The alcohol-abuse deterrent disulfiram (DSF) is shown to have a highly selective toxicity against melanoma in culture, inducing a largely apoptotic response, with much lower toxicity against several other cell lines. Melanoma cell lines derived from different stages (radial, vertical, and metastatic phase) were all sensitive to DSF treatment in vitro; melanocytes were only slightly affected. A required role of extracellular Cu is demonstrated for DSF toxicity. Low concentrations of DSF alone decreased the number of viable cells, and the addition of CuCl2 significantly enhanced the DSF-induced cell death to less than 10% of control. Significantly, the intracellular Cu concentration of melanoma cells increased rapidly upon DSF treatment. Both the intracellular Cu uptake and the toxicity induced by DSF were blocked by co-incubation with bathocuproine disulfonic acid (BCPD, 100 μM), a non-membrane-permeable Cu chelator. Chemical studies demonstrated a complicated, extracellular redox reaction between Cu(II) and DSF, which forms the complex Cu(deDTC)2 in high yield, accompanied by oxidative decomposition of small amounts of disulfiram. The Cu complex has somewhat higher activity against melanoma and is suggested to be the active agent in DSF-induced toxicity. The redox conversion of DSF was unique to Cu(II) and not engendered by the other common biological metal ions Fe(II or III), Mn(III), and Zn(II). The implications of this work are significant both in the possible treatment of melanoma as well as in limiting the known side-effects of DSF, which we propose may be diminished by cotreatment to decrease adventitious Cu.
Nuclear factor kappa B (NFκB) is an essential regulator of gene transcription for hundreds of genes, including many critically involved in apoptosis. NFκB complexes containing cRel generally activate pro‐apoptotic genes, while those with RelA activate anti‐apoptotic genes. We have previously shown that NFκB binding by RelA is constitutively elevated in human metastatic melanoma cultures relative to normal melanocytes. Here we extended our investigation to immunohistochemical analysis of human tissue biopsies. We found that RelA expression is significantly elevated in melanocytes of human naevi and melanomas relative to normal skin, but expression of its inhibitor IκB‐α is significantly lower in metastatic melanomas than in intradermal naevi. Antibodies specific for the nuclear localization signal of RelA also showed significantly increased staining in metastatic melanoma biopsies. Notably, in melanomas and in naevi, we also found that RelA is phosphorylated at serine 529, and this activated form accumulates in the nuclei of melanomas. This suggests that increased expression and phosphorylation of RelA occurs at the stage of the benign naevus, but IκB‐α is able to sequester RelA in the cytoplasm and regulate RelA transcriptional transactivation. We also found that antibodies against cRel show a progressive increase in staining from naevi to melanoma. However, staining for IκB‐ɛ, which primarily inhibits the nuclear localization of cRel was also progressively increased, and cRel expression was predominantly cytoplasmic in melanomas. These results confirm that the altered expression of RelA found in metastatic melanoma cells in tissue culture is relevant to human tumors and offer new insights into the deregulation of NFκB signaling.
A 47-year-old woman sought medical attention after 1 week of progressive fatigue, scleral icterus, and dark urine. She had no fever, cough, or shortness of breath. She had no history of HIV, cancer, autoimmune disease, recent infection, diarrhea, or new medication/supplements. In addition, she had no history of out-of-state travel. On examination at the time of admission, she had mild scleral icterus, normal spleen size, no evidence of petechiae or purpura, and a normal neurological examination. Laboratory tests revealed a white blood cell count of 4.8 K/µL, hemoglobin level of 11.5 g/dL, and platelet count of 8 K/µL. She was admitted and tested positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by nasopharyngeal swab via reverse transcription polymerase chain reaction (RT-PCR). Additional laboratory values were as follows: total bilirubin, 1.7 mg/dL; creatinine, 0.87 mg/dL; lactate dehydrogenase (LDH), 708 U/L; reticulocytes, 1.22%; haptoglobin, <9 mg/dL; prothrombin time, 13.1; and partial thromboplastin time (PTT), 26.6. mg/dL; fibrinogen, 389 mg/dL; and C-reactive protein, 0.1 mg/dL. D-dimer was elevated at 3.9 µg/mL. Normal values for test results are given in Table 1. A direct antiglobulin test (DAT) was negative. The results of a urine pregnancy test were negative. She had extensive negative workup for nonimmune
No abstract
