Trends in TransplantationTransplant. 2012;6:4-16 2012;6

Hand Transplantation

Stefan Schneeberger1,2, Theresa Hautz1, Robert Sucher1, WP Andrew Lee2, Johann Pratschke1 and Gerald Brandacher2

1Center for Operative Medicine, Department of Visceral, Transplant and Thoracic Surgery, Innsbruck Medical University, Innsbruck, Austria; 2Department of Plastic and Reconstructive Surgery, Johns Hopkins University School of Medicine, Baltimore, MD, USA

Abstract

Composite tissue originated during World War II, when skin allografts were first used to provide a soft-tissue coverage for severely burned patients. Composite tissue, and in particular skin allotransplantation, was limited by the stringent immune response towards the skin and reconstructive transplantation only became a clinical reality after more powerful immunosuppressants were developed in the 1980s and 1990s and after large animal trials indi- cated that survival was plausible under use of high-dose multidrug immunosuppression. Reconstructive transplantation has provided the successful restoration of partial and complete faces, hands, forearms, arms, abdominal walls, larynx, diaphyseal bone, bone-joint complexes, flexor tendon constructs, and, in one case, a penis. The surgical procedures are time-intensive and technically demanding and postoperative care and monitoring require a high degree of compliance. Hand transplantation has been the most frequently performed human composite tissue allotransplantation, with more than 50 upper extremity based transplants done worldwide. The functional and cosmetic outcome as well as patient satisfaction has been convincing in most cases, but skin rejection occurs in the majority of cases and requires adequate topical and systemic treatment. The intense immunosuppressive therapy usage in reconstructive transplantation has also resulted in loss of one patient after a combined bilateral hand and face transplantation as well as a high morbidity with metabolic side effects, infections, and malignancies. Recent advances in transplant immunology are shifting the focus from immunosuppression towards immunoregulation, making reconstructive transplantation with novel and less toxic immunosuppressive regimens a possibility. While early results are promising, further experimental and clinical trials are needed for better definition of suitable immunomodulatory protocols in reconstructive transplantation, and careful oversight and individualized screening procedures will be required as patients seeking improved quality of life through human composite tissue allotransplantation come to accept a certain level of risk in these experimental procedures. In summary, reconstructive transplantation offers to advance transplant medicine and reconstructive surgery, but more patients and science are needed before complex tissue defects can routinely be treated with composite tissue allografts. (Trends in Transplant. 2012;6:4-16) Corresponding author: Stefan Schneeberger, [email protected]

Correspondence to: Stefan Schneeberger Department of Visceral, Transplant and Thoracic Surgery Innsbruck Medical University Anichstrasse, 35 A-6020 Innsbruck, Austria E-mail: [email protected]

4 Stefan Schneeberger, et al.: Hand Transplantation

Key words

Reconstructive transplantation. Hand transplantation. Outcome. Rejection. Basic science. Cell therapy.

in the majority of patients14-19 (www.handreg- ntroduction I istry.com). Side effects of immunosuppression included viral, bacterial and fungal infections, Surgical reconstruction of complex tissue diabetes mellitus, nephrotoxicity, osteonecrosis, defects, e.g. after surgical excision of tumors, leucopenia, hypertension, posttransplant lym- accidents, or to treat congenital malformation, is phoproliferative disease, and hyperlipidemia1-7,20 in high demand. While small tissue defects can and the need for long-term immunosuppres- often be treated by transfer of autologous tissue, sion limited its wider application in hand trans- large and complex tissue defects leave the plantation. We herein summarize the achieve- patient with significant deficits in function, body ments in human hand transplantation and integrity, and appearance. Reconstructive trans- highlight interesting findings in basic science. plantation (RT) using allogeneic tissue from a brain-dead organ donor represents a valid solu- tion and has been performed in over 100 patients Hand transplantation – worldwide with success1-9 (www.a-s-r-t.com). Clinical aspects

