In December of 2008, our institution performed a near total face transplant. The patient was monitored for signs of rejection assessed by paired skin and mucosa biopsies. The results of histological review of 120 biopsies collected during the first 4 years posttransplant are discussed. All biopsies were stained with hematoxylin and eosin, periodic acid-Schiff, immunohistochemical and TUNEL assays and graded using the Banff 2007 classification. Grade III rejection was diagnosed clinically at weeks 45 and 66, posttransplant; week 45 was determined as folliculitis while the erythema episode at week 66 confirmed an acute rejection (AR) that required hospitalization. The mucosa frequently showed interface inflammation without clinical signs of rejection and was not present in skin biopsies. In all, 34 of the 45 mucosal biopsies (75%) showed these interface changes. Clinical symptoms concurred with skin pathology in two grade III rejections. The mucosa showed histologic signs of rejection more frequently, which may indicate: increased mucosal sensitivity to rejection, a different type or subtype of AR that is specific to the mucosa, or a nonspecific process such as a drug effect. With more data and world experience, the diagnosis of face transplant rejection will be better defined and the Banff classification enhanced.
hemotoxin and eosin
terminal deoxynucleotidyl transferase
terminal deoxynucleotidyl transferase dUTP nick end labeling
vascularized composite allograft
Advances in immunosuppression and surgical techniques facilitated the engineering of a new class of grafts known as vascularized composite allografts (VCA). In December of 2008, a multidisciplinary team of Cleveland Clinic physicians performed clinical VCA—a near total face transplant. It was the most complex and extensive procedure of this kind, at that time . The pathology team at Cleveland Clinic was responsible for monitoring the transplant tissue for signs of acute rejection (AR), chronic rejection, infection and general histologic changes as part of their effort to preserve facial graft function. In this article, we discuss the histologic results of all (n = 120) skin and mucosal biopsies obtained during the first 4 years after the patient's face transplant. Specifically, we emphasize the correlation of biopsy grading with changes in immunosuppressive therapy, clinical events of rejection, infection and patient hospitalization.
Materials and Methods
The Cleveland Clinic IRB committee approved face allograft transplantation on November 15, 2004. The patient signed informed consent for the procedure on August 7, 2008.
The patient was a 45-year-old woman who had suffered a gunshot wound to the face in 2004, resulting in extensive injuries to the central part of her face including, but not limited to skin, lower eyelids, entire nose, upper lip, teeth, underlying bones (maxilla, zygomas) and oral cavity with hard palate . This severe multitissue loss impacted the patient's ability to breathe, eat, smell or speak without a slur. More than 23 reconstructive procedures were performed to correct these defects but were unable to restore form and function. At this point, a decision was made to proceed with facial allotransplantation. The composite graft included skin, mucosa, bone, nerves, vessels, cartilage, muscle and teeth, as well as functional units of nose, eyelids and lip. Extra skin was left at the margin of the graft to allow for the collection of edematous fluid and to act as a bank of aesthetically unimportant tissue for future biopsies, taking the place of a sentinel patch. Maintenance immunosuppression for year one consisted of tacrolimus, mycophenolate mofetil (MMF), prednisone and topical application of clobetasol cream . Maintenance immunosuppression for years 1, 3 and 4 consisted of tacrolimus and sirolimus, prednisone, topical application of clobetasol cream and tacrolimus ointment. Rejection surveillance consisted of clinical follow-up and skin and mucosa biopsies.
Biopsy specimens and immunopathomorphologic assessment
Between December 2008 and December 2012, a total of 120 biopsies were collected from the skin (n = 75) and oral mucosa (n = 45). Paired biopsies were taken weekly for 2 months, then biweekly for 1 month, monthly and annually (Figure 1A and B). Additional biopsies were taken if rejection was suspected. The biopsies were taken from the buccal mucosa and lateral cheek. Biopsy specimens were processed with formalin fixation and paraffin embedding. For each biopsy, two hematoxylin and eosin (H&E) stains and one periodic acid-Schiff (PAS) stain were prepared. The Banff working group classification for VCA pathology , combined with our additions which included assessment of mucosal lesions and clinical signs of rejection (Table 1), was used to grade rejection.
