Delivering the precision oncology paradigm: reduced R&D costs and greater return on investment through a companion diagnostic informed precision oncology medicines approach.

BACKGROUND: Precision oncology medicines represent a paradigm shift compared to non-precision oncology medicines in cancer therapy, in some situations delivering more clinical benefit, and potentially lowering healthcare costs. We determined whether employing a companion diagnostic (CDx) approach during oncology medicines development delivers effective therapies that are within the cost constraints of current health systems. R&D costs of developing a medicine are subject to debate, with average estimates ranging from $765 million (m) to $4.6 billion (b). Our aim was to determine whether precision oncology medicines are cheaper to bring from R&D to market; a secondary goal was to determine whether precision oncology medicines have a greater return on investment (ROI). METHOD: Data on oncology medicines approved between 1997 and 2020 by the US Food and Drug Administration (FDA) were analysed from the Securities and Exchange Commission (SEC) filings. Data were compiled from 10-K, 10-Q, and 20-F financial performance filings on medicines' development costs through their R&D lifetime. Clinical trial data were split into clinical trial phases 1-3 and probability of success (POS) of trials was calculated, along with preclinical costs. Cost-of-capital (CoC) approach was applied and, if appropriate, a tax rebate was subtracted from the total. RESULTS: Data on 42 precision and 29 non-precision oncology medicines from 56 companies listed by the National Cancer Institute which had complete data available were analysed. Estimated mean cost to deliver a new oncology medicine was $4.4b (95% CI, $3.6-5.2b). Costs to bring a precision oncology medicine to market were $1.1b less ($3.5b; 95% CI, $2.7-4.5b) compared to non-precision oncology medicines ($4.6b; 95% CI, $3.5-6.1b). The key driver of costs was POS of clinical trials, accounting for a difference of $591.3 m. Additional data analysis illustrated that there was a 27% increase in return on investment (ROI) of precision oncology medicines over non-precision oncology medicines. CONCLUSION: Our results provide an accurate estimate of the R&D spend required to bring an oncology medicine to market. Deployment of a CDx at the earliest stage substantially lowers the cost associated with oncology medicines development, potentially making them available to more patients, while staying within the cost constraints of cancer health systems.

Interferon-Inducible MicroRNA miR-128 Modulates HIV-1 Replication by Targeting TNPO3 mRNA.

The HIV/AIDS pandemic remains an important threat to human health. We have recently demonstrated that a novel microRNA (miR), miR-128, represses retrotransposon long interspaced element 1 (L1) by a dual mechanism, namely, by directly targeting the coding region of the L1 RNA and by repressing a required nuclear import factor (TNPO1). We have further determined that miR-128 represses the expression of all three TNPO proteins (transportins TNPO1, TNPO2, and TNPO3). Here, we establish that miR-128 also influences HIV-1 replication by repressing TNPO3, a factor that regulates HIV-1 nuclear import and viral; replication of TNPO3 is well established to regulate HIV-1 nuclear import and viral replication. Here, we report that type I interferon (IFN)-inducible miR-128 directly targets two sites in the TNPO3 mRNA, significantly downregulating TNPO3 mRNA and protein expression levels. Challenging miR-modulated Jurkat cells or primary CD4+ T-cells with wild-type (WT), replication-competent HIV-1 demonstrated that miR-128 reduces viral replication and delays spreading of infection. Manipulation of miR-128 levels in HIV-1 target cell lines and in primary CD4+ T-cells by overexpression or knockdown showed that reduction of TNPO3 levels by miR-128 significantly affects HIV-1 replication but not murine leukemia virus (MLV) infection and that miR-128 modulation of HIV-1 replication is reduced with TNPO3-independent HIV-1 virus, suggesting that miR-128-indued TNPO3 repression contributes to the inhibition of HIV-1 replication. Finally, we determine that anti-miR-128 partly neutralizes the IFN-mediated block of HIV-1. Thus, we have established a novel role of miR-128 in antiviral defense in human cells, namely inhibiting HIV-1 replication by altering the cellular milieu through targeting factors that include TNPO3.IMPORTANCE HIV-1 is the causative agent of AIDS. During HIV-1 infection, type I interferons (IFNs) are induced, and their effectors limit HIV-1 replication at multiple steps in its life cycle. However, the cellular targets of INFs are still largely unknown. In this study, we identified the interferon-inducible microRNA (miR) miR-128, a novel antiviral mediator that suppresses the expression of the host gene TNPO3, which is known to modulate HIV-1 replication. Notably, we observe that anti-miR-128 partly neutralizes the IFN-mediated block of HIV-1. Elucidation of the mechanisms through which miR-128 impairs HIV-1 replication may provide novel candidates for the development of therapeutic interventions.

