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Microregional fluctuations in perfusion within human tumours detected using laser Doppler flowmetry
rt&ADIOTHERAPY ONCOLOGY t- I S I- V I F R
Radiotherapy and Oncology 40 (1996) 45-50
Microregional fluctuations in perfusion within human tumours detected using laser Doppler flowmetry K.H. Pigott*, S.A. Hill, D.J. Chaplin, M.I. Saunders The Marie
Curie Research Wing for Microcirculation
Oncology, Mount Vernon Centre for Cancer Treatment, Group, The Gray Laboratory, Northwood, Middlesex,
Mount Vernon Hospital HA6 2RN. UK
and Tumour
Received 6 April 1995; revised 29 January 1996; accepted 12 February 1996
Purpose:Transientfluctatioosin erythrocyte flux consistentwith perfusiondriven hypoxia havebeenpreviouslyreportedusing experimentaltumour models.The presentstudy wasdesignedto establishwhethersuchchangesare a commonfeatureof human tumours.Merhodr and Materials: A multi-channellaserDoppler systemwasusedto monitor microregionalchangesin flow in human tumours. Eight individual tumours were investigated, two primary and one locally recurrent breast carcinoma,two metastaticskin depositsandthreemetastaticlymph nodes.Sixcustomdesigned microprobes(diameterof 300pm), eachmonitoring a nominalsamplingvolumeof approximatelylo-’ mm3wereinsertedinto the tumour and perfusionmonitoredover a periodof 60min. Resuirs: The resultsshowthat in 54%of the regionsmonitoredtherewasa change in microregionalblood flow by a factor of 1.5 or more. Over the whole6knin period, 19%of the changes were reversed, with a time course of 4-44 min. Conclusions: Thisfindingdemonstrates that microregionalfluctuationsin perfusionoccurfrequently in humantumours.Furthermore,the observation that 19%of the changeswerereversedimpliesthat at leastsomeof the cellsare subjectto transientacute hypoxia. Keyworuk
Tumour blood flow; LaserDoppler; Transienthypoxia
1. Introduction
Hypoxic cells are radioresistant, requiring up to three times the dose of radiation required by aerated cells. Their presence within a tumour is believed to be one of the factors contributing to treatment failure following radical radiotherapy. The result of a meta-analysis of trials employing methods to overcome hypoxia, has demonstrated a small but signilicant improvement in local tumour control and survival [ 131. Additionally, there is now clinical evidence that hypoxic cells can influence response to radiotherapy [7,9]. These two factors have contributed to renewed clinical interest in hypoxia. Two different mechanisms for hypoxia induction have been shown to exist in experimental tumours. Chronic hypoxia, first described in 1955 by Thomlinson and Gray, arises because of the diffusion limitation of oxy-
l
Corresponding author.
gen [14]. In 1979, Brown postulated that acute hypoxia or perfusion-limited hypoxia also occurred in tumours, due to the temporary closure of blood vessels [2]. Confirmation that such hypoxia occurs in experimental murine tumours was obtained utilising novel flow cytometric and histological techniques [3,15]. These techniques, which involved the administration of fluorescent perfusion stains, are not clinically applicable and thus the relative importance of perfusion-limited hypoxia in human malignancies could not be assessed. A multi-channel laser Doppler system has made possible the monitoring of microregional blood flow in tumours over time. This system has been used successfully in transplantable murine tumours to study the occurrence of microregional changes in blood flow [5,8]. The studies have indicated the potential of these probes to provide real-time spatial flow mapping of microregional erythrocyte flux within tumours. The size of the probe enabled the red blood cell flux to be monitored in a discrete sampling volume (- 10m2 mm3) containing only a small number of capillaries. In the present study
0167-8140/96/$15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved PII SOl67-8140(96)01730-6
46
KH. Pigott et al. /Radiotherapy and Oncology 40 (19%) 4.5-50
we have utilised this technology to determine whether temporal changes in microregional perfusion are a feature of human malignancies. Such information may aid in the design of optimal treatment schedules to eliminate or reduce tumour hypoxia.
2,Materialsandmethod 2.1. Patients In all cases, informed consent was gained prior to entry into the study. Only patients who had tumours which were easily accessible, such as a breast carcinoma or skin deposits, were suitable (see Table 1). Measurements were performed with the patient lying comfortably on a couch. Since laser Doppler flowmetry is subject to movement artefact it was important to ensure that the patient was requested to remain still during recording. 2.2. Laser Doppler jlowmetry Microvascular perfusion was measured using the Oxford Array Multiple Channel Laser Doppler System (Oxford Optronix Ltd., Oxford, UK). Laser Doppler flowmetry generates a signal that is proportional to the mean erythrocyte velocity multiplied by the number of moving erythrocytes within the sampling volume. This signal is termed the red cell flux. In addition, a signal which is proportional to the total amount of light detected by the probe, termed the backscatter signal, is also recorded. This signal can detect probe movement during a recording as an alteration in the region being sampled results in an abrupt change in the backscatter signal due to a change in the cellular microstructure adjacent to the probe tip. Any change in perfusion accompanied by an abrupt change in the backscatter signal was excluded from the analysis. Custom made microprobes were used which measured 25 mm in length and had a diameter of 300 pm. The mean sampling depth for laser Doppler has been estimated in a number of tissues for a range of probes geometries [11,12]. Taking the active area of the probe as 300 pm and assuming a cylindrical sampling volume, the nominal sampling volume can be calculated as approximately IO-* mm3.
