Isolated Limb Perfusion and External Beam Radiotherapy for Soft Tissue Sarcomas of the Extremity: Long-Term Effects on Normal Tissue According to the LENT-SOMA Scoring System

Background With the combined treatment procedure of isolated limb perfusion (ILP), delayed surgical resection and external beam radiotherapy (EBRT) for locally advanced soft tissue sarcomas (STS) of the extremities, limb salvage rates of more than 80% can be achieved. However, long-term damage to the healthy surrounding tissue cannot be prevented. We studied the late effects on the normal tissue using the LENT-SOMA scoring system.
In the 1960s and 1970s, the effectiveness of (neo)adjuvant isolated limb perfusion (ILP) with various cytostatics was explored in the limb salvage treatment of extremity sarcomas.1 The practice of performing extremity perfusions for sarcomas ended after Rosenberg showed the effectiveness of adjuvant radiation treatment in limb salvage. Amputations or exarticulations of the affected limb did not result in higher survival rates.2,3
There was a renewed interest in neoadjuvant ILP in the early 1990s, when Lejeune and co-workers added tumor necrosis factor alpha (TNFα) to Melphalan in the ILP treatment of locally advanced extremity melanomas and sarcomas. This resulted in high local response rates and high limb salvage rates with an acceptable local and systemic toxicity.4,5
Currently, an established limb salvage strategy for primarily irresectable soft tissue sarcomas (STSs) of the limbs consists of induction treatment with hyperthermic ILP with TNFα and Melphalan, followed by delayed surgical resection and, in case of marginal or non-radical resection, adjuvant external beam radiotherapy (EBRT). With this combined treatment modality, short-term limb salvage rates of more than 80% are achieved, with long-term rates of 60%.5,6 However, as the number of long-term cancer survivors increases, late complications of therapy will become an increasingly important concern.7 Long-term damage to the healthy surrounding tissue cannot be prevented. Toxic effects of pre- or postoperative EBRT and intra-arterial chemotherapy have been extensively described in previous literature and include edema, fibrosis, neuropathy, limited mobility, impaired wound healing and, less commonly, pain, bone fractures and second tumors.3,8–11 Moreover, during ILP, normal tissue in the limb—skin, subcutaneous tissue, muscle, nerves, blood vessels, bone and cartilage—is exposed to the same concentrations of cytostatic agents active against the tumor, causing regional toxic effects, which lead to comparable late complications.12 Recently, we described the late vascular complications that occur after the TNF ILP treatment procedure.13 Functional morbidity and symptoms such as edema and pain can also be extremely invalidating, sometimes necessitating aggressive treatment. To evaluate all toxic long-term effects, we used the uniform, generally accepted toxicity scoring system “LENT-SOMA”. This system not only allows late damage to be classified in different gradations, but also allows data acquisition using three different methods: (1) subjective, the injury is recorded as perceived by the patient; (2) objective, morbidity is assessed during a clinical examination; and (3) management, indicates the active steps made to ameliorate the symptoms.14 In order to provide an overview of all late toxic effects on the limb treated with ILP, delayed surgical resection and adjuvant high-dose EBRT, a retrospective study was conducted to evaluate long-term morbidity using the LENT-SOMA scoring system.
Patients and Methods
Between 1991 and 2003, 73 patients with locally advanced STSs underwent 77 perfusions with a combination of TNFα and Melphalan, with (n = 19) or without (n = 58) interferon-gamma (IFNγ), at the Division of Surgical Oncology of the University Medical Center Groningen (UMCG). The perfusion technique has been previously described extensively.15,16 Currently, 39 patients are still alive and in follow-up. There were 7 patients who were unable to participate because of severe or advanced (co-)morbidity (n = 2) or non-medical reasons (n = 5). Consequently, 32 patients, 14 males (44%) and 18 females (56%), median age 47 (range 14–71) years, could be evaluated (response rate 82%). Of all the 32 patients, 5 had STSs of the upper extremity (16%), treated with an axillary perfusion. In the remaining 27, they were located in the lower limb (84%) and were treated with iliacal (n = 13, 41%), femoral (n = 5, 16%) or popliteal (n = 9, 28%) perfusion. There were 27 primary (84%) and 5 recurrent (16%) STSs.
After a median post-perfusion duration of 8 (range 6–12) weeks, all patients underwent a delayed local resection of the tumor remnant. Of the patients, 25 had clear microscopic surgical margins (R0 resection, 78%) and 7 had histologically positive microscopic margins (R1 resection, 22%). No patient had macroscopic residual disease. A total of 24 patients (75%) with marginal (≤1 cm, n = 17) or microscopically positive resection (n = 7) margins received adjuvant high dose (60–70 Gy) postoperative EBRT in 25 fractions of 2 Gy, followed by a boost of 10–20 Gy. One patient with a recurrent STS received EBRT at the time of treatment of the primary tumor, but not after ILP and resection of the recurrence. Adjuvant systemic chemotherapy was given to 7 patients: 2 due to the definite histopathological classification of the tumor (one embryonal rhabdomyosarcoma and one extraosseal osteosarcoma); 3 as part of an EORTC trial (EORTC 62931);17 and 2 in a palliative setting because of distant metastasis.
All patients received a tailored post-surgical rehabilitation program, during and after the radiation treatment, until a final result was achieved. All patients were treated after informed consent was obtained according to institutional guidelines. A total of 12 different histological types of STS were distinguished. The pathological grade and stage of the tumor were scored according to the criteria of Coindre et al. and the American Joint Committee on Cancer (AJCC), respectively.18,19 Patient and tumor characteristics are summarized in Table 1.
Table 1Characteristics of patients. PNET primitive neuroectodermal tumor, PUS pleiomorphic undifferentiated sarcoma, MPNST malignant peripheral nerve sheath tumor, FU follow-up in months, na not availableAge (years)SexHistologyGradeAJCC stageLevelEBRTAmputationFU18FemalePrimaryEmbryonal rhabdomyosarcoma33IliacalNoNo15944FemaleRecurrent Myxoid liposarcoma11IliacalYesNo15243MalePrimarySynovial sarcoma33IliacalYesNo15118MalePrimaryMyxoid chondrosarcoma23Popliteal YesYes14948FemalePrimaryWell-differentiated liposarcoma11IliacalYesNo14356FemalePrimaryPNET33IliacalYesYes13750FemaleRecurrent PUS33Popliteal NoNo13525FemalePrimarySynovial sarcoma23Popliteal YesNo13244MalePrimaryMyxoid liposarcoma11IliacalYesNo13124MaleRecurrent Synovial sarcoma23Popliteal YesNo12937FemalePrimaryMPNST23Axillary YesNo12148MalePrimaryMyxoid liposarcoma23IliacalYesNo11163FemaleRecurrent PUS23Popliteal NoYes10737MalePrimaryMyxoid liposarcoma11IliacalYesNo10558MalePrimaryPUS23IliacalYesNo9871FemalePrimaryLeiomyosarcoma11Femoral NoNo9445FemalePrimaryPUS33IliacalNoNo8256FemalePrimaryPUS33Popliteal YesNo6163MalePrimaryMyxoid chondrosarcomana3Femoral YesNo5037FemalePrimaryLeiomyosarcoma11Femoral YesNo3628MalePrimarySynovial sarcoma23Femoral YesNo3657FemalePrimaryPUS33Femoral YesNo3542FemalePrimarySynovial sarcoma23IliacalYesYes3247FemalePrimaryPUS33Axillary YesNo3058FemalePrimaryPUS33Popliteal YesNo2627MalePrimaryEpithelioid sarcoma34Axillary YesNo2671MaleRecurrent Myxoid fibrosarcoma11Axillary NoNo2456FemalePrimaryPUS33Axillary NoNo2214MalePrimaryPUS33Popliteal YesNo2065FemalePrimarySynovial sarcoma23Popliteal NoYes1863MalePrimarySynovial sarcoma33IliacalYesNo1771MalePrimaryRhabdomyosarcoma33IliacalYesNo17
Local status of the treated limb was first globally assessed using a checklist including the following items: lymphedema, color, stiffness, hypesthesia, paresthesia, muscle atrophy, pain, scar status, function and use of aids.
Late effects on surrounding tissue were graded according to the subjective, objective and management portions of the LENT-SOMA scales, as proposed by the EORTC and RTOG Late Effects Working Group in 1995.14,20,21 Four LENT-SOMA scales were used: muscle/soft tissue, peripheral nerves, skin/subcutaneous tissue and vessels.22 Not all patients could be scored on all four items, since in 5 patients (16 %) the affected limb was amputated.
Results
After a median follow-up of 88 (range 17–159) months, 14 patients (44%) mentioned one or more locoregional complaints in the treated leg, mainly consisting of pain, spasm and limitation of mobility. Scoring the local status using a checklist, deviating symptoms and signs were documented and are summarized in Table 2. Only 15 patients (47%) did not use any aids; the remaining 17 (53%) needed elastic compression stockings, braces, prostheses or crutches.
Table 2Local status in 32 patients: deviating symptoms and signsSymptomNo.%Lymphedema1444Discoloration1856Stiffness1650Atrophy2784Pain516Hypesthesia1753Paresthesia1341Function  - Limited1856  - Severely limited413
LENT-SOMA Scales
Results of scoring late tissue effects according to the LENT-SOMA scoring system are listed below and illustrated in Table 3. Grade 1 represents the most minor symptoms, which do not require treatment. Grade 2 represents moderate symptoms, requiring only conservative treatment. Grade 3 represents severe symptoms, which have a significant negative impact on daily activities and require more aggressive treatment. Grade 4 represents irreversible functional damage, necessitating major therapeutic intervention. SOMA stands for: S, subjective complaints; O, objective symptoms; and M, management and therapy.
