Ultrasound in Med. & Bto/. Vol. 19, No. 1, pp. 67-74, 1993 0301-5629/93 $6.00 + .00 Printed in the USA © 1993 Pergamon Press Ltd.

OOriginal Contribution

H I S T O L O G I C A L C H A N G E S I N R A T L I V E R T U M O U R S T R E A T E D W I T H H I G H - I N T E N S I T Y F O C U S E D U L T R A S O U N D

LILI CHEN, ? IAN RIVENS, t GAIL TER HAAR,t SHARON RIDDLER,*

C. R. HILL* a n d J. P. M. BENSTED* *Joint Department of Physics, Institute of Cancer Research and Royal Marsden Hospital, *Biological Services

Unit and *Department of Histopathology, Institute of Cancer Research, Sutton, Surrey SM2 5PT, UK

(Received 11 February 1992; in final form 24 July 1992)

Abstract--This paper reports the histological changes found in rat liver turnouts treated with high-intensity focused ultrasound. HSN fibrosarcoma, implanted subcapsularly in the livers of CBH rats, were treated using an array of ultrasound exposures. At predetermined times following treatment, the rats were sacrificed and tissue specimens were examined histologically. Evident tissue damage was confined to regions that had been given high ultrasound exposures. Within these regions ("lesions") there was no evidence of intact cells whereas in the sharply demarcated surrounding tissue there was no evidence of cell damage. Where individual ultrasound lesions had been placed in sufficiently close proximity, there was correspondingly continuous and complete cell destruction. There is suggestive evidence that tissue damage may arise through two different mechanisms: direct, primarily thermal, damage and indirect damage resulting from compromised blood supply. Under the same exposure condi- tions, normal liver cells appear to lose their morphological structure more readily than do tumour cells.

Key Words: Ultrasound surgery, Tissue ablation, Focused ultrasound surgery, Rat liver tumours, H S N fibrosar- coma, Histological changes, Tumour vascularity, Tumour cell survival, Local control of tumour.

INTRODUCTION

The potential of high-intensity focused ultrasound for spatially selective tissue destruction has been investi- gated by a number of workers over the years. It has been applied to "noninvasive" surgery in the treat- ment of glaucoma and ocular tumours in ophthalmol- ogy (Coleman et al. 1985, 1986, 1988), in the brain for neurological studies (Borrelli et al. 1986; Fry and Dunn 1956; Fry and Johnson 1978; Fry et al. 1970) and in experimental treatments of brain tumours (Britt et al. 1982; Robinson and Lele 1969).

Curtis (1965, 1972) reviewed early work carried out on liver and other abdominal tissues. The liver work in particular has been carried out with spatial peak intensities generally no higher than the range l-100 W/cm 2, although Linke et al. (1973) reported tissue destruction by high-intensity focused ultra- sound on normal rat and rabbit liver, and rabbit kid- ney (using focal intensity: 300-900 W/cm2). None of this work has concerned liver tumours.

Address correspondence to: Lili Chen.

We are carrying out research directed towards the treatment of discrete hepatic metastases (ter Haar et al. 1989). These tumours arise from other major cancers elsewhere in the body, and patients are often debilitated and unsuitable for the major operations necessary to remove the metastases. As ultrasound energy can be sharply focused within well-defined predetermined volumes, without damage to overlying tissue, this allows the possibility of using an external source of ultrasound to damage tumour cells in a strictly defined volume of the liver, without resorting to laparotomy. Experiments have been carried out on excised normal pig liver and other animal models (ter Haar et al. 1989, 1991a, 1991b), and the relationship between ultrasonic treatment parameters and lesion volume has been studied. Results show that the vol- ume of a single lesion is predictably determined by exposure time and intensity (ter Haar et al. 1989). Based on these data, ultrasound treatment of im- planted tumours in the rat liver has been performed using an array of exposures to cover the tumour vol- ume. As a result of such treatment, tumour growth delay has been observed (ter Haar et al. 199 lb). In this paper, we report the corresponding histological

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68 Ultrasound in Medicine and Biology Volume 19, Number 1, 1993

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Fig. 1. Schematic diagram of an ultrasound treatment system.

changes in rat liver tumours and surrounding normal tissue.