Six microprobes were inserted into the tumour in order to make simultaneous readings of red blood cell flux from several sites. In fungating tumours no local anaesthetic was employed. However a subcutaneous injection of local anaesthetic (lignocaine 1%) was used to anaesthetise the skin in the remaining cases. It was assumed that the use of local anaesthetic was unlikely to
affect the results: firstly, because the probes were 25 mm in length and measurements were therefore made some distance below the skin; secondly, similar perfusion fluctuations had been observed in anaesthetised and unanaesthetised mice, although the actual readings were lower in the anaesthetised animals [5]. Since the microprobes have a blunt end, a 20 gauge cannula was used to make a nick in the skin; the probes were then advanced down the cannula into the tumour. Care was taken to make sure that the microprobes were advanced well beyond the tip of the cannula, and that they were secure in their position. Once a stable backscatter reading was obtained from each probe indicating that no probe movement was occurring, perfusion was then recorded over a period of 60 min. 2.4. Data analysis Each of the six channels recorded 20 flow readings per second. An average value for each 2-n& interval recorded was calculated and plotted against time for the separate channels. Since laser Doppler does not record an absolute measure of blood flow, arbitrary units of flow were used and data were analysed in terms of fold changes in blood flow. Each channel was looked at separately and the number, magnitude and duration of change noted. A proportion of the laser Doppler signal is due to a biological zero [1,6]. This signal is, in part, a result of the Brownian motion of free red blood cells in front of the probe. Based on animal studies a 30% correction was applied to our results [5]. This was done by calculating 30% of the last 2-min average recorded for each trace and subtracting that from the whole trace.
3.Results Seven patients were entered into this study, live female and two male with a median age of 67 years and a range of 55-80 years. In total eight sets of readings were obtained, one patient with bilateral groin nodes, had measurements of tumour perfusion performed on both sides of the groin, on two separate occasions. Blood flow measurements could only be undertaken in tumours which were easily accessible and large enough to allow the insertion of six microprobes (Fig. 1). The masses studied included primary and locally recurrent breast carcinomas, as well as metastases to regional lymph nodes and skin, from a variety of different histological tumours (Table 1). Six traces of microregional blood flow were obtained from each tumour, resulting in a total of 48 traces. Two traces were excluded, one because the probe was outside the tumour volume and the other because of probe movement throughout the 60-min recording period. In total 46 traces were available for analysis. Fluctuations
K.H.
Pigott
et al. /Radiotherapy
and Oncology
40 (1996)
45-50
Fig. I. Photograph of a tumour with the six microprobes inserted.
in blood flow were apparent in many of the traces, with some demonstrating more than one change over the observation period (Table 2). Furthermore, within the same tumour, changes in blood flow occurred independently in the different areas measured, with some areas showing no change and others a decrease or increase in perfusion (Fig. 2). A change in blood flow by at least a factor of 1.5 was seen in 25 of the 46 (54%) traces. In total, 37 fluctuations were observed: 24 increases and 13 decreases (Table 3). Patient no. 5 had a low grade non-Hodgkin lymphoma and a low platelet count (40 x lOgA) due to bone marrow involvement. Five of his six traces demonstrated a rise in blood flow, which we believe may have been a reflection of haemorrhage in front of the probe, resulting from his low platelet count. Since tumour perfusion was monitored continuously,
it was possible to study the time over which changes occurred. The duration of change was defined as the time from any maximum to any minimum blood flow reading or vice versa. Of the traces which changed by a factor of 1.5 or more, 54% of the changes occurred over 20 min or less. In a number of traces, 19% (7 of 37) reversal of the change in blood flow was seen during the 60-min observation period, with three of the traces showing more than one reversal of change. The median time taken for this change to occur was 13 min with a range of 4-44 min. 4. Discussion This study demonstrates the feasibility of using laser Doppler microprobes to detect temporal changes in microregional perfusion in human malignancies. Fur-
Table 1 Patient and tumour characteristics Case no. Age
Site
Size
Histology
1
80
Primary breast
Ductal carcinoma
2 3
61 13
4
19
5 6
59 67 55
Skin deposit Local breast recurrence Metastatic right groin node Metastatic left groin node Left groin node Primary breast Skin deposit
Whole breast replaced by tumour and involving skin 7 x 6 cm nodule involving skin 8 x 6 cm fungating mass 4 x 3 cm mass no skin involvement 7 x 8 cm mass involvmg skin 10 x 6 cm mass involving skin 10 x 10 cm mass no skin involvement 4 x 4 cm mass involving skin
I
Adenocarcinoma of the lung Ductal carcinoma Squamous cell carcinoma of the penis Squamous cell carcinoma of the penis Low grade lymphoma Ductal carcinoma Adenocarcinoma of the pancreas
ELfi. Pigott et al. /Radiotherapy and Oncology 40 (19%) 45-50
48
Table 2 Fold changes in blood flow for each trace of blood flow recorded
caseno.
