Immune-Related Adverse Events in PD-1 Treated Melanoma and Impact Upon Anti-Tumor Efficacy: A Real World Analysis.

BACKGROUND: Anti-PD-1 immune checkpoint inhibitor (ICI) therapy has revolutionized the treatment of melanoma by producing durable long-term responses in a subset of patients. ICI-treated patients develop unique toxicities - immune related adverse events (irAEs) - that arise from unrestrained immune activation. The link between irAE development and clinical outcome in melanoma and other cancers is inconsistent; and little data exists on the occurrence of multiple irAEs. We sought to characterize development of single and multiple irAEs, and association of irAE(s) development with clinical variables and impact upon outcomes in advanced melanoma patients treated with anti-PD-1 ICIs. METHODS: We conducted a retrospective study of 190 patients with metastatic melanoma treated with single-agent anti-PD-1 ICI therapy between June 2014 and August 2020 at a large integrated network cancer center identified through retrospective review of pharmacy records. irAEs were graded based on the Common Terminology Criteria for Adverse Events (CTCAE) version 5.0. RESULTS: 190 patients were evaluated of whom 114 patients (60.0%) experienced ≥1 irAE, including 30 (15.8%) with grade 3/4 irAEs. The occurrence of any irAE was strongly associated with the development of investigator-assessed response to anti-PD-1 therapy (p < 0.0001); whether evaluated by current (p=0.0082) or best (p=0.0001) response. In patients with ≥2 irAEs, distinct patterns were observed. Median progression-free survival (PFS) and overall survival (OS) were greater in those with any irAE compared to those without (PFS, 28 months vs. 5 months, p < 0.0001; OS, not reached vs. 9 months, p < 0.0001). Development of ≥2 irAEs had a trend towards improved PFS and OS compared to those who developed a single irAE, although this did not reach statistical significance (p=0.2555, PFS; p=0.0583, OS). Obesity but not age or gender was distinctly associated with irAE development. CONCLUSIONS: In this study, we demonstrated that irAE occurrence was significantly associated with response to anti-PD-1 therapy and improved PFS/OS. Those who developed multiple irAEs had a trend towards improved PFS and OS compared to those who developed only a single irAE. Increased BMI but neither age nor gender were associated with irAE development. Distinct patterns of irAEs observed suggest shared etiopathogenetic mechanisms.

Neoadjuvant ipilimumab (3 mg/kg or 10 mg/kg) and high dose IFN-α2b in locally/regionally advanced melanoma: safety, efficacy and impact on T-cell repertoire.

BACKGROUND: Neoadjuvant immunotherapy utilizing novel combinations has the potential to transform the standard of care for locally/regionally advanced melanoma. We hypothesized that neoadjuvant ipilimumab in combination with high dose IFNα2b (HDI) is safe and associated with durable pathologic complete responses (pCR). METHODS: Patients with locally/regionally advanced melanoma were randomized to ipilimumab 3 or 10 mg/kg × 4 doses bracketing definitive surgery, then every 12 weeks × 4. HDI was given concurrently. We evaluated the safety and efficacy of the combination with ipilimumab 3 or 10 mg/kg. The impact on T-cell fraction and clonality were investigated in tumor and blood. RESULTS: Thirty patients (age 37-76), 15 each at 3 and 10 mg/kg, 18 male and 12 female were treated. Considering immune related adverse events (irAEs) of interest, more grade 3/4 irAEs were seen with ipilimumab 10 mg/kg versus 3 mg/kg (p = 0.042). Among 28 evaluable patients, 11 relapsed, of whom 5 died. Median follow-up for 17 patients who have not relapsed was 32 months. The radiologic preoperative response rate was 36% (95% CI, 21-54); 4 patients at ipilimumab 3 mg/kg and 6 at 10 mg/kg and 2 (at 10 mg/kg) later relapsed. The pCR was 32% (95% CI, 18-51); 5 patients at ipilimumab 3 mg/kg and 4 at 10 mg/kg and one (at 3 mg/kg) had a late relapse. In patients with pCR, T-cell fraction was significantly higher when measured in primary melanoma tumors (p = 0.033). Higher tumor T-cell clonality in primary tumor and more so following neoadjuvant therapy was significantly associated with improved relapse free survival. CONCLUSIONS: Neoadjuvant ipilimumab-HDI was relatively safe and exhibited promising tumor response rates with an associated measurable impact on T-cell fraction and clonality. Most pCRs were durable supporting the value of pCR as a primary endpoint in neoadjuvant immunotherapy trials. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01608594 . Registered 31 May 2012.

