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1Cambridge Breast Unit, and 2Department of Nuclear Medicine, Addenbrooke's Hospital, Cambridge; 3Department of Physiological Medicine, St. George's Hospital Medical School, London, United Kingdom
Submitted 1 April 2005 ; accepted in final form 14 August 2005
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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99mTc-immunoglobulin; proteolysis
The majority of women (
75%) undergoing axillary lymph node resection never develop BCRL (15). Although the compensatory protective mechanisms are unknown, it has been suggested that they include the opening up of anatomical lymphovenous communications in the arm (1), rerouting of lymph through lympholymphatic communications distal to excised nodes (4, 9) and increased macrophage-mediated tissue proteolysis, allowing the escape of low-molecular weight peptides and protein fragments via the blood (5, 6).
Most studies of the effects of axillary lymph node clearance surgery have been on patients with established BCRL. Early pathophysiological responses to surgery, however, may provide important clues as to the cause of the condition. The aim of the present study, therefore, was to investigate the effects of surgery on the transport or mobilization of extravascular protein into local blood of the operated, ipsilateral arm as well as into central blood.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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The study population consisted of 16 women with recently diagnosed breast cancer ranging in age from 39 to 76 yr (mean 58 yr) (Table 1). Each patient was studied before and after axillary lymph node clearance surgery. The period between the first study and surgery ranged from 1 to 31 days (mean 12 days) and between surgery and the second study from 70 to 119 days (mean 90 days). Of the 16 patients, 11 had mastectomy, whereas 5 had wide local excision. None developed any infection, but one patient had a seroma. Five patients received radiotherapy to the breast and seven to the chest wall (of whom, 3 also had radiotherapy to the supraclavicular region). Ten patients received tamoxifen, two neoadjuvant chemotherapy, and four adjuvant chemotherapy. By the time of 3-yr follow up, four patients had developed clinical evidence of BCRL, defined as an arm volume of 10% or greater compared with the contralateral arm (allowing for preoperative differences).
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Radiolabeling of HIgG
99mTc-HIgG was prepared by the addition of 99mTc-pertechnetate to kits that contained 1 mg of 2-iminothiolane-derivatized HIgG and 8 mg of stannous chloride (Technescan HIG, Mallinkrodt Medical BV, Petten, Holland), as previously described (16–19). Radiochemical purity at the time of injection was >90%. For labeling with 111In, human immunoglobulin (Sandoglobulin, Novartis Pharmaceuticals UK, Frimley, Surrey, UK) was derivatized with diethylenetriamine penta-acetic acid cyclic anhydride as described by Hnatowich et al. (10). Before injection, 99mTc-HIgG and 111In-HIgG were diluted to 10 and 5 MBq/ml, respectively, with 0.1 M sodium bicarbonate containing 5 mg/ml HIgG, and 0.2-ml volumes were drawn into tuberculin syringes with 25-gauge needles. Radiochemical purity at the time of injection was >90%.
Depot Clearance
In 10 patients, 99mTc-HIgG was injected subcutaneously in the second dorsal web space of the hand on the affected side and 111In-HIgG was injected at the same site in the opposite unaffected hand, each in a volume of 0.2 ml, before and after surgery. In six patients, 111In-HIgG was injected on the affected side and 99mTc-HIgG on the normal side, again before and after surgery. Clearance rates from each depot were measured with a collimated sodium iodide scintillation detector, as previously described (16–19).
Venous Sampling
Twenty-gauge venous cannulae were positioned in the medial cubital vein at the antecubital fossa bilaterally. Blood samples of 5 ml each (after a 2-ml discard from the line) were taken 15, 30, 45, 60, 75, 90, 105, 120, 150, and 180 min after depot injection. All samples were prepared for counting in an automatic gamma counter (LKB-Wallac 1282 CompuGamma) as previously described (16–19).
Data Analysis
Depot clearance rate. The count rates recorded at each time point at the depot of injected radiolabeled HIgG were fitted with a single exponential, and the rate constant (k) was calculated. The value of k is dependent on several processes, including diffusion in the interstitial fluid away from the depot and possible direct access to local blood capillaries, but is generally assumed to be largely dependent on local lymph flow (13), quantification of which is routinely based on k in clinical lymphoscintigraphy (26).