EQUIPMENT

A schematic diagram of the experimental ar- rangement employed in this work is shown in Fig. 1. The high intensity 1.7 MHz ultrasound focused field is produced by a plane, 10 cm diameter piezo-electric ceramic transducer (PZT4) and focused by a bi-con- cave perspex lens with focal length of 14 cm. Varying the electrical input to the transducer from 57 to 154 W results in spatial peak pressures in the range 6.4- 10.24 MPa (peak focal intensities 1.4-3.5 kW/cm2). The acoustic field has been mapped using a calibrated polyvinylidene fluoride (PVDF) membrane hydro- phone (GEC Marconi, Chelmsford, Essex, UK) (Ri- vens 1992; ter Haar et al. 1989, 1991b), and corre- sponding exposure values are given below in terms of in situ spatial peak intensity. The transducer, lens, and the sample to be treated are suspended in a bath of degassed water. A laser pointer is used to position the area to be treated at the point of peak intensity in the beam (on the beam axis). The focal region is cigar shaped and is about 18 mm long by 1.6 mm wide (FWHM: full width, half pressure maximum). The relationship between beam properties and lesion size in rat liver tissue has been reported by Rivens (1992).

METHOD

As described in detail in a previous paper (ter Haar et al. 1991 b), HSN fibrosarcoma cells were im- planted into 46 adult female CBH rats by subeapsular injection of 106 cells suspended in 0.05 mL of Dul- becco's Modified Eagle's Medium and foetal calf serum (10%) into the left lobe. One week after im- plantation, laparotomy was performed under general anaesthesia using intraperitoneal Diazepam (0.05 mL/100 g) and intramuscular Hypnorm (0.1 mL/100 g). The tumour dimensions (about 8 × 8 x 5 mm) were measured on the front and back faces of the liver lobe using vernier calipers. Twenty-nine rats were treated with high-intensity focused ultrasound and the rest were used as controls (i.e., the rats underwent the same surgical procedures, the tumour dimensions were measured but no ultrasound treatment was per- formed).

The rat to be treated was mounted vertically in a Perspex holder with a thin Mylar window of dimen- sions 5 cm x 9 cm facing the transducer. The holder was filled with degassed phosphate buffered saline at 37°C. The rat head was held well above the water with its airway maintained. The holder was placed in a tank of degassed water, as shown in Fig. 1. The tu- mour-bearing liver lobe was exposed to a single shot of 1.7 MHz focused ultrasound at an intensity in the

Fig. 2. Sections of rat liver tumour 24 h after ultrasound treatment (intensity: 3.5 kW/cm2; focal depth: 6 mm; exposure duration: 10 s; exposure separation: 2 mm). (a) PMNs around the boundary region between treated (left) and untreated (right) normal liver area (magnification: x 240). (b) Ultrasonically damaged tumour showing cell

debris, pyknotic nuclei and ultrasonically induced "implosion cysts" (IC) (magnification: x 240).

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Histological changes in rat liver tumours • L. CHiN et al. 71

range 1.4 to 3.5 kW/cm 2, with exposure duration from 5 to 15 s. These conditions were chosen to en- able comparison with our own early work (ter Haar et al. 1989). Depending on the ultrasound intensity, the beam focus was set at a depth between 0 and 6 mm below the liver surface in order to ensure that the le- sion covered both the front and back surfaces. The lateral exposure separation was set at 2 mm, a value derived from our previous experimental data (ter Haar et al. 199 lb). A margin of normal tissue about 2 mm wide around the visible tumour was also treated. During the ultrasound treatment the skin behind the exteriorized liver was protected from damage using a cushion and a reflector. The cushion comprised a thin rubber glove finger containing degassed water. The reflector was made from several sealed layers of air- filled aluminium foil wrapped in damp gauze. The liver surface was flattened using a Mylar window. After the treatment, the liver was replaced into the abdomen. The abdominal wound was closed (with a 4-0 chromic suture) and the animal was allowed to recover.

Rats (both treated and control) were sacrificed from 1 day to 2 weeks following treatment. The livers were removed and fixed in methacarn. Three to four ~m thick histological cross-sections, taken at 1 mm intervals through the lobe, were stained with haemo- toxylin and eosin (H & E) and examined microscopi- cally. In histological examination particular attention has been paid to evidence of cell integrity and of mito- sis (taken as indicative of cell viability).

RESULTS

Gross changes

The tumour nodules could be seen clearly before the ultrasound treatment. The treated normal tissue around the tumour usually showed tissue blanching of varying degrees, as did the treated tumour, and it could be difficult to distinguish treated from the un- treated tumour by naked eye. In some cases, however, the treated area did not show complete blanching. This usually meant that the lesion array had not cov- ered the whole treatment area. Sometimes ultrasoni- cally induced holes (about 0.5-1.5 mm in diameter) were seen in the treated normal liver area around the tumour, whereas holes in the treated tumour area were uncommon.

