Immuno-oncology approaches in uveal melanoma: tebentafusp and beyond.

Uveal melanoma (UM) is the most common ocular malignancy in adults, associated with the poorest prognosis, with metastatic disease occurring in up to 50% of patients. In contrast to metastatic cutaneous melanoma, the use of immune checkpoint inhibitors is associated with poor outcomes in metastatic uveal melanoma (mUM). Tebentafusp, a bispecific molecule, has recently become the first treatment in decades to improve overall survival for mUM. This review summarises the existing and emerging immuno-oncology approaches for the treatment of mUM, and biomarkers of response and resistance to the same. Finally, we propose future research directions that could maximise treatment benefit to a wider pool of patients with UM.

Short KIR haplotypes in pygmy chimpanzee (Bonobo) resemble the conserved framework of diverse human KIR haplotypes.

Some pygmy chimpanzees (also called Bonobos) give much simpler patterns of hybridization on Southern blotting with killer cell immunoglobulin-like receptor (KIR) cDNA probes than do either humans or common chimpanzees. Characterization of KIRs from pygmy chimpanzees having simple and complex banding patterns identified nine different KIRs, representing seven genes. Five of these genes have orthologs in the common chimpanzee, and three of them (KIRCI, KIR2DL4, and KIR2DL5) also have human orthologs. The remaining two genes are KIR3D paralogous to the human and common chimpanzee major histocompatibility complex A- and/or -B-specific KIRs. Within a pygmy chimpanzee family, KIR haplotypes were defined. Simple patterns on Southern blot were due to inheritance of "short" KIR haplotypes containing only three KIR genes, KIRCI, KIR2DL4, and KIR3D, each of which represents one of the three major KIR lineages. These three genes in pygmy chimpanzees or their corresponding genes in humans and common chimpanzees form the conserved "framework" common to all KIR haplotypes in these species and upon which haplotypic diversity is built. The fecundity and health of individual pygmy chimpanzees who are homozygotes for short KIR haplotypes attest to the viability of short KIR haplotypes, indicating that they can provide minimal, essential KIRs for the natural killer and T cells of the hominoid immune system.

Identification of unknown human remains by comparison of antemortem and postmortem radiographs.

The identification of unknown human remains by comparison of ante mortem and post mortem radiographs has found wide acceptance in recent years. This report documents two recent cases in Hong Kong in which unequivocal positive identifications were achieved by comparison of radiographs. In both cases, comparison of radiographs was the only available means of establishment of personal identity. The first case involved the identification of a very badly burned body of a 22-year-old male by the matching of the ante mortem and post mortem radiographic images of the cranial base (in particular the sella turcica), hard palate, paranasal air sinuses, mastoid air cells, the lambdoid suture, and the upper molar teeth as revealed on comparable lateral skull films. The second was the identification of a deliberately mutilated torso of a 28-year-old female by comparison of the radiographic images of the lumbar spine, pelvis, and head of the femur as revealed on plain abdominal films taken before and after death. While evidence of injury or surgery did not figure in either of the identifications, the presence of a mild scoliosis and the absence of the twelfth ribs aided identification in the latter case. Emphasis is placed in this account on the value of radiographic comparison as a means of identification because it draws upon the matching of intricate and highly individual patterns of external and internal bony anatomy that are stable over long periods of time. In cases where ante mortem radiographs and photographs are available, establishment of personal identity by comparison of radiographs is deemed preferable to identification by photographic superimposition because radiographic comparison permits the matching of a potentially larger number of unique anatomical features and is less time consuming and technically exacting.

Fatal massive upper respiratory tract haemorrhage: an unusual complication of localized amyloidosis of the larynx.

Localized amyloidosis of the larynx is usually described as a non-bleeding lesion. We report a patient with localized laryngeal amyloidosis who developed a massive upper respiratory tract haemorrhage and died. This potentially fatal complication of localized amyloidosis of the larynx merits recognition as the disease could be controlled in most instances by surgical excision of the amyloid deposit.

A study on the correlation of blood and vitreous humour alcohol levels in the late absorption and elimination phases.

By using the urine:blood alcohol level ratio as the indicator, the correlation of blood alcohol level (B) and vitreous humour alcohol level (V) in the late absorption and elimination phases was studied. It was found to be good (r = 0.98) and B = 0.76V + 4.7. It is suggested that this equation can safely be used to estimate the minimum blood alcohol level where cadaveric blood is unsuitable or unavailable for analysis and that the B/V ratio can be used to infer the phase in which death occurred where urine is not available.

