Determinants of overall survival in patients with metastatic uveal melanoma.

BACKGROUND: Despite improvements in the treatment of primary uveal melanoma (UM), patients with metastatic disease continue to exhibit poor survival. METHODS: A retrospective review of metastatic UM patients at Yale (initial cohort) and Memorial Sloan Kettering (validation cohort) was conducted. Cox proportional hazards regression was used to determine baseline factors that are associated with overall survival, including sex, Eastern Cooperative Oncology Group (ECOG) Performance Status Scale, laboratory measurements, metastasis location, and use of anti-CTLA-4 and anti-PD-1 therapies. Differences in overall survival were analyzed using Kaplan-Meier analysis. RESULTS: A total of 89 patients with metastatic UM were identified; 71 and 18, in the initial and validation cohorts, respectively. In the initial cohort, median follow-up was 19.8 months (range, 2-127 months) and median overall survival was 21.8 months (95% CI, 16.6-31.3). Female sex, anti-CTLA-4, and anti-PD-1 therapy were associated with better survival outcomes with adjusted death hazard ratios (HRs) of 0.40 (95% CI, 0.20-0.78), 0.44 (0.20-0.97), and 0.42 (0.22-0.84), respectively, whereas development of hepatic metastases and ECOG score ≥1 (per 1 U/L) were associated with worse survival outcomes with HRs of 2.86 (1.28-7.13) and 2.84 (1.29-6.09), respectively. In both the initial and validation cohorts, use of immune checkpoint inhibitors was associated with improved overall survival after adjusting for sex and ECOG score, with death HRs of 0.22 (0.08-0.56) and 0.04 (0.002-0.26), respectively. CONCLUSIONS: Development of extrahepatic-only metastases, ECOG of 0, immune checkpoint therapy, and female sex were each associated with more than 2-fold reductions in risk of death. PLAIN LANGUAGE SUMMARY: Metastatic uveal melanoma patients face limited treatment options and poor survival rates. Results from this retrospective analysis indicate that immune checkpoint inhibitors, such as anti-CTLA-4 and anti-PD-1 therapies, were associated with improved survival outcomes. Factors such as extrahepatic-only metastases, better baseline performance status, and female sex contributed to a more than 2-fold reduction in death risk. These findings highlight the potential of immunotherapy in treating metastatic uveal melanoma.

Epigenetic dysregulation from chromosomal transit in micronuclei.

Chromosomal instability (CIN) and epigenetic alterations are characteristics of advanced and metastatic cancers1-4, but whether they are mechanistically linked is unknown. Here we show that missegregation of mitotic chromosomes, their sequestration in micronuclei5,6 and subsequent rupture of the micronuclear envelope7 profoundly disrupt normal histone post-translational modifications (PTMs), a phenomenon conserved across humans and mice, as well as in cancer and non-transformed cells. Some of the changes in histone PTMs occur because of the rupture of the micronuclear envelope, whereas others are inherited from mitotic abnormalities before the micronucleus is formed. Using orthogonal approaches, we demonstrate that micronuclei exhibit extensive differences in chromatin accessibility, with a strong positional bias between promoters and distal or intergenic regions, in line with observed redistributions of histone PTMs. Inducing CIN causes widespread epigenetic dysregulation, and chromosomes that transit in micronuclei experience heritable abnormalities in their accessibility long after they have been reincorporated into the primary nucleus. Thus, as well as altering genomic copy number, CIN promotes epigenetic reprogramming and heterogeneity in cancer.

Effect of pH on the Early Development of the Biofouling Ascidian Ciona robusta.

Ocean acidification (OA) impacts the survival, fertilization, and community structure of marine organisms across the world. However, some populations or species are considered more resilient than others, such as those that are invasive, globally distributed, or biofouling. Here, we tested this assumption by investigating the effect of pH on the larval development of one such tunicate, Ciona robusta, which is currently exposed to a wide range of pH levels. Consistent with our hypothesis, C. robusta larvae developed and metamorphosed at a rate comparable to control (pH 8.0) at modest near-future conditions (pH 7.7) over a 58-hour period. However, development was stunted at the extreme low pH of 6.8 such that no embryo progressed beyond late cleavage after 58 hours. Interestingly, piecewise regression of the proportion of embryos at the most advanced stage at a given time point against pH identified a breakpoint with the highest pH (~pH 7.6) at around hatching. The variation in breakpoint pH throughout ontogeny highlighted that the sensitivity to decreasing pH differs significantly between developmental stages. More broadly, our results show that even a cosmopolitan, biofouling, invasive species could be negatively impacted by decreasing pH.

