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Kim K, Schuetz C, Elias N, Veillette GR, Wamala I, Varma M, Smith RN, Robson SC, Cosimi AB, Sachs DH, Hertl M. Up to 9-day survival and control of thrombocytopenia following GalT-KO swine liver xenotransplantation in baboons. Xenotransplantation 2012; 19: 256–264.. © 2012 John Wiley & Sons A/S. Abstract: Background: With standard miniature swine donors, survivals of only 3 days have been achieved in primate liver-transplant recipients. The recent production of alpha1,3-galactosyl transferase knockout (GalT-KO) miniature swine has made it possible to evaluate xenotransplantation of pig organs in clinically relevant pig-to-non-human primate models in the absence of the effects of natural anti-Gal antibodies. We are reporting our results using GalT-KO liver grafts. Methods: We performed GalT-KO liver transplants in baboons using an immunosuppressive regimen previously used by our group in xeno heart and kidney transplantation. Post-operative liver function was assessed by laboratory function tests, coagulation parameters and histology. Results: In two hepatectomized recipients of GalT-KO grafts, post-transplant liver function returned rapidly to normal. Over the first few days, the synthetic products of the donor swine graft appeared to replace those of the baboon. The first recipient survived for 6 days and showed no histopathological evidence of rejection at the time of death from uncontrolled bleeding, probably caused by transfusion-refractory thrombocytopenia. Amicar treatment of the second and third recipients led to maintenance of platelet counts of over 40 000 per μl throughout their 9- and 8-day survivals, which represents the longest reported survival of pig-to-primate liver transplants to date. Both of the last two animals nevertheless succumbed to bleeding and enterococcal infection, without evidence of rejection. Conclusions: These observations suggest that thrombocytopenia after liver xenotransplantation may be overcome by Amicar therapy. The coagulopathy and sepsis that nevertheless occurred suggest that additional causes of coagulation disturbance must be addressed, along with better prevention of infection, to achieve long-term survival.

Induction of donor-specific tolerance has been an ultimate goal in organ transplantation. Although numerous regimens for the induction of allograft tolerance have been developed in rodents, their application to primates has been limited. The approaches that have been successfully applied in primates can be divided into (i) use of total lymphoid irradiation, (ii) costimulatory blockade, (iii) profound depletion of recipient T cells, (iv) infusion of regulatory cells and (v) donor bone marrow (DBM) infusion/transplantation. Among these approaches, successful allograft tolerance has been achieved in clinical kidney transplantation using DBM transplantation.

Background: Histological recurrence of the hepatitis C virus (HCV) occurs in the majority of persons transplanted for cirrhosis as a result of HCV. Herein we analyze our experience with the use of both conventional and pegylated (PEG) interferon (IFN) in combination with ribavirin (RBV) in liver transplant recipients with recurrent HCV. Methods: Patients transplanted between 1992 and 2001 with post-orthotopic liver transplantation (OLT) histological recurrence of HCV, and who were treated with at least 6 months of IFN or PEG-IFN in combination with RBV were included in this analysis. A retrospective chart review was performed. Results: A total of 31 patients were included. Fifteen were treated with IFN/RBV and 16 with PEG-IFN/RBV. Of these 16, 11 had been begun on IFN/RBV and were changed to PEG-IFN/RBV because of persistent viremia. Three patients (20%) in the IFN/RBV group and six patients (37.5%) in the PEG-IFN/RBV group experienced a virologic response (VR) on therapy. Of the six patients experiencing VR in the PEG-IFN/RBV group, three (50%) were IFN/RBV non-responders. There were two sustained VRs (SVR). The 65.6% of all patients experienced a biochemical response (BR) on therapy. Seven deaths were observed. Dose modifications of IFN or PEG-IFN (87.1%) and RBV (80.6%) and the requirement for hematopoietic growth factors were frequent. Conclusions: Treatment of recurrent HCV infection with combination of IFN or PEG-IFN and RBV produced an on-therapy VR in 29% and BR in 65% of patients. Hematologic toxicity and dose modifications were frequent. Our experience with antiviral therapy for HCV post-OLT remains disappointing but PEG-IFN + RBV appears to produce VR in a sizable portion of IFN + RBV non-responders.

