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To determine the oncological outcomes of cervical esophageal cancer (CEC) treated primarily with surgery.A systematic review and meta-analysis was performed according to the PRISMA guidelines.A total of 868 patients were included from 18 studies. Estimated pooled Overall Survival (OS) rates (95% Confidence Interval, CI) at 1 and 5 years were 74.4% (66.5-83.3), and 26.6% (20.3-34.7), respectively. Larynx non-preserving surgery (n = 229) showed an estimated pooled OS rates (95% CI) at 1 and 5 years of 59.3% (51.5-68.2) and 14.6% (8.8-24.3), respectively. On the other hand, larynx preserving surgery (n = 213) showed an estimated pooled OS rates (95% CI) at 1 and 5 years of 83.6% (78.2-89.4) and 35.1% (24.9-49.6), respectively.Primary larynx-preserving surgery remains a valuable option for the management of CEC, with similar survival outcomes compared to primary chemoradiotherapy (CRT). On the other hand, larynx non-preserving surgery showed a significantly reduced survival, that may reflect the more advanced T classification of these tumors. Further studies are mandatory to directly compare primary surgery and primary CRT, distinguishing larynx preserving and non-preserving surgery.

In a previous study, we performed a meta-analysis of the oncological outcomes of patients suffering from cervical esophageal squamous cell carcinoma treated with definitive chemoradiotherapy. Further analysis was performed, and a random effect modeling showed a pooled local-regional failure rate of 41.4% (95% CI 32.2-50.8), and a pooled distant failure rate of 21.6% (95% CI 17.0-26.5). The included studies used a median radiotherapy (RT) dose of 61.2 Gy (95% CI 60.0-62.0, range 56.0-66.0), but we measured a non-significant impact of the RT dose on the pooled overall survival (OS), suggesting that an increased RT dose might not be related to an improved OS (p = 0.23). Further research should be conducted to define predictors and prognostic categories that may select the best treatment option for each patient.

Background Desmoid-Type Fibromatosis (DTF) is a rare mesenchymal neoplasm with a locally invasive pattern and high risk of local recurrence after surgery. Historically, the standard treatment for DTF was surgical resection. However, considering the difficulty of achieving surgical eradication, the possible unnecessary morbidity and the unpredictability of the natural history, a wait-and-see approach has been proposed for asymptomatic DTF. Methods We analyzed 87 consecutive patients with histologically-proven sporadic primary DTF, first recurrence or residual disease managed at our institution between 2000 and 2018. Patients and tumor-related variables were reviewed and analyzed. Two different treatment strategies were adopted according to different time periods: in the “early period” (2000–2010) patients underwent surgical treatment irrespective of the clinical presentation, whereas in the “late period” (2012–2018) asymptomatic patients used to undergo a wait-and-see strategy. The event-free survival (EFS) was compared trough a pre-post comparison. Results In the early period, surgery was performed in 51 (94.4%) patients and watchful waiting in 3 (5.6%). In the late period, the watchful waiting group accounted for 24 (72.7%) patients and the surgical group for 9 (27.3%). No statistically independent prognostic factors were found. EFS did not show statistically significant differences between early and late period groups. Conclusion Wait-and-see policy has shown to be equivalent to upfront surgery in terms of EFS; therefore, a conservative approach is recommended in asymptomatic patients diagnosed with DTF that can be followed through watchful waiting.

To determine the oncological outcomes of cervical esophageal squamous cell carcinoma (CESCC) treated with definitive chemoradiotherapy (CRT).A systematic review and meta-analysis was performed according to the PRISMA guidelines.A total of 1222 patients (median age: 63.0 years, 95% CI 61.0-65.0) were included from 22 studies. The median follow-up time was 34.0 months (n = 1181, 95% CI 26.4-36.0). Estimated pooled OS rates (95% CI) at 1, 3, and 5 years were 77.9% (73.9-82.2), 48.4% (43.2-54.3), and 35.3% (29.7-41.9), respectively. The median OS (95% CI) was 33.4 months (25.8-42.2). Estimated pooled PFS rates (n = 595; 95% CI) at 1, 3, and 5 years were 64.1% (57.9-71.0), 38.0% (33.3-45.5), and 29.8% (23.9-37.1), respectively. The median PFS (95% CI) was 19.8 months (14.9-26.6).Definitive CRT is a valuable first-line treatment for the management of CESCC. Further studies should focus on survival predictors able to define stage-based clinical guidelines.

To underline discrepancies between the Union for International Cancer Control (UICC) and the American Joint Committee on Cancer (AJCC) Tumor-Node-Metastasis (TNM) classifications in oral cavity cancer. Comparison between the UICC and AJCC TNM classifications of oral cavity cancer in their 8th editions and following versions. The most important update was the introduction of the depth of infiltration (DOI), which reflects the proximity of the tumor to the underlying lymphovascular tissues and was associated to the presence of nodal metastases. Since the first publication of the 8th edition of the AJCC Cancer Staging Manual on March 30, 2017, two further versions have been published, while the UICC TNM classification was left unchanged until a document containing modifications to the 8th edition of the UICC TNM Classification of Malignant Tumours was published online on October 6, 2020. Different versions of the TNM classification can be confounding for the scientific community. Citing the 8th edition of the UICC TNM Classification of Malignant Tumours or the AJCC Cancer Staging Manual without specifying the precise version used for classification may be insufficient. Clinicians and researchers are invited to always refer to the latest update of each classification.

BACKGROUND: Although antibody-mediated rejection (ABMR) has been long recognized as a leading cause of allograft failure after kidney transplantation, the cellular and molecular processes underlying the induction of deleterious donor-specific antibody (DSA) responses remain poorly understood. METHODS: Using high-dimensional flow cytometry, in vitro assays, and RNA sequencing, we concomitantly investigated the role of T follicular helper (T(FH)) cells and B cells during ABMR in 105 kidney transplant recipients. RESULTS: There were 54 patients without DSAs; of those with DSAs, ABMR emerged in 20 patients, but not in 31 patients. We identified proliferating populations of circulating T(FH) cells and activated B cells emerging in blood of patients undergoing ABMR. Although these circulating T(FH) cells comprised heterogeneous phenotypes, they were dominated by activated (ICOS(+)PD-1(+)) and early memory precursor (CCR7(+)CD127(+)) subsets, and were enriched for the transcription factors IRF4 and c-Maf. These circulating T(FH) cells produced large amounts of IL-21 upon stimulation with donor antigen and induced B cells to differentiate into antibody-secreting cells that produced DSAs. Combined analysis of the matched circulating T(FH) cell and activated B cell RNA-sequencing profiles identified highly coordinated transcriptional programs in circulating T(FH) cells and B cells among patients with ABMR, which markedly differed from those of patients who did not develop DSAs or ABMR. The timing of expansion of the distinctive circulating T(FH) cells and activated B cells paralleled emergence of DSAs in blood, and their magnitude was predictive of IgG3 DSA generation, more severe allograft injury, and higher rate of allograft loss. CONCLUSIONS: Patients undergoing ABMR may benefit from monitoring and therapeutic targeting of T(FH) cell–B cell interactions.