Validation of the Prognostic Usefulness of the Gene Expression Profiling Test in Patients with Uveal Melanoma.

PURPOSE: To validate the prognostic usefulness of gene expression profile (GEP) testing in patients with uveal melanoma. To determine whether combining tumor size with the GEP classification provides additional prognostic value. DESIGN: Retrospective analysis. PARTICIPANTS: Patients with a diagnosis of choroidal melanoma examined at Yale New Haven Hospital; University of California, San Diego; and Memorial Sloan Kettering Cancer Center. METHODS: Patients' demographic and clinical data and tumor characteristics were collected. Univariate and multivariate Cox hazard regression analysis were used to assess the association between tumor characteristics and GEP classification with metastasis as an outcome. MAIN OUTCOME MEASURES: Metastasis-free survival (MFS). RESULTS: Of the 337 individuals included in the study, 87 demonstrated metastases. The mean follow-up time was 37.2 (standard deviation [SD], 40.2) months for patients with metastases and 55.0 (SD, 49.3) months for those without metastases. Tumors of larger thickness and GEP class 2 (vs. class 1) were associated significantly with increased risk of metastasis. Tumor thickness showed better prognostic usefulness than GEP classification (Wald statistic, 40.7 and 24.2, respectively). Class 2 tumors with a thickness of 7.0 mm or more were associated with increased risk of metastasis than tumors with a thickness of < 7.0 mm (hazard ratio [HR], 3.23; 95% confidence interval [CI], 1.61-6.51), whereas class 1 tumors with a thickness of 9.0 mm or more were associated with increased risk of metastasis than tumors with a thickness of < 9.0 mm (HR, 2.07; 95% CI, 0.86-4.99). No difference in MFS was found between patients with class 1A tumors compared with those with class 1B tumors (P = 0.8). Patients with class 2 tumors showed an observed 5-year MFS of 47.5% (95% CI, 36.0%-62.8%). CONCLUSIONS: Tumor size was the most significant predictor of metastasis and provided additional prognostic value independent of GEP classification. In addition, rates of metastasis for class 2 tumors were lower than estimates reported by Castle Bioscience, and no difference in rates of metastasis were found between class 1A and 1B tumors. This indicates that tumor size should be accounted for when relying on GEP for prognostication and that patients with GEP class 1A or 1B tumors may benefit from the same metastatic surveillance protocols. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Apoptotic effects of platelet factor VIII on megakaryopoiesis: implications for a modified human FVIII for platelet-based gene therapy.

BACKGROUND: Ectopically expressed B-domainless factor VIII in megakaryocytes is stored in α-granules, is effective in a number of murine hemostatic models, and is protected from circulating inhibitors. However, this platelet (p) FVIII has different temporal-spatial availability from plasma FVIII, with limited efficacy in other murine hemostatic models. OBJECTIVES AND METHODS: We sought to improve pFVIII hemostatic efficacy by expressing canine (c) FVIII, which has higher stability and activity than human (h) FVIII in FVIII(null) mice. RESULTS AND CONCLUSIONS: We found that pcFVIII was more effective than phFVIII at restoring hemostasis, but peak pcFVIII antigen levels were lower and were associated with greater megakaryocyte apoptosis than phFVIII. These new insights suggest that pFVIII gene therapy strategies should focus on enhancing activity rather than levels. We previously showed that modification of the PACE/furin cleavage site in hFVIII resulted in secretion of hFVIII primarily as a single-chain molecule with increased biological activity. In megakaryocytes, this variant was expressed at the same level as phFVIII with a lentiviral bone marrow transplant approach to reconstitute FVIII(null) mice, but was more effective, resulting in near-normal hemostasis in the cremaster laser injury model. These studies may have implications for pFVIII gene therapy in hemophilia A.

