LBA9514 Background: Advanced Merkel cell carcinoma (MCC) often responds to anti-PD-(L)1, but patients (pts) with immune checkpoint inhibitor (ICI)-refractory disease have poor outcomes and few therapy options. MCC has defects in G1 checkpoint control, proliferates rapidly, and is reliant on S/G2 checkpoints such as ATR (ataxia telangiectasia mutated and Rad3-related). Inhibiting ATR can promote immunogenic cell death. MATRiX (MCC refractory to immunotherapy treated with ATR inhibitor ± avelumab) is a multicenter, NCI-sponsored, randomized, open-label phase II trial evaluating tuvusertib, an ATR inhibitor, alone (Arm 1) or in combination with avelumab (Arm 2) in anti-PD-(L)1-refractory MCC, NCT05947500. Methods: Pts were randomized 1:1 using a permuted-block design. Arm 1 received tuvusertib 180 mg PO on days 1-14 of each 21-day cycle. Arm 2 received tuvusertib as in Arm 1, plus avelumab 1600 mg IV every 21 days. Pts with disease progression in Arm 1 were eligible to cross over to Arm 2. The planned sample size (N=50; 25 per Arm) provided >80% power to observe a statistically significant (1-sided level of 0.15) difference in the primary endpoint, progression-free survival (PFS), assuming a hazard ratio of 0.5. Secondary endpoints were overall response rate (ORR), duration of response, and overall survival (OS). Data cutoff: January 20, 2026. Results: From 5/2024 to 9/2025, 35 subjects (median age 73) received therapy: Arm 1, n=10; Arm 2, n=25. One pt was deemed ineligible post-randomization and excluded from all analyses. 18 pts (53%) had primary ICI-refractory disease, and 22 (65%) had ≥2 prior systemic therapies. Arm 1 met prespecified futility criteria (0/10 responses) and was closed early. No objective responses were observed among 4 pts who crossed over to Arm 2. Among 24 eligible pts in Arm 2, 1 complete and 4 partial responses [ORR 20.8% (95% CI: 7.1-42.2%)] were observed, including 4 primary and 1 acquired ICI-resistant cases. 4 of 5 responders remained progression-free at cutoff (182-273 days from treatment initiation); 1 relapsed at 479 days. One-year estimates were PFS 0% (Arm 1) and 26.4% (Arm 2; 95% CI: 10.8-45.1%) and OS 29.2% (Arm 1; 1.5-69.8%) and 36.5% (Arm 2; 7.8-67.2%), respectively. Grade ≥3 treatment-emergent adverse events occurred in 80% of pts in Arm 1 and 58% in Arm 2; the most common were lymphopenia (20% vs 21%), anemia (50% vs 17%), fatigue (20% vs 17%), pain (10% vs 17%), and infection (0% vs 17%). Safety profiles were consistent with previous reports for these agents. Conclusions: Tuvusertib with avelumab demonstrated antitumor activity in anti-PD-(L)1-refractory MCC, inducing durable clinical benefit in ICI-resistant metastatic pts with limited salvage options. No signal of anticancer efficacy was observed with ATRi monotherapy. These findings warrant further investigation of ATRi in combination with immunotherapy in ICI-refractory MCC. Clinical trial information: NCT05947500 .