Supplemental Digital Content is available in the text, Background: Regorafenib, a multitargeted tyrosine kinase inhibitor, proved to be active in patients with soft tissue sarcomas (STS). Methods: We conducted an open-label, non-randomized, single-center phase II study in advanced pretreated STS patients. Patients received regorafenib 160 mg daily on days 1 enrule 21 of a 28-day cycle. The primary endpoint was the progression-free survival (PFS) at 8 weeks. Toxicity was registered. Results: Between April 2015 and November 2016, 21 patients were enrolled in the trial. A total of 13 out of 21 evaluable patients (61.9%) were progression-free at 8 weeks. Median PFS was 3.8 months (95% CI: 2.1–9.4). Median overall survival was 14.8 months (95% CI: 7.7–27.8). In the intention-to-treat population, we reported a PFS of 66.7% at 3 months (95% CI: 40.4–83.4) and 16.7% at 12 months (95% CI: 4.1–36.5). As per the RECIST criteria, the response rate was 4.7% (1 partial response out of 21 evaluable patients) with a clinical benefit rate of 61.9%; no complete response was observed. Treatment was well tolerated. Conclusion: Regorafenib shows signs of clinical activity in patients with advanced STS. Clinical Trial Registration: ClinicalTrials.gov NCT02307500.

Rhabdomyosarcoma (RMS) represents more than 50% of paediatric soft tissue tumours. Conversely, it is extremely rare among adults, where it shows peculiar biological and clinical features that are still poorly investigated. RMS patients should be referred to a Sarcoma Centre, where the contribution of experienced radiologists plays a relevant role in the diagnostic assessment of the disease, including precise localisation, staging, image-guided biopsy, response evaluation after treatment and follow-up. Besides CT and MRI, hybrid imaging including positron emission tomography (PET)/CT and PET/MRI are giving an increasing contribution to provide functional insights about tumour biology and to improve the diagnostic accuracy of the imaging work-up. This review paper provides a revision of the pathology, clinical and radiological features of adult RMS, with a particular focus on the growing role of hybrid PET-based imaging.

HLA matching at the epitope level offers new opportunities to identify suitable donors for transplant patients. The International HLA Epitope Registry (www.Epregistry.com.br) describes for the various HLA loci, repertoires of eplets including those that correspond to epitopes experimentally verified with specific antibodies. There are also many eplets which have remained as theoretical entities because no informative antibodies have been found. Which of them have immunogenic potential or conversely, might be considered as non-epitopes that cannot elicit specific antibody responses? This question is important for the application of epitope-based HLA matching in clinical transplantation. Correct predictions of B-cell epitopes on antigenic proteins are essential to the effective design of microbial vaccines and the development of specific antibodies used in immunotherapy and immunodiagnostics but prediction programs based on structural and physiochemical properties of amino acid residues are generally ineffective. Recent prediction programs based on three-dimensional structures of antigen-antibody complexes are more promising. One such program is called ElliPro developed by Ponomarenko. This report describes studies demonstrating that ElliPro can predict alloantibody responses to HLA-ABC eplets. Antibody-verified eplets have amino acid residues with much higher ElliPro scores than eplets for which no specific antibodies have been found. The latter group includes residues with very low ElliPro scores; they appear to represent eplets that might be classified as non-epitopes. In conclusion, ElliPro offers a new approach to characterize epitope repertoires that are clinically relevant in HLA matching.

Objective: To assess the impact of adjuvant volumetric modulated arc therapy (VMAT) compared with three-dimensional conformal radiation therapy (3DCRT) in terms of toxicity and local control (LC) in patients with soft tissue sarcoma of the extremities. Methods: From 2004 to 2016, 109 patients were treated, initially using 3DCRT and subsequently with VMAT. Clinical outcome was evaluated by contrast-enhanced MRI, thoracic and abdominal CT 3 months after treatments and then every 6 months. Toxicity was evaluated with Common Terminology Criteria for Adverse Events scale v. 4.3. Results: Patients presented Stage III soft tissue sarcoma disease (77%), localized tumor (95%) at the lower extremity (87%), adipocytic histotype (46%). Surgical resection was performed in all patients, followed by adjuvant 3DCRT in 38, and VMAT in 71. The median total dose was 66 Gy/33 fractions (range 60–70 Gy;25–35 fractions). More successful bone sparing was recorded using VMAT (p < 0.001). Median follow-up was 61 months, 93 and 58 months for 3DCRT and VMAT group, respectively. The 2- and 5 year LC were 95.3±2.1%, and 87.4±3.4% for the whole cohort, 92.0±4.5%, 82.9±6.4% for 3DCRT, 97.1±2.0%, 89.6±4.1% for VMAT (p = 0.150). On univariate and multivariate analysis the factors recorded as conditioning LC were the status of the surgical resection margins (p = 0.028) and the total dose delivered (p = 0.013). Conclusion: The availability of modern radiotherapy technique permit a better conformity on the target with maximum sparing of normal tissue and acceptable side-effects. VMAT is a safe and feasible treatment with limited rate of toxicity, compared to 3DCRT. Results on LC of VMAT are encouraging. Advances in knowledge: Soft tissue sarcoma of the extremities can benefit from the use of VMAT, with a reduction of the high dose to bones to avoid radiation osteonecrosis. An adequate total dose of at least 66 Gy and a radical surgical margin allow a good local control.

The concept that HLA antibodies recognize epitopes is leading to new approaches of HLA matching at the epitope level. HLA-DQ plays an important role and many studies have identified structurally defined DQ epitopes specifically recognized by antibodies; they have been recorded in the International HLA Epitope Registry http://www.epregistry.com.br but the list is still incomplete. Pregnancy offers an attractive model to study antibody responses to HLA epitopes. The current analysis was done on 42 DQ-reactive post-pregnancy sera tested in binding assays with a panel of DQ heterodimers. The reactivity of 29 sera corresponded fully to the presence of antibody-verified DQA and DQB epitopes recorded in the Registry. Analysis of the remaining 13 sera led to the identification of additional antibody-defined DQB and DQA epitopes. We have designed the first version of an eplet map for DQ alleles which includes antibody-defined DQA and DQB epitopes and shows sequence positions with polymorphic residues which can be used in HLA epitology studies to identify new antibody-defined DQ epitopes.

Trabectedin, a marine-derived DNA-binding antineoplastic agent, has been registered by the EMA and recently also by the FDA for the treatment of patients with advanced soft-tissue sarcoma (STS), a rare and heterogeneous disease.The antitumor activity of trabectedin is related both to direct effects on cancer cells, such as growth inhibition, cell death and differentiation, and indirect effects related to its anti-inflammatory and anti-angiogenic properties. Furthermore, trabectedin is the first compound that targets an oncogenic transcription factor with high selectivity in mixoid liposarcomas. This peculiar mechanism of action is the basis of its clinical development. The clinical pharmacology of trabectedin, the subsequent phase I, II and III trials are summarized and put into perspectives in this review.Trabectedin is a relevant pleiotropic antitumoral agent within the complex scenario of the management of STS. It can be used in advanced STS, either after failure of anthracyclines and ifosfamide or in patients unfit for these drugs, especially when reaching a high-tumor control and a long-term benefit is a priority. Toxicity profile is acceptable and manageable with no reported cumulative toxicities. Therefore, trabectedin has become one relevant therapeutic option in metastatic STS, especially in selected histologies.

Background The optimal treatment of leiomyosarcoma (LMS) of the inferior vena cava (IVC) is still unclear, especially in the metastatic and/or recurrent setting. We herein evaluated the long-term outcome after aggressive management. Methods Eleven patients underwent surgery for primary LMS of the IVC between 2000 and 2012. The clinical, pathological, and survival data were reviewed. Results The IVC was managed by graft replacement in four cases, primary repair in four, and ligation in three. The R0 resection rate was 64%. The median follow-up was 60 months. Nine patients developed distant relapse, two of them concomitant local recurrence; no exclusive local recurrence was observed. The 3- and 5-year distant recurrence free survival were 30% and 10%, respectively. The 3- and 5-year overall-survival (OS) were 77.8%. The presence of residual disease after surgery (P = 0.024) and the time to recurrence (P = 0.033) were associated with the OS in a univariate analysis. The time to recurrence was related to the post-metastases survival (P = 0.032). Conclusions An adequate surgery minimizes the risk of local recurrence and remains the main treatment for primary LMS of the IVC. Nevertheless, the rate of distant metastases remains extremely high. An aggressive surgical policy may be of benefit to selected patients with metastatic disease. J. Surg. Oncol. © 2016 Wiley Periodicals, Inc.

