Tumor eradication by triplet therapy with BRAF inhibitor, TLR 7 agonist, and PD-1 antibody for BRAF-mutated melanoma.

Programmed death 1 (PD-1)/programmed death-ligand 1 inhibitors are commonly used to treat various cancers, including melanoma. However, their efficacy as monotherapy is limited, and combination immunotherapies are being explored to improve outcomes. In this study, we investigated a combination immunotherapy involving an anti-PD-1 antibody that blocks the major adaptive immune-resistant mechanisms, a BRAF inhibitor that inhibits melanoma cell proliferation, and multiple primary immune-resistant mechanisms, such as cancer cell-derived immunosuppressive cytokines, and a Toll-like receptor 7 agonist that enhances innate immune responses that promote antitumor T-cell induction and functions. Using a xenogeneic nude mouse model implanted with human BRAF-mutated melanoma, a BRAF inhibitor vemurafenib was found to restore T-cell-stimulatory activity in conventional dendritic cells by reducing immunosuppressive cytokines, including interleukin 6, produced by human melanoma. Additionally, intravenous administration of the Toll-like receptor 7 agonist DSR6434 enhanced tumor growth inhibition by vemurafenib through stimulating the plasmacytoid dendritic cells/interferon-α/natural killer cell pathways and augmenting the T-cell-stimulatory activity of conventional dendritic cells. In a syngeneic mouse model implanted with murine BRAF-mutated melanoma, the vemurafenib and DSR6434 combination synergistically augmented the induction of melanoma antigen gp100-specific T cells and inhibited tumor growth. Notably, only triplet therapy with vemurafenib, DSR6434, and the anti-PD-1 antibody resulted in complete regression of SIY antigen-transduced BRAF-mutated melanoma in a CD8 T-cell-dependent manner. These findings indicate that a triple-combination strategy targeting adaptive and primary resistant mechanisms while enhancing innate immune responses that promote tumor-specific T cells may be crucial for effective tumor eradication.

Recent treatment and prognosis in 643 patients with extramammary Paget's disease.

Information about extramammary Paget's (EMPD) treatment is limited because of the rarity of the disease. The prognosis differs between in situ EMPD and invasive EMPD; therefore, therapy should be planned according to the disease stage. We collected data on 643 EMPD cases treated between 2015 and 2019 in Japan and assessed recent trends in EMPD treatment and prognosis based on the EMPD-oriented TNM staging. Among the 643 patients, 317 had stage 0 (49.3%), 185 had stage I (28.8%), 51 had stage II (7.9%), 18 had stage IIIA (2.8%), 48 had stage IIIB (7.5%) and 24 had stage IV (3.7%) disease. Each stage showed a distinct survival curve, with the exception of stages II and IIIA. Curative surgery was most common in patients with stage 0-III disease. Chemotherapy was the first-line therapy, mainly in patients with stage IIIB and IV disease, most commonly with docetaxel (DTX), followed by DTX + tegafur gimeracil oteracil potassium (TS-1) and TS-1. Patients with local disease exhibited a 4.4% recurrence rate. Univariate analysis revealed no prognostic differences according to age, sex or primary tumour site. SLNB was not related to disease-specific survival. In multivariate analysis, female sex significantly predicted local relapse in stage 0-I (HR 3.09; 95% CI, 1.13-8.43), and initial treatment with curative surgery was significantly protective in terms of disease-specific survival in stage II-IIIA (HR, 0.17; 95% CI, 0.04-0.71) and stage IIIB-IV (HR 0.16; 95% CI, 0.05-0.51). Further clinical studies are needed to improve the prognosis of patients with stage II-IV EMPD.

Persistent alteration of the skin microbiome in patients with skin rash after receiving EGFR inhibitor treatment.

The pathological mechanism responsible for EGFR inhibitor (EGFRI)-induced skin rash remains unclear. Recent studies reveal associations between skin dysbiosis and skin inflammatory diseases. This study aimed to examine whether skin dysbiosis is associated with EGFRI-induced skin rash. Bacterial swabs were taken from the forehead of 17 cancer patients at baseline and at several time points after EGFRIs initiation, as well as from 20 healthy controls. The skin microbiome was analysed using 16S rRNA sequencing. The severity of the skin rash was assessed using the rash grade. Skin surface parameters (pH, water capacitance, and sebum level) were also measured. Compared with baseline, the abundance of Cutibacterium acnes decreased in 13 of 15 cases, and that of Staphylococcus aureus, Corynebacterium spp., Staphylococcus epidermidis or Proteobacteria increased in 13 of 15 cases after EGFRIs initiation. Skin pH increased significantly in parallel with a decrease in water capacitance after EGFRI initiation. Also, the composition of the skin microbiome of patients with severe rash was significantly different from that of healthy controls. In addition, the skin dysbiosis did not return to baseline during EGFRIs treatment for >1 year. These longitudinal observations indicate that skin dysbiosis is associated with development of skin rash.