A unique gene expression signature is significantly differentially expressed in tumor-positive or tumor-negative sentinel lymph nodes in patients with melanoma.

The purpose of this study was to learn whether molecular characterization through gene expression profiling of node-positive and node-negative sentinel lymph nodes (SLNs) in patients with clinical stage I and II melanoma may improve the understanding of mechanisms of metastasis and identify gene signatures for SLNs/SLNs that correlate with diagnosis or clinical outcome. Gene expression profiling was performed on SLN biopsies of 48 (24 SLN and 24 SLN) patients (T3a/b-T4a/b) who underwent staging of SLNs using transcriptome profiling analysis on 5 µm sections of fresh SLNs. U133A 2.0 Affymetrix gene chips were used. Significance analysis of microarrays was used to test the association between gene expression level and SLN status. Genes with fold change more than 1.5 and q value less than 0.05 were considered differentially expressed. Pathway analysis was performed using Ingenuity Pathway Analysis. The Benjamini and Hochberg method was used to adjust for multiple testing in pathway analysis. We identified 89 probe sets that were significantly differentially expressed (1.5-27-fold; q<0.05). Upon performing the pathway analysis, it was found that 25 genes were common among the most significant and biologically relevant canonical pathways. The molecules and pathways that achieved differential expression of highest statistical significance were notably related to melanoma and its microenvironment and to signaling pathways implicated in immunosuppression and development of cancer. A 25-gene signature is significantly differentially expressed between SLN and SLN and is related to melanoma oncogenesis and immunosuppression. The identified expression profile provides a signature of melanoma nodal involvement. These findings warrant further investigation into the mechanisms of metastasis, melanoma metastasis diagnosis, and prediction of outcome.

A Pilot Study to Validate the Burn Center Pediatric Early Warning Score Tool in Clinical Practice.

The pediatric early warning score (PEWS) tool helps providers to detect subtle clinical deterioration in non-intensive care unit pediatric patients and intervene early to prevent significant adverse outcomes. Although widely used in general pediatrics, limited studies report on its validation; none report on use with burn-injured patients. New York-Presbyterian/Weill Cornell Medical Center modified a general PEWS system to a burn-specific PEWS and integrated its use into standard practice. This study investigated the external validity of the PEWS process in clinical practice. Fifty cases of patients aged 0 to 15.9 years admitted between January 2012 and June 2013, whose length of stay (LOS) more than 3 days were selected for review from this cohort of n equal to 187. Demographics, total PEWS and score changes, and compliance with PEWS documentation and with resultant interventions were reviewed. Continuous variables are presented as mean ± SD, P less than 0.05. Mean age, burn size, and LOS were 3.2 ± 3.3 years, 4.8 ± 5.7%, and 9.8 ± 7.0 days; 26% required grafting, and 50% were male. No mortalities occurred. One thousand six hundred and twelve PEWS from 1745 opportunities were documented (92.4%). For all PEWS (n = 1612) and PEWS greater than 0 (n = 912), means were 0.9 ± 1.2 and 1.6 ± 1.2, respectively. Among the 162 PEWS increase events, intake (54.1%) and output (4.5%) parameters increased most commonly. Of these, 129 PEWS increases (79.6%) were followed by an intervention that most commonly included text notation of score increase (93.7%), physician/physician assistant notification (70.5%), and feeding-tube insertion (25.6%). Patients with PEWS greater than 0 had similar age, LOS, and larger burn size (5.2% vs 1.4%, P < 0.05) than those with PEWS equal to 0. Compliance with PEWS performance and resultant actions based on score increases are high. Data support that even small changes in burn-injury specific PEWS stimulate provider discussion and intervention and support its validation; further studies on its effect on practice are warranted.

Tumor associated PD-L1 expression pattern in microscopically tumor positive sentinel lymph nodes in patients with melanoma.

