Breast cancer is the most common malignancy in women and, each year, more than 1 million
new cases of breast cancer are diagnosed worldwide (McPherson et al, 2000). Radical
and modified radical mastectomy including axillary lymph node dissection has long
been regarded as appropriate therapies. During the last two decades, significant advances
have been made in the development of early detection modalities and therapeutic methods.
Breast conservation surgery, combined with radiotherapy, chemotherapy, and hormonal
therapy, is performed with increasing frequency in patients with early-stage breast
cancer. The more from the mastectomy toward breast conservation therapy has not changed
long-term survival rates of patients with breast cancer (Veronesi et al, 1993; Fisher
et al, 1995; Jacobson et al, 1995; Curran et al, 1998).
In spite of this progress, much remains to be achieved, and major new therapies will
be required to keep on the fight against breast cancer. Recently, cryoablation (Staren
et al, 1997), laser (Robinson et al, 1998; Dowlatshahi et al, 2000), radiofrequency
(Bohm et al, 2000; McGahan et al, 2000), as minimally invasive modalities, have been
studied to explore the possibility of ablating breast lesions. However, these alternative
treatments require at least percutaneous access and only destroy small lesion in the
management of breast tumours.
High-intensity focused ultrasound (HIFU) is a noninvasive technique for the thermal
ablation of solid tumours. Ultrasound (US) beam can be focused and transmitted through
solid tissues within the body. This allows the possibility of using an extracorporeal
source of US for therapeutic purpose. With real-time image guidance, HIFU could noninvasively
induce complete coagulative necrosis of a target tumour, without requiring surgical
exposure or insertion of instruments into the lesion. These advantages make it one
of the most attractive potential therapies for the localised treatment of tumour.
To our knowledge, most of the studies on HIFU have been dealing with animal experiments
in which HIFU has induced target lesions at depth in tumour tissues and normal organs,
including liver, kidney, prostate, bladder, and soft tissue (Chapelon et al, 1992;
Chen et al, 1993; Sibille et al, 1993; Prat et al, 1995; Rowland et al, 1997).
Up to now, in the clinical application of HIFU ablation for human breast tumour, Hynynen
et al (2001) reported that 11 breast fibroadenomas in nine patients were treated with
HIFU. Eight of the 11 lesions treated with HIFU demonstrated complete or partial ablation
response. No adverse effects were detected, except for one case of transient oedema
in the pectoralis muscle 2 days after therapy. Huber et al (2001) used MRI-guided
HIFU to treat a 56-year-old patient with breast cancer. Postprocedural pathologic
examination indicated that HIFU induced lethal and sublethal tumour ablation without
damage to the surrounding healthy tissue or systemic effects. However, to our knowledge,
no randomised clinical trial exists that explores the efficacy and safety of HIFU
in the treatment of patients with breast cancer, with emphasis on the assessment of
histological changes in the treated tumour. In this paper, we studied the efficacy
and safty of HIFU treatment in a randomised controlled trial for the ablation of patients
with localised breast cancer. The purpose of this study was to investigate the efficacy,
safety, and feasibility of HIFU ablation in the treatment of patients with localised
breast cancer. This paper reports the work in progress on this noninvasive modality,
and it is not our intention to compare it with other breast conservation therapies
for the treatment of patients with early breast cancer at present.
MATERIALS AND METHODS
HIFU therapeutic system
As described in detail previously (Wu et al, 2001a, 2002), the HIFU therapeutic system
used in this clinical trial of breast cancer treatment principally consists of a diagnostic
US device, units for computer automatic control, six-direction movement, and a therapeutic
planning system, a US generator, integrated US therapy transducers, and a degassed
water circulation unit (see Figure 1
Figure 1
JC-HIFU therapeutic system for tumour (Chongqing HAIFU™ Technology Company, People's
Republic of China).
). The therapeutic US beam is produced by a 12-cm diameter PZT-4 piezo-ceramic transducer
with a focal length of 90 mm, operating at a frequency of 1.6 MHz. The focal region
is ellipsoid, with dimensions of 3.3 mm along the beam axis and 1.1 mm in the transverse
direction.