miR-128 Restriction of LINE-1 (L1) Retrotransposition Is Dependent on Targeting hnRNPA1 mRNA.

The majority of the human genome is made of transposable elements, giving rise to interspaced repeats, including Long INterspersed Element-1s (LINE-1s or L1s). L1s are active human transposable elements involved in genomic diversity and evolution; however, they can also contribute to genomic instability and diseases. L1s require host factors to complete their life cycles, whereas the host has evolved numerous mechanisms to restrict L1-induced mutagenesis. Restriction mechanisms in somatic cells include methylation of the L1 promoter, anti-viral factors and RNA-mediated processes such as small RNAs. microRNAs (miRNAs or miRs) are small non-coding RNAs that post-transcriptionally repress multiple target genes often found in the same cellular pathways. We have recently established that miR-128 functions as a novel restriction factor inhibiting L1 mobilization in somatic cells. We have further demonstrated that miR-128 functions through a dual mechanism; by directly targeting L1 RNA for degradation and indirectly by inhibiting a cellular co-factor which L1 is dependent on to transpose to new genomic locations (TNPO1). Here, we add another piece to the puzzle of the enigmatic L1 lifecycle. We show that miR-128 also inhibits another key cellular factor, hnRNPA1 (heterogeneous nuclear ribonucleoprotein A1), by significantly reducing mRNA and protein levels through direct interaction with the coding sequence (CDS) of hnRNPA1 mRNA. In addition, we demonstrate that repression of hnRNPA1 using hnRNPA1-shRNA significantly decreases de novo L1 retro-transposition and that induced hnRNPA1 expression enhances L1 mobilization. Furthermore, we establish that hnRNPA1 is a functional target of miR-128. Finally, we determine that induced hnRNPA1 expression in miR-128-overexpressing cells can partly rescue the miR-128-induced repression of L1's ability to transpose to different genomic locations. Thus, we have identified an additional mechanism by which miR-128 represses L1 retro-transposition and mediates genomic stability.

miR-128 inhibits telomerase activity by targeting TERT mRNA.

Telomerase is a unique cellular reverse transcriptase (RT) essential for maintaining telomere stability and required for the unlimited proliferation of cancer cells. The limiting determinant of telomerase activity is the catalytic component TERT, and TERT expression is closely correlated with telomerase activity and cancer initiation and disease progression. For this reason the regulation of TERT levels in the cell is of great importance. microRNAs (miRs) function as an additional regulatory level in cells, crucial for defining expression boundaries, proper cell fate decisions, cell cycle control, genome integrity, cell death and metastasis. We performed an anti-miR library screen to identity novel miRs, which participate in the control of telomerase. We identified the tumor suppressor miR (miR-128) as a novel endogenous telomerase inhibitor and determined that miR-128 significantly reduces the mRNA and protein levels of Tert in a panel of cancer cell lines. We further evaluated the mechanism by which miR-128 regulates TERT and demonstrated that miR-128 interacts directly with the coding sequence of TERT mRNA in both HeLa cells and teratoma cells. Interestingly, the functional miR-128 binding site in TERT mRNA, is conserved between TERT and the other cellular reverse transcriptase encoded by Long Interspersed Elements-1 (LINE-1 or L1), which can also contribute to the oncogenic phenotype of cancer. This finding supports the novel idea that miRs may function in parallel pathways to inhibit tumorigenesis, by regulating a group of enzymes (such as RT) by targeting conserved binding sites in the coding region of both enzymes.