To identify cellular components specific for normal and affected tissues, the immunohistochemical (IHC) stains were performed for the presence of T-lymphocytes: CD3, CD4, CD8, FOXP3, B-lymphocytes: CD20, activated cells: CD30 and HLA-DR, macrophages: CD68, Factor XIIIa, Langerhans cells: CD1a, S100, proliferative index: Ki67, melanocytes and vessel endothelium: CD31, CD34. Biopsies taken at the time of AR episodes throughout the 4-year period were checked for the presence of cytomegalovirus (CMV). All antibodies used for IHC staining are listed in Table 2. The Ventana automated immunohistochemistry system was used to perform immunological studies. If rejection was suspected based on the H&E and PAS stains, a C4d IHC stain was performed to assess for the presence of antibody-mediated rejection.
|Detected antigen||Clone||Product of:|
|CD3||2GV6||Ventana Medical Systems (VMS), Tucson, AZ|
|CD4||1F6||Leica Microsystems, Buffalo Grove, IL|
|CD8||1A5||Biogenex Laboratories Inc., Fremont, CA|
|CD20||L26||Dako, Carpinteria, CA|
|CD30||Ber-H2||Dako, Carpinteria, CA|
|HLA-DR||TAL.1B5||Dako, Carpinteria, CA|
|CD68||KP-1||Ventana Medical Systems (VMS), Tucson, AZ|
|Factor XIIIa||AC-1A1||Cell Marque|
|CD1a||O10||Serotec, Raleigh, NC|
|S100||Polyclonal||Dako, Carpinteria, CA|
|Ki67||Rabbit monoclonal 30-9||Ventana Medical Systems (VMS), Tucson, AZ|
|Melanocytes (gp100)||HMB45||Biogenex Laboratories Inc., Fremont, CA|
|CD31||JC70A||Dako, Carpinteria, CA|
|CD34||QBEnd/10||Cell Marque Corp., Rocklin, CA|
|Cytomegalovirus (CMV)||DDG9+CCH2||Dako, Carpinteria, CA|
|C4d||A24-T||American Research Products, Belmont, MA|
TUNEL assay for apoptosis
Sections were processed for in situ IHC localization of nuclei exhibiting DNA fragmentation, using a TUNEL assay (Apoptag Plus; Chemicon International, Temecula, CA). Briefly, sections were deparaffinized, rehydrated with xylene and ethanol and permeabilized with 20 µg/mL Proteinase K (Gibco, Grand Island, NY). Endogenous peroxidase was inhibited with 3% H2O2, sections were immersed for 60 min in TdT buffer at 37°C, incubated for 30 min with anti-digoxygenin peroxidase conjugate followed by peroxidase substrate diaminobenzidine. Finally, sections were counterstained with methyl green. Standard sections of rat mammary gland were used as positive controls. Parallel sections of tissue were stained but not immersed in TdT—these acted as negative controls. The presence of apoptotic cells was determined by microscopic assessment at 400× magnification.
The density of the TdT positive cells was graded by cell counting in five high power fields (HPF) based on pathologic severity scores for acute and chronic GvHD [3, 4]. The grading was as follow: grade 0 = no cells, grade 1 = 1–5 TdT+ cells/5 HPF, grade 2 = 6–10/TdT+ cells/5 HPF and grade 3 >10 TdT+ cells/5 HPF.
Histopathologic and clinical correlation
The first cutaneous biopsy was unremarkable. However, the mucosal biopsy contained neutrophilic debris. At week 3, the cutaneous biopsy contained a segment of entrapped salivary gland with associated inflammation, but there were no signs of rejection (Figure 1C). The mucosal biopsy from week 3 contained a mild interface inflammation and interface mucositis with basal vacuolar change, but no obvious signs of apoptosis. There was a complete lack of submucosal perivascular inflammation.