Table 3Modified LENT-SOMA scoresMaximum grade of toxicity01234Muscle/ soft tissue (n = 31)  Subjective619%929%26%1445%00%  Objective00%00%1548%1652%00%  Management2065%13%929%13%00%Peripheral nerves (n = 30)  Subjective620%310%930%1240%00%  Objectivea621%1243%725%311%00%  Management2377%13%413%27%00%Skin/subcutaneous tissue (n = 27)  Subjective830%1556%27%27%00%  Objective14%622%1556%519%00%  Management2385%00%00%27%27%Vessels (n = 25)  Subjective1664%416%416%14%00%  Objective1352%832%28%14%14%  Management1872%416%00%14%28%a n = 28.
Muscle/Soft Tissue
Of the patients, 6 experienced no subjective toxic effects (19%), 9 scored maximum grade 1 (29%), 2 scored grade 2 (6%) and 14 had a maximum of grade-3 toxicity (45%). When scored objectively, a maximum of grade-2 toxicity was found in 15 patients (48%) and grade 3 in 16 patients (52%). No patient had only minor or no symptoms. No treatment was required in 20 patients (65%), 1 patient (3%) scored grade 1 (occasional physiotherapy), 9 scored grade 2 (29%) and 1 (3%) needed continued medical intervention (grade 3) for edema and extremity malfunction. A detailed overview of all items is provided in Table 4.
Table 4LENT-SOMA scores for muscle/soft tissue (n = 31)Grade of toxicity01234NASubjective  - Pain2065%723%26%26%00%00%  - Function619%929%26%1445%00%00%Objective  - Edema1445%1032%413%26%00%13%  - Mobility and extremity function619%929%1135%516%00%00%  - Fibrosis13%310%1548%1032%00%00%  - Atrophy00%310%1652%1032%00%26%  - Contraction1858%00%1239%00%00%13%Management  - Pain2581%310%310%00%00%00%  - Edema2477%00%516%13%00%13%  - Mobility and extremity function2787%13%26%13%00%00%  - Fibrosis2994%13%00%00%00%13%  - Atrophy2994%00%13%00%00%13%
Peripheral Nerves
No subjective toxic effects were observed in 6 patients (20%), 3 scored maximum grade-1 toxicity (10%), 9 scored grade 2 (30%) and 12 grade-3 toxicity (40%). When scored objectively, 6 patients experienced no toxicity (21%), and 12 scored grade-1 (43%), 7 grade-2 (25%) and 3 grade-3 (11%) toxic effects. Of the patients, 23 did not require any treatment (77%), 1 (3%) scored grade-1 (occasional non-narcotic), 4 (13%) grade-2 (regular non- narcotic) and 2 (7%) grade-3 (physical or medical intervention because of severe motor dysfunction) toxicity. A detailed overview is provided in Table 5.
Table 5LENT-SOMA scores for peripheral nerves (n = 30)Grade of toxicity01234NASubjective  - Pain2067%723%13%27%00%00%  - Strength723%13%1240%827%00%27%  - Sensory1033%1137%27%620%00%13%  - Motor paresis1447%827%517%27%00%13%Objective  - Motor dysfunction723%1137%723%27%13%27%  - Sensory dysfunction1240%1240%310%13%00%27%  - Reflex1757%723%310%00%00%310%Management  - Pain2273%310%413%00%00%13%  - Motor dysfunction2583%00%00%27%00%310%  - Sensory dysfunction2790%00%00%00%00%310%  - Sensory2790%00%00%00%00%310%
Skin/Subcutaneous Tissue
No subjective symptoms were observed in 8 patients (30%), 15 scored maximum grade-1 toxicity (56%), 2 grade 2 (7%) and 2 (7%) grade 3 (scaliness/roughness requiring constant attention). Only 1 patient experienced no objective toxicity (4%), while 6 scored grade 1 (22%), 15 grade 2 (56%) and 5 grade 3 (19%). No treatment was required in 23 patients (85%), but 2 (7%) required medical intervention because of edema and dryness (grade-3 toxicity) and 2 (7%) scored grade-4 toxicity—surgical intervention due to massive fibrosis and a persistent ulcer requiring surgical therapy. A detailed overview is provided in Table 6.
Table 6LENT-SOMA scores for skin/subcutaneous tissue (n = 27)Grade of toxicity01234NASubjective  - Scaliness/ roughness933%1659%00%27%00%00%  - Sensation2074%519%27%00%00%00%Objective  - Edema1141%830%519%27%00%14%  - Alopecia (scalp)933%1348%311%14%00%14%  - Pigmentation change622%830%1141%14%00%14%  - Ulcer/ necrosis2593%14%14%00%00%00%  - Teleangiectasia1659%726%311%00%00%14%  - Fibrosis/ scar27%1970%622%00%00%00%  - Atrophy/ contraction27%1556%622%27%00%27%Management  - Dryness2385%14%14%14%00%14%  - Sensation2696%00%00%00%00%14%  - Ulcer2696%00%00%00%14%00%  - Edema2593%00%00%27%00%00%  - Fibrosis/ scar2696%00%00%00%14%00%
Vessels
No subjective symptoms were observed in 16 patients (64%), while 4 scored maximum grade-1 (16%), 4 grade-2 (16%) and 1 grade-3 (4%) toxicity: clinical symptoms of ischemia at rest. Objective symptoms were absent in 13 patients (52%), but 8 patients scored grade-1 toxicity (32%), 2 scored grade 2 (8%), 1 grade 3 (intense ischemia) (4%) and 1 grade 4 (necrosis) (4%). No form of management was needed in 18 patients (72%). However, 4 patients scored grade 1 (16%), 1 (4%) grade 3 (conservative surgery because of arterial disease) and 2 (8%) required amputation (grade-4 toxicity) because of critical leg ischemia. A detailed overview is provided in Table 7.
Table 7LENT-SOMA scores for vessels (n = 25)Grade of toxicity01234NASubjective  - Arterial2080%14%312%14%00%00%  - Venous2080%416%14%00%00%00%Objective  - Arterial2288%00%14%14%14%00%  - Venous1664%832%14%00%00%00%Management  - Arterial2288%00%00%14%28%00%  - Venous2184%416%00%00%00%00%
Overall, outcomes from the Subjective and Objective symptoms are different from those of the Management part of the LENT-SOMA scales. According to the subjective and objective parts of the scale, two-thirds of patients had serious late toxic side effects concerning any of the surrounding normal tissue. A grade-3 toxicity was scored by 17 patients (53%) on one or more items of the subjective parts of the four used scales. They experienced their long-term morbidity as severe and with a negative impact on daily activities. Grade-3 or -4 toxicity was scored by 18 patients (56%) on the objective parts of the scoring scales, showing severe or irreversible damage during a clinical examination. However, according to the management parts, 16 patients (50%) scored no toxicity (grade 0) on all management items of the four used LENT-SOMA scales and did not require any treatment. Moreover, 9 patients (28%) scored a maximum grade-1 or -2 toxicity and only needed conservative treatment. In less than a quarter of patients (n = 7, 22%), aggressive treatment, e.g., continuous medical intervention or surgery, was necessary due to long-term effects of the combined treatment procedure.
Discussion
The limb-sparing treatment strategy of ILP using TNF and Melphalan—with or without adjuvant EBRT—is increasingly being pursued for locally advanced STS of the extremities after the publication of the results of a European multi-center trial performed in the 1990s. Using this combined treatment modality, excellent short-term limb salvage rates of more than 80% and long-term rates of 60% can be achieved with no negative impact on survival.5,6 While, at first, selected groups of patients were often excluded from undergoing this procedure, limb preservation has now become a major challenge for all patients with a locally advanced STS.
It seems to be a safe and highly effective procedure in elderly patients, for whom every attempt to avoid an amputation that may end their independence must be considered.23 It can also be used in a palliative setting for patients who present with distant metastasis at the time of diagnosis, as better local control improves the quality of life.24 In patients with multifocal primary or multiple recurrent extremity sarcomas, ILP also provides excellent local control and limb salvage rates of more than 80% are achieved.25
However, acute toxicity and long-term morbidity due to the combined treatment procedure of ILP, surgical resection and EBRT cannot be prevented. In ILP, normal tissue in the limb is exposed to the same concentrations of cytostatic agents as the tumor. The effects of the perfusate on normal tissue vary widely among individuals and long-term morbidity also varies widely in severity.
As far as we know, this is the first study to assess late functional morbidity with the LENT-SOMA scales in patients treated for STS with ILP, surgical resection and EBRT.
Neurotoxicity
Neuropathy is particularly important and is mainly seen after the use of specific cytostatic perfusion agents, such as cisplatin, carboplatin and doxorubicin.26–28 Furthermore, nerve damage can be caused by initial tumor swelling or pressure from the tight tourniquet.29,30 A prophylactic fasciotomy can prevent the occurrence of a persistent neurotoxicity with motor-sensory neuropathy and is routinely performed in our series.31 Radiotherapy also plays an important role, especially when patients have received a boost.32 All our patients received a boost of 10– 20 Gy EBRT. Despite the fact that neurotoxicity was subjectively found to be severe in 40% of patients, only in 11% could it be objectified, and in only 7% was physical or medical intervention needed. None of the patients had paralysis or complete anesthesia or needed surgical intervention.
Functional Morbidity
Functional outcome after limb-sparing treatment with ILP has been analyzed previously, but mostly in melanoma patients and not using a uniform scoring system. Other studies show limitations in ankle joint mobility in 25–40% of patients, compared with no significant objective limitations in previous investigations in our study population.12,33 The routinely performed fasciotomy, combined with an intensive physiotherapy program, might explain these better outcomes because the regional toxic effects of ILP are hereby reduced.34 In this study, we found muscle atrophy in almost all patients (96%), which may be due to an extensive surgical resection having been performed after ILP for deep-seated STSs, in contrast to the mostly superficial resection in melanoma patients. Only 4 patients (13%) still needed medical intervention: occasional or regular physiotherapy. However, 14 patients (44%) found that their limb function interfered with daily activities. In only 6 patients (19%) could no functional impairment be objectified.