Channel 1
Channel 2
I
I x2.1
X
Channel 3
Channel 4
Channel 5
Channel 6
t x II
I x 1.8 1 x 1.8
1x 1x 1x 1x t x I x 1x I x 1x
1.6 2.1 1.8 1.5 2.1 1.8 1.7 1.7 2.0
I I 1 t
1.9 1.6 1.5 1.7
I x 12 2 3
f x 1.9
4
t x 1.9
I x 4.0
1 x 1.8
t x 1.9
1 x 2.0 1 x 1.7
t x 1.9 X
5 6
1 x 1.5 1 x 1.5
I x 2.5
7
I I I t I
x x x x x
2.9 1.g 2.0 1.9 1.9
x x x x
I x 2.5 t x 1.8
X-trace excluded from analysis. Patient I, channel 2 - probe outside the tumour. Patient 6, channel 4 - backscatter signal not stable indicating probe movement. -: no changes in perfusion greater than 1.5.fold observed.
Patient 4 5000
-
4000
-
n
5000-
s
4000-
‘E
3000-
3000-
3
2000-
2000-
2
Probe
lOOO-
2
1
---....-.................==
O-d-
10
.r;
Time
aL
5000.
-
4000. 30
ii
30
40
50
60
-==-=-=-..-...--.............~. 0’
01
10
20 Tim
-
,000.
4
(m in.)
Probe2
3000 2000.
5
lOOO-
..--
20
Probe
5000
-
4000
-
Probe
30
40
n~l..............=--=-==-=-
Od
10
20 Tim
30
40
5
J-7
2000
-
,000. 50
60
60
e (m in .)
3000-
n .-.
50
1.1. ..=- r
n =...... 04
1 0
10
20
e (m in .)
Tim
30
‘I
40
50
60
e (m in .)
t 00
5000-
L, a
40003000.
cn
m J
Probe
50004000. 3 000
3
.--
2000-
l.
lOOO-
01
0
. ...= 10
20 Tim
2000.
2====== 30
40
e (m in .)
60
. . . . . . --‘I-
n
-=
iooo50
. J-9
m..======
.-i .m......
Probe6
- n >
0'
1
0
'
10
20 Time
Fig. 2. Example of six traces of blood flow from patient number 4.
30
40 (m in.)
50
60
K. H. Pigott et al. /Radiotherapy and Oncology 40 (19%) 45-M Table 3 Changes in microregional perfusion during 60-min observation period Factor of change
% of traces showing change
Direction of change
No. of traces
>I.5 >lS
54 50
241 131 191 121
46 4cP
BPatient no. 5 excluded due to low platelet count and probable haemorrhaging around the probe.
thermore, it provides the first direct evidence that such changes are a frequent occurrence in human tumours. Over the 6Omin observation period used in the study, an increase or a decrease in flow by a factor of 1.5 was seen in 54% of all traces, with each tumour evaluated demonstrating a change in at least one microregion sampled. Twenty-eight percent of the regions sampled demonstrated a change in erythrocyte flux by at least a factor of two. It is not possible, in the absence of detailed morphological data, to accurately estimate the number of capillaries sampled in front of the probe. If, for example, we assume that 10 capillaries are sampled in front of a microprobe, then a reduction in flux by a factor of two could reflect an equal reduction in all capillaries or a situation where flow has ceased in five capillaries. The former would decrease the oxygen diffusion distance and thus increase the rim of hypoxic cells; the latter would create regional foci of hypoxia. Although it is not possible to discriminate between these potential causes, the example does indicate that perfusion-driven changes in oxygen delivery could contribute significantly to the occurrence of hypoxia in human tumours. Of some concern was the fact that, in the current study, there was a trend to see more increases than decreases in microregional red blood cell flux. Such a trend has also been identified in the CaNT tumour in mice but not in the SaF tumour (S.A. Hill and D.J. Chaplin, unpublished observations). One explanation for such an occurrence is that haemorrhaging into the region in front of a probe can occur over the 60-min observation period. This hypothesis is supported by the fact that one of the patients (no. 5) with a low platelet count and, thus, a tendency to bleed, displayed a gradual increase in blood flow in five of the six microregions sampled, with the sixth showing no change. We are confident that the variation in tumour blood flow seen in this study does not reflect variation in skin perfusion. Skin blood flow in a number of patients has been recorded using surface probes and, for comparison, only 11% of the traces varied by a factor of 1.5 or more compared with 54% of the tumour blood flow traces (S.A. Hill and D.J. Chaplin, unpublished observations).
