Genetic and Genomic Potential in Liver Transplantation

Human Leukocyte Antigen Typing

The major histocompatibility complex (MHC) is a genetic region defined initially by the rejection of skin grafts in genetically incompatible mouse strains. In humans the MHC, known as the human leukocyte antigen (HLA) region , comprises about 3 Mb located on the short arm of chromosome 6 ( Fig. 102-2 ). This region contains approximately 200 genes, many of which are involved in immune responses and some of which exhibit extensive genetic polymorphism. The genes encoding the HLA class I (A, B, and C) and class II (DR, DQ, DP) molecules are the most polymorphic loci in the human genome, with some loci (i.e., HLA-B or DRB1) having more than 300 alleles.

HLA typing methods were originally based on the detection of expressed HLA molecules on the surface of separated T cells (HLA class I) and B cells (HLA class II) using panels of antisera, usually obtained from multiparous women or patients who received multiple blood transfusions in a complement-dependent cytotoxicity test. Such “serological” HLA typing suffers from a number of drawbacks. Live lymphocytes are required, and lymphocyte counts can be low in some transplant patients. Panels of antisera must be maintained, although commercial kits are now available. Finally, the typing resolution obtainable from serological methods is low.

The development of polymerase chain reaction (PCR) amplification enabled the study of the allelic diversity at the HLA loci, as well as the development of simple and rapid DNA-based typing methods. In general, sequence-based typing methods allow a more accurate and much more precise method of typing than serological or cellular methods. For example, there are more than 300 alleles at the DRB1 locus but only 17 distinct serological specificities or serotypes. Although good serological studies may provide a level of resolution adequate for solid organ transplant HLA typing, they are inadequate for stem cell transplant matching and therefore have been largely superseded by DNA-based typing in clinical HLA laboratories. However, serological typing still has a useful role as an adjunct to DNA-based typing, for example, to determine whether a particular HLA allele is actually expressed at the cell surface. A number of such nonexpressed “null” HLA alleles are now known. DNA-based typing methods offer a number of advantages over serological typing methods. Live lymphocytes are not required, and DNA is easily extracted from any nucleated cell, although peripheral blood lymphocytes are the usual source. DNA is easily stored, allowing repeat sample testing when required. A number of different DNA-based HLA typing methods are in everyday use in clinical HLA typing laboratories, all of which are based on PCR amplification of target sequences in the HLA genes under investigation. PCR primers and oligonucleotide probes can be designed and validated in house or purchased commercially. As such, unlike antisera, they are a renewable resource.

Additionally, recent studies have indicated that the DNA typing of loci that cannot be typed serologically (e.g., HLA-DP), as well as DNA typing that distinguishes alleles within a serotype, can have clinical relevance in cadaveric kidney transplants. For solid organ transplants the degree of HLA matching, in terms of the number of mismatched A, B, and DR antigens, significantly affects the survival of kidney grafts, but does not seem to play as big a role in liver transplants. There is, however, emerging data that indicate that donor-specific antibody against these HLA mismatches leads to poor graft outcomes in liver transplants.

Antibody Identification

The Luminex assay is currently considered the gold standard for antibody screening and identification. Separate HLA molecules are synthesized in vitro using DNA recombinant technology. These molecules are then bound to polystyrene particles. Beads can be coated with either single or multiple HLA molecules depending on the assay. Beads are then mixed with patient serum to allow antibody binding. The individual polystyrene particles are loaded with a single type or multiple types of HLA molecules, each stained with varying amounts of different fluorochromes, giving a unique but reproducible gated position that provides HLA specificity. A second antihuman antibody linked to a reporter molecule is then added to the reaction mixture, and the reporter fluorescence is measured with another laser, giving a semiquantitative level of antibodies in the patient sample.

The detection of donor-specific antibody by Luminex microspheres is associated with significantly higher rates of graft dysfunction and immunological events in kidney transplant recipients. Although no significant beneficial or detrimental effects associating the degree of HLA donor-recipient matching and liver graft survival have been observed, there is evidence to support the role of HLA matching and antibody screening in certain populations such as those with hepatitis B and those undergoing liver retransplantation.

Biomarkers of Rejection—Kidneys as a Template for Liver Transplantation?

A critical gap in transplantation is the lack of available tests or biomarkers to indicate when a patient has become tolerant to his or her graft and early noninvasive markers of rejection. The current standard is to frequently monitor the serum creatinine level, which is obviously correlative to acute rejection episodes, because a rise in the serum creatinine level is usually the first available indication of allograft dysfunction. This is less than ideal because by the time an elevation in the serum creatinine level has been observed it is a relatively late development in the course of a rejection episode and invariably indicates the presence of significant histological damage. The histological examination of the percutaneous core needle transplant biopsy specimen remains the gold standard for the diagnosis of acute rejection; however, it is not without its fair share of complications, including massive hemorrhage. Therefore less invasive biomarkers are needed that could diagnose rejection earlier and be able to identify mechanisms as well, such as acute cellular versus antibody-mediated rejection.