An AU3 US imaging device (Esaote, Genoa, Italy) was used as the real-time imaging
unit of the system. This imaging probe (3.5–5.0 MHz) is situated at the centre of
the HIFU transducer for real-time guidance during HIFU procedure. The US beams of
the therapeutic transducer and the imaging probe completely overlap, so that the longitudinal
axis of HIFU beams is in the 2D US imaging plane. The integrated transducer is attached
to electric motors, and can be moved smoothly in the six directions with millimetric
precision. Through computer control, the imaging transducer was placed either against
the skin or at a distance from the skin in the water for pretreatment imaging. However,
during HIFU ablation, the imaging probe was usually situated in the water apart from
the skin.
Patients
A total of 48 women with biopsy-proven breast cancer were selected for this clinical
trial phase II. Before the beginning of this study, the protocol design was approved
by the ethics committee at our university. In accordance with the specification stipulated
by the Helsinki Committee, an informed consent form was signed by each patient at
the time of enrollment after the principles of HIFU thermal ablation had been completely
described. The selection criteria were as follows: histologically proven invasive
breast cancer (T1-2, N0-2, M0); single palpable tumours no greater than 6 cm in diameter;
the lesion boundaries visualised with color Doppler US imaging, circumscribed at least
more than 0.5 cm from skin or rib cage, and more than 2 cm from nipple. All patients
were older than 18 years and had no breast implants. They had stable haematogenic
parameters, and no history of active myocardial infarction within the past 6 months.
They were randomised to two treatment groups: the control group (n=25), in which modified
radical mastectomy was performed without any intervention prior to surgery; or the
HIFU group (n=23), in which extracorporeal in situ ablation of the breast cancer with
HIFU was followed by modified radical mastectomy within 1–2 weeks. A 2-week follow-up
after HIFU treatment was performed to evaluate the potential side effects of HIFU
such as skin burns, local pain or discomfort, mammary oedema, haemorrhage or infection,
and fever. After surgery, all patients received relevant doses of local radiation
therapy, conventional chemotherapy, and hormonal therapy, as part of their adjuvant
treatment.
Preoperative clinical assessments included the patient’s history, a physical examination,
haemotology tests, electrolytes, renal, and liver function tests. Color Doppler US
imaging of the diseased breast, with a 10 MHz linear array probe (Q-2000, Siemens,
Erlangen, Germany), was performed by a radiologist. Direct visualisation of blood
flow within the lesion was imaged by adjusting color setting for optimal slow flow
detection. If a patient was recruited to receive the thermal ablation on the basis
of the images, US-guided core needle biopsy was performed to confirm histological
diagnosis. Technetium bone scanning and a chest radiograph were required for the each
case. A comparison of the eligible patients’ characteristics in both groups, including
age distribution, tumour size, histologic diagnosis, node status, and TNM classification,
is shown in Table 1
Table 1
Characteristics of eligible patients in both groups
Characteristics
Control group
HIFU group
No. of patients
25
23
Age (years)
45.5±1.2
46.5±1.7
Tumour diameter (cm)
3.5±0.23 (1.8–5.6)
3.1±0.79 (2.0–4.7)
Histology
Invasive
21
21
Noninvasive
4
2
Node status
N0
13
12
N1
6
5
N2
6
6
TNM
I
2
2
II
22
21
III
1
0
. Three of 23 patients in HIFU group received one pre- and postprocedural magnetic
resonance imaging (1.0-T scanner, Impact, Siemens, Erlangen, Germany). The MRI protocol
included transverse conventional spin echo (SE) T1-weighted image (WI), FSE T2-WI
with fat saturation and dynamic contrast-enhanced (Gadolinium, 0.2 ml kg−1, Magnevist,
Berlex Laboratories, Wayne, USA). Two radiologists reviewed the pre- and postprocedural
MR imaging, and reached a consensus in each patient.
HIFU 3D conformal treatment
In this study, real-time US imaging was utilised to monitor the HIFU ablation procedure.
