Dermoscopy of external ear melanoma (EEM).

External ear melanoma (EEM) belongs to extremely rare melanoma locations. So far, only single cases of EEM have been described in terms of dermoscopic presentations. This case study report presents dermoscopic patterns of EEM in six patients. The retrospective case study was based on medical documentation (epidemiological, anamnestic, clinical, videodermoscopic, and histopathologic) of consecutive patients who were diagnosed with melanoma located on the external ear between January 2013 and May 2021 in three diagnostic dermatologic centers. In four of six cases, the melanoma was placed on the helix. The histopathological diagnoses included 1/6 lentigo maligna and 5/6 invasive melanomas. The dermoscopic pattern of facial melanoma (FM) was present in 3/6 cases, 1/6 exhibited the typical superficial spreading pattern (one with nodular invasion), 1/6 the multicomponent asymmetric pattern, and 1/6 the hypomelanotic type. Five melanomas presented numerous (3-6) dermoscopic structures characteristic for each dermoscopic subtype. In conclusion, dermoscopy has proved effective for detection of both difficult and easy-to-diagnose EEM, but also in differentiating their dermoscopic subtypes.

Single-agent anti-PD-1 or combined with ipilimumab in patients with mucosal melanoma: an international, retrospective, cohort study.

BACKGROUND: Mucosal melanoma (MM) is a rare melanoma subtype with distinct biology and poor prognosis. Data on the efficacy of immune checkpoint inhibitors (ICIs) are limited. We determined the efficacy of ICIs in MM, analyzed by primary site and ethnicity/race. PATIENTS AND METHODS: A retrospective cohort study from 25 cancer centers in Australia, Europe, USA and Asia was carried out. Patients with histologically confirmed MM were treated with anti-programmed cell death protein 1 (PD-1) ± ipilimumab. Primary endpoints were response rate (RR), progression-free survival (PFS), overall survival (OS) by primary site (naso-oral, urogenital, anorectal, other), ethnicity/race (Caucasian, Asian, Other) and treatment. Univariate and multivariate Cox proportional hazards model analyses were conducted. RESULTS: In total, 545 patients were included: 331 (63%) Caucasian, 176 (33%) Asian and 20 (4%) Other. Primary sites included 113 (21%) anorectal, 178 (32%) urogenital, 206 (38%) naso-oral and 45 (8%) other. Three hundred and forty-eight (64%) patients received anti-PD-1 and 197 (36%) anti-PD-1/ipilimumab. RR, PFS and OS did not differ by primary site, ethnicity/race or treatment. RR for naso-oral was numerically higher for anti-PD-1/ipilimumab [40%, 95% confidence interval (CI) 29% to 54%] compared with anti-PD-1 (29%, 95% CI 21% to 37%). Thirty-five percent of patients who initially responded progressed. The median duration of response (mDoR) was 26 months (95% CI 18 months-not reached). Factors associated with short PFS were Eastern Cooperative Oncology Group (ECOG) performance status (PS) ≥3 (P < 0.01), lactate dehydrogenase (LDH) more than the upper limit of normal (ULN) (P = 0.01), lung metastases (P < 0.01) and ≥1 previous treatments (P < 0.01). Factors associated with short OS were ECOG PS ≥1 (P < 0.01), LDH >ULN (P = 0.03), lung metastases (P < 0.01) and ≥1 previous treatments (P < 0.01). CONCLUSIONS: MM has poor prognosis. Treatment efficacy of anti-PD-1 ± ipilimumab was similar and did not differ by ethnicity/race. Naso-oral primaries had numerically higher response to anti-PD-1/ipilimumab, without difference in survival. The addition of ipilimumab did not show greater benefit over anti-PD-1 for other primary sites. In responders, mDoR was short and acquired resistance was common. Other factors, including site and number of metastases, were associated with survival.

Analysis of efficacy and safety of vismodegib therapy in patients with advanced basal cell carcinoma - real world multicenter cohort study.

