A comparison of ink-directed and traditional whole-cavity re-excision for breast lumpectomy specimens with positive margins.

BACKGROUND: Excising a breast tumor with negative margins minimizes local recurrence. With a positive margin, the standard re-excision consists of excising the whole cavity and all surrounding breast tissue. By marking the sides of the lumpectomy specimen with six different colored inks, the surgeon can limit the re-excision to the involved margin. We compared the local recurrence rate after these two re-excision methods. METHODS: Records were reviewed of 527 women (546 breasts) treated with lumpectomy at two institutions. The log-rank test was used to compare the local recurrence-free survival. RESULTS: Of 546 tumors, 245 (45%) had negative margins on the initial lumpectomy and were not re-excised. Fifty-five percent had a positive or close margin; 181 underwent whole-cavity re-excision, and 120 had ink-directed re-excision. The mean follow-up time was 3.4 years. There was no significant difference in local recurrence for the patients whose initial margin was negative (3.7%) compared with the 243 patients with initially positive margins who underwent a re-excision (3.3%). Eleven of 181 (6%) patients undergoing a whole-cavity re-excision developed a local recurrence, compared with none of 120 (0%) patients with an ink-directed re-excision (P = not significant). Tissue mass excised was significantly smaller in the ink-directed group (23 vs. 83 g, P < .05). CONCLUSIONS: Ink-directed re-excision of lumpectomy specimens with positive margins minimizes the amount of breast tissue removed without increasing the incidence of local recurrence and is therefore preferable to the standard whole-cavity method.

Xenogeneic endothelial cells activate human prothrombin.

BACKGROUND: Delayed xenograft rejection is characterized by platelet activation and fibrin deposition and is thought to occur independently of complement activation. We have therefore investigated the potential for xenogeneic endothelial cells (EC) to regulate the conversion of prothrombin to thrombin, a central component of the final common pathway of coagulation and an important platelet agonist. METHODS AND RESULTS: Quiescent porcine aortic EC (PAEC) were found to convert high levels of human prothrombin to thrombin (0.234+/-0.019 IU/ml) when compared with human aortic EC (0.017+/-0 IU/ml, 30-min time point, chromogenic assay; P<0.001). PAEC activation by human complement resulted in comparable levels of thrombin generation. Prothrombin conversion by PAEC as determined by generation of F1+2 (1.909+/-0.119 nmol/L) and formation of thrombin-antithrombin III complexes (125.611+/-6.373 microg/L) was significantly greater than the matched human aortic EC values (F1+2: 1.539+/-0.03 nmol/L, P<0.001; thrombin-antithrombin III: 1.833+/-0.104 microg/L, P<0.001). Sequential analysis of prothrombin activation by PAEC indicated generation of the intermediate meizothrombin followed by autolytically accelerated thrombin formation. Subsequent experiments established important cross-species' incompatibilities with respect to porcine thrombomodulin interaction with human thrombin and protein C in that PAEC had a reduced capacity to generate activated human protein C in vitro. CONCLUSION: These observations indicate a potentially important molecular barrier involving blood coagulation that may impact on the planned clinical application of porcine transgenic organs.

Effect of repetitive high-dose treatment with soluble complement receptor type 1 and cobra venom factor on discordant xenograft survival.

Hyperacute xenograft rejection may be modified by the activation and depletion of complement (C) using cobra venom factor (CVF). This method of prolonging xenograft survival is toxic and associated with systemic inflammation, which may potentially contribute to the pathologic features of delayed xenograft rejection. Soluble complement receptor type 1 (sCR1) inhibits both the classical and alternative C pathways and thus limits the production of proinflammatory products such as the anaphylatoxins. Hence, we investigated the effects of various sCR1 and CVF regimens, and combinations thereof, in the discordant guinea pig-to-Lewis rat cardiac xenograft model. Mean graft survival time (MST) was significantly prolonged with repetitive dosing (MST=22 hr) or continuous infusion of sCR1 (MST=32 hr) as compared with unmodified controls (MST=15 min). However, sCR1 did not prevent intragraft deposition of C3 or neutrophil infiltration and resulted in only partial inhibition of C-mediated hemolytic activity in vitro. Grafts in rats treated with a single dose of CVF (MST=67 hr) or repetitive doses of CVF (MST=69 hr) survived significantly longer than those treated with sCR1 alone, and lacked C3 deposition or neutrophil accumulation. Sera from these animals were completely depleted of C-mediated hemolytic activity. Animals treated with a single dose of CVF, or sCRI plus a single dose of CVF (MST=64 hr), had similar xenograft survival times. However, immunohistologic studies showed that addition of sCR1 to a single dose of CVF resulted in decreased macrophage activation and reduced levels of cytokines (tumor necrosis factor-alpha and interleukin-1beta) within xenografts as compared with that in recipients treated with CVF alone. Such decreased macrophage activation may result from the binding of C4b by sCR1, since combination therapy was associated with decreased intragraft C4b as compared with either therapy alone. High doses of sCR1 were well tolerated by rats and significantly prolonged discordant xenograft survival (MST=32 hr), although not to the same extent as CVF. The modification of the intragraft immune responses seen with CVF/sCR1 combination therapy may augment further therapeutic manipulations to achieve discordant xenograft survival without the attendant toxicity associated with repeated CVF administration.