Scoring Systems
Most previous studies evaluating functional outcome after treatment for STS included no ILP, but consisted of wide local excision with or without postoperative EBRT and/or adjuvant chemotherapy. Gerrand et al. found a significant decrease in functional scores when the revised Musculoskeletal Tumor Society Rating Scale (MSTS) and Toronto Extremity Salvage Score (TESS) were used after treatment of deep-seated tumors35. In two studies, the LENT-SOMA scales were used to assess functional morbidity after wide local excision and EBRT sometimes combined with chemotherapy for STS. The LENT-SOMA scales were found to be time-consuming, but reliable for a detailed description of late toxicity.32,36
To evaluate the long-term morbidity in our study, we used this generally accepted LENT-SOMA scoring system. According to Denekamp et al., no overall LENT score is calculated but all criteria are separately described in order to prevent the high scores being filtered out, thus giving a falsely optimistic picture.37 Although primarily developed to analyze late radiation effects, it can also be used to score the toxicity of chemotherapy.21 In this study, 25 of the 32 patients analyzed (78%) received EBRT. The 3 patients who scored grade-4 toxicity on one or more items all received EBRT. Overall, there was no patient who did not experience any late toxic effect.
Quality of Life
All functional complaints can have a substantial negative impact on daily functioning, which brings into question whether expectations of the time-consuming technically demanding limb-sparing treatment are upheld. Previous studies showed no improved quality of life with limb-sparing surgery compared with that after amputation.38,39 The long rehabilitation period is a disadvantage of a limb-sparing treatment strategy, but the impact of a prosthesis should not be underestimated. Thijssens et al. recently showed a worse physical functioning and more role limitations in amputated patients in this study population. Attention to collaborative decision making and communication with the patient seem very important.40
Future Perspectives
Every effort should be made to prevent long-term morbidity in the future. As long-term morbidity seems to be correlated to acute regional toxic reactions, these acute reactions should be reduced as far as possible.30 Improved perfusion technique and enhanced technical insights have led to the following suggestions: reduce the doses of TNFα, decrease systemic leakage, reduce maximum temperatures and strictly regulate perfusion pressures.15,30,41 To prevent the significant toxicity related to muscle and nerve damage, a prophylactic fasciotomy should be performed in all cases and the isolating tourniquet should not be applied too tightly.30,31 Furthermore, all patients should receive an individualized rehabilitation program to achieve an optimum functional situation.
Finally, a lot of long-term morbidity after EBRT is subscribed to the occurrence of radiation-induced fibrosis, which was thought to be irreversible.7 Thanks to the pioneering work of Delanian et al., there is growing knowledge that radiation-induced fibrosis is at least partially reversible by administering drugs with antioxidant and anti-fibrotic properties, such as pentoxifylline and vitamin E. Large randomized trials are, however, needed to confirm these findings.32
In conclusion, the Subjective, Objective, and Management parts of the LENT-SOMA scoring system confirm late toxic effects on surrounding normal tissue in all patients treated with a combined treatment modality for locally advanced STS of the extremity. Two-thirds of the patients have severe Subjective or Objective damage, but half of all patients do not require any Management for their symptoms. To prevent long-term morbidity in the future, efforts should be made to reduce acute toxic reactions after ILP, and a fasciotomy and a customized rehabilitation program are recommended. Furthermore, anti-fibrotic therapies might be considered.

perfusion sarcoma lent-soma radiation complications


DNA-PK contributes to the phosphorylation of AIRE: Importance in transcriptional activity

The autoimmune regulator (AIRE) protein is a key mediator of the central tolerance for tissue specific antigens and is involved in transcriptional control of many antigens in thymic medullary epithelial cells (mTEC). Mutations in the AIRE gene cause a rare disease named autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). Here we report using GST pull-down assay, mass-spectrometry and co-immunoprecipitation that a heterotrimeric complex of DNA-Dependent Protein Kinase (DNA-PK), consisting of Ku70, Ku80 and DNA-PK catalytic subunit (DNA-PKcs), is a novel interaction partner for AIRE. In vitro phosphorylation assays show that the residues Thr68 and Ser156 are DNA-PK phosphorylation sites in AIRE. In addition, we demonstrate that DNA-PKcs is expressed in AIRE positive mTEC cell population and that introduction of mutations into the AIRE phosphorylation sites decrease the capacity of AIRE to activate transcription from reporter promoters. In conclusion, our results suggest that phosphorylation of the AIRE protein at Thr68 and Ser156 by DNA-PK influences AIRE transactivation ability and might have impact on other aspects of the functional regulation of the AIRE protein.
1
Introduction
AIRE (autoimmune regulator) is a defective gene in APECED (autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy, OMIM #240300), the genetic autoimmune disease, which manifests as autoimmunity to multiple endocrine glands. In human tissues, AIRE is expressed in the thymus, spleen, and lymph nodes [1–3].
The AIRE protein (57.5 kDa) has several features which indicate that it might function in transcriptional regulation. AIRE shares similar domain architecture with the Sp100 family proteins (Sp140, Sp110 and Sp100C), which all have HSR (for homogenously staining region), SAND (for Sp100, AIRE-1, NucP41/75 and DEAF-1) and PHD (for plant homeodomain) domains. HSR is central for AIRE di- or oligomerization [4,5]. The SAND domain is a DNA binding domain found in several mammalian nuclear proteins. NMR spectroscopy studies of the Sp100B SAND domain have shown that a conserved amino acid motif KDWK is essential for DNA recognition [6]. Although AIRE lacks the KDWK motif, its SAND domain has also been shown to bind DNA in vitro
[4]. The PHD fingers are found in nuclear proteins that are often involved in chromatin-mediated transcriptional regulation and appear to mediate protein–protein interactions. The zinc-binding cysteine-rich regions of the PHD finger form two flexible loops and two protein ligands have been proposed to bind one PHD finger simultaneously [7]. It has been suggested that one of the ligands can be specific for each PHD. For example, the PHD finger of ING2 can bind phosphoinositides whereas PHD of the KAP-1 repressor binds to Mi2α, a component of the NuRD chromatin remodelling complex [8,9]. Another ligand, which might be common for all PHD fingers, has been long suggested to be chromatin, and recently, PHD domains of ING2 and BPTF proteins were reported to bind tri-methylated lysine 4 of histone H3 [10,11]. However, despite the determination of its 3-D structure, no conclusive ligand of the AIRE PHD finger has yet been identified [12].
We and others have shown that AIRE can activate reporter genes from the interferon beta promoter or in a system where AIRE is tethered via heterologous GAL4 DNA binding domain to the reporter [5,13]. The transcriptional activation region of AIRE has been initially mapped to the two C-terminal PHD fingers as the mutations in these domains severely decreased the transactivation capacity [13,14]. More recent reports have shown that several other APECED-causing mutations in the HSR or SAND domain also decrease the AIRE transactivation ability [15,16].
So far, the only known protein partner for AIRE is a general transcriptional co-regulator and histone acetyltransferase, CREBP binding protein (CBP). CBP has been reported to bind directly to AIRE in vitro, it colocalizes with AIRE into the nuclear bodies in monocytic cell cultures and it enhances the transcription of reporter genes in collaboration with AIRE [5,16]. The mechanisms how AIRE in collaboration with CBP activates its target genes are largely unknown.
DNA-dependent protein kinase (DNA-PK) is a Ser/Thr kinase that belongs to the phosphatidylinositol 3-kinase (PI3K) family. It is formed as holoenzyme in the presence of DNA by two Ku regulatory subunits, Ku70 (70 kDa) and Ku80 (86 kDa), and a large catalytic subunit DNA-PKcs (465 kDa). With very few exceptions, DNA-PKcs is active only when it is in hetero-trimeric complex with Ku proteins and in interaction with DNA or RNA [17,18]. The main function of DNA-PK is to recognize double stranded DNA breaks and to catalyze a repair process known as non-homologous end joining (NHEJ). In a similar way, DNA-PK is crucial for V(D)J recombination in developing T and B cells. Concordantly, DNA-PKcs or Ku deficient mice are severely immunodeficient, with elevated radiosensitivity and susceptible for tumor development [17,19].
In addition to the role of DNA-PK in chromatin repair, DNA-PK has been shown to phosphorylate many proteins involved in cell cycling and transcriptional regulation, such as p53, Sp1, Myc, Fos, Jun, TBP, TFIIB, RNA Polymerase II [17] and several nuclear receptors, for example, the glucocorticoid [20], progesterone [21] and androgen receptors [22]. DNA-PK can positively and negatively modulate transcription. As a repressor, DNA-PK binds directly to a specific DNA sequence element within the mouse mammary tumor virus (MMTV) promoter [20,23]. Similarly, the whole DNA-PK complex has been shown to bind to the E-box/TATA like elements and to suppress the human xanthine oxidoreductase (hXOR) gene expression [24]. At the same time, the Ku proteins have been described as transcriptional recycling co-activators of androgen receptor [22]. In addition to these examples, DNA-PK can modulate gene expression via RNA-dependent phosphorylation of hnRNP proteins [18].
In order to further define molecular mechanisms of AIRE functioning, we searched for novel protein partners of AIRE using the GST pull-down approach combined with mass spectrometry. We found that AIRE interacts with DNA-PK complex proteins DNA-PKcs, Ku70 and Ku80. Further in vitro phosphorylation assays demonstrated that DNA-PK phosphorylates the AIRE protein in vitro at the residues Thr68 and Ser156. In addition, we show that the mutation of either of the phosphorylation sites significantly decreases AIRE transcriptional activity.
2
Materials and methods
2.1
Plasmids
Plasmid pGST-PHD1 was generated by insertion of PCR-amplified sequence encoding human AIRE-1 amino acids 293–354 into the NcoI and KpnI restriction sites of the pETM-30 vector (EMBL-Heidelberg protein expression and purification laboratory). To generate pGST-AIRE-SPP (SAND/PHD1/PHD2), the sequence encoding AIRE amino acids 178–482 was cloned into EcoRI and XhoI sites of pGEX1λT-SH3 vector (a gift from K. Saksela, University of Tampere, Finland). pGST AIRE-T68A, pGST-AIRE-S156A and pGST-AIRE-V80L mutation constructs were generated by altering amino acid Thr68 or Ser156 to alanines and V80 to leucine in pGST-AIRE [5] constructs by PCR-based site-directed mutagenesis. The control plasmid pGEX-2T-p53 was a gift from T. Punga (Uppsala University, Sweden). pSI-AIRE-T68A and pSI-AIRE-S156A were prepared by further cloning the mutated AIRE fragments from pGST-AIRE-T68A and pGST-AIRE-S156A into EcoRI and SalI sites of pSI vector (Promega). To generate luciferase reporter plasmids pBL-INV and pBL-LOR, the promoter areas of involucrin (3737 nt) and loricrin (2217 nt) genes were cut out from the plasmids pTZhINV-nlbgal and pTZhLOR1 (gifts from A. Männik, FitBiotech, Estonia) and cloned into the HindIII site of pBL-KS (a gift from K. Saksela, University of Tampere, Finland). The cloned plasmids were verified by sequencing.