Purpose Before the wait-and-see policy became the standard approach in abdominal wall desmoid tumors, surgery was performed on a systematic basis. Surgery remains indicated for progressing tumors but its extent is debatable. The abdominal wall is a common site of origin of sporadic desmoids, usually associated with a favorable prognosis. We analyzed the results of surgery at this specific site. Methods Data from 33 patients affected by sporadic desmoid tumors of the abdominal wall (31 primary, 2 recurrent) consecutively treated at our cancer center between January 2000 and September 2013 were retrospectively studied. Results Twenty-nine patients underwent surgery upfront and 1 after progression during the initial wait-and-see period. Prosthetic reconstruction of the abdominal wall was required in 28 patients. The average hospital stay was 5 days. Three patients developed surgical complications. Local recurrence-free survival was 90% at 5 and 10 years. Three patients had an uneventful childbirth during the follow-up after surgery. Conclusions Desmoid tumors of the abdominal wall have a favorable prognosis after surgical resection, which remains a safe and effective treatment. Wild-type tumors are common, whereas the incidence of S45F mutation in the beta-catenin gene is lower than in other anatomic sites. Upfront surgery may be considered in selected women who wish to bear a child.

The International Registry of HLA Epitopes (http://www.epregistry.com.br) is a website-based resource for HLA epitopes important in transplant rejection and platelet transfusion refractoriness. Its primary goal is to document epitopes that are verified experimentally with specific antibodies. Such epitopes can be defined by single eplets and by eplets paired with certain polymorphic residues within a 15-A radius, the dimension of the corresponding structural epitope. This report is an update of the HLA-ABC repertoire including descriptions of 72 antibody-verifications of epitopes defined by eplets and/or eplet pairs. The newly updated version 2.0 EpRegistry shows also the polymorphic residue compositions of structural epitopes corresponding to eplets shared between groups of alleles. At present, 151 eplets have not been antibody-verified, and we ranked them with a so-called ElliPro score as a potential predictor of immunogenicity. Sixty eplets with low ElliPro scores might be considered non-epitopes incapable of inducing specific antibodies.

Donor-specific alloantibodies (DSA) to HLA-DP may cause antibody-mediated rejection (AMR), especially in re-transplants. We describe the immunization history of a patient who received 3 kidney transplants; the 3rd kidney was completely matched except at DPA1 and DPB1. Prior to the 3rd transplant, single antigen bead analysis (SAB) showed DSA reactivity against DPA1 shared by the 1st and 3rd donors, but B and T flow crossmatch (FXM) results were negative. Within 11 days the 3rd transplant underwent acute C4d+ AMR which coincided with the presence of complement (C1q)-binding IgG1 DSA against donor DPA1 and DPB1. Using HLAMatchmaker and SAB, we provide evidence that eplet (epitope) spreading on DPA1 and eplet sharing on differing DPB1 alleles of the 1st and 3rd transplants was associated with AMR. Since weak DSA to DPA1/DPB1 may induce acute AMR with negative FXM, donor DPA1/DPB1 high resolution typing should be considered in sensitized patients with DP-directed DSA.

The International Registry of Antibody-Defined HLA Epitopes ( http://www.epregistry.com.br) has been recently established as a tool to understand humoral responses to human leukocyte antigen (HLA) mismatches. These epitopes are defined structurally by three-dimensional molecular modeling and amino acid sequence differences between HLA antigens. So-called eplets represent essential components of HLA epitopes and they are defined by polymorphic residues. A major goal is to identify HLA epitopes that have been verified experimentally with informative antibodies. Our analysis has also included data in many publications. As of 1 November 2013, 95 HLA-ABC antibody-verified epitopes have been recorded, 62 correspond to eplets and 33 are defined by eplets paired with other residue configurations. The Registry is still a work-in-progress and will become a useful resource for HLA professionals interested in histocompatibility testing at the epitope level and investigating antibody responses to HLA mismatches in transplant patients.

This study addresses the reactivity patterns of human cytotoxic HLA class I epitope-specific monoclonal antibodies in Ig-binding and complement component C1q-binding Luminex assays in comparison with complement-dependent lymphocytotoxicity data reported at the 13th International HLA Workshop. Some monoclonal antibodies reacted similarly with epitope-carrying alleles in all three assays but others showed different reactivity patterns. These reactivity differences were analyzed with HLAMatchmaker and we incorporated the concept that eplets are essential parts of structural epitopes which can contact the six Complementarity Determining Regions (CDRs) of antibody. The data show that technique-dependent reactivity patterns are associated with distinct differences between polymorphic amino acid configurations on eplet-defined structural epitopes. The findings have been viewed in context of antigen-antibody complex formation that results in the release of free energy necessary to stabilize binding and to induce conformational changes in the antibody molecule to expose the C1q binding site, the first step of complement activation. Moreover the amount of free energy should be sufficient to induce a conformational change of C1q thereby initiating the first stages of the classical complement cascade leading to lymphocytotoxicity. The complement-fixing properties of HLA antibodies require not only specific recognition of eplets but also depend on interactions of other CDRs with critical amino acid configurations within the structural epitope. Eplet-carrying alleles that lack such configurations may only bind with antibody. This concept is important to our understanding whether or not complement-fixing donor-specific HLA antibodies can initiate antibody-mediated rejection.

The determination of HLA mismatch acceptability at the epitope level can be best performed with epitopes that have been verified experimentally with informative antibodies. The website-based International Registry of HLA Epitopes (http://www.epregistry.com.br) has a list of 81 antibody-verified HLA-ABC epitopes but more epitopes need to be added. Pregnancy offers an attractive model to study antibody responses to mismatched HLA epitopes which can be readily determined from the HLA types of child and mother. This report describes a HLAMatchmaker-based analysis of 16 postpregnancy sera tested in single HLA-ABC allele binding assays. Most sera reacted with alleles carrying epitopes that have been antibody-verified, and this study focused on the reactivity of additional alleles that share other epitopes corresponding to eplets and other amino acid residue configurations. This analysis led in the identification of 16 newly antibody-defined epitopes, seven are equivalent to eplets and nine correspond to combinations of eplets in combination with other nearby residue configurations. These epitopes will be added to the repertoire of antibody-verified epitopes in the HLA Epitope Registry.

BACKGROUND: Atrial fibrillation is the most common cardiac arrhythmia, and no current therapy is ideal for control of this condition. Experimental studies suggest that angiotensin II-receptor blockers (ARBs) can influence atrial remodeling, and some clinical studies suggest that they may prevent atrial fibrillation. METHODS: We conducted a large, randomized, prospective, placebo-controlled, multicenter trial to test whether the ARB valsartan could reduce the recurrence of atrial fibrillation. We enrolled patients who were in sinus rhythm but had had either two or more documented episodes of atrial fibrillation in the previous 6 months or successful cardioversion for atrial fibrillation in the previous 2 weeks. To be eligible, patients also had to have underlying cardiovascular disease, diabetes, or left atrial enlargement. Patients were randomly assigned to receive valsartan or placebo. The two primary end points were the time to a first recurrence of atrial fibrillation and the proportion of patients who had more than one recurrence of atrial fibrillation over the course of 1 year. RESULTS: A total of 1442 patients were enrolled in the study. Atrial fibrillation recurred in 371 of the 722 patients (51.4%) in the valsartan group, as compared with 375 of 720 (52.1%) in the placebo group (adjusted hazard ratio, 0.97; 96% confidence interval [CI], 0.83 to 1.14; P = 0.73). More than one episode of atrial fibrillation occurred in 194 of 722 patients (26.9%) in the valsartan group and in 201 of 720 (27.9%) in the placebo group (adjusted odds ratio, 0.89; 99% CI, 0.64 to 1.23; P = 0.34). The results were similar in all predefined subgroups of patients, including those who were not receiving angiotensin-converting-enzyme inhibitors. CONCLUSIONS: Treatment with valsartan was not associated with a reduction in the incidence of recurrent atrial fibrillation. (ClinicalTrials.gov number, NCT00376272.) Copyright © 2009 Massachusetts Medical Society.