It can accomplish three separate functions, including real-time targeting of the tumour
to be treated, guidance of US energy deposition within the treated region, and rapid
real-time assessment of the volume of coagulation necrosis during therapy. High-intensity
focused ultrasound treatment was performed in the patients under intravenous sedation
(N=4) or general anaesthesia (N=19). During and following suitable anaesthesia, the
patient was monitored to track the blood pressure, pulse, respiration rate, temperature,
and peripheral oxygenation. Then, the patient was placed prone and carefully positioned,
so that the skin overlaying to the lesion to be treated was easily in contact with
degassed water.
The coaxial US imaging device was used to establish the 3D image of the whole tumour.
For therapeutic purposes, the whole tumour was divided into slices with 5 mm separation
using US images. By scanning the HIFU beam in successive sweeps from the deep to the
shallow regions of the tumour, the targeted regions on each slice were completely
ablated. This process was repeated slice by slice to achieve complete tumour ablation,
in a manner resembling that of cutting away slices of bread. In this study, the extent
of HIFU treatment was larger than the tumour extent, in order to ensure the ablation
of any adjacent microsatellites and to obtain a sufficient tumour-free margin. It
included the breast lesion and its marginal breast tissue about 1.5–2.0 cm around
the visible tumour. This is a principle that is routinely employed in conventional
surgery. During the HIFU treatment, each slice of US images taken before and immediately
after the treatment of each slice were compared. These differences between treated
and untreated areas were used for monitoring the therapeutic effect. In this study,
the target tissue was exposed at acoustic focal peak intensities from 5000 to 15 000 W cm−2.
The scanning speed ranged 1–3 mm s−1, and the track length was 20 mm. High-intensity
focused ultrasound treatment time ranged from 45 min to 2.5 h (median, 1.3 h).
Pathologic examination
In the clinical trial, modified radical mastectomy was performed in all of the eligible
patients. In all, 23 patients in HIFU group underwent the operation within 1–2 weeks
after the thermal ablation. All removed specimens including breast tumour and normal
breast tissue were evaluated by gross and histological observations. They were serially
sectioned at approximately 5 mm intervals and the slice thickness was measured. Gross
observations were recorded in a protocol containing the overall appearance, size,
and shape. By using a calipers, the visible lesions on each slice were measured in
both parallel and perpendicular axes, and volume was calculated by multiplying the
area by slice thickness. Representative sections were prepared for routine microscopic
analysis. They were fixed in 10% phosphate-buffered formalin (pH=7), embedded in paraffin,
cut at distances of 1 mm in 4 μm-thick slices, and stained with haematoxylin and eosin
(H&E). To study the possibility of tumour vascular wall destruction induced by HIFU
ablation, elasticity fibrin and collagen fibrin double staining (Victoria blue and
ponceau's histochemical staining) were performed.
Immunohistochemistry
Immunohistochemistry was performed in all removed breast tissue from eligible patients.
Proliferating cell nuclear antigen (PCNA), cell adhesion molecule CD44v6, and matrix
metalloproteinase-9 (MMP-9) are molecular indicators that represent malignant behaviours
of breast cancer cell such as proliferation, invasion, and metastasis, respectively.
They were identified using the biotin–streptavidin–peroxidase immunohistochemical
technology in treated breast cancer in the HIFU group, breast cancer in the control
group, and normal breast tissue from the HIFU group, respectively. The paraffin-embedded
breast specimens were sectioned in 5-μm-thick slices.
The slices were deparaffinised in xylene, and rehydrated. Endogenous peroxidase was
blocked with 0.3% hydrogen peroxide in methanol for 20 min. Antigen retrieval was
performed using microwave heating. Subsequently, sections were preincubated with 1%
bovine serum albumin. The primary antibodies used were mouse monoclonal anti-human
PCNA, anti-CD44v6, and anti-MMP-9. All of them were obtained from Santa Cruz Biotechnology
(Santa Cruz, CA, USA). Sections were incubated at room temperature with three kinds
of the primary antibody, respectively, followed by biotinylated second antibody incubation.