Turbulent pipe flow at extreme Reynolds numbers.

Both the inherent intractability and complex beauty of turbulence reside in its large range of physical and temporal scales. This range of scales is captured by the Reynolds number, which in nature and in many engineering applications can be as large as 10(5)-10(6). Here, we report turbulence measurements over an unprecedented range of Reynolds numbers using a unique combination of a high-pressure air facility and a new nanoscale anemometry probe. The results reveal previously unknown universal scaling behavior for the turbulent velocity fluctuations, which is remarkably similar to the well-known scaling behavior of the mean velocity distribution.

Measurement of local dissipation scales in turbulent pipe flow.

Local dissipation scales are a manifestation of the intermittent small-scale nature of turbulence. We report the first experimental evaluation of the distribution of local dissipation scales in turbulent pipe flows for a range of Reynolds numbers: 2.4x10(4)

Brain metastasis from a primary liposarcoma of the digit: case report.

Soft-tissue sarcomas of the digit are uncommon. We herein report on a patient with a de-novo subungual right thumb liposarcoma with subsequent failure in the brain. The pertinent literature and recommendations for management are presented.

Characterization of ricin heterogeneity by electrospray mass spectrometry, capillary electrophoresis, and resonant mirror.

Electrospray mass spectrometry (ES/MS), capillary-zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), and multianalyte resonant mirror are used to evaluate the heterogeneity of samples of ricin toxins extracted from five horticultural varieties of Ricinus communis seeds: R. communis zanzibariensis, carmencita, impala, sanguineus, and gibsonii. The investigation is also extended to the geographical provenance of the beans. Combining mass spectrometry, CE techniques, and resonant mirror results in a powerful analytical tool capable to characterize and differentiate between different varieties of ricin toxins. Each technique complements the others, adding another level of information. This study reveals a large extent of heterogeneity for each cultivar, demonstrating that ricin toxins consist of a series of glycosylated proteins most likely originating from a multigene family. By combining these techniques, it is possible to differentiate between zanzibariensis and the other four varieties, and that variations in the functional characteristics may be observed between the different cultivars. This study demonstrates that knowledge of the variety of R. communis beans used and their geographical provenance is essential before any type of investigation of ricin toxins is carried out. Consequently, any unusual behavior observed can only be attributed to that particular cultivar studied and not automatically extended to include all R. communis varieties.

Comparison of the quality of protection elicited by toxoid and peptide liposomal vaccine formulations against ricin as assessed by markers of inflammation.

Ricin is a very toxic substance which inhibits protein synthesis and produces severe tissue damage and inflammation. It is very potent when inhaled as an aerosol and protection has been examined in a series of studies using vaccine candidates including a formaldehyde inactivated ricin toxoid and the A chain of ricin, a polypeptide equivalent to half of the toxin molecule. Initially, subcutaneous injections of both compounds were found to protect against inhaled ricin but not without some subsequent adverse signs. Intra-pulmonary vaccination using liposomal formulations of these compounds was investigated with a view to improving lung condition following challenge. Using the humoral and local pulmonary immune responses as indices of vaccine performance, no significant difference between toxoid or peptide vaccines was found. In the third and current study, the quality of lung protection by vaccines was assessed using markers of inflammation. Thus, the profiles of inflammatory cell and protein influx into the lung were determined following intratracheal (i.t.) challenge with ricin of rats treated with liposomal vaccine formulations. Results showed that liposomal ricin toxoid offered a better quality of protection with a significantly lower influx of polymorphonuclear leucocytes (neutrophils) and little pulmonary oedema compared with the A chain/liposome formulation. Further, there was no significant difference between the quality of protection offered by the A chain when administered subcutaneously or locally into the lung by i.t. instillation. Liposomal ricin toxoid is a good candidate vaccine and optimised pulmonary delivery by inhalation should be further examined.

Local and systemic responses against ricin toxin promoted by toxoid or peptide vaccines alone or in liposomal formulations.