Context.—The inhibition of oncogenic kinase signaling is a successful strategy to treat both hematologic and solid malignancies. Patients with chronic myelogenous leukemia, lung adenocarcinoma, renal cell carcinoma, and gastrointestinal stromal tumors are experiencing tremendous clinical benefits from targeted therapies in the form of kinase inhibitors. These drugs marked a revolution in cancer treatment, not only for their safety and efficacy, but also because they continue to expand our knowledge of the pathophysiology of cancer. Objective.—To provide a summary of the biologic predictors of gastrointestinal stromal tumor behavior and response to targeted therapies that currently help guide clinical decision making. Data Sources.—Published articles pertaining to the diagnosis, molecular genetics, prognostication, clinical behavior, and treatment of gastrointestinal stromal tumors, as well as experiences in a multidisciplinary sarcoma clinic. Conclusions.—In gastrointestinal stromal tumors, the strongest predictor of response to targeted therapies is the mutational status of KIT or PDGFRA. Patients whose tumors harbor a KIT exon 11 mutation benefit the most from imatinib mesylate therapy, in terms of response rate, progression-free survival, and overall survival. Conversely, tumors without detectable mutations in either gene (“wild-type” gastrointestinal stromal tumors) are generally not responsive to imatinib mesylate.

Although the determination of human leukocyte antigen (HLA) antibody specificity has traditionally been directed toward HLA antigens, there is now increasing attention to structurally defined HLA epitopes. An understanding of the HLA epitope repertoire is important to acceptable mismatching for sensitized patients and to a new epitope-based matching algorithm aimed to reduce antibody-mediated rejection. There are two strategies to determine the HLA epitope repertoire. Terasaki's group has used an empirical method to analyze the reactivity of single allele Luminex panels with mouse monoclonal antibodies (mAbs) and absorbed/eluted alloantibodies with a computer program based on shared residues in the amino acid sequences of reactive alleles. HLAMatchmaker is a theoretical algorithm that predicts HLA epitopes on the HLA molecular surface from stereochemical modeling of epitope-paratope interfaces of antigen-antibody complexes. Our epitope repertoire is based on so-called 'eplets' representing 3-A patches of at least one polymorphic residue on the molecular surface. A comparative analysis has shown that 81/103 Terasaki's HLA class I epitopes are equivalent to individual eplets (n = 50) or pairs of eplets (n = 31) separated far enough to serve as potential contact sites for two complementarity-determining regions of antibody. An additional 12 Terasaki's epitopes (TerEps) correspond to eplets with permissible residue combinations that do not seem to affect epitope specificity. We could not identify corresponding eplets for the remaining 10 TerEps, including 8 that might be considered xeno-epitopes defined by mouse mAbs. Conversely, HLAMatchmaker has 38 additional eplets in well-exposed surface positions that do not have equivalent TerEps, and for many of them, we have found specific antibodies. These findings strengthen the concept that eplets are essential basic units of HLA epitopes and that they provide a better understanding of HLA immunogenicity (i.e. ability to induce an antibody response) and antigenicity (i.e. reactivity with specific antibody).

This report describes a HLAMatchmaker-based antibody analysis of post-pregnancy sera with antibodies against child-specific HLA-DR epitopes. These sera were reactive in IgG-binding assays with single allele bead (SAB) panels on a Luminex platform. The antibody specificity analysis focused on DRB epitopes that have been recorded in the International HLA Epitope Registry (http://www.epregistry.com.br) as experimentally verified with informative antibodies but we also considered other eplets that predict potential epitopes. The SAB panel has in several instances two or more alleles corresponding to the same serologically defined DR antigen and we selected six sera were with different reactivity patterns with DR1, DR4, DR13 and/or DR52 alleles. We demonstrate here how amino acid differences between these alleles can provide useful information in the determination of new epitope specificities of antibodies in these sera. Eight newly antibody-verified epitopes were identified including three that correspond to eplets paired with self-residue configurations. Epitope specificity information appears to be useful in the prediction of mismatch acceptability of non-SAB alleles within serological DR antigen groups.

HLAMatchmaker is a computer algorithm that assesses human leukocyte antigen (HLA) compatibility at the structural level by intralocus and interlocus comparisons of polymorphic amino acid triplets in antibody-accessible sequences of HLA class I molecules. This program permits the identification of mismatched HLA antigens that share all of their polymorphic triplets with the patient's HLA antigens and, therefore, could be considered fully compatible. The validity of this algorithm has been verified by analyzing the antibody specificity patterns of 127 well-characterized sera (panel reactive antibody [PRA] > 80%) that had been screened by direct complement-dependent and/or anti-human globulin augmented lymphocytotoxicity testing with large HLA-typed cell panels. A 2 x 2 table-based Chi-square analysis program was applied to determine positive and negative correlations between serum reactivity and the presence HLA triplets assigned from the HLA types in the cell panel. The results indicate that high PRA patients do not produce antibodies to shared triplets on mismatched HLA antigens. Moreover, this serum analysis has permitted the identification of triplets with different degrees of immunogenicity as indicated by the frequencies of positive and negative correlations of serum reactivity with the HLA-typed cell panel. Mismatching for triplets with low immunogenicity provides further opportunities for identifying donors with acceptable HLA mismatches for highly sensitized patients.

The International Registry of HLA Epitopes (http://epregistry.com.br) has been recently established as a tool to understand antibody responses to HLA mismatches. These epitopes are defined structurally by three-dimensional molecular modelling and amino acid sequence differences between HLA antigens. A major goal was to identify HLA epitopes that have been verified experimentally with informative antibodies. This report addresses the identification of MICA epitopes. Our analysis included published information about MICA antibody reactivity in sera from sensitized patients as well as data from our own laboratories. This report describes twenty-one MICA epitopes verified with antibodies which have primarily been tested in Luminex assays with single alleles. The epitopes correspond to distinct eplets that are often defined by single residues. The Registry is still a work-in-progress and will become a useful resource for HLA professionals interested in histocompatibility testing at the epitope level and investigating antibody responses to HLA mismatches in transplant patients.

The International Registry of Antibody-Defined HLA Epitopes (http://www.epregistry.com.br) has been recently established as a tool to understand humoral responses to HLA mismatches. These epitopes can be structurally defined as eplets by three-dimensional molecular modeling and amino acid sequence differences between HLA antigens. A major goal is to identify HLA eplets that have been verified experimentally with informative antibodies. This report addresses class II epitopes encoded by genes in the HLA-D region. Our analysis included reviews of many publications about epitope specificity of class II reactive human and murine monoclonal antibodies and informative alloantibodies from HLA sensitized patients as well as our own antibody testing results. As of July 1, 2014, 24 HLA-DRB1/3/4/5, 15 DQB, 3 DQA and 8 DPB antibody-verified epitopes have been identified and recorded. The Registry is still a work-in-progress and will become a useful resource for HLA professionals interested in histocompatibility testing at the epitope level and investigating antibody responses to HLA mismatches in transplant patients.