Hepatitis C virus (HCV) is currently the leading indication worldwide for orthotopic liver transplantation. However, the majority of patients receiving transplant for HCV eventually develop histopathologic evidence of recurrent allograft HCV and approximately 10% die or require retransplantation within the first 5 post-operative years because of accelerated graft injury and cirrhosis. Traditional induction immunosuppressive regimens and intensive immunosuppression used to treat episodes of acute cellular rejection (ACR) are associated with enhanced viral replication and higher likelihood and severity of recurrent HCV. At our institution, therefore, we have used low-dose steroid therapy in an effort to limit HCV replication. However, this practice has been associated with frequent early presentations consistent with ACR. Here, we present three cases consistent with histologic ACR treated with conventional antirejection therapy that improved transiently, but evolved rapidly to progressive HCV. A fourth patient with a similar presentation experienced dramatic improvement in aminotransferases when treated solely with interferon and ribavirin. We propose that histologic characteristics traditionally associated with ACR may, in fact, represent early recurrent HCV as both processes share common immunopathogenetic mechanisms, or alternatively, that both ACR and recurrent HCV may be present simultaneously. We conclude that in cases suggestive of ACR, careful consideration should be given to treatment for recurrent HCV in lieu of or in concert with intensive immunosuppression.

BACKGROUND Sentinel lymph node biopsy following radioisotope labeling is a recently developed, minimally invasive surgical staging procedure used in the management of primary cutaneous malignant melanoma. If histologic analysis reveals melanoma metastasis in the sentinel lymph node, completion lymphadenectomy is performed and adjuvant therapy considered. The routine pathologic assessment of the sentinel lymph node consists of bisecting the lymph node along its long axis and histologic examination of one hematoxylin and eosin–stained section of each cut surface. METHODS In this study, the authors reexamined 235 sentinel lymph nodes reported as negative for melanoma metastasis following routine histologic examination, from 94 patients with American Joint Committee on Cancer (AJCC) Stage I and II cutaneous melanoma. RESULTS Deeper sections into the lymph node and immunohistochemical stains with antibodies to S-100, HMB-45, NK1C3, and MART-1 led to the identification of microscopic metastases in 11 sentinel lymph nodes from 11 patients and capsular nevi in 9 sentinel lymph nodes from 8 patients. CONCLUSIONS Deeper serial sections and immunohistochemical stains detected microscopic metastases in approximately 12% of cases that would be reported as negative for metastasis by routine pathologic analysis. These techniques also allowed for the identification of capsular melanocytic nevi in the sentinel lymph nodes of 9% of patients. [See editorial on pages 551–2, this issue.] Cancer 1999;86:617–27. © 1999 American Cancer Society.

Prior studies from our laboratory have demonstrated that a nonmyeloablative conditioning regimen can induce transient mixed chimerism and renal allograft tolerance between MHC disparate cynomolgus monkeys. We have also shown that this preparative regimen can be extended to a concordant baboon to cynomolgus xenograft model by adding, to the post transplant protocol, therapy designed to prevent antibody production. Here we examine the use of brequinar (BQR) for this purpose and the efficacy of two new reagents developed to demonstrate the establishment of chimerism in the xenograft recipients. The cynomolgus recipients were conditioned with WBI (300 cGy), TI (700 cGy), ATG, cyclosporine, and brequinar sodium. To detect engraftment of the donor marrow, we prepared a polyclonal cynomolgus anti-baboon antibody (CABA) and a monoclonal antibody (215.1), which distinguish baboon and cynomolgus lymphocytes and granulocytes. We employed flow cytometry analysis to detect multilineage chimerism in the xenograft recipients. Five of the six recipients monitored using our new reagents (CABA and 215.1) developed detectable chimerism and only one of these animals lost its kidney to rejection. However, other complications have not permitted assessment of long-term outcome. The features of the multilineage chimerism included the detection of donor granulocytes (1.8-77.4%) and lymphocytes (2.4-22.2%) for 9 to 37 days. Our new reagents permit the detection of multilineage mixed chimerism, which may be a predictor of xenograft tolerance. We also conclude that brequinar may be effective in preventing antibody formation, but because of its toxicity, it is probably not the drug of choice for extension of the mixed chimerism protocol to concordant xenografts.

