Viewing the immune checkpoint VISTA: landscape and outcomes across cancers.

BACKGROUND: Optimizing immune checkpoint inhibitor (ICI) therapy may require identification of co-targetable checkpoint pathways via immune profiling. Herein, we analyzed the transcriptomic expression and clinical correlates of V-domain immunoglobulin suppressor of T-cell activation (VISTA), a promising targetable checkpoint. PATIENTS AND METHODS: RNA sequencing was carried out on 514 tissues reflecting diverse advanced/metastatic cancers. Expression of eight immune checkpoint markers [lymphocyte-activation gene 3 (LAG-3), tumor necrosis factor receptor superfamily 14 (TNFRSF14), programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), programmed death-ligand 2 (PD-L2), B- and T-lymphocyte attenuator (BTLA), T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), cytotoxic T-lymphocyte antigen 4 (CTLA-4)], in addition to VISTA, was analyzed, along with clinical outcomes. RESULTS: High VISTA RNA expression was observed in 32% of tumors (66/514) and was the most common highly expressed checkpoint among the nine assessed. High VISTA expression was independently correlated with high BTLA, TIM-3, and TNFRSF14, and with a diagnosis of pancreatic, small intestine, and stomach cancer. VISTA transcript levels did not correlate with overall survival (OS) from metastatic/advanced disease in the pan-cancer cohort or with immunotherapy outcome (progression-free survival and OS from the start of ICI) in 217 ICI-treated patients. However, in ICI-treated pancreatic cancer patients (n = 16), median OS was significantly shorter (from immunotherapy initiation) for the high- versus not-high-VISTA groups (0.28 versus 1.21 years) (P = 0.047); in contrast, VISTA levels were not correlated with OS in 36 pancreatic cancer patients who did not receive ICI. CONCLUSION: High VISTA expression correlates with high BTLA, TIM-3, and TNFRSF14 checkpoint-related molecules and with poorer post-immunotherapy survival in pancreatic cancer, consistent with prior literature indicating that VISTA is prominently expressed on CD68+ macrophages in pancreatic cancers and requiring validation in larger prospective studies. Immunomic analysis may be important for individualized precision immunotherapy.

Intrachromosomal genomic instability in human sporadic colorectal cancer measured by genome-wide allelotyping and inter-(simple sequence repeat) PCR.

We have used genome-wide allelotyping with 348 polymorphic autosomal markers spaced, on average, 10 cM apart to quantitate the extent of intrachromosomal instability in 59 human sporadic colorectal carcinomas. We have compared instability measured by this method with that measured by inter-(simple sequence repeat) PCR and microsatellite instability assays. Instability quantitated by fractional allelic loss rates was found to be independent of that detected by microsatellite instability analyses but was weakly associated with that measured by inter-(simple sequence repeat) PCR. A set of seven loci were identified that were most strongly associated with elevated rates of fractional allelic loss and/or inter-(simple sequence repeat) PCR instability; these seven loci were on chromosomes 3, 8, 11, 13, 14, 18, and 20. A lesser association was seen with two loci flanking p53 on chromosome 17. Coordinate loss patterns for these loci suggest that at least two separate sets of cooperating loci exist for intrachromosomal genomic instability in human colorectal cancer.

Canine pulmonary angiotensin-converting enzyme. Physicochemical, catalytic and immunological properties.

Antiontensin-converting enzyme (peptidyldipeptide hydrolase, EC 3.4.15.1) has been solubilized from canine pulmonary particles and purified to apparent homogeneity. A value of approx. 140000 was estimated for the molecular weight of the native and the reduced, denatured forms of the enzyme. No free NH2-terminal residue was detected by the dansylation procedure. Carbohydrate accounted for 17% of the weight of the enzyme, and the major residues were galactose, mannose and N-acetylglucosamine with smaller amounts of sialic acid and fucose. Removal of sialic acid residues with neuraminidase did not alter enzymatic activity. The enzyme contained one molar equivalent of zinc. Addition of this metal reversed stimulation and inhibition of activity observed in the presence of Co2+ and Mn2+, respectively. Immunologic homology of pure dog and rabbit enzymes was demonstrable with goat antisera. Fab fragments and intact IgG antibodies displayed similar inhibition dose vs. response curves with homologous enzyme, whereas the fragments were poor inhibitors of heterologous activity compared to the holoantibodies. The canine glycoprotein was much less active than the rabbit preparation in catalyzing hydrolysis of Hip-His-Leu. In contrast, the two enzymes exhibited comparable kinetic parameters with angiotensin I as substrate.