The chromogen was 3,3-diaminobenzidine tetrahydrochloride (brown). Sections were incubated
with phosphate-buffered saline, which served as a negative control instead of the
primary antibody.
The number of immunostained cells was counted in a microscopic grid, 0.5 × 0.5 mm2
in size (0.25 mm2), using a microscopic field of × 200. A total of 10 areas with the
most abundant distribution were chosen in each case for this observation. Two independent
observers judged the distribution of these three variables and counted the positive
cells. If the results were inconsistent, the two observers discussed to reach a consensus
on the semi-quantitative scoring. For the PCNA, we used the labelling index of PCNA
in cancer cells to evaluate the result. The number of PCNA+ cancer cells was counted
among 100 cancer cells using a × 200 microscopic field.
Statistical analysis
All observed data are displayed as the mean value plus or minus the standard deviation.
The statistical significance of any observed difference between the mean values of
control and treatment groups was evaluated using an unpaired Student's t-test. The
variability in data was compared with the F test for variance. The differences in
percentage data were analysed by using the Fisher's exact test. Statistical significance
was defined as a P-value of less than 0.05.
RESULTS
Short-term follow-up
Among 23 patients, 16 cases underwent surgery at 7–10 days after HIFU ablation, and
the remaining seven patients were operated at 11–14 days. After HIFU, oedema was noted
in the mammary tissue surrounding the treated tumour. In all cases, the treated area
included the tumour and a 1.5–2.0 cm margin of normal breast tissue around the tumour.
At 7–10 days postoperatively, the oedema gradually disappeared. In total, 14 patients
experienced mild local pain, warmth, and sensation of heaviness in the diseased breast.
But only four patients were given 3–5 days prescription for oral analgesics. One patient
had minimal skin burn, which had entirely recovered by 10 days post-HIFU. There was
no incidence of bleeding or infection of the treated breast requiring intervention.
Three patients received one MRI examination after HIFU treatment. Compared with preprocedural
images, enhanced MRI revealed the absence of contrast enhancement in the treated region
including tumour and 1.5–2.0 cm normal breast tissue surrounding the tumour (see Figure
2
Figure 2
Contrast-enhanced MRI of a left breast cancer (arrowed) in a 36-year-old woman before
(A) and 1 week after (B) HIFU ablation. Note the absence of contrast uptake in ablated
volume, including tumour and a margin of treated normal breast tissue about 1.5–2.0 cm
around the cancer. Oedema is observed in the mammary tissue surrounding the treated
region.
), indicative of coagulative necrosis.
Pathologic examination
All treated tumours in the 23 patients were confirmed by gross observation after removal
of the diseased breast. Neither bleeding of the treated regions nor injury of intervening
tissues was identified, indicating the safety of HIFU ablation. Macroscopic examination
showed that HIFU treatment induced complete coagulative necrosis of the target tissue,
which included the tumour and a mean margin of 1.80±0.58 cm (range from 1.5 to 2.2 cm)
of an obviously normal breast tissue surrounding the tumour. This felt firmer on palpation
than normal tissue. At the margin between the treated and untreated regions, there
was a rim of congestion that represented an inflammatory reaction to thermal ablation.
Histological examination revealed homogeneous coagulative necrosis, including the
tumour and normal breast tissue within the target region. Figure 3
Figure 3
Microscopic changes of tumour cells in patients with breast cancer treated by HIFU.