Ongoing studies at our center on facilitating transplantation of discordant xenogeneic organs are focused on tolerance induction. To abrogate hyperacute rejection, we have used adsorption methods to eliminate natural anti-Gal(alpha)1-3Gal (alphaGal) antibodies from the circulation of baboons. We have analyzed data concerning antibody removal in baboons that were 1) immunologically naive, 2) receiving conventional pharmacologic immunosuppressive therapy (IS), and 3) treated with a conditioning regimen for tolerance induction. We compared the efficiency of removing alphaGal antibody 1) by perfusion of whole blood through an alphaGal affinity column (CP; n=5) with 2) perfusion of plasma (separated from cellular components by apheresis) through an alphaGal column (CPA; n=39). Our studies demonstrate that 1) CP and CPA are equally effective in removing anti-alphaGal antibody, 2) CPA is the method of choice if multiple adsorptions are required, 3) CPA in naive animals transiently affects levels of total IgG and IgM, 4) four CPAs repeated at 2-4 day intervals in association with heavy IS reduce the pool of anti-alphaGal antibody and total Ig, and 5) splenectomy and/or IS delay the return of anti-alphaGal antibody.

BACKGROUND Melanoma can remain clinically quiescent for decades before regional or distant recurrence appears. This protracted disease free interval challenges the concept of a "cure" for melanoma. METHODS To understand this prolonged dormancy better, the authors retrospectively studied patients who developed recurrent melanoma 15 years or longer after their initial diagnosis ("ultra-late" recurrence). These cases were identified from 2766 melanoma diagnoses available in the Cancer Registry at the Massachusetts General Hospital (MGH). Histologic features of the primary lesion were also included when possible. RESULTS Twenty cases were retrieved from the MGH database. There were equal numbers of women and men, although women were younger at the time of initial diagnosis (mean age of women: 29.8 years vs. 43.0 years for men). No patients had more than one primary cutaneous melanoma. The trunk was the most common primary site (35%), although there was no predominant anatomic localization. The average disease free interval was 17.3 years for women, 20.0 years for men, 18.1 years for patients with regional recurrence, and 19.0 years for patients with distant metastases. Distant recurrence was the most common type of recurrence (50% of women and 60% of men). The estimated probability of survival (5 years after recurrence) was 0.8 for regional disease and 0.2 for distant disease. With the available histologic records, it appears that almost all tumors were Clark Level III or IV with thicknesses ranging from 0.8-2.3 mm. In contrast to the published cases, this study did not find that women with lower extremity melanomas were at higher risk for developing ultra-late recurrence. CONCLUSIONS Ultra-late recurrence of melanoma, although uncommon, can occur in any patient without identifiable risk factors. Because many prognostically favorable melanomas (thin melanomas on extremities) can recur after prolonged disease free intervals, the possibility of delayed recurrence remains and must be kept in mind. Cancer 1997; 79:2361-70. © 1997 American Cancer Society.

Long-term survival of discordant xenografts will require control of both humoral and cellular aspects of the immune response. Because cellular responses to xenografts appear to be more potent than those encountered by allografts, recipients of xenogeneic tissues are likely to require more immunosuppression, with potential for unacceptably high complication rates. We have therefore directed our attention toward the induction of tolerance to some or all of the xenogeneic antigens recognized in the primate anti-pig cellular immune response, utilizing mixed lymphohematopoietic chimerism as the means for tolerance induction. We report here our initial series of 16 animals in which the conditions for application of this treatment regimen were established. In 14 cynomolgus monkey recipients, induction therapy consisted of low dose whole body irradiation, thymic irradiation, and ATG, followed by infusion of pig bone marrow and a pig kidney transplant. Other aspects of the regimen included splenectomy and removal of monkey anti-porcine natural antibodies by extracorporeal perfusion of the recipient's blood through a pig liver. Two control animals received either no treatment or extracorporeal perfusion but no additional induction therapy. Five of the experimental animals were treated posttransplant with an anti-IgM monoclonal antibody, five with Cyclosporin A, and two with a combination of both immunosuppressants. Both IgM and IgG natural antibodies were removed effectively by liver perfusion in all but one monkey, as determined by flow cytometry. Antibody liters remained low for 5–7 days, but increased progressively thereafter. The longest kidney survival in this series was 13 days, in an animal which maintained excellent kidney function for the first 11 days posttransplant. Peripheral chimerism was detected only transiently on day 10 in the peripheral blood of this recipient. We conclude that extracorporeal perfusion by this technique removes natural antibodies and prevents hyperacute rejection, permitting maintenance of excellent renal xenograft function for at least 11 days. Additional manipulations appear to be required to achieve mixed chimerism and tolerance of the cellular immune system in this model.