Histological changes Twenty-four hours after the ultrasound treat-

ment, the treated area could be distinguished from the untreated area by colour on a histological section. The lesion was "paler" (i.e., it had taken up less stain). Inflammatory changes appeared with the presence of polymorphonuclear leucocytes (PMNs) around the boundary between the treated and untreated area (Fig. 2a). The ultrasonically damaged tumour area showed cell debris, pyknotic nuclei, distorted tumour cells and the presence of "implosion cysts" (Fig. 2b). These were irregularly shaped holes of about 0.02-0.2 mm diameter, appearing mainly in the central por- tion of a lesion of single exposure. The mechanism for the formation of these "implosion cysts" is thought to be acoustic cavitation (i.e., they are produced by cavi- tation within the tissue, with consequent disintegra- tion of nearby tissues, the resulting void then becom- ing filled by liquid). It could also, however, have been the result of vaporisation within the tissue because of the high temperatures achieved (for intensities 1.35- 2.14 kW/cm 2 and exposure duration 5-10 s, the tem- perature at focal peak in the tumour is 70°C-100°C, measured using a 0.2 mm diameter needle thermo- couple). In some cases, a few large holes (0.5-1.5 mm in diameter) were also seen in the treated area and corresponded to the holes observed on the liver sur- face during the treatment. Normal liver tissue treated with ultrasound showed dissociated lobular structure with the presence of "implosion cysts." Pyknotic nu- clei, cell debris, and injured hepatic cells with cyto- plasmic vacuolation were also seen. Compared with damaged tumour cells, damaged normal liver cells had taken up stain less well. In some cases, small is- lands of surviving tumour were found within the treated tumour volume, which showed an arrange- ment of morphologically intact tumour cells with mi- totic activity.

Seven days after treatment, granulation tissue was formed with the presence of young fibroblasts and new capillaries in the boundary region between the ultrasonically treated and the untreated zone (Fig. 3a). PMNs migrated from the boundary region to- wards the centre of the damaged area. Large numbers of dead PMNs appeared in the treated volume (Fig. 3b). Cell nuclei were generally recognisable in dam- aged tumour areas but not in damaged normal liver areas. No intact cells were found in the ultrasonically

Fig. 3. Sections of rat liver tumour one week after ultrasound treatment. (a) Young fibroblasts and new capillaries in the boundary between treated (left) and untreated (right) normal liver area (intensity: 1.7 kW/cm2; focal depth: 3.0 mm; exposure duration: 10 s; exposure separation: 2.0 ram; magnification: × 150). (b) Large numbers of dead PMNs in the treated volume (intensity: 2.14 kW/cm2; focal depth: 3.0 mm; exposure duration: 5 s: exposure

separation: 2.0 mm; magnification: × 240).

Histological changes in rat liver tumours • L. C-MEN et al. 73

treated normal tissue volume but, in some cases, small islands of tumour cells in the treated tumour volume appeared to be morphologically intact. Divid- ing tumour cells were observed among the intact tu- mour cells, and scattered "implosion cysts" could still be found.

Two weeks after treatment, the boundary region was generally replaced by proliferative repair tissue (Fig. 4a), while the damaged area was only partially so replaced. The number of inflammatory cells was markedly decreased, and nuclei of damaged tumour cells were still visible in the damaged tumour area. Again, in some cases, small islands of viable tumour could be seen in the treated volume (Fig. 4b) and di- viding tumour cells could be observed. The ultrasoni- cally induced "implosion cysts" were still seen in the treated volume. Substantial tumour growth delay was achieved in most animals treated compared with the control group.

DISCUSSION AND CONCLUSIONS

It has been shown that small regions of tumour cells can be completely ablated by single ultrasound exposures. For the destruction of a large tumour vol- ume, however, it is necessary to place multiple lesions side by side. In this work, the liver tumours were treated using an array of exposures. Following some treatments, small islands of cells in the treated tu- mour volume appeared to remain viable. This may be due to failure of the lesion arrays to cover the whole tumour volume. In a separate investigation on ex- cised pig liver (Chen et al. 1993a), it has been found that if the lesion separation is large, some tissue is left untreated and if the lesions are too close to each other, their length is reduced and they migrate towards the surface. That is, one lesion can affect the production of the next lesion, if the separation is too small. We term this phenomenon "lesion-to-lesion interaction" (LLI). So far no ideal separation value has been found in excised pig liver. Because the ultrasound lesion is cigar-shaped it is difficult to define an appropriate value for the exposure separation with which the treatment volume can be covered completely by le- sions, whilst avoiding LLI. However, some rat treat- ments have resulted in complete destruction of liver tumours. This reduced effect of LLI on the in vivo treatment is believed to be a consequence of compro- mised blood supply (see below).