Simplified methods for large scale enzymatic synthesis of oligoribonucleotides.

We report simplified methods for large scale enzymatic synthesis of oligoribonucleotides using polynucleotide phosphorylase. The main features of the method are use of RPC-5 chromatography, including chromatography at two pH values to deal with the problem of primer phosphorolysis, rapid dialysis for large scale desalting, simplified methods for enzyme removal, and high resolution 1H and 31P NMR for product identification and demonstration of purity. The capacity of the method is adequate to allow beginning with grams of material in the first polymerization step, so that product yields of several milligrams, sufficient for many physical studies, are possible after as many as three separate polymerization reactions.

Intrathoracic vagus nerve neurofibroma and sudden death in a patient with neurofibromatosis.

A 21 year old man with type 1 neurofibromatosis was found dead in the middle of the night. Postmortem examination revealed a large neurofibroma arising from the right intrathoracic vagus nerve, which might have contributed to his sudden death.

Isolation of a classical MHC class I cDNA from an amphibian. Evidence for only one class I locus in the Xenopus MHC.

The amphibian Xenopus is an ectothermic vertebrate in which the MHC has been studied extensively at the functional, biochemical, and genetic levels. A cDNA clone corresponding to the MHC class la gene (Xela-UAA1f) of Xenopus laevis was isolated by screening a cDNA phage library with oligonucleotides based on NH2-terminal protein sequence. Three pieces of evidence support its status as a class la gene: 1) Previous biochemical data suggested that only one polymorphic class la molecule is expressed per MHC haplotype in X. laevis. NH2-terminal sequencing of the class I protein encoded by the f haplotype showed a single unambiguous sequence of the first 22 amino acids; the deduced protein sequence of the cDNA clone matches precisely to this peptide sequence; 2) Genes that hybridized to the cDNA clone segregated perfectly with the serologically typed MHC in two family studies; and 3) There is a strong conservation of amino acids in the peptide-binding region that have been shown in mammals to dock peptides at their NH2- and COOH-termini. In contrast to all other species that have been examined, there appears to be only one class I locus present in the MHC of X. laevis. Xenopus speciates by allopolyploidization, and there are Xenopus species with different levels of ploidy (2n-12n). Functionally, the MHC has been shown to be "diploidized" in most Xenopus species. As in previous studies with MHC class II and HSP70 probes, there is a trend toward maintaining a diploid number of class la genes in all Xenopus species regardless of their chromosome number, probably accomplished through a deletional mechanism. Thus, there is a strong pressure in Xenopus to maintain very few MHC-linked class I genes, exemplified both by the number of class I genes per MHC haplotype and by the number of class la genes per organism.

Residue 3 of beta2-microglobulin affects binding of class I MHC molecules by the W6/32 antibody.

Previous studies of class I MHC molecules have shown that the owl monkey (Aotus) possesses at least two variants of the beta2-microglobulin (beta2m) protein. These two variants have different isoelectric points, and exhibit differential reactivity with the monoclonal antibody W6/32. We report cDNA sequences of the B2m gene, from W6/32-positive and W6/32-negative Aotus cell lines. The two beta2m variants we identified exhibit a single amino acid difference at position three. An arginine residue at position 3 was correlated with W6/32 reactivity, whereas histidine was associated with non-reactivity. W6/32 reactivity was conferred to a W6/32-negative Aotus cell line when it was transfected with the B2m from the W6/32-positive cell line. Residue 3 of beta2m is located at the surface of the class I molecule. It is also close to position 121 of the MHC class I heavy chain, which has previously been shown to influence W6/32 antibody binding. We conclude that W6/32 binds a compact epitope on the class I molecule that includes both residue 3 of beta2m and residue 121 of the heavy chain. We examined the distribution of the two beta2m motifs in a sample Aotus population using an allele-specific polymerase chain reaction assay. The pattern of beta2m segregation we observed matches that which was defined previously by serology. Additionally, we identified laboratory-born hybrid animals who possess both variants of beta2m.

Conserved organization of the ILT/LIR gene family within the polymorphic human leukocyte receptor complex.