BACKGROUND: Basal cell carcinoma (BCC) is the most frequent non-melanoma skin cancer. The basis of treatment is surgical resection. The treatment of locally advanced and metastatic disease is currently based on sonidegb or vismodegib, small molecule inhibitors of the hedgehog signalling pathway. OBJECTIVES: The study aimed to retrospectively analyse the efficacy and safety of treatment with vismodegib in 108 patients with locally advanced or metastatic disease treated from August 1st, 2017 to December 31st, 2020. The primary objective was to evaluate the objective response rate (ORR), overall survival (OS) and progression-free survival rates. The secondary aims of the study were the disease control rate, the incidence of adverse events (AEs) and the estimation of the factors that potentially impact the treatment outcome and patient survival. METHODS: Patients treated in national drug programme were enrolled into this retrospective cohort study. Evaluation of the treatment efficacy was performed according to CT/MRI scans and by the response evaluation criteria in solid tumours (RECIST) 1.1. The safety evaluation was performed according to the Common Terminology Criteria for Adverse Events v. 5.0 (CTCAE) classification and severity assessment. RESULTS: The median duration of treatment was 14 months (range 1-94 months). The median progression-free survival reached 30.5 months (95% CI; 24.8-36.3), and the progression-free survival rate after 6, 12 and 24-months were 92%, 78% and 61%, respectively. The median overall survival was 41.5 months (95% CI; 31.6-51.3), and the overall survival rate after 1, 2 and 3 years accordingly 86%, 73% and 60%. The univariant and multivariant analysis indicated that the female gender is an independent positive prognostic factor of progression-free survival. CONCLUSIONS: The response to treatment is the prognostic factor for response maintenance and better overall survival. The therapy was well tolerated with the safety profile consistent in general with known from previous studies.

Intramolecular immunological signal hypothesis revived--structural background of signalling revealed by using Congo Red as a specific tool.

Micellar structures formed by self-assembling Congo red molecules bind to proteins penetrating into function-related unstable packing areas. Here, we have used Congo red--a supramolecular protein ligand--to investigate how the intramolecular structural changes that take place in antibodies following antigen binding lead to complement activation. According to our findings, Congo red binding significantly enhances the formation of antigen-antibody complexes. As a result, even low-affinity transiently binding antibodies can participate in immune complexes in the presence of Congo red, although immune complexes formed by these antibodies fail to trigger the complement cascade. This indicates that binding of antibodies to the antigen may not, by itself, fulfill the necessary conditions to generate the signal which triggers effector activity. These findings, together with the results of molecular dynamics simulation studies, enable us to conclude that, apart from the necessary assembling of antibodies, intramolecular structural changes generated by strains which associate high- affinity bivalent antibody fitting to antigen determinants are also required to cross the complement activation threshold.

Intramolecular signaling in immunoglobulins -- new evidence emerging from the use of supramolecular protein ligands.

The postulated intramolecular signaling in immunoglobulins generated by antigen binding has been controversial for years. The high heterogeneity of immune complexes as signaling systems and the requirement of the immobilized antigen form for efficient triggering of effector activity is likely the reason for the lack of clarity. Here we present new evidence supporting the notion of intramolecular signaling, based on the use of supramolecular dyes that bind to signal-derived specific sites in immunoglobulins.

Egg yolk platelet proteins from Xenopus laevis are amyloidogenic.

The association of amphibian (Xenopus laevis) egg yolk platelet proteins, represented predominantly by lipovitellin, was studied as a model of the formation of amyloid deposits. Two kinds of molecular organization formed by this protein material - native and heat-denatured - were found to exhibit amyloid properties although they differ significantly in structural organization. The first consisted in protein molecules arranged in the natural, physiological, net-like platelet organization, with a tendency to orient uni-directionally. The second was obtained by the gradual removal of Congo red from lipovitellin denatured by heating in an excess of dye. This procedure produced the twisted fibrillar organization of molecules typical for amyloids, represented predominantly by end-to-end associated major polypeptide chains of lipovitellin. Both native and denatured structural forms bind Congo red and produce a green birefringence effect, confirming the near parallel alignment of the complexed Congo red molecules. However, a dye(1,4-bis(1-amino-4-sulfonaphtyl-2-azo)phenylene) closely related to Congo red but with a very weak self-assembling tendency appeared inactive when the spectral shift was studied in a cross-polarization system, indicating in this way that dye supramolecularity is an extra factor which may determine binding to amyloid proteins and specific spectral effects.

Heat-induced formation of a specific binding site for self-assembled Congo Red in the V domain of immunoglobulin L chain lambda.