The objective of this study was to develop a vaccine which would ultimately protect man from the lethal effects of inhaled ricin toxin. Porton rats have previously been protected from lethal quantities of inhaled ricin by subcutaneous (s.c.) ricin toxoid vaccine, but not without lung damage. This situation might be improved by an alternative vaccine such as the A chain of ricin, already known to protect against inhaled ricin. Another option would be to improve respiratory tract immunity by local vaccination in conjunction with liposomal formulation with a view to enhancing lung secretion of immune IgA. While boosted s.c. doses of ricin toxoid or A chain produced indistinguishable systemic immune responses 3 weeks later, when delivered by the intratracheal (i.t.) route, the A chain failed to elicit a specific immune response, unlike ricin toxoid. This situation was overcome by liposomal formulations and although ricin toxoid was readily encapsulated in liposomes, A chain was not. However, by simply mixing A chain and liposomes in the same weight ratio determined for liposomal toxoid, systemic immune responses for each formulation were indistinguishable 1 week after boosting. Ricin antibody responses in lung fluid monitored 1, 3, 7 and 14 days after i.t. challenge with ricin were statistically indistinguishable, but the group vaccinated with liposomal toxoid secreted 28.7% IgA compared with 0.9-14.9% for the A chain liposomal group. From this, it might be anticipated that the lungs would be better protected by liposomally-encapsulated ricin toxoid than by the A chain-liposome mixture.

Liposomally-encapsulated ricin toxoid vaccine delivered intratracheally elicits a good immune response and protects against a lethal pulmonary dose of ricin toxin.

A small study was performed to examine whether the instillation of ricin toxoid vaccine into the lungs of Porton rats offered protection from lethal effects of subsequent intratracheal challenge with ricin toxin. Further the immune response to liposomally-encapsulated vaccine and the protection offered was compared with vaccine either adsorbed to Alhydrogel adjuvant or as a simple aqueous solution. The formaldehyde-treated ricin toxin vaccine (RTV) was administered at two dose levels, 500 and 100 micrograms kg-1 body weight to groups of rats, on two occasions by intratracheal instillation. Liposomally-encapsulated vaccine (LRTV) produced a higher titre of ricin-specific antibodies than Alhydrogel-vaccine (ARTV) and vaccine solution. When challenged with 3 LD50 of ricin by intratracheal instillation 7 weeks after the second vaccine instillation, all rats in both LRTV dose groups survived with minimal signs of incapacitation. Analysis of antibody secretion by spleen cells, 14 days post challenge, showed that the IgG isotype in the LRTV group was significantly higher than that in the ARTV and RTV groups and also that the proportion of specific IgA in lung fluid was higher in the LRTV group than in the ARTV and RTV groups. The results of this study indicate that effective vaccinations against inhaled ricin could be achieved with liposomally-encapsulated ricin toxoid, via the lung and should be investigated further.

Dibenzoxepinone hydroxylamines and hydroxamic acids: dual inhibitors of cyclooxygenase and 5-lipoxygenase with potent topical antiinflammatory activity.

Hydroxylamine and hydroxamic acid derivatives of a known nonsteroidal antiinflammatory dibenzoxepine series display both cyclooxygenase (CO) and 5-lipoxygenase (5-LO) inhibitory properties. Many of these new dual CO/5-LO inhibitors also exhibit potent topical antiinflammatory activity in the arachidonic acid-induced murine ear edema model. On the basis of their promising profile of in vitro and in vivo activities, hydroxamic acids 24h, 3-(6,11-dihydro-11-oxodibenz[b,e]oxepin-2-yl)-N-hydroxy-N-++ +methylpropanamide (HP 977), and 25, 3-(6,11-dihydrodibenz[b,e]oxepin-2-yl)-N-hydroxy-N- methylpropanamide (P10294), were selected as developmental candidates for the topical treatment of inflammatory skin disorders.

Protection against inhalation toxicity of ricin and abrin by immunisation.