In the area of observable tissue damage (both in normal and tumour tissue) two different appearances can be seen. One area, which may be immediately outside the treated volume, is characterized by dead cells, which are relatively evenly distributed, uniform in shape and apparently intact but less well stained. The morphological features of the liver lobular struc- ture could be seen clearly, with no sign of ultrasoni- cally induced "implosion cysts" in this damaged area. The other area shows more evidence of cell disruption or destruction, together with the presence of "implo- sion cysts." We believe that the latter type of damage is a direct result of the ultrasonically induced local temperature rise and cavitation, whilst the former may result "indirectly" from compromised blood supply after the ultrasound treatment. This indirect ultrasound damage is similar to that reported by Linke et al. (1973) who observed areas of secondary infarction in normal rat liver treated with high-inten- sity focused ultrasound (focal intensity: 312 W/cm 2, frequency: 2 MHz).

There have been reports on the effect of blood perfusion and heat transfer in the hyperthermic treat- ment of cancer (Billard et al. 1990). It was shown that the advancing margins of the tumour have good vascu- laxity, perhaps even better than normal tissue, and thus with high oxygenation, good nutrition, and rapid heat transfer (Short and Turner 1980). These periph- eral tumour cells are the most rapidly growing part of the cancer and have the best cooling.

In this study, a cushion was placed between the exteriorized liver and the animal skin, and a Mylar window was used to flatten the liver surface during the ultrasound treatment. The blood flow in this case was reduced to 15% of the normal value (measured nonin- vasively using a laser Doppler blood flow monitor). It has been found, however, from comparisons between the lesion size without blood flow (i.e., both the blood hepatic artery and portal vein were ligated) and with reduced blood flow (i.e., only the hepatic artery was clamped and the blood flow was reduced to 15% of the normal value), that with the ultrasound exposure con- ditions chosen for this study the effect of this reduced blood flow on lesion size is negligible (Chen et al. 1993b).

There have been reports that certain experimen- tal tumours and some spontaneously occurring tu- mours may be more sensitive to hyperthermia than surrounding normal tissue (Cavaliere et al. 1967).

Fig. 4. Sections of rat liver tumour two weeks after ultrasound treatment (intensity: 2.7 kW/cm2; focal depth: 5.0 mm; exposure duration: 10 s; exposure separation: 2.0 ram). (a) The boundary between the treated (left) and untreated (right) normal liver area which has generally been replaced by proliferative repair tissue (magnification:

× 150). (b) Intact tumour cells within a treated area (magnification: × 240).

74 Ultrasound in Medicine and Biology Volume 19, Number 1, 1993

O u r exper iments , however , d o not conf i rm this sug- gestion. O n the cont ra ry , we have obse rved that , close to the b o u n d a r y be tween the t rea ted a n d un t r ea t ed tissue, there appea r s to be a region o f i n t e r m e d i a t e level o f exposure where, a l though no n o r m a l l iver cells r emain , there is ev idence (both f rom m o r p h o l - ogy and cell d iv is ion) tha t some t u m o u r cells m a y survive. The reason for this a p p a r e n t grea ter res i l ience o f t u m o u r cells is no t clear.

In s u m m a r y , h igh- in tens i ty focused u l t r a sound has been used to d a m a g e ra t l iver t umour s . Large areas o f u l t rasonica l ly d a m a g e d t u m o u r cells can be

ident i f ied one day af ter t r ea tmen t , bu t smal l i s lands o f morpho log ica l ly in tac t t u m o u r cells have been found wi th in the t rea ted v o l u m e f rom l to 14 days af ter

t r ea tmen t , though a b n o r m a l mi to t i c figures can be seen, suggesting a l imi t ed l i fe-span for these cells. Cell d ivis ion has been observed a n d t aken as a measu re o f t u m o u r survival . T u m o u r growth de lay has been achieved in the t rea ted v o l u m e fol lowing u l t r a sound exposure . " I n d i r e c t " d a m a g e to n o r m a l l iver ou ts ide the t r e a t m e n t v o l u m e m a y a p p e a r as a resul t o f lack o f b l o o d supply. Sat is fac tory assessment o f the ex ten t o f long- te rm local con t ro l by this t r e a t m e n t has not, how- ever, been possible with ou r presen t a n i m a l t u m o u r model , which exhibi ts r ap id onse t o f severe me tas t a t i c disease.

Acknowledgements--This work was supported by programme funding jointly from the UK Cancer Research Campaign and Medi- cal Research Council. We are grateful to Sue Clinton for her expert technical support.

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