MicroRNA miR-128 represses LINE-1 (L1) retrotransposition by down-regulating the nuclear import factor TNPO1.

Repetitive elements, including LINE-1 (L1), comprise approximately half of the human genome. These elements can potentially destabilize the genome by initiating their own replication and reintegration into new sites (retrotransposition). In somatic cells, transcription of L1 elements is repressed by distinct molecular mechanisms, including DNA methylation and histone modifications, to repress transcription. Under conditions of hypomethylation (e.g. in tumor cells), a window of opportunity for L1 derepression arises, and additional restriction mechanisms become crucial. We recently demonstrated that the microRNA miR-128 represses L1 activity by directly binding to L1 ORF2 RNA. In this study, we tested whether miR-128 can also control L1 activity by repressing cellular proteins important for L1 retrotransposition. We found that miR-128 targets the 3' UTR of nuclear import factor transportin 1 (TNPO1) mRNA. Manipulation of miR-128 and TNPO1 levels demonstrated that induction or depletion of TNPO1 affects L1 retrotransposition and nuclear import of an L1-ribonucleoprotein complex (using L1-encoded ORF1p as a proxy for L1-ribonucleoprotein complexes). Moreover, TNPO1 overexpression partially reversed the repressive effect of miR-128 on L1 retrotransposition. Our study represents the first description of a protein factor involved in nuclear import of the L1 element and demonstrates that miR-128 controls L1 activity in somatic cells through two independent mechanisms: direct binding to L1 RNA and regulation of a cellular factor necessary for L1 nuclear import and retrotransposition.

Interferons Induce Expression of SAMHD1 in Monocytes through Down-regulation of miR-181a and miR-30a.

SAMHD1 is a phosphohydrolase maintaining cellular dNTP homeostasis but also acts as a critical regulator in innate immune responses due to its antiviral activity and association with autoimmune disease, leading to aberrant activation of interferon. SAMHD1 expression is differentially regulated by interferon in certain primary cells, but the underlying mechanism is not understood. Here, we report a detailed characterization of the promotor region, the 5'- and 3'-untranslated region (UTR) of SAMHD1, and the mechanism responsible for the cell type-dependent up-regulation of SAMHD1 protein by interferon. We demonstrate that induction of SAMHD1 by type I and II interferons depends on 3'-UTR post-transcriptional regulation, whereas the promoter drives basal expression levels. We reveal novel functional target sites for the microRNAs miR-181a, miR-30a, and miR-155 in the SAMHD1 3'-UTR. Furthermore, we demonstrate that down-regulation of endogenous miR-181a and miR-30a levels inversely correlates with SAMHD1 protein up-regulation upon type I and II interferon stimulation in primary human monocytes. These miRNAs are not modulated by interferon in macrophages or dendritic cells, and consequently protein levels of SAMHD1 remain unchanged. These results suggest that SAMHD1 is a non-classical interferon-stimulated gene regulated through cell type-dependent down-regulation of miR-181a and miR-30a in innate sentinel cells.

Donor-Specific HLA Antibodies: A Review of Data Published in 2016.

The results from continued research regarding the role of anti-human leukocyte antigen (anti-HLA) antibodies and donor-specific antibodies (DSA) in transplantation has strengthened the association between DSA and allograft rejection. The formation of de novo DSAs is particularly detrimental to allograft function and survival. Paradoxically, grafts of patients without DSA may fail and patients with DSA may continue to have extended post-transplant graft function. An explanation for this inconsistency in outcomes must be found to utilize anti-HLA DSA as a biomarker of allograft rejection. This review highlights multiple studies regarding DSA and secondary characteristics of DSA, including C1q-binding, mean fluorescence, and immunoglobulin G subtype, which have been useful in stratifying DSA-positive patients into low- and high-risk groups of allograft rejection.

miR-128 represses L1 retrotransposition by binding directly to L1 RNA.

