PracticeUpdate Conference Series: IID 2018

ISSN 2208-150X (Print) ISSN 2208-1518 (Online)

INTERNATIONAL INVESTIGATIVE DERMATOLOGY 2018 16–19 MAY 2018 • ORLANDO, FLORIDA, USA

THE BEST OF IID 2018 MEETING Results of Two Studies Point to Potential Treatments for Alopecia Areata • Cutaneous Bacterial Presence May Provoke Activated Immune Response in Hidradenitis Suppurativa • Neutralization of Interleukin 17 Implicated in Inflammatory Skin Diseases Overall and in Psoriasis Specifically • CD8-Positive T Cells Predict Disease Progression and Drive Visible Inflammation in Cutaneous T-Cell Lymphoma

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References: 1. Gittler JK, et al. J Allergy Clin Immunol 2012;130(6):1344–1354. 2. Leung DYM, et al. J Clin Invest 2004;113(5):651–657. 3. Suárez Fariñas M, et al. J Allergy Clin Immunol 2011;127(4):954–964. 4. De Benedetto A, et al. J Allergy Clin Immunol 2011;127(3):773–786. 5. Mollanazar NK, et al. Clinic Rev Allerg Immunol 2015. doi:10.1007/s12016-015-8488-5. ©2018 Sanofi. Sanofi-Aventis Australia Pty Ltd trading as Sanofi Genzyme, ABN 31 008 558 807. Talavera Corporate Centre, Building D 12-24 Talavera Road, Macquarie Park, NSW 2113. www.sanofigenzyme.com.au. GZANZ.DUP.17.10.0192 Date of preparation May 2018. SAG0151.

Sanofi Genzyme and Regeneron are committed to providing resources to advance research in dermatology in areas of unmet medical needs among patients with poorly controlled moderate-to-severe atopic dermatitis.

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Contents

IID 2018 • 16–19 May 2018 • Orlando, Florida, USA BY THE PRACTICEUPDATE EDITORIAL TEAM

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4 Results of Two Studies Point to Potential Treatments for Alopecia Areata 6 Cutaneous Bacterial Presence May Provoke Activated Immune Response in Hidradenitis Suppurativa 8 The OX40 Inhibitor GBR 830 Induces Progressive and Sustained Changes in Biomarkers of Eczema 9 Results of Two Preclinical Studies Point to Potential Therapeutic Targets in Cutaneous T-Cell Lymphoma 10 Neutralization of Interleukin 17 Implicated in Inflammatory Skin Diseases Overall and in Psoriasis Specifically 12 CD8-Positive T Cells Predict Disease Progression and Drive Visible Inflammation in Cutaneous T-Cell Lymphoma

14 BLZ-100 (Tozuleristide) Shown to Discriminate Between Subtle Molecular Subtypes of Basal Cell Carcinoma 14 Secukinumab Reduces CD4-Positive Th17, As Well As CD8- and CD4-Positive Treg Cells in Patients With Plaque Psoriasis 16 Vitamin C and Its Derivatives Suppress Melanogenesis 18 Results of Two Studies Point to Janus Kinase Inhibition for Alopecia Areata 18 After 52 Weeks, Ixekizumab Is Linked to Consistently Higher Responses Than Ustekinumab Across Patient Subgroups With Moderate to Severe Plaque Psoriasis 20 Response to IL-23 Blockade With Guselkumab Associated With HLA-Cw6 Status in Patients With Psoriasis 22 Two Topical Preparations Show Promise for Eczema

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Results of Two Studies Point to Potential Treatments for Alopecia Areata The selective interleukin 2/9/15 inhibitor BNZ-1 has shown promise as a therapy for alopecia areata, reports a preclinical and a clinical study presented at IID 2018.

BNZ-1 P aul Frohna, MD, PhD, PharmD, of Bioniz in Irvine, California, explained that immune pathways required for autoreactive T cell activation in alopecia areata have been recently proposed to involve the common γ chain cytokines interleukins 2, 9, and 15. These interleu- kins have been shown to be upregulated in animal models of alopecia areata and in human biopsies of alopecia areata lesions. BNZ-1 is a PEGylated peptide that binds to the γ chain receptor and selectively inhib- its interleukin 2, 9, and 15 signaling. This inhibition shuts off the JAK-STAT/P13K/Akt/ mTOR and Raf/ERK/MAPK pathways.

BNZ-1 has exhibited efficacy in reversing hair loss in a humanized mouse model of alopecia areata. BNZ-1 has undergone extensive preclinical safety testing to initiate an investigational new drug appli- cation with the FDA. BNZ1-CT-102 was a randomized, single-blind, placebo-controlled, multiple-dose study that characterized the safety, pharmacokinetics, and pharmacodynamics of intravenous BNZ-1 administered to healthy adults (n=5 per dose) once weekly at 0.5, 1, or 1.5 mg per kilogram of body weight. Overall safety was good, with no serious or severe adverse effects, infusion reac- tions, or dose-limiting toxicity observed. In all, 11 of 24 patients (46%) who received BNZ-1 experienced at least 1 adverse effect. A total of 19 adverse effects were reported. A total of 3 of 7 (43%) patients administered placebo experienced at least 1 adverse effect, with 9 adverse effects reported. Adverse effects that occurred in more than 1 BNZ-1-treated subject were

Dr. Paul Frohna

" …strong associations were shown between alopecia areata and mental health disorders and emergencies in this cohort of hospitalized patients. "

