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Gianmarco M, Carolina P, Gregorio M, Michela V, Monica P, Claire GG, Michele M, Giulia M, Roberta M, Cinzia A, Lorena B, Marcello T, Fabiana P, Roberta M. Circulating tumor DNA monitoring in advanced mutated melanoma (LIQUID-MEL). THE JOURNAL OF LIQUID BIOPSY 2025; 8:100295. [PMID: 40276578 PMCID: PMC12019447 DOI: 10.1016/j.jlb.2025.100295] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/27/2025] [Revised: 04/07/2025] [Accepted: 04/08/2025] [Indexed: 04/26/2025]
Abstract
Introduction Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of metastatic melanoma, but a percentage of patients did not show benefit. Circulating tumor DNA (ctDNA) has emerged as a potential non-invasive tool for monitoring disease evolution and treatment response. The present study aimed to evaluate the clinical utility of ctDNA dynamics in patients with metastatic melanoma receiving ICIs, while exploring its role in the oncological course. Materials and methods The LIQUID-MEL study is a prospective, single-centre pilot study including patients with BRAF/NRAS-mutant metastatic melanoma. ctDNA was quantified using digital droplet PCR (ddPCR) at four different time points. Uni- and multivariable Cox regression models were used to assess the correlation between shedding and progression-free survival (PFS), and overall survival (OS). Results Overall, 23 patients were included. At baseline, ctDNA was detectable in 5/23 (21.7 %) cases. Baseline ctDNA shedding was associated with shorter PFS (3.88 months vs. 0.69 months, p=0.012). A strong numerical trend was observed also in OS (12.66 months vs. 2.53 months, p=0.287). Shedding at baseline did not demonstrate independent prognostic or predictive value in the uni- and multivariable analysis. The longitudinal analysis revealed intriguing patterns of ctDNA shedding in individual patients. Conclusion ctDNA detectability and its dynamic changes during treatment may have potential clinical utility in patients with metastatic melanoma, offering a valuable non-invasive tool for monitoring disease and treatment response. The small sample size limited the statistical power of the analysis. Further studies with larger cohorts are needed to validate its role in routine clinical practice.
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Affiliation(s)
| | - Palazzi Carolina
- Medical Oncology Unit, University Hospital of Parma, Parma, Italy
| | - Monica Gregorio
- Department of Medicine and Surgery, University of Parma, Parma, Italy
| | - Verzè Michela
- Medical Oncology Unit, University Hospital of Parma, Parma, Italy
- Department of Medicine and Surgery, University of Parma, Parma, Italy
| | - Pluchino Monica
- Medical Oncology Unit, University Hospital of Parma, Parma, Italy
- Department of Medicine and Surgery, University of Parma, Parma, Italy
| | | | - Maffezzoli Michele
- Department of Medicine and Surgery, University of Parma, Parma, Italy
- Portsmouth Hospital University NHS Trust, Portsmouth, United Kingdom
| | - Mazzaschi Giulia
- Medical Oncology Unit, University Hospital of Parma, Parma, Italy
- Department of Medicine and Surgery, University of Parma, Parma, Italy
| | - Manuguerra Roberta
- Pathology Unit, Department of Medicine and Surgery, University of Parma, Parma, Italy
| | - Azzoni Cinzia
- Pathology Unit, Department of Medicine and Surgery, University of Parma, Parma, Italy
| | - Bottarelli Lorena
- Pathology Unit, Department of Medicine and Surgery, University of Parma, Parma, Italy
| | - Tiseo Marcello
- Medical Oncology Unit, University Hospital of Parma, Parma, Italy
- Department of Medicine and Surgery, University of Parma, Parma, Italy
| | - Perrone Fabiana