The human leukocyte receptor complex (LRC) at Chromosome 19q13.4 encodes Ig superfamily proteins which regulate the function of various hematopoietic cell types. We investigated characteristics of the Ig-like transcript (ILT)/leukocyte Ig-like receptor (LIR) group of LRC genes in comparison with the other major LRC loci encoding the killer cell Ig-like receptors (KIRs). In direct contrast to KIR genes, the ILT/LIR loci of ethnically diverse individuals did not display haplotypic variations in gene number. Investigation of gene expression identified novel cDNA sequences related to the ILT2/LIR1, ILT4/LIR2, ILT3/LIR5, and ILT7 loci, while phylogenetic analysis revealed two distinct lineages of ILT/LIR genes. These two lineages differ in both the nature and extent of their sequence polymorphism. The presence of certain transcription factor-related motifs in the 5' untranslated region of ILT/LIR cDNAs correlates with the specific cell types in which particular ILT/LIR genes are expressed. Although extensive gene duplications and conversion events have apparently forged the LRC, our results indicate striking conservation in the organization of the ILT/LIR genes when compared with the related and closely linked KIR genes. This suggests the evolutionary maintenance of a significant function consistent with the cellular distribution of the ILT/LIR proteins.

Population pharmacokinetic analysis of mycophenolic acid in renal transplant recipients following oral administration of mycophenolate mofetil.

AIM: To develop a population pharmacokinetic model for mycophenolic acid in adult kidney transplant recipients, quantifying average population pharmacokinetic parameter values, and between- and within-subject variability and to evaluate the influence of covariates on the pharmacokinetic variability. METHODS: Pharmacokinetic data for mycophenolic acid and covariate information were previously available from 22 patients who underwent kidney transplantation at the Princess Alexandra Hospital. All patients received mycophenolate mofetil 1 g orally twice daily. A total of 557 concentration-time points were available. Data were analysed using the first-order method in NONMEM (version 5 level 1.1) using the G77 FORTRAN compiler. RESULTS: The best base model was a two-compartment model with a lag time (apparent oral clearance was 27 l h(-1), and apparent volume of the central compartment 98 l). There was visual evidence of complex absorption and time-dependent clearance processes, but they could not be successfully modelled in this study. Weight was investigated as a covariate, but no significant relationship was determined. CONCLUSIONS: The complexity in determining the pharmacokinetics of mycophenolic acid is currently underestimated. More complex pharmacokinetic models, though not supported by the limited data collected for this study, may prove useful in the future. The large between-subject and between-occasion variability and the possibility of nonlinear processes associated with the pharmacokinetics of mycophenolic acid raise questions about the value of the use of therapeutic monitoring and limited sampling strategies.

A novel type of class I gene organization in vertebrates: a large family of non-MHC-linked class I genes is expressed at the RNA level in the amphibian Xenopus.

A Xenopus class I cDNA clone, isolated from a cDNA expression library using antisera, is a member of a large family of non-classical class I genes (class Ib) composed of at least nine subfamilies, all of which are expressed at the RNA level. The subfamilies are well conserved in their immunoglobulin-like alpha 3 domains, but their peptide-binding regions (PBRs) and cytoplasmic domains are very divergent. In contrast to the great allelic diversity found in the PBR of classical class I genes, the alleles of one of the Xenopus non-classical subfamilies are extremely well conserved in all regions. Several of the invariant amino acids essential for the anchoring of peptides in the classical class I groove are not conserved in some subfamilies, but the class Ib genes are nevertheless more closely related in the PBR to classical and non-classical genes linked to the MHC in mammals and birds than to any other described class I genes like CD1 and the neonatal rat intestinal Fc receptor. Comparison with the Xenopus MHC-linked class Ia protein indicate that amino acids presumed to interact with beta 2-microglobulin are identical or conservatively changed in the two major class I families. Genomic analyses of Xenopus species suggest that the classical and non-classical families diverged from a common ancestor before the emergence of the genus Xenopus over 100 million years ago; all of the non-classical genes appear to be linked on a chromosome distinct from the one harboring the MHC. We hypothesize that this class Ib gene family is under very different selection pressures from the classical MHC genes, and that each subfamily may have evolved for a particular function.

CK-1, a putative chemokine of rainbow trout (Oncorhynchus mykiss).