Objectives: Although MHC class II molecules can be expressed on dendritic cells (DC), macrophages and B-cells in lymph nodes and have the potential to activate CD4 T cells, it is believed that the dendritic cells are the major antigen presenting cells for T lymphocytes. They are located at portals of antigen entrance and when located in paracortical areas are more efficient antigen processors than macrophages and B-cells whose antigen-presenting role is limited by their location away from the paracortex. Here we focus on DC expression of MHC-class II molecules and the interaction with OPD4 + activated T cell in same nodal quadrants. We also evaluated the overall CD68 + macrophage density in sentinel nodes (SN) and non-sentinel nodes (NSN). Methods: Eighteen sets of metastasis-free SN and NSN from melanoma patients were evaluated for HLA-DR + DC and OPD4 + activated T lymphocytes. Immunohistochemical single staining was performed for OPD4 + activated T-cells and cocktail-triple staining to distinguish HLA-DR + DC from CD20 + B cells and CD68 + macrophages. The densities, area occupied by HLA-DR + /CD68 ) CD20 ) DC and OPD4 + activated T-cells, CD68 + macrophages and coefficient of variation of density of HLA-DR + /CD68 ) CD20 ) DC and OPD4 + activated T-cells by quadrants were measured by a Simple-PCI imaging system. We randomly subdivided the SN and NSN into quarters and evaluated the areas of five maximum density of HLA-DR + /CD68 ) CD20 ) DC in each quarter for both HLA-DR + /CD68 ) CD20 ) DC and OPD4 + T lymphocytes. Results obtained: The area occupied by HLA-DR + /CD68 ) CD20 ) DC was 5.30 ± 6.98% in SN and 11.95 ± 5.79% in NSN (P ¼ 0.017); the density of HLA-DR + /CD68 ) CD20 ) DC was 112.45 ± 100.42 in SN and 231.55 ± 49.22 in NSN (P ¼ 0.002). The coefficient of variation of HLA-DR + / CD68 ) CD20 ) DC density was significant higher among the four quarters in SN compared with in NSN (44.49 ± 19.58, 27.55 ± 14.87, P ¼ 0.012). The density of OPD4 + activated T-cells was significant lower in SN than in NSN (1329.1 ± 680.9, 1546.7 ± 699.1; P ¼ 0.012). The area occupied by OPD4 + activated T-cells was lower in SN but the difference was not significant (15.19 ± 12.66 and 17.94 ± 13.65%; P ¼ 0.832). The coefficient of variation of OPD4 + T-cell density by quadrants (32.5 ± 39.3 in SN, 27.0 ± 24.3 in NSN, P ¼ 0.417) was not significant. Overall the density of D68 + macrophages was 105.12 ± 59.6 in SN and 136.18 ± 82.7 in NSN (P ¼ 0.081); while the area occupied by CD68 + macrophages was significant lower in SN rather than in NSN (0.69 ± 0.46%, 1.09 ± 0.66, P ¼ 0.015). Conclusion: Lymph nodes influenced by melanoma are immune modulated. The antigen presentation by APCs via MHC-class II to CD4 T-cells is inhibited in SN compared with NSN. Higher variations in DC density between the different quadrants of SN suggests that the area of the SN receiving lymph via the tumor-derived afferent lymphatic may be immune-suppressed prior to seeding of metastases.
Melanoma tumors have an unusually high rate of metaluptake and a poor ability to mediate oxidative stress, both of which may be attributed to constitutive changes in their intracellular melanin. Melanin pigments are fundamentally composed of polymeric catechols, and as such have inherent redox equilibria between quinole, semi-quinone and quinone oxidation states. This redox reactivity is inherent in the ability of melanins to act as buffers against oxidative and photochemical stress in vivo. We have investigated the speciation, metal-binding affinity and redox reactivity of these catecholic subunits in synthetic melanins formed from dihydroxyindole. The binding of various metal ions can generate a pro-oxidant response, making the melanin more easily oxidized, and susceptibility to reaction with oxygen to form superoxide and other cytotoxic reactive oxygen species. Similar pro-oxidant behavior has been seen in melanoma cell lines, and we hypothesized that this activity is due to the melanin within the melanoma cells. In line with this idea, several families of lipophilic metal compounds with antimelanoma activity have been identified. Our initial screening of the pro-oxidant response of synthetic melanin to various metal ions has identified new potential candidates for metalbased chemotherapy. IL-11ANTIOXIDANT AND PHOTOPROTECTIVE PROPER-TIES OF HUMAN IRIDIAL MELANIN. T. Objectives: To analyze the effects of human iridial melanin on formation and decay of reactive oxygen species, and to determine whether antioxidant properties of the iridial melanin change with age of donors and color of their irises. Methods: Content and type of melanin in human and bovine iris homogenates was determined by electron paramagnetic resonance (EPR) spectroscopy and HPLC detection of characteristic melanin degradation products. Human eyes were obtained from Wisconsin Lion Eye Bank. Iridial samples were pooled into four age groups (8-34; 37-54; 57-75 and 76-98 yr) and four color groups (blue, medium light, medium light and brown). Antioxidant and photoprotective properties of radial melanin were analyzed by EPR-asymmetry, EPR-spin trapping, oxidize electrode and iodometric assay of lipid hydroperoxides. Reactive oxygen species, in the absence and presence of iridial homogenates, were generated by UV radiation and photosensitized oxidation reactions. Results: On average, human brown irises contain about 50% more melanin than human blue irises. Human iridial melanin, regardless the iris color and age of donors, is predominantly eumelanin. Age of donors seems to have little effect on either the content or type of the iridial melanin. Irradiation of iridial homogenates with light, particularly with UV radiation, induced the formation of superoxide anion and hydrogen peroxide. Such an aerobic photoreactivity of the iridial melanin was independent of age of donors and color of their irises; however, it could be enhanced by exogenous ascorbate. Iridial melanin decomposed, in a dose-dependent manner, hydrogen peroxide and inhibited peroxidation...