Long interspersed element 1 (LINE-1 or L1) retrotransposons compose 17% of the human genome. Active L1 elements are capable of replicative transposition (mobilization) and can act as drivers of genetic diversity. However, this mobilization is mutagenic and may be detrimental to the host, and therefore it is under strict control. Somatic cells usually silence L1 activity by DNA methylation of the L1 promoter. In hypomethylated cells, such as cancer cells and induced pluripotent stem cells (iPSCs), a window of opportunity for L1 reactivation emerges, and with it comes an increased risk of genomic instability and tumorigenesis. Here we show that miR-128 represses new retrotransposition events in human cancer cells and iPSCs by binding directly to L1 RNA. Thus, we have identified and characterized a new function of microRNAs: mediating genomic stability by suppressing the mobility of endogenous retrotransposons.

Effect of amino acid mismatch in the UNOS dataset.

This study began with the 2010 UNOS data-set of 181,653 deceased donor kidney transplant cases and 92,577 living donor cases. Cases with ambiguous or missing HLA typing were excluded, and the remaining cases were split into subgroups by the number of previous transplants and ethnic groups of donor-patient pairs. 41,128 Caucasian donor-patient pairs that were primary living-donor transplant cases were used as the pilot population to identify potential epitope groups that have a negative effect on graft outcome. Sixty four of the most common HLA-A and -B antigens were selected. Amino acid sequences of the most frequently corresponding allele in the Caucasian population were used to build the starting theoretical epitope table. Amino acids of the 115 polymorphic positions, analyzed in one, two or three positions, resulted in 15,801,920 combinations. After eliminating combinations shared by no allele or by all 64 alleles, the table was trimmed to 1,635,044. Grouping combinations according to their antigen list (antigens that share the combinations/epitopes), 40,830 epitope groups were left. Based on the distances between amino acid positions of each epitope, and the requirement that each epitope must be shared by at least one allele, but not all 64, the number of theoretical epitopes was reduced to 39,670 and 3,703 epitope groups of unique antigen lists. The pilot population was composed of 41,128 primary living-donor transplants with Caucasian donor-patient pairs. For each of the 40,830 epitope groups in the non-distance-restricted table, 15-year death-censored graft survival was computed for epitope-group mismatches--i.e., cases with a BMQ0001 mismatch, with a BMQ0002 mismatch, etc.. Results were compared, using the log rank test, with average graft survival. Of the 3,703 epitope groups, 2,487 appeared in over 1000 cases, but only 88 of them had significant p-values, which ranged from 0.006 to 0.049, with 76 of the 88 significantly below average, 12 above average (Fig. 1). We then ran survival analyses taking the 76 epitope groups that were below average two at a time--i.e., cases with mismatches of the first and second epitope group, the first and third, first and fourth, etc. Of more than 2,500 pairs, 148 resulted in significantly (p < 0.01) lower than average survival in those primary living-donor cases. The effect of the 76 epitope-group mismatches that showed below-average results was then analyzed for other transplant populations--Caucasian donor-patient pair cases with deceased donors , Caucasian donor-patient pair cases with re-transplant living donors, Caucasian donor-patient pair cases with re-transplant deceased donors, and African-American donor-patient pair cases with primary living donors. None of these four populations exhibited any significant effect due to the 76 epitope- group mismatches. Likewise, the effect of the 148 epitope-group combination mismatches detailed in paragraph 5, above, was analyzed for the other four other transplant populations, detailed in paragraph 6. That significant effect was absent in all four. The analyses were repeated on the 40,830 epitope groups without the 27 angstrom distance constraint. Of the 40,830 epitope groups, 439 exhibited a significantly lower 15-year graft survival, with p-value ranging from 0.0053 to 0.0498. Again, none of these had any negatively significant effects on graft survival for the other four transplant populations. In the pilot population, the negative effect of the epitope group mismatches was clearly seen, but the significant differences did not carry across to the other four populations. That absence may be partially explained by the allele level differences in the HLA-A and -B typing of the donors and patients. Past studies indicate that the appearance of DSA has a negative effect on the graft outcome, so the mismatch of epitopes recognized by these DSA could also have similar negative effects. With the data available at present, and with the currently available assays for antibody detection, we are not able to analyze the impact of specific epitope mismatches. We will need the development of new methods of antibody detection that specifically indicate the exact epitope to which an antibody binds before we can continue this effort to determine the negative impact on graft survival due to epitope mismatches.

