A phase Ib study of interleukin-2 plus pembrolizumab for patients with advanced melanoma.

Introduction: High-dose interleukin-2 (HD IL-2) and pembrolizumab are each approved as single agents by the U.S. F.D.A. for the treatment of metastatic melanoma. There is limited data using the agents concurrently. The objectives of this study were to characterize the safety profile of IL-2 in combination with pembrolizumab in patients with unresectable or metastatic melanoma. Methods: In this Phase Ib study, patients received pembrolizumab (200 mg IV every 3 weeks) and escalating doses of IL-2 (6,000 or 60,000 or 600,000 IU/kg IV bolus every 8 hours up to 14 doses per cycle) in cohorts of 3 patients. Prior treatment with a PD-1 blocking antibody was allowed. The primary endpoint was the maximum tolerated dose (MTD) of IL-2 when co-administered with pembrolizumab. Results: Ten participants were enrolled, and 9 participants were evaluable for safety and efficacy. The majority of the evaluable participants (8/9) had been treated with PD-1 blocking antibody prior to enrollment. Patients received a median of 42, 22, and 9 doses of IL-2 in the low, intermediate, and high dose cohorts, respectively. Adverse events were more frequent with increasing doses of IL-2. No dose limiting toxicities were observed. The MTD of IL-2 was not reached. One partial response occurred in 9 patients (11%). The responding patient, who had received treatment with an anti-PD-1 prior to study entry, was treated in the HD IL-2 cohort. Discussion: Although the sample size was small, HD IL-2 therapy in combination with pembrolizumab appears feasible and tolerable. Clinical trial registration: ClinicalTrials.gov, identifier NCT02748564.

A phase 1b single-arm trial of intratumoral oncolytic virus V937 in combination with pembrolizumab in patients with advanced melanoma: results from the CAPRA study.

BACKGROUND: CAPRA (NCT02565992) evaluated Coxsackievirus A21 (V937) + pembrolizumab for metastatic/unresectable stage IIIB-IV melanoma. METHODS: Patients received intratumoral V937 on days 1, 3, 5, and 8 (then every 3 weeks [Q3W]) and intravenous pembrolizumab 2 mg/kg Q3W from day 8. Primary endpoint was safety. RESULTS: Median time from first dose to data cutoff was 32.0 months. No dose-limiting toxicities occurred; 14% (5/36) of patients experienced grade 3‒5 treatment-related adverse events. Objective response rate was 47% (complete response, 22%). Among 17 responders, 14 (82%) had responses ≥ 6 months. Among 8 patients previously treated with immunotherapy, 3 responded (1 complete, 2 partial). Responses were associated with increased serum CXCL10 and CCL22, suggesting viral replication contributes to antitumor immunity. For responders versus nonresponders, there was no difference in baseline tumor PD-L1 expression, ICAM1 expression, or CD3+ infiltrates. Surprisingly, the baseline cell density of CD3+CD8- T cells in the tumor microenvironment was significantly lower in responders compared with nonresponders (P = 0.0179). CONCLUSIONS: These findings suggest responses to this combination may be seen even in patients without a typical "immune-active" microenvironment. TRIAL REGISTRATION NUMBER: NCT02565992.

Acquired methemoglobinemia presenting to the pediatric emergency department: a clinical challenge.