Inhibition of horseradish peroxidase activity by specific antibody: determinant specificity of anticatalytic antibodies.

Rabbit antisera specific for horseradish peroxidase inhibit the catalytic activity of the enzyme. All antibodies prepared against the holoenzyme react with the peroxidase apoenzyme. However, only a minority (30-45%) of the total antiperoxidase pool cross react with reduced and alkylated apoenzymes. The antibodies inhibiting peroxidase activity do not bind to S-carboxymethyl of S-carboxamidomethylated apoenzyme derivatives as measured by absorption and competition of inhibition experiments. Glycopeptides derived from horseradish peroxidase also failed to bind anticatalytic antibodies. Antibodies that inhibit enzyme activity have specificity for noncarbohydrate conformation dependent antigenic determinants of horseradish peroxidase. Additional experiments probed the mechanism by which inhibitory antibody decreases the catalytic activity of horseradish peroxidase. Absorption spectra of horseradish peroxidase that has bound Fab fragments sufficient to cause 90% inhibition of the enzyme activity determined that the enzyme retained the ability to bind hydrogen peroxide. Thus, anticatalytic antibodies do not prevent the formation of the first enzyme-substrate intermediate but mediate their inhibitory effects by disrupting a later step in the reaction mechanism.

The effect of alternate substrates and substrate concentration on the antibody-mediated inhibition of horseradish peroxidase.

Inhibition of the catalytic activity of horseradish peroxidase by rabbit antisera was measured using alternate enzyme substrates. The same general inhibition curve patterns were obtained regardless of the electron donor or hydroperoxide used. In all cases typical biphasic inhibition patterns were found and complete inhibition of enzyme activity was never observed. Measurement of the degree of inhibition as a function of substrate concn revealed a dependence of anticatalytic activity on hydroperoxide concn. As the concn of hydrogen peroxide in the assay mixture increased, there was a corresponding increase in the inhibition observed. On the other hand, the degree of inhibition was not dependent on the concn of electron donor (dianisidine) in the assay mixture. Spectrophotometric experiments with an electron donor analogue demonstrated that antibodies do not inhibit peroxidase activity by excluding electron donor molecules from enzyme binding sites. The results have suggested possible mechanisms for the antibody-mediated inhibition of peroxidase activity.

Heterogeneity in anticatalytic activity of antibody specific for horseradish peroxidase.

Rabbit antibodies specific for horseradish peroxidase are heterogeneous in their ability to inhibit enzyme activity. Heterogeneity was demonstrated by fractionation of the total antiperoxidase pool by differential ammonium sulfate precipitation and differential elution of antibody from enzyme affinity columns. Both fractionation methods yielded antibody subpopulations that differed in anticatalytic activity. Some antibody subpopulations decreased enzyme activity almost completely at low molar ratios of antibody to peroxidase. Other subpopulations were not effective inhibitors even at great molar excess. Admixture experiments demonstrated that inefficient antibody pools decreased the anticatalytic effect of highly inhibitory antibody. The degree of inhibition observed with unfractionated antiserum is a reflection of the interaction of various antibody subpopulations with the enzyme. No correlation was found between the immunoglobulin class of antiperoxidase and anticatalytic efficiency in analyses of numerous antisera. The determinant specificity of an antiperoxidase molecule determines its anticatalytic ability. A peptide fragment (mol. wt 22,500) of peroxidase prepared by partial tryptic digestion bount 60 to 70% of the total antiperoxidase in a number of antisera. However, the peptide did not bind inhibitory antibodies.