Biopsy results were unremarkable from weeks 4–6. At week 7, the mucosal biopsy assessed by routine H&E and TdT staining demonstrated interface inflammation and interface mucositis with clumps of apoptotic epithelial cells diagnosed as grade III AR. The patient was treated with 1 g of methylprednisolone. Follow-up biopsies from week 8 showed a mild perivascular infiltrate in the skin, interface inflammation and interface mucositis with vacuolar changes without apoptosis in the mucosa. By week 10, multiple foci of interface mucositis were observed (Figure 1D). There was a complete lack of submucosal perivascular inflammation.
The mucosa continued to show interface mucositis without apoptosis in the biopsies from weeks 12 and 14, diagnosed as grade II rejection. At week 23, two separate mucosal biopsies showed interface mucositis with apoptosis classified as grade III AR. The biopsy at week 26 was unremarkable, then at weeks 30, 36 and 39, the mucosal biopsies showed interface mucositis without apoptosis, diagnosed as grade II rejection.
At week 44, the cutaneous biopsy showed a mild perivascular inflammation without interface inflammation, diagnosed as grade I AR. The mucosal biopsy showed interface mucositis with apoptosis, but without submucosal inflammation. At week 44, the patient developed a small red acneiform papule (Figure 2A). Histologically, it contained a moderate perivascular inflammation in the superficial and deep dermis. This was classified as grade II AR (Figure 2B). The line diagnosis, however, was descriptive and suggested the possibility of a chronic folliculitis. The mucosal biopsy from week 44 again showed grade III rejection. Topical clobetasol ointment to the upper lip area was stopped due to suspicion of a drug effect, and face washing twice daily with cetaphil was recommended. Over the next week, a faint rash appeared in the area surrounding the papule. A biopsy of this area showed a perivascular, perifollicular infiltrate that filled the superficial and middle dermis and contained scattered eosinophils. The differential diagnosis of a grade II rejection or acneiform rash lesion/drug eruption was considered. The patient was started on topical clindamycin 1% lotion to the upper lip area, and the acneiform rash resolved.
At week 66, biopsies were accompanied by reports that the graft appeared more pigmented, possibly due to a recent episode of unprotected sun exposure (Figure 2C). Histologically, both the skin and mucosa biopsies demonstrated changes consistent with grade III AR (Figure 2D and D1). Since the graft had become more erythematous, the patient was admitted to the hospital and treated with methylprednisolone 1000 mg for 2 days, an adjustment in tacrolimus (to 15 mg), and the addition of sirolimus (2–4 mg) and topical tacrolimus.
Biopsies at weeks 67–69 continued to show grade II rejection but with gradual improvement. By week 71, the episode of rejection had subsided. The skin biopsies were normal, and there was grade II focal interface inflammation in the mucosa. The tacrolimus, sirolimus and prednisone were gradually tapered until a low dose combination of these medications became the patient's standard immunosuppression regimen, at that time, in addition to topical tacrolimus and clobetasol cream.
At week 74, there was a mild perivascular infiltrate suggestive of grade I rejection in skin, and interface mucositis, without apoptosis, consistent with grade II rejection in the mucosa. During this period the immunosuppressive regimen was changed, slowly discontinuing sirolimus in preparation for the elective removal of redundant skin. At week 81, a mild to moderate perivascular lymphocytic infiltrate in the skin biopsies was seen. The mucosal biopsies showed a dense interface mucositis with apoptosis consistent with grade III rejection. Clinically, the patient had no symptoms between weeks 74–81.
During the 84th week posttransplantation, the patient underwent a planned surgical removal of excess skin and tissues. These tissues included fragments of bone (ethmoid and nasal), parotid gland, native skin, graft skin and mucosa. Each of the five excised portions of skin showed a mild perivascular and periadnexal lymphocytic infiltrate consistent with grade I rejection. The mucosa showed an interface mucositis consistent with grade II rejection. The native skin that was removed was histologically unremarkable.
At week 106, there were two skin biopsies demonstrating perivascular inflammation consistent with grade II rejection, and two mucosal biopsies with interface mucositis and clusters of apoptotic keratinocytes consistent with grade III rejection, without clinical signs of rejection.
