HPV-EM: an accurate HPV detection and genotyping EM algorithm.

Accurate HPV genotyping is crucial in facilitating epidemiology studies, vaccine trials, and HPV-related cancer research. Contemporary HPV genotyping assays only detect < 25% of all known HPV genotypes and are not accurate for low-risk or mixed HPV genotypes. Current genomic HPV genotyping algorithms use a simple read-alignment and filtering strategy that has difficulty handling repeats and homology sequences. Therefore, we have developed an optimized expectation-maximization algorithm, designated HPV-EM, to address the ambiguities caused by repetitive sequencing reads. HPV-EM achieved 97-100% accuracy when benchmarked using cell line data and TCGA cervical cancer data. We also validated HPV-EM using DNA tiling data on an institutional cervical cancer cohort (96.5% accuracy). Using HPV-EM, we demonstrated HPV genotypic differences in recurrence and patient outcomes in cervical and head and neck cancers.

Utility of protocol kidney biopsies for de novo donor-specific antibodies.

There is limited information about the role of protocol kidney biopsies for de novo donor-specific antibodies (dnDSA) in kidney transplant recipients, especially in those with stable graft function. We initiated a routine posttransplant DSA monitoring and surveillance biopsy program for dnDSA since 2014. We identified 45 kidney transplant recipients with dnDSA detected between January 2014 and February 2017 who underwent kidney biopsy within 60 days of detection of dnDSA. Twenty-nine (64%) had stable graft function and 16 (36%) had impaired graft function at the time of dnDSA detection. Even in the group with stable graft function, we found a high rate of rejection (53%) on biopsy. Eighty-eight percent of patients with impaired graft function had rejection. Those patients with impaired graft function had significantly lower estimated glomerular filtration rate at 12 months postbiopsy and at last follow-up. Those with impaired graft function had more graft failures; however, this result was not statistically significant. The high rate of asymptomatic rejection, and the fact that outcomes in asymptomatic patients are poor, is in support of the utility of surveillance biopsies in patients with dnDSA.

Virtual HLA Crossmatching as a Means to Safely Expedite Transplantation of Imported Pancreata.

BACKGROUND: Imported pancreata accumulate cold ischemia time (CIT), limiting utilization and worsening outcomes. Flow cytometric crossmatching (FXM) is a standard method to assess recipient and donor compatibility, but can prolong CIT. Single-antigen bead assays allow for detection of recipient donor-specific HLA antibodies, enabling prediction of compatibility through a "virtual crossmatch" (VXM). This study investigates the utility and outcomes of VXM after transplantation of imported pancreata. METHODS: We retrospectively compared outcomes of 153 patients undergoing pancreas transplantation at our institution over a 3.5-year period. RESULTS: Three patient groups were analyzed based on geographic source of the pancreas graft and the type of prospective crossmatch performed: (1) imported VXM-only, n = 39; (2) imported VXM + FXM, n = 12; and (3) local VXM + FXM, n = 102. There were no episodes of hyperacute rejection and 1 episode of early antibody-mediated rejection (<90 days) in the imported VXM group. Death-censored graft survival, patient survival, and rejection rates were comparable among the recipient groups. For pancreata imported from United Network of Organ Sharing regions 3 and 4, proceeding to surgery without an FXM reduced CIT by 5.1 hours (P < 0.001). The time from organ arrival at the hospital to operation start was significantly shorter in the VXM-only group compared with the VXM + FXM group (P < 0.001). CONCLUSIONS: Virtual crossmatch helps minimize CIT without increasing rejection or adversely affecting graft survival, making it a viable method to increase pancreas graft utilization across distant organ sharing regions.

Current outcomes of chronic active antibody mediated rejection - A large single center retrospective review using the updated BANFF 2013 criteria.