LBA9504 Background: Merkel cell carcinoma (MCC) with lymph node (LN) metastases (mets) is historically associated with high risk of relapse and mortality despite surgery and/or radiation therapy (RT). Previously reported adjuvant trials (ADMEC-O and STAMP) have not shown a clear benefit with adjuvant PD-(L)1 blockade. Methods: ADAM trial (NCT03271372) is an investigator-initiated, phase III, multi-center, double-blinded, placebo-controlled study. We enrolled 100 patients (pts) with MCC and LN mets (stage III) treated definitively with surgery and/or RT, and without radiologic evidence of residual MCC. Pts were randomized (1:1), stratified by primary tumor/LN status and RT status, to receive IV avelumab (A; 800 mg/dose) or placebo (P), for up to 2 years (yrs) on a de-escalating schedule. The primary endpoint was relapse-free survival (RFS). 100 patients provide 86% power to observe a statistically significant (2-sided 5%) difference in RFS assuming true hazard ratio (HR) for RFS failure of 0.4. Secondary endpoints included distant metastasis-free survival (DMFS), disease-specific survival (DSS), overall survival (OS), and safety. Point estimates were obtained using Kaplan-Meier or cumulative-incidence methods, and Cox regression (stratified, with pre-specified adjustment of confounding variables as needed) was used to estimate HRs of failure. Results: 100 eligible pts (pathologic stage IIIB - 52; IIIA - 48) were randomized to A (N=48) vs P (N=52) between 12/2017 and 01/2024. At the data cutoff date (01/15/2026), median follow-up among relapse-free survivors was 4.2 yrs [range 1.9-7.1]. Median age was 70 yrs overall (A-72 [50-86], P- 67 [35-83]) and 93% of pts had received adjuvant RT. The HRs for RFS, relapse, DMFS, DSS and OS are listed in the Table below. The point estimates for MCC relapse for A vs P arms, respectively, were 12.8% vs 40.4% at 1 year, 21.3% vs 42.3% at 2 years and 28.3% vs 44.5% at 3-, 4- and 5-year timepoints. Grade 3/4 treatment-related adverse events (TRAE) rate was 15% (n=7) in A and 0% in P arms; no grade 5 TRAEs occurred. Conclusion: In this study, adjuvant avelumab was associated with reduced risk of MCC relapse in pts with LN mets. High MCC-specific survival in both arms points to effectiveness of PD-(L)1 blockade in adjuvant and metastatic settings. These data will inform future discussions regarding adjuvant therapy in clinical practice. Clinical trial information: NCT03271372 . Efficacy endpoints. Endpoint HR for Failure, Avelumab vs Placebo (95% CI, p-value)Stratified /Adjusted stratified Number of eventsAvelumab/Placebo RFS 0.61 (0.32-1.16, p=0.132) /0.54 (0.28-1.05, p=0.069) 16/24 Relapse* 0.51 (0.26-1.00) /0.47 (0.23-0.94) 13/23 DMFS 0.93 (0.44-1.97) /0.89 (0.41-1.94) 13/15 DSS 1.81 (0.43-7.55) /** 5/3 OS 2.37 (0.73-7.73) /** 9/5 *Not a pre-specified analysis; **Not enough events to accommodate adjustment.

Surfactin, as one of the most effective biosurfactants, holds excellent emulsifying, stability and antibacterial properties. However, low TYP index (titer, yield and productivity), as well as the difference activity of homologues have presented challenges in application of pharmaceutical, cosmetics and food industries. To solve this challenge, surfactin biosynthesis and the central carbon metabolism is systematically reprogrammed to build a Bacillus subtilis chassis with high TYP index and tailor C14/C15 homologue proportions. Firstly, by redesigning the native promoter to ease regulatory intensity and improve expression of surfactin synthetase, we achieved a remarkedly accumulation of surfactin up to 21.6 g/L in 7.5-L fermenter. Secondly, a "push-pull-promote-block" approach efficiently optimized growth and production fluxes, and reduced carbon overflow and carbon loss. Finally, implementation of these genetic strategies led us to obtain a B. subtilis strain with superior TYP index. Surfactin titer in 7.5-L fermenter was increased from 9.8 g/L to 52.6 g/L, the yield was improved from 0.11 to 0.34 g/g sucrose, and the productivity was increased from 0.54 to 1.75 g/L/h. The TYP index is the highest levels ever reported, and the yield reached 75 % of the theoretical pathway yield (0.45 g/g sucrose). Additionally, enlarging odd or even chain fatty acid pools by feeding with isovaleric acid, threonine, or isobutyric acid, respectively increased the C15-surfactin or C14-surfactin analog ratio exceeds 73 %. This work demonstrates that a maximization of the synergy between cell growth and surfactin production is crucial for enhancing the efficiency of cell factories.

Precise intraoperative pathological diagnosis is crucial for effective surgical management. However, conventional method based on hematoxylin-eosin (H&E) staining of frozen sections often suffers from accuracy limitations due to a variety of factors. In this study, we developed a novel and efficient method utilizing a lipid droplets (LDs)-targeted fluorescent probe, named NA-LD, for intraoperative pathological diagnosis of breast cancer. Owing to the intramolecular charge transfer (ICT) effect, NA-LD exhibited ultrasensitive fluorescence responses to environmental polarity changes, enabling high-fidelity imaging of LDs. Interestingly, the probe was capable of differentiating breast cancer cells from normal ones by visualizing the difference of LDs characteristics, including polarity, number and size. Furthermore, using NA-LD the discrimination of tumor in a mouse model was achieved. Most importantly, it has been successfully applied in determining the benign or malignant nature of the intraoperative breast lesion, as well as in discriminating ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC). Therefore, this work may provide novel insights for the precise diagnosis and personalized treatment of breast cancer patients.