Determining the Tsallis parameter via maximum entropy.

The nonextensive entropic measure proposed by Tsallis [C. Tsallis, J. Stat. Phys. 52, 479 (1988)] introduces a parameter, q, which is not defined but rather must be determined. The value of q is typically determined from a piece of data and then fixed over the range of interest. On the other hand, from a phenomenological viewpoint, there are instances in which q cannot be treated as a constant. We present two distinct approaches for determining q depending on the form of the equations of constraint for the particular system. In the first case the equations of constraint for the operator Ô can be written as Tr(F(q)Ô)=C, where C may be an explicit function of the distribution function F. We show that in this case one can solve an equivalent maxent problem which yields q as a function of the corresponding Lagrange multiplier. As an illustration the exact solution of the static generalized Fokker-Planck equation (GFPE) is obtained from maxent with the Tsallis enropy. As in the case where C is a constant, if q is treated as a variable within the maxent framework the entropic measure is maximized trivially for all values of q. Therefore q must be determined from existing data. In the second case an additional equation of constraint exists which cannot be brought into the above form. In this case the additional equation of constraint may be used to determine the fixed value of q.

Associations between ERCC2 polymorphisms and gliomas.

Xeroderma pigmentosum complementation group D/excision repair cross-complementing in rodents 2 (ERCC2) encodes a protein that is part of the nucleotide excision repair pathway and the transcription factor IIH transcription complex. Mutations in this gene have been shown to cause three distinct clinical diseases including xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. Several ERCC2 polymorphisms, the effects of which on gene function are not known, have been described. To investigate whether constitutive sequence variations might be associated with adult onset gliomas, blood specimens from a case-control study (187 cases and 169 controls) were genotyped for seven previously described polymorphisms (R156R, I199M, H201Y, D312N, A575A, D711D, and K751Q). A novel R616C polymorphism was also identified. Cases were significantly more likely than controls to be homozygous for the silent AA variant at codon 156 (odds ratio, 2.3; 95% confidence interval, 1.3-4.2). Although this was observed for patients in each of three histological subgroups of cases, (glioblastoma multiforme, astrocytoma, and oligoastrocytoma) compared with controls, the association was strongest for patients with oligoastrocytoma (odds ratio, 3.2; 95% confidence interval, 1.1-9.5). In contrast, cases were somewhat less likely than controls to carry variants at D312N, D711D, and K751Q, but not significantly so overall or for any subgroup after adjustment for age and gender. Individuals with variant nucleotides at D312N, D711D, and K751Q were significantly more likely to carry a variant at another of those three codons and less likely to carry a variant nucleotide at R156R, regardless of case or control status. Although the pattern of association observed here is consistent with a role of ERCC2 variants in the prevention or causation of glioma, these results are also consistent with the possibility that another gene linked to ERCC2 may be involved. This seems especially so because the strongest association was observed with a silent nucleotide variation.

Immunoquantitation of carnitine palmitoyl transferase in skeletal muscle of 31 patients.

We studied 31 patients suspected of having muscle carnitine palmitoyl transferase 2 (CPT2) deficiency. The catalytic activity of CPT2 was measured in muscle biopsies by the isotope exchange method and CPT2 immunoreactivity was quantitated by an enzyme-linked immunosorbent assay. Nine patients had normal enzyme activity and immunoreactivity. Eight patients had significant deficiencies in catalytic activity (> 3 S.D. below reference mean) of which six were also deficient in immunoreactivity. An additional nine patients were significantly deficient in immunoreactivity with normal catalytic activity and five patients had partial deficiencies in both. At least two categories of alterations in CPT may exist which lead to a deficiency based on the data presented: (1) a regulatory defect in CPT which only alters the enzyme active site; and (2) a structural defect due to altered synthesis, increased degradation, or changes in the immunoreactive site. It may prove to be of diagnostic importance to combine the analysis of enzyme activity and immunoreactivity in patients suspected of having a CPT deficiency and to further investigate the condition of partial CPT deficiency.