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Alopecia areata carries a substantial mental health burden V ivek Singam, BLA, of the Feinberg School of Medicine at Northwestern University in Chicago, explained that alopecia areata can be distressing psychologically and can impair quality of life. Studies of mental health comorbidities of alopecia areata, however, are limited. Dr. Singam and colleagues sought to identify mental health disorders and emergencies associated with alopecia areata. They examined data from the National Inpatient Sample, which includes an approx- imately 20% sample of all US hospitalizations (n=87,053,155 admissions). They constructed multivariable logistic regression models with each mental health condition as the dependent variable and alopecia areata, as well as age, sex, race/ethnicity, and insurance status as independent variables. Overall, 1167 children and adults were identified with an inpatient diagnosis of alopecia areata. Alopecia areata was significantly associated with any mental health disorder (adjusted odds ratio 2.292, 95% CI 1.974–2.661). These disorders included: ƒ ƒ Attention deficit hyperactivity disorder and conduct disorders (8.109, 95% CI 5.162–12.739) ƒ ƒ Impulse control disorders (4.946, 95% CI 1.601–15.275) ƒ ƒ Adjustment disorders (4.795, 95% CI 2.586–8.889) ƒ ƒ Developmental disorders (4.345, 95% CI 2.748–6.869) ƒ ƒ Personality disorders (3.815, 95% CI 2.282–6.370) ƒ ƒ Suicide or intentional self-inflicted injury (2.773, 95% CI 1.659–4.634) ƒ ƒ Substance abuse (2.585, 95% CI 1.973–3.385) ƒ ƒ Anxiety disorders (2.456, 95% CI 1.905–3.165) ƒ ƒ Mood disorders (2.178, 95% CI 1.805–2.628) ƒ ƒ Psychotic disorders (2.063, 95 % CI 1.375–3.097) ƒ ƒ History of mental health disorder or substance abuse (1.718, 95% CI 1.415–2.085) ƒ ƒ Alcohol abuse (1.479, 95% CI 1.052–2.080) ƒ ƒ Other mental health disorders (2.914, 95% CI 1.717–4.946) Alopecia areata was not associated with cognitive or pediatric mental health disorders. Dr. Singam concluded that strong associations were shown between alopecia areata and mental health disorders and emergencies in this cohort of hospitalized patients. Studies are needed to address the causes and ideal interventions for mental health disorders surrounding alopecia areata.

sore throat, headache, and rhinorrhea. Exposure increased dose-proportionally with an accumulation of approximately 40–60% and approximately 10–20% with once weekly and once every other week dosing, respectively. BNZ-1 elimination half-life was approxi- mately 4–5 days. BNZ-1 was associated with exposure- dependent decreases in Tregs (interleukin 2), natural killer cells (interleukins 2 and 15), and CD8-positive central memory T cells (interleukin 15) as measured by flow cytometry of peripheral bloodmononuclear cells. Tregs and central memory T cells reached maximum reduction at day 29. Natural killer cells dropped initially and tended to recover by day 29 except in the 1.5 mg once-weekly and 3 mg every- other-week cohorts. T cells, B cells, and monocytes were unaffected. Dr. Frohna concluded that the favorable safety and potent pharmacodynamic activity of BNZ-1 support the planned phase II trial in patients with moderate to severe alopecia areata.

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IID 2018 • PRACTICEUPDATE CONFERENCE SERIES

Cutaneous Bacterial Presence May Provoke Activated Immune Response in Hidradenitis Suppurativa Cutaneous bacterial presence may provoke an activated immune response in hidradenitis suppurativa, and these organisms may become resistant to antibiotics, results of a genomic and proteomic study show.

M ichelle A. Lowes, MD, PhD, of Rockefeller University, and Lauren K. Hoffman, BA, of Albert Einstein College of Medicine in Bronx, New York, set out to determine the skin and blood transcriptomeof patientswithhidradenitis suppurativa. She analyzed patient data from two 2016 studies by Blok et al. The female-to-male ratio was 13:4 and age range 20–53 years. A total of 13 patients suffered from Hurley stage II, and 4 from stage III disease. Data of these patients were compared with that of 10 healthy volunteers. The reanalysis employed a mixed effect model with random intercept to determine: ƒ ƒ Differentially expressed genes and fold change >2.0 and false discovery rate <0.05 ƒ ƒ Differentially expressed proteins as fold change >1.5 and false discovery rate <0.05 In the hidradenitis suppurativa skin transcriptome (lesional vs nonlesional skin), an abundance of immunoglobulins, antimicrobial peptides, and an interferon signature were observed. CIBERSORT analysis of the hidradenitis suppu- rativa skin transcriptome revealed a significantly increased proportion of plasma cells in lesional skin. Gene sets related Notch signaling and interferon pathways were activated differentially in hidrade- nitis suppurativa lesional skin vs nonlesional skin. In the hidradenitis suppurativa skin and blood tran- scriptome and blood proteome, gene sets related to the complement system changed significantly (false discovery rate <0.05), with dysregulation of complement-specific differentially expressed genes and differentially expressed proteins. Ms. Hoffman concluded that the data pointed to an activated immune response that may be respond- ing to a cutaneous bacterial presence. The results raised the possibility that this activated immune response may drive hidradenitis suppurativa dis- ease progression.