1. Abrin and ricin are highly toxic plant proteins which are very similar in structure and function and inhibit protein synthesis in eukaryotes. 2. Rats have been immunised against either toxin using formaldehyde-toxoids by three subcutaneous injections at intervals of 3 weeks. For abrin, serum titres in 14 out of 15 rats were raised to between 1:12800 and 1:51200 after two injections, 6 weeks from the start of the experiment. Titres of between 1:256 and 1:1024 were also measured in lung washes after challenge with active abrin toxin. 3. The three major antibody classes, IgG, IgM and IgA were present in the immune sera but IgG and IgA only were detected in lung washes. The proportion of IgA to IgG was higher in the lung fluid than in sera. Rats immunised by abrin toxoid were protected against 5 LCt50's of abrin by inhalation but others exposed to ricin were not. 4. For ricin, serum titres ranged from 1:800 to 1:25600 after two injections and after a third injection the titre range was the same but population samples were weighted towards the higher titres. All rats immunised with ricin toxoid survived the challenge of 5 LCt50's of ricin toxin by inhalation over the observation period of 28 days post-challenge. 5. Representative immunised rats (abrin toxoid) were taken at various times post-exposure, humanely killed and tissues were examined for pathological changes. It was concluded that an apparently severe lung lesion occurred at a later time than in non-immunised, toxin challenged rats. This damage was not lethal over the experimental observation periods. 6. Immunisation by the sub-cutaneous route therefore protects against lethality from challenge by inhalation of ricin or abrin toxins but does not prevent significant lung damage.

Antipeptide antibodies as probes of the recombinant and endogenous murine erythropoietin receptors.

The binding of erythropoietin (Epo) to its plasma membrane receptor activates signal pathways that result in erythroid cell proliferation and differentiation. To elucidate the structural features of the receptor that are important for hormone binding and signaling, we have developed a series of site-specific antibody probes. These antibodies were raised against synthetic peptides homologous to six exoplasmic domains and one cytoplasmic domain of the murine receptor and were affinity-purified by binding to their respective peptide antigen, immobilized on agarose. Western blot analyses demonstrated that the recombinant receptor expressed transiently in COS-7 cells is synthesized as three protein species of 62, 64, and 66 kd, consistent with previous observations. Importantly, probing the endogenous receptor in both virally transformed erythroleukemia cells and normal erythroid cells demonstrated similar 62- to 66-kd receptor species. The affinity-purified antibodies also recognized several antigenically related proteins. An examination of the capacity of the antireceptor antibodies to block receptor activation by Epo revealed that antibodies to five of the six exoplasmic domains blocked the receptor. This was reversed with excess Epo. Inhibition of receptor activation by antibody probes to five discrete hydrophilic domains suggests that receptor function may be critically dependent on the structural integrity (conformation) of the entire exoplasmic portion.

Erythropoietin induces cytosolic protein phosphorylation and dephosphorylation in erythroid cells.

Erythropoietin, the prime regulator of red blood cell growth and differentiation, causes rapid changes in the phosphorylation of several integral plasma membrane proteins (Choi, H-S., Wojchowski, D. M., and Sytkowski, A. J. (1987) J. Biol. Chem. 262, 2933-2936; Choi, H-S., Bailey, S. C., Donahue, K. A., Vanasse, G. J., and Sytkowski, A. J. (1990) J. Biol. Chem. 265, 4143-4148). In the present study we have demonstrated that erythropoietin's signal is transduced rapidly to the cytosol resulting in specific phosphorylation/dephosphorylation events. Erythropoietin treatment of Rauscher murine erythroleukemia cells previously labeled with [32P]orthophosphate results in a rapid increase in phosphorylation of two cytosolic proteins, designated pp96 and pp80, and a decrease in phosphorylation of another protein, designated pp90. The relative molecular mass and pI of pp80 are virtually identical to those reported for the protein kinase C substrate p80, or "MARCKS protein." Treatment of the cells with 12-O-tetradecanoylphorbol-13-acetate also increases pp80 but not pp96 phosphorylation, suggesting that erythropoietin triggers a protein kinase C-dependent pathway to pp80 and a protein kinase C-independent pathway to pp96. The effect of erythropoietin on pp96 phosphorylation was also shown in nontransformed erythroid cells isolated from the spleens of phenylhydrazine-treated mice. In contrast, almost no 32P labeling of pp80 or pp90 was detected, and pp80 and pp90 protein were nearly absent from these normal cells. These differences in expression and phosphorylation of erythropoietin-sensitive phosphoproteins may be related to the growth factor independence or dependence of the erythroid cells.