BACKGROUND: The updated BANFF 2013 criteria has enabled a more standardized and complete serologic and histopathologic diagnosis of chronic active antibody mediated rejection (cAMR). Little data exists on the outcomes of cAMR since the initiation of this updated criteria. METHODS: 123 consecutive patients with biopsy proven cAMR (BANFF 2013) between 2006 and 2012 were identified. RESULTS: Patients identified with cAMR were followed for a median of 9.5 (2.7-20.3) years after transplant and 4.3 (0-8.8) years after cAMR. Ninety-four (76%) recipients lost their grafts with a median survival of 1.9 years after diagnosis with cAMR. Mean C4d and allograft glomerulopathy scores were 2.6 ± 0.7 and 2.2 ± 0.8, respectively. 53.2% had class II DSA, 32.2% had both class I and II, and 14.5% had class I DSA only. Chronicity score >8 (HR 2.9, 95% CI 1-8.4, p=0.05), DSA >2500 MFI (HR 2.8, 95% CI 1.1-6.8, p=0.03), Scr >3mg/dL (HR 3.2, 95% CI 1.6-6.3, p=0.001) and UPC >1g/g (HR 2.5, 95% CI 1.4-4.5, p=0.003) were associated with a higher risk of graft loss. CONCLUSIONS: cAMR was associated with poor graft survival after diagnosis. Improved therapies and earlier detection strategies are likely needed to improve outcomes of cAMR in kidney transplant recipients.

Acute Rejection in 6-Antigen HLA-Matched Kidney Transplant Recipients: Risk Factors and Outcomes from the Wisconsin Allograft Recipient Database (WisARD).

Acute rejection in human leukocyte antigen (HLA)-matched kidney transplant recipients is uncommon and the mechanisms involved are not well understood. Data from 6-antigen HLA-matched recipients transplanted between 1994 and 2014 were analyzed to identify the incidence, risk factors, and outcomes associated with biopsy-proven acute rejection (BPAR). A total of 278 HLA-matched recipients were identified, of which, 155 (55.8%) received a graft from a sibling donor. Ten patients (3.6%) experienced BPAR over a median follow-up of 10 years (0.41 cases per 100 person-years). Median time to rejection was 36.5 months (standard deviation ±56.4). Recipients who experienced rejection did not differ from those who did not in terms of age, gender, ethnicity, induction agent, panel reactive antibody, or sensitizing events. Acute cellular, antibody-mediated, and mixed rejection occurred in 5, 3, and 2 patients, respectively. The most common biopsy classification was Banff IA (n=4). Four out of 10 patients had documented nonadherence to maintenance immunosuppression. Thirty percent of HLA-matched recipients who rejected had graft loss and 10% died, compared to 30.8% graft loss and 28.4% deaths in non-rejectors (p=0.57 and 0.20, respectively). In conclusion, acute cellular and antibody-mediated rejection are infrequent in HLA-matched kidney transplant recipients. Nonadherence appears to be relatively common among those experiencing rejection. Acute rejection was not associated with higher graft loss or death. The pathogenesis of acute rejection in HLA-matched recipients remains to be determined.

Older kidney transplant patients experience less antibody-mediated rejection: a retrospective study of patients with mild to moderate sensitization.

BACKGROUND: There is a paucity of data stratifying by age the incidence of antibody-mediated rejection (AMR) in kidney transplant patients. METHODS: We performed a retrospective study of all adult, renal transplant recipients at a single institution between December 12, 2009, and March 16, 2011. Mildly and moderately sensitized patients were defined as patients with positive donor-specific antibody (DSA) and negative flow cross-match. RESULTS: One hundred and forty-six patients were determined to have mild to moderate pre-transplantation sensitization. Thirty percent of patients younger than 40 yr of age experienced AMR vs. 15% in the older group (p = 0.04). There was a disproportionate increase in DSA for younger patients at 12 months, particularly for antibodies to class II. Histologic presence of C4d deposition was independently associated with AMR on multivariate analysis. CONCLUSIONS: Following renal transplantation, moderately sensitized patients aged 40 yr old and older were less likely to develop AMR when compared with younger patients.

C1q Binding Activity of De Novo Donor-specific HLA Antibodies in Renal Transplant Recipients With and Without Antibody-mediated Rejection.