2.2
Expression and purification of GST fusion proteins
The GST-tagged proteins were expressed either from Section 2.1. or previously described [5] AIRE GST-fusion constructs, pGST-AIRE (1–545), pGST-R257X (1–256), pGST-AIRE293 (1–293), pGST-AIRE348 (1–348), pAIRE-SAND (175–298) in Escherichia coli BL21-DE3 strain by induction with 0.4 mM IPTG for 4 h at room temperature. Full-length GST-AIRE and PHD-containing fusion proteins were expressed in the presence of 0.1 mM ZnCl2. The proteins were purified using Glutathione Sepharose 4B (AmershamBiosciences) according to the manufacturer's instructions except that 1% N-laurylsarcosine and 3% Triton X-100 were added to increase solubility of the proteins and 50 μM ZnCl2 was included when GST-AIRE and PHD-containing fusion proteins were purified. The purified proteins were verified on SDS-PAGE and Coomassie Blue staining.
2.3
GST pull-down and mass spectrometry
50–100 μg of nuclear extract, prepared from human monocyte cell line THP-1 according to [25], was incubated with 20–25 μg of GST-fusion proteins bound to 25 μl of packed sepharose beads in buffer B1 (10 mM HEPES pH 8.0, 150 mM NaCl, 0.7 mM MgCl2, 12.5% glycerol, 0.1 mM EDTA, 25 μM ZnCl2, 0.5 mM DTT and proteinase inhibitor mix) over-night at 4 °C. The beads were washed intensively with buffer B1, the bound proteins were eluted and separated on SDS-PAGE. The specific protein bands were cut out and analyzed with mass spectrometry.
Mass mapping of the peptides generated was performed with an Ultraflex™MALDI-TOF/TOF mass spectrometer (Bruker-Daltonics, Bremen, Germany) equipped with a nitrogen laser in a positive ion reflector mode using á-cyano-4-hydroxycinnamic acid as the matrix. The MALDI spectra were externally calibrated with the standard peptide mixture from Bruker-Daltonics (Bremen, Germany). In case of a peptide fragmentation analysis, a peptide from above mass mapping analysis was selected as a precursor ion and subjected for further MS/MS fragmentation in the MALDI-TOF/TOF lift-mode. Database searches were carried out by either Mascot peptide map fingerprint or Mascot MS/MS ions search (http://www.matrixscience.com/).
2.4
Transfections and immunoprecipitations
The transfections were carried out with ExGen500 in vitro reagent (Fermentas) according to the manufacturer's protocol. For immunoprecipitations, approximately 2 × 106 HEK293 cells were transfected with 10 μg of pcAIRE [3] or either pcDNA3.1B-myc/his (Invitrogen) or pdEYFP-N1 (Clontech) vector. After 46 h, a whole-cell extract was prepared by lysis with 0.25 ml of lysis buffer (20 mM Tris pH 7.5, 1% NP-40, 0.3 M NaCl, 0.1 mM ZnCl2, 1.5 mM MgCl2, 0.5 mM DTT, 0.2 mM EDTA, 25% glycerol, protease inhibitors) for 30 min on ice. The lysates were treated five times with the 25G syringe to shear genomic DNA, centrifuged at 13 500 rpm, 15 min at 4 °C. The supernatants were diluted 5 times with the lysis buffer lacking NP-40 and immunoprecipitations were performed with mouse monoclonal anti-DNA-PK 4F10C5 (BD Pharmingen), anti-AIRE 6.1 [3], anti-HuR 3A2 (a gift from J. Steitz, Yale University, USA), anti-HA sc-7392 (SantaCruzBiotech) anti-GAPDH 6C5 (Ambion), anti-c-myc 9E10 (Sigma) and rabbit polyclonal anti-GST sc-459 (SantaCruzBiotech) antibodies. 1.5 μg of each antibody was added to 700–900 μg of whole-cell extracts and incubated for 4 h at 4 °C, then 15 μl of packed protein G-Sepharose beads (Amersham Pharmacia Biotech) were included and incubation was continued for 1 h at 4 °C. The immunoprecipitates were washed 4 times with the IP buffer (20 mM Tris pH 7.5, 0.2% NP-40, 0.3 M NaCl, 0.1 mM ZnCl2, 1.5 mM MgCl2, 0.5 mM DTT, 0.2 mM EDTA, 25% glycerol, protease inhibitors) and eluted from the beads with 2× SDS loading buffer, separated on SDS-PAGE and subjected to Western blot with anti-AIRE 6.1 or DNA-PK antibody (1:1000) and ECL detection. Transfer DNA-PK was carried out with 0.01 M CAPS buffer, pH 11.0, supplemented with 20% methanol in semi-dry blotting system for 90 min at 1 mA/cm2.
Where indicated, RNaseA/RNaseT mix (Fermentas) with final concentrations 4 μg/ml and 10 u/ml, respectively, was added. Ethidium bromide (EtBr) and micrococcal nuclease (MNase) treatments were performed as demonstrated earlier [26]. Either 0.4 mg/ml EtBr was added to the extract or 1 u of MNase (Fermentas) in 50 μl of digestion buffer (10 mM HEPES pH 7.0, 4 mM CaCl2, 50 mM NaCl, 0.1 mM ZnCl2) was used. Digestion efficiency of MNase was tested in the separate control experiments, using the same conditions. On agarose gels, only the mononucleosomal fraction of DNA was detected when whole nuclei were treated and no DNA was left when free DNA was digested.
2.5
Computer prediction of AIRE phosphorylation
Scansite 2.0 program at http://scansite.mit.edu/cgi-bin/motifscan_seq
[27] and NetPhos 2.0 program at http://www.cbs.dtu.dk/services/NetPhos
[28] were used to predict AIRE phosphorylation sites.
2.6
Phosphorylation assays
The kinase assays were performed with SignaTECT DNA-PK Kinase Assay System kit (Promega) essentially according to the manufacturer's protocol. The phosphorylation reactions were carried out in the presence of γATP-32P for 7 min at 30 °C and either 25 μg of biotinylated-p53 peptide (EPPLSQEAFADLWKK, Promega) or 4–5 μg of GST fusion proteins were used as the substrates. Efficiencies of 32P labeling of the substrates were measured by radioactivity counter (1414 Guardian, Wallac). Nuclear extracts and immunoprecipitations were made as described in Sections 2.3 and 2.4. Either 600 μg (Fig. 3A) or 15–20 μg (all other figures) of nuclear extracts were used per reaction. Alternatively, 10 U of purified DNA-PK complex (Promega) in the presence of 250 ng of calf thymus DNA was used. 50 μM LY294002 hydrochloride (Sigma) was added when indicated. In assays using GST fusion proteins as the substrates, the GST proteins bound to 10 μl of packed glutathione beads were pre-washed with the kinase reaction buffer (SignaTECT kit buffer supplemented with 0.1 mM ZnCl2 and 0.2 mM Na3VO4). After the kinase reaction, the beads were washed three times with ice cold washing buffer (25 mM HEPES pH 7.5, 150 mM NaCl, 0.1% BSA, 0.2% NP-40, 0.1 mM ZnCl2 and 0.2 mM Na3VO4) before measuring the radioactivity.
2.7
Mice, thymic stromal cell isolation, cell sorting and RT-PCR
C57BL/6J background wild type mice were maintained at the mouse facility of the Institute of Molecular and Cell Biology, Tartu University. Thymuses from 4- to 6-week-old mice were used. Thymic stromal cell isolation, cell sorting and RT-PCR were carried out as described previously [29].
2.8
Luciferase reporter assays
Luciferase assays were performed using Luciferase Assay System kit (Promega) according to the manufacturer's protocol. 4 × 104 HEK293 cells in 24-well plates were transfected with 0.1 μg of INV-pBL or LOR-pBL and 0.3 μg pSI-AIRE, pSI-AIRE-T68A or pSI-AIRE-S156A for 46 h. In Western blot, anti-AIRE 6.1 antibody was use and 0.3 μg of pSI-AIRE, pSI-AIRE-T68A or pSI-AIRE-S156A were transfected for 46 h.
3
Results
3.1
DNA-PK complex proteins co-purify with AIRE PHD domain
In order to find new AIRE interacting partners, we expressed the first PHD domain (PHD1) of AIRE as a GST-fusion protein (Fig. 1A) and performed the GST pull-down using the nuclear extract prepared from monocytic THP-1 cells. The interacting proteins were separated on SDS-PAGE and visualized by Coomassie staining. Three protein bands with molecular weight of about 70, 80 and over 250 kDa were consistently seen after the SDS-PAGE analyses (Fig. 1B). Next, the protein bands were excised from the gel and analyzed by mass spectrometry. The MALDI-TOF mass map analysis demonstrated that the two bands with molecular masses of about 70 and 80 kDa belong to Ku70 and Ku80, respectively, whereas the protein with the mass over 250 kDa was presumed to be the catalytic subunit of DNA-PK (DNA-PKcs) (Table 1). Mass map identifications with low sequence coverage were further verified by MALDI-TOF/TOF fragmentation of three tryptic peptides (Table 1). Thus, these results identified a trimeric complex of DNA-PK as a candidate AIRE interacting partner.