Semiautomated clone verification by real-time PCR using molecular beacons.

Conventional, high-throughput PCR analysis of common elements utilizing numerous primer sets and template DNA requires multiple rounds of PCR to ensure optimal conditions. Laborious gel electrophoresis and staining is then necessary to visualize amplification products. We propose novel multicolor molecular beacons, to establish a high-throughput, PCR-based sequence tagged site (STS) detection system that swiftly and accurately confirms marker content in template containing common repeat elements. A simple, one-tube, real-time PCR assay system was developed to specifically detect regions containing CA and GATA repeats. Ninety-six samples can be confirmed for marker content in a closed-tube format in 3 h, eliminating product confirmation on agarose gels and avoiding crossover contamination. Multiple STSs can be detected simultaneously in the same reaction tube by utilizing molecular beacons labeled with multicolor fluorophores. Template DNA from 260 RPCI-11 bacterial artificial chromosome (BAC) clones was examined for the presence of CA and/or GATA repeats using molecular beacon PCR and compared with conventional PCR results of the same clones. Of the 205 clones containing CA and GATA repeats, we were able to identify 129 clones (CA, n = 99; GATA, n = 30) by using molecular beacons and only 121 clones (CA, n = 92; GATA, n = 29) by conventional PCR amplification. As anticipated, 55 clones that contained sequences other than CA or GATA failed molecular beacon detection. Molecular beacon PCR, employing beacons specific for tandem repeat elements, provides a fast, accurate, and sensitive multiplex detection assay that will expedite verification of marker content in a multitude of template containing these repeats.

Parental origin of De Novo chromosome 9 deletions in del(9p) syndrome.

Parental origin of de novo deletions in the short arm of chromosome 9 in patients with a clinical diagnosis of del(9p) syndrome was assessed in 13 patients using polymerase chain reaction (PCR) analysis of highly polymorphic dinucleotide repeat microsatellite markers located in the putative deleted region. The deletion was found to be of paternal origin in 9 cases and of maternal origin in the remaining 4 cases, suggesting that the molecular event resulting in the deletion occurs in both male and female gametogenesis and that genomic imprinting does not appear to play a role in the pathogenesis of del(9p) syndrome.

Molecular and cytogenetic analysis of familial Xp deletions.

Five families in which an Xp deletion is segregating and two families in which an X chromosome rearrangement including a deletion of the short arm is segregating were ascertained for study. Normal fertility was seen in all families. Members from 5 of the 7 families manifested short stature (height <5th centile), while normal height was present in two families. Studies of both the FMR-1 and the androgen receptor loci using PCR based X-inactivation analysis demonstrated that in all families analyzed, there is preferential inactivation of one X chromosome. Molecular cytogenetic analysis showed that members of 3 of the 7 families share a common breakpoint in an approximate 2-3 Mb region at Xp22.12, suggesting a possible hotspot for chromatin breakage. Previous genotype-phenotype correlations and deletion mapping have indicated that a gene for stature resides within the pseudoautosomal region in Xp22.33. Our findings indicate that the loss of this region is not always associated with short stature, suggesting that other factors may be involved.

Clinical spectrum and molecular diagnosis of Angelman and Prader-Willi syndrome patients with an imprinting mutation.

Recent studies have identified a new class of Prader-Willi syndrome (PWS) and Angelman syndrome (AS) patients who have biparental inheritance, but neither the typical deletion nor uniparental disomy (UPD) or translocation. However, these patients have uniparental DNA methylation throughout 15q11-q13, and thus appear to have a mutation in the imprinting process for this region. Here we describe detailed clinical findings of five AS imprinting mutation patients (three families) and two PWS imprinting mutation patients (one new family). All these patients have essentially the classical clinical phenotype for the respective syndrome, except that the incidence of microcephaly is lower in imprinting mutation AS patients than in deletion AS patients. Furthermore, imprinting mutation AS and PWS patients do not typically have hypopigmentation, which is commonly found in patients with the usual large deletion. Molecular diagnosis of these cases is initially achieved by DNA methylation analyses of the DN34/ZNF127, PW71 (D15S63), and SNRPN loci. The latter two probes have clear advantages in the simple molecular diagnostic analysis of PWS and AS patients with an imprinting mutation, as has been found for typical deletion or UPD PWS and AS cases. With the recent finding of inherited microdeletions in PWS and AS imprinting mutation families, our studies define a new class of these two syndromes. The clinical and molecular identification of these PWS and AS patients has important genetic counseling consequences.