Inhibition of platelet integrin GPIIbIIIa prolongs survival of discordant cardiac xenografts.

The integrin GPIIbIIIa is known to be crucial to the formation of platelet aggregates and potentiates adhesion to subendothelial matrices via fibrin(ogen), von Willebrand factor, and vitronectin. Given the demonstration by us and others of widespread platelet aggregation during xenograft rejection, we hypothesized that platelet thrombi might contribute to graft dysfunction during development of hyperacute rejection (HAR), as well as during what we have termed delayed xenograft rejection (DXR), e.g., as seen in complement-depleted rat recipients of guinea pig cardiac xenografts. We therefore tested the effects of a specific GPIIbIIIa antagonist (SDZ GPI 562) during xenograft rejection. Lewis rats received heterotopic guinea pig cardiac xenografts and were treated with GPI 562 alone (HAR model) or in combination with cobra venom factor (CVF) (DXR model). A high (0.5 mg/kg) or a low dose (0.1 mg/kg) of GPI 562 was administered perioperatively and then given twice daily in the same dose until rejection. CVF was given daily until rejection. Plasma drawn after the first dose of GPI 562 and at the time of rejection was tested for the ability to inhibit ADP-stimulated platelet aggregation in vitro. Rejected grafts were analyzed by immunohistology. Plasma from animals in the high-dose group completely inhibited platelet aggregation in vitro, whereas plasma from the low-dose group resulted in only partial inhibition. Similarly, whereas low-dose GPI 562 failed to prolong graft survival, high-dose GPI 562 showed a statistically significant increase in graft survival in both HAR and DXR groups. Immunohistologic studies of HAR showed little effect of GPI 562 on platelet aggregation or activation and no effect on fibrin deposition. However, the combination of high-dose GPI 562 and CVF resulted in a significant decrease in intragraft platelet aggregation, P-selectin expression, and leukocyte infiltration compared with CVF alone. In conclusion, GPIIbIIIa antagonist therapy can inhibit platelet aggregation in vitro and prolong xenograft survival. The diminution of intragraft platelet microthrombi formation and leukocyte infiltration suggests an important role for platelet-dependent mechanisms in leukocyte recruitment during DXR.

Thrombin inhibition in an ex vivo model of porcine heart xenograft hyperacute rejection.

Prominent components of vascularized xenograft rejection such as platelet activation and microvascular thrombosis may be dependent upon thrombin generation in vivo. To study potential therapeutic benefits of a synthetic low-molecular-weight thrombin inhibitor, SDZ MTH 958, in hyperacute porcine heart rejection by human blood ex vivo, a working model of hyperacute rejection of porcine by fresh, heparinized (6 microM/ml) human blood with or without 1 microM SDZ MTH 958 was used. Thrombin-antithrombin complexes (TAT) and prothrombin fragment F1.2 levels as markers of thrombin activation were determined, and biopsies from rejected hearts were analyzed by immunohistopathology. Control porcine hearts (n=8) underwent a rapid and consistent decline in cardiac output, ceasing function by 60 min. Experimental cardiac output values of 14 ml/g (SEM 1.2) were significantly higher than seen in controls (5 ml/g SEM 0.6) after 5 min of cardiac work, and prolonged survival times up to 120 min were noted (P<0.05). Activity of SDZ MTH 958 was confirmed by functional assays throughout perfusion. Levels of TAT and F1.2 increased consistently in control samples when compared with plasma samples containing SDZ MTH 958. Immunohistopathological examination confirmed diminished fibrin deposition, reduced leukocyte adherence to endothelium, impaired diapedesis and less tissue necrosis in the hearts perfused with SDZ MTH 958. SDZ MTH 958, in this xenoperfusion model, prolonged survival, enhanced function of the explanted organ, and improved histological features at the time of rejection. Effective and specific antagonism of thrombin may be useful as an adjunct therapy to complement inhibition for xenograft rejection