49
The occurrence of perfusion-driven changes in tumour oxygenation has important implications for the strategies that need to be employed to circumvent hypoxia-induced radioresistance. Methods which provide an increase in the oxygen-carrying capacity of the blood would not be expected to be effective in reoxygenating regions where the blood flow has temporarily ceased. To reoxygenate such regions, agents are required which would prevent the fluctuations in microregional perfusion. One such agent, nicotinamide, has been shown to reduce the occurrence of transient reductions in microregional flow in a number of animal tumour systems [4,10]. The use of nicotinamide with carbogen breathing as an approach to improve radiation response in the clinic is being studied at several centres worldwide, including Mount Vernon Hospital. In summary, the present study supports the hypothesis that spontaneous and potentially reversible changes in microregional blood flow can occur within human tumours. These findings have potentially important implications for the design of therapeutic approaches to overcome or eliminate hypoxia. References 111 Abbot, N.C. and Beck, J.S. Biological zero in laser Doppler measurements in normal ischemic and inflamed human skin. Int. J. Microcirc. Clin. Exp. 12: 89-98, 1993. VI Brown, J.M. Evidence for acutely hypoxic cells in mouse tumours and a possible mechanism for reoxygenation. Br. J. Radiol. 52: 650-656, 1979. I31 Chaplin, D.J., Olive, P.L. and Durand, R.E. Intermittent blood flow in a murine tumour: Radiobiological effects. Cancer Res. 47: 597-601, 1987. 141 Chaplin, D.J., Horsmann, M.R. and Trotter, M.J. Effect of nicotinamide on the microregional heterogeneity of oxygen delivery within a murine tumour. J. Natl. Cancer. Inst. 82: 672-676, 1990. ISI Chaplin, D.J. and Hill, S.A. Temporal heterogeneity in microregional erythrocyte flux in experimental solid turnours. Br. J. Cancer 71: 1210-1213, 1995. 161 Colantuoni, A., Bertuglia, S. and Intagietta, M. Biological zero of laser Doppler fluxmetry-microcirculatory correlates in the hamster-cheek pouch during flow and no flow conditions. Int. J. Micro&c. Clin. Exp. 13: 125-136, 1993. [71 Gatenby, R.A., Kessler, H.B., Rosenblum, J.S., Coia, L.R., Moldofsky, P.J., Hartz, W.H. and Broder, G.J. Oxygen distribution in squamous cell carcinoma metastases and its relationship to outcome of radiation therapy. Int. J. Radiat. Oncol. Biol. Phys. 14: 831-838, 1988. 181 Hill, S.A. and Chaplin, D.J. Detection of microregional tluctuations in erythrocyte flow using laser Doppler microprobes.Oxygen transport in tissue. Adv. Exp. Med. Biol. XVIII: 367-372, 1996. B., [91 Hockel, M., Knoop, C., Schilenger, K., Vomdran, Baumann, E., Mitze, M., Knapstein, PG. and Vaupel, P. Intratumoral p0zpredict.s survival in advanced cancer of the uterine cervix. Radiother. Oncol. 26: 45-50, 1993. 1101Horsman, M.R., Brown, D.M., Hirst, V.K., Lemmon, M.J., Wood, P.J., Dunphy, E.P. and Overgaard, J. Mechanism of action of the selective tumour radiosensitiser nicotinamide. Int. J. Radiat. Oncol. Biol. Phys. 15: 685-690, 1988.
50 [ll]
K.H. Pigott et al. /Radiotherapy and Oncology 40 (19%) 45-50
Jakobsson, A. and Nilsson, G.E. Prediction of the sampling depth and photon path length in laser Doppler flowmetry. Med. Biol. Eng. Comput. 31: 301-307, 1993. [12] Johansson, K., Jakobsson, A., Lindahl, K., Lindhage, J., Lundgren, 0. and Nilsson, G.E. Influence of tibre diameter and probe geometry on the measuring depth of laser Doppler flowmetry in the gastrointestinal application. Int. J. Microcirc. Clin. Exp. IO: 219-229, 1991. 113) Overgaard, J. Importance of tumour hypoxia in radiotherapy. A
meta-analysis of controlled clinical trials. Radiother. Oncol. Suppl. 1, 24: s64, 1992. [14] Thomhnson, R.H. and Gray, L.H. The histological structure of some human lung cancers and the possible implications for radiotherapy. Br. J. Cancer. 9: 539-549, 1955. [15] Trotter, M.J., Chaplin, D.J., Durand, R.E. and Olive, P.L. The use of fluorescent probes to identify regions of transient perfusion in murine tumours. Int. J. Radiat. Oncol. Biol. Phys. 16: 93 I-935. 1989.