Since this episode of mucosal grade III AR at week 106, and continuing through the end of year 4 following face transplantation, we have observed no significant changes in the skin or mucosa. Only mild to moderate perivascular infiltrations, without keratinocyte apoptosis, were seen in the examined skin and mucosa.
Review of histology
A review of all H&E stains revealed that 27 of the 75 (36%) cutaneous biopsies showed signs consistent with a diagnosis of AR (Table 3). Eleven of these biopsies were associated with two specific occasions where rejection was clinically suspected. The first occurred at week 45 when a red acneiform papule that clinically resembled folliculitis developed, and at week 66, when the whole graft became erythematous. Prominent interface activity, keratinocyte apoptosis or vacuolar changes were identified in H&E stained sections during the second episode, at week 66.
A review of 45 mucosal biopsies revealed that 22 (49%) showed signs consistent with a diagnosis of grade II rejection and 9 (20%) with a diagnosis of grade III rejection. None of the mucosal biopsies contained a prominent submucosal infiltrate. All of the 30 biopsies with a diagnosis of rejection contained interface mucositis. Twenty-four of these mucosal biopsies with interface mucositis did not have accompanying clinical symptoms of rejection or mucosal changes. The nine specimens diagnosed with grade III rejection contained a cluster of two or more apoptotic cells in the H&E stained specimens. Mucosal biopsies from weeks 7 and 23, histologically assessed as grade III rejection, contained apoptotic keratinocytes confirmed by positive TdT staining (Figure 3). According to our grading scale for TdT positive cells, the density of apoptotic cells corresponded with grade 3 at week 7 and with grade 1 at week 23.
IHC studies were performed on both the mucosal and skin biopsies. In all biopsies, CD1a and S-100 stains highlighted epidermal and dermal Langerhans cells that were qualitatively and quantitatively similar to native skin. A normal number of melanocytes was identified by HMB-45 stain. The epidermal proliferative fraction was identified by basal nuclear and follicular epithelial staining to Ki-67 and was quantitatively similar to native skin. Between 70% and 95% of the lymphocytes were CD3 positive T cells in each of the biopsies, with a CD4:8 ratio between 1.5:1 and 3:1 (Figure 4A and B). CD20 identified only scattered dermal B-cells with the exception of weeks 41, 43, 66–68 and 106 in which several small aggregates were present (Figure 4C). FOXP3 expression identified regulatory T cell lymphocytes varying from 1% to 15% (Figure 4D). HLA-DR expression was detected on the small perivascular aggregates, on the vessel endothelial cells, and on the single keratinocytes in the skin. Focal expression of the HLA-DR antigen was seen in the mucosal epithelium (Figure 4E and F) and was detected in the specimens histologically consistent with AR. CD30 identified rare scattered, activated lymphocytes. CD68 and Factor XIIIa stains revealed normal numbers of dermal histiocytes. Dermal and submucosal vessel endothelial cells stained positively for CD31 and were unremarkable.
Neither C4d deposition or CMV protein expression was identified by IHC staining in any of the examined skin or mucosa biopsies.
This face transplant was only the fourth procedure of its kind and the most extensive in regard to the variety of tissue and percent of face transplanted, at the time it was performed. To date, 24 face transplants have been performed worldwide. Most of these cases have had positive outcomes, increasing the likelihood that face transplantation will become more common in the foreseeable future [1, 5-8]; however, there are still limited reports regarding the pathology of face transplant [9-12] and thus, we introduce here, one of the most comprehensive overview of 120 skin and mucosal biopsy reports, in a face transplant patient, over a 4-year period.