The first attempt to transplant a human Immunosuppression hand was carried out in Ecuador in 196410. in reconstructive transplantation After rejection and re-amputation of the hand, the second attempt was only carried out in Vascularized composite tissue allografts 1998 in Lyon, France11. A series of small and with a skin component are considered an im- large animal studies indicated the efficacy of munological challenge as the skin is highly modern immunosuppression in limb trans- immunogenic21. Immunosuppressive regimens plantation and led us to believe that hand loss used in RT are in conformity with protocols used could now be prevented12-14. And this turned in solid . Most hand and out to be the case. Over 60 hand transplants patients received either poly- have since followed and one-year graft survival clonal (anti-thymocyte globulins, ATG) or mono- has exceeded 90%. The satisfactory functional clonal (alemtuzumab, basiliximab) antibody outcome also encouraged surgeons to proceed infusions for induction followed by tacrolimus, with forearm, arm, and face transplants. mycophenolate mofetil (MMF) and steroids­ triple-drug combination for maintenance20. Despite good return of function and Further, steroid-sparing protocols, mammalian high patient satisfaction, episodes of acute skin target of rapamycin (mTOR) inhibitors, and rejection were observed in most patients and use of topical steroids and tacrolimus ointments some hands were lost when immunosuppres- have been applied with success. The treatment sion was withdrawn or reduced in an uncon- used is efficient in preventing graft loss, but trolled fashion upon non-compliance or lack was not sufficient to prevent acute skin rejec- of funding14-19. Monoclonal or polyclonal anti­ tion the majority of cases. bodies were used for induction, and tacroli- mus, prednisone, and mycophenolic acid Donor bone marrow cell infusions have comprised maintenance immunosuppression been added to the induction regimens in hand 5 Trends in Transplantation 2012;6

transplantation in one center. The early outcome Skin rejection can present either in a focal or suggests that bone marrow infusion is safe and diffuse pattern of maculopapular lesions of enhances the chance of reducing maintenance diverse intensity3,5-8,18. In atypical cases of re- immunosuppression22-24. Data also indicate that jection, specially reddening of the palm and implementation of cell-based therapies could changes of the nails have been described27 help to induce immunomodulation subsequent Histopathological and immune cellular charac- to composite tissue allotransplantation (CTA) teristics of skin rejection in RT have been well and thus further optimize the outcomes. characterized and a uniform Banff grading sys- tem has been developed3,28,29. Grading of rejec- tion also reflect interesting dynamics of cellular Reconstructive trafficking. Rejection first appears in the perivas- transplantation rejection cular space of the dermis. If rejection progress- es, the infiltrate spreads to the interphase be- Intrinsic differences make RT rejection tween dermis and epidermis and/or adnexal both interesting and challenging. Per definition structures3,28. A cellular infiltrate within the epi- of composite tissue allografts, they comprise dermis is typical for moderate rejection, with the a set of different tissues conjoint to build a immunologic response reaching the outermost functional, but not immunological, unit. Experi­ layer. When progressing further, necrosis of mental work has indicated that the skin might single keratinocytes can be observed, resulting be the immunologically most challenging tis- in focal dermal-epidermal separation and finally sue and it has long been believed that human necrosis and loss of the epidermis3,28. skin allotransplantation cannot be performed successfully under conventional immunosup- Immunohistochemical markers for cellular pression. Early work investigating the immune infiltration and adhesion molecule expression in response to RT has revealed an interesting human hand transplant skin rejection were phenomenon: the sum of tissues comprising a analyzed in a set of 174 skin biopsies from five RT induces a weaker immune response that the human hand transplant recipients29. Acute re- individual tissues transplanted separately25. The jection was characterized by a predominantly mechanisms responsible for this, however, have CD3+ cell infiltrate that began in the perivas- not been adequately analyzed and it hence cular areas and spread from the dermis to the remains speculative if the quantity or the qual- epidermis as severity of rejection increased. ity of tissue has a prominent role in RT. A similar phenomenon has been described in Among the CD3+ cells, CD8+ cells are combined transplantation of solid organs. more prominent than CD4+ cells. Ten to 50% of cells stain positive for CD68 (histiocytic/ Rana, et al. recently found that com- macrophage linage). Very few cells are posi- bined simultaneous transplants of heart, liver, tive for CD20. and kidneys protect and are themselves protected from rejection. Also, this analysis Antibody mediated rejection revealed that a higher antigen load, such as Analysis of C4d depositions has been in double-lung or double-, used as the prime tool in the search for also reduces the risk of rejection26. antibody mediated rejection (AMR) in RT. Complement staining, however, is an unspecific Rejection of the individual tissues and indirect diagnostic marker for AMR and it remains to be proven if absence or insignifi- External location and ease of sampling cance of C4d staining indicates absence of allow for close monitoring of skin rejection in RT. antibody mediated processes in RT. 6 Stefan Schneeberger, et al.: Hand Transplantation