OBJECTIVES: Acquired methemoglobinemia (MetHb) is an uncommon presentation of cyanosis in the pediatric emergency department (ED), making its diagnosis and management a clinical challenge. Through this case series we hope to improve clinician ability to recognize the potential for MetHb in pediatric ED patients and to avoid overlooking this important cause of cyanosis. METHODS: This was a case series using a health records review, investigating patients diagnosed with MetHb at our pediatric ED during 2007-2018. We included only cases with methemoglobin saturation ≥5%. RESULTS: Ten patients were diagnosed with MetHb in our pediatric ED during the study period. Five had an underlying hematologic disease who received a pharmacologic trigger known to induce MetHb as well (four dapsone, one rasburicase). The other five patients were previously healthy, who presented with a clinical picture of hemolytic anemia, all of whom were diagnosed with previously unknown glucose-6-phosphate dehydrogenase (G6PD) deficiency. Two of the patients received methylene blue, and five patients needed packed red blood cells. All of the patients survived the acute MetHb episode. CONCLUSION: Acquired MetHb in the pediatric ED is a rare but important cause of cyanosis. Diagnosis and management of acute, acquired MetHb in the ED requires a high level of suspicion, and a background knowledge of the common precipitants and underlying conditions associated with this condition. We hope this case series will help ED physicians to consider MetHb in pediatric patients presenting with cyanosis and persistent hypoxia. Exposure to known precipitants (e.g., medications and foods), particularly in the setting of active treatment for malignancy or with symptoms of hemolytic anemia should further increase suspicion.

Avelumab Immunotherapy: Management of Adverse Events Associated With New Treatment for Merkel Cell Carcinoma.

BACKGROUND: Metastatic Merkel cell carcinoma (mMCC) is a rare skin cancer with poor prognosis. Avelumab is the first approved treatment option for patients with mMCC. Immune checkpoint inhibitors, such as avelumab, are associated with unique toxicities that can be effectively addressed with prompt recognition and appropriate management. OBJECTIVES: This article discusses the use of avelumab for the treatment of mMCC and management of associated toxicities. METHODS: Literature on mMCC disease state and clinical trial data for avelumab were reviewed. FINDINGS: Avelumab has been investigated in patients with mMCC either following disease progression after one or more prior lines of chemotherapy or no prior systemic therapy. These patients experience clinically meaningful benefit. About 70% of patients receiving avelumab experience treatment-related adverse events. Given the limited benefit of chemotherapy, managing symptoms related to avelumab is key to administering this effective treatment to patients with mMCC.

Functions of stone cells and oleoresin terpenes in the conifer defense syndrome.

Conifers depend on complex defense systems against herbivores. Stone cells (SC) and oleoresin are physical and chemical defenses of Sitka spruce that have been separately studied in previous work. Weevil oviposit at the tip of the previous year's apical shoot (PYAS). We investigated interactions between weevil larvae and trees in controlled oviposition experiments with resistant (R) and susceptible (S) Sitka spruce. R trees have an abundance of SC in the PYAS cortex. SC are mostly absent in S trees. R trees and S trees also differ in the composition of oleoresin terpenes. Transcriptomes of R and S trees revealed differences in long-term weevil-induced responses. Performance of larvae was significantly reduced on R trees compared with S trees under experimental conditions that mimicked natural oviposition behavior at apical shoot tips and may be attributed to the effects of SC. In oviposition experiments designed for larvae to feed below the area of highest SC abundance, larvae showed an unusual feeding behavior and oleoresin appeared to function as the major defense. The results support a role for both SC and oleoresin terpenes and possible synergies between these traits in the defense syndrome of weevil-resistant Sitka spruce.

Quantitative Detection and Resolution of BRAF V600 Status in Colorectal Cancer Using Droplet Digital PCR and a Novel Wild-Type Negative Assay.

A need exists for robust and cost-effective assays to detect a single or small set of actionable point mutations, or a complete set of clinically informative mutant alleles. Knowledge of these mutations can be used to alert the clinician to a rare mutation that might necessitate more aggressive clinical monitoring or a personalized course of treatment. An example is BRAF, a (proto)oncogene susceptible to either common or rare mutations in codon V600 and adjacent codons. We report a diagnostic technology that leverages the unique capabilities of droplet digital PCR to achieve not only accurate and sensitive detection of BRAF(V600E) but also all known somatic point mutations within the BRAF V600 codon. The simple and inexpensive two-well droplet digital PCR assay uses a chimeric locked nucleic acid/DNA probe against wild-type BRAF and a novel wild-type-negative screening paradigm. The assay shows complete diagnostic accuracy when applied to formalin-fixed, paraffin-embedded tumor specimens from metastatic colorectal cancer patients deficient for Mut L homologue-1.