Immunoregulation of the mixed lymphocyte reaction by macrophage-derived factors: functional and biochemical separation of enhancing and inhibitory factors.

Positive and negative immunoregulation of the mixed lymphocyte reaction (MLR) occurred through release of macrophage(MO)-derived, soluble enhancing and inhibitory factors. Macrophages, sonicated or cultured in low concentrations, produced nondialyzable, soluble factor(s) capable of enhancing the MLR; but the culture supernatant had no biologically detectable levels of Interleukin 1, Interleukin 2, or interferon. Production of enhancing supernatants was not affected by pretreatment of MO with trypsin or anti-Thy 1 antibody plus complement. In contrast, MO cultured in high concentrations yielded an inhibitory supernatant factor(s) which suppressed MLR reactivity even when MO were cultured in the presence of indomethacin. Culturing MO with proteolytic enzyme inhibitors increased the yields of the inhibitory and enhancing factors. Both factors were precipitable with ammonium sulfate and could be separated into several biologically active fractions using anion exchange chromatography.

The allele frequency of mutations in four genes that confer enhanced susceptibility to venous thromboembolism in an unselected group of New York State newborns.

The frequencies of Factor V G1691A (FVL), prothrombin (PT) G20210A, 5'10'methylenetetrahydrofolate reductase (MTHFR) C677T, and methionine synthase (MS) A2756G (four mutations associated with an increased risk of venous thromboembolism [VTE]) were determined in a sample of approximately 1500 New York State residents. Dried blood spots from approximately equal numbers of Caucasians, African-Americans and Hispanics were anonymously obtained from the New York State Department of Health Newborn Screening Program. Following PCR amplification of dried blood spot DNA, allele-specific oligonucleotide hybridization was used to detect mutant alleles. The total number of individuals at increased genetic risk for VTE was 271 (17.5%) of the 1553 persons tested. Increased genetic risk was defined as the heterozygous state for FVL or PT and the homozygous state for the MTHFR or MS polymorphisms. Sixteen individuals had more than one genetic risk factor. The MS gene variant allele frequencies for African-Americans and Hispanics are the first to be reported. This report also provides an estimate of the variant PT allele in the largest group of Hispanics studied to date.

Selection of anesthesiology residents.

BACKGROUND: CARCS (computer-assisted resident candidate selection) is a database application developed in 1983 at the Department of Anesthesiology of the Medical University of South Carolina to deal with the greatly increased quantity of applicant information. This article relates a representative sample of CARCS data to the process of selecting residents in general. METHOD AND RESULTS: CARCS files were analyzed for 1985-86, 1986-87, 1990-91, and 1991-92, and data for each year were derived as simple averages and percentages for two groups: (1) the entire pool of residency applicants (approximately 200 per year) and (2) the eight residency candidates per year who actually matched with the program through the National Resident Matching Program, Analyses showed that the standardized test scores, grades, and class ranks of the matched candidates were not significantly higher than those of the applicants; however, the matched candidates' scores for letters of reference and for interviews were consistently higher than those for the applicant pool. CONCLUSIONS: The results support the view of medical educators that the traditional academic criteria are not sufficiently predictive of clinical performance or interpersonal skills. Research relating residents' performances to personality, learning style, and other neuropsychological factors may provide needed alternatives to knowledge testing by developing combined cognitive-noncognitive profiles. The anesthesiology clerkship experience is now almost universal among applicants and could be structured to provide pertinent information about potential residents through direct observation as well as behavioral testing.

