with the treatment

groups when compared with the control group.

Animal model,Tumour

A rat fibrosarcoma tumour called HSN was used. This cell type was

chosen because it causes an aggressive, locally invasive tumour

that does not widely metastasise or encapsulate. This means that

there should be a reasonably high local recurrence rate if

tumours are removed without wide excision. The animals used were

Chester-Beatty hooded rats, in which this tumour grows.

Tumour preparationFibrosacroma cells were obtained from the Chester-Beatty research

laboratory, The Royal Marsden Hospital, Fulham Road, London.

These were inoculated into the dorsal spine musculature of

Chester Beatty hooded (CBH) rats, in order to grow a solid

tumour. Using a freshly killed donor animal with tumour

previously grown, the tumour periphery (containing viable, non-

necrotic tumour) was excised. Small pieces of tumour

approximately 1mm x 1mm x 1mm were formed by dividing up the

donor tissue. These were then implanted under direct vision into

the flank musculature of further adult CBH rats, under sedation

with Midazolam. Both flank areas were inoculated. Following this

the skin was closed with clips, and the animals were recovered.

Photosensitiser

The photosensitizing drug used was meta-

tetra(hydroxyphenyl)chlorin, mTHPC, or FoscanR. This drug is a

second generation photosensitiser, created to have enhanced

properties over 1st generation compounds. The drug dose of FoscanR

used was 1.0 milligrammes per kilogramme (mg/kg) body weight

delivered intravenously via the tail vein. This compares with the

human dose of 0.15 – 0.3 mg/kg, the higher dose in rats being a

function of their surface area to weight ratio. FoscanR was

obtained from Scotia Pharmaceuticals, Stirling, U.K., as a gift.

It was stored as a sterile powder in a fridge, and reconstituted

fresh with 1g ethanol and 1g polyethylene glycol 400 made up to 5

ml with sterile water for injection, to make up a solution of 0.5

mg/ml. When reconstituting the drug for injection, meticulous

attention was taken to ensure that the drug had gone into

solution. Invariably this meant 2 or 3 minutes of vigorous

shaking, ensuring no residue was visible on the bottom of the

tube used for reconstitution. The interval between injection of

the drug and delivery of activating laser light (the drug-light

interval, DLI) was 96 hours, as this appears to be a good drug-

light interval (DLI) for normal tissue selectivity and a powerful

tumouricidal effect (Ris et al 1993, Van Geel et al 1993).

Light parametersThe light dose given for photoactivation was 20 joules/square

centimetre (j/cm2) at an intensity of 100 milliwatts/cm2 (mW/cm2).

Laser light was used, at a wavelength of 652 nm, corresponding

with a high peak of absorption for FoscanR. Red light at this

wavelength has good tissue penetration characteristics, which are

beneficial when treating cancer, so as to treat a large volume of

potentially involved tissue. The light intensity was measured

using a calibrated integrating sphere (type INS250), a photodiode

with a flat response filter (type SED100) and a radiometer (type

IL1700), all from International Light Inc., Mass., USA, and was

delivered via a flexible quartz fibre and microlens (Quadra Logic

Technologies, Vancouver, Canada). The laser used was a copper

vapour CU15 system pumping a Rhodamine dye laser, both from

Oxford Lasers Ltd., Abingdon Rd., Oxford, U.K. These light

parameters were used in the first studies with the mTHPC, which

showed excellent necrosis of malignant tissue (Ris et al 1991).

Surgical Technique

Under a general anaesthetic of Hypnorm and Midazolam a single

midline incision was made on the dorsum, between the 2 tumours.

Each tumour was identified and measured separately. Using a No.15

scalpel blade fitted to a scalpel blade holder, the tumour was

excised close to, but not macroscopically involving, its margin.

Macroscopic clearance was confirmed by eye in all cases before

the study could progress. Following adjunctive treatment, the

skin was closed with clips and the animal recovered.

Experimental studies

There were 2 preliminary and 1 definitive studies

The aims of the 2 preliminary studies were to determine:

1) What the feasible maximum size of the tumours was before the

perioperative mortality rate became unacceptable. This was

determined since size of tumour is likely to be directly

proportional to recurrence rate. We wanted as high a target

recurrence rate in controls as possible.

2) What the growth characteristics of the tumour were, in order

to predict the day at which tumours would reach the previously

determined optimum size.