Moderate heating (40-50 degrees C) of immunoglobulins makes them accessible for binding with Congo Red and some related highly associated dyes. The binding is specific and involves supramolecular dye ligands presenting ribbon-like micellar bodies. The L chain lambda dimer, which upon heating disclosed the same binding requirement with respect to supramolecular dye ligands, was used in this work to identify the site of their attachment. Two clearly defined dye-protein (L lambda chain) complexes arise upon heating, here called complex I and complex II. The first is formed at low temperatures (up to 40-45 degrees C) and hence by a still native protein, while the formation of the second one is associated with domain melting above 55 degrees C. They contain 4 and 8 dye molecules bound per L chain monomer, respectively. Complex I also forms efficiently at high dye concentration even at ambient temperature. Complex I and its formation was the object of the present studies. Three structural events that could make the protein accessible to penetration by the large dye ligand were considered to occur in L chains upon heating: local polypeptide chain destabilization, VL-VL domain incoherence, and protein melting. Of these three possibilities, local low-energy structural alteration was found to correlate best with the formation of complex I. It was identified as decreased packing stability of the N-terminal polypeptide chain fragment, which as a result made the V domain accessible for dye penetration. The 19-amino acid N-terminal fragment becomes susceptible to proteolytic cleavage after being replaced by the dye at its packing locus. Its splitting from the dye-protein complex was proved by amino acid sequence analysis. The emptied packing locus, which becomes the site that holds the dye, is bordered by strands of amino acids numbered 74-80 and 105-110, as shown by model analysis. The character of the temperature-induced local polypeptide chain destabilization and its possible role in intramolecular antibody signaling is discussed.

Why Congo red binding is specific for amyloid proteins - model studies and a computer analysis approach.

BACKGROUND: The complexing of Congo red in two different ligand forms - unimolecular and supramolecular (seven molecules in a micelle) - with eight deca-peptides organized in a b-sheet was tested by computational analysis to identify its dye-binding preferences. Polyphenylananine and polylysine peptides were selected to represent the specific side chain interactions expected to ensure particularly the stabilization of the dye-protein complex. Polyalanine was used to verify the participation of non-specific backbone-derived interactions. MATERIAL AND METHODS: The initial complexes for calculation were constructed by intercalating the dye between the peptides in the middle of the beta-sheet. The long axis of the dye molecule (in the case of unimolecular systems) or the long axis of the ribbon-like micelle (in the case of the supramolecular dye form) was oriented parallel to the peptide backbone. This positioning maximally reduced the exposure of the hydrophobic diphenyl (central dye fragment) to water. In general the complexes of supramolecular Congo red ligands appeared more stable than those formed by individual dye molecules. Specific interactions (electrostatic and/or ring stacking) dominated as binding forces in the case of the single molecule, while non-specific surface adsorption seemed decisive in complexing with the supramolecular ligand. RESULTS: Both the unimolecular and supramolecular versions of the dye ligand were found to be likely to form complexes of sufficient stability with peptides. The low stability of the protein and the gap accessible to penetration in the peptide sheet seem sufficient for supramolecular ligand binding, but the presence of positively charged or hydrophobic amino acids may strengthen binding significantly. CONCLUSIONS: The need for specific interaction makes single-molecule Congo red binding rather unusual as a general amyloid protein ligand. The structural feature of Congo red, which enables specific and common interaction with amyloid proteins, probably derives from the ribbon-like self-assembled form of the dye.

Evidence that supramolecular Congo red is the sole ligation form of this dye for L chain lambda derived amyloid proteins.

The mechanism of Congo red binding to amyloid protein was studied in order to establish which of two structural dye versions present in water solutions--unimolecular and supramolecular--represent its actual ligation form. Immunoglobulin L chain lambda of amyloidogenic nature, expressed by Congo red binding and easy gel formation, was used as the model amyloid protein. Congo red was coassembled with rhodamine B, designed to be a marker of the Congo red micellar organisation in complexation with protein. The particular suitability of rhodamine B for this role results from significant difference in its binding affinity to Congo red and to protein. It associates readily with Congo red, becoming incorporated into its micellar organisation, but as homogenous dye it shows an almost complete inability to bind to protein. In view of these properties, Congo red was used as a vehicle to draw rhodamine B into complexation with protein, at the same time supplying evidence of its supramolecular ligation form. The results show that both soluble amyloid precursor L chain and the derived gel material attach rhodamine B coassembled with Congo red but not the homogenous rhodamine B. Despite its dynamic, supramolecular character, Congo red participates in complexation with amyloid proteins as an integral ligand unit.

The conformational characteristics of Congo red, Evans blue and Trypan blue.

The structures of the closely related bis-azo dyes Evans blue, Trypan blue and Congo red, which appeared to have different self-assembly properties and correspondingly different abilities to form complexes with amyloids and some other proteins, were compared in this work. Ab initio and semi-empirical methods were used to find the optimal structures and partial charge distributions of the dyes. The optimal structures were searched using different widely used programs. The structures of Congo red and evans blue were found to be planar, except for the torsion on the central diphenyl bond connecting the two halves of the dye. Both symmetrical parts of the molecules appeared very close to planarity. However, Trypan blue exhibits non planarity on the di-azo bonds, as well as on the central bond between the symmetrical parts of the dye. In a consequence, the non planarity of this molecule is higher than in the case of its isomer, Evans blue and Congo red as well. The extra rotation around the azo bonds extorted by the close proximity of the sulfonic groups may be the direct cause of its poor self-assembling and complexation properties versus Evans blue.