HLA antibody (Ab) detection and characterization is a critical step in transplantation. With high sensitivity and allele level Ab identification, the Luminex-based single beads assay is an essential tool in all HLA labs. One of the challenges with the current testing is the potential detection of cryptic or not-physiologically expressed epitopes. Here we report an interesting case of cryptic epitopes and describe the decision making process in preparation for virtual crossmatch requests. A 2 y/o female patient being evaluated for small bowel transplant was tested for presence of HLA Ab. Her class II profile was positive for all DR except DR10, with strongest reactions to alleles with epitopes 98E (DR4,7,9) and 48YQ6 (DR53). The patient typed as DR7,10,53 and the sample was immediately flagged as a possible false pattern of reactivity. Single antigen beads from 2 different vendors showed similar profiles; the same reactivity pattern was also detected by multi-antigen beads. Upon dilution the Ab profile was weaker but similar to the undiluted sample (figure). The pattern “all DR but DR10” could be explained by the patient carrying a rare DR7 allele or a non-expressed DR7 variant. However, no patient cells were available for serological DR7 detection. Molecular typing by SSO and/or SSP may detect a rare DR7 but could not exclude the possibility of a SNP or frameshift mutations creating a premature stop codon behind exon 2. Therefore, the sample was tested by NGS. By sequencing past exon 2 we confirmed that the recipient expresses DRB1∗07:01:01 and no premature stop codons were present in exons 3 or 4. To determine the functional characteristics of the Ab, surrogate donors were tested by flow cytometry crossmatch (FCXM). One donor was DR7 homozygous and the other was DR4, DR7. No reactivity was detected against these donors’ DR antigens, despite the presence of 4 potential targets (figure). This case demonstrates the usefulness of additional testing by FCXM to confirm the presence of false pan-DR reactivity, probably due to a denatured epitopes. Download : Download high-res image (533KB) Download : Download full-size image

Aim To determine the impact of pre-treatment donor specific HLA antibody (DSA) titer on the effectiveness of carfilzomib-based therapy (CFZ) for antibody mediated rejection (AMR) in lung transplant recipients (LTRs). Methods Sixteen adult LTRs were treated with plasma exchange, IVIG and CFZ for AMR. DSA evaluation was performed using Luminex single antigen beads before and after CFZ. DSA titer was determined prior to CFZ using neat and 4-fold serial dilutions until the DSA MFI drop was >75% of neat IgG MFI. C1q reactivity was tested pre- and post-CFZ. Patients were considered responders and non-responders based on the change in DSA neat IgG MFI, C1q reactivity and change in FEV1 post CFZ. Normality was tested, and descriptive statistics and univariate parametric and nonparametric tests were used to assess outcomes where appropriate; risk was assigned with univariable logistic regression. Results The 16 LTRs underwent 19 courses of CFZ for 28 DSAs. DSA were 68.8% DQ, 12.5% DR, 18.7% DQ+DR. 67% of LTRs responded to CFZ. Median DSA C1q MFI fell from 3187 (IQR 629-13907) to 0 after therapy (p = 0.002) and two weeks later (p = 0.006). 11/12 responders had DSA IgG MFI drop >75% of neat MFI, with a mean drop of 91.4% (range 55–100%) at 1:256 dilution. In contrast, for non-responders, the mean drop in DSA IgG MFI was only 25% (range 0–52%) at 1:256 dilution, and DSA was still positive (MFI >1500) at 1:2048 dilution. DSA MFI drop >75% at 1:64 and 1:256 was significantly associated with response (p = 0.003; p > 0.001, respectively). Furthermore, 8/12 responders were treated within 1–7 months after DSA detection, while 3/3 non-responders had persistent DSA >12 months prior to treatment. DSA duration 2 = 0.728). DSA neat MFI >8000 did not predict non-response, and explained very little variance in non-responders (HR 2.0, 95%CI 0.5–8.0, p = 0.33, r 2 = 0.09). Conclusions Response to CFZ-based AMR therapy was impacted by pre-treatment DSA titer and length of time from DSA detection to treatment. Importantly, only DSA titer, but not DSA neat IgG MFI, predicted CFZ responsiveness.

Aim Human intravenous immunoglobulin (IVIG) is frequently used for treatment of antibody mediated rejection. Little is known regarding the risk of passively transferred HLA antibody (Ab) in these products. Our aim was to test commercial IVIG preparations for presence and level of HLA Ab and specificity. Methods Thirty IgG preparations derived from plasma collected in the US, Europe and Canada (CSL Behring: 6 Hizentra, 20% protein; 24 Privigen, 10% protein) were tested for HLA class I/II Ab in Luminex single antigen bead assays. Reagents were treated with Adsorb Out, then tested at various dilutions. Testing was performed with 2 vendor kits. Furthermore, preparations were tested on the C1q screening assay and after acid treatment. Results Initial testing showed HLA Ab reactivity (MFI >2000) at 1:10 but negative by 1:50. Regardless of protein concentration, IgG preparations had similar reactivity patterns. Following acid reduction, most class I beads remained positive with one vendor kit, suggesting the remaining reactivity was towards denatured and not native HLA molecules. As shown in the Table, class I Ab reactivity after acid treatment was limited to a few HLA specificities. Class I results with a second vendor kit were negative. Class II initial testing with one kit showed a similar reactivity pattern in 20/30 samples, mainly DR4+DR18, plus a few additional positive reactions; testing with the second kit gave MFI values Conclusions Ab reactivity in IgG preparations was low titer regardless of the percentage protein. All positive reactions were observed with One Lambda and not Immucor kits, and most positive class I reactions were towards denatured HLA molecules. Class II reactivity appeared to be non-specific. We conclude that these IgG preparations do not pose a risk for passively transferred HLA Ab that can potentially be harmful to the allograft. Research support and test materials provided by CSL Behring, LLC. Class I Luminex reagents provided by Immucor. M. Berger: Employee; Company/Organization; CSL Behring, LLC. M. Spycher: Employee; Company/Organization; CSL Behring AG. A. Zeevi: Grant/Research Support; Company/Organization; CSL Behring, LLC.

The concept that HLA antibodies are specific for epitopes rather than HLA antigens is important not only for the determination of mismatch acceptability for sensitized patients but also for a better understanding of the antibody response to an HLA mismatch. Numerous publications describe epitope-specific antibodies, but there is no standardized information about the repertoire of clinically relevant HLA epitopes. Under auspices of the 16th IHIW, we have developed a website-based registry of antibody-verified HLA epitopes. Epitope notations are based on HLA molecular modelling of amino acid residues in polymorphic sequence positions. Informative epitope-specific antibodies had been induced by a transplant, transfusion or pregnancy and were monoclonal antibodies or eluates of sera absorbed with single HLA alleles. Antibody reactivity was determined in binding assays with single-allele panels. Antibody producer/immunizer HLA types enhanced the characterization of specific epitopes. The Registry also includes epitopes described in original research publications. Based on the extent of antibody reactivity information, we assigned epitope status as confirmed (well documented) or provisional (more data are needed). At present, the Registry has 69 HLA-ABC, 53 DRB1/3/4/5, 17 DQ, 8 DP and 22 MICA antibody-verified epitopes and will be updated on a quarterly basis. Laboratories worldwide continue to submit data about previously unreported antibody-specific epitopes. For each epitope, the website shows its amino acid composition and HLA alleles that share the epitope. Links show antibody reactivity patterns, sensitization information and references. Other links show molecular modelling of corresponding structural epitopes and polymorphic residue information for epitope-carrying alleles. The website will also have a link to epitope frequency information in different populations. Search functions will list mismatched epitopes on mismatched alleles for selected HLA types. The HLA Epitope Registry will become a valuable resource for researchers interested in HLA compatibility at the epitope level and investigating antibody responses to HLA mismatches.