(A) Untreated viable breast cancer cells. (B–D): Necrosed tumour cells represented
by pyknotic nuclei (B); nuclear disruption (C); and nuclear disappearance (D). H&E
staining × 400.
shows that the tumour cells of breast cancer treated with HIFU were very abnormal,
with the appearance of irreversible cell death, as demonstrated by pyknotic nuclei,
nuclear disruption, and disappearance. Variable amounts of granulation tissue were
noted with the presence of immature fibroblasts, inflammatory cells, and new capillaries
in the boundary region. Compared with blood vessels in the control group, vascular
structure in the HIFU group was seriously damaged. Appearances were of indistinct
cellular margins, endothelial disruption, and nuclear disappearance, and destruction
of tunica media, indicative of coagulative necrosis (Figure 4
Figure 4
Microscopic changes of tumour vascularity in patients with breast cancer treated by
HIFU. (A) Untreated tumour blood vessels; (B) destruction of tumour vascularity with
disruption of the endothelium and tunica media, H&E staining × 400. (C) untreated
tumour vessel wall; (D) destroyed tumour blood vessels with the collapse and disruption
of vascular elasticity fibrin and collagen fibrin, Victoria blue and ponceau's histochemical
staining × 400.
). The size of the destroyed vessels was less than 2 mm in diameter, including the
arteries and veins. Scattered intravascular thrombi were often observed in the destroyed
vessels. Victoria blue and ponceau's histochemical staining showed that vascular elasticity
fibrin and collagen fibrin were significantly collapsed and disrupted, as shown in
Figure 4.
Immunohistochemical staining
Figure 5
Figure 5
Immunohistochemistry for PCNA (the first row), CD44v6 (the second row), and MMP-9
(the third row) in the patients with breast cancer treated by HIFU (S-P staining,
× 400). The immunoreactivity is shown by the color brown. From left to right, each
separated morphology is represented by a normal breast tissue in the HIFU group, untreated
cancer cells in the control group, and treated cancer in the HIFU group, respectively.
Compared with untreated cancer cells, no positive cells of PCNA, CD44v6, and MMP-9
within the treated cancer cells were observed in the HIFU group.
showed immunohistochemical staining for PCNA, CD44v6, and MMP-9 in breast cancer in
the control group, normal mammary tissue, and treated breast cancer in the HIFU group,
respectively. The percentages of the PCNA labelling index in the normal breast tissue,
breast cancer, and HIFU-treated tumour were 6, 44, and 0%. CD44v6-positive cells were
present in 56% of tumour samples in the control group (n=14), and 4.5% of normal breast
tissue (n=1). But, in the HIFU group, CD44v6-positive cells were not detected within
the treated breast cancer (n=0). CD44v6+ cell density was significantly higher in
tumour than in the normal breast tissue (P< 0.01), but in the HIFU group the proportion
of these membrane-positive cancer cells was 0%. Compared with 60% of MMP-9 within
breast cancer in the control group (n=15), no MMP-9+ cancer cells were detected in
either normal breast cancer or treated breast cancer (n=0), as shown in Table 2
Table 2
Results of immunohistochemical staining in both groups
PCNA labelling index (%)
CD44v6 (%)
MMP-9 (%)
Normal breast tissue
6
4.5
0
Untreated breast cancer
44
56
60
Treated breast cancer
0
0
0
.
DISCUSSION
The energy deposition of focused US on the target tissue causes coagulative necrosis
of tumours (Chapelon et al, 1992; Chen et al, 1993; Sibille et al, 1993; Prat et al,
1995; Rowland et al, 1997). In this study, pathological examination revealed that
both breast cancer cells and normal tissues within the treated region presented a
typical appearance of coagulative necrosis with severe nuclear damage. Furthermore,
tumour vessels were severely damaged in the patients with breast cancer, resulting
in significant vascular disruption and vessel occlusion. By using immunohistochemical
staining, we found that no expression of PCNA, CD44v6, and MMP-9 of the treated tumour
cells was identified in the HIFU group, unlike those in the control group. These findings
indicated that the treated breast cancer cells presented had not only undergone coagulative
necrosis in histology, but also the absence of malignant behaviours such as proliferation,
invasion, and metastasis.
The goal of our clinical study is to explore the efficacy, safety, and feasibility
of extracorporeal HIFU ablation for primary breast cancer. To achieve this purpose,
we hypothesise that both tumour and some normal breast tissue surrounding the tumour
should be completely destroyed with focused US energy. This larger ablation extent
would guarantee an adequate ‘surgical’ HIFU margin surrounding the tumour, and would
be required if HIFU therapy is considered as an alternative to surgical procedure.
