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.

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.

Balanced translocation 46,XY,t(2;15)(q37.2;q11.2) associated with atypical Prader-Willi syndrome.

The lack of normally active paternal genes in 15q11-q13, as an outcome of either a paternal deletion or maternal disomy, accounts for >95% of all patients with Prader-Willi syndrome. Other mechanisms, including imprinting mutations and unbalanced translocations involving pat 15q11-q13, have been described elsewhere. In this study, we present a patient with a rare balanced, de novo translocation-46,XY,t(2;15)(q37.2;q11.2)-involving breakage within the Prader-Willi/Angelman syndrome region of the paternal homologue, without an apparent deletion. The patient demonstrated several manifestations of the Prader-Willi syndrome but was clinically atypical. Cytogenetic and molecular studies of this case demonstrated the translocation breakpoint to be between SNRPN and IPW, with mRNA expression of SNRPN and PAR-5 but absence of IPW and PAR-1 expression. These results suggest that disruption of either IPW expression or a nearby gene by an upstream break may contribute to the Prader-Willi syndrome phenotype and that expression of SNRPN or other upstream genes is responsible for other aspects of the classical Prader-Willi syndrome phenotype.

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.

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.

Management of cancer pain.

Pain is often the first symptom of cancer. Pain and fear of pain are concerns of most cancer patients and their families. The therapeutic options available to control pain are numerous, allowing practitioners to better individualize treatment. We present an overview of the pathophysiology of cancer pain, the current theories of pharmacologic opioid management, advantages of adjuvant drugs, and the range of invasive and noninvasive procedures that practitioners can offer their patients who have cancer-related pain.

Postoperative analgesia after major shoulder surgery with interscalene brachial plexus blockade: etidocaine versus bupivacaine.

Postoperative pain is commonly treated with significant doses of narcotics, occasionally resulting in side effects including nausea, pruritus, and respiratory depression. One potential advantage of regional anesthesia is profound postoperative analgesia that reduces exposure to potent narcotics. To evaluate the efficacy of two long-acting local anesthetics, bupivacaine and etidocaine, in providing pain relief after major shoulder surgery, we randomized 20 patients to receive either bupivacaine or etidocaine for brachial plexus block as the primary anesthetic for shoulder surgery. Surgeons, patients, and the acute pain service were blinded as to drug selection. After the patient was sedated, an interscalene block was placed with the use of a nerve stimulator to facilitate proper needle placement. Forty milliliters of either 0.5% bupivacaine or 0.75% etidocaine containing 5 micrograms/mL epinephrine was injected into the brachial plexus sheath. An additional 8 mL of local anesthetic was administered for superficial cervical plexus blockade. Intraoperative sedation was accomplished with an intravenous infusion of methohexital as needed. After surgery, patients received a standard patient-controlled analgesia protocol providing incremental doses of morphine. The degree of postoperative analgesia resulting from residual local anesthetic effect was expressed as the time until first morphine requirement and the total dose of morphine required during the first 24 hours postoperatively. We found no statistically significant intergroup differences either in time of initial use of morphine or in the total dose of morphine required in the first 24 hours. Both etidocaine and bupivacaine provide prolonged analgesia after major shoulder surgery when injected into the brachial plexus. Bupivacaine, however, possesses significant cardiotoxicity and has a relatively delayed onset in peripheral neural blockade. Etidocaine is less cardiotoxic and also has a more rapid onset of effect. Thus etidocaine may be a preferable agent for interscalene block for major shoulder surgery.

Acquired methemoglobinemia from multiple oxidants.

Phenazopyridine has been associated with methemoglobinemia in patients who have received an overdose, have decreased renal function, or are discovered to be unusually susceptible to the drug (ie, they may have an undetected NADH methemoglobin reductase deficiency). The case we have presented is unusual in that normal doses of phenazopyridine were given, no renal dysfunction was evident, and our patient had previously been given this drug without complication. Methemoglobinemia appeared to be the result of metabolic overload by multiple oxidants (phenazopyridine and lidocaine) of the normal reductase pathways in the erythrocytes. We saw no evidence to indicate that bupivacaine contributed to its development. Enzyme pathway changes induced by chemotherapy should be considered, though few studies have linked alterations of enzyme levels and pathways with chemotherapy and malignancy.