Dr. Michelle A. Lowes

Bacteriamay become resistant to antibiotics used for hidradenitis suppurativa H eather Ma, BS, of the University of California in San Francisco, explained that long courses of antibiotics are commonly used to treat hidradenitis suppurativa. These prolonged courses raise concern about antibiotic resistance. Ms. Ma, Joslyn Sciacca Kirby, MD, and Paul Leiphart, BA, of the Penn State College of Medicine in Hershey, and Haley B. Naik, MD, of the University of California in San Francisco, set out to examine the effect of antiseptic washes on the frequency of antibiotic resistance in hidradenitis suppurativa lesions. Dr. Naik told Elsevier’s PracticeUpdate , “High- quality evidence to support therapeutic interventions for hidradenitis suppurativa is lacking, partly due to a poor understanding of the patho- genesis of the disorder. More effective, targeted therapies for hidradenitis suppurativa are needed.” She continued, “Antimicrobial therapy and antisep- tic washes have been a mainstay of hidradenitis suppurativa management for decades, owing to the purulent and malodorous nature of lesions and the presence of bacteria identified by con- ventional culture methods in approximately 50% of

Dr. Lauren K. Hoffman

Ms. Heather Ma

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lesions. Few data, however, support the use of antiseptic washes, both to manage hidradenitis suppurativa and to prevent antibiotic resistance.” “Our aim,” she asserted, “in conducting this study was to better understand the role that antiseptic washes may play in mitigating antibiotic resistance.” The team assessed patients with hidrad- enitis suppurativa at two tertiary care centers from 2012–2017. They abstracted data from the electronic records of 80 patients with a total of 121 total wound or tissue culture specimens from hidradenitis suppurativa lesions. The study included culture results and the use of oral or top- ical antibiotics and antiseptic cleansers such as chlorhexidine, dilute bleach baths, and benzoyl peroxide concomitantly or within 4 weeks prior to culture. Dr. Sciacca Kirby told Elsevier’s PracticeUpdate , “While topical antimicro- bial washes are frequently discussed for hidradenitis suppurativa, this study found they were part of the treatment plan in about 38% of patients.” Staphylococcus aureus was the most common bacterium cultured from

Lower resistance rates to penicillins were observed in patients not using antibiotics or concomitant antiseptic wash vs penicil- lins + a concomitant antiseptic wash (no antibiotics/antiseptics 37.7% vs penicillins + antiseptic cleanser 51.8%, P = .02). Ms. Ma, Mr. Leiphart, andDrs. Sciacca Kirby and Naik concluded that taking antibiot- ics may contribute to antibiotic-resistant organisms in hidradenitis suppurativa lesions. Further study is needed to deter- mine whether antiseptic washes affect resistance rates and their role in the management of hidradenitis suppurativa. Dr. Naik noted, “Results of this small ret- rospective study are compelling because they indicate that prolonged and likely repetitive courses of antibiotics for hidradenitis suppurativa management may contribute to antibiotic resistance. They also suggest a possible role of anti- septics in mitigating resistance to specific antibiotics used to manage the disorder. Antiseptics may potentially ensure anti- biotic efficacy. Large prospective studies are needed to confirm and expand our findings.”

hidradenitis suppurativa lesions. It exhib- ited lower resistance rates to tetracyclines (15.9%) and trimethoprim-sulfamethoxa- zole (4.4%) and higher resistance rates to penicillins (61.6%) and cephalosporins (35.3%) (P < .0001). Across cultures, the resistance rate for penicillins (43.1%) was the highest of all cultures and sensitivities performed. Clindamycin, tetracyclines, and cephalo- sporins all exhibited resistance rates of 13% or higher. The researchers investigated whether rates of resistance to these antibiotics differed when patients were treated with an antibiotic alone, an antibiotic + an anti- septic wash, an antiseptic wash alone, or none of these. The team found a difference in rates of resistance across groups to penicillins and cephalosporins (P = .02). Specifically, cephalosporin resistance was lower in patients who used antibiotics + a con- comitant antiseptic wash (4 of 44 patients, 9.1%); the wash alone (1 of 15 patients, 6.7%); or neither (5 of 60 patients, 8.3%); than in those using an antibiotic alone (9 of 27 patients, 33.3%).

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TheOX40 Inhibitor GBR830 Induces Progressive and SustainedChanges inBiomarkers of Eczema The first-in-class, humanized, monoclonal immunoglobulin G1 antibody specific for inhibiting OX40, a costimulatory receptor on activated T-cells, has been shown to induce significant, progressive, and long-lasting changes in lesional biopsies up to day 71 in patients with eczema, a proof-of-concept, placebo-controlled trial shows.

E mma Guttman-Yassky, MD, PhD, of the Mount Sinai Health System in New York, investigated the effects of GBR 830 on biomarkers of eczema, and generated the first clinical evidence of biological activity. OX40 is a costimulatory tumor necrosis factor R-superfamily immune checkpoint receptor. OX40 is expressed almost exclusively on activated T lym- phocytes, and not on naïve or resting memory cells. Costimulatory signals are essential for T cell activity, and binding between OX40 and OX40L is a biomarker of autoimmune disease severity. Activation of this pathway leads to conversion of activated T cells into memory T cells, which pro- mote inflammation. Regulatory T cells also contribute to inflammation, and OX40 signaling by these cells downregulates immune-suppressing functions. It is believed that GBR 830 may inhibit the dual activities of OX40 and OX40L binding in both activated T cells and regula- tory T cells, thus potentially reducing inflammation associated with symptoms of atopic dermatitis. OX40 signaling can enhance proliferation, sur- vival, and effector functions of T cells with a more pronounced effect on late, chronic, and memory responses. Results of numerous studies have indicated that OX40 may play a central role in various T cell- mediated pathological immune responses, implying therapeutic potential of antagonistic antibodies targeting OX40. Adults with affected body surface area ≥10%, Eczema Area and Severity Index score ≥12, SCORing Atopic Dermatitis ≥20, immunoglobulin A ≥3, and history of inadequate response to topical treatments were randomized 3:1 to GBR 830 (10 mg per kilogram of body weight intravenously at baseline and day 29 or placebo). In all, 63% (39/62) of patients with eczema expe- rienced at least 1 treatment-emergent adverse effect; these were distributed similarly between the GBR 830 and placebo groups. Evaluable skin biopsies were available for 40 patients at baseline, for 39 patients at day 29 (n=28, GBR 830; n=11 placebo). Evaluable skin biopsies were available for 29 patients at day 71 (n=22, GBR 830; n=7 placebo). Significant reductions from baseline (P < .001) were found with GBR 830, but not placebo, in