BACKGROUND: Complement fixation by donor-specific HLA antibodies (DSA) is a primary mechanism for antibody-mediated damage of organ allografts. Using a recently developed kit that measures C1q binding to distinguish complement fixing and nonfixing antibodies, studies showed that C1q + DSAs have a higher risk of rejection and graft loss compared to C1q-DSA. The objective of this study was to assess the ability of the C1q-binding assay to identify clinically significant de novo DSA in renal transplant recipients and to define the properties of DSA that confer C1q binding ability. METHODS: The DSA-positive sera from 34 kidney recipients, 19 with biopsy-proven antibody-mediated rejection (AMR) + and 15 who were AMR-, were assayed in C1q-binding assays (C1q Screen; One Lambda, Inc. Canoga Park, CA). The correlation between C1q-binding activity, presence of AMR, DSA mean fluorescence intensity (MFI) values, and immunoglobulin G isotype was determined. RESULTS: Fifty-three percent (10/19) of sera from AMR+ patients had C1q + DSA, whereas only 13% (2/15) of sera from AMR- patients contained C1q + DSA. C1q + DSA exhibited significantly higher MFI values regardless of whether they were from AMR+ or AMR- patients (16,118 ± 6698 vs 6429 ± 4003; P < 0.0001). C1q + DSA converted to C1q - when diluted to a comparable MFI level as the C1q - DSA from AMR- patients, and some C1q - antibodies converted to C1q + when concentrated to MFI levels comparable to those observed for AMR+/C1q + sera. CONCLUSIONS: The C1q binding activity by de novo DSA in patients with AMR largely reflects differences in antibody strength. The C1q assay does not appear to distinguish functionally distinct DSA with clinical significance.

The influence of human leukocyte antigen matching on outcomes in pediatric heart transplantation.

Previous adult heart transplantation studies have demonstrated that donor-recipient human leukocyte antigen (HLA) matching results in reduced graft failure and improved patient survival. No study has examined these effects in children. This study investigated the effect of HLA matching on outcomes in pediatric heart transplantation. All pediatric heart transplantation data for patients 0-18 years of age available from the United Network for Organ Sharing Transplant Registry from 1987 to 2009 were analyzed retrospectively. Donor-recipient HLA matching at loci A, B, and DR (0-6) was compared with graft survival and recipient survival. For this study, 3,751 pediatric cardiac transplantation events with complete HLA matching data were identified and grouped as having 0 to 2 matches (3,416 events) or 3 to 6 matches (335 events). The 3- to 6-match group had less graft failure than the 0- to 2-match group (28.7% vs 34.4%; p = 0.035) and greater patient survival by 5 years (81% vs 72%; p = 0.045) and 10 years (66% vs 55%; p = 0.005) after transplantation. The HLA-DR matching alone resulted in less graft failure (p = 0.038) and improved patient survival (p = 0.017). A higher degree of HLA matching in pediatric heart transplantation is associated with decreased graft failure and improved patient survival. In this study, decreased graft failure rates and superior survival also were seen with DR matching alone.

Antithymocyte globulin is associated with a lower incidence of de novo donor-specific antibodies in moderately sensitized renal transplant recipients.

BACKGROUND: Recent evidence suggests that de novo donor-specific antibodies (dnDSA) are associated with antibody-mediated rejection (ABMR) and graft failure after kidney transplantation. The effects of induction immunosuppression on dnDSA are unknown. METHODS: The study population comprised 114 consecutive moderately sensitized (positive DSA and negative flow crossmatch) recipients who received deceased donor renal transplants between December 2009 and November 2011. Patients were divided into two groups based on induction immunosuppression: antithymocyte globulin (ATG) (n=85) or basiliximab (n=29) and were followed up for 36 months. RESULTS: Patients in the ATG group received a mean dose of 4.98 mg/kg ± 7.9 mg/kg, had a significantly higher PRA, and received more plasmapheresis and IVIG at the time of transplant. The incidence of dnDSA (P=0.02, HR=0.33, 95% CI 0.09-1.24) and ABMR (P=0.002, HR=0.2, 95% CI 0.04-0.87) was significantly lower in the ATG group. In multivariate regression analyses, ATG induction was the single most important variable associated with both ABMR and dnDSA. CONCLUSIONS: In moderately sensitized deceased donor renal transplant recipients, induction with ATG is associated with a reduction in the occurrence of dnDSA and ABMR when compared with basiliximab.

