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.

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.

Quantitative analysis of the BRAF V600E mutation in circulating tumor-derived DNA in melanoma patients using competitive allele-specific TaqMan PCR.

BACKGROUND: BRAF V600E is a common mutation in melanoma, and BRAF inhibitors are effective in treating of BRAF mutation-positive melanoma. DNA carrying this mutation is released from melanoma cells into the circulation. As such, circulating tumor-derived DNA (ctDNA) in peripheral blood represents a novel biomarker for evaluating tumor features in cancer patients. However, ctDNA is present in the peripheral blood at very low levels, which makes the detection of specific mutations in this DNA a challenge. Competitive allele-specific TaqMan PCR (castPCR), a straightforward commercially available assay, is a sensitive technique for quantitating a small amount of DNA. METHODS: The level of BRAF V600E ctDNA was quantified by castPCR in 26 consecutive plasma samples from six melanoma patients. RESULTS: The castPCR assay was performed using a mixture of BRAF V600E DNA and BRAF wild DNA and found to be able to detect BRAF V600E at a fractional abundance of ≥0.5 % in 2- to 10-ng samples of genomic DNA. Cell-free DNA was then extracted from peripheral blood samples collected from six patients with melanoma harboring the BRAF V600E mutation. BRAF V600E ctDNA was detected in three patients, at a fractional abundance of between 1.28 and 58.0 % of total BRAF cell-free DNA. The abundance of BRAF V600E ctDNA correlated with tumor burden, as determined by computed tomography imaging. In two cases, an increase in the level of BRAF V600E ctDNA preceded exacerbation of clinical symptoms. CONCLUSION: The castPCR assay can detect and quantitate small amounts of BRAF V600E ctDNA in samples containing large amounts of BRAF wild cell-free DNA. Thus, we suggest that the castPCR assay is suitable for monitoring ctDNA in the plasma of melanoma patients.

Clinical features of pagetoid spread: Analysis of perianal lesions associated with anal canal adenocarcinoma and perianal primary extramammary Paget disease.

Secondary extramammary Paget disease (s-EMPD) represents anal canal and rectal, bladder, and gynecological cancers, which horizontally extend within the epidermis of the anal and vulvar skin. It is necessary to distinguish this condition from primary extramammary Paget disease (p-EMPD), which occurs primarily in genital and perianal areas. This study aimed to investigate the clinical and histopathological features of these two conditions in the perianal skin and to identify useful features for differentiation. We retrospectively analyzed 16 patients who visited Shinshu University Hospital from 2009 to 2022 and presented with perianal skin lesions and suspected EMPD. Six patients had p-EMPD and 10 had s-EMPD derived from anal canal adenocarcinoma. Regarding clinical features, nine of 10 (90%) of the s-EMPD cases had symmetric skin lesions, whereas all of the p-EMPD cases had asymmetrical lesions (p = 0.0004). Furthermore, assessment of symmetry around the anus showed that s-EMPD had a significantly smaller coefficient of variation than p-EMPD (0.35 and 0.62, respectively; p = 0.048), suggesting that s-EMPD was more symmetric around the anus. The frequency of raised lesions, such as foci or nodules, was nine of 10 (90%) for s-EMPD and one of six (16%) for p-EMPD (p = 0.003). Well-defined tumor borders on the lateral margins were identified in s-EMPD (5/10, 50%); however, they were not identified in p-EMPD (0/6, 0%). The borders tended to be clearer in s-EMPD; however, the difference was not significant (p = 0.078). Based on these findings, we recommend consideration of s-EMPD when anal skin lesions are symmetrical, well-defined, or raised.

Detecting copy number alterations of oncogenes in cell-free DNA to monitor treatment response in acral and mucosal melanoma.

BACKGROUND: Reliable biomarkers are necessary for assessment of treatment responses. Acral and mucosal melanomas are commonly associated with copy number (CN) alterations rather than specific point mutations, with CN alterations inKIT, CDK4, and CCND1 occurring frequently. Cell-free DNA is released to peripheral blood by both normal and tumor cells, and therefore contains the same genetic alterations present in the source tumor. OBJECTIVE: To investigate the usefulness of detecting CN alterations in oncogenes in cell-free DNA for monitoring treatment response in acral and mucosal melanomas. METHODS: We isolated cell-free DNA from peripheral blood and assessed the CN alterations in the cell-free DNA. Using droplet digital PCR, we examined CN alterations ofKIT, CDK4, and CCND1 in tumors from 37 melanoma patients (acral, n = 27; mucosal, n = 10) and peripheral blood from 24 melanoma patients (acral, n = 17; mucosal, n = 7). RESULTS: CN gain was detected in at least one of the genes examined in 62.9 % (17/27) of acral melanomas and 70 % (7/10) of mucosal melanomas. CN gains were also detected in the plasma of some patients. Furthermore, plasma CN ratio was correlated with clinical condition. This correlation was especially clear in patients with high CN ratios in tumors and high tumor burdens. CONCLUSION: Plasma CN ratios may be useful for evaluating treatment responses in patients with acral and mucosal melanoma.

Clinical effects of stereotactic radiation surgery in patients with metastatic melanoma.

We examined the effectiveness of stereotactic radiation surgery (SRS) in 14 patients with brain metastasis in our hospital. The age of the patients ranged 45-85 years old (mean, 65). Brain metastasis was detected by neurological symptoms in seven patients and by regular imaging examination in the remaining patients. The number of metastatic lesions in the brain before SRS ranged 1-11 (median, 2). The treatment number of SRS was 1-4 times (median, 2). Six of 14 patients had neurological symptoms before SRS. Overall survival (OS) after SRS was 1.7-21.2 months (median, 8.2). The progression-free survival (PFS) after SRS was 0.9-10.5 months (median, 2.2). The result of univariate analysis showed that the application of two or more courses of SRS was significantly related to OS (P = 0.005). Single metastatic lesion (P = 0.051) and no extracranial lesion (P = 0.055) showed a slight tendency to be related to disease-free survival (DFS). Neither lactate dehydrogenase nor neurological symptoms were significantly related to OS or DFS. Although OS and DFS after SRS were not very long, the treatment of brain metastases has the potential to prevent neurological events. Repeating SRS may be accepted as a local therapy in the multimodal approach including new molecular targeting drugs for metastatic melanoma.