treatment-specific biomarkers (OX40-positive T-cells, OX40L-positive dendritic cells). Patients who received GBR 830 also exhibited significant reductions from baseline (P < .001) in measures of hyperplasia (epidermal thickness, keratin 16 mRNA and protein expression, and Ki67 cell counts). GBR 830 was associated with significant reduc- tions from baseline (P < .01) in eczema biomarkers, including Th2 chemokines (CCL17, CCL11, TSLPR), Th1/interferon markers (interferon γ, CXCL10), and Th17/Th22-associated products (interleukin 23p19, S100A9/S100A12). Clinically, a greater proportion of GBR830-treated patients achieved Eczema Area and Severity Index score 50 (≥50% reduction in score from baseline) vs placebo at day 29 (43.6% vs 20.0%, P = .11) and day 71 (76.9% vs 37.5%, P = .04). Dr. Guttman-Yassky concluded that GBR 830 induced significant, progressive, and long-lasting changes in lesional biopsies up to day 71 and was well tolerated in this cohort of patients with eczema. A phase IIb clinical trial of GBR 830 for mod- erate-to-severe atopic dermatitis will begin enrollment in June 2018. In this double-blind, placebo-controlled trial, approximately 392 patients will be randomized across four dosing arms to receive GBR 830 or placebo. The trial’s primary objective will be to compare the effectiveness of GBR 830 on reducing the severity of atopic dermatitis vs placebo, as measured by Investigator’s Global Assessment. Secondary efficacy measures will include patients with >75% improvement in disease severity, as measured by the Eczema Area and Severity Index. Other measures will include disease activity using validated assessment tools such as Eczema Area and Severity Index response and Scoring Atopic Dermatitis. The trial will also assess safety, as well as biomark- ers relevant to the disease and unique mechanism of GBR 830. GBR 830 is also being explored to treat other inflammatory autoimmune conditions where dys- regulation of OX40 overexpression is implicated in disease activity.

Dr. Emma Guttman-Yassky

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Results of Two Preclinical Studies Point to Potential Therapeutic Targets inCutaneous T-Cell Lymphoma Combined inhibition of the BET family proteins + histone deacetylases, as well as the topic antimicrobial gentian violet, may hold promise in targeting cutaneous T-cell lymphoma, results of two preclinical studies show. Combined inhibition of the BET family proteins + histone deacetylases activation-induced cell death is an effective way to kill CTCL cells in vitro and ex vivo.

" …the superior antitumor effects of JQ1/SAHA in vitro and ex vivo provide a

Dr. Wu and colleagues set out to identify novel small molecules that induce extrinsic apoptosis in CTCL. They used the HH CTCL line in an enzyme- linked immunosorbent assay to detect cleaved caspase 8 induced by 1710 unique compounds in the National Institutes of Health Clinical Collection, Selleck Kinase Inhibitor Library and Prestwick Chemical Library. As assessed by annexin V/PI flow cytome- try of the HH cell line, the non-prescription topical antimicrobial gentian violet induced more total apoptosis than mechlorethamine. Mechlorethamine is approved as topical therapy for CTCL. Gentian violet induced approximately 4 to 6 times greater apoptosis in CTCL lines MyLa, SeAx, Hut78, HH, and SZ4 than in normal keratinocytes. This pronounced effect suggested a favorable topical toxicity profile. In addition to increasing caspase 8, gentian violet also upregulated death receptors DR4, DR5, and the TRAIL and FasL ligands. This upregulation was consistent with induction of extrinsic apoptosis via the TRAIL and Fas pathways. Increased phosphorylation of phospholipase C-γ 1, Ca++ influx, and reactive oxygen species were also detected in CTCL lines, which indicated that the mechanism of FasL upregulation involves key ele- ments of the activation-induced cell death pathway. Methotrexate increased gentian violet apop- tosis significantly in Sezary blood cells and all CTCL lines evaluated except Fas-null SeAx. This increase suggested the role of Fas in this process. In ex vivo studies, 1 μm gentian violet induced up to 90% CTCL apoptosis in Sezary blood cells. Gentian violet also reduced expression of anti-apoptotic MCL-1 and proproliferative NFkB components, while increasing IkB levels that pro- mote NFkB catabolism. Furthermore, gentian violet inhibited cell prolifera- tion and induced cell cycle arrest. Though purple at neutral pH, gentian violet can be rendered colorless by altering its pH. Dr. Wu concluded that these preclinical findings broaden the knowledge about the antineoplastic effects of gentian violet and provide a rationale for clinical studies of gentian violet as a novel, inexpensive topical therapy for CTCL.