3.2
Full-length AIRE interacts with DNA-PK
To further confirm the pull-down results and to demonstrate that the full-length AIRE protein is able to interact with DNA-PK, we carried out co-immunoprecipitation experiments. So far, no AIRE endogenous expression on protein level has been detected in immortalized cell-lines. As subcellular localization of transfected AIRE in immortalized cell lines is similar to the AIRE localization in thymus in vivo, as well as AIRE is able to activate transcription in those cells, we transfected HEK293 cells with the plasmid expressing full-length AIRE or empty vector as a negative control. The whole-cell extracts were prepared from transfected cells and used in co-immunoprecipitations with anti-DNA-PKcs, anti-AIRE or various control antibodies. The precipitates were separated on SDS-PAGE and immunoblotted with anti-AIRE antibody. As seen in Fig. 2A, anti-DNA-PKcs and anti-AIRE antibodies (as a positive control) were able to precipitate AIRE protein whereas control antibodies against GST, GAPDH and HuR (a nuclear mRNA-binding protein) could not. Similarly, AIRE and DNA-PKcs interaction was detectable when the immunoprecipitation with anti-AIRE antibody followed by the Western with anti-DNA-PKcs antibody was carried out (Fig. 2B).
Since the whole-cell extracts used in immunoprecipitations contained remarkable amounts of chromatin and RNA (data not shown), we next wanted to test whether the interaction between AIRE and DNA-PK is DNA and RNA dependent or independent. Previously, a DNA intercalator ethidium bromide (EtBr) has been shown to efficiently disrupt the binding of Ku proteins to Oct2 and DNA-PK [26,30], whereas the micrococcal nuclease has been widely used for the disruption of chromatin as well as for the digestion of contaminating DNA from cell lysates and immunoprecipitates [26,31]. Consequently, we transfected the HEK293 cells with the plasmid expressing full-length myc-tagged AIRE or empty vector as a negative control and performed co-immunoprecipitations with anti-DNA-PKcs antibodies in the presence of EtBr, RNaseA/T mix or treated the immunoprecipitates with the micrococcal nuclease (Fig. 2C, top panel). As a positive control, to show that the same level of the AIRE protein was present in each lysate, the immunoprecipitations were also performed with the anti-myc antibodies (Fig. 2C, bottom panel). Fig. 2C top panel reveals that DNA-PKcs still co-immunoprecipitates AIRE from the cell extracts treated with EtBr or RNaseA, which indicates that AIRE does not require DNA or RNA to interact with DNA-PKcs. However, when we treated the co-immunoprecipitates with micrococcal nuclease, we observed a weaker interaction between AIRE and DNA-PKcs (Fig. 2C, top panel), which suggests that in the cell lysates containing DNA and chromatin, a fraction of AIRE binds DNA-PK through the DNA and/or chromatin. In conclusion, the co-immunoprecipitation results together confirm that the AIRE protein interacts with DNA-PK.
3.3
DNA-PK phosphorylates the AIRE protein
DNA-PK is a nuclear serine/threonine protein kinase that has been reported to phosphorylate many nuclear targets. To study whether the AIRE protein is one of the targets of DNA-PK, we used several different assays which enabled quantitation of DNA-PK kinase activity. We first studied whether the AIRE protein co-immunoprecipitates DNA-PK kinase activity. Co-immunoprecipitations from nuclear extracts prepared from HEK293 cells transfected with myc-tagged AIRE were carried out with antibodies recognizing DNA-PKcs, myc tag and GAPDH (as a negative control). The co-immunoprecipitates were further incubated with biotinylated p53 derived peptide as a DNA-PK specific substrate [32] in the presence of DNA-PKcs activating dsDNA and 32P-γATP. As a positive control, HEK293 nuclear extract was used instead of the co-immunoprecipitates. Fig. 3A shows that a similar amount of 32P was incorporated into the p53 peptide when AIRE and DNA-PKcs co-immunoprecipitates were applied, whereas no activity was detectable with GAPDH co-immunoprecipitate. The relatively high phosphorylation efficiency achieved with AIRE co-immunoprecipitate was most likely due to the reason that the co-immunoprecipitation with anti-myc antibody is more efficient than that of with DNA-PK antibody. Since the p53 peptide used in the assay is a DNA-PK specific substrate [32], these data show that the AIRE/DNA-PK complex has kinase activity and suggest that AIRE, too, might be phosphorylated by DNA-PK.
To further investigate whether AIRE can be phosphorylated by DNA-PK, we carried out three different in vitro kinase assays where recombinant GST-AIRE, GST-p53 and GST alone were used as the substrates. First, we performed the experiments using the nuclear extract from HEK293 cells as a source of kinases in the absence or presence of a specific inhibitor for PI3 family kinases, LY294002 (Fig. 3B). Second, we did the kinase assay using the nuclear extracts either from DNA-PKcs positive (MO59K) or DNA-PKcs negative (MO59J) cell lines (Fig. 3C). Third, we carried out kinase reactions using a purified HeLa DNA-PK complex, again, in the absence or presence of LY294002 (Fig. 3D). As a result, we observed that the phosphorylation level of the AIRE protein reached up to 50–80% of that of the p53 protein in all three experimental approaches, whereas only background phosphorylation of GST alone was detected (Fig. 3B–D). In concordance, the kinase activity was decreased, although moderately, when LY294002 inhibitor was added (Fig. 3B) or when the nuclear extract from DNA-PKcs negative MO59J cells was used (Fig. 3C), indicating that besides DNA-PKcs other kinases can phosphorylate the AIRE protein.
In order to identify which region of the AIRE protein is phosphorylated, we expressed different AIRE domains as GST fusion proteins and tested their phosphorylation using the nuclear extracts as a source of kinases. The domain structure of the AIRE protein is given in Fig. 4A. Fig. 4B shows that the HEK293 nuclear extract phosphorylated more efficiently the GST-fusions of N-terminal AIRE containing the first 256 (1–256), 293 (1–293) or 348 (1–348) amino acids (Fig. 4B) while about 2 times less phosphorylation was seen with the GST-fusions of AIRE C-terminal fragments containing SAND (175–298) or SAND-PHD1-PHD2 (178–482) regions. A similar pattern of AIRE phosphorylation was observed when the phosphorylation assay was performed with DNA-PKcs competent MO59K or DNA-PKcs deficient MO59J cell extracts (Fig. 4C). Thus, although in pull-down experiments, the first PHD of AIRE alone was able to interact with DNA-PK, the main DNA-PK target seems to be the N-terminal part of AIRE indicating that AIRE and DNA-PK make multiple contacts. Taken together, these results demonstrate that DNA-PK can phosphorylate AIRE in vitro and that the N-terminal region of AIRE is the predominant target of DNA-PK.
3.4
T68 and S156 are the phosphorylation sites on the AIRE protein
The N-terminal region of AIRE contains the HSR domain that mediates di- or tetramerization of the protein [4,5]. Interestingly, previous in vitro experiments have indicated that AIRE dimerization may occur as a result of phosphorylation [4]. We next aimed to find the DNA-PK specific phosphorylation sites on AIRE. Using the Scansite 2.0 and NetPhos 2.0 programs, we found two putative DNA-PK phosphorylation sites: T68 within and S156 close to the HSR domain of the AIRE protein (Figs. 4A and 5A). To verify the prediction, we changed either Thr68 or Ser156 to alanines, both in wild type GST-AIRE and truncated GST-AIRE (1–256) constructs, and tested the influence of these mutations on the phosphorylation efficiency. Fig. 5B demonstrates that compared to GST-AIRE, the phosphorylation levels of the mutant proteins were decreased about 30–50% when the cell extracts from either HEK293 or MO59K cells were used as source of cellular kinases. Finally, we performed the phosphorylation assays using purified DNA-PK complex (Fig. 5C). In agreement with the previous experiments, a reduction of about 30% in the phosphorylation levels of the T68A and S156A mutants was detected, whereas the APECED patient mutation V80L did not negatively affect the AIRE phosphorylation level. Thus, we conclude that amino acids Thr68 and Ser156 of the AIRE protein are the phosphorylation sites of DNA-PK.
3.5
DNA-PK is expressed in thymic medullary epithelial cells (mTEC)
DNA-PK is a ubiquitously expressed protein which levels are enhanced by induction of DNA double strand brakes [17]. To study whether the DNA-PK and AIRE interaction might occur in vivo, we studied whether DNA-PK is also present in thymic medullary epithelial cells (mTEC): the only subset of cells where the AIRE protein expression is detected thus far [1–3]. Thus, we purified the thymic mTEC based on the cell surface marker EpCAM [29] and analyzed the expression of Aire, DNA-PK and two AIRE target genes; involucrin and loricrin. The involucrin and loricrin genes are highly expressed during the terminal differentiation of epidermal keratinocytes [33] and are downregulated in mTEC of Aire knock-out mouse [34]. Fig. 6, right panel demonstrates that DNA-PKcs mRNA is highly expressed in thymic epithelial cells, including the AIRE-enriched mTEC fraction. As characteristic to Aire-regulated genes in the thymus, the expression levels of involucrin and loricrin were relatively low, remarkable lower than those of DNA-PKcs and Aire in mTEC subpopulation (Fig. 6, left panel).
3.6
T68A and S156A mutations downregulate the transactivation activity of AIRE
Previously, it has been demonstrated that several missense mutations in the HSR domain affect the dimerization, cellular localization and the transactivation ability of AIRE [13–15,35]. Thus, we next tested whether the mutation of AIRE phosphorylation sites influences the AIRE transactivation activity. We cloned AIRE with T68A and S156A mutations into mammalian expression vector and performed luciferase assays using the lysates from HEK293 cells transfected with wild type AIRE or the mutant constructs. As AIRE target genes, human loricrin and involucrin gene promoters fused to the luciferase gene were used. Fig. 7A demonstrates that when the HEK293 cells were transfected with AIRE T68A and AIRE S156A mutation constructs, significantly lower transactivation activity compared to the wild type AIRE was seen. Western blot of the transfected lysates with anti-AIRE antibody shows that both mutants are expressed even in higher level than the wild type AIRE construct (Fig. 7B). In conclusion, the luciferase assays suggest that the phosphorylation status of AIRE at T68 and S156 is important for the transactivation activity.