The effect of 6% hydroxyethyl starch and desmopressin infusion on von Willebrand factor: ristocetin cofactor activity.

Hydroxyethyl starch is commonly used as a plasma volume expander in the surgical patient. Although it is generally considered a safe plasma substitution, some reports of an acquired von Willebrand's disease-like syndrome have been documented. To examine this further, von Willebrand factor: ristocetin cofactor activity (RCoF) was measured in two groups of patients perioperatively, following hydroxyethyl starch infusion and at 30, 60, and 240 minutes following either deamino-8-D-arginine vasopressin (DDAVP) (group I, n = 12) or saline (group II, n = 11). Following hydroxyethyl starch infusion, ristocetin cofactor activity decreased to 58 percent (group I) and 55 percent (group II) of their respective baseline values. After infusion of DDAVP, mean ristocetin cofactor activity in group I increased significantly to 95 percent at 30 minutes and 100 percent of baseline at 60 minutes. Mean ristocetin cofactor activity levels in group II, however, remained decreased, 69 percent and 57 percent of baseline at the same time points. There was no statistical difference between groups before or immediately after hydroxyethyl starch administration or at 240 minutes post-DDAVP or saline infusion. It is our conclusion that DDAVP is a safe therapy for the mild coagulapathy infrequently associated with hydroxyethyl starch administration.

Vertical transmission of HIV in New York State: a basis for statewide testing of newborns.

Infants (n = 313) of HIV-infected mothers were enrolled (mean age 1.9 weeks, range 0-8 weeks) in a 3-year prospective study of vertical transmission. Fifty-six infants (17.9%) had laboratory and clinical evidence of HIV infection. Polymerase chain reaction (PCR) provided early and reliable identification of infected infants. Thirty-one of the 56 infected infants had specimens submitted when the infants were 4 weeks of age or less and 30 (97%) tested PCR positive. This percentage increased to 100% by 8 weeks of age when 51 of the 56 infected infants had specimens tested for that time period. Immune complex dissociation (ICD) antigen testing was a sensitive method for diagnosis of infection but only in infants older than 1 month. p24 antigen testing, although free of false positives, is less sensitive than either of the other methods. Among surrogate markers of HIV infection, elevation of soluble CD8 levels precedes an increase in immunoglobulin levels or a decline in CD4 T lymphocytes. Vertical transmission is significantly lower in Central and Western New York State than other regions. Transmission is significantly higher in low birthweight babies and in infants whose mothers have CD4 counts < 500. This study provided the basis for establishing a Pediatric HIV PCR Testing Service for the early diagnosis of HIV infection in neonates.

A comparison of wound instillation and caudal block for analgesia following pediatric inguinal herniorrhaphy.

Regional analgesia, in a variety of forms, has been shown to afford effective postoperative pain relief after pediatric inguinal hernia repair. This study compares the efficacy of wound instillation with 0.25% bupivacaine (n = 20), caudal block with 0.25% bupivacaine (n = 35), and a control group (n = 15). Outcome parameters examined include total operating room time, time to extubation, postoperative objective pain scales, and requirement for supplemental analgesics. Patients who received caudal blocks had significantly decreased emergence times (P < .002), exhibited fewer pain-related behaviors postoperatively (P < .0025), and required less narcotic to maintain normal hemodynamics (P < .05). Operating room time was not statistically different between the three groups. The use of perioperative analgesic blocks resulted in quicker awakening, a more comfortable postoperative course, and potentially earlier discharge from same-day surgery.

The anesthetic management of the pediatric strabismus patient.

Postoperative nausea and vomiting continues to be a common perioperative complication for pediatric strabismus patients. Postoperative pain management and the choice of general anesthetic can increase the incidence of perioperative nausea. Current techniques for induction of general anesthesia and selection of agents, prevention and treatment of postoperative pain, and options for antiemetic therapy will be reviewed.