Supramolecularity creates nonstandard protein ligands.

Congo red and a group of structurally related dyes long used to stain amyloid proteins are known to associate in water solutions. The self-association of some dyes belonging to this group appears particularly strong. In water solutions their molecules are arranged in ribbon-like micellar forms with liquid crystalline properties. These compounds have recently been found to form complexes with some native proteins in a non-standard way. Gaps formed by the local distribution of beta-sheets in proteins probably represent the receptor sites for these dye ligands. They may result from higher structural instability in unfolding conditions, but also may appear as long range cooperative fluctuations generated by ligand binding. Immunoglobulins G were chosen as model binding proteins to check the mechanism of binding of these dyes. The sites of structural changes generated by antigen binding in antibodies, believed to act as a signal propagated to distant parts of the molecule, were assumed to be suitable sites for the complexation of liquid-crystalline dyes. This assumption was confirmed by proving that antibodies engaged in immune complexation really do bind these dyes; as expected, this binding affects their function by significantly enhancing antigen binding and simultaneously inhibiting C1q attachment. Binding of these supramolecular dyes by some other native proteins including serpins and their natural complexes was also shown. The strict dependence of the ligation properties on strong self-assembling and the particular arrangement of dye molecules indicate that supramolecularity is the feature that creates non-standard protein ligands, with potential uses in medicine and experimental science.

Supramolecular ligands: monomer structure and protein ligation capability.

The aim of this work was to define the chemical structure of compounds self-assembling in water solutions, which appear to interact with proteins as single ligands with their supramolecular nature preserved. For this purpose the ligation to proteins of bis azo dyes, represented by Congo red and its derivatives with designed structural alterations, were tested. The three parameters which characterize the reactivity of supramolecular material were determined in the same conditions for all studied dyes. These were: A) stability of the assembly products; B) binding to heat-denatured protein (human IgG); and C) binding to native protein (rabbit antibodies in the immune complex) measured by the enhancement of hemagglutination. The structural differences between the Congo red derivatives concerned the symmetry of the molecule and the structure of its non-polar component, which occupies the central part of the dye molecule and is thought to be crucial for self-assembly. Other dyes were also studied for the same purpose: Evans blue and Trypan blue, bis-ANS and ANS, as well as a group of compounds with a structural design unlike that of bis azo dyes. Compounds with rigid elongated symmetric molecules with a large non-polar middle fragment are expected to form a ribbon-like supramolecular organization in assembling. They appeared to have ligation properties related to their self-assembling tendency. The compounds with different structures, not corresponding to bis azo dyes, did not reveal ligation capability, at least in respect to native protein. The conditions of binding to denatured proteins seem less restrictive than the conditions of binding to native molecules. The molten hydrophobic protein interior becomes a new binding area allowing for complexation of even non-assembled molecules.

Effect of self association of bis-ANS and bis-azo dyes on protein binding.

A correlation was found between the ability of dyes (ANS, bis-ANS, Congo red, Evans blue) to form self-associated supramolecular structures in water and their tendency to form complexes with proteins. The self-association ability of dyes was measured as the resistance of a molecular sieve to their penetration. Quantitative evaluation of dye-protein interaction involved measuring the effect of dye on antibodies that agglutinate sheep red blood cells. Enhancement of agglutination by dye was assumed to represent its protein complexation ability. The results confirm that, relative to monomers, self-associated ligands also have altered protein binding properties.

The use of congo red as a lyotropic liquid crystal to carry stains in a model immunotargeting system--microscopic studies.

The lyotropic liquid crystal dye-Congo Red was used as a carrier in a model immunotargeting system constructed from sheep red blood cells (SRBC) representing the antigen target and rabbit IgG anti-SRBC as the specific driving immunoglobulin. Rhodamine B and Hemin stains were chosen as example chemicals carried to the target. The carried stains were introduced to the micellar organization of Congo Red by intercalation. Preserving its supramolecular organization, Congo Red binds spontaneously and selectively to antibodies that have altered structure extorted by interaction with the antigen in the immune complex. The functionality of the studied immunotargeting model was verified by fluorescence and electron microscopy. The results indicate that the supramolecular nature of protein ligands offers new ligation capabilities possibly useful for carrying stains or drugs in immune-oriented systems.