The pathology team was responsible for monitoring the patient for AR, chronic rejection, infection and general histological changes. CMV infection or reactivation is one of the risk factors contributing to the development of acute or chronic rejection leading to allograft dysfunction [13, 14]. It is unclear whether CMV infection promotes AR or if augmented immunosuppression used for AR prevention is responsible for CMV infection. In addition, CMV treatment protocols after transplantation include reduction of immunosuppression, which may increase the risk of allograft rejection. Our CMV-seronegative patient was at a higher risk of CMV infection since she received face transplant from the CMV-seropositive donor. When she developed a CMV infection, it was always confirmed by the CMV DNA copies detected in the peripheral blood (shown in Table 3); however, intragraft CMV protein expression was not identified by IHC staining in any of the examined skin or mucosa biopsies. Moreover, we have not detected intranuclear inclusions within dermal endothelial cells, which represent the histological hallmark of CMV infection . During the first posttransplant year, the presence of CMV DNA copies in peripheral blood was associated with grade II histological lesions in the graft mucosa; however, no inflammatory infiltrates were seen in skin biopsies and there was no evidence of clinical symptoms of rejection. Episodes of grade III AR in the graft mucosa were preceded by positive CMV copies in the peripheral blood. During the first posttransplant year, there was no correlation between CMV-viremia and skin or mucosa lesions, whereas in the second year, significant AR episodes (grade III) occurred without detectable CMV copies in the peripheral blood. These observations confirm that in our face transplant patient, the correlation between CMV infection and AR was not always predictable compared to reports in solid organ transplants . In June 2009, our patient received the CMX vaccine and in April 2011 converted to a CMV positive status. Since that time we have not observed any grade III rejections or clinical symptoms of CMV infection.
Signs of chronic rejection were never seen. This lack of chronic rejection is consistent with previous reports assessing biopsies after hand and face VCA . In contrast to hand allograft recipients, where unexplained C4d positive staining was detected at 1 year posttransplantation , we have not identified any C4d depositions in the vessel walls and donor-specific antibodies have never been detected in our face transplant patient over the entire 4-year follow-up period, which is consistent with previous reports . However, histological changes suggestive of AR have been frequent. In total, 58 of 120 biopsies (48%) had histologic changes that were assessed as AR. In summary, out of 120 skin and mucosa biopsies only 18 pairs were concordant in their histological assessment, showing either grade 0 or mild inflammatory perivascular infiltrates. Only at week 66, concordant skin and mucosa specimens were diagnosed as grade III AR. In contrast, 24 paired skin and mucosa biopsies were found discordant; mucosa was always assessed as either grade II or III AR, whereas skin biopsies were unremarkable or assessed as grade I.
We noted that in mucosal biopsies, there was frequent interface mucositis that was not present in the skin biopsies. These features led us to question whether the histological picture observed on the mucosal biopsies truly represented AR. The diagnosis of AR grade III was confirmed after expert consultation ruled out other causes of interface inflammation.
We used the Banff working group classification for VCA to grade signs of transplant rejection . This classification system was developed through a collaboration of scientists and physicians involved in VCA cases. At the time this grading system was developed, only three face transplants had been reported, versus 28 hand transplants, nine abdominal walls and several knees [2, 9, 10]. Thus, few mucosal biopsies were available for evaluation during the formation of this classification system. Our findings are consistent with those of Kanitakis et al.  and note a different histological picture in the skin and the mucosa. It is known that each of the transplanted tissues has its own antigenicity . The varying antigenicity of the VCA components may lead to a process known as split tolerance, as introduced in the swine experimental model, where musculoskeletal portions of VCA showed fewer signs of rejection than cutaneous components . Likewise, in our experience and in the literature [10, 12], mucosal biopsies generally show more severe signs of rejection than the skin biopsies and are consistently more sensitive but frequently do not correspond with clinical signs of AR in the graft. Moreover, if these mucosal changes do represent true rejection, the manifestation and progression of rejection in the skin and mucosal biopsies examined differ.
Apoptosis is a hallmark of AR in tissue and cell allotransplantation. None of the previous studies has evaluated the presence of apoptosis by detection of DNA fragmentation in VCA [2, 10]. TUNEL results have shown higher sensitivity for apoptotic cell detection than other available histochemical approaches , and have clearly confirmed the presence of apoptotic cells in grade III rejection. Moreover, detection of TdT positive cells in the affected tissues may help in assessment of pathological severity scores.
Rejection in cutaneous biopsies begins with mild, superficial perivascular inflammation that progresses to a moderate perivascular infiltrate and then proceeds to involve the epidermis. Most of the mucosal biopsies had multiple foci of lymphocytic interface inflammation without accompanying submucosal changes.