- Medical Oncology Unit, University Hospital of Parma, Parma, Italy
| | - Minari Roberta
- Medical Oncology Unit, University Hospital of Parma, Parma, Italy
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Sorino C, Iezzi S, Ciuffreda L, Falcone I. Immunotherapy in melanoma: advances, pitfalls, and future perspectives. Front Mol Biosci 2024; 11:1403021. [PMID: 39086722 PMCID: PMC11289331 DOI: 10.3389/fmolb.2024.1403021] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2024] [Accepted: 05/16/2024] [Indexed: 08/02/2024] Open
Abstract
Cutaneous melanoma is the deadliest and most aggressive form of skin cancer owing to its high capacity for metastasis. Over the past few decades, the management of this type of malignancy has undergone a significant revolution with the advent of both targeted therapies and immunotherapy, which have greatly improved patient quality of life and survival. Nevertheless, the response rates are still unsatisfactory for the presence of side effects and development of resistance mechanisms. In this context, tumor microenvironment has emerged as a factor affecting the responsiveness and efficacy of immunotherapy, and the study of its interplay with the immune system has offered new promising clinical strategies. This review provides a brief overview of the currently available immunotherapeutic strategies for melanoma treatment by analyzing both the positive aspects and those that require further improvement. Indeed, a better understanding of the mechanisms involved in the immune evasion of melanoma cells, with particular attention on the role of the tumor microenvironment, could provide the basis for improving current therapies and identifying new predictive biomarkers.
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Guo GH, Xie YB, Jiang T, An Y. Droplet digital polymerase chain reaction assay for methylated ring finger protein 180 in gastric cancer. World J Gastrointest Oncol 2022; 14:2038-2047. [PMID: 36310700 PMCID: PMC9611431 DOI: 10.4251/wjgo.v14.i10.2038] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/20/2022] [Revised: 08/21/2022] [Accepted: 09/02/2022] [Indexed: 02/05/2023] Open
Abstract
BACKGROUND Gastric cancer (GC) is one of the most prevalent malignant tumors that endangers human health. Early diagnosis is essential for improving the prognosis and survival rate of GC patients. Ring finger protein 180 (RNF180) is involved in the regulation of cell differentiation, proliferation, apoptosis, and tumorigenesis, and aberrant hypermethylation of CpG islands in the promoter is strongly associated with the occurrence and development of GC. Thus, methylated RNF180 can be used as a potential biomarker for GC diagnosis.
AIM To use droplet digital polymerase chain reaction (ddPCR) to quantify the methylation level of the RN180 gene. A reproducible ddPCR assay to detect methylated RNF180 from trace DNA was designed and optimized.
METHODS The primer and probe were designed and selected, the conversion time of bisulfite was optimized, the ddPCR system was adjusted by primer concentration, amplification temperature and amplification cycles, and the detection limit of ddPCR was determined.
RESULTS The best conversion time for blood DNA was 2 h 10 min, and that for plasma DNA was 2 h 10 min and 2 h 30 min. The results of ddPCR were better when the amplification temperature was 56 °C and the number of amplification cycles was 50. Primer concentrations showed little effect on the assay outcome. Therefore, the primer concentration could be adjusted according to the reaction system and DNA input. The assay required at least 0.1 ng of input DNA.
CONCLUSION In summary, a ddPCR assay was established to detect methylated RNF180, which is expected to be a new diagnostic biomarker for GC.