Blood radiolabeled HIgG recovery. Blood concentrations of radiolabeled HIgG, sampled from the antecubital vein contralateral to the side of injection, were recorded as percentage of administered activity per liter of blood. The total amount of circulating radioactivity, expressed as a percentage of administered activity, was obtained by multiplication of the blood concentration in each sample by the subject's blood volume in liters, estimated from height, weight, gender, and age using a standard conversion equation (20). The total blood accumulation rate (bcontra), as determined from contralateral sampling, was essentially linear and fitted by linear regression analysis to give a slope with units of percent of administered dose per minute.
Blood concentrations of radiolabeled HIgG, sampled from the ipsilateral antecubital vein, were recorded as percentage of administered activity per liter of blood. The contralateral time-concentration curve was subtracted from the ipsilateral curve to record a curve that is corrected for recirculating activity. Using the principle of indicator dilution (i.e., principle of conservation of mass as in the Stewart-Hamilton equation for measurement of blood flow), the recirculation-corrected ipsilateral time-concentration curve was then integrated over 3 h (estimating concentrations at 135 and 165 min by interpolation) and compared with an assumed value for the local forearm blood flow that contributed to the dilution of radioprotein to obtain an estimate for total amount of radioactivity (M) accumulating in local ipsilateral blood as a function of time (18):
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where t is time. Effective forearm blood flow, which is not necessarily the same as total forearm blood flow, was conservatively assumed to be 20 ml/min, based on a forearm volume estimate of 1 liter and a forearm blood perfusion of 2 ml·min–1·100 g–1 (22). As for contralateral sampling, the profile of radioactivity accumulating ipsilaterally was fitted by linear regression analysis to give the rate of ipsilateral protein accumulation (bipsi) in units of percentage of administered activity per minute.
It has previously been shown in normal subjects that 111In-HIgG and 99mTc-HIgG give the same values of k and bcontra. Differences between the two tracers, however, were recorded from ipsilateral blood sampling (18). This was almost certainly because ipsilateral sampling is highly sensitive to small amounts of protein-free tracer; radioactivity associated with solutes of small molecular size preferentially enter local blood vessels instead of lymphatics, and this was more of a problem with 111In-HIgG than with 99mTc-HIgG. The bipsi based on 111In-HIgG was not therefore included for analysis.
Tissue-to-blood transport. The amount of radioactivity that had accumulated in central blood (as measured by contralateral sampling) and in the local vasculature (as measured by ipsilateral sampling and restricted to 99mTc) at 3 h was divided by the amount of 99mTc that had left the depot at the same time to give central and local tissue-to-blood (TB) transport, respectively, as previously described (17, 18).
Statistical Analysis
All variables described in Data Analysis were expressed as means ± SE. Associations between variables were quantified using Pearson's correlation coefficient. A P value of <5% was regarded as statistically significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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There was no difference between k, respectively, based on 111In and 99mTc in normal, contralateral arms, so the data based on the separate radionuclides were pooled. Mean k in the affected arm was 0.12 ± 0.007%/min, not significantly different from the mean value after surgery, which was also 0.12 ± 0.014%/min, although there was a wide range of changes (post/pre: 0.36–2.3). There was less variation in k before than after surgery, with respective coefficients of variation (standard deviation divided by mean) of 24 and 44% (Fig. 1).
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As for k, there was no difference between bcontra, respectively, based on 111In and 99mTc in normal, contralateral arms, so the data based on the separate radionuclides were pooled. Mean bcontra in the affected arm was 0.068 ± 0.009%/min, which was not significantly different from the mean value after surgery, which was 0.053 ± 0.0.011%/min As with k, there was a wide range of changes (post/pre: 0.15–2.2) and more variation after surgery, with respective coefficients of variation before and after of 49 and 73%, respectively (Fig. 2). There was a significant correlation in bcontra between the two arms before surgery (r = 0.72, P < 0.01) but not after.