Immunopathomorphology revealed that interface mucositis was associated with the expression of HLA-DR antigens on the infiltrated cells and aberrant expression on oral mucosa epithelial cells—a hallmark sign of the activation process. Upregulation of HLA-DR expression is associated with migration of inflammatory cells to the site with local aberrant HLA-DR expression and is associated with episodes of AR as presented in pancreas, kidney and hand transplants [16, 20, 21].
Possible explanations for different presentations of AR in the skin and mucosa include lack of adnexa and variable numbers of dendritic cells and regulatory T cells. Recently, it has been hypothesized that semimature dendritic cells cause graft tolerance through the expression of low levels of CD40, CD80 and CD86, and a lack of expression of proinflammatory cytokines IL-1, IL-6 and IL-12. Furthermore, they cause up-regulation of CD4+/CD25+ regulatory T cells, which promotes graft tolerance by the secretion of IL-10 and other tolerance-promoting cytokines . We did not notice a significant difference in the dendritic cell or the FOXP3-positive lymphocyte populations between the mucosa and the skin. Some authors have questioned whether the adnexal structures, which have been proposed to be less antigenic, may play a protective role in the skin . This remains an unproven, but interesting conclusion.
Immunopathomorphologic assessment plays an important role in evaluating the normal architecture of the different tissue components included in the transplanted VCA. Our studies confirmed the presence of a normal representation of proliferating cells (Ki67+) in the basal layer of the epidermis, within the skin component and within the epithelial cells of the oral mucosa. In addition, a normal representation of Langerhans cells and dermal histiocytes was observed. These findings confirmed normal regeneration process in different tissues and components of facial VCA.
It is also important to consider tissue lesions caused by medication-related maintenance immunosuppression. During the single colonoscopic evaluation of our patient, multiple biopsies showed histological changes suggestive of mycophenolate (MPA)-induced injury. In recent years, a growing body of evidence has shown that MPA can cause a GVHD pattern of injury to the bowel through its inhibition of the de novo pathway of purine biosynthesis . MPA is also associated with injury and apoptosis to the squamous esophagus . At week 66 posttransplant, sirolimus was added to the immunosuppression protocol. Mouth ulcers are a common side effect of sirolimus, further complicating the differential diagnosis .
The decision of when to treat the patient and when to observe and wait was challenging due to the significant number of discordant skin and mucosa biopsies that, to make the decision even more difficult, did not correlate with clinical signs of rejection. Thus, we decided to treat all episodes where clinical signs of rejection were present and were confirmed by concordant skin and mucosa biopsies to represent grade III AR.
Based on clinical signs, two episodes of rejection were suspected in our patient. One of these episodes presented as a perinasal acneiform and likely represented folliculitis that was not treated by any changes in immunosuppression. The second episode (week 66) presented clinically with whole facial allograft erythema and histologically, grade III AR was confirmed by concordant skin and mucosa biopsies. This episode resulted in hospitalization and gradually improved over the following 3 weeks as the patient's anti-rejection medication regimen was adjusted. By the end of the second month posttransplant, we added topical immunosuppression based on the favorable results reported in hand transplant recipients treated topically during lower grades of AR .
We found the BANFF classification to be a valuable guide in grading our patient's skin biopsies in addition to mucosa biopsies and clinical features of rejection. Clinical and histological correlation is necessary to determine AR. Initially, only histological changes were used to determine AR. Later, it was found that the diagnosis of AR requires both clinical changes in the graft and concordance with histological changes. Perhaps the interface inflammation that we observed represents a different type or sub-type of AR which is specific to the mucosa or represents a therapy effect. In the next review of the BANFF classification system, it is our hope that new data confirmed in all 45 mucosal biopsies will bring a greater understanding of differences in the presentation of AR between the skin and mucosa components of facial allograft and that these new pathological and clinical findings will be reflected in future Banff classification.
The authors would like to thank Cheryl Smith for help with the editorial work on the final version of the manuscript.
No external funding sources were used for this study. The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.