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Affiliation(s)
- Guang-Hong Guo
- Department of Laboratory Medicine, The First Medical Center of Chinese PLA General Hospital, Beijing 100853, China
| | - Yi-Bin Xie
- Department of Pancreatic and Gastric Surgery, National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China
| | - Tao Jiang
- Medicine Innovation Research Division of Chinese PLA General Hospital, Beijing 100853, China
| | - Yang An
- Faculty of Hepato-Pancreato-Biliary Surgery, The Sixth Medical Center of Chinese PLA General Hospital, Beijing 100048, China
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Gouda MA, Duose DY, Lapin M, Zalles S, Huang HJ, Xi Y, Zheng X, Aldesoky AI, Alhanafy AM, Shehata MA, Wang J, Kopetz S, Meric-Bernstam F, Wistuba II, Luthra R, Janku F. Mutation-Agnostic Detection of Colorectal Cancer Using Liquid Biopsy-Based Methylation-Specific Signatures. Oncologist 2022; 28:368-372. [PMID: 36200910 PMCID: PMC10078907 DOI: 10.1093/oncolo/oyac204] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2022] [Accepted: 08/29/2022] [Indexed: 11/12/2022] Open
Abstract
Detection of methylation patterns in circulating tumor DNA (ctDNA) can offer a novel approach for cancer diagnostics given the unique signature for each tumor type. We developed a next-generation sequencing (NGS)-based assay targeting 32 CpG sites to detect colorectal cancer-specific ctDNA. NGS was performed on bisulfite-converted libraries and status dichotomization was done using median methylation ratios at all targets. We included plasma samples from patients with metastatic colorectal (n = 20) and non-colorectal cancers (n = 8); and healthy volunteers (n = 4). Median methylation ratio was higher in colorectal cancer compared with non-colorectal cancers (P = .001) and normal donors (P = .005). The assay detected ctDNA in 85% of patients with colorectal cancer at a specificity of 92%. Notably, we were able to detect methylated ctDNA in 75% of patients in whom ctDNA was not detected by other methods. Detection of methylated ctDNA was associated with shorter median progression-free survival compared to non-detection (8 weeks versus 54 weeks; P = .027).
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Affiliation(s)
- Mohamed A Gouda
- Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, USA.,Department of Investigational Cancer Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, USA.,Department of Clinical Oncology and Nuclear Medicine, Faculty of Medicine, Menoufia University, Shebin Al-Kom, Egypt
| | - Dzifa Y Duose
- Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, USA
| | - Morten Lapin
- Department of Hematology and Oncology, Stavanger University Hospital, Stavanger, Norway
| | - Stephanie Zalles
- Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, USA
| | - Helen J Huang
- Department of Investigational Cancer Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, USA
| | - Yuanxin Xi
- Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, USA
| | - Xiaofeng Zheng
- Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, USA
| | - Amira I Aldesoky
- Department of Clinical Oncology and Nuclear Medicine, Faculty of Medicine, Menoufia University, Shebin Al-Kom, Egypt
| | - Alshimaa M Alhanafy
- Department of Clinical Oncology and Nuclear Medicine, Faculty of Medicine, Menoufia University, Shebin Al-Kom, Egypt
| | - Mohamed A Shehata
- Department of Clinical Oncology and Nuclear Medicine, Faculty of Medicine, Menoufia University, Shebin Al-Kom, Egypt
| | - Jing Wang
- Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, USA
| | - Scott Kopetz
- Department of Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, USA
| | - Funda Meric-Bernstam
- Department of Investigational Cancer Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, USA
| | - Ignacio I Wistuba
- Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, USA
| | - Rajyalakshmi Luthra
- Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, USA
| | - Filip Janku
- Department of Investigational Cancer Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, USA
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Dobre EG, Constantin C, Neagu M. Skin Cancer Research Goes Digital: Looking for Biomarkers within the Droplets. J Pers Med 2022; 12:jpm12071136. [PMID: 35887633 PMCID: PMC9323323 DOI: 10.3390/jpm12071136] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2022] [Revised: 07/12/2022] [Accepted: 07/12/2022] [Indexed: 12/24/2022] Open
Abstract
Skin cancer, which includes the most frequent malignant non-melanoma carcinomas (basal cell carcinoma, BCC, and squamous cell carcinoma, SCC), along with the difficult to treat cutaneous melanoma (CM), pose important worldwide issues for the health care system. Despite the improved anti-cancer armamentarium and the latest scientific achievements, many skin cancer patients fail to respond to therapies, due to the remarkable heterogeneity of cutaneous tumors, calling for even more sophisticated biomarker discovery and patient monitoring approaches. Droplet digital polymerase chain reaction (ddPCR), a robust method for detecting and quantifying low-abundance nucleic acids, has recently emerged as a powerful technology for skin cancer analysis in tissue and liquid biopsies (LBs). The ddPCR method, being capable of analyzing various biological samples, has proved to be efficient in studying variations in gene sequences, including copy number variations (CNVs) and point mutations, DNA methylation, circulatory miRNome, and transcriptome dynamics. Moreover, ddPCR can be designed as a dynamic platform for individualized cancer detection and monitoring therapy efficacy. Here, we present the latest scientific studies applying ddPCR in dermato-oncology, highlighting the potential of this technology for skin cancer biomarker discovery and validation in the context of personalized medicine. The benefits and challenges associated with ddPCR implementation in the clinical setting, mainly when analyzing LBs, are also discussed.