The HLA-matching effect in different cohorts of kidney transplant recipients: 10 years later.

Almost all the HLA-matching effects found by the 2000 analysis were confirmed by this study. The only HLA-matching effect found in the 2000 analysis that disappeared were those of "small matching effect" found in sub-populations of type I diabetes (PRA < 10%, donor age 20-35). The 2000 analysis found a lack of HLA matching effect in non-African American kidney transplant patients with type I diabetes between 1987 and 2000. The 2000 analysis found that a patients' ethnic group was a factor in graft survival; African American patients were found to have a significantly lower 10-year graft survival in the 5 or 6 mismatched group (27%) compared to Caucasian patients (40%). In addition, Asian patients (42%) had higher graft survival compared to that of Caucasian patients. In this study, we observe a similar pattern with death-censored graft analysis for all ethnic groups with 10-year graft survivals at 72.9% for Asians, 69.5% for Caucasians, and 49.3% for African Americans. There was an overall lack of HLA-matching effect on patient survival in the 2000 analysis. In our current analysis, the patient survivals remained virtually the same despite moderate increase in graft survival over the same period of time. The HLA-C locus mismatch was found to have additive effect to the 10-year graft survival trends observed in A and B mismatch cases. HLA-DQ mismatch on the other hand, showed limited HLA-matching effect and did not show the same additive effect as C. There are various possible issues in the DQ mismatch analysis, from the consistency of DQ typing results, lack of diversity in the DQ antigen, to the possibility of DQ mismatch having little effect on the graft survival. Utilizing kidney transplant cases performed from 1995 through 2000, the 2000 analysis projected 10-year survivals of 64% and 47% for the 0 ABDR mismatch and 5 or 6 ABDR mismatched cases respectively; the 2000 projection only missed actual death-censored survivals by 9% lower for the 0 mismatch and 17% lower for the 5 or 6 mismatch cases. Utilizing the transplant cases of 2005 through 2009, we projected their 10-year graft survivals for year 2020. The 10-year graft survival for 0 ABDR mismatched patients is expected to be over 85% and nearly 70% for 5 or 6 ABDR mismatched patients. The general upward trend of graft survival we have observed in the last 10 years has been dependent upon the development of novel transplant protocols and use of novel immunomodulatory reagents. This trend is likely to continue given the promise of new drugs and personalized healthcare. The decreasing range of the differences in the 10-year graft survival between best matched and worst matched HLA groups is also likely to continue. One interesting trend that is clearly evident is the increasing difference between the best and worst HLA-matching in terms of the associated graft half-life. The positive HLA-matching effect on long-term graft survival is clearly evident and should be taken into consideration for all kidney transplants.

Abrogation of anti-HLA antibodies via proteasome inhibition.

BACKGROUND: Current treatments for autoantibody-mediated diseases (i.e., systemic lupus erythematosus) and alloantibodies (in transplant) are minimally effective. Although they deplete naïve B cells, plasmablasts, and transiently reduce antibody concentrations, they are minimally effective against long-lived, antibody-producing plasma cells. In transplantation, plasma cells produce antibodies directed against human leukocyte antigen (HLA) antigens causing poor allograft survival. We report the first clinical experience with a plasma cell depleting therapy, bortezomib, to abrogate anti-HLA antibodies in transplantation (outside of rejection) in an attempt to improve long-term allograft survival. METHODS: Eleven patients with anti-HLA alloantibodies were treated with bortezomib. All patients underwent plasmapheresis to aid in removal of antibodies and to determine the effect of bortezomib. Serial measurements of anti-HLA antibody levels were conducted weekly by single antigen bead on Luminex platform. RESULTS: Bortezomib treatment elicited substantial reduction in both donor-specific antibody (DSA) and non-DSA levels. Antibodies were directed against DSA in 8 of 11 cases. Mean time to antibody appearance was 2 months posttransplant. Within 22 days (median) from treatment initiation, 9 of 11 patients' antibody levels dropped to less than 1000 mean fluorescence intensity. Of two patients without successful depletion, all had peak mean fluorescence intensity more than 10,000. At a mean follow-up of approximately 4 months posttreatment, all patients have stable graft function. Minimal transient side effects were noticed with bortezomib in the form of gastrointestinal toxicity, thrombocytopenia, and paresthesias. CONCLUSIONS: Bortezomib therapy effectively abrogates anti-HLA antibodies. Hence, removal of antibodies, by proteasome inhibition, represents a new treatment strategy for transplantation and may have benefit in autoimmune-related disease.