Prevalence of Psoriatic Arthritis in Nagano Prefecture, Japan, and Efficacy of the Psoriasis Epidemiology Screening Tool: A Real-World Survey.

Pain, stiffness, and swelling are the main joint symptoms of psoriatic arthritis (PsA); however, they are also common symptoms of other joint diseases. Therefore, it is challenging to distinguish PsA from other joint diseases. To evaluate the prevalence of PsA and the frequency of joint symptoms in psoriasis patients, we conducted a prefecture-wide survey using the Psoriasis Epidemiology Screening Tool (PEST), a patient questionnaire for screening PsA to assess joint symptoms. Data were collected from 764 psoriasis patients, all of whom visited hospitals (55.1%) or clinics (44.9%) in Nagano Prefecture, Japan. The proportion of psoriasis patients with PsA was 6.5% (50 of 764); four patients (1.2%) with PsA were treated in clinics, while 46 patients (10.9%) were treated in hospitals. Based on the responses to the PEST, 18.1% of patients with psoriasis had joint symptoms. In contrast, 73.2% of psoriasis patients with joint symptoms did not have PsA. The PEST showed 52% sensitivity and 93.4% specificity for PsA. In addition, fingernail alterations were common in PsA. The proportion of the population with PsA was lower than reported previously in Japan. This may have been due to the enrollment of a large number of patients treated in clinics. Many patients with PsA were treated at hospitals, which likely reflects the tendency of patients with joint symptoms to receive intensive treatment in hospitals. In addition, based on the lower sensitivity of the PEST in this study, further studies are necessary to establish the validity of the PEST.

Usefulness of monitoring circulating tumor cells as a therapeutic biomarker in melanoma with BRAF mutation.

BACKGROUND: While molecularly targeted therapies and immune checkpoint inhibitors have improved the prognosis of advanced melanoma, biomarkers are required to monitor drug responses. Circulating tumor cells (CTCs) are released from primary and/or metastatic tumors into the peripheral blood. We examined whether CTCs have potential as biomarkers by checking the number of CTCs, as well as the BRAF genotype of individual CTCs, in melanoma patients undergoing BRAF/MEK inhibitor treatment. METHODS: CTCs were isolated from peripheral blood using a high-density dielectrophoretic microwell array, followed by labeling with melanoma-specific markers (MART-1 and/or gp100) and a leukocyte marker (CD45). The numbers of CTCs were analyzed in fifteen patients with stage 0-III melanoma. Furthermore, changes in CTC numbers were assessed in five patients with stage IV melanoma at four time points during BRAF/MEK inhibitor treatment, and the BRAF genotype was analyzed in CTCs isolated from one patient. RESULTS: We examined CTCs in patients with stage 0-III (five samples per stage: stage 0-I, stage II, and stage III), and detected CTCs even in patients with early disease (stage 0 and I). Interestingly, recurrence occurred in the lymph nodes of one stage I patient 2 years after the detection of a high number of CTCs in the patient's blood. The total number of CTCs in four of five patients with stage IV melanoma fluctuated in response to BRAF/MEK inhibitor treatment, suggesting that CTC number has potential for use as a drug response marker in advanced disease patients. Interestingly, one of those patients had CTCs harboring seven different BRAF genotypes, and the mutated CTCs disappeared upon BRAF/MEK inhibitor treatment, except for those harboring BRAFA598V. CONCLUSIONS: CTCs are present even in the early stage of melanoma, and the number of CTCs seems to reflect patients' responses to BRAF/MEK inhibitor treatment. Furthermore, genetic heterogeneity of BRAF may contribute to resistance to BRAF/MEK inhibitors. Our findings demonstrate the usefulness of CTC analysis for monitoring responses to targeted therapies in melanoma patients, and for understanding the mechanism of drug resistance.

Ligand Bound Fatty Acid Binding Protein 7 (FABP7) Drives Melanoma Cell Proliferation Via Modulation of Wnt/ß-Catenin Signaling.