Radiation exposure from medical exposure, occupational exposure, or accidental incidents, increases significant risks to cardiovascular health. Small molecule metabolites have attracted widespread attention owing to the role in cardiovascular cell metabolism, immune response and inflammatory injury. This article reviews the classification and characteristics of small molecule metabolites and their multiple roles in radiation-induced cardiovascular inflammatory injury. This article explored the potential of small molecule metabolites as biomarkers in the diagnosis and prognosis of cardiovascular diseases, and the potential as therapeutic targets. Based on the relevant research in recent years, this article summarizes the relationship between small molecule metabolites and cardiovascular inflammatory injury, as well as the mechanisms on the cardiovascular system under radiation, aiming to understand the potential mechanism of small molecule metabolites in radiation-induced cardiovascular diseases to develop new prevention and treatment strategies.

e15071 Background: Studies indicate that Merkel cell polyomavirus (MCPyV) infection is a causative factor for the development of virus-positive Merkel cell carcinoma (VP MCC). This neuroendocrine neoplasm (NEN) is sometimes undiagnosed or misdiagnosed when MCPyV infection is not properly identified; accurate diagnosis is essential, as research suggests the utility of immunotherapy in the treatment of MCC compared to other NENs. Consequently, we seek to establish whole exome next-generation sequencing (WES) as a reliable method for finding MCPyV in tumors. Specifically, we propose a novel assay incorporating MCPyV detection into standard-of-care molecular cancer profiling, therefore ensuring proper diagnosis of VP MCC. Methods: WES was validated on DNA extracted from previously diagnosed VP MCC tumor samples submitted to Caris Life Sciences utilizing NovaSeq 6000 technology (Illumina, Inc.). A hybrid pull-down panel of sequencing baits was used to provide enhanced 1500x depth of coverage for 720 cancer-related genes and 500x coverage for the remaining exome. Given bait availability, the panel was also designed to cover the maximal possible amount of the MCPyV genome—82.4%. The presence of virus was suggested if ≥1,000 sequencing reads specific to MCPyV were detected. Orthogonal IHC testing using CM2B4 antibody (Santa Cruz Biotechnology, Inc.) was used to confirm the MCPyV infection status detected by WES. Clinical utility of the sequencing assay was demonstrated by testing primary and metastatic tumor specimens previously classified as NENs not otherwise specified, so as to identify potentially undiagnosed or misdiagnosed MCC. Results: We performed WES on 835 NEN specimens that were not previously diagnosed as MCC. MCPyV was detected in 10/835 (1.2%), suggesting that these cases might have been undiagnosed or misdiagnosed. To verify the validity of the sequencing results, we performed WES on 70 samples with a known diagnosis of MCC, and MCPyV was detected in 49 tumors. Conversely, orthogonal IHC testing identified virus in 45 of the 70 specimens. The presence of viral sequences was confirmed by the Basic Local Alignment Search Tool (BLAST, National Institutes of Health) in 49 of the cases, including the four tumors discordant by IHC, indicating that WES is more sensitive in detecting the virus (100% sensitivity [95% CI: 91.4-100%] and 84.0% specificity [95% CI: 63.9-95.5%]) compared to IHC. Conclusions: This study presents a robust method for identifying MCPyV in VP MCC utilizing standard-of-care cancer genomic profiling for precision oncology. By confirming the presence of viral sequences within neoplastic tissue specimens, we have demonstrated the superior sensitivity of WES in detecting MCPyV compared to IHC. Moreover, our sequencing assay can assist in properly classifying tumors, therefore ensuring that patients receive appropriate treatment based on the diagnosis of MCC.

There are three FAM98 family proteins (FAM98A/B/C) in humans and mice. Their physiological functions remain largely unknown. We have previously reported that Fam98a interacts with Plekhm1 in murine osteoclasts and functions in lysosome trafficking/secretion and bone resorption in osteoclasts in vitro. In this study, we found that all three Fam98 genes were expressed in precursor and mature osteoclasts. While the knockdown of Fam98c by a specific short-hairpin RNA (shRNA) in osteoclast precursors attenuated osteoclastogenesis, depletion of Fam98b by an shRNA specifically disrupted osteoclast lysosome trafficking and bone resorption with phenotypes similar to Fam98a shRNA-knockdown in our previous study. Loss of Fam98a in myeloid osteoclast precursors was dispensable for trabecular and cortical bone mass in mice, as well as osteoclastogenesis/bone resorption in vitro, possibly due to compensation by increased Fam98b expression in Fam98a-null osteoclasts. These findings indicate that the three Fam98 proteins play distinct roles in osteoclastogenesis and osteoclast function and need further investigation in future studies.