4
Discussion
In this study, we demonstrate that AIRE interacts with and is phosphorylated by the DNA-PK complex. The best DNA-PK substrates are the DNA associated proteins and the most effective phosphorylation occurs when DNA-PK is colocalized with the same DNA molecule as its target proteins [36]. Binding to the Ku proteins improves the affinity of DNA-PKcs for DNA ends about 100-fold [37]. This is in keeping with the finding showing that AIRE is able to bind DNA via its SAND domain [4]. In addition to the double strand brakes, the DNA-PK and/or the Ku proteins are known to bind telomeres and sequence-specific promoter elements [17,24], single stranded DNA [38], nucleosomes [39], RNA [18] and base unpairing regions (BURs) which typically are found in nuclear matrix attachment regions (MARs) [40]. Ku70 and Ku80 also colocalize to RNA polymerase II elongation sites [41,42]. Interestingly, AIRE has been reported to associate with nuclear matrix [43] and has been proposed to regulate gene clusters [44]. In addition, the AIRE protein contains PHD domains, which recently have been shown to recognize specifically modified histones within the chromatin [10,11]. Thus, AIRE appears to be a chromatin and DNA-binding protein. In this study, we observed that although AIRE is able to bind DNA-PK without DNA, most likely the DNA or chromatin is contributing to the AIRE and DNA-PK interaction (Fig. 2C). This is in concordance with DNA-PK ability to phosphorylate its substrates more efficiently at the presence of DNA [17]. At least two possibilities can be considered how DNA-PK, AIRE and DNA or chromatin may interact. First, DNA or chromatin, or even certain DNA sequence elements or specifically modified chromatin, may increase the affinity of AIRE for DNA-PK. Second, posttranslationally modified AIRE subpopulations may bind DNA-PK in different conditions. As we cannot exclude that this type of experiments can be influenced by the overexpression of AIRE, further studies are needed to find out the role of chromatin in AIRE and DNA-PK interaction in vivo, especially in the thymic environment.
Phosphorylation of AIRE by cAMP dependent protein kinases A (PKA) and C (PKC) has been reported earlier [4], though, the phosphorylated domain of AIRE remained unknown. We here demonstrate that in vitro the AIRE protein is mainly phosphorylated at the N-terminal region and define two residues, Thr68 and Ser156, as DNA-PK specific target sites. Thr68 resides within and Ser156 is located not far from the HSR domain. Both these phosphorylation sites are located outside the PHD1 finger, the domain of AIRE that was initially shown to pull-down DNA-PK, indicating that multiple different contacts can occur between AIRE and the DNA-PK complex. However, as previously reported [4], our in vitro phosphorylation studies also indicate that other kinases may phosphorylate the AIRE protein, and that phosphorylation sites other than Thr68 and Ser156 should exist within AIRE.
The HSR domain of AIRE has been shown to be responsible for the dimerization and tetramerization of the protein [4,5] and several, but not all, APECED mutations within the HSR decrease AIRE transactivation activity and influence AIRE cellular localization [15,16]. In addition, previous in vitro experiments have indicated that AIRE dimerization may occur as a result of phosphorylation [4]. As shown in this study, conversion of Thr68 and Ser156 amino acids to alanines suppresses AIRE transactivation activity. However, we did not observe any influence of these mutations to AIRE dimerization and cellular localization (data not shown). Thus, since the mutations that do not impact AIRE dimerization and the dot formation ability can downregulate AIRE transactivation activity, the N-terminal part of AIRE appears to be involved in transcriptional regulation not only via its dimerization activity. It is possible that status of AIRE phosphorylation at N-terminus influences AIRE interactions with other protein partners or with DNA.
In response to ionizing radiation, DNA-PK can have indirect effects on transcriptional regulation. It has been shown that phosphorylation of interferon regulated factor 3 (IRF-3) and pancreatic duodenal homeobox-1 (PDX-1) protein by DNA-PK in response to ionizing radiation directs these proteins to degradation by the proteasome and therefore downregulates IRF-3 and PDX-1 target genes [45,46]. Alternatively, phosphorylation of transcription factor Oct-1 by DNA-PK in response to ionizing radiation leads to stabilization but disrupts transactivation activity of Oct-1. As a result, the expression of Oct-1 target genes U6 and Histone H2B is downregulated [47]. We also studied whether UV irradiation changes AIRE cellular localization and expression level in various cell cultures but we could not detect remarkable differences (data not shown). Thus, it seems that DNA-PK phosphorylates AIRE independently of its function in DNA repair.
Based on research on AIRE deficient mice, it is evident that AIRE upregulates the expression level of many genes in thymus [48,49]. Studies in tissue culture cells confirm that AIRE indeed activates several promoters [5,13,15,16]. Thus, although in vivo the AIRE protein expression is only detected in thymic medullary epithelial cells [1–3], the transfected AIRE protein has similar transactivating properties, and nuclear localization pattern as AIRE in the thymus [14,16]. Despite these studies, the mechanisms of how AIRE regulates its target genes are still unclear. Posttranscriptional modifications, like phosphorylation, acetylation and ubiquitination, are widely used to regulate activity of transcription factors and regulators. In this study, we first describe the phosphorylation sites of AIRE and propose that its phosphorylation status is important for its transactivation activity. However, several questions remain, including what are the signals that trigger AIRE phosphorylation and how AIRE phosphorylation is connected with its function in the thymus. The differentiation of AIRE positive thymic epithelial cells was recently shown to be dependent on RANK-mediated signaling [50], which is known to activate several protein kinases [51]. As we showed in this study, DNA-PK is expressed in medullary epithelial cells (Fig. 6). Whether the thymic epithelial differentiation process correlates with the activation of DNA-PK and other possible AIRE-specific kinases needs further studies.

phosphorylation
apeced
phd finger
hsr domain


Does Microvascular Invasion Affect Outcomes After Liver Transplantation for HCC? A Histopathological Analysis of 155 Consecutive Explants

Macroscopic vascular invasion (macroVI) is associated with poor outcomes after liver transplantation (LT) for hepatocellular carcinoma (HCC). Whether microvascular invasion (microVI) is associated with the same adverse prognosis is unclear. One hundred and fifty-five consecutive patients with confirmed HCC after LT from March 1991 to 2004 at our institution were reviewed. Patients had to satisfy Milan criteria to be accepted for LT. They were followed with surveillance images every 3 months while on the waiting list. Disease-free survival (DFS) and overall survival (OS) were evaluated by Kaplan–Meier analysis. Demographic, tumor, and histopathologic characteristics were tested for their prognostic significance. Median follow-up after LT was 30 months. Overall graft survival rates were 87, 74, and 65% at 1, 3, and 5 years, respectively. All recurrences (22/155, 14%) developed within 4 years after LT with an overall 5-year DFS of 79%. Vascular invasion, either microVI or macroVI, was more likely in patients with multicentric HCC (n ≥ 3, p < 0.001) and larger tumor size >4 cm (p = 0.04). Tumor size >5 cm (p = 0.04), advanced pathological TMN stage (p = 0.007), microVI (p = 0.001), and macroVI (p < 0.001) predicted poor tumor-free survival on univariate analysis, but only macroVI was significant in multivariate analysis (hazard ratio 54.2, 95% confidence interval 11, 266). Furthermore, only macroVI was a significant predictor of mortality after LT (p = 0.01). Macrovascular invasion is strongly associated with high rates of recurrence and diminished survival after LT whereas microVI is not an independent risk factor.
Introduction
In 1996, Mazzaferro et al.1 documented excellent survival results after liver transplantation (LT) in a highly selected group of patients with hepatocellular carcinoma (HCC) with a single tumor <5 cm or as many as three tumors, each smaller than 3 cm. Recent studies have shown that selected patients who do not meet these criteria can still be cured with a transplant; the challenge now is to decide which factors, other than size and number, carry a sufficiently poor prognosis to deny transplantation.2–5
Gross vascular invasion or radiological evidence of tumor invasion in major veins is a known determinant of poor outcome after resection or transplantation for HCC and is an absolute contraindication to LT.4–13 Macrovascular invasion (macroVI) or gross vascular invasion of major portal or hepatic veins evident visually at time of transplant or on pathological evaluation may also be a predictor of recurrence after LT.14 Whether microvascular invasion (microVI), defined as microscopic tumor invasion in smaller intrahepatic vessels identified on pathologic analysis, or macroVI should also be considered a contraindication to LT is controversial. Both types of tumor invasion are difficult to determine pre-LT; therefore, their significance remains uncertain.
Several published studies have found histopathological factors, namely, poorly differentiated grade, microVI, and macroVI, to be independent predictors of poor survival after LT.4,11,15–17 We have previously shown that microVI is an independent predictor of early recurrence after resection for HCC.5 The purpose of this study was to determine if microVI, macroVI, and other pathological factors are associated with tumor recurrence after LT and examine the outcomes thereafter. If these factors are predictors of poor tumor-free survival, then how can we attempt to identify these variables before transplant?
Patients and Methods
One hundred fifty-nine consecutive patients with confirmed HCC on liver explant pathology after LT from October 1991 to October 2004 at our institution were reviewed from a prospective database. The cohort includes 32 patients (20%) with incidental tumors. Patients had to satisfy Milan criteria and have no radiologic evidence of gross vascular invasion to be accepted for LT. Four patients were omitted from analysis for recurrent hepatitis B virus (HBV) before antiviral therapy; after 1991, hepatitis B immune globulin, lamivudine, or combination therapy was used. Patients with known HCC were followed every 3 months while on the waiting list with either an ultrasound or triphasic computed tomography (CT). Patients with HCC were eligible for deceased donor whole organs (n = 149) and split (n = 0) or living donor LT (n = 6).