Study 1

Initial studies were performed to determine the optimum size of

tumour in terms of local recurrence rate after macroscopic

excision, and survival of the animal. This was necessary because

the larger the tumour, the more likely it is to recur locally due

to inadequacy of excision, even if the margins are

macroscopically clear. However, resection of very large tumours

will result in a critical loss of fluid, and death of the animal.

Therefore it was necessary to know what the safe maximum size of

the tumours was. Tumours were grown bilaterally until they

reached a size of 1cm, 2cm or 3cm largest diameter as measured

externally with the animal awake. At this stage a general

anaesthetic was administered, and both tumours removed locally,

but with full macroscopic clearance (i.e. no visible tumour

left). The skin was then closed and the animals recovered.

Animals were closely monitored postoperatively for signs of

distress. If any were shown the animals were immediately killed

by a schedule 1 technique. Other animals succumbed shortly after

surgery. All non-survivors were recorded. The survivors were

killed 2 weeks after surgery and the surgical sites examined for

signs of local recurrence, which was recorded.

Study 2

This was performed to determine the predictability of tumour

growth prior to photosensitisation. Once the optimum tumour size

had been determined in study 1, it was important to have an idea

regarding the growth rate of the tumour, since the decision had

been taken to sensitise 4 days prior to treatment. Clearly, a

tumour measured at the safe maximum size at injection would be

significantly above that size 96 hours later, given a doubling

time of x hours, where x is any number less than around 300. Ten

tumours were grown in 10 animals, the same way as above, but

unilaterally. Tumours were implanted on day zero. Alternate daily

measurements of the maximum tumour diameter were taken.

Measurements were taken with the animal awake using a steel ruler

with 1mm gradations. Once the tumour reached around 20mm diameter

(the chosen size for the 3rd experiment on the basis of the first

preliminary study), the animal was killed.

Once this data had been gathered, a blind, prospective controlled

study (Study 3) was performed to test the hypothesis that AIOPDT

with mTHPC reduces significantly local recurrence after

macroscopic surgical excision in the HSN rat fibrosarcoma model:

Study 3

4 groups of 30 animals were included in the experiment. These

were:

1) Surgery and AIOPDT.

2) Surgery, mTHPC diluent and light only at the above parameters

(no drug).

3) Surgery and drug only

4) Surgery only.

Two independent tumours were grown in each animal, on the back,

to the left and right side of the midline, well separated, using

the method described above. After 10 days, the animals were

admitted into one of the 4 treatment groups. They were kept in

cages of up to 5 animals each, cages were coded according to the

treatment group each animal was in. Each cage was coded following

randomn selection by biological services staff. Those animals due

for drug injection (groups 1 and 2) were weighed and injected

intravenously via the tail vein with 1mg/kg mTHPC that had been

freshly prepared from powder. 4 days later a general anaesthetic

was administered to all animals and the tumours exposed. The

largest diameter was measured. Any tumour of 20mm +/- 2mm largest

diameter was included in the study and resected so that no

macroscopic tumour was left. Tumours beyond these criteria were

not used. At that stage animals due for light administration

(groups 1 and 2) were identified and 20 joules/cm2 of 652 nm

laser light of 100 milliwatts/cm2 intensity was given. The light

was produced by a copper vapour laser pumping a rhodamine dye

laser (Cu 15, Oxford Lasers, Abingdon Rd., Oxford, U.K.).

In all cases a 2.5 cm diameter laser spot was used with the

original tumour site in the middle, giving a margin of normal

tissue treatment, which was approximately 15 % of the original

tumour size. The laser spot diameter was measured using a steel

ruler with 1 mm gradations. The microlens was held steady over

the treatment site by holding it with a clamp fixed to a retort

stand. Following this the skin was closed and the animals were

recovered. At this stage the animals were numbered by puncturing

and marking the ears with Evans Blue dye. Postoperatively the

animals were observed closely. Any animal in distress was killed

immediately and this was recorded. Any animal succumbing to the

immediate effects of surgery was also recorded.