Interpretation of HLA single antigen bead assays.

The introduction of single antigen bead (SAB) assays for detection and quantitation of HLA antibodies has improved our ability to identify and manage allosensitized transplant candidates and recipients and to improve organ allocation, and was critical to the creation of national paired kidney exchanges. The principal limitations of the technology have been detailed in the literature and include artifacts resulting in non-specific background, variability, lack of standardization, and interpretive challenges. Accurate interpretation of SAB assays requires consideration of a number of factors, including identification of epitope reactivity patterns, mean fluorescence intensity (MFI) values, patient history, and appreciation of individual bead and assay nuances. The MFI value provides an estimate of relative HLA antibody levels although limited by saturation and epitope distribution effects. A better understanding of SAB assays and MFI values will be necessary to ensure appropriate application of these assays clinically and a higher quality of antibody data used in support of published clinical studies.

Pretransplant donor-specific anti-HLA antibodies as predictors of early allograft rejection in ABO-compatible liver transplantation.

The significance of preexisting donor-specific HLA antibodies (HLA-DSAs) for liver allograft function is unclear. Our previous studies have shown that humoral alloreactivity frequently accompanies acute cellular rejection (ACR). In the present study, we set out to determine whether pretransplant HLA-DSAs correlate with clinically significant ACR in the first 90 days after transplantation and, if so, to determine their predictive values. Class I HLA-DSAs and class II HLA-DSAs were determined by single-antigen bead flow cytometry for 113 consecutive adult transplants. A statistical analysis was performed for data from 109 consecutive patients with graft survival greater than or equal to 90 days. All patients who developed biochemical graft dysfunction underwent liver biopsy for hematoxylin-eosin and complement component 4d staining. Cox proportional hazards models and associated hazard ratios revealed a significant association of pretransplant HLA-DSAs with clinically significant ACR: this association started with a mean fluorescence intensity (MFI) as low as 300 for both class I (hazard ratio = 2.7, P < 0.01) and class II (hazard ratio = 6.0, P < 0.01). Pretransplant HLA-DSAs were associated with an increased risk of ACR: P < 0.01 for class I (42% versus 18%), P < 0.001 for class II (37% versus 7%), and P < 0.001 for either class I or II (36% versus 3%). Class I or II HLA-DSAs with an MFI ≥ 1000 had the best positive predictive value for clinically significant ACR at 46%, whereas class I or II HLA-DSAs with an MFI ≥ 300 had the best negative predictive value at 97.1%. Although our study was based on consecutive patients, it was limited by the relatively low number of single-center subjects. In conclusion, the present study indicates that pretransplant HLA-DSAs, even at low levels of allosensitization, correlate with the risk of clinically significant ACR. Our findings suggest that anti-human leukocyte antigen antibodies could serve as donor-specific markers of immunoreactivity to the liver graft.

Increased C4d in post-reperfusion biopsies and increased donor specific antibodies at one-week post transplant are risk factors for acute rejection in mild to moderately sensitized kidney transplant recipients.

In order to define the intensity of immunosuppression, we examined risk factors for acute rejection in desensitization protocols that use baseline donor-specific antibody levels measured as mean fluorescence intensity (MFImax). The study included 146 patients transplanted with a negative flow crossmatch and a mean follow-up of 18 months with the majority (83%) followed for at least 1 year. At the time of transplant, mean-calculated panel-reactive antibody and MFImax ranged from 10.3-57.2% and 262-1691, respectively, between low- and high-risk protocols. Mean MFImax increased significantly from transplant to 1 week and 1 year. The incidence of acute rejection (mean 1.65 months) as a combination of clinical and subclinical rejection was 32%, including 14% cellular, 12% antibody-mediated, and 6% mixed rejection. In regression analyses, only C4d staining in post-reperfusion biopsies (hazard ratio 3.3, confidence interval 1.71-6.45) and increased specific antibodies at 1-week post transplant were significant predictors of rejection. A rise in MFImax by 500 was associated with a 2.8-fold risk of rejection. Thus, C4d staining in post-reperfusion biopsies and an early rise in donor specific antibodies after transplantation are risk factors for rejection in moderately sensitized patients.