Background/Objectives: Immune checkpoint inhibitors (ICIs) are frontline treatment for advanced Merkel Cell Carcinoma (MCC), regardless of viral status. Frontline ICIs provide durable benefit to only half of patients, highlighting a need for alternative therapies. In this study, the objective is to leverage whole exome sequencing (WES) and transcriptome sequencing (WTS) to distinguish genomic alterations associated with ICI response. Investigate differential genomic alterations between virus-positive (VP) and virus-negative (VN)-MCC to identify novel therapeutic targets. Methods: A total of 95 MCC cases underwent WES and WTS. Utilizing computational pipelines applied to WES, we identified viral status and tumor mutational burden (TMB). RNA-seq data was used to characterize the immune microenvironment. Results: Of 95 MCC cases, 57 (60%) were VP-MCC and 38 (40%) were VN-MCC. Median TMB was higher in VN-MCC (27.5 vs. 1 Muts/Mb). Mutations in TP53, RB1, NOTCH1, KMTD2, KMT2C, and PIK3CA were primarily found in VN-MCC. MAPK Pathway Activity Score, NK cell infiltration, and the immune checkpoint gene CD276 in VN-MCC tumors were upregulated. No overall survival (OS) difference was identified between VP and VN-MCC, even after ICIs. Conclusions: MCC oncogenesis and treatment response transcend viral status. While mutational analysis confirms previous findings, assessment of the transcriptome and tumor microenvironment suggests alternate therapeutic targets.

Merkel cell carcinoma (MCC) is a rare but aggressive neuroendocrine carcinoma, and immune checkpoint inhibitors (ICIs) are the only approved therapy; nonetheless, resistance is notable and there is a critical need for novel effective therapies. Recently, CD276 was identified as a promising therapeutic target in human cancers. In preclinical studies, a modified CD276 antibody-drug conjugate (ADC) with pyrrolobenzodiazepine (m276-SL-PBD) elicited more potent anti-tumor effects than two CD276 ADCs currently in clinical trials. Here, we uncover notable CD276 expression in MCC patient tumors, and demonstrate m276-SL-PBD efficacy against MCC preclinical models. Complete eradication is observed in all xenografts bearing CD276 expression, with 82% achieving 180-day tumor-free survival after 4 or 5 weekly doses, and m276-SL-PBD remained efficacious against relapsed tumors. Of clinical relevance, m276-SL-PBD retains its potency in MCC-bearing humanized mice. Importantly, no detectable adverse effects were observed. Thus, m276-SL-PBD is a promising therapy for patients unsuitable or resistant to ICIs.

Background:Extensive laboratory research and clinical trial results have shown that herpes simplex virus-thymidine kinase/ganciclovir (HSV-TK/GCV) system has a therapeutic effect on various tumours. This study focused on the role of HSV-TK/GCV system therapy for lung adenocarcinoma. Methods:Cell proliferation, migration, invasion, and premature senescence were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and colony formation assay, wound-healing assay, invasion assay, and β-galactosidase staining, respectively. Cells were transfected with adeno-associated virus (AAV) vector expressing HSV-TK (AAV-TK) or green fluorescent protein (GFP) (AAV-GFP, control). A murine xenograft model of Lewis cells was established to investigate the effect of HSV-TK/GCV system on tumour growth. Protein expression related to cell proliferation and senescence in tumour was analysed by immunohistochemical staining. Results:AAV-TK transfection combined with GCV treatment significantly inhibited the proliferation, migration and invasion of A549 and Lewis cells compared with AAV-GFP transfection. β-galactosidase activity assay indicated that the premature senescence of cells was enhanced after AAV-TK/GCV treatment. In-vivo tumour growth was significantly inhibited after intratumour injection of AAV-TK/GCV. Immunohistochemical staining showed that the expression of p16 protein significantly increased while proliferating cell nuclear antigen (PCNA) expression decreased in tumour tissue after AAV-TK administration, which conformed that the proliferation of tumour cells was inhibited by AAV-TK/GCV treatment. Conclusions:HSV-TK/GCV system could significantly inhibit the growth and metastasis of lung adenocarcinoma, accompanied by cell premature senescence.