In a recent systematic analysis of AMR much less dramatic when compared with the in a rat limb transplant model, an increase in effect of transplanting tissues as a composite. antibody against third party following multiple This phenomenon was first described in a land- rejections was observed, but no clear evidence mark article by Lee, et al. in 199125. In an at- of an antibody mediated alloresponse was tempt to investigate the relative antigenicity of found30. This is in agreement with most case tissues, different timing and intensity of the reports, where mild and unspecific C4d stain- rejection of individual tissues was observed. ing at the vascular endothelium was found in More importantly, the whole-limb allograft elici­ some biopsy specimens in presence and ab- ted less immune response than did the indi- sence of cellular rejection. The significance vidual components. This evidence, however, remains unclear, this but did not correlate with derived from systematic analysis in animal rejection in any of the patients investigated6-8. models in which no or little immunosuppres- sion was used. When RT in humans was initi- Deposits of C4d were found in capillaries ated under conventional immunosuppressive not only accompanying cellular rejection, but regimens, early observations in individual cas- also in the absence of clinical rejection. es seemed to confirm the theory of a diverse immune response to the individual tissue com- The adhesion molecules most signifi- ponents. Individual case reports and cumula- cantly associated with severity of rejection were tive evidence from the International Registry on intercellular adhesion molecule (ICAM)-1 and Hand and Composite Tissue Transplantation E-selectin. An E- and P-selectin inhibitor, efo- indicate that the vast majority of patients ex- mycine M, administered in the subcutaneous perience one (> 85%) or multiple (56%) epi- tissues of the graft, facilitated allograft accep- sodes of skin rejection. Tissues other than the tance in a rat RT model when combined with skin remained unaffected or were affected to ATG and tacrolimus29. These studies demon- a lesser extent2-8,20,32. Probably the best indi- strate the feasibility of investigating early mark- vidual example was obtained from the first hand ers of rejection and testing their functional transplant of this series, where the patient re- significance in rodent RT models. quested re-amputation of the transplant after progressive skin rejection32. The systematic Cell adhesion and trafficking, however, histopathological assessment of the hand re- only represents one of many mechanistic ele- vealed erosive and necrotic areas over the skin, ments of skin rejection. After invasion of allore- but only mild inflammation in muscles and ten- active cells, a complex cascade of cell com- dons and no changes in bones and joints. Ad- munication and interaction can be monitored ditional evidence from deep-tissue biopsies during rejection. Similar to solid organ rejection, performed occasionally confirmed a dominance some of these markers might be interesting tar- of the immune response towards the skin, but gets for intervention (manuscript in preparation). also revealed cellular infiltrates in muscle and connective tissue. The predominant role of the skin in RT rejection was also observed in large Effect of allograft mass animal trials in which survival of all tissues except the skin was achieved after immuno- Little is known about the relevance of modulation with donor cells. In summary, there tissue mass on the alloimmune response. In a is good evidence that a composite of vascu- recent experimental trial, Ususal, et al. dem- larized tissue induces less immune response onstrated an advantageous effect of a larger than the individual components, convincing vascularized skin tissue mass on velocity and data supporting the concept that the skin elicits severity of rejection31. The effect, however, was the strongest immune response, but only weak 7 Trends in Transplantation 2012;6

evidence that tissue mass has a major impact atrophy with macrophage infiltration was seen on allograft rejection. Furthermore, little is known early after transplantation, vasculopathy was about the difference in the mechanisms of only observed later. rejection in tissues other than the skin. In par- ticular, it has not been analyzed if dynamics and trafficking patterns, such as those de- Basic science in reconstructive scribed for the skin, exist in other tissues. transplantation