Chemokines are small inducible proteins that direct the migration of leukocytes. While chemokines are well characterised in mammals, they have yet to be identified in fish. We have isolated a cDNA clone from rainbow trout (Oncorhynchus mykiss) which encodes a protein (CK-1) having structural features typical of chemokines. Amino-acid residues that define the beta-chemokines of mammals are conserved in CK-1, including the paired cysteine motif, CC. Further similarities are shared with the C6 subfamily of beta-chemokines. In contrast, the organisation of the CK-1 gene is closer to that of mammalian alpha-chemokine genes than beta-chemokine genes. The CK-1 gene is present in all four salmonid species examined and the nucleotide sequences of the exons are highly conserved. CK-1 has characteristics in common with mammalian alpha and beta-chemokine genes, suggesting that this salmonid chemokine gene preserves traits once present in the ancestral chemokine gene from which modern mammalian chemokine genes evolved.

Unexpected beta2-microglobulin sequence diversity in individual rainbow trout.

For mammals beta2-microglobulin (beta2m), the light chain of major histocompatibility complex (MHC) class I molecules, is invariant (or highly conserved) and is encoded by a single gene unlinked to the MHC. We find that beta2m of a salmonid fish, the rainbow trout (Oncorhynchus mykiss), does not conform to the mammalian paradigm. Ten of 12 randomly selected beta2m cDNA clones from an individual fish have different nucleotide sequences. A complex restriction fragment length polymorphism pattern is observed with rainbow trout, suggesting multiple beta2m genes in the genome, in excess of the two genes expected from the ancestral salmonid tetraploidy. Additional duplication and diversification of the beta2m genes might have occurred subsequently. Variation in the beta2m cDNA sequences is mainly at sites that do not perturb the structure of the mature beta2m protein, showing that the observed diversity of the trout beta2m genes is not primarily a result of pathogen selection.

Comparison of chimpanzee and human leukocyte Ig-like receptor genes reveals framework and rapidly evolving genes.

The leukocyte receptor complex (LRC) on human chromosome 19 contains related Ig superfamily killer cell Ig-like receptor (KIR) and leukocyte Ig-like receptor (LIR) genes. Previously, we discovered much difference in the KIR genes between humans and chimpanzees, primate species estimated to have approximately 98.8% genomic sequence similarity. Here, the common chimpanzee LIR genes are identified, characterized, and compared with their human counterparts. From screening a chimpanzee splenocyte cDNA library, clones corresponding to nine different chimpanzee LIRs were isolated and sequenced. Analysis of genomic DNA from 48 unrelated chimpanzees showed 42 to have all nine LIR genes, and six animals to lack just one of the genes. In structural diversity and functional type, the chimpanzee LIRs cover the range of human LIRs. Although both species have the same number of inhibitory LIRs, humans have more activating receptors, a trend also seen for KIRs. Four chimpanzee LIRs are clearly orthologs of human LIRs. Five other chimpanzee LIRs have paralogous relationships with clusters of human LIRs and have undergone much recombination. Like the human genes, chimpanzee LIR genes appear to be organized into two duplicated blocks, each block containing two orthologous genes. This organization provides a conserved framework within which there are clusters of faster evolving genes. Human and chimpanzee KIR genes have an analogous arrangement. Whereas both KIR and LIR genes can exhibit greater interspecies differences than the genome average, within each species the LIR gene family is more conserved than the KIR gene family.

Exon-intron organization of Xenopus MHC class II beta chain genes.

The amphibian Xenopus laevis is the most primitive vertebrate in which the major histocompatibility complex (MHC) has been defined at the biochemical, functional, and molecular genetic levels. We previously described the isolation and characterization of cDNA clones encoding X. laevis MHC class II beta chains. In the present study, genomic clones encoding class II beta chains were isolated from X. laevis homozygous for the MHC f haplotype. Three class II beta chain genes, designated Xela-DAB, Xela-DBB, and Xela-DCB, were identified. Sequence analysis of these genes showed that Xela-DBB and Xela-DCB correspond to the previously characterized cDNA clones F3 and F8, respectively, whereas Xela-DAB encodes a third, hitherto unidentified class II beta chain of the MHC f haplotype. As a representative of X. laevis class II beta chain genes, the Xela-DAB gene underwent detailed structural analysis. In addition, the nucleotide sequence of Xela-DABf cDNA clones was determined. The Xela-DAB gene is made up of at least six exons, with an exon-intron organization similar to that of a typical mammalian class II beta chain gene. The 5'-flanking region of the Xela-DAB gene contains transcriptional control elements known as X1, X2, and Y, but lacks typical TATA or CCAAT boxes. A notable feature of the X. laevis class II beta chain genes is that the sizes of the introns are larger than those of their mammalian counterparts. As assessed by northern blot analysis, the three class II beta chain genes had similar expression patterns, with the highest level of transcription detected in the intestine. Identification of the Xela-DAB, -DBB, and -DCB genes is consistent with our previous observations, which suggested that the MHC of the tetraploid frog X. laevis is diploidized at the genomic level and contains three class II beta chain genes per haplotype that cross-hybridize to one another under reduced stringency conditions.