Once obvious macroscopic and potentially distressing tumour

recurrence had occurred in one animal (the experimental end

point), all animals in the groups treated in that session were

killed. This occurred up to 4 weeks post surgery. The flank areas

were opened and examined for tumour recurrence. Any suspicious

areas that were not obviously tumour were biopsied and sent for

histological confirmation of the result, otherwise the diagnosis

of tumour recurrence was made by sight alone. At this stage and

during recording of the results the examining surgeon was blind

to the group the animals were in, only the experiment had been

completed were the codes broken.

Number ofrecurrences

Surgery(%)

Laser Light alone (%)

Photosensitising drug alone (%)

Surgery and drug-light combination = AIOPDT (%)

0 6 (21) 10 (30) 6 (18) 18 (50)

1 13 (44)

9 (27) 12 (36) 10 (28)

2 8 (28) 13 (39) 15 (45) 5 (14)

Death 2 (6) 1 (3) 0 3 (8)

Total 29 33 33 36

Statistical Analysis

A Kruskal-Wallis one way analysis of variance of ranks was used

to compare the 4 treatment groups. There were significant

differences between the 4 groups (p=0.01). Examination of the

distribution of outcome in the 4 groups suggested that the first

3 groups had similar outcomes. A Kruskal-Wallace anova test was

used to compare these first 3 groups and found no significant

difference (p=0.6). The 4th group (AIOPDT) was then compared with

these 3 groups combined. The Kruskal-Wallace anova test result

demonstrated a significant difference (p=0.002) between the

AIOPDT group and the other 3 groups combined.

A rank transformation was also used on the data, and a two way

analysis of variance on the ranks with the factors “treatment

group” and “day” was performed. There were no significant

differences between the 7 days on which the study was performed

(p=0.7).

Discussion

Similar adjunctive intraoperative preclinical studies have been

performed in the past (Davis et al 1990, Van Hillsberg et al,

Abulafi et al). These were different to this study, both in terms

of study design, and tumour type and sensitiser used. In

particular, the studies by Davis, and Van Hillsberg did not have

control groups as in this study. Abulafi used a different tumour

model and photosensitiser have shown this theory to be true,

although the structure of these studies was not fully blind, and

some potential control groups were missing.

The results show a statistically significant reduction in the

local tumour recurrence rate with the treatment group as compared

to the control groups. Although there were more deaths

postoperatively in this group, this was not a significant

finding, and may just represent the relatively small numbers

tested. The variation of numbers within the groups represents

excluded tumours that were outside the treatment parameters. The

control groups tested the hypothesis that the drug and diluent is

toxic to tumours without light (drug only group), although even

in this group there will have been a mild photodynamic effect due

to background theatre illumination and theatre lights during the

excision. However, the total light dose given during the

relatively short (approximately 5 minutes) time that it took for

excision of both tumours is so low that this effect is unlikely

to have been significant. The control groups also tested the

hypothesis that the drug diluent is an active photosensitising

agent (light and diluent only group), although the hypothesis

that 652 nm laser light at non-thermal intensity could be

cytotoxic was not independently tested. This is extremely

unlikely however since there are many publicly available lasers

at around this wavelength (laser light pointers) that have been

shown to be totally safe except when shone directly into the

eyes. The fact that there was no significant difference between

any of the control groups including the surgery alone means that

these hypotheses are invalid.

A study such as this is only valid if the principle that local

tumour recurrence is due to residual disease is agreed. There are

other theories regarding why tumours recur in the excision bed,

such as further metastases from the primary area, or new primary

disease growing in the tumour bed. Studies have shown that after

a macroscopically complete excision, if the tumour bed is washed

with cell growth medium and incubated in the correct conditions

for growth, viable tumour cells can be grown (Harris and Smith

1960). These cells almost certainly come from the excised tumour

area, either due to "burst and spill" or unwittingly cutting

through tumour during the excision, and contaminating the

surgical instruments (Beahrs and Barber 1962). What is not known

is whether these are viable or not in the clinical situation.

Disease recurrence due to residual tumour can also occur because

of involvement of vital structures rendering the tumour

inoperable, or because tumour margins or not clear due to the

disease growing to the defined limits of the operation (Olcott et

al 1981).

Other adjunctive intraoperative methods have been tried in order

to reduce the local recurrence rate of tumour after Head and Neck

surgery. These include intraoperative external beam radiotherapy,

which was logistically very difficult, time consuming and

damaging to the carotid artery (Freeman et al 1990), and washing

the tumour bed with cytotoxic agents, including distilled,

sterile water which has not been shown to be effective,

presumably because there is little penetration of effect into the

microscopic cracks and crevices on the tumour bed where viable

tumour clumps may lodge.