Lei Zhao, PhD, of the Wisconsin School of Medicine and Public Health in Madison, Wisconsin, explained that advanced-stage cutaneous T-cell lymphoma (CTCL) is often a fatal malignancy despite optimal use of available treatments. Dr. Zhao and colleagues evaluated a novel therapy for CTCL in preclinical in vitro and ex vivo experiments. Their goal was to evaluate combination therapy with JQ1 (a prototypical BET bromodomain inhibitor (BET) + the HDAC inhibitor SAHA, approved to treat CTCL. In five CTCL lines, the IC50 was 0.5–1 µM for both compounds. The JQ1/SAHA combination proved to be uniformly synergistic against cell viability at 1 µM concentrations (Cl <1). The combination also induced G0/G1 cell cycle arrest. Propidium iodide/annexin V flowcytometry showed all CTCL lines exhibited significantly greater apop- tosis (approximately 40–90%) with combination treatment than with either agent alone. Relative to single agents, combination treatment showed significantly greater activation of apop- totic caspases 3, 8, and 9. This greater activation was confirmed by immunoblot analysis. Combination treatment induced significant increases in extrinsic apoptotic pathway death receptors and ligands (FasL, DR4, DR5, TRAIL, and tumor necrosis factor R1) that were more consistent across the CTCL lines than with the single agents. Ex vivo studies of leukemic CTCL cells obtained from four patients with Sezary syndrome showed the highest levels of apoptosis were in response to JQ1/SAHA combination treatment and ranged from 71% to 93%. In contrast, combination treatment of normal CD4- positive T cells induced only minimal increases in apoptosis (2–9%). Dr. Zhao concluded that the superior antitumor effects of JQ1/SAHA in vitro and ex vivo provide a rationale for clinical trials exploring BET inhibition/ HDAC inhibition in combination for CTCL. Gentian violet Jianqiang Wu, MD, PhD, also of the Wisconsin School of Medicine and Public Health, stated that in 2015 his team showed previously that triggering the extrinsic apoptotic pathway by upregulating

rationale for clinical trials exploring BET inhibition/ HDAC inhibition in combination for CTCL. "

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Neutralization of Interleukin 17 Implicated in Inflammatory Skin Diseases Overall and in Psoriasis Specifically Interleukin 17C was explored as a checkpoint in innate skin immunology, and interleukin 17A in terms of molecular transformation. These two presentations were delivered IID 2018.

Interleukin 17C: A checkpoint in innate skin immunology F elix Lauffer, MD, of the Technische Universität München in Munich, Germany, explained that inflammatory skin diseases are frequent and exert a major impact on quality of life. While the importance of interleukin 17A is well known, other members of the interleukin 17 family, such as inter- leukin 17C, remain poorly investigated.

Cell-free supernatant of keratinocytes stimulated with interleukin 17C enhanced the migratory poten- tial of neutrophil granulocytes to a comparable level as CXCL8. To assess the relevance of interleukin 17C in a complex model of human disease, Dr. Lauffer’s team finally cultured human skin biopsies of pso- riasis and atopic dermatitis with an interleukin 17C-neutralizing antibody. Compared with untreated controls, neutralization of interleukin 17C led to significant downregulation of pro-inflammatory cytokines and antimicrobial peptides, for example, interleukin 36G (DEFB4A). This downregulation demonstrated the crucial role of interleukin 17C in human inflammatory conditions. Dr. Lauffer concluded that interleukin 17C is broadly expressed in human skin pathology, is induced by innate immune stimulative activation of NFкB, and forms a pro-inflammatory feedback loop with TNFα. Dr. Lauffer concluded, “We were able to demon- strate that interleukin 17C potentiates the inflammatory response of keratinocytes in vitro and that specific neutralization of interleukin 17C decreases inflammation in murine and human models of psoriasis and atopic dermatitis. Inhibition of interleukin 17C signaling is a promising, novel approach to treat inflammatory skin diseases.”

Dr. Lauffer told Elsevier’s PracticeUpdate , “Inflammatory skin disease are frequent and impact quality of life severely. While new therapeutic strat- egies for psoriasis have been discovered with great success, targeted therapies in other inflammatory skin diseases, such as atopic dermatitis, are still needed. We performed this study to understand how the epithelial- derived cytokine interleukin 17C can influence skin inflammation.” Dr. Lauffer and colleagues detected high numbers of interleukin 17C-positive cells in diverse inflam- matory, autoimmune, and infectious skin diseases. In keratinocytes, interleukin 17C was induced by interleukin 1β, flagellin, and tumor necrosis factor α (TNFα) via upregulation of p65, phospho-p65, and IкBα. Expression of interleukin 17C transcripts was dependent on NFкB and ERK1/2. Stimulation of interleukin 17C led to enhanced expression of antimicrobial peptides in primary human keratino- cytes. This effect was potentiated synergistically by costimulation with TNFα.

Dr. Felix Lauffer

Dr. Trine Bertelsen

PRACTICEUPDATE CONFERENCE SERIES • IID 2018 10

" …the results provided insight into molecular transformation during anti-interleukin 17A treatment. They provide an explanation of the molecular “scar” that is generated when psoriasis is in remission. "

Molecular transformation during interleukin 17A treatment T rine Bertelsen, MD, of Aarhus University in Denmark, explained that antagonists of inter- leukin 17 have proven to be highly effective for psoriasis. Many mechanisms underlying this efficacy, however, have yet to be determined. Moreover, mechanisms underlying molecular remission of psoriasis remain elusive. Dr. Bertelsen and colleagues set out to investigate molecular complexity in psoriatic patients during treatment with an anti-interleukin 17A agent. Fourteen patients treated with secukinumab (anti-in- terleukin 17A) were included. Skin biopsies and blood samples were collected at day 0, 4, 14, 42, and 84. Clinical scores such as Psoriasis Affected Surface Area, Physician's Global Assessment, and body surface area were registered at each visit. Samples were then processed for microar- ray, quantitative polymerase chain reaction, and immunohistochemistry. Inflammatory mediators such as CXCL8, interleukin 19, DEFB4, and interleukin 36A were downregu- lated early, at day 4 after the start of treatment. Other mediators such as S100A7, interleukin 20, and CCL20 were downregulated at day 14. Moreover, when comparing nonlesional skin with previous lesional skin at day 84, expression of sev- eral molecular markers was altered, even though psoriasis had cleared.