A simplified method for screening siblings for HLA identity using short tandem repeat (STR) polymorphisms.

Identifying an HLA-matched sibling donor for hematopoietic stem cell transplantation (HSCT) is time-consuming and expensive, and often limited by reimbursement caps imposed by insurance providers. To improve the effectiveness and efficiency of screening for HLA-matched siblings, we developed an assay for determining HLA identity using a panel of nine informative short tandem repeat (STR) loci located throughout the HLA complex. The STR panel was assessed for accuracy in identifying HLA-matched siblings in 88 family workups comprising a total of 132 related donor and recipient typing comparisons. All sibling pairs with identical STR alleles were also HLA identical. Of the 48 pairs mismatched at one or more STR alleles, all were genotypically HLA non-identical at one or more loci. The sensitivity and specificity of STR analysis for identifying HLA-matched siblings were 91% and 100%, respectively. Three false negatives occurred due to an STR mutation or possible HLA-DPB1/DQB1 recombination. Additionally, STR genotyping provided additional information allowing determination of the extent of HLA identity in families where HLA haplotype inheritance was ambiguous, due to extensive homozygosity or shared parental haplotypes. The HLA STR assay is a reliable and rapid test that can be used to inexpensively screen potential sibling donors for HLA identity.

Diagnostic accuracy of solid phase HLA antibody assays for prediction of crossmatch strength.

Solid phase antibody assays are increasingly used to provide quantitative measures of donor-specific HLA antibodies for assessment of pretransplant risk, although cell-based crossmatches continue to serve as gold standards for determination of donor HLA antibody strength. This study determined the ability of HLA antibody solid phase assays to predict the strength of cell-based flow cytometric (FC) and complement-dependent cytotoxicity (CDC) crossmatches. Eighty-two recipient/donors pairs were analyzed using receiver operating characteristic (ROC) curve analyses to determine the accuracy of donor-specific median fluorescence intensity values (Σ MFI) from single antigen bead assays for predicting strong FC and CDC crossmatches. Diagnostic sensitivity and specificity of optimal Σ MFI values were highest for predicting strong T cell FCs. Σ MFI values showed good sensitivity for predicting positive direct and AHG-augmented CDC crossmatches (91% and 94%, respectively), but with lower specificity (67% each). Specificity and sensitivity for predicting positive B cell CDC crossmatches were 73% and 84%. Σ MFI values derived from single antigen bead assays can predict strong flow and positive CDC crossmatches, but with tradeoffs between sensitivity and specificity. The results support the use of solid phase assays for quantitative virtual crossmatching and as a replacement for cell-based crossmatching.

Effects of mismatching for minor histocompatibility antigens on clinical outcomes in HLA-matched, unrelated hematopoietic stem cell transplants.

Several studies in HLA-matched sibling hematopoietic stem cell transplantation (HSCT) have reported an association between mismatches in minor histocompatibility antigens (mHAg) and outcomes. We assessed whether single and multiple minor mHAg mismatches are associated with outcomes in 730 unrelated donor, HLA-A, B, C, DRB1, and DQB1 allele-matched hematopoietic stem cell transplants (HSCT) facilitated by the National Marrow Donor Program (NMDP) between 1996 and 2003. Patients had acute and chronic leukemia or myelodysplastic syndrome (MDS), received myeloablative conditioning regimens and calcineurin inhibitor-based graft-versus-host-disease (GVHD) prophylaxis, and most received bone marrow (BM; 85%). Donor and recipient DNA samples were genotyped for mHAg including: HA-1, HA-2, HA-3, HA-8, HB-1 and CD31(125/563). Primary outcomes included grades III-IV acute GVHD (aGVHD) and survival; secondary outcomes included chronic GVHD (cGVHD), engraftment, and relapse. Single disparities at HA-1, HA-2, HA-3, HA-8, and HB-1 were not significantly associated with any of the outcomes analyzed. In HLA-A2-positive individuals, single CD31(563) or multiple mHAg mismatches in the HVG vector were associated with lower risk of grades III-IV aGVHD. Based on these data, we conclude that mHAg incompatibility at HA-1, HA-2, HA-3, HA-8, HB-1, and CD31 has no detectable effect on the outcome of HLA matched unrelated donor HSCT.