Now that we are in the era of the human genome, molecular biomarkers have been a clinical reality for some time, especially in the field of oncology. Carcinoembryonic antigen was one of the earliest molecular biomarkers identified by Joseph Gold. Identifying more sensitive parameters (genes, mRNA transcripts, microRNAs [miRNAs], peptides, proteins, and metabolites) has been investigated by monitoring these parameters in allograft biopsy, blood, and urine. To date, several such potential biomarkers have been reported, yet no novel biomarkers have been validated in a large multicenter clinical trial for either clinical practice or drug development. Most of the current work has been done in kidney allografts, where urine and peripheral blood cell profiles offer a noninvasive means of predicting the development of acute rejection; these profiles have been shown to be diagnostic of biopsy-confirmed acute rejection. Currently mRNA transcripts of granzyme B, perforin, serine proteinase inhibitor-9 (PI-9), granulysin, FOXP3, IP-10, CXCR3, NKG2D, TIM-3, FasL, and CD103 have been shown to be higher in the urine during acute rejection in kidney transplants. Similar transcripts have been validated in peripheral blood of kidney recipients and shown to be predictive of acute rejection as well. Certainly immune response directed at infections could confound the diagnostic utility of inflammatory gene–based signatures of acute rejection, and a number of investigators have addressed this important concern. Investigators have shown that granzyme B mRNA levels are not increased in renal allograft recipients with bacterial urinary tract infection, granzyme B mRNA levels in urine are not increased in renal allograft recipients with bacteriuria, and that both granzyme B and granulysin levels in urine distinguish acute rejection from bacteriuria. It was also found that perforin mRNA levels distinguish acute rejection from cytomegalovirus (CMV) infection. However, it has been shown that kidney allograft dysfunction due to BK polyomavirus nephropathy is associated with many overlaps of these biomarkers, so much so that an important goal in this area is to investigate whether acute rejection could even be distinguished from BK polyomavirus nephropathy noninvasively.

In addition to studying gene expression a group of research efforts have been focused on studying urinary proteomics as a novel approach to assessing early detection of acute renal transplant rejection. Analysis of proteins offers a greater promise to capture cellular events more accurately because of the prime role proteins play in cellular activities. Gene expression may not necessarily correlate with the corresponding level of protein. Furthermore, the number of protein products far outweighs the known number of genes because of the splice variants of genes that are manifest as different protein products as well as the posttranslation modifications (i.e., sulfation, phosphorylation, glycosylation, methylation,). The application of surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) offers a novel, noninvasive, sensitive, highly predictive, reproducible, and rapid method for the prediction of acute renal injury. SELDI-TOF-MS is a protein analysis tool capable of detecting protein profile differences between biological samples. Its low-resolution ‘‘mass fingerprinting” and its issues in identifying the actual corresponding peptides have contributed to less enthusiasm toward the use of this platform in biomarker discovery efforts.

To identify proteins for discovery purposes, liquid chromatography coupled with mass spectrometry (LC/MS)-based shotgun proteomics has shown the most promise because of its ability to identify the majority of proteins in urine. In a recent report Nagaraj and Mann ^12a used LC-MS/MS to analyze urine collected from seven normal human donors for 3 consecutive days and reported that there are about 500 urine proteins that can be referred as common and abundant in human urine. One of the major hurdles, however, is depleting the high-abundance protein (e.g., albumin) so as to increase the sensitivity of biomarker identification.

miRNAs are small noncoding RNAs approximately 22 nucleotides long that regulate gene expression by inducing translational repression, mRNA degradation, or transcriptional inhibition ( Fig. 102-3 ). Primary miRNAs (pri-miRNAs) are cleaved by the ribonuclease III enzyme Drosha to approximately 70-nucleotide–long stem-loop precursors called precursor microRNAs (pre-miRNAs) (see Fig. 102-3 ). These precursor microRNAs are exported into the cytoplasm by Exportin-5 and then are processed by Dicer, an endonuclease, to form mature double-stranded miRNAs. These are then incorporated into an RNA-induced silencing complex (RISC), in which the passenger strand is selectively degraded. Along with Argonaute 2 and other RISC factors, the mature, approximately 20- to 22-nucleotide-long miRNAs bind to complementary sites on messenger RNA transcripts to induce transcript degradation. A single miRNA has the ability to regulate the expression of numerous mRNAs. miRNAs have been shown to control processes such as cellular survival, development, differentiation, and proliferation and to modulate both innate and adaptive immunity. The hypothesis that urinary cell or peripheral blood cell miRNA expression profiles are predictive, diagnostic, or prognostic biomarkers of allografts is worthy of investigation. A 2009 study by Anglicheau et al showed that miRNA profiles can accurately distinguish acute rejection from a normal biopsy specimen. In addition, the miRNA profiles predicted allograft function. These overexpressed miRNAs were also overexpressed in the peripheral blood and could be used as a potential noninvasive biomarker. In fact, it has recently been demonstrated that two liver-enriched miRNAs (miR-122 and miR-192) are promising biomarkers for acetaminophen-induced acute liver injury, and this is the first evidence for the potential use of miRNAs as biomarkers of human drug-induced liver injury.

Certainly the field of noninvasive biomarkers is more developed and well studied in the field of kidney transplantation. How any of these biomarkers can be used in liver transplantation remains to be seen. The ability to discriminate between rejection and other infectious processes will be a critical factor. A number of studies have begun to search for biomarkers that try to identify patients who are tolerant and who are at risk for rejection, infection, tumor, and hepatitis C virus (HCV) recurrence.