IкBζ, encoded by the NFKBIZ gene, is a key reg- ulator in psoriasis through its role in mediating interleukin 17A-driven effects. Interestingly, NFKBIZ mRNA expression was downregulated during anti-interleukin 17A treatment and correlated with clinical scores. Therefore, signaling pathways involved in interleu- kin 17A-induced IкBζ expression were investigated further in human keratinocytes. By using siRNA knockdown and quantitative polymerase chain reaction, Dr. Bertelsen’s team demonstrated IкBζ to be mediated through an Act1/p38 MAPK/c-Jun/ NF-кB- dependent mechanism. Dr. Bertelsen concluded that the results provided insight into molecular transformation during anti-interleukin 17A treatment. They provide an explanation of the molecular “scar” that is gener- ated when psoriasis is in remission. Furthermore, the results presented IкBζ as an important player in interleukin 17A-driven effects in psoriasis.

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IID 2018 • PRACTICEUPDATE CONFERENCE SERIES 11

CD8-Positive T Cells Predict Disease Progression and Drive Visible Inflammation in Cutaneous T-Cell Lymphoma Two studies employing skin biopsy analysis pointed to a predictor of disease progression and a driver of visible inflammation in cutaneous T-cell lymphoma. Results were reported at IID 2018.

CD8-positive T cells as predictors of disease progression E dward Seger, MS, of the State University of New York Downstate Medical Center in Brooklyn, working in the laboratory of John O’Malley, MD, PhD, at the Dana-Farber/Brigham and Women’s Cancer Center in Boston, Massachusetts, explained that among the challenges of caring for patients with mycosis fungoides, the most common of the cutaneous T-cell lymphomas, is early identification of patients who will develop progressive disease. Mr. Seger explained to Elsevier’s PracticeUpdate , “Risk-stratifying patients with cutaneous T-cell lymphoma has remained a challenge. While many will experience an indolent disease course and a normal life expectancy, a small subset will progress to more advanced and potentially lethal disease. We are attempting to identify these patients early in their disease course so we can treat them more appropriately.” Mr. Seger and colleagues hypothesized that the balance between antitumor CD8-positive T cells and anti-inflammatory FoxP3-positive T cell regulatory cell infiltration in the initial cutaneous T-cell lymphoma biopsy would be predictive of disease progression. The cohort included 33 patients with cutaneous T-cell lymphoma (n=28 stage I, n=5 stage IIB). Median follow-up duration was 78 months. The cohort was divided into two groups depending on whether they developed progressive disease. They studied their skin biopsy specimens by multispectral immunofluorescence based on staining for CD4, CD8, FoxP3, programmed death 1, and DAPI expression. These examinations were followed by image deconvolution and automated cell analysis. Patients with greater CD8-positive T cell infiltration experienced significantly improved progres- sion-free survival in univariate (HR 0.6, CI 0.4–0.97, P = .04) and multivariate analysis (HR 0.4, CI 0.2–0.7, P = .01). When the team restricted the analysis to early-stage patients, this finding remained significant (HR 0.6, CI 0.4–0.98, P = .04) and did not correlate with malignant clone frequency or age.

Mr. Edward Seger

Dr. Pablo Augusto Vieyra-Garcia

PRACTICEUPDATE CONFERENCE SERIES • IID 2018 12

" …the results are exciting because we show inherent differences in the microenvironment early in the disease course, when patients present clinically very similarly. "

He continued, “On that same note, the results are exciting because we show inherent differences in the microenviron- ment early in the disease course, when patients present clinically very similarly.” “The results are very encouraging in terms of risk stratification of cutaneous T-cell lymphoma,” Mr. Seger added. “We are in the process of validating these findings in a much larger patient cohort. Additionally, we are interested in the significance of the proximity of these cytotoxic T cells to malignant immune cells. We are utilizing gene expression profiling and multispec- tral immunofluorescence to identify these close proximity cells and assess their functional capacity.” Benign T cells as drivers of visible inflammation P ablo Augusto Vieyra-Garcia, PhD, MSc, of the Medical University of Graz in Austria, explained that skin lesions of mycosis fungoides contain both malignant and benign infiltrating T cells. Dr. Vieyra-Garcia told Elsevier’s PracticeUpdate , “Understanding the sig- nificance of inflammatory signals at play, and the predominance of the malignant clone in tissue, is the main focus of our efforts.” Dr. Vieyra-Garcia and colleagues set out to study mycosis fungoides skin before and after psoralen and ultraviolet A ther- apy using high-throughput T-cell receptor sequencing, gene expression profiling, and immunostaining. Though all patients improved clinically, 30% of patients experienced improve- ment of clinical inflammation (Composite Assessment of Index Lesion Severity, modified Severity Weighted Assessment Tool) despite the continued presence of many malignant T cells in skin. Improved Composite Assessment of Index Lesion Severity scores correlated with turnover of benign T-cell clones as measured by High-throughput T cell receptor sequencing (R=0.8) but not with reduction in malignant cells (R=0.3). NanoString profiling demonstrated that benign T cell-associated genes differed markedly before and after treatment.