OBJECTIVE:This study aimed to investigate the effects of low-dose radiation on the abdominal aorta of mice and vascular endothelial cells. METHODS:Wild-type and tumor-bearing mice were exposed to 15 sessions of low-dose irradiation, resulting in cumulative radiation doses of 187.5, 375, and 750 mGy. The effect on the cardiovascular system was assessed. Immunohistochemistry analyzed protein expressions of PAPP-A, CD62, P65, and COX-2 in the abdominal aorta. Microarray technology, Gene Ontology analysis, and pathway enrichment analysis evaluated gene expression changes in endothelial cells exposed to 375 mGy X-ray. Cell viability was assessed using the Cell Counting Kit 8 assay. Immunofluorescence staining measured γ-H2AX levels, and real-time polymerase chain reaction quantified mRNA levels of interleukin-6 (IL-6), ICAM-1, and Cx43. RESULTS:Hematoxylin and eosin staining revealed thickening of the inner membranes and irregular arrangement of smooth muscle cells in the media membrane at 375 and 750 mGy. Inflammation was observed in the inner membranes at 750 mGy, with a clear inflammatory response in the hearts of tumor-bearing mice. Immunohistochemistry indicated increased levels of PAPP-A, P65, and COX-2 post-irradiation. Microarray analysis showed 425 up-regulated and 235 down-regulated genes, associated with processes like endothelial cell-cell adhesion, IL-6, and NF-κB signaling. Cell Counting Kit 8 assay results indicated inhibited viability at 750 mGy in EA.hy926 cells. Immunofluorescence staining demonstrated a dose-dependent increase in γ-H2AX foci. Reverse transcription quantitative PCR results showed increased expression of IL6, ICAM-1, and Cx43 in EA.hy926 cells post 750 mGy X-ray exposure. CONCLUSION:Repeated low-dose ionizing radiation exposures triggered the development of pro-atherosclerotic phenotypes in mice and damage to vascular endothelial cells.

Mutations in PLEKHM1 cause osteopetrosis in humans and rats. The germline and osteoclast conditional deletions of Plekhm1 gene in mice lead to defective osteoclast bone resorption and increased trabecular bone mass without overt abnormalities in other organs. As an adaptor protein, pleckstrin homology and RUN domain containing M1 (PLEKHM1) interacts with the key lysosome regulator small GTPase RAB7 via its C-terminal RUBICON homologous (RH) domain. In this study, we have conducted a structural-functional study of the PLEKHM1 RH domain and RAB7 interaction in osteoclasts in vitro. The single mutations of the key residues in the Plekhm1 RH predicted from the crystal structure of the RUBICON RH domain and RAB7 interface failed to disrupt the Plekhm1-Rab7 binding, lysosome trafficking, and bone resorption. The compound alanine mutations at Y949-R954 and L1011-I1018 regions decreased Plekhm1 protein stability and Rab7-binding, respectively, thereby attenuated lysosome trafficking and bone resorption in osteoclasts. In contrast, the compound alanine mutations at R1060-Q1068 region were dispensable for Rab7-binding and Plekhm1 function in osteoclasts. These results indicate that the regions spanning Y949-R954 and L1011-I1018 of Plekhm1 RH domain are functionally important for Plekhm1 in osteoclasts and offer the therapeutic targets for blocking bone resorption in treatment of osteoporosis and other metabolic bone diseases.

Skin Exposure of skin to ionizing radiation can induce acute or chronic biological effects, resulting in radiation-induced skin injury (RSI). Premature cellular senescence, caused by oxidative stress and/or DNA damage from chemical or physical agents, leads to the decrease of cellular proliferation and physiological function. Persistent DNA damage and accumulation of senescent cells are associated with the progression of radiation-induced injury. Atopic dermatitis and RSI have similar inflammatory symptoms. The treatment of tacrolimus (TAC) in atopic dermatitis may be associated with premature cellular senescence. TAC can prevent the onset of cellular senescence by inactivating the p38 mitogen-activated protein kinase (p38 ​MAPK). The activation of p38 ​MAPK can induce the senescence-associated secretory phenotype (SASP) by enhancing the transcriptional activity of nuclear factor kappa-B (NF-κB), which ultimately leads to premature cellular senescence. FK506 binding protein 51 (FKBP51) exhibits resistance to ionizing radiation, but the mechanism of TAC regulation of ionizing radiation-induced premature senescence still needs further study. This review discusses the mechanism of cellular senescence in RSI and the role of TAC in both dermatitis and RSI.