This study addresses the concept that human leukocyte antigen (HLA) class I-specific alloantibodies are specific for epitopes that correspond to HLAMatchmaker-defined eplets. Eplets are essential parts of so-called structural epitopes that make contact with the 6 complementarity determining regions of an antibody. From published molecular models of crystallized protein antigen-antibody complexes, we have calculated that contact residues on structural HLA epitopes should reside within a 15-Å radius of a mismatched eplet. This study addresses the structural basis of high-frequency HLA class I epitopes reacting with human monoclonal antibodies (mAbs) derived from women sensitized during pregnancy. All mAbs were tested in Luminex assays with single HLA allele panels. The HLAMatchmaker algorithm was used to determine their specificity in context with eplet sharing between the immunizing allele and antibody-reactive alleles. To assess the autoreactive B cell origin of these antibodies, we have applied the recently developed nonself-self paradigm of epitope immunogenicity to analyze residue differences between the immunizer and the alleles of the antibody producer. A total of 9 mAbs were specific for epitopes associated with the 41T, 80NRG, 163LW, 69AA, or 80ERILR eplets. In each case, the immunizing allele had within 15 Å of the mismatched eplet, no residue differences with 1 of the alleles of the antibody producer. This observation is consistent with the concept that these mAbs originated from B cells with self HLA immunoglobulin receptors. Eplet-carrying alleles exhibited different levels of reactivity, which, when compared with the immunizing allele, ranged from high to intermediate to very low. In many cases, lower reactivities were associated with differences from self to nonself residues in surface locations within 15 Å of the specific eplet. Apparently, such locations may serve as critical contact sites for the antibody. In other cases, other residue differences did not appear to affect binding with the antibody, suggesting that these locations do not play a major role in antibody binding. For these mAbs we did not obtain convincing evidence that residue differences in hidden positions below the molecular surface had significant effects on antibody binding. These findings have increased our understanding of the structural basis of the immunogenicity and antigenicity of HLA class I epitopes and provide a basis for interpreting HLA antibody reactivity patterns in Luminex assays with single alleles.

This report summarizes the 8-year experience of the DR survey program designed to evaluate the performance of histocompatibility laboratories in the serologic typing of cell specimens for HLA-DR and HLA-DQ polymorphisms and the HLA class II antibody identification and B-cell crossmatching of serum specimens. The number of participants increased from 45 in 1985 to 214 in 1992. Although the performance criteria are based on laboratory consensus, the availability of DNA typing since 1990 has enabled a critical assessment of the reliability of serologic HLA-DR, DQ typing. The survey results shows that unsplit HLA class II antigens DR1-DR8, DR52/53, and DQ1-3 are generally correctly identified in over 90% of the participating laboratories. DR9 and DR10 have not yet been tested and testing for DQ4 has not yet achieved this level on consensus. In contrast, the assignments of serologic subtypes of HLA-DR and HLA-DQ are less consistent and frequently unreliable. Although the B-cell crossmatches show generally high laboratory consensus rates, the serum screening results show frequently inconsistent results regarding HLA class-II-specific antibody identification.

The Histocompatibility Survey Program was organized in 1982 as a joint project by the ASHI and CAP to evaluate laboratory performance in HLA typing, lymphocytotoxicity crossmatching, and antibody analysis. This report summarizes the experience with the HS surveys on HLA class I serology. During a 12-year period, the number of participating laboratories increased from 150 to 285 and HLA typing was done with 90 survey specimens representing 20 HLA-A and 35 HLA-B antigens. Most unsplit antigens were correctly identified in more than 90% of the laboratories. For many antigens, a high percentage of participants reported a split and there was generally a high consensus of a correct assignment. Nevertheless, several antigens were difficult to define, as shown by low consensus rates. During recent years, the assignments of Bw4/6 and HLA-C antigens have significantly improved. Lymphocytotoxicity crossmatching was analyzed for 138 cell-serum combinations tested by an average of 143 laboratories. Comparisons between four techniques (basic NIH, Amos modified, LI, and AHG) showed consistent results (greater than 90% crossmatch compatibility or incompatibility) for 71% of the cell-serum combinations. The crossmatch results with the remaining combinations were more variable for one or more of the crossmatch techniques. Serum antibody identification showed a continued improvement during recent years, and the average consensus for assigning acceptable antibody specificity reached 88%. A performance grading system based on a 90% consensus rate among participants is used to satisfy requirements for laboratory accreditation.

This case report summarizes the spectrum of anti–human leukocyte antigen (HLA) antibody reactivity determined by single-allele Luminex immunoglobulin G and C1q binding assays before transplant, during an episode of antibody-mediated rejection (AMR), and following treatment in a sensitized pediatric heart transplant (Tx) recipient. We were able to discriminate between complement- and non-complement-binding epitope-specific antibodies present against a single donor antigen (HLA-A2) during the progression of AMR and its resolution. Our findings illustrate the usefulness of determining antibody specificities against epitopes using various Luminex-based assays.

Although the determination of human leukocyte antigen (HLA) antibody specificity has traditionally been directed toward HLA antigens, there is now increasing attention to structurally defined HLA epitopes. An understanding of the HLA epitope repertoire is important to acceptable mismatching for sensitized patients and to a new epitope-based matching algorithm aimed to reduce antibody-mediated rejection. There are two strategies to determine the HLA epitope repertoire. Terasaki's group has used an empirical method to analyze the reactivity of single allele Luminex panels with mouse monoclonal antibodies (mAbs) and absorbed/eluted alloantibodies with a computer program based on shared residues in the amino acid sequences of reactive alleles. HLAMatchmaker is a theoretical algorithm that predicts HLA epitopes on the HLA molecular surface from stereochemical modeling of epitope-paratope interfaces of antigen-antibody complexes. Our epitope repertoire is based on so-called 'eplets' representing 3-A patches of at least one polymorphic residue on the molecular surface. A comparative analysis has shown that 81/103 Terasaki's HLA class I epitopes are equivalent to individual eplets (n = 50) or pairs of eplets (n = 31) separated far enough to serve as potential contact sites for two complementarity-determining regions of antibody. An additional 12 Terasaki's epitopes (TerEps) correspond to eplets with permissible residue combinations that do not seem to affect epitope specificity. We could not identify corresponding eplets for the remaining 10 TerEps, including 8 that might be considered xeno-epitopes defined by mouse mAbs. Conversely, HLAMatchmaker has 38 additional eplets in well-exposed surface positions that do not have equivalent TerEps, and for many of them, we have found specific antibodies. These findings strengthen the concept that eplets are essential basic units of HLA epitopes and that they provide a better understanding of HLA immunogenicity (i.e. ability to induce an antibody response) and antigenicity (i.e. reactivity with specific antibody).