Patient, tumor, operative, and treatment characteristics were evaluated using a prospective clinical database and review of all pathological explants. The stage, size, and histopathology of the tumor were determined by analysis of the liver explant. Patients were staged based on the explanted specimens using the TNM staging classification of the American Liver Tumor Study Group18. Tumor size was measured as the largest diameter of the major tumor in centimeters. MacroVI was defined as gross vascular invasion into major portal vessels or hepatic veins identified either intraoperatively or on pathologic explant, whereas microVI was determined on pathologic analysis as microscopic vascular invasion of small vessels within the parenchyma of the liver. Pretransplant therapy was used in selective cases; if waiting time was determined to be longer than 6 months, patients commonly underwent radio frequency ablation as a bridge to LT. This was performed in an attempt to keep patients within Milan criteria while on the LT waiting list. Postoperative immunosuppression was similar in all patients and consisted of cyclosporine or tacrolimus and steroids.
After LT, patients were followed with Q3 monthly alpha-fetoprotein (AFP) and CT along with standard post-LT evaluation. Recurrences were defined as new nodules diagnosed by CT with confirmed biopsy in most cases. Overall survival (OS) was death as a result of any cause after LT. Patients were followed until death or study closure (arbitrarily denoted as October 1, 2004). Data was collected until May 1, 2005 to ensure at least 6 months of follow-up for all patients.
Statistical Package for Social Sciences software (SPSS, Inc., Chicago, IL) was used for data analysis. Statistical comparison of categorical and continuous variables was performed using the χ2 test and Mann–Whitney U test, respectively. All data was reported as median with range, mean ± SD or interquartile range (IQR) when appropriate. Analysis of patient OS and disease-free survival (DFS) was performed according to the Kaplan–Meier method. Patient survival in different groups was compared using the log-rank test. All variables with a p value less than 0.1 were then included in a multivariate analysis applying the Cox multiple backward stepwise model.
Results
Patient Characteristics
Of the 1,070 LTs performed during the 13-year period from 1991 to 2004, 159 patients (14%) had HCC. After omission of 4 patients because of recurrent HBV before the antiviral therapy era, 155 patients were included in this analysis (Table 1). The average waiting time for LT was 7 months. There was no perioperative mortality. The median age was 57 years (range 28–70) and the majority of patients were men (79%). The median age of all patients who underwent LT for any cause was 51 years (range 16–71). The most common causes of end-stage liver disease and HCC were hepatitis C virus (HCV) (n = 79; 51%), alcoholic liver disease (n = 34; 22%), and HBV (n = 25; 16%). Selected patients underwent pre-LT therapy, most commonly radio frequency or percutaneous ethanol ablation (ablation, 23%), but the majority of patients received no treatment before LT (72%). Use of ablation did not result in any adverse outcomes in this series of patients who underwent eventual LT.
Table 1Pretransplant Demographics of 155 Patients with HCC Who Underwent LTPretransplant CriteriaN = 155p ValueMedian age (range)57 (28–70)0.48Sex0.38 Male123 (79%) Female32 (21%)Cause of liver disease0.10 HCV79 (51%) HBV25 (16%) Alcohol34 (22%) Cryptogenic7 (4.5%) Alpha-1 antitrypsin6 (3.9%) NASH2 (1.3%) PBC2 (1.3%)Pretransplant therapy0.67 Ablation36 (23%) Resection6 (3.9%) TACE1 (0.6%) EBRT1 (0.6%) None111 (72%)The p values were determined by log-rank test as predictor of DFS after Kaplan–Meier analysis.NASH = nonalcoholic steatohepatitis, PBC = primary biliary cirrhosis, EBRT = external beam radiation therapy
Histopathological Analysis
The pathologic features for the 155 explants are shown in Table 2. The median number of tumors was 2 (range 1–20) and 18% were bilobar. The median size of the largest tumor was 2.6 cm and most tumors were graded as well or moderately differentiated (74%). Gross macroVI was evident in 3.9% (6/155) of the explants on pathologic examination, whereas 21% (33/155) of explants had the presence of microVI. Using the pathological TNM classification, 31 patients (20%) had stage I tumors, 69 patients (44%) had stage II tumors, 26 patients had stage III tumors (17%), and 29 patients had stage IV tumors (19%). Patterns of advanced stage were most often because of multifocal HCC or three or greater in number (40%).
Table 2Pathologic Characteristics of 155 Liver ExplantsCharacteristicN = 155p ValueHR (95% CI)No. of tumors Median (range, cm)2 (1–20)0.23 <3126 (81%) >329 (19%)Bilobar32 (21%)0.15Size Median (range; cm)2.6 (.1–16)0.040.47 (0.14,1.61) <5 cm145 (94%) >5 cm15 (10%)Stage I31 (20%)0.0071.17 (0.39,3.50) II69 (44%) III26 (17%) IV29 (19%)Positive lymph nodes3 (2.0%)0.31Vascular invasiona Microscopic33 (21%)0.0013.16 (0.92,10.93) Macroscopic6 (4%)<0.00154.2 (11.03,266.4) None121 (78%)b1.0cGrade Well/mod115 (74%)0.36 Poor13 (9%) N/Ad27 (17%)Margins Positive2 (1%)0.25 Negative153 (99%)Incidental tumor34 (22%)0.19The p values were determined by chi-square test or log-rank test of variables after Kaplan–Meier analysis (univariate). HR (95% CI) represents multivariate analysis of factors affecting recurrence after resection.aFive of six patients had characteristics of both microvascular and macrovascular tumor invasion.bReference category for comparisoncReference category for each categorical variable is assigned HR = 1.0.dNot available in the analysis
Vascular invasion was associated with both the number and size of tumors. Liver explants, with either microVI or macroVI, had more tumors (3.8 vs 2.0, p < 0.001) and were larger in size (3.6 cm vs 2.8 cm, p = 0.04) compared to those without vascular invasion. Specifically, if microVI or macroVI was present on histopathology, 64 and 100% of the tumors, respectively, were outside of the Milan criteria or were TNM stage III or IV. Histological grade was not associated with either type of vascular invasion; 85% of patients with either type of vascular invasion had favorably differentiated tumors. Ablation did not affect the rate of vascular invasion, either microVI or macroVI, in this series. Eleven of 33 patients (33%) with vascular invasion on explant analysis had undergone ablation of their tumor.
Predictors of Recurrence and Survival
The 5-year disease-specific survival was 79% (Fig. 1a). All recurrences developed within 44 months after LT. The 1-, 3-, and 5-year overall graft OS rates were 87, 74, and 65%, respectively (Fig. 1b). Only 22 patients (14%) developed tumor recurrence after LT with a median follow-up of 30 months (range 6–144 months). Eighty-six percent of patients (122/155) are currently alive and free of cancer. There was no difference in OS in patients with incidental tumors compared to those with known HCC (data not shown), but a significant difference was observed in DFS (5-year OS rate 94 vs 74%, p = 0.02).
Figure 1(A) Disease-free survival after LT for HCC of 155 patients. (B) Overall graft survival after LT for HCC of 155 patients.
The median time to recurrence for the 22 patients who developed recurrent HCC was 16.3 months (IQR 8.0–28). Tumor size >5 cm (p = 0.04), pathological TMN stage (p = 0.007), microVI (p = 0.001), and macroVI (p < 0.001) were found to be independent predictors of tumor-free survival. Patients with tumor size of 5 cm or larger had a 5-year DFS of 48 vs 82% for those with tumors smaller than 5 cm. Advanced TMN stage was also associated with poor recurrence-free survival after LT (Fig. 2). Twenty-one of the 22 patients (95%) who developed HCC recurrence were found to have either microVI (n = 15) or macroVI (n = 6) on pathological analysis. This accounts for 68% (15/22) of all patients with microVI and 100% (6/6) of patients with macroVI in the entire study. Patients with macroVI had a median DFS of only 7.1 months compared to a more favorable DFS in patients with microVI or no vascular invasion (median not achieved) (Fig. 3). No significant differences in DFS based on age, sex type, hepatitis status, tumor grade, bilobar disease, tumor number, ablative therapy, or type of transplant were found on univariate analysis. Pre-LT therapy did not result in any improvement in DFS or OS after LT (data not shown).
Figure 2The effect of TNM stage on DFS after LT for HCC.Figure 3The effect of vascular invasion on recurrence-free survival after LT for HCC.
In a multivariate analysis including factors with an influence on DFS, only macroVI (hazard ratio [HR] 54.2, 95% CI 11.03–266.4) was identified to be predictive (Table 2). This was confirmed with a Cox backward stepwise model of multivariate analysis (data not shown).
Discussion
In this study, we report our experience with LT for HCC with a specific focus on pathological factors affecting long-term outcomes. Overall survival rates of LT for HCC were 87, 74, and 65% at 1, 3, and 5 years, respectively, with a median follow-up time of 30 months. The overall recurrence rate was 14% with 79% 5-year disease-specific survival. Patients with incidental tumors had similar OS rates as those with known tumors consistent with previous reports,8,9,14,15,19 but in this study, a significant difference was found in DFS. Our studies suggest that large tumor size (>4 cm) and multiple tumors (≥3) correlate with an increased incidence of vascular invasion and may provide a surrogate marker for entities that are often difficult to detect before LT.
Recurrence rates after LT may not simply reflect only size and number as suggested in the initial Milan series, but may be a complex interplay of host- and tumor-related factors, which are still largely unknown.20 This report suggests that tumor grade, size, number, and microVI do not influence outcome after LT for HCC; only the presence of macroVI appears to be associated with poor outcomes on multivariate analysis. MacroVI and microVI were more commonly found in multicentric (three or more) and large (>4 cm) HCC. Furthermore, 21 of 22 recurrences had evidence of either microVI or macroVI on pathologic examination. Because we are unable to identify these biologic factors preoperatively, markers of histopathologic or biologic variables that predict poor outcomes are extremely important.14,20–22
MacroVI was shown to be an independent predictor of tumor recurrence after LT in some studies.4,9,10,14 The Pittsburgh group found that microVI and major vascular invasion was associated with increased risk of recurrence by multivariate analysis.4,8 In a report of 344 patients with HCC treated by LT, microVI and macroVI were associated with 4.4- and 15-fold increased risk of recurrence, respectively.8 Shetty et al.14 found that macroVI, but not microVI, was a significant predictor of DSF and OS after LT for HCC. In this study, microVI is associated with higher stage and recurrent tumors, but does not appear to be an independent factor for survival.