PURPOSE: Fatty acid-binding protein 7 (FABP7) involved in intracellular lipid dynamics, is highly expressed in melanomas and associated with decreased patient survival. Several studies put FABP7 at the center of melanoma cell proliferation. However, the underlying mechanisms are not well deciphered. This study examines the effects of FABP7 on Wnt/ß-catenin signaling that enhances proliferation in melanoma cells. METHODS: Skmel23 cells with FABP7 silencing and Mel2 cells overexpressed with wild-type FABP7 (FABP7wt) and mutated FABP7 (FABP7mut) were used. Cell proliferation and migration were analyzed by proliferation and wound-healing assay, respectively. Transcriptional activation of the Wnt/ß-catenin signaling was measured by luciferase reporter assay. The effects of a specific FABP7 inhibitor, MF6, on proliferation, migration, and modulation of the Wnt/ß-catenin signaling were examined. RESULTS: FABP7 siRNA knockdown in Skmel23 decreased proliferation and migration, cyclin D1 expression, as well as Wnt/ß-catenin activity. Similarly, FABP7wt overexpression in Mel2 cells increased these effects, but FABP7mut abrogated these effects. Pharmacological inhibition of FABP7 function with MF6 suppressed FABP7-regulated proliferation of melanoma cells. CONCLUSION: These results suggest the importance of the interaction between FABP7 and its ligands in melanoma proliferation modulation, and the beneficial implications of therapeutic targeting of FABP7 for melanoma treatment.

Recent advances in molecular targeted therapy for unresectable and metastatic BRAF-mutated melanoma.

The clinical outcome of BRAF-mutated advanced melanoma has been improved by both molecular targeted therapies and immune checkpoint inhibitors. Long-term follow-up data reveal durable clinical responses in patients receiving first-line combinations of BRAF inhibitors plus MEK inhibitors, particularly those showing a complete response. Clinical outcomes are also associated with the lactate dehydrogenase levels and the number of metastatic organs. Although brain metastasis is frequently difficult to control, systemic therapy is preferred in cases with small and asymptomatic brain metastases associated with progressive extra-cranial disease. Control of intra-cranial disease with BRAF inhibitors plus MEK inhibitors is comparable with that of immune checkpoint inhibitors, although immune checkpoint inhibitors are superior to targeted therapies with respect to survival. The BRAF inhibitors plus MEK inhibitors regimen is well-tolerated, and toxicities are usually manageable and reversible, but differ according to the specific regimen used. Guidelines in the United States, Europe, and Japan recommend targeted therapy for patients who need early tumor responses. A meta-analysis of retrospective data shows that the baseline lactate dehydrogenase level is significantly higher in patients treated with BRAF inhibitors plus MEK inhibitors than in those treated with immune checkpoint inhibitors, suggesting that clinicians tend to use BRAF inhibitors plus MEK inhibitors for more advanced disease. Since there is insufficient efficacy and safety data on the use of targeted therapies for acral and mucosal melanoma, a retrospective analysis may be useful. The combination of molecular targeted therapy plus immune checkpoint inhibitors is expected to elicit further improvement. The results of several trials using combination or sequential therapies will be available in the next few years.

Involvement of local renin-angiotensin system in immunosuppression of tumor microenvironment.

To improve current cancer immunotherapies, strategies to modulate various immunosuppressive cells including myeloid derived suppressor cells (MDSC) which were shown to be negative factors in immune-checkpoint blockade therapy, need to be developed. In the present study, we evaluated the role of the local renin-angiotensin system (RAS) in the tumor immune-microenvironment using murine models bearing tumor cell lines in which RAS was not involved in their proliferation and angiogenetic ability. Giving angiotensin II receptor blockers (ARB) to C57BL/6 mice bearing murine colon cancer cell line MC38 resulted in significant enhancement of tumor antigen gp70 specific T cells. ARB administration did not change the numbers of CD11b+ myeloid cells in tumors, but significantly reduced their T-cell inhibitory ability along with decreased production of various immunosuppressive factors including interleukin (IL)-6, IL-10, vascular endothelial growth factor (VEGF), and arginase by CD11b+ cells in tumors. ARB also decreased expression of immunosuppressive factors such as chemokine ligand 12 and nitric oxide synthase 2 in cancer-associated fibroblasts (CAF). Last, combination of ARB and anti-programmed death-ligand 1 (PD-L1) antibodies resulted in significant augmentation of anti-tumor effects in a CD8+ T cell-dependent way. These results showed that RAS is involved in the generation of an immunosuppressive tumor microenvironment caused by myeloid cells and fibroblasts, other than the previously shown proliferative and angiogenetic properties of cancer cells and macrophages, and that ARB can transform the immunosuppressive properties of MDSC and CAF and could be used in combination with PD-1/PD-L1 immune-checkpoint blockade therapy.

NRAS mutations in primary and metastatic melanomas of Japanese patients.