Radiotherapy and Oncology 40 (1996) 45-50
Microregional fluctuations in perfusion within human tumours detected using laser Doppler flowmetry K.H. Pigott*, S.A. Hill, D.J. Chaplin, M.I. Saunders The Marie
Curie Research Wing for Microcirculation
Oncology, Mount Vernon Centre for Cancer Treatment, Group, The Gray Laboratory, Northwood, Middlesex,
Mount Vernon Hospital HA6 2RN. UK
and Tumour
Received 6 April 1995; revised 29 January 1996; accepted 12 February 1996
Purpose:Transientfluctatioosin erythrocyte flux consistentwith perfusiondriven hypoxia havebeenpreviouslyreportedusing experimentaltumour models.The presentstudy wasdesignedto establishwhethersuchchangesare a commonfeatureof human tumours.Merhodr and Materials: A multi-channellaserDoppler systemwasusedto monitor microregionalchangesin flow in human tumours. Eight individual tumours were investigated, two primary and one locally recurrent breast carcinoma,two metastaticskin depositsandthreemetastaticlymph nodes.Sixcustomdesigned microprobes(diameterof 300pm), eachmonitoring a nominalsamplingvolumeof approximatelylo-’ mm3wereinsertedinto the tumour and perfusionmonitoredover a periodof 60min. Resuirs: The resultsshowthat in 54%of the regionsmonitoredtherewasa change in microregionalblood flow by a factor of 1.5 or more. Over the whole6knin period, 19%of the changes were reversed, with a time course of 4-44 min. Conclusions: Thisfindingdemonstrates that microregionalfluctuationsin perfusionoccurfrequently in humantumours.Furthermore,the observation that 19%of the changeswerereversedimpliesthat at leastsomeof the cellsare subjectto transientacute hypoxia. Keyworuk
Tumour blood flow; LaserDoppler; Transienthypoxia
1. Introduction
Hypoxic cells are radioresistant, requiring up to three times the dose of radiation required by aerated cells. Their presence within a tumour is believed to be one of the factors contributing to treatment failure following radical radiotherapy. The result of a meta-analysis of trials employing methods to overcome hypoxia, has demonstrated a small but signilicant improvement in local tumour control and survival [ 131. Additionally, there is now clinical evidence that hypoxic cells can influence response to radiotherapy [7,9]. These two factors have contributed to renewed clinical interest in hypoxia. Two different mechanisms for hypoxia induction have been shown to exist in experimental tumours. Chronic hypoxia, first described in 1955 by Thomlinson and Gray, arises because of the diffusion limitation of oxy-
l
Corresponding author.
gen [14]. In 1979, Brown postulated that acute hypoxia or perfusion-limited hypoxia also occurred in tumours, due to the temporary closure of blood vessels [2]. Confirmation that such hypoxia occurs in experimental murine tumours was obtained utilising novel flow cytometric and histological techniques [3,15]. These techniques, which involved the administration of fluorescent perfusion stains, are not clinically applicable and thus the relative importance of perfusion-limited hypoxia in human malignancies could not be assessed. A multi-channel laser Doppler system has made possible the monitoring of microregional blood flow in tumours over time. This system has been used successfully in transplantable murine tumours to study the occurrence of microregional changes in blood flow [5,8]. The studies have indicated the potential of these probes to provide real-time spatial flow mapping of microregional erythrocyte flux within tumours. The size of the probe enabled the red blood cell flux to be monitored in a discrete sampling volume (- 10m2 mm3) containing only a small number of capillaries. In the present study
0167-8140/96/$15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved PII SOl67-8140(96)01730-6
46
KH. Pigott et al. /Radiotherapy and Oncology 40 (19%) 4.5-50
we have utilised this technology to determine whether temporal changes in microregional perfusion are a feature of human malignancies. Such information may aid in the design of optimal treatment schedules to eliminate or reduce tumour hypoxia.