A multivariate analysis was performed on 20 clinical and histologic variables from 327 Stage I prospectively studied melanoma patients who underwent elective regional lymph node dissection (ERLD). Primary tumor thickness, microscopic satellites, and the elapsed interval between diagnosis and ERLD, were selected as the combination of variables that were most highly associated with clinically occult regional lymph node metastases (P = 10(-15), model chi-square). Microscopic satellites were defined as tumor nests, greater than 0.05 mm in diameter, in the reticular dermis, panniculus, or vessels beneath the principal invasive tumor mass but separated from it by normal tissue on the section in which the Breslow measurement was taken. The probability of finding nodal metastases for melanomas less than 0.75 mm thick was 0% (0/41 patients); for those 0.76-1.50 mm, 4% (4/108); 1.51-3.0 mm, 14% (14/102); and greater than 3.0 mm, 39.5% (30/76). Primary melanomas greater than 1.50 mm thick with microscopic satellites were more often associated with nodal metastases than those of similar thickness without satellites (30/57 (53%) versus 14/121 (12%), P = 0.01). Some satellites probably represent intraspecimen metastases, while others do not. Any predictive model for occult regional lymph node metastases based on data from ERLD done less than 50 days after diagnosis may underestimate the prevalence of metastases.

Between 1976 and 1980, 136 patients were evaluated for primary treatment of cutaneous melanoma. Forty-nine lesions were invasive to Clark's Level II (38 patients) or III (II patients) with a thickness of 0.3 to 1.2 mm. Conservative re-excision was advised as definitive therapy for these patients. The margin of resection was defined as the maximum excision that would allow primary closure of the wound. Pathology reports of the re-excised specimens revealed the narrowest margins to be 0.7 to 4 cm. Unexpected residual tumor was present in 2 specimens and melanocytic hyperplasia in 12 specimens. After a follow-up period of 2.5 to 7.0 years, there have been no local recurrences. One patient developed regional lymph node metastases 16 months and, then central nervous system (CNS) metastases 25 months after primary treatment. A second patient died with pulmonary metastases 4.5 years after initial therapy. Melanomas that are not deeply invasive do not require wide excision and skin grafting for local control. Occasionally these thin lesions do produce systemic metastases, emphasizing the need for long-term follow-up of even “low-risk” patients. Cancer53:1256–1259, 1984. Cancer 53:1256-1259, 1984.

Fifteen variables were tested for their value in predicting recurrent disease in 46 clinical Stage I melanoma patients with metastases to regional nodes. A stepwise proportional hazards general linear model (Cox multivariate analysis) separated these melanoma patients with regional node metastases into at least two risk groups. Twenty patients in the relatively low-risk group had a five-year disease-free survival of 80% (in spite of having nodal metastases). This compares to a five-year disease-free survival of 17.5% for 26 patients in the high-risk group (P less than 0.001, Lee-Desu Statistic). Criteria for the high-risk group required that a patient have only one of the following two values: (1) The number of regional lymph nodes that contained tumor divided by the total number of nodes removed x 100% (percentage of positive nodes) greater than or equal to 20%; or (2) a primary tumor thickness of greater than 3.5 mm (regardless of node percentage). Conversely, patients in the low-risk group had neither of the above features. The high-risk group could further be stratified by the lymphocytic response at the base of the tumor. These findings have direct immediate application to the elective regional node dissection controversy and to adjuvant therapy studies containing these patients.