BACKGROUND: Characterization of PD-L1 expression within clinically/radiologically negative but microscopically tumor positive sentinel lymph nodes (SLN) is important to our understanding of the relevance of this immune checkpoint pathway for adjuvant therapy. METHODS: Patients included had primary cutaneous melanoma, Breslow thickness of 2.01-4.0 or >4 mm with or without tumor ulceration (T3a, T3b, T4a, T4b). All patients had microscopically tumor positive SLN. Hematoxylin and eosin (H&E) staining was performed, followed by PD-L1 immunohistochemical (IHC) staining using a preliminary IHC assay with anti-PD-L1 antibody clone 22C3. The slides were separately evaluated by two pathologists (JY and CG). Samples containing metastatic melanoma lesions were scored separately for PD-L1 expression in intratumoral and peritumoral locations, by utilizing two scoring methods. RESULTS: Twenty-four patients where metastatic melanoma presence in the SLN was confirmed by H&E review of the cut sections were included in the final analysis of PD-L1 expression. SLN tumor size ranged from 1 to 2 mm. For three patients, the melanin content was too high to confidently assign a PD-L1 score. For the remaining 21 patients, all had some evidence of either intratumoral or peritumoral PD-L1 expression. The frequency of intratumoral tumor-associated PD-L1 expression was: 0 % of tumor cells (3 pts, 14 %); <1 % (5 pts, 24 %); 1-10 % (6 pts, 29 %) and >10 % (7 pts, 33 %). CONCLUSIONS: Tumor-associated PD-L1 expression is readily detectable within melanoma micrometastases in the SLN of the majority of patients. These results support the testing of a therapeutic role for PD1/PD-L1 inhibition in the adjuvant setting, targeting melanoma micrometastases.

Adaptation of a commonly used, chemically defined medium for human embryonic stem cells to stable isotope labeling with amino acids in cell culture.

Metabolic labeling with stable isotopes is a prominent technique for comparative quantitative proteomics, and stable isotope labeling with amino acids in cell culture (SILAC) is the most commonly used approach. SILAC is, however, traditionally limited to simple tissue culture regimens and only rarely employed in the context of complex culturing conditions as those required for human embryonic stem cells (hESCs). Classic hESC culture is based on the use of mouse embryonic fibroblasts (MEFs) as a feeder layer, and as a result, possible xenogeneic contamination, contribution of unlabeled amino acids by the feeders, interlaboratory variability of MEF preparation, and the overall complexity of the culture system are all of concern in conjunction with SILAC. We demonstrate a feeder-free SILAC culture system based on a customized version of a commonly used, chemically defined hESC medium developed by Ludwig et al. and commercially available as mTeSR1 [mTeSR1 is a trade mark of WiCell (Madison, WI) licensed to STEMCELL Technologies (Vancouver, Canada)]. This medium, together with adjustments to the culturing protocol, facilitates reproducible labeling that is easily scalable to the protein amounts required by proteomic work flows. It greatly enhances the usability of quantitative proteomics as a tool for the study of mechanisms underlying hESCs differentiation and self-renewal. Associated data have been deposited to the ProteomeXchange with the identifier PXD000151.

Transcript profiling reveals mechanisms for lipid conservation during diapause in the mosquito, Aedes albopictus.

The Asian tiger mosquito, Aedes albopictus, is a medically important invasive species whose geographic distribution has expanded dramatically during the past 20 years, and one of the key elements of its success is its capacity to survive long distance transport as a diapausing pharate first instar larva, encased within the chorion of the egg. We report that pharate larvae entering diapause are larger and contain 30% more lipid than their nondiapausing counterparts. To improve our understanding of the molecular regulation of lipid metabolism during diapause, we assessed the relative mRNA abundance of 21 genes using qRT-PCR. Elevated expression of lipid storage droplet protein 2 during embryonic development likely contributes to the higher amounts of lipid we noted in diapausing individuals. The conservation of lipids during diapause is reflected in downregulation of genes involved in lipid catabolism, including lipase 2, lipase 3, lipase 4, acyl-CoA dehydrogenase 4, and isovaleryl-CoA dehydrogenase. Two genes involved in fatty acid synthesis and modification, Δ(9)-desaturase, and fatty acyl-CoA elongase, were both upregulated in diapausing pharate larvae, suggesting roles for their gene products in generating unsaturated fatty acids to enhance membrane fluidity at low temperatures and generating precursors to the surface hydrocarbons needed to resist desiccation, respectively. Together, the results point to substantial distinctions in lipid metabolism within the embryo as a consequence of the diapause program, and these differences occur both before the actual onset of diapause as well as during the diapause state.