HLAMatchmaker is a matching algorithm that can be used to characterize antibodies specific for structurally defined epitopes. Under the auspices of the 15(th) International Histocompatibility Workshop, we are conducting a multilaboratory collaborative project to characterize these epitopes and also to determine how often they induce specific antibodies in patients with rejected kidney transplants. This report addresses the reactivity of post-allograft nephrectomy sera tested for DRB antibodies with Luminex assays using single alleles. This analysis was performed for 19 informative kidney transplant cases contributed by 13 laboratories worldwide. There were 11 cases with a single DR2 mismatch (DR15 or DR16), and nine of them (82%) showed antibodies with both DR2 and DR1. Although these antigens might share an epitope recognized by these antibodies, this interpretation is incorrect. The HLAMatchmaker analysis offers a clearly different explanation that involves antibodies induced by DR51, which commonly associates with DR2. DR51 has an epitope defined by the 96EV eplet, which is also present on DR1 but no other DR antigen. This means that the reactivity with DR51 and DR1 reflects the presence of 96EV-specific antibodies. Conversely, we analyzed eight patients sensitized by a single DR1 mismatch that has no associated DR51. All of these patients reacted also with DR51, and this could be explained only by antibodies against the shared 96EV eplet. These findings demonstrate that 96EV represents a highly immunogenic epitope that can induce cross-sensitization between antigens encoded by the different DRB loci, and also that DR51 is important in determining DRB mismatch acceptability of potential donors. This analysis has also demonstrated that antibody responses are restricted to a few epitopes on these immunizing DR antigens. For DR2 they are 142M3 (unique for DR2), 71QAA (shared with DB5*02), and 96QV (shared with DR10). DR51 mismatches appear to have three immunogenic eplets: 96EV (shared with DR1), 108T3 (unique for DR51), and 40HFD (shared with DR9). Immunogenic eplets on DR1 are 12LKF2 (unique for DR1), 14FEH (shared with DR9 and DR10), and 25HRL (shared with DR10).

108 seropositive homosexual men were examined for associations between HLA phenotype and progression of human immunodeficiency virus type 1 (HIV-1) infection. Among men of predominantly European ethnic origin, 49 with very rapid 2-year declines in CD4 + lymphocyte counts showed significant differences in antigen frequencies from 59 men matched for ethnic background, study centre, and initial CD4 + cell count but with little or no decline in CD4 + cells. Relations of varying strength (odds ratios 6·1-10·3) were seen with several HLA antigens often linked in the A1-Cw7-B8-DR3 haplotype. The strongest relation was with the A1, Cw7, B8 combination (odds ratio 10·3). Associations between these antigen combinations and development of AIDS were weaker. The frequency of HLA A24 was also significantly higher in rapid than in slow decliners (odds ratio 4·3). These findings strengthen the suggested link between the product of a gene in the A1-Cw7-B8-DR3 haplotype and HIV-1-related disease.

Context.—This report presents results of the serum antibody analysis and crossmatch challenges in the proficiency testing program for histocompatibility testing jointly sponsored by the American Society for Histocompatibility and Immunogenetics and the College of American Pathologists. Objective.—To obtain information about consensus rates among participating laboratories that reported antibody screening and crossmatch results by direct complement-dependent lymphocytotoxicity (CDC) and/or anti-human globulin (AHG)–augmentation methods. Design.—We analyzed responses from approximately 165 laboratories participating in 32 surveys during 1993–2000. Most of the testing was done by CDC methods, but increasing proportions of laboratories are using AHG augmentation of these techniques; almost one half of the serum screenings and crossmatches were done by AHG. Results.—A total of 40 serum specimens were screened to determine the percent panel-reactive antibody (PRA) and identify HLA-specific antibodies. Participants often reported very wide ranges of PRA values. Panel-reactive antibody ranges exceeded 60 percentage points for 16 (40%) of the serum screening results by CDC and for 31 (77%) of the results by AHG. The interlaboratory variability of PRA values suggests that in many laboratories, the CDC or AHG procedures were often too insensitive or overly sensitive. The antibody identification results revealed inconsistent patterns among the participants performing CDC or AHG screening. Most participants reported the same primary antibody specificities by both methods. The consensus levels were generally high for the monospecific sera. On the other hand, there was much less agreement among the participants if the sera reacted with 2 or more HLA antigens. Participants using the more sensitive AHG method reported additional antibody specificities in many specimens, but invariably the consensus levels were rather low. A total of 192 serum-cell combinations were used for the crossmatch challenges. There was considerable interlaboratory variability; 21% of the CDC crossmatches and 36% of AHG crossmatches failed to reach the 90% consensus threshold. Conclusions.—This experience demonstrates considerable inconsistencies in serum screening and crossmatching among laboratories participating in the American Society for Histocompatibility and Immunogenetics/College of American Pathologists surveys. A lack of uniformity in test results may limit the efficient application of these methods in a clinical setting. Standardization of crossmatch and antibody screening techniques is highly desirable.

Aim Antibody mediated rejection (AMR) after lung transplantation (LTx) is associated with early and severe chronic rejection and death. Conventional therapies consisting of plasma exchange and IVIG with and without Rituximab are partially effective. Newer therapies with proteasome inhibitors (Carfizomib (CFZ) and Bortezomib (BTZ)) that target both B and plasma cells may be more effective in removing the donor specific antibodies (DSA). CFZ, unlike BTZ, irreversibly inhibits the activity of 20S proteasome and results in less neuropathic side effects. Method We evaluated the impact of CFZ therapy in 7 LTx on DSA level and function and correlated with clinical outcome as measured by pulmonary function test. All 7 patients had DSA IgG specificity and titer measured by single antigen bead Luminex assay and DSA function of complement binding by C1q-screen at baseline and at 1 month and 3 months post-treatment. Results The specificity, strengths and C1q reactivity pre- and post-therapy is summarized in Table below. All 7 patients had class II DSA and 6/7 were DQ specific with C1q reactivity. 5/7 LTx responded to initial treatment with a drop in IgG DSA level, with negative C1q DSA reactivity ( 15,000 MFI the IgG DSA was inhibited (“prozone effect”), however IgG DSA significantly increased in the presence of EDTA (LTx 3, 5, 6, 7). Conclusion In this small cohort of LTx who mostly failed conventional therapies and have persistent DSA, CFZ based approach appears to be useful, initially. However, 5/7 LTx recovered their C1q DSA reactivity upon longer follow-up suggesting that one cycle of CFZ it may not be effective to impact the rebound of circulating DSA.

Aim To perform a retrospective analysis of the 3rd kidney transplant rejection with exclusive disparity at HLA-DP. Methods The 1st transplant, nearly completely mismatched, functioned well for 7 years but was rejected due to chronic allograft nephropathy. The 2nd transplant, completely matched except at DRB3 and DPB1, failed after 1 year with acute and chronic rejection. A 3rd kidney was completely matched except at DPA1*02:01 and DPB1*01:01. Although single antigen bead (SAB) analysis showed donor-specific alloantibodies (DSA) directed to DPA1*02:01 (3496-4024 MFI), both B and T flow crossmatches (FXM) were negative. Within 10 days the re-transplant underwent aggressive C4d+ acute antibody-mediated rejection (AMR; Banff 2a). Despite thymoglobulin therapy and 4 cycles of plasmapheresis, kidney failed within 6 months. At 30 days post-transplant, SAB assay revealed that DSA (directed to donor DPA1*02:01 and DPB1*01:01 bead) was IgG (9511 MFI) of IgG1 subclass (8664 MFI) with C1q binding function (22800 MFI). Results Epitope analysis showed that the 1st transplant shared DPA1*02:01 with the 3rd transplant while the 1st transplant DPB1*11:01 shared two epitopes (35YA, 84DEAV) with the 3rd donor DPB1*01:01. DSA response directed to just one epitope (111R) on DPA1*02:01 spread to other epitopes (83A, 51RA, 127P)(Fig. 1). Conclusions (1) DPA1 and DPB1 alleles may induce an aggressive acute AMR resulting in graft failure; (2) DSA directed to donor DPA1 allele may be undetected by FXM; (3) DPA1 and DPB1 alleles should be identified in donors of re-transplanted patients with DPA1/DPB1-directed Abs; and 4) epitope spreading and epitope sharing enhance FXM-undetectable DSA during acute AMR [Fig. 1].