2,Materialsandmethod 2.1. Patients In all cases, informed consent was gained prior to entry into the study. Only patients who had tumours which were easily accessible, such as a breast carcinoma or skin deposits, were suitable (see Table 1). Measurements were performed with the patient lying comfortably on a couch. Since laser Doppler flowmetry is subject to movement artefact it was important to ensure that the patient was requested to remain still during recording. 2.2. Laser Doppler jlowmetry Microvascular perfusion was measured using the Oxford Array Multiple Channel Laser Doppler System (Oxford Optronix Ltd., Oxford, UK). Laser Doppler flowmetry generates a signal that is proportional to the mean erythrocyte velocity multiplied by the number of moving erythrocytes within the sampling volume. This signal is termed the red cell flux. In addition, a signal which is proportional to the total amount of light detected by the probe, termed the backscatter signal, is also recorded. This signal can detect probe movement during a recording as an alteration in the region being sampled results in an abrupt change in the backscatter signal due to a change in the cellular microstructure adjacent to the probe tip. Any change in perfusion accompanied by an abrupt change in the backscatter signal was excluded from the analysis. Custom made microprobes were used which measured 25 mm in length and had a diameter of 300 pm. The mean sampling depth for laser Doppler has been estimated in a number of tissues for a range of probes geometries [11,12]. Taking the active area of the probe as 300 pm and assuming a cylindrical sampling volume, the nominal sampling volume can be calculated as approximately IO-* mm3.
Six microprobes were inserted into the tumour in order to make simultaneous readings of red blood cell flux from several sites. In fungating tumours no local anaesthetic was employed. However a subcutaneous injection of local anaesthetic (lignocaine 1%) was used to anaesthetise the skin in the remaining cases. It was assumed that the use of local anaesthetic was unlikely to
affect the results: firstly, because the probes were 25 mm in length and measurements were therefore made some distance below the skin; secondly, similar perfusion fluctuations had been observed in anaesthetised and unanaesthetised mice, although the actual readings were lower in the anaesthetised animals [5]. Since the microprobes have a blunt end, a 20 gauge cannula was used to make a nick in the skin; the probes were then advanced down the cannula into the tumour. Care was taken to make sure that the microprobes were advanced well beyond the tip of the cannula, and that they were secure in their position. Once a stable backscatter reading was obtained from each probe indicating that no probe movement was occurring, perfusion was then recorded over a period of 60 min. 2.4. Data analysis Each of the six channels recorded 20 flow readings per second. An average value for each 2-n& interval recorded was calculated and plotted against time for the separate channels. Since laser Doppler does not record an absolute measure of blood flow, arbitrary units of flow were used and data were analysed in terms of fold changes in blood flow. Each channel was looked at separately and the number, magnitude and duration of change noted. A proportion of the laser Doppler signal is due to a biological zero [1,6]. This signal is, in part, a result of the Brownian motion of free red blood cells in front of the probe. Based on animal studies a 30% correction was applied to our results [5]. This was done by calculating 30% of the last 2-min average recorded for each trace and subtracting that from the whole trace.
3.Results Seven patients were entered into this study, live female and two male with a median age of 67 years and a range of 55-80 years. In total eight sets of readings were obtained, one patient with bilateral groin nodes, had measurements of tumour perfusion performed on both sides of the groin, on two separate occasions. Blood flow measurements could only be undertaken in tumours which were easily accessible and large enough to allow the insertion of six microprobes (Fig. 1). The masses studied included primary and locally recurrent breast carcinomas, as well as metastases to regional lymph nodes and skin, from a variety of different histological tumours (Table 1). Six traces of microregional blood flow were obtained from each tumour, resulting in a total of 48 traces. Two traces were excluded, one because the probe was outside the tumour volume and the other because of probe movement throughout the 60-min recording period. In total 46 traces were available for analysis. Fluctuations
K.H.
Pigott
et al. /Radiotherapy
and Oncology
40 (1996)
45-50
Fig. I. Photograph of a tumour with the six microprobes inserted.
in blood flow were apparent in many of the traces, with some demonstrating more than one change over the observation period (Table 2). Furthermore, within the same tumour, changes in blood flow occurred independently in the different areas measured, with some areas showing no change and others a decrease or increase in perfusion (Fig. 2). A change in blood flow by at least a factor of 1.5 was seen in 25 of the 46 (54%) traces. In total, 37 fluctuations were observed: 24 increases and 13 decreases (Table 3). Patient no. 5 had a low grade non-Hodgkin lymphoma and a low platelet count (40 x lOgA) due to bone marrow involvement. Five of his six traces demonstrated a rise in blood flow, which we believe may have been a reflection of haemorrhage in front of the probe, resulting from his low platelet count. Since tumour perfusion was monitored continuously,
it was possible to study the time over which changes occurred. The duration of change was defined as the time from any maximum to any minimum blood flow reading or vice versa. Of the traces which changed by a factor of 1.5 or more, 54% of the changes occurred over 20 min or less. In a number of traces, 19% (7 of 37) reversal of the change in blood flow was seen during the 60-min observation period, with three of the traces showing more than one reversal of change. The median time taken for this change to occur was 13 min with a range of 4-44 min. 4. Discussion This study demonstrates the feasibility of using laser Doppler microprobes to detect temporal changes in microregional perfusion in human malignancies. Fur-
Table 1 Patient and tumour characteristics Case no. Age
Site
Size
Histology
1
80
Primary breast
Ductal carcinoma
2 3
61 13
4
19
5 6
59 67 55
Skin deposit Local breast recurrence Metastatic right groin node Metastatic left groin node Left groin node Primary breast Skin deposit
Whole breast replaced by tumour and involving skin 7 x 6 cm nodule involving skin 8 x 6 cm fungating mass 4 x 3 cm mass no skin involvement 7 x 8 cm mass involvmg skin 10 x 6 cm mass involving skin 10 x 10 cm mass no skin involvement 4 x 4 cm mass involving skin
I
Adenocarcinoma of the lung Ductal carcinoma Squamous cell carcinoma of the penis Squamous cell carcinoma of the penis Low grade lymphoma Ductal carcinoma Adenocarcinoma of the pancreas
ELfi. Pigott et al. /Radiotherapy and Oncology 40 (19%) 45-50
48
Table 2 Fold changes in blood flow for each trace of blood flow recorded
caseno.