A yeast phenomic model for the gene interaction network modulating CFTR-ΔF508 protein biogenesis.

BACKGROUND: The overall influence of gene interaction in human disease is unknown. In cystic fibrosis (CF) a single allele of the cystic fibrosis transmembrane conductance regulator (CFTR-[increment]F508) accounts for most of the disease. In cell models, CFTR-[increment]F508 exhibits defective protein biogenesis and degradation rather than proper trafficking to the plasma membrane where CFTR normally functions. Numerous genes function in the biogenesis of CFTR and influence the fate of CFTR-[increment]F508. However it is not known whether genetic variation in such genes contributes to disease severity in patients. Nor is there an easy way to study how numerous gene interactions involving CFTR-[increment]F would manifest phenotypically. METHODS: To gain insight into the function and evolutionary conservation of a gene interaction network that regulates biogenesis of a misfolded ABC-transporter, we employed yeast genetics to develop a "phenomic" model, in which the CFTR-[increment]F508-equivalent residue of a yeast homolog is mutated (Yor1-[increment]F670), and where the genome is scanned quantitatively for interaction. We first confirmed that Yor1-[increment]F undergoes protein misfolding and has reduced half-life, analogous to CFTR-[increment]F. Gene interaction was then assessed quantitatively by growth curves for all ~5000 double mutants, based on alteration in the dose response to growth inhibition by oligomycin, a toxin extruded from the cell at the plasma membrane by Yor1. RESULTS: From a comparative genomic perspective, yeast gene interaction influencing Yor1-[increment]F biogenesis was representative of human homologs previously found to modulate processing of CFTR-[increment]F in mammalian cells. Additional evolutionarily conserved pathways were implicated by the study, and a [increment]F-specific pro-biogenesis function of the recently discovered ER Membrane Complex (EMC) was evident from the yeast screen. This novel function was validated biochemically by siRNA of an EMC ortholog in a human cell line expressing CFTR-[increment]F508. The precision and accuracy of quantitative high throughput cell array phenotyping (Q-HTCP), which captures tens of thousands of growth curves simultaneously, provided powerful resolution to measure gene interaction on a phenomic scale, based on discrete cell proliferation parameters. CONCLUSION: We propose phenomic analysis of Yor1-[increment]F as a model for investigating gene interaction networks that can modulate cystic fibrosis disease severity. Although the clinical relevance of the Yor1-[increment]F gene interaction network for cystic fibrosis remains to be defined, the model appears to be informative with respect to human cell models of CFTR-[increment]F. Moreover, the general strategy of yeast phenomics can be employed in a systematic manner to model gene interaction for other diseases relating to pathologies that result from protein misfolding or potentially any disease involving evolutionarily conserved genetic pathways.

Synthetic lethality of cohesins with PARPs and replication fork mediators.

Synthetic lethality has been proposed as a way to leverage the genetic differences found in tumor cells to affect their selective killing. Cohesins, which tether sister chromatids together until anaphase onset, are mutated in a variety of tumor types. The elucidation of synthetic lethal interactions with cohesin mutants therefore identifies potential therapeutic targets. We used a cross-species approach to identify robust negative genetic interactions with cohesin mutants. Utilizing essential and non-essential mutant synthetic genetic arrays in Saccharomyces cerevisiae, we screened genome-wide for genetic interactions with hypomorphic mutations in cohesin genes. A somatic cell proliferation assay in Caenorhabditis elegans demonstrated that the majority of interactions were conserved. Analysis of the interactions found that cohesin mutants require the function of genes that mediate replication fork progression. Conservation of these interactions between replication fork mediators and cohesin in both yeast and C. elegans prompted us to test whether other replication fork mediators not found in the yeast were required for viability in cohesin mutants. PARP1 has roles in the DNA damage response but also in the restart of stalled replication forks. We found that a hypomorphic allele of the C. elegans SMC1 orthologue, him-1(e879), genetically interacted with mutations in the orthologues of PAR metabolism genes resulting in a reduced brood size and somatic cell defects. We then demonstrated that this interaction is conserved in human cells by showing that PARP inhibitors reduce the viability of cultured human cells depleted for cohesin components. This work demonstrates that large-scale genetic interaction screening in yeast can identify clinically relevant genetic interactions and suggests that PARP inhibitors, which are currently undergoing clinical trials as a treatment of homologous recombination-deficient cancers, may be effective in treating cancers that harbor cohesin mutations.