PURPOSE:Lipidomics is an important tool for triaging exposed individuals, and helps early adoption of prevention and control strategies. The purpose of this study was to screen significantly perturbed lipids between pre- and post-irradiation of human plasma samples after total body irradiation (TBI) and explore potential radiation biomarkers for early radiation classification. METHODS:Plasma samples were collected before and after irradiation from 22 hospitalized cases of acute myeloid leukemia (AML) prepared for bone marrow transplantation. Acute total-body γ irradiation was performed at doses of 0, 4, 8, and 12 Gy. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with multiple reaction monitoring (MRM) method was utilized. Self-paired studies before and after irradiation were performed to screen potential lipid categorization markers and markers of dose-response relationships for radiation perturbation in humans. Based on the screened potential markers, a human TBI dose estimation model was developed. RESULTS:In total, 426 individual lipids from 14 major classes were quantified and 152 potential biomarkers with categorical characteristics were screened. A total of 80 lipids (32 TGs, 29 SMs, 9 FAs, 5 CEs, 5 PIs) were upregulated at 4 Gy, and a total of 91 lipids (39 SMs, 18 TGs, 15 HexCers, 7 CEs, 6 Cers, 3 LacCers, 2 LPEs, 1 PI) were upregulated at 12 Gy. Comparison of the ROC curves between the non-exposed and exposed groups at different doses showed AUC values ranging from 0.807 to 0.876. The metabolic pathways of potential lipid markers are mainly sphingolipid and glycerolipid metabolism, unsaturated fatty acid biosynthesis, fatty acid degradation and biosynthesis. Among the 13 dose-dependent radiosensitive lipids, CE (20:5), CE (18:1) and PI (18:2/18:2) were gradually incorporated into the TBI dose estimation model. CONCLUSION:This study suggested that it was feasible to acquire quantitative lipid biomarker panels using targeted lipidomics platforms for rapid, high-throughput triage. Lipidomics strategies for radiation biodosimetry in humans were established with lipid biomarkers with good dose-response relationship.

IMPORTANCE Merkel cell carcinoma (MCC) is a rare and highly aggressive cutaneous neuroendocrine carcinoma with increasing incidence. Cytotoxic chemotherapy and checkpoint inhibitors provide treatment options in the metastatic setting; however, there are no approved or standard of care targeted therapy treatment options. OBJECTIVE To identify actionable alterations annotated by the OncoKB database therapeutic evidence level in association with tumor mutation burden (TMB). DESIGN, SETTING, AND PARTICIPANTS This is a retrospective, cross-sectional study using data from the American Association for Cancer Research Genomics Evidence Neoplasia Information Exchange, a multicenter international cancer consortium database. Patients with MCC were enrolled in participating institutions between 2017 and 2022. Data from version 11.0 of the database were released in January 2022 and analyzed from April to June 2022. MAIN OUTCOMES AND MEASURES The main outcome was the percentage of patients with high TMB and OncoKB level 3B and 4 alterations. RESULTS A total of 324 tumor samples from 313 patients with MCC (107 women [34.2%]; 287 White patients [91.7%]; 7 Black patients [2.2%]) were cataloged in the database. The median (range) number of alterations was 4.0 (0.0-178.0), with a mean (SD) of 13.6 (21.2) alterations. Oncogenic alterations represented 20.2% of all alterations (862 of 4259 alterations). Tissue originated from primary tumor in 55.0% of patients (172 patients) vs metastasis in 39.6% (124 patients). TMB-high (>= 10 mutations per megabase) was present in 26.2% of cases (82 patients). Next-generation sequencing identified 55 patients (17.6%) with a level 3B variation for a Food and Drug Administration-approved drug for use in a biomarker-approved indication or approved drug in another indication. An additional 8.6% of patients (27 patients) had a level 4 variation. Actionable alterations were more common among high TMB cases, with 37 of 82 patients (45.1%) harboring level 3 alterations compared with only 18 of 231 patients (7.8%) with low TMB. The most common level 3B gene variants included PIK3CA (12 patients [3.8%]), BRCA1/2 (13 patients [4.2%]), ATM (7 patients [2.2%]), HRAS (5 patients [1.6%]), and TSC1/2 (6 patients [1.9%]). The most common level 4 variants include PTEN (13 patients [4.1%]), ARID1A (9 patients [2.9%]), NF1 (7 patients [2.2%]), and CDKN2A (7 patients [2.2%]). Copy number alterations and fusions were infrequent. In 61.0% of cases (191 cases), a PanCancer pathway was altered, and 39.9% (125 cases) had alterations in multiple pathways. Commonly altered pathways were RTK-RAS (119 patients [38.0%]), TP53 (103 patients [32.9%]), cell cycle (104 patients [33.2%]), PI3K (99 patients [31.6%]), and NOTCH (93 patients [29.7%]). In addition, oncogenic DNA mismatch repair gene alterations were present in 8.0% of cases (25 patients). CONCLUSIONS AND RELEVANCE In this cross-sectional retrospective study of alterations and TMB in MCC, a minority of patients had potentially actionable alterations. These findings support the investigation of targeted therapies as single agent or in combination with immunotherapy or cytotoxic chemotherapy in selected MCC populations.