As outlined above, over the past decade Chronic rejection RT such as hand and face transplantation has become a clinical reality and a clear treatment While chronic rejection – or composite option for those patients in need, suffering from tissue vasculopathy and degeneration – has complex tissue injuries or defects not amen­ not been an eminent threat in human hand or able to conventional reconstruction. Despite face transplantation, proliferation and/or degen- the fact that early and intermediate functional erative processes after RT remain a concern. A outcomes are highly encouraging, rejection and high rate of acute rejection episodes as fre- the need for high-dose multidrug immunosup- quently seen in RT was speculated to be myo- pressive treatment continues to be the bane intimal proliferation at the graft vessels, but little of vascularized composite allotransplantation evidence has risen from the case reports pub- (VCA), preventing wider clinical application. lished. It remains unclear at this point if (i) skin Thus, any reconstructive measures to improve rejection correlates with vasculitis and endo- these non-life-threatening conditions must ad- thelial cell damage, (ii) if the vessels of a hand dress a delicate balance of risks and benefits. or face are equally susceptible to vascular changes as e.g. coronary arteries, and (iii) if Therefore, research in this novel field mechanisms other than an alloimmune response needs to focus on both basic and translational may contribute to myointimal proliferation. studies related to the unique immunological features of VCA, to investigate the basic mech- Recently, a transplanted hand was lost anisms of an alloimmune response in VCA as at nine months after transplantation in Louis- well as to develop novel strategies and pro- ville as a result of myointimal proliferation in tocols for immunomodulation and tolerance allograft vessels and subsequent malperfu- induction after VCA without the need for long- sion and necrosis of the transplanted hand33. term immunosuppression. Another major sci- So far, this is the only report on chronic rejec- entific emphasis currently is centered around tion in RT; however, long-term follow-up and the question of how to establish new treatment evaluation for vascular endothelial prolifera- options to improve and enhance nerve regen- tion and myointimal thickening as a risk factor eration and hence functional outcome in VCA. for graft failure and graft loss are still needed.

Transplant vasculopathy has not been Animal models systematically investigated in a rat RT model30. After multiple treated episodes of acute rejec- To date, preclinical experimental animal tion, significant vascular lesions along with models have substantially propelled VCA re- skin and muscle atrophy, upregulation of pro- search and paved the way to study basic fibrotic gene expression and fibrosis was ob- immunology, functional recovery, and technical served. The bone became sclerotic, weak and feasibility of RT procedures. Orthotopic hind- prone to malunion and nonunion. While muscle limb transplantation in the rat has been the 8 Stefan Schneeberger, et al.: Hand Transplantation preferred rodent model ever since; however, marrow cells) established stable chimerism novel super-microsurgical techniques34,35 fa- and induced tolerance to vascularized com- cilitated VCA in even smaller species like the posite allografts46. In addition, Prabhune, et al. mouse36. In addition, different swine37 and showed that transplantation of donor bone primate38 models have been applied to study marrow cells into conditioned hosts immediate- acute and chronic rejection as well as tolerance ly after hind-limb transplantation combined with induction on the basis of different immunosup- tacrolimus and MMF resulted in stable mixed pressive protocols. chimerism and limb allograft tolerance47.

In 1984, cyclosporine (CsA) monotherapy Posited by the striking homology be- was successfully used to prevent hind-limb tween the mouse H2 and the human HLA allograft rejection between major histocompat- system, the widespread availability of genet­ ibility complex (MHC)-mismatched Buffalo and ically defined inbred and knockout strains and Lewis rats39 and several years later the com- the applicability of specific monoclonal anti- bination of both CsA and MMF was shown to bodies, murine orthotopic and heterotopic be synergistically effective40. Since orthotopic transplant models have been developed only limb transplantation is a rather traumatic op- recently48. Similar to the rat models, orthotopic erative procedure, requiring the removal of the murine hind-limb transplant models seem to limb at the recipient site to gain room for an be best suited to study functional outcome allograft, non-functional heterotopic hind-limb and nerve regeneration in VCA, whereas the transplant procedures have been developed to technically less demanding heterotopic cervi- overcome the surgical and technical difficulties cal model may be used to investigate basic of orthotopic transplantation41. Accordingly, immunology and clinically relevant questions these mo