The type II collagen N-propeptide, PIIBNP, inhibits cell survival and bone resorption of osteoclasts via integrin-mediated signaling.

OBJECTIVE: Type IIB procollagen is characteristic of cartilage, comprising 50% of the extracellular matrix. The NH(2)-propeptide of type IIB collagen, PIIBNP, can kill tumor cells via binding to integrins α(V)ß(3) and α(V)ß(5). As osteoclasts rely on α(V)ß(3) integrins for function in bone erosion, we sought to determine whether PIIBNP could inhibit osteoclast function. METHODS: We undertook in vitro and in vivo experiments to evaluate both osteoblast and osteoclast functions in the presence of recombinant PIIBNP. Adhesion of osteoclasts to PIIBNP was analyzed by staining of attached cells with crystal violet. PIIBNP-induced cell death was evaluated by counting Trypan Blue stained cells. The mechanism of cell death was evaluated by DNA fragmentation, TUNEL staining and western blotting to detect cleaved caspases. To determine the role of α(V)ß(3) integrin, osteoclasts were pretreated with α(V) or ß(3) integrin specific siRNA before the treatment with PIIBNP. To explore PIIBNP function in vivo, a lipopolysaccharide-induced mouse calvaria lysis model was employed. RESULTS: Osteoclasts adhered to PIIBNP via an RGD-mediated mechanism. When osteoclasts were plated on extracellular matrix proteins, PIIBNP induced apoptosis of osteoclasts via caspase 3/8 activation. Osteoblasts and macrophages were not killed. Reduction of α(V) or ß(3) integrin levels on osteoclasts by siRNA reduced cell death in a dose-dependent manner. In vivo, PIIBNP could inhibit bone resorption. CONCLUSION: We conclude that PIIBNP can inhibit osteoclast survival and bone resorption via signal transduction through the α(V)ß(3) integrins. Because of this property and the cell specificity, we propose that PIIBNP may play a role in vivo in protecting cartilage from osteoclast invasion and also could be a new therapeutic strategy for decreasing bone loss.

Type IIB procollagen NH(2)-propeptide induces death of tumor cells via interaction with integrins alpha(V)beta(3) and alpha(V)beta(5).

Cartilage is resistant to tumor invasion. In the present study, we found that the NH(2)-propeptide of the cartilage-characteristic collagen, type IIB, PIIBNP, is capable of killing tumor cells. The NH(2)-propeptide is liberated into the extracellular matrix prior to deposition of the collagen fibrils. This peptide adheres to and kills cells from chondrosarcoma and cervical and breast cancer cell lines via the integrins alpha(v)beta(5) and alpha(v)beta(3). Adhesion is abrogated by blocking with anti alpha(v)beta(5) and alpha(v)beta(3) antibodies. When alpha(v) is suppressed by small intefering RNA, adhesion and cell killing are blocked. Normal chondrocytes from developing cartilage do not express alpha(v)beta(3) and alpha(v)beta(5) integrins and are thus protected from cell death. Morphological, DNA, and biochemical evidence indicates that the cell death is not by apoptosis but probably by necrosis. In an assay for invasion, PIIBNP reduced the number of cells crossing the membrane. In vivo, in a tumor model for breast cancer, PIIBNP was consistently able to reduce the size of the tumor.

Synthetic lethal genetic interactions that decrease somatic cell proliferation in Caenorhabditis elegans identify the alternative RFC CTF18 as a candidate cancer drug target.