Purpose: UVB exposure accelerates skin aging and age-associated pigmentation, but their relationship remains unclear. UVB induces premature senescence and melanin production within melanocytes, accompanied by the up-regulation of p53 and cellular tyrosinase (TYR). As a tumor suppressor gene, p53 can keep the genome intact by modulating cell apoptosis and growth arrest during DNA injury. P53 also relates to age-associated pigmentation, directly or indirectly regulating pigment-related gene expression. Melatonin effectively regulates tyrosinase activity and resists aging. This study focused on the regulation of p53 on TYR to understand the association between premature senescence and senescence-associated pigmentation and determine the mechanism behind melatonin affecting UVB stimulated melanin production.Methods: Primary melanocytes were extracted and identified from the male foreskin. The primary melanocytes were transduced using lentivirus pLKD-CMV-EGFP-2A-Puro-U6-TYR to knock down TYR expression. The melanin content was determined using the NaOH method, the oxidation of 3,4-Dihydroxy-L-phenylalanine (L-DOPA) into dopachrome to determine TYR activity, and Western blotting was used to detect the level of TYR protein. After being pretreated with Nutlin-3 or PFT-α to up or down-regulate p53 levels or melatonin for 12 h, primary melanocytes were under UVB irradiation at 80 mJ/cm2. The senescence-associated beta-galactosidase (SA-β-gal) kit analyzed premature senescence. At the same time, the level of p53, p-p53, and TYR protein were detected using the automated capillary electrophoresis western analysis in melanocytes at 72 h after UVB irradiation. Wild-type and TYR (–/–) or TYR (+/–) knockout C57BL/6J mice were used to verify the regulatory role of TYR on melanin synthesis in vivo . Moreover, the effect of melatonin on skin erythema and pigmentation induced by UVB irradiation was illustrated in vivo.Results: Primary melanocytes showed a deep black color after L-DOPA staining, higher TYR protein, and mRNA expression. Tyrosinase activity and melanin levels induced by UVB irradiation were significantly alleviated after being infected with pLKD-CMV-EGFP-2A-Puro-U6-TYR (P <0.05). Premature senescence, tyrosinase activity, and melanin levels were increased under UVB irradiation induction. Furthermore, there was a dramatic increase following the Nutlin-3 treatment while significantly inhibited after being treated with PFT-α (P <0.05) in the primary melanocytes. Melatonin inhibited UVB-induced premature senescence, associated with decreased p53 level and phosphorylation on serine-15 position, decreased UVB-induced tyrosinase activity and melanin levels, and reduced TYR expression. TYR (–/–) knockout mice were recognized through white hair colors, whiskers, paws, and pigment loss in the eyes. The tyrosinase activity and melanin levels in the whiskers follicles of TYR (–/–) knockout mice were also significantly decreased (P <0.05) relative to the wild-type (WT) counterparts. Skin erythema and melanin pigmentation induced by UVB irradiation were reduced in the dorsal and ear skin of C57BL/6J mice topically pretreated with 2.5% melatonin.Conclusion: Melanin synthesis induced by UVB irradiation is dependent on TYR in primary melanocytes and C57BL/6J mice. Moreover, p53 links the UVB irradiation-induced premature senescence and senescence-associated pigmentation in the primary melanocytes, and directly regulates TYR in primary mel