Channel 1
Channel 2
I
I x2.1
X
Channel 3
Channel 4
Channel 5
Channel 6
t x II
I x 1.8 1 x 1.8
1x 1x 1x 1x t x I x 1x I x 1x
1.6 2.1 1.8 1.5 2.1 1.8 1.7 1.7 2.0
I I 1 t
1.9 1.6 1.5 1.7
I x 12 2 3
f x 1.9
4
t x 1.9
I x 4.0
1 x 1.8
t x 1.9
1 x 2.0 1 x 1.7
t x 1.9 X
5 6
1 x 1.5 1 x 1.5
I x 2.5
7
I I I t I
x x x x x
2.9 1.g 2.0 1.9 1.9
x x x x
I x 2.5 t x 1.8
X-trace excluded from analysis. Patient I, channel 2 - probe outside the tumour. Patient 6, channel 4 - backscatter signal not stable indicating probe movement. -: no changes in perfusion greater than 1.5.fold observed.
Patient 4 5000
-
4000
-
n
5000-
s
4000-
‘E
3000-
3000-
3
2000-
2000-
2
Probe
lOOO-
2
1
---....-.................==
O-d-
10
.r;
Time
aL
5000.
-
4000. 30
ii
30
40
50
60
-==-=-=-..-...--.............~. 0’
01
10
20 Tim
-
,000.
4
(m in.)
Probe2
3000 2000.
5
lOOO-
..--
20
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Fig. 2. Example of six traces of blood flow from patient number 4.
30
40 (m in.)
50
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K. H. Pigott et al. /Radiotherapy and Oncology 40 (19%) 45-M Table 3 Changes in microregional perfusion during 60-min observation period Factor of change
% of traces showing change
Direction of change
No. of traces
>I.5 >lS
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241 131 191 121
46 4cP
BPatient no. 5 excluded due to low platelet count and probable haemorrhaging around the probe.
thermore, it provides the first direct evidence that such changes are a frequent occurrence in human tumours. Over the 6Omin observation period used in the study, an increase or a decrease in flow by a factor of 1.5 was seen in 54% of all traces, with each tumour evaluated demonstrating a change in at least one microregion sampled. Twenty-eight percent of the regions sampled demonstrated a change in erythrocyte flux by at least a factor of two. It is not possible, in the absence of detailed morphological data, to accurately estimate the number of capillaries sampled in front of the probe. If, for example, we assume that 10 capillaries are sampled in front of a microprobe, then a reduction in flux by a factor of two could reflect an equal reduction in all capillaries or a situation where flow has ceased in five capillaries. The former would decrease the oxygen diffusion distance and thus increase the rim of hypoxic cells; the latter would create regional foci of hypoxia. Although it is not possible to discriminate between these potential causes, the example does indicate that perfusion-driven changes in oxygen delivery could contribute significantly to the occurrence of hypoxia in human tumours. Of some concern was the fact that, in the current study, there was a trend to see more increases than decreases in microregional red blood cell flux. Such a trend has also been identified in the CaNT tumour in mice but not in the SaF tumour (S.A. Hill and D.J. Chaplin, unpublished observations). One explanation for such an occurrence is that haemorrhaging into the region in front of a probe can occur over the 60-min observation period. This hypothesis is supported by the fact that one of the patients (no. 5) with a low platelet count and, thus, a tendency to bleed, displayed a gradual increase in blood flow in five of the six microregions sampled, with the sixth showing no change. We are confident that the variation in tumour blood flow seen in this study does not reflect variation in skin perfusion. Skin blood flow in a number of patients has been recorded using surface probes and, for comparison, only 11% of the traces varied by a factor of 1.5 or more compared with 54% of the tumour blood flow traces (S.A. Hill and D.J. Chaplin, unpublished observations).