Benign T cells were strongly associated with a CD4/Th2 signature (CCL18, CSF1) before but with a CD8/Tc1 (CXCL9, 10, 11) signature after treatment. In all, 25 of 29 downregulated genes in untreated mycosis fungoides vs healthy skin mapped to the posttreatment CD8 cell signature. This mapping suggested that psoralen and ultraviolet A recruits a CD8 population present in healthy skin but absent from untreated mycosis fungoides. The CD8 signature also correlated with clearance of malignant cells. Changes in score on the Composite Assessment of Index Lesion Severity were correlated to changes in score across the data- set to identify genes that drive clinical inflammation. Reduced clinical inflammation was linked to loss of the pretreatment benign T-cell signature and acquisition of the post- treatment benign T-cell signature, but was unrelated to the malignant T cell sig- nature. This observation suggested that benign T cells drive visible inflammation in mycosis fungoides. OX-40/OX-40L interactions are known to drive chronic T cell stimulation. Malignant T cells expressed OX-40L and benign T cells expressed OX-40 before, but not after, psoralen and ultraviolet A. Dr. Vieyra-Garcia concluded that malig- nant and benign Th2-infiltrating T cells create an inflammatory circuit that is dis- rupted when psoralen and ultraviolet A induces Th2 cells to leave and replaces them with CD8 T cells capable of killing malignant T cells. "We are very excited with our findings,” Dr. Vieyra-Garcia said. “Together, they clearly indicate that benign T cells not only play an important role in the onset of this disease but they are also implicated in therapeutic mechanisms of well- established treatments like psoralen and ultraviolet A.” “We have many additional questions await- ing answers,” he noted, “that will bring us closer to providing better, personalized care of these patients.”

The number of FoxP3-positive regulatory T cells, programmed death 1-positive T cells, and total CD4-positive T cells were not associated with improved survival. Ratios between these cell types were also examined for potential associations with prognosis. An increased ratio of CD4-positive T cells to FoxP3-positive T cells was associated with worse pro- gression-free survival (HR 1.02, CI 1.0–1.03, P = .01), but this result did not remain sig- nificant in multivariate analysis. Mr. Seger’s group is in the process of confirming this finding in a larger cohort. Mr. Seger concluded that CD8-positive T cell density in the lesional skin of patients with cutaneous T-cell lymphoma is predictive of disease progression and may be a useful adjunct in risk-stratifying early-stage patients. “The results carry tremendous clinical sig- nificance,” Mr. Seger noted. “Using initial skin biopsies and staining for common cellular markers, we are using a technique that can easily be implemented into clin- ical practice.”

www.practiceupdate.com/c/68300

IID 2018 • PRACTICEUPDATE CONFERENCE SERIES 13

BLZ-100 (Tozuleristide) Shown to Discriminate Between Subtle Molecular Subtypes of Basal Cell Carcinoma The first in-human study of BLZ-100 (tozuleristide), which selectively detects malignant tissue to enable more complete and precise surgical resection, has shown that the compound can discriminate between subtle molecular subtypes of basal cell carcinoma. M iko Yamada, PhD, of the University of Queensland in Brisbane, Australia, explained that skin as more precise and complete surgical removal of cancer. Surrounding normal tissue is spared. In this first in-human study, BLZ 100 was administered intravenously to 21 adult patients 2 days prior to excision of known/ suspected skin cancers.

cancer is often curable with complete excision. Wide margins are needed, how- ever, particularly for cancers on the face. BLZ-100 (tozuleristide) is an intraoperative fluorescent imaging agent (tumor paint) that selectively detects malignant tissue to enable more complete and precise surgical resection. Surgery is first-line therapy for most solid tumor cancers. Tumor paint products intend to improve cancer surgery by providing real-time, high-resolution visual- ization of cancer cells throughout surgery. The ability to see cancer cells in real time and high resolution throughout surgery should enable better detection as well

The first tumor paint product candidate, BLZ-100, is in phase I clinical trials for brain and breast cancers. Additional potential applications of BLZ-100 include prostate, lung, colorectal, and other solid tumor cancers. BLZ-100 is administered by IV injection, circulates within the body, and “light ups” cancer cells. It consists of an Optide, which binds and internalizes into cancer cells, and a fluorescent dye, which emits light in the near-infrared range. Preclinical utility of BLZ-100 was demon- strated in a wide range of cancer types, including brain, breast, prostate, lung, colorectal, skin, and sarcomas.

Doses were 1, 3, 6, 12, and 18 mg, with 3 to 6 patients per cohort. Fluorescence imaging was conducted before and up to 48 h after dosing. BLZ-100 was tolerated well. No serious adverse effects were observed. For intermediate dose levels (3–12 mg), 9 of 10 malignant lesions were considered positive for BLZ-100 fluorescence. This positivity led Dr. Yamada and col- leagues to explore differential gene expression profiles in basal cell carci- noma tumors, the most homogeneous of the examined lesions.

SecukinumabReduces CD4-Positive Th17, AsWell As CD8- andCD4-Positive Treg Cells inPatientsWith Plaque Psoriasis

In SCULPTURE, Psoriasis Area and Severity Index 75 responders at week 12 were randomized to double-blind main- tenance treatment of 300 or 150 mg of secukinumab, given in either a 4-week fixed-interval regimen or in a retreatment- as-needed regimen. Patients who completed 52 weeks of the SCULPTURE study were eligible to continue the same dose and regimen in the extension study (n=642). The primary objective of the extension study was to assess the long-term safety and tolerability of secukinumab in patients with moderate to severe plaque psoriasis. Efficacy measures included proportion of patients achieving Psoriasis Area and Severity Index 75/90/100.