Mimetic human leukocyte antigen epitopes: shown by monoclonal antibodies and extra antibodies produced on transplantation.

BACKGROUND: Transplant patients often produce human leukocyte antigen (HLA) antibodies against their donors and produce more specificities than can be accounted for by HLA antigen mismatches. We theorize that the presence of extra, otherwise unexplainable specificities could be accounted for if antibodies reacted to more than one epitope (primary and mimetic) on distinct HLA molecules. The theory states that mimetic epitopes consist of the same three amino acids that comprise the primary, sterically placed approximately the same distance apart as are the corresponding amino acids of the primary. METHODS: A mimetic epitope table containing all primary epitopes and corresponding mimetic epitopes was built. Then, the HLA specificities of monoclonal and single patient antibodies were determined. These specificities that could not be defined by unique position or amino acid epitopes alone were then used to query the mimetic epitope table. RESULTS: A single antibody from a transplant patient and three monoclonal antibodies produced reactions that can best be explained as the result of one antibody reacting to the same amino acids at two distinct sites on the molecule. Those position and amino acid combinations (pos/aa) are the primary and mimetic epitopes. Using computerized methods of searching, mimetic epitopes were found in five additional kidney transplant patients who produced nondonor-specific antibodies in addition to donor-specific antibodies. CONCLUSIONS: Epitopes on the HLA molecule that mimic the primary epitope have been found. We suggest that these mimetic epitopes explain the additional antibodies often found on HLA immunization resulting from allograft rejections, pregnancies, and transfusions.

Using the mimetic epitope concept to find a possible way to widen the graft-survival difference between matched and mismatched renal transplants.

1. Immunological responses cause antibodies to be produced, and transplant patients often produce HLA antibodies against their donors' HLA. But in many cases, more antibodies are produced than just those against mismatched HLA. Many patients with zero mismatched transplants also produce antibodies, and some of these grafts fail. This study used graft survival analysis that contemplated the primary epitopes against the donors' alleles and the corresponding mimetic epitopes, less those shared with the patients' alleles. 2. A table of primary epitopes and corresponding mimetic epitopes was built in a previous study. For each case studied, here, a subset of the table was selected for all of the donor's HLA alleles. The subset was reduced by eliminating all primary and mimetic epitopes shared by the patient's own HLA alleles. The remaining mimetic and primary epitopes-the maximum pool of epitopes (MaxPEpi)-produced epitope frequency (Efr) for each allele. Using a zero-HLA-AB-mismatch subset (n = 16) of the Greenville and Holland kidney transplant data set (n = 451), the Efr for each allele in the MaxPEpi of each case was tallied and compared with the corresponding observed antibody specificities. The average frequencies were 2,869 and 1,975, respectively, and the t-test was significant at the p-value of less than 0.00001, indicating that the higher the frequency, the more likely that the corresponding allele appears as a part of the specificity. 3. The trends were used to perform survival analysis on zero-mismatch kidney transplant cases in the 2007 UNOS data file. Different variables of MaxPEpi parameters and cutoff points were tried with half of the deceased-donor cases in the file, survival rates for those below and above each cutoff point were compared by the log-rank test. The average Efr with a cutoff of 3,700 yielded the best 10-year graft survival in this subgroup of the 2007 UNOS cases. The results were supported and verified by analyzing the second half of the deceased-donor cases and the living-donor cases in the 2007 UNOS data set. 4. With the 2007 file living-donor cases, 10-year graft survival rate was 86.2% for cases below the cutoff, and 85.4% for those above, and the difference was significant at a p-value less than 0.01. A larger difference was observed for 2007 living-donor cases, excluding identical sibling transplants, but the difference was not statistically significant due to the low number of cases. 5. The same trends were found for 10-year survivals with cases in the 2008 data, but were not statistically significant. With living donors, the rates below and above the cutoff were 85.7% and 85.2%, respectively, and with deceased donors, 72.7% and 72.1%, respectively, p < 0.125. As with the 2007 data, cases in the 2008 file with living donors--excluding identical sibling transplants--had survival rates of 91.7% and 79.0%, respectively, but the 12.7% difference was not statistically significant because of the low number of cases.