Background Merkel cell carcinoma (MCC) is a rare, aggressive neuroendocrine cancer with rapid progression and mortality rates of 33–46%.1 The majority of MCC is caused by Merkel Cell Polyomavirus (MCPyV) with the remainder induced by UV-mediated damage.1 2 Regardless of virus positivity or tumor mutational burden (TMB), immune checkpoint inhibitors (ICIs) are first line treatment for MCC1 with half of patients not responding or developing resistance. Few options exist for those refractory to immunotherapy.3 There is a need to identify the MCC-specific factors driving resistance and to identify alternate molecular targets. Methods 205 MCC tumors were analyzed using next-generation sequencing (592, NextSeq; WES, NovaSeq) and WTS (NovaSeq) (Caris Life Sciences, Phoenix, AZ). TMB was measured by totaling somatic mutations per tumor (TMB-H: >10 mutations/MB). MCPy viral (MCPyV) status was determined for 68 WES profiled cases using a cut-off of 1000 reads after concordance testing with IHC. Immune cell infiltrates were estimated by Quantiseq. Significance was determined using Chi-square and Mann-Whitney U tests and adjusted for multiple comparisons (q-value <0.05). Results The majority (89.3%) of MCPyV-negative MCC tumors were TMB-high (>10 mutations/Mb), with 96.4% having mutations in TP53 and 80.8% in RB1. Other gene mutations included NOTCH1 (37%), KMT2C (28.6%), TERT (17.9%), FAT1 (14.3%), and PIK3CA (14.3%). In contrast, MCPyV-positive tumors were frequently TMB-low (100%) and rarely harbored mutations in TP53 and RB1 (10.3% and 2.6%, respectively). Immune checkpoint gene (CD80, CD86, CD274, PD1, PD1L, and CTLA4) expression was similar between MCPyV-positive and -negative tumors. Estimated NK cell infiltration was significantly higher in MCPyV-negative tumors. MCPyV-negative MCC also had significantly higher expression of a MAPK pathway activation signature (MPAS). Conclusions MCPyV-positive and -negative MCC represent two classes of molecularly distinct tumors and can be differentiated based on their TMB and mutational profile. The significantly increased NK cell infiltration seen in MCPyV-negative MCC represents a potential therapeutic pathway with the efficacy of NK cell-stimulating agents currently under investigation in the Quilt-3.063 trial.4 MPAS up-regulation in MCPyV-negative MCC suggests that MAPK inhibitors could be used as an alternative to ICIs, which is supported by preclinical data.3 5 MCPyV-negative and -positive MCC are distinct tumor subtypes whose molecular and immune cell profiles warrant further investigation to optimize use of current ICIs and identify therapeutic targets. References Park SY, Doolittle-Amieva C, Moshiri Y, Akaike T, Parvathaneni U, Bhatia S, Zaba LC, Nghiem P. How we treat Merkel cell carcinoma: within and beyond current guidelines. Future Oncol. 2021 Apr;17(11):1363−1377. Knepper TC, Montesion M, Russell JS, Sokol ES, Frampton GM, Miller VA, Albacker LA, McLeod HL, Eroglu Z, Khushalani NI, Sondak VK, Messina JL, Schell MJ, DeCaprio JA, Tsai KY, Brohl AS. The Genomic Landscape of Merkel Cell Carcinoma and Clinicogenomic Biomarkers of Response to Immune Checkpoint Inhibitor Therapy. Clin Cancer Res. 2019 Oct 1;25(19):5961−5971. Fang B, Kannan A, Zhao S, Nguyen QH, Ejadi S, Yamamoto M, Barreto JC, Zhao H, Gao L. Inhibition of PI3K by copanlisib exerts potent antitumor effects on Merkel cell carcinoma cell lines and mouse xenografts. Sci Rep. 2020 Jun 1;10(1):8867. ImmunityBio,Inc. QUILT-3.063: A Study of N-803, haNK and Avelumab in Patients With Merkel Cell Carcinoma That Has Progressed After Checkpoint Therapy. ClinicalTrials.gov identifier: NCT03853317. Updated June 18, 2023. Accessed June 27, 2023. Temblador A, Topalis D, Andrei G, Snoeck R. Synergistic targeting of the PI3K/mTOR and MAPK/ERK pathways in Merkel cell carcinoma. Tumour Virus Res. 2022 Dec;14:200244. Ethics Approval This study was conducted in accordance with guidelines of the Declaration of Helsinki, Belmont report, and U.S. Common rule utilizing retrospective, deidentified clinical data in keeping with 45 CFR 46.101(b)(4). Therefore, this study is considered Institutional Review Board (IRB) exempt and no consent was necessary from the subjects.

Circulating tumor DNA (ctDNA) is a subset of circulating cell-free DNA released by lysed tumor cells that can be characterized by its shorter strand length and tumor genome-specific information. The relatively short half-life of ctDNA allows it to provide a real-time measure of tumor burden which has potential prognostic and surveillance value as a tumor biomarker. Merkel cell carcinoma (MCC) is a rare neuroendocrine skin cancer that requires close monitoring due to the high risk of relapse. There are currently no good tumor biomarkers for MCC patients, especially those who are negative for Merkel cell polyomavirus. ctDNA shows promise for improving the prognoses of MCC patients by monitoring tumor burden, identifying minimal residual disease (MRD), and stratifying patients by their likelihood of response to immune checkpoint inhibition or risk of relapse. In particular, bespoke ultra-sequencing platforms allow for the creation of patient-specific mutation panels that improve ctDNA detection, especially for patients with rare or uncharacteristic mutations. Leveraging bespoke ctDNA assays may improve physicians’ ability to alter treatment plans for non-responsive or high-risk patients. In addition, ctDNA MRD monitoring may allow physicians to treat relapses early before clinically evident disease is present.