Aim The significance of donor-specific HLA antibodies (DSA) in lung transplantation (LTx) has emerged with the introduction of solid phase assays for antibody (Ab) detection. In the present study we evaluated 1) frequency of DSA in LTx using Luminex (L)-based methods for specificity and functional characterization and 2) the impact of de novo DSA on LTx outcome. Methods LTx from 2011 to 2012 and screened prospectively with L-PRA beads were included in the analysis. Testing was done pre- and post-Tx at 1 month and then every 2 months with surveillance and clinical bronchoscopies. DSA specificity/strength (MFI) determined with single antigen beads, and complement binding with the L-C1q assay, was associated with cellular (ACR) and Ab mediated rejection (AMR). Results 161 consecutive LTx with minimum follow-up of 6 months and negative CDC T- and B-cell crossmatch were studied. 10% had DSA pre-Tx and 19% had de-novo DSA. 54% of LTx had high grade ACR; de novo DSA was higher in ACR (27/87, 31%) vs. no ACR (10/74, 13.5%). The most frequent (67%) de novo DSA were HLA-DQ including Ab towards DQA,DQB/DQA pair and DQB (fig). In LTx with persistent, severe ACR (n=4), increased levels of de novo DSA with C1q reactivity were associated with AMR that required treatment with PP/IVIG and Ritux. In contrast, LTx (n=9) with persistent ACR and low level or declining DSA with transient C1q reactivity did not develop AMR. Similarly, no AMR was diagnosed in LTx who had one or more ACR and transient DSA. DSA detected without ACR were also low level, transient and without C1q reactivity. Conclusions Persistent and high grade ACR with C1q binding class II HLA-DQ specific DSA was associated with AMR. [figure1]

Aim While it is generally accepted that HLA antibodies (Abs) represent significant risk factors for transplant rejection and graft failure, it has become apparent that Abs are specific for epitopes (Eps), not HLA antigens. A distinction between HLA Eps and antigens is important not only for the determination of mismatch acceptability for sensitized patients but also for a better understanding of the Ab response to an HLA mismatch. Numerous publications describe Ep-specific Abs, but there is no standardized information about the repertoire of clinically relevant HLA Eps. Under auspices of the 16 th IHIW we developed a website-based registry of Ab-verified HLA Eps. Ep notations are based on HLA molecular modeling of amino acid residues in polymorphic sequence positions. Methods Informative Ep-specific Abs were induced by transplant, transfusion or pregnancy, or were monoclonal Abs or eluates of sera absorbed with single HLA alleles. Ab reactivity was determined in binding assays with single allele panels. Ab producer/immunizer HLA types enhanced the characterization of specific Eps. The registry also includes Eps described in original research publications. Based on the extent of Ab reactivity information, we assigned Ep status as confirmed (well-documented) or provisional (more data needed). At present, the registry has 77 HLA-ABC, 52 DRB1/3/4/5, 17 DQAB, 8 DPAB and 6 MICA Ab-verified Eps. Laboratories worldwide continue to submit data about previously unreported Ab-specific Eps. Results For each Ep, the website shows its amino acid composition and HLA alleles that share the Ep. A link shows Ab reactivity, sensitization information and references. Another link shows molecular modeling of corresponding structural Eps and polymorphic residue information for Ep-carrying alleles. The website will also have a link to Ep frequency information in different populations. Conclusions The HLA Epitope Registry website will become a valuable resource for researchers investigating Ab responses to HLA mismatches.

Aim Complement-fixing (CF) HLA antibodies (Ab) play an important role in transplantation because they can initiate inflammatory processes within the graft that lead to rejection. The Luminex Ig-binding assay (LUM-Ig) is now the principal method for HLA Ab testing. The C1q-binding assasy (LUM-C1q) can distinguish between CF and non-CF Abs. We show here evidence that the differential reactivity of CF HLA Ab depends on the structural composition of epitope (Ep)-carrying alleles (Al). Methods Human cytotoxic class I Ep-specific monoclonal antibodies (mAb) were tested in LUM-Ig and LUM-C1q (OneLambda). Results were compared to CDC reactivity with informative cells from the 13th International Workshop. Results Two reactivity patterns were observed. For some mAb, Ep-carrying Al reacted similarly in all assays. But certain mAb reacted with more Al in LUM-Ig than in LUM-C1q and CDC. Reactivity was analyzed with HLAMatchmaker, incorporating the concept that eplets are essential parts of structural Eps which can contact the 6 Complementarity Determining Regions (CDRs) of Ab. The data show that LUM-Ig mAb are specific for eplets but that LUM-C1q and CDC reactivity require an additional polymorphic amino acid configuration on eplet-carrying Al. Conclusions The findings must be viewed in context of formation of an antigen-Ab complex resulting in release of free energy necessary to stabilize binding and to induce conformational change in the Fc part of the Ab structure to expose the C1q receptor, the first step of Complement activation. The CF properties of HLA Ab require not only specific recognition of an eplet but also depend on interactions of other CDRs with critical amino acid configurations within the structural Ep. Eplet-carrying Al that lack such critical configurations will bind with Ab but the amount of free energy is insufficient to expose the C1q-binding site and there is no CF. This concept is important to our understanding whether or not CF donor-specific HLA Ab can initiate Ab-mediated rejection.

Aim HLA-A2, B57 and B58 share a well-defined epitope (Ep) which corresponds to the 62GE eplet on the α1 helix, but not all antibodies (Ab) bind equally to these alleles (Al). Methods Six 62GE-specific human monoclonal antibodies (mAb) were tested in Luminex single Al assays with OneLambda (OL) and Gen-Probe (GP) kits according to manufacturer’s instructions. Both have A∗02:01/A∗02:03/B∗57:01/B∗58:01. Only OL has A∗02:06 and B∗57:03; only GP has A∗02:02 and A∗02:05. Results Three mAbs reacted well with all 62GE Al. MFI values were 16688 ± 3565(OL), 11843 ± 2993(GP); other Al had low MFI (range 4-89(OL), 2-211(GP)). Three mAbs had much lower MFI with 62GE Al: 7580 ± 3521(OL), 3107 ± 4228(GP). The table shows kit-related differences: [Fig. 1] For WK1D3 and VN2F1, A∗02:01 and ∗02:03 were OL-positive but GP-negative; note that A∗02:05(GP) reacted well. WIM1B3 reacted with all 62GE Al except B∗57:01(GP). Reactivity (Rx) patterns were reproducible upon repeat testing. Download : Download full-size image Conclusions Differences between Al Rx in OL and GP can be explained by the concept that peptides bound to the groove affect binding of some HLA Ep-specific Ab. Mulder et al demonstrated “peptide-dependent” HLA Ab (J. Immunol. 175:5950, 2005), anti-HLA-A2 mAb with binding differences to recombinant A∗02:01 molecules loaded with different peptides. Also, Rx of some mAb with mouse class I antigens has been shown to be governed by a limited peptide repertoire. The 62GE eplet and peptides in the groove are in close enough proximity to serve as additional Ab contact sites. Discrepancies between OL and GP Ab Rx patterns may reflect the possibility that Al preparations have different peptide profiles related to different vendor manufacturing processes.

Two transplant candidates sensitized during pregnancy by a B*44:02 mismatch showed antibodies that reacted with an epitope defined by the 145R+82LR eplet pair shared by all Bw4 antigens in single allele Luminex panels except B13. Both eplets are on one or more alleles of the antibody producer and according to HLAMatchmaker, they are considered intralocus and interlocus matches which should not induce antibodies. The recently developed nonself-self paradigm for HLA epitope immunogenicity has offered a ready explanation why the pair of self-145R and self-82LR eplets on B*44:02 induced specific antibodies. This finding is consistent with the concept that alloantibody responses originate from B-cells with self-HLA immunoglobulin receptors.