This tumour model bears a reasonable relationship to the proposed

clinical treatment, namely metastatic neck disease treated by

radical neck dissection. Neither are primary tumour sites, the

animal model being an implanted tumour graft, the clinical model

being a proliferating clump of tumour cells shed from the primary

site into lymphatic channels and caught in the draining lymph

glands. The main difference apart from site of growth is in the

fact that the clinical model lies in a lymph gland, the animal

model in normal surrounding muscle, although this is important

since the tumour is growing in its natural environment, being a

connective tissue tumour (Bown S.G. pers. comm.). It thus is a

more natural model than others used for AIOPDT studies, such as

neuroblastoma in muscle (Davis et al 1990) or adenocarcinoma in

subcutaneous tissue (Abulafi et al 1994). However, by the time

most secondary tumours in the neck have been detected, they have

reached a size of at least 1cm diameter, and have usually

destroyed the lymph node they lie in. The adjacent tissue to most

of the lymph nodes in the neck is mainly muscle, particularly in

the lymph node bed, and fat, which tends to lie superficially.

Thus even in the human situation, the tumour lies in close

proximity to the muscle. This is even more so following radical

neck dissection, since the superficial tissue (mainly fat,

internal jugular vein and the sternocleidomastoid muscle) and

tumour is removed, the deep musculature is left untouched. Thus

the treatment site for AIOPDT in both cases would be a muscle

bed.

The tumour type used, HSN fibrosarcoma was also similar to the

human model: squamous cell carcinoma of the upper aerodigestive

tract. This is because it is a locally invasive, non-

encapsulating tumour (see picture). Clearly an encapsulating

tumour would be easy to shell out with little chance of local

recurrence, whereas a tumour microscopically invading into the

surrounding tissue will recur unless a wide block of apparently

normal tissue is removed with the specimen, which was not done in

this case. The tumour was removed very close to the main tumour

lump, although macroscopic clearance was complete in all cases.

Therefore it is more likely that microscopic tumour residue

occurs, leading to a local recurrence rate in the control groups

of around 70%. The initial studies on survival after excision

also showed this - it would have been useful to have had an even

higher local recurrence rate as occurred with the 3 cm diameter

tumours, but the postoperative mortality rate made this

unacceptable.

The scope of procedures suitable for AIOPDT in surgery is much

greater than just radical neck dissection . It may also have a

role in general surgery (Herrera-Ornelas et al 1986, Nambisan

R.L. et al 1988, Abulafi et al 1993), neurosurgery (Muller and

Wilson 1995) and thoracic surgery (Ris et al 1993). Conservation

surgery in the neck, in vogue since it seems to reduce

postoperative morbidity (Bocca 1975) could be made safer by

treating those retained structures such as the accessory nerve

with PDT to mop up any residual tumour around such structures.

The fact that red light penetrates tissue significantly means

that structures such as the accessory nerve can be completely

treated by illuminating onto and through the nerve. The same may

be true of other vital structures in the neck such as the carotid

tree, although the fact that this is of quite large diameter and

filled with blood means that complete penetration and treatment

of the medial surface is unlikely to be effective. Prior to

clinical studies, safety work is therefore needed, particularly

regarding the effect of PDT on arterial structures.

Conclusion

The scope of adjunctive intraoperative photodynamic therapy is

large, in the Head and Neck as well as other areas. This and

other studies have successfully demonstrated, to the satisfaction

of statisticians, the principle that this treatment reduces the

local recurrence rate of tumour following macroscopically clear

excision to be true on a preclinical basis.

References

(INCOMPLETE)

Abulafi A.M., DeJode M.L, Allardice J.T., Ansell J.K, Williams N.S. (1997) Adjuvant intraoperative photodynamic therapy in experimental colorectal cancer using a new photosensitiser. Br. J. Surg.;84(3):368-71

Van Hillegersberg R., Hekking-Weijma J.M., Wilson J.H.,

Edixhoven-Bosdijk A. and Kort W.J. (1995) Adjuvant intraoperative

photodynamic therapy diminishes the rate of local recurrence in a

rat mammary tumour model. Br. J. Cancer;71(4):733-737