Modes of salmonid MHC class I and II evolution differ from the primate paradigm.

Rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) represent two salmonid genera separated for 15--20 million years. cDNA sequences were determined for the classical MHC class I heavy chain gene UBA and the MHC class II beta-chain gene DAB from 15 rainbow and 10 brown trout. Both genes are highly polymorphic in both species and diploid in expression. The MHC class I alleles comprise several highly divergent lineages that are represented in both species and predate genera separation. The class II alleles are less divergent, highly species specific, and probably arose after genera separation. The striking difference in salmonid MHC class I and class II evolution contrasts with the situation in primates, where lineages of class II alleles have been sustained over longer periods of time relative to class I lineages. The difference may arise because salmonid MHC class I and II genes are not linked, whereas in mammals they are closely linked. A prevalent mechanism for evolving new MHC class I alleles in salmonids is recombination in intron II that shuffles alpha 1 and alpha 2 domains into different combinations.

Human diversity in killer cell inhibitory receptor genes.

The presence and expression of killer inhibitory receptor (KIR) and CD94:NKG2 genes from 68 donors were analyzed using molecular typing techniques. The genes encoding CD94:NKG2 receptors were present in each person, but KIR gene possession varied. Most individuals expressed inhibitory KIR for the three well-defined HLA-B and -C ligands, but noninhibitory KIR genes were more variable. Twenty different KIR phenotypes were defined. Two groups of KIR haplotypes were distinguished and occurred at relatively even frequency. Group A KIR haplotypes consist of six genes: the main inhibitory KIR, one noninhibitory KIR, and a structurally divergent KIR. Allelic polymorphism within five KIR genes was detected. Group B comprises more noninhibitory KIR genes and contains at least one additional gene not represented in group A. The KIR locus therefore appears to be polygenic and polymorphic within the human population.

Rapid evolution of NK cell receptor systems demonstrated by comparison of chimpanzees and humans.

That NK cell receptors engage fast-evolving MHC class I ligands suggests that they, too, evolve rapidly. To test this hypothesis, the structure and class I specificity of chimpanzee KIR and CD94:NKG2 receptors were determined and compared to their human counterparts. The KIR families are divergent, with only three KIR conserved between chimpanzees and humans. By contrast, CD94:NKG2 receptors are conserved. Whereas receptors for polymorphic class I are divergent, those for nonpolymorphic class I are conserved. Although chimpanzee and human NK cells exhibit identical receptor specificities for MHC-C, they are mediated by nonorthologous KIR. These results demonstrate the rapid evolution of NK cell receptor systems and imply that "catching up" with class I is not the only force driving this evolution.

A divergent non-classical class I gene conserved in salmonids.

Complementary DNA for two class I genes of the rainbow trout, Oncorhynchus mykiss, were characterized. MhcOnmy-UBA*01 is similar to Onmy-UAC32 and the classical major histocompatibility complex class I genes of other fish species, whereas Onmy-UAA*01 is divergent from all class I genes so far characterized. Onmy-UAA*01 is expressed at lower levels than Onmy-UBA*01. Although Onmy-UAA*01 exhibits restriction fragment length polymorphism on Southern blotting, the encoded protein is highly conserved. Two allotypes, which differ only by substitution at amino acid position 223 of the alpha 3 domain, have been defined. Onmy-UAA*01 has an exon-intron organization like other class I genes and contains a Tc1-like transposon element in intron III. Orthologues of Onmy-UAA*01 have been characterized in four other species of salmonid. Between four species of Oncorhynchus, UAA*01 proteins differ by only 2-6 amino acids, whereas comparison of Oncorhynchus with Salmo trutta (brown trout) reveals 14-16 amino acid differences. The Onmy-UAA*01 gene has properties indicative of a particularly divergent non-classical class I gene.