The role of microVI on posttransplant recurrence and survival outcomes for HCC still remains unclear. Several published studies have found poorly differentiated histological grade or microVI to be independent predictors of impaired survival after LT.11,15–17 Jonas et al.11 found vascular invasion and histological grade to be the only statistically significant independent predictors of poor survival after LT in 120 patients. In their study, only poorly differentiated tumors larger than 5 cm predicted the presence of microVI. But other studies involving LT and HCC were not able to corroborate poor results regarding microVI.3,14,23,24 The close relationship between histological grade and microVI may explain why microVI is often eliminated in multivariate models for analyzing tumor recurrence prognostic factors that include histological grading and vice versa.4,8,11,15,23,25,26 Multiple tumors, larger tumors, and higher grade of differentiation have all been shown to be associated with microVI after resection for HCC. Esnaola et al.13 reported that tumor size greater than 4 cm and poorly differentiated/undifferentiated histopathologic grade increased the odds of microVI by 3 and 6.3-fold, respectively, but these tumors were primarily in Child class A cirrhotic.
The degree of fibrosis and scarring of the liver may play a significant role in the biological behavior and significance of microVI and macroVI. We have previously shown that microVI was a significant predictor of early recurrence and death after resection for HCC in cirrhotic patients.5 Most recurrences were intrahepatic and away from the staple line, suggesting that liver mobilization and manipulation may cause progression of microVI or a new tumor has developed in the presence of ongoing oncogenic stimulus from cirrhosis. For these reasons, prognostic factors for LT from resection studies should be interpreted with caution and a possible rationale why microVI is so important after resection but not after LT.12,13 Lack of manipulation of the liver and intrahepatic dissection may be a potential explanation for the lack of importance of microVI with LT.
Because of the importance of histological features of HCC, some have advocated pre-LT biopsy to examine grade, vascular invasion, and genetic typing.13,15,20 Complications of needle biopsy such as tumor tract seeding and lack of sensitivity have made routine biopsy unfavorable.27 In our experience, needle biopsy was a poor predictor of microVI or macroVI, and therefore was not considered in our pre-LT work-up for HCC (data not shown).
There are several limitations to this study and therefore some of the results should be interpreted with caution. The need to standardize grading systems for HCC has long been recognized and would allow us to determine if tumor grade is indeed an important prognostic marker for recurrence and survival. Few tumors in this study were graded as poorly differentiated; moreover, in 27 patients, their grade was not determined. Results from histopathological analysis are often met with inherent biases from the pathologist and comprehensive evaluation of the whole liver explant may vary among pathologists and institutions. Finally, with very few tumors containing macroVI, strong conclusions about prognostic characteristics concerning macroVI cannot be made in this report. Whether microVI is a harbinger of macroVI or in some way correlated with a more aggressive form of HCC remains unclear.
The use of pathological and biological features of the tumor may allow us to identify those patients who are at increased risk of recurrence; then these patients should be considered for adjuvant therapy before evidence of a recurrence. Because vascular invasion is more common in multicentric (>3) HCC or large tumors (>4 cm), we propose shortening the interval of pre-LT imaging in patients with these tumors to identify vascular invasion or rapid growth of the tumor before LT. Genetic testing of tumors before LT may be a novel method to predict other prognostic factors affecting recurrence.20 Several studies have reported improved posttransplant survival in HCC patients with transcatheter arterial chemoembolization (TACE) or systemic therapy,28–31 but overall results for adjuvant chemotherapy post-LT were disappointing.32 Because of small numbers, this would be best done in a multicenter-randomized trial. We are currently evaluating the role of tumor size and number in our allocation system in LT for HCC to determine their respective predictive value for prognosis.
In summary, LT for HCC can be performed with acceptable survival outcomes. A single tumor characteristic alone does not appear to determine prognosis or outcome. In the present study, macroVI alone was associated with very poor outcomes after LT. Extending criteria of LT for advanced HCC is possible only with better patient selection using improved pre-LT staging and identification of histopathological biological markers such as macroVI that would preclude LT.

microvascular invasion
vascular invasion
liver transplantation
hepatocellular carcinoma
recurrence


Endovascular treatment of an open cervical fracture with carotid artery tear

The dilemma of how to treat penetrating wound injuries to the neck, which involve a combination of a common carotid artery rupture and a cervical spinal fracture, is presented in this case report.
Introduction
Penetrating injuries of the carotid artery can pose a significant challenge to the vascular surgeon. This type of vascular trauma accounts for only 3% of patients studied retrospectively in most trauma centers [1, 3, 4, 6, 9, 10, 13]. The severity of injury varies from complete transection to formation of a pseudoaneurysm or initimal flap formation with or without occlusion. Traditionally, the treatment of these lesions has been surgical. However, limitations to obtain satisfactory vascular control led to the search for other alternative treatment options [1, 4, 13]. Recently, the management armamentarium has expanded with the advent of more sophisticated endovascular techniques [3, 9].
Pseudoaneurysm of the common carotid artery after gunshot injury is a relatively infrequent finding. We present a case of a common carotid artery pseudoaneurysm managed by a commercially flexible covered stent.
Case history
A 16-year-old adolescent was transferred to our institution from a different hospital for stabilization of a cervical spinal fracture. Six hours prior to his transfer he had sustained an isolated gunshot wound to his neck. He was quadraplegic and unconscious on his admission to the referring hospital and was therefore intubated and ventilated. CT scan of the cervical spine showed a blow-out fracture of the body of C6 with involvement of the left vertebral foramen (Fig. 1). CT scans of the brain and chest were within normal limits.
Fig. 1CT scan at the C6 level demonstrating a vertebral body fracture extending into the left vertebral foramen
Upon admission to our institution his hemodynamic condition was stable with a blood pressure of 115/70 mmHg and pulse of 60 beats/minute. He was ventilated and had a SaO2 of 100% with FiO2 of 0.4. Physical examination of his neck area demonstrated an entry wound in the posterior neck to the left of the midline and an exit wound in the left side of his neck. A large hematoma on the left side of the neck was demonstrated and palpated. A duplex scan of the left carotid artery was performed and was interpreted as a probable false aneurysm of common carotid artery. CT angiogram of the neck demonstrated anterior and posterior leak of contrast from the common carotid artery, 1 cm proximal to the bifurcation (Fig. 2). The left vertebral artery was shown to be intact. The left internal jugular vein was compressed by the hematoma. The patient was transferred to the angiography suite and the presence of false aneurysms was confirmed. Under local anesthesia, through a transfemoral approach a catheter was inserted into the aneurysmatic area. The aneurysms were treated by the insertion of a covered stent (Jostent 8 (× 38 mm2), which also occluded the origin of the external carotid artery (Fig. 2). No change in his neurological status after the insertion of the stent was seen (Fig. 3). No anticoagulation was given. His neck fracture was stabilized by an external fixation device and he was transferred back to the referring hospital.
Fig. 2Selective left common carotid artery angiography shows traumatic pseudoaneurysm 1 cm below bifurcationFig. 3Selective common carotid artery angiography demonstrates endovascular stent—extending proximal and distal to the borders of pseudoaneurysm with complete exclusion of pseudoaneurysm and external carotid artery
One year later, duplex US follow-up showed good hemodynamics without stenosis. Clinically, no cranial lesions were found.
Discussion
The use of endovascular stent-grafts to address arterial tears was first introduced by Dotter in 1969 [2]. Over the past decade, endovascular grafting has been evaluated as an alternative to conventional surgical repair in the management of aneurysms and other vascular lesions. The application of this form of therapy to the management of vascular trauma offers many potential advantages. Angiography assists with the diagnosis of traumatic lesions such as intimal dissections [12]. In addition, endovascular techniques can be utilized to help with vascular control, as an adjunct to surgery in regions where obtaining proximal or distal control is technically difficult. The use of endovascular stent-grafting for the definitive repair of traumatic lesions has been shown to be associated with a decrease in anesthetic requirement, blood loss and extent of dissection [7]. High-velocity missiles can produce a significant amount of tissue damage, even if not suspected on initial examination [11]. The inherent problems of graft placement in the setting of severe tissue damage, gross contamination and scar tissue formation make the use of a remote insertion site particularly advantageous in the trauma patient [5, 8].
Injury to the carotid artery constitutes only 3% of all arterial injuries [1, 3, 4, 9, 10, 13]. In spite of the rarity of carotid injuries, this trauma presents significant challenges to the vascular surgeon. Gunshot wounds account for approximately half the injuries [9]. Injury of the carotid artery is frequently associated with other organ injuries [1, 3, 9]. Clinical examination may be unreliable due to confounding factors such as destructive injuries of spine, chest, abdomen, fractures [1, 3, 4, 6, 9, 10, 13]. Therefore, imaging evaluation is essential for the identification and localization of carotid trauma [1, 3, 9].
In contrast to blunt carotid injury, treatment of penetrating injuries of the carotid artery has been primarily surgical [4, 10, 13]. Depending on the described clinical scenario the surgeon is left with several options, which are observation, anticoagulation, ligation of the carotid artery, balloon occlusion of the artery, resection with graft interposition or bypass procedure. However, the best definitive management remains controversial, as no prospective studies regarding the management of penetrating carotid injuries have been reported [1, 3, 4, 6, 9, 10, 13]. Only few case reports on internal carotid artery aneurysm and pseudoaneurysm treated with endovascular stent and coiling procedure have been published [3, 9]. In case of carotid trauma, surgical treatment would be difficult because of problems of exposing the bleeding carotid and to get enough vascular access.
The combined evidences from all cases published in the medical literature support endovascular intervention for pseudoaneurysm of the carotid artery as a good clinical treatment choice. In our patient, we chose to use device graft to ensure complete exclusion of the pseudoaneurysm and external carotid artery without the need for endovascular coil placement. Further studies to investigate the role of endovascular treatment for penetrating carotid artery injury are indicated.

endovascular treatment
cervical fracture
carotid artery tear
penetrating injury