BACKGROUND: Characterization of the MAPK signaling pathway in melanoma has led to the development of MEK inhibitors for the treatment of NRAS-mutated melanoma. The success of molecular-targeted therapies underscores the need to identify mutations in target genes. Most of the current data on genetic mutations have been obtained from Caucasian melanoma patients, and screenings of Asian populations are limited. OBJECTIVE: The aim of the present study was to examine NRAS mutations in primary and metastatic lesions of Japanese melanoma patients. METHODS: Clinical melanoma specimens were collected from 127 Japanese patients, including primary (n = 67), metastatic (n = 25) and paired primary and metastatic lesions (n = 35). NRAS mutations in exons 1 and 2 were assessed by polymerase chain reaction and Sanger sequencing. RESULTS: The incidence of NRAS mutations was 7.1 %. NRAS (Q61) was the predominant genetic alteration (77.8 %). NRAS mutations were most frequently detected in acral melanomas (9.3 %), followed by melanomas without chronic sun-induced damage (7.0 %) and mucosal melanomas (4.8 %), and were not detected in melanomas with chronic sun-induced damage. In addition, NRAS mutations were more prevalent in the extremities than in other sites. The NRAS sequence in metastatic lesions did not match that of the primary tumor in one case. CONCLUSION: The frequency of NRAS mutations is lower in the Asian population than in Caucasian patients. The observed heterogeneity of melanoma suggests that genotyping of both primary and metastatic lesions is important to identify candidate patients for molecular-targeted therapies.

Toll-like receptor 4 signaling promotes the migration of human melanoma cells.

Immune cell Toll-like receptors (TLRs) recognize conserved microbial components, leading to immune and inflammatory responses. However, TLRs are also expressed in cancer cells, including melanoma cells, which express TLR2-4. TLR4 ligands have received attention as immunotherapies; therefore, we assessed the expression of TLR4 in human melanoma specimens (29 primary lesions and 28 metastatic lesions) representing different types of melanoma. A high percentage (≥ 90%) of melanoma lesions expressed TLR4, as judged by immunohistochemistry. Next, the role of TLR4 in cell proliferation and migration was assessed using the TLR4-positive (TLR4(+)) melanoma cell lines 501mel and 888mel, and TLR4-negative (TLR4(‒)) 928mel melanoma cells. Lipopolysaccharide (LPS), a TLR4 agonist, increased the proliferation of TLR4(+) melanoma cells but not of TLR4(‒) 928mel cells. The proliferation-inducing effect of LPS in 888mel cells was abolished by blockade of TLR4 signaling via treatment with short interfering RNA (siRNA) targeting TLR4 or myeloid differentiation primary response gene 88 (MyD88), a molecule downstream of TLR4. However, knockdown of TLR4 or MyD88 expression did not affect the LPS-induced proliferation of 501mel cells, suggesting that residual TLR4 signaling is sufficient to maintain cell proliferation. By contrast, LPS increased the migration of TLR4(+) melanoma cells, and this effect was substantially inhibited by TLR4 or MyD88 knockdown. Furthermore, TLR4 knockdown decreased cell migration even in the absence of LPS, suggesting the presence of an endogenous TLR4 ligand(s) in melanoma cells. TLR4 signaling may contribute to melanoma progression, and caution should be exercised when using TLR4 ligands as adjuvant therapy for cancer.

Recurrent GATA3 P409Afs*99 Frameshift Extension Mutations in Sweat-gland Carcinoma With Neuroendocrine Differentiation.

Sweat-gland carcinoma with neuroendocrine differentiation (SCAND) was recently proposed as a new cutaneous adnexal neoplasm with neuroendocrine differentiation; however, its genetics are not well known. Herein, we performed clinicopathologic and genetic analyses of 13 SCAND cases and 5 control cases of endocrine mucin-producing sweat gland carcinoma (EMPSGC). The SCAND group included 11 males and 2 females with a median age of 68 years (range, 50 to 80 y). All SCAND lesions occurred in the ventral trunk or genital area. Of the 13 SCAND cases, 9 and 5 exhibited lymph node and distant metastases, respectively. Three (23.1%) patients with SCAND died of the disease. In contrast, neither metastasis nor mortality was confirmed in the EMPSGC cases. Immunoexpression of the androgen receptor, c-Myb, and MUC2 was limited in SCAND, whereas EMPSGC frequently expressed these immunomarkers. GATA3 P409Afs*99 extension mutations were detected in 7 (53.8%) of the 13 SCAND cases, using Sanger or panel sequencing. All 7 SCAND cases with GATA3 mutations were located in the genital, inguinal, or lower abdominal regions, whereas 5 of the other 6 SCAND cases were located in the anterior upper to mid-trunk. No GATA3 mutations were detected in the EMPSGC cases (0/5, 0%). These clinicopathologic and genetic findings support SCAND as a tumor entity distinguishable from EMPSGC. In addition, the characteristic frameshift extension mutations in GATA3 contribute to the establishment of the tumor-type concept of SCAND.