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There was no difference between central TB transport based on 111In and central TB transport based on 99mTc in normal, contralateral arms, so the data based on the separate radionuclides were pooled. Before surgery, central TB transport was 0.56 ± 0.049, not significantly different from the postsurgical mean value, which was 0.49 ± 0.043. As with bcontra, there was a significant side-to-side association before (r = 0.68, P < 0.01) but not after surgery.
Ipsilateral Blood Recovery
Ipsilateral blood 99mTc concentration was higher postoperatively than preoperatively in all patients at all time points (Fig. 4). Accordingly, bipsi was higher after surgery in all seven patients in whom it could be measured, increasing from a mean preoperative value of 0.018 ± 0.006 to 0.038 ± 0.007%/min after surgery (P < 0.05). Cumulative ipsilateral activity expressed as a quotient of contralateral activity also increased after surgery in all seven patients so prominently that to facilitate comparison the ratio was log transformed (Fig. 5). Indeed in five of the seven subjects, cumulative ipsilateral activity exceeded contralateral activity, a finding that was not recorded in any patient preoperatively, in any of the patients in whom 99mTc-HIgG was injected into the unaffected arm or in any of our previously studied normal controls.
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Local TB transport increased from a mean preoperative value of 0.17 ± 0.046 to a mean postoperative value of 0.41 ± 0.083 (n = 7; P < 0.02; Fig. 6).
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When values for cumulative ipsilateral activity were compared with corresponding contralateral activities up to all individual blood sampling time points before surgery, strong positive correlations were seen at early time points (strongest at 30 min) with a slight decrease in the coefficient at later time points (Fig. 7A). Surgery, however, completely abolished these correlations and in fact reversed them all to negative correlations, although, unlike the presurgical correlations, they were not significant.
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Cumulative ipsilateral activities up to individual time points before surgery correlated significantly with the postoperative values at the corresponding times (Fig. 7B). The correlations, however, between correspondingly timed contralateral activities before and after surgery were all weak and negative.
Relation Between Lymphatic Function and BCRL
There was no clear association between the development of BCRL and any of the pre- or postoperative indexes of lymphatic function, either on the affected or unaffected side. It is noteworthy, however, that none of the seven patients in whom there was increases in bipsi and local TB transport developed BCRL.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Protein is normally transported in lymph from the interstitial space of the arm to central blood via the lymphovenous communications in the neck. It is believed that microvascular fluid flows from capillary lumen to interstitial space, but not in the reverse direction, even into venules (3). According to this belief, lymph flow must be equal to capillary fluid filtration. Because it is also believed that macromolecular transport from blood capillary lumen to interstitial space is overwhelmingly through convection, there is, under normal circumstances, no significant protein transport through the capillary from interstitial space to blood (14). If after surgery, however, protein was cleared from the extravascular space by transport into local blood vessels, it could be achieved either via peripheral anatomical lymphovenous communications that open up after surgery or directly across the blood capillary endothelium, possibly mediated through an increase in interstitial pressure resulting from surgery. Moreover, this could be facilitated by enzymatic degradation of protein into fragments with higher diffusibility (5, 6).
There is evidence of lymphovenous communications in both humans (1) and experimental animals (8, 7, 21). There is also evidence that protein can transfer directly across endothelium from interstitial fluid to capillary lumen, even physiologically (11, 12, 22), and indeed significant radioactivity was detected in ipsilateral blood preoperatively in the current patients. Although we cannot confirm that it was protein bound in the current patients, and although ipsilateral sampling is heavily influenced by even small levels of protein-free activity, our laboratory has previously shown that a significant proportion of the activity in ipsilateral blood in normal subjects is protein bound (17, 18), especially with respect to 99mTc-HIgG. The present results are therefore particularly interesting because they suggest that, after surgery, protein appears to be diverted toward direct local vascular access. Although the mechanism is not clear, this would clearly tend to offer protection against edema. Indeed none of the seven patients in whom ipsilateral 99mTc activity increased developed BCRL, as opposed to four for the entire population (25%).