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.

Postoperative pulmonary toxicity associated with mitomycin-C therapy.

As more patients survive cancer, and as more sophisticated multidrug antineoplastic protocols are developed, the chances of an anesthesiologist's coming into contact with patients who have been treated with such protocols are increasing. The anesthesiologist who must administer anesthesia to a patient who has had chemotherapy must be cognizant of the particular antineoplastic agents that have the potential for producing occult pulmonary dysfunction. Anesthetic management of these cases must be carefully planned and titrated to prevent further lung injury.

Colloid oncotic pressure as a guide for the anesthesiologist in directing fluid therapy.

One useful but underused parameter of fluid replacement is colloid oncotic pressure. Colloid oncotic pressure (COP) is one of the Starling forces that maintain a balance between intravascular and extravascular fluid. Systemic and pulmonary circulations exhibit differences that limit the usefulness of COP manipulation in the treatment of pulmonary edema, especially that associated with hypoxic damage or pulmonary contusion. Systemic transcapillary fluid transport, however, is governed significantly by COP, and serial measurements of COP can serve as useful guides for colloid replacement. In this paper we present instances in which COP determinations were found to be clinically helpful, and discuss colloid replacement during surgery.

Macrophage-derived prostaglandin E modulation of the mixed-lymphocyte reaction: an anomaly of increased production and decreased T-cell susceptibility during tumor growth.

One-way mixed-lymphocyte reactions (MLR) were used to assess macrophage (M phi)-derived factor-mediated modulation of normal and tumor-bearing host (TBH) T-cell immune responsiveness. Normal and TBH M psi culture supernatants contained the inhibitory substance prostaglandin E (PGE) in concentrations of 10(-8) to 10(-9) M, with TBH M phi supernatant containing approximately twice the amount of PGE as its normal counterpart. Normal and TBH MLR reactivity were both suppressed by the addition of normal host M phi supernatant. However, TBH T cells were less inhibited by TBH M phi supernatant (55%) as compared to normal host T cells (73%). Although dialyzed M phi supernatants were less inhibitory (17-19%) on normal host T-cell MLR reactivity, TBH T-cell responses were enhanced (20-46%). Indomethacin or eicosatetraynoic acid treatment of M phi reduced PGE levels in the supernatants and in general enhanced MLR reactivity. When PGE1 and PGE2 were titrated in the MLR, normal host T lymphocytes were more susceptible to inhibition than were TBH. Concentrations of PGE1 and PGE2 comparable to that found in normal host M phi supernatants caused approximately 38% inhibition whereas whole M phi supernatants decreased MLR reactivity by greater than 70%, suggesting that another factor(s) was necessary to account for the additional M phi-mediated suppression of lymphocyte function. Isoelectric focusing was used to fractionate normal host M phi supernatant. Two factors with isoelectric points in the pH ranges 7.0-8.5 and 4.5-5.0 were inhibitory in the MLR. An enhancing factor was also identified with an pI in the range of pH 6.0-7.0. These data suggest that TBH M phi-derived PGE production was increased over its normal counterpart, but that TBH T cells were less susceptible to its effect and an additional factor(s) was working in concert with PGE.

An enzyme-linked immunosorbent assay for zein and other proteins using unconventional solvents for antigen adsorption.

An enzyme-linked immunosorbent assay (ELISA) performed in polystyrene microtiter plates that can detect and quantitate the maize prolamin zein is described. The assay yields positive reactions with as little as 1 ng of antigen and uses solvents not ordinarily employed in ELISA methods. A systematic investigation of zein adsorption to polystyrene in various solvents supports the hypothesis that antigen binding occurs through nonpolar interactions. The method was also used to determine structural relationships among three zein polypeptides differing in size and charge. Additional experiments indicate that a number of soluble proteins are absorbed to polystyrene in the denaturing agent urea and retain immunological reactivity. The retention of antigen reactivity after solubilization in 6-8 M urea suggests that ELISA methods may be applicable to other proteins which are insoluble, or rendered insoluble, in aqueous buffers.