favorable outcome than those whose nodes are negative by both approaches. Thus, there is a strong likelihood that, in the future, molecular approaches will be used in parallel with standard histology. Conclusion: The sentinel node technique is the most accurate staging technique currently available for melanoma patients and provides predictive information on non-sentinel node tumor status, likelihood of recurrence and death from melanoma. It is also applicable to other tumors. The identification of a tumor-free sentinel node spares a majority of patients unnecessary nodal surgery. The effect of the approach on survival will be known when outcomes from current studies are evaluated. Source of funding: CA, 29605.
The COVID-19 pandemic brought with it TX changes for many patients (pts) with AMEL, as it did for other pts with cancer. The long-term impacts of mandated area lockdowns, social distancing, medical society guidelines, and patient preference will not be fully understood for some time. The first step to learning from the pandemic is to assess how AMEL care was rendered in 2020. We performed a retrospective analysis of systemic TX for AMEL in KPNC, an integrated community healthcare system with approximately 4 million pts and about 150 de novo diagnoses of AMEL annually. We performed a chart review of pts with AMEL who were treated with standard of care systemic therapy, either immune-checkpoint inhibitors (ICI) or BRAF/MEK inhibitors (BRAF/MEKi), from January 1 to March 15, 2020, as a control group, and between March 15 and May 20, during the first wave of the COVID-19 pandemic in California with follow-up through November 4, 2020. Between January 1 and March 15, 26 pts started palliative ICI of whom 11 started combination PD1 (PD1i) and CTLA4 inhibitors. Among 15 pts who started on single-agent PD1i, 14 pts received short-interval TX (SIT), while 1 started long-interval TX (LIT). All 21 pts who started perioperative PD1i pre-pandemic, started on SIT. Between March 15 and May 20, 21 pts started palliative ICI, of whom only 3 started combination TX. Among pts who started palliative single-agent PD1i 40% started on LIT in this initial phase of the pandemic. 27 pts started perioperative ICI during this time. We found 3 started with neoadjuvant therapy and 78% started on LIT. Among 78 pts who were already on palliative single-agent ICI at the start of the COVID-19 pandemic, 15% remained on SIT and 24% changed to LIT. Sixteen pts (21%) also interrupted palliative ICI between March 15 and April 15 after a median time on TX of 45 weeks and for 63% the cited reason for interruption on chart review was the COVID 19 pandemic. Three of these pts who stopped ICI changed to BRAF/MEKi, the remainder continue in active follow-up as of November 2020. Among 72 pts already receiving perioperative ICI in March 2020, 19% remained on SIT, 35% changed to LIT, and 11% were already on LIT. 39% of pts interrupted perioperative ICI after a median time of 20 weeks on TX and 46% of these cited COVID 19 as the reason for interruption. Three pts have since resumed peri-operative TX, but the others remain in active follow-up off therapy. Between 3/15 and 5/30/2020, we noted a 325% increase in pts started on BRAF/MEKi; 69% of pts received therapy for palliative intent. The start of the COVID-19 pandemic saw many different changes in AMEL TX in KPNC, with increased use of single-agent ICI, LIT, and oral therapy, in line with public health guidance, oncology societal guidelines and patient preference. It will be important to assess the long-term outcomes relating to these changes, including the impact of early discontinuation of ICI, to help guide future Melanoma care during and after the pandemic. Citation Format: Juraj Kavecansky, Nina Shah, Angeles Price, Frank Hsieh, Alice Kengla, Belinda Ark, Jahan Tavakoli, Christine Kaiser, Mingqing Li, Sejal Jhatakia, Mala Reddy, Dazhi Cen, Philip Sardar, Stephen Wang, Ashok Pai, Andrea Harzstark, Tatjana Kolevska, Thach-Giao Truong. Treatment (TX) of advanced melanoma during the Coronavirus Disease 2019 (COVID-19) pandemic in Kaiser Permanente Northern California (KPNC) [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2021 Feb 3-5. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(6_Suppl):Abstract nr P15.
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