Somatic mutations causing chromosome instability (CIN) in tumors can be exploited for selective killing of cancer cells by knockdown of second-site genes causing synthetic lethality. We tested and statistically validated synthetic lethal (SL) interactions between mutations in six Saccharomyces cerevisiae CIN genes orthologous to genes mutated in colon tumors and five additional CIN genes. To identify which SL interactions are conserved in higher organisms and represent potential chemotherapeutic targets, we developed an assay system in Caenorhabditis elegans to test genetic interactions causing synthetic proliferation defects in somatic cells. We made use of postembryonic RNA interference and the vulval cell lineage of C. elegans as a readout for somatic cell proliferation defects. We identified SL interactions between members of the cohesin complex and CTF4, RAD27, and components of the alternative RFC(CTF18) complex. The genetic interactions tested are highly conserved between S. cerevisiae and C. elegans and suggest that the alternative RFC components DCC1, CTF8, and CTF18 are ideal therapeutic targets because of their mild phenotype when knocked down singly in C. elegans. Furthermore, the C. elegans assay system will contribute to our knowledge of genetic interactions in a multicellular animal and is a powerful approach to identify new cancer therapeutic targets.

Prospective ranking of the sonographic markers for aneuploidy: data of 2143 prenatal cytogenetic diagnoses referred for abnormalities on ultrasound.

OBJECTIVE: To design a scheme to rank sonographic anomalies as indicators of aneuploidy and record the distribution of data from 2143 prenatal amniotic fluid/chorionic villous sample diagnoses referred for karyotyping because of fetal anomalies detected with ultrasound. METHODS: In all cases the records of sonographic anomalies were obtained prior to karyotyping. A cascade of seven prospective categories of ultrasound anomalies was chosen and the data were included in the highest compatible sonography category. The categories were in descending order: (I) combined central nervous system (CNS)/cranial shape and cardiac anomalies (excluding spina bifida and anencephaly); (II) key anomaly present (exomphalos/ intrauterine growth restriction/duodenal atresia/cystic hygroma/fetal hydrops/talipes--with other multiple anomalies); (III) CNS +/- other abnormality (excluding choroid plexus cyst, spina bifida, anencephaly); (IVa) increased nuchal translucency--first trimester +/- other abnormality; (IVb) increased nuchal thickening--second trimester +/- other abnormality; (V) cardiac anomaly +/- other abnormality; (VI) other markers of aneuploidy (pyelectasis/two vessel cord/echogenic bowel/short femur); and (VII) other (mostly isolated) malformations. RESULTS: There were 412/2143 (19.2%) chromosome abnormalities detected in this sonographically abnormal group. Overall, the prevalence of aneuploidy significantly ranged from 51 to 3% according to the above I-VII ultrasound categories and from approximately 1-80% for individual ultrasound anomalies. Likelihood ratios were derived for many ultrasound anomalies for several aneuploidy groups: trisomies of 13; 18; and 21; 45,X and 45,X mosaics; triploidy; other autosomal duplications and/or deletions; and other (than 45,X) sex chromosomal aneuploidies. CONCLUSION: It is suggested this data could be used to assist pre-procedural counselling of patients after the ultrasound scan in tertiary referral centres for prenatal cytogenetic diagnosis.

A second case of intrauterine growth retardation and primary hypospadias associated with a trisomy 22 placenta but with biparental inheritance of chromosome 22 in the fetus.

We report a case of severe intrauterine growth retardation (IUGR) and hypospadias in association with trisomy 22 diagnosed following chorionic villus sampling (CVS). Subsequent analysis of amniotic fluid cultures showed a normal male karyotype, 46,XY. As a previous case had been reported with similar abnormalities, in association with maternal uniparental disomy (UPD) 22, molecular studies were also performed. Microsatellite marker studies showed biparental inheritance. Follow-up studies after delivery showed a normal cell line in lymphocytes with the trisomy appearing to be confined to the placenta. The present case concurs with other earlier reports that maternal UPD for chromosome 22 has no impact on the phenotype. The features seen in the fetus are most likely the result of placental dysfunction due to trisomy, tissue-specific mosaicism and/or the effects of local growth restriction.