Effect of amino acid mismatch in the UNOS 2007 dataset.

1. The study began with the 2007 UNOS dataset of 270,690 kidney transplant, from which were selected post-1995 first transplants with Caucasian donor/patients and available ABDR typing data, yielding 87,616 cases. These were split into cadaver donor (n=46,927) and living donor (n=40,689) populations. 2. Cases with broad antigens such as A9, A10, B12 and B17 were eliminated, as were cases that had failed within 30 days. That left 28,264 cadaver donor and 26,211 living donor cases. 3. We looked at every theoretical mismatch between donors and patients in the living donor population. Overall, 405 single, 21,269 double, and 391,325 triple position/amino acid mismatches were included in the analysis. Two tallies for each mismatch were generated: "function" and "fail" based on patient's associated graft survival. 4. We generated a list of "fail" single, double, and triple position/amino acid mismatches and computed 10-year survival curves for each of the mismatches, comparing them with the average 10-year survival curve using the log-rank test. Based on the log-rank statistics, a ranking of the bad mismatches was established. 5. We looked at the long-term graft survival of additive single, double, and triple "fail" mismatches in cadaver donor transplants. 6. Survival curves of transplants with position/ amino acid mismatches were generated and compared with the survival curves of the traditional standards: 0 ABDR mismatch; 1 A, 1 B and 1 DR mismatch; and full 6 ABDR mismatches. 7. The greatest effect was seen in first transplants with a male recipient, but that were not Caucasian-to-Caucasian. Up to 125 double mismatches resulted in a 10-year survival 29% lower than 0 ABDR mismatches. 8. In first transplants that were not Caucasian-to-Caucasian 1154 out of 1177 (98%) pos/aa mismatches (single, double, or triple) had lower 10-year survival than cases with one mismatch, each, in A, B and DR. Looking at re-grafts, we see that 498 out of 499 with single, double and triple mismatches had lower 10-year survival than that of cases with one mismatch, each, in A, B and DR. 9. Overall, position/amino acid mismatches had consistently lower 10-year survival than 1 A, 1 B, 1 DR mismatches. We believe our selection of "fail" pos/aa mismatches provide a good starting point for establishing a list of mismatches to be looked for and avoided in future transplants in order to give them a better chance of survival.

HLA matching by amino acid/positions in double and triple combinations.

1. Began with the 2006 UNOS dataset and then selected for Caucasian donor/patients (post-1995 transplant and first transplant kidney only with full ABDR typing data). This was then split into cadaver donor (n = 20,542) and living donor (n = 21,890) populations. 2. Looked at every theoretical mismatch between donor and patients in the living donor population. Overall, 167 single, 4601 double, and 83,655 triple position/amino acid mismatches were included in the analysis. Two tallies for each mismatch were generated: "good" and "bad" based on patient's associated graft survival. 3. Generated a list of "bad" single, double, and triple position/ amino acid mismatches. 4. Looked at associated long-term graft survivals of patient/donor pairs with additive single, double, and triple "bad" mismatches in the cadaver donor population. 5. Survival curves of transplant pairs with associated mismatches were generated and compared with traditional standards of 0ABDR mismatch, 1 or more ABDR mismatch, and full 6ABDR mismatch survival curves. 6. The greatest effect was seen in re-grafted male patients where up to 50 double mismatches were associated with a 10-year survival 27% lower than 0ABDR mismatch. 7. Many of the "bad" position/amino acid mismatches identified were the same as those identified by generation of antibodies against Class I and Class II epitopes. This is evidence that antibody-defined epitopes and those functioning as transplantation epitopes influencing long-term graft survival are the same. 8. Overall, we are disappointed that position/ amino acid mismatches were not associated with more markedly lower survivals. It appears that current immunosuppression can largely negate the effect of mismatching for immunogenic epitopes.