The possibility needs to be considered that ipsilateral radioactivity concentration was increased not as a result of increased transport of intact or degraded radioprotein but as a result of a reduction in the blood flow diluting the radioactivity that leaves the depot to enter blood vessels. This blood flow is not identical to forearm blood flow but is presumably closely related to it. This explanation, however, seems highly unlikely from earlier work based on Doppler ultrasound showing that upper limb blood flow increases after axillary lymph node clearance both early and late after surgery, whether or not edema develops (25, 27). Moreover, from our own work in progress, it has recently been shown (Bennett Britton T, Wilkinson I, Peters AM, and Purushotham AD, unpublished observations) that forearm blood flow measured from strain-gauge venous occlusion plethysmography tended to increase 1–3 mo after axillary lymph node clearance surgery from 2.8 to 3.1 ml·min–1·100 ml–1 (n = 13; P > 0.05).
Increased proteolysis as a protective mechanism against BCRL was proposed by Casley-Smith and colleagues (5, 6). It would not only explain the present findings but also those of Bates et al. (3), who found a reduced interstitial protein concentration in epifascial interstitial fluid of patients with established BCRL. On the other hand, we have not found high ipsilateral levels, comparable to those reported here, in our own more recently studied patients with BCRL despite the fact that the radioprotein remains in the injection depot longer as a result of impaired local lymph flow (and low values of k).
The significant preoperative correlations observed between 99mTc activities respectively accumulating in central and ipsilateral blood and the significant correlations between 99mTc activities accumulating in ipsilateral blood, respectively, before and after surgery but with an overall doubling of activity after surgery support the view that local protein transport from interstitial space to blood does take place. We have previously shown that, when there is substantial protein-free activity in ipsilateral blood, the correlation between cumulative ipsilateral and contralateral blood contents tends to be negative (18), presumably because free activity is cleared rapidly from the central circulation once it arrives there. The inverse correlations observed after surgery suggests either that ipsilateral protein clearance tends to compensate for poor protein transport via lymphatics or that there is local proteolysis.
Whereas the effect of surgery on ipsilateral local protein transport was quite consistent from patient to patient (at least qualitatively), there was a highly variable response with respect to k and bcontra. These two latter variables, however, were closely correlated, making measurement error unlikely as the sole cause of their variability.
In conclusion, we have found a consistent increase in local mobilization of subcutaneously injected protein in response to axillary node resection that might be expected to blunt the tendency to develop BCRL. This increased mobilization is most likely the result of increased local transport from interstitial fluid to blood either directly across the vascular endothelium or through new lymphovenous communications. It may also be due to increased local proteolysis, but the net effect would be the same. Further prospective work is required on larger patient populations to relate this mobilization to the development or otherwise of BCRL and to establish to what extent it is the result of removal of intact protein or increased local proteolysis.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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-pyrone. N Engl J Med 329: 1158–1163, 1993.This article has been cited by other articles:
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  K. N. Lane, L. B. Dolan, D. Worsley, and D. C. McKenzie Upper extremity lymphatic function at rest and during exercise in breast cancer survivors with and without lymphedema compared with healthy controls J Appl Physiol, September 1, 2007; 103(3): 917 - 925. [Abstract] [Full Text] [PDF]
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) and after (
) axillary lymph node clearance surgery (99mTc and 111In combined), expressed as the percentage of the initial count rate. Values are mean ± SE of all patients for each time point. Note the wider SE values (vertical bars) after surgery.
) axillary lymph node clearance surgery. Before surgery, the correlations were all positive and mostly statistically significant, but after surgery, in contrast, they were all negative, although, individually, not significantly. Insets: relations recorded at 30 min, significant preoperatively but not postoperatively. B: correlation coefficient between the amounts of 99mTc that had accumulated respectively before and after axillary lymph node clearance surgery is shown for all corresponding sampling time points after injection into the affected upper limb. 
, accumulation in central blood. Correlations were all positive for ipsilateral accumulation (several statistically significantly) but in contrast were all negative (but not significantly) with respect to accumulation in central blood. Insets: relations recorded at 30 min, significant preoperatively but not postoperatively.