49
The occurrence of perfusion-driven changes in tumour oxygenation has important implications for the strategies that need to be employed to circumvent hypoxia-induced radioresistance. Methods which provide an increase in the oxygen-carrying capacity of the blood would not be expected to be effective in reoxygenating regions where the blood flow has temporarily ceased. To reoxygenate such regions, agents are required which would prevent the fluctuations in microregional perfusion. One such agent, nicotinamide, has been shown to reduce the occurrence of transient reductions in microregional flow in a number of animal tumour systems [4,10]. The use of nicotinamide with carbogen breathing as an approach to improve radiation response in the clinic is being studied at several centres worldwide, including Mount Vernon Hospital. In summary, the present study supports the hypothesis that spontaneous and potentially reversible changes in microregional blood flow can occur within human tumours. These findings have potentially important implications for the design of therapeutic approaches to overcome or eliminate hypoxia. References 111 Abbot, N.C. and Beck, J.S. Biological zero in laser Doppler measurements in normal ischemic and inflamed human skin. Int. J. Microcirc. Clin. Exp. 12: 89-98, 1993. VI Brown, J.M. Evidence for acutely hypoxic cells in mouse tumours and a possible mechanism for reoxygenation. Br. J. Radiol. 52: 650-656, 1979. I31 Chaplin, D.J., Olive, P.L. and Durand, R.E. Intermittent blood flow in a murine tumour: Radiobiological effects. Cancer Res. 47: 597-601, 1987. 141 Chaplin, D.J., Horsmann, M.R. and Trotter, M.J. Effect of nicotinamide on the microregional heterogeneity of oxygen delivery within a murine tumour. J. Natl. Cancer. Inst. 82: 672-676, 1990. ISI Chaplin, D.J. and Hill, S.A. Temporal heterogeneity in microregional erythrocyte flux in experimental solid turnours. Br. J. Cancer 71: 1210-1213, 1995. 161 Colantuoni, A., Bertuglia, S. and Intagietta, M. Biological zero of laser Doppler fluxmetry-microcirculatory correlates in the hamster-cheek pouch during flow and no flow conditions. Int. J. Micro&c. Clin. Exp. 13: 125-136, 1993. [71 Gatenby, R.A., Kessler, H.B., Rosenblum, J.S., Coia, L.R., Moldofsky, P.J., Hartz, W.H. and Broder, G.J. Oxygen distribution in squamous cell carcinoma metastases and its relationship to outcome of radiation therapy. Int. J. Radiat. Oncol. Biol. Phys. 14: 831-838, 1988. 181 Hill, S.A. and Chaplin, D.J. Detection of microregional tluctuations in erythrocyte flow using laser Doppler microprobes.Oxygen transport in tissue. Adv. Exp. Med. Biol. XVIII: 367-372, 1996. B., [91 Hockel, M., Knoop, C., Schilenger, K., Vomdran, Baumann, E., Mitze, M., Knapstein, PG. and Vaupel, P. Intratumoral p0zpredict.s survival in advanced cancer of the uterine cervix. Radiother. Oncol. 26: 45-50, 1993. 1101Horsman, M.R., Brown, D.M., Hirst, V.K., Lemmon, M.J., Wood, P.J., Dunphy, E.P. and Overgaard, J. Mechanism of action of the selective tumour radiosensitiser nicotinamide. Int. J. Radiat. Oncol. Biol. Phys. 15: 685-690, 1988.
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K.H. Pigott et al. /Radiotherapy and Oncology 40 (19%) 45-50
Jakobsson, A. and Nilsson, G.E. Prediction of the sampling depth and photon path length in laser Doppler flowmetry. Med. Biol. Eng. Comput. 31: 301-307, 1993. [12] Johansson, K., Jakobsson, A., Lindahl, K., Lindhage, J., Lundgren, 0. and Nilsson, G.E. Influence of tibre diameter and probe geometry on the measuring depth of laser Doppler flowmetry in the gastrointestinal application. Int. J. Microcirc. Clin. Exp. IO: 219-229, 1991. 113) Overgaard, J. Importance of tumour hypoxia in radiotherapy. A
meta-analysis of controlled clinical trials. Radiother. Oncol. Suppl. 1, 24: s64, 1992. [14] Thomhnson, R.H. and Gray, L.H. The histological structure of some human lung cancers and the possible implications for radiotherapy. Br. J. Cancer. 9: 539-549, 1955. [15] Trotter, M.J., Chaplin, D.J., Durand, R.E. and Olive, P.L. The use of fluorescent probes to identify regions of transient perfusion in murine tumours. Int. J. Radiat. Oncol. Biol. Phys. 16: 93 I-935. 1989.