Unexpected frequencies of HLA antibody specificities present in sera of multitransfused patients.

1. The resolution of single antigen beads is high enough to distinguish between a patient's own HLA phenotype and antibody specificity down to the allele level. 2. Mismatched antigens generally produce an antibody specific to that antigen. Therefore, one would naturally expect antibody specificity frequency to correlate with phenotype frequencies for any given population (e.g., rare phenotypes would rarely have an antibody generated against it). We called this the antigen mismatch model. 3. When testing the sera of 367 highly sensitized patients with single antigen beads, we found results which did not correlate with the antigen mismatch model. Antibodies specific for rare antigens occurred at surprisingly high levels within the study population. 4. We postulate that these high frequencies are due to mismatches on epitopes as defined by absorption/elution experiments (9) or NEpis. Thus, a mismatch on a common antigen could produce an antibody specific for a rare antigen as long as the two antigens share a NEpi (unique position/amino acid combination). 5. The probability of mismatching on 58 NEpis based on published phenotype frequencies was calculated. We called this the NEpi mismatch model. 6. Both the antigen mismatch model and the NEpi mismatch model were compared to observed antibody specificity frequencies in our study population. Overall, the NEpi mismatch model correlated with observed frequencies much more accurately than the traditional antigen mismatch model. 7. In conclusion, we present a probable explanation for higher-than-expected antibody specificity frequencies and propose that mismatching on NEpis has further potential clinical implications.

Unexpected frequencies of HLA antibody specificities in the sera of pre-transplant kidney patients.

1. We utilize single-antigen beads to test 103 pre-transplant patients to discern HLA antibody specificity frequencies. 2. Overall, we found much higher levels of immunization to HLA than anticipated, particularly rare specificities. We postulate that the presence of epitopes and mismatching on epitopes are responsible for these higher levels of immunization. 3. Two models were generated: (A) The first was an antigen mismatch model representing the traditional theory that higher rates of immunization to an antigen would occur with a higher incidence of the antigen within the population. This model was based on published gene frequencies (4), which were converted into antigen frequencies. (B) The second model was an epitope mismatch model based on published gene frequencies (4) as well as experimentally proven epitopes (3). 4. These two models were then compared with observed antibody specificity frequencies. The antigen mismatch model consistently underestimated observed frequencies. On the other hand, the epitope mismatch model seems to be a much better representation of observed frequencies. 5. In conclusion, we bring attention to the occurrence of higher-than-expected frequencies of antibodies against HLA, especially rare antigens like A43 and B76, and we show that mismatched epitopes might provide a plausible explanation for these observed high frequencies.

System for combined three-dimensional morphological and molecular analysis of thick tissue specimens.

We present a new system for simultaneous morphological and molecular analysis of thick tissue samples. The system is composed of a computer-assisted microscope and a JAVA-based image display, analysis, and visualization program that allows acquisition, annotation, meaningful storage, three-dimensional reconstruction, and analysis of structures of interest in thick sectioned tissue specimens. We describe the system in detail and illustrate its use by imaging, reconstructing, and analyzing two complete tissue blocks that were differently processed and stained. One block was obtained from a ductal carcinoma in situ (DCIS) lumpectomy specimen and stained alternatively with Hematoxilyn and Eosin (H&E), and with a counterstain and fluorescence in situ hybridization (FISH) to the ERB-B2 gene. The second block contained a fully sectioned mammary gland of a mouse, stained for histology with H&E. We show how the system greatly reduces the amount of interaction required for the acquisition and analysis and is, therefore, suitable for studies that require morphologically driven, wide-scale (e.g., whole gland) analysis of complex tissue samples or cultures.