This principle is routinely used in conventional surgery in order to ensure the removal
of adjacent microsatellites, and to allow for uncertainty that frequently exists concerning
the exact location of definite tumour margins. In the clinical study, histological
examination revealed that HIFU induced coagulative necrosis of the target tumour and
its surrounding normal tissue by about 1.5–2.2 cm (mean 1.80±0.58 cm), which corresponded
closely with the pretreatment extent (1.5–2.0 cm around the tumour) determined in
the therapeutic protocol before HIFU procedure. This result indicated that, with real-time
US guidance, HIFU can precisely cause a complete necrosis of target tissue, including
the tumour and normal tissue.
In clinical applications, real-time imaging modalities including both US (Vallancien
et al, 1996; Beerlage et al, 1999; Chapelon et al, 1999; Gelet et al, 1999; Wu et
al, 2001a, 2002; Uchida et al, 2002) and MRI (Huber et al, 2001; Hynynen et al, 2001)
can guide HIFU ablation of solid tumours. These imaging techniques have been used
for targeting the lesion to be treated, monitoring the therapeutic procedure, assessing
the thermal effects on target tissue, and controlling the ultrasonic energy. In this
study, the reasons for choosing real-time US for the guidance of HIFU treatment were
as follows: low cost, flexibility, speed of image processing, and extensive availability.
During the HIFU procedure, obvious changes in grey scale within the target tissue
were immediately detected on the US image after HIFU ablation. Our previous studies
indicated that these hyperechoic zones corresponded well to the extent of coagulative
necrosis, and that there was a close relationship between the extent of necrosis as
measured by gross examination and the hyperechoic extent measured on the US image
immediately after FUS in both in vivo and in vitro tissues (Wang et al, 1997; Wu et
al, 1998, 2000, 2001b; Bai et al, 1999). In the clinical treatment of patients with
breast cancer, we also found that the hyperechogenic regions were regular in shape
and size, and conformed to the extent of coagulative necrosis induced by HIFU ablation
(Liu et al, 1998; Cao et al, 2001; Wu et al, 2001; Zhou et al, 2001). Although the
mechanism for tissue grey-scale change is still unclear, US effects on tissue such
as heating and cavitation are involved in the formation of coagulative necrosis, and
real-time diagnostic US guidance is seen to be useful in the detection of the coagulative
necrosis of target tissue during HIFU procedures.
As a noninvasive method, HIFU has a potential application in the treatment of patients
with breast cancer. However, some problems still exist with this technique. First,
to our knowledge, neither MRI nor diagnostic US have satisfactory sensitivity for
precise visualisation of tumour margins. A completely precise imaging modality for
pretreatment demonstration of tumour extent is not currently available. Therefore,
in our clinical study, image-detected breast cancer and 1.5–2.0 cm normal tissue surrounding
the cancer were completely ablated to allow for the uncertainty that frequently exists
concerning the exact location of definite tumour margins. However, for some superficial
lesions close to the skin, there is a possibility of causing skin burns, or leaving
residual viable tumour cells if the overlying skin is left undamaged. A second problem
is that a palpable firm lump would persist in the treated region for a period of time
after HIFU treatment. This lump may not be satisfactory or cosmetically desirable
for the patient, although the tissue would revert to benign status. This could also
cause potential problems with follow-up images, in distinguishing whether there is
local recurrence postoperatively. Finally, a suitable indication of HIFU ablation
for breast cancer is essential if this technique can be used in clinical practice.
Breast tumours with undefined margins, scattered multiple foci, and tumours close
to the nipple should be excluded as potential targets for HIFU.
In conclusion, this study demonstrates for the first time that US-guided HIFU is effective,
safe, and feasible in the treatment of localised breast cancer with well-demarcated
margins. However, much supplementary investigation is necessary to explore these above
problems, and large controlled clinical trials must be performed before extracorporeal
HIFU can be recommended as a conserving breast modality in the treatment of breast
cancer.