The alleles of PECAM-1.

Previous studies have reported the existence of eleven different single nucleotide polymorphisms (SNPs) within human PECAM-1 mRNA, several of which have recently been associated with disease. Though SNPs in the PECAM-1 gene have been known for some time, the genetic background on which they exist, and their association into distinct allelic isoforms has not yet been established. To identify the major allelic isoforms of PECAM-1, we determined the nucleotide sequence of individual full-length cloned cDNAs derived from anonymous, unrelated volunteer individuals. Initial sequence analysis of 34 alleles from 17 individuals confirmed the presence of two distinct human PECAM-1 alleles (L(98)S(536)R(643) and V(98)N(536)G(643)) within the human population. Each of these were found, upon more detailed analysis, to be superimposed on a previously unreported a2479g nucleotide polymorphism within the 3' untranslated region (3'UTR) that occurred on both allelic isoforms - yielding a total of four major alleles. Multiplex Luminex bead analysis of an additional 259 individuals allowed identification of 117 individuals homozygous for either the L(98)S(536) or V(98)N(536) allele, and sequence analysis around the R643G and a2479g polymorphic sites permitted accurate determination of significant differences in the gene frequencies of LSRa, LSRg, VNGa, and VNGg among Caucasian individuals. Identification of these PECAM-1 allelic isoforms should facilitate future detailed examination of PECAM-1-related disease associations, and may help resolve previously disparate results.

Multiplex genotyping of human minor histocompatibility antigens.

Minor histocompatibility antigens (mHAg) induce major histocompatibility complex-restricted, T cell-mediated immune responses that may contribute to increased risk of graft-versus-host disease and graft-versus-leukemia effects. Unlike human leukocyte antigen genes, mHAg are encoded by genetically and functionally unrelated genes located throughout the chromosome. The role of mHAg in stem cell transplantation and the population frequencies of mHAg alleles remain unknown due in part to the lack of suitable high throughput methods for genotyping these diverse genes. Here we describe the development and utility of a multiplexed Luminex assay for genotyping human mHAg, including HA-1, HA-2, HA-3, HA-8, HB-1, CD31(125), and CD31(563). The assay uses a multiplexed, allele-independent, gated amplification of mHAg genes followed by differential detection of allele-specific primer extension products using the MultiCode PLx system (EraGen Biosciences, Madison, WI). The alleles are interrogated using a multiplex allele-specific primer extension reaction using primers tagged with EraCodes. The products are hybridized to Luminex beads and the hybridization duplexes are detected using streptavidin-phycoerythrin. The assay resolved the mHAg genotypes of 259 Caucasian donors and provided population estimates of mHAg gene and phenotypic frequencies. All mHAg alleles evaluated in this study exhibited Hardy-Weinberg equilibrium, although some mHAg phenotypes were present in large majority of individuals tested (HA-2, HB-1). This assay will provide a valuable tool for determining mHAg frequencies in other ethnic populations, as well as for establishing the clinical importance of mHAg disparities in stem cell transplantation.

Flow cytometric assessment of HLA alloantibodies.

Antibodies against HLA molecules are formed in response to exposure to foreign HLA molecules, which can occur as a result of blood transfusion, pregnancy, or transplant. Blood components, particularly those containing cellular elements, are the most common cause of HLA antibodies. This unit describes technical aspects of the flow cytometric crossmatch (FCXM), flow cytometric microparticle assays, and cell-based flow cytometric screening assays. The collective goal for these assays is to clearly identify the presence of HLA antibody, determine the titer of antibody, and elucidate the specificities (i.e., HLA antigens) to which they will react. Knowledge of this information is critical for organ allocation and accurate assessment of the immunological risk for a patient at the time of transplantation. In addition, the identification of HLA antibodies in blood components may be useful in planning appropriate transfusion support strategies for selected patients.