Secukinumab has been found to reduce not only CD4-positive Th17, but also CD8- and CD4-positive T-regulatory (Treg) cells in patients with plaque psoriasis. This finding of a flow cytometry study to characterize changes in immune cell populations in psoriatic lesions treated with secukinumab was reported at IID 2018. E ric J. Yang, MD, of the University of California in San Francisco, explained that psoriasis is a chronic inflamma- demonstrated broad changes that occurred in lesioned skin.

Secukinumab, approved for plaque pso- riasis in 2015, is a recombinant human monoclonal IgG1/κ antibody that binds specifically to interleukin 17A. In 2017, 5-year data were reported on secukinumab for moderate to severe plaque psoriasis. The 5-year extension study was a multicenter, double-blind, open-label, 5-year extension to the core phase 3 SCULPTURE study.

tory skin disease that involves widespread immune dysregulation. Cell populations implicated in psoriasis include kerati- nocytes, dendritic cells, and Th17 cells. These produce excess tumor necrosis factor α, and interleukins 17 and 23. Studies evaluating the impact of biolog- ics on the molecular profile of psoriasis have used bulk skin tissue and have

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Gene expression profiles of BLZ 100-positive and -negative tumors were substantially similar. Significantly, differ- entially expressed genes were cytosolic, extracellular, and cell death genes. These modest differences were correlated to a binary staining profile that suggested that BLZ 100 can discriminate between subtle molecular subtypes of basal cell carcinoma, and possibly, melanoma. Dr. Yamada concluded that the results support the safety of BLZ 100 at doses up to 18 mg. Ongoing studies are evaluating BLZ 100 safety and tissue tissue uptake in patients with other solid tumors. Over an extended treatment period from year 1 (week 52) to the end of year 5 (week 260), Psoriasis Area and Severity Index 75/90/100 response rates remained consistent. At year 1, 89% and 69%, respectively, of 168 patients with psoriasis achieved Psoriasis Area and Severity Index 75 and 90. This high rate was maintained at year 5 with 126 patients observed (89% and 66%, respectively). In addition, 44% of patients with psoriasis achieved completely clear skin (Psoriasis Area and Severity Index 100) at year 1. This rate was maintained to year 5 (41%). The safety profile continued to exhibit a favorable and consistent safety profile. In the present study, Dr. Yang and col- leagues used a novel flow cytometry protocol based on protein expression to analyze individual cell population activ- ity in skin disease. They characterized changes in immune cell populations in psoriatic lesions treated with secuki- numab, an interleukin 17A inhibitor. as an aid to surgery has tremendous potential… " " Successful surgery is a foundation of treatment and extent of resection is the single best predictor of survival in pediatric patients with brain tumors. I believe BLZ-100

An open-label phase I dose escalation and expansion study is evaluating BLZ- 100 in pediatric subjects with primary central nervous system tumors. The dose escalation part of the study has enrolled 15 pediatric patients at five prespecified dose levels to evaluate the safety and tolerability of BLZ-100 and provide clin- ical proof of principle data for BLZ-100 to detect tumors in pediatric subjects. The dose expansion part of the study is ongoing. Lead investigator Sarah Leary, MD, of Seattle Children’s Hospital, said, in a press

release, “We are continually inspired by our pediatric patients and their families, and aim to provide the best outcomes possible for these children.” She continued, “Successful surgery is a foundation of treatment and extent of resection is the single best predictor of survival in pediatric patients with brain tumors. I believe BLZ-100 as an aid to surgery has tremendous potential and look forward to further clinical testing in the pediatric population.”

www.practiceupdate.com/c/68650

" From weeks 0–12, the percentage of interleukin 17-expressing CD4-positive T effector cells in lesional skin decreased from 11.6% to 5.8%, vs a baseline percentage of 7.5% in healthy controls. "

Lesional skin biopsies of 11 patients with psoriasis were obtained at weeks 0, 2, 4, and 12 of secukinumab treatment. They were analyzed via flow cytometry and compared to healthy control skin. At week 12, mean Psoriasis Area and Severity Index improvement form week 0 of treatment was 87.1%, with 100% of patients achieving at least 75% improve- ment in mean Psoriasis Area and Severity Index. From weeks 0–12, the percentage of interleukin 17-expressing CD4-positive T effector cells in lesional skin decreased from 11.6% to 5.8%, vs a baseline percent- age of 7.5% in healthy controls. The percentage of interleukin 17-express- ing CD8-positive T cells decreased from 13.5% to 7.6%, vs 2.0% in controls.

The percentage of CD4-positive Treg cell-expressing interleukin 17 decreased from 3.6% to 0.9%, vs to 1.6% among controls. Dr. Yang concluded that secukinumab treatment reduced the percentage of interleukin 17-expressing immune cells greatly in lesioned skin. The proportion of interleukin 17-expressing CD4-positive T effector and Treg cells decreased to levels similar to or below that of healthy controls. Not only CD4-positive Th17 cells, but also CD8- and CD4-positive Treg cells produce interleukin 17 in psoriasis. Secukinumab reduces these significantly.

www.practiceupdate.com/c/68649

15 IID 2018 • PRACTICEUPDATE CONFERENCE SERIES