Congo red-stabilized intermediates in the lambda light chain transition from native to molten state.

Disruption of tertiary interaction makes a protein accessible to penetration by different small molecular compounds. Their interaction may stabilize the altered protein conformation. Congo red is proposed here as a stabilizer of the molten globule state and also of highly reversible intermediates in the transition from native to molten state. Human immunoglobulin lambda light chain (dimer) was used. Two protein-Congo red complexes were found after heating lambda chain in the presence of Congo red. They differed in the amount of attached dye molecules. The binding of dye was interpreted as a two-step dye penetration process involving the peripheral parts of the protein in the first step (at lower temperatures). It was concluded that the liquid crystal properties of Congo red enable it to form specific complexes with proteins, which have become accessible to penetration by ligands after global or local disruption of tertiary interaction. This dye may thus be used as a stabilizer of unfolding intermediates in the step preceding the molten globule state.

The detection of specific acute phase serum protein complexes and immune complexes by congo red binding.

The study concerns the problem of selective binding of Congo Red dye to serum proteins. The dye-binding structural motifs were assumed to appear in proteins as the result of local disruptions of tertiary interaction associated with biological activities. Functional structures of serum proteinase inhibitors and haptoglobins induced by interaction of these proteins with their natural substrates were found to bind Congo Red. Soluble immune complexes formed by bivalent hapten-bridged anti-TNP rabbit antibodies also appeared able to interact with the dye. The possible use of these effects for diagnostic analysis was suggested.

Bis azo dye liquid crystalline micelles as possible drug carriers in immunotargeting technique.

Lyotropic liquid crystal mesophases, represented in this work by Congo Red bis azo dye solution, were proposed as systems to carry drugs to immuno-selected targets. The possible use of Congo Red for this aim arises from its liquid crystalline features, enabling it to attach to immune complexes as polymolecular, ordered conglomerates and simultaneously to incorporate many organic compounds into its mesophase, largely independent of the water with a specific but adaptive molecular organization. Molecules with planar rigid structure, and/or large hydrophobic fragments, especially those with a positively charged group in the molecule, are found to be incorporated best. Rhodamine B, Rhodamine 6G and adriamycine, which have the assumed binding features, were tested as model compounds and were found to be readily engaged into Congo Red mesophase. The effect of hydrophobicity on ligand binding was evaluated following the incorporation of homologic 10, 12, 14, 16 carbon chain organic acids and the effect of charge using small mobile tandem molecules of pI differing by a few pH units (lysine-norleucine, tyrosine-tyramine). Positive charge seems to affect binding especially by influencing the organization of molecules and the shape of the micelle simultaneously. Congo Red immobilized to heat-aggregated immunoglobulins and antibodies in the immune complex was found to retain its binding ability, confirming its possible usefulness for drug transport.

Heat-induced structural changes in the Fab fragment of IgG recognized by molecular dynamics stimulation--implications for signal transduction in antibodies.

Molecular dynamics simulation was used to identify the conformational alterations in the Fab fragment (Kol), driven by heating at 300, 320, and 340 K. Comparison of heat-modified VH,CH1 and VL,CL domain structures with the corresponding crystal conformations revealed specific differences, most definitely expressed in the CH1 domain. These are dislocations of predominantly peripheral peptide loops exposed to the V-C interdomain interface, comprising in particular the 175-185 amino acids of the heavy chain, as well as the 112-123 amino acids of the interdomain hinge fragment. The deviations, limited to peripheral domain regions at 300 and 320 K, spread at 340 K. The resulting relaxation of the tertiary packing in the protein (including the hydrophobic core) initiates global melting of the domain. These theoretical results were supported by experimental findings concerning penetration and binding of dyes (Congo Red, Trypan Blue, ANS) to the protein. Packing relaxation of the CH1 domain is turned on after disruption of the specific peptide arrangement formed at the V-C interdomain interface basically at the hinge portion (117-122) and at fragments of adjacent peptide loops (149-154, 171-179) of CH1 origin, most probably playing the role of a switching mechanism. The dislocations also comprise the 131-141 amino acids of the loops accommodated at the CH1-CH2 interface. However, the lack of crystallographic data concerning the Fab-Fc interface limits discussion of this effect to speculation. It was concluded that the unconcerted movements of the V and C parts of the Fab fragment are an intrinsic driving mechanism, introducing structural alterations into the C domains. It is suggested that the domain relaxation, induced by heating or mechanical constraints, allows for intermolecular interactions, affecting in this way the stability of the immune complex.