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Affiliation(s)
- Elena-Georgiana Dobre
- Faculty of Biology, University of Bucharest, Splaiul Independentei 91–95, 050095 Bucharest, Romania;
- Correspondence:
| | - Carolina Constantin
- Immunology Department, “Victor Babes” National Institute of Pathology, 050096 Bucharest, Romania;
- Pathology Department, Colentina Clinical Hospital, 020125 Bucharest, Romania
| | - Monica Neagu
- Faculty of Biology, University of Bucharest, Splaiul Independentei 91–95, 050095 Bucharest, Romania;
- Immunology Department, “Victor Babes” National Institute of Pathology, 050096 Bucharest, Romania;
- Pathology Department, Colentina Clinical Hospital, 020125 Bucharest, Romania
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Aleotti V, Catoni C, Poggiana C, Rosato A, Facchinetti A, Scaini MC. Methylation Markers in Cutaneous Melanoma: Unravelling the Potential Utility of Their Tracking by Liquid Biopsy. Cancers (Basel) 2021; 13:6217. [PMID: 34944843 PMCID: PMC8699653 DOI: 10.3390/cancers13246217] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2021] [Revised: 12/02/2021] [Accepted: 12/08/2021] [Indexed: 01/19/2023] Open
Abstract
Malignant melanoma is the most serious, life-threatening form of all dermatologic diseases, with a poor prognosis in the presence of metastases and advanced disease. Despite recent advances in targeted therapy and immunotherapy, there is still a critical need for a better understanding of the fundamental mechanisms behind melanoma progression and resistance onset. Recent advances in genome-wide methylation methods have revealed that aberrant changes in the pattern of DNA methylation play an important role in many aspects of cancer progression, including cell proliferation and migration, evasion of cell death, invasion, and metastasization. The purpose of the current review was to gather evidence regarding the usefulness of DNA methylation tracking in liquid biopsy as a potential biomarker in melanoma. We investigated the key genes and signal transduction pathways that have been found to be altered epigenetically in melanoma. We then highlighted the circulating tumor components present in blood, including circulating melanoma cells (CMC), circulating tumor DNA (ctDNA), and tumor-derived extracellular vesicles (EVs), as a valuable source for identifying relevant aberrations in DNA methylation. Finally, we focused on DNA methylation signatures as a marker for tracking response to therapy and resistance, thus facilitating personalized medicine and decision-making in the treatment of melanoma patients.
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Affiliation(s)
- Valentina Aleotti
- Immunology and Molecular Oncology Unit, Veneto Institute of Oncology IOV-IRCCS, 35128 Padua, Italy; (V.A.); (C.C.); (A.F.); (M.C.S.)
| | - Cristina Catoni
- Immunology and Molecular Oncology Unit, Veneto Institute of Oncology IOV-IRCCS, 35128 Padua, Italy; (V.A.); (C.C.); (A.F.); (M.C.S.)
| | - Cristina Poggiana
- Immunology and Molecular Oncology Unit, Veneto Institute of Oncology IOV-IRCCS, 35128 Padua, Italy; (V.A.); (C.C.); (A.F.); (M.C.S.)
| | - Antonio Rosato
- Immunology and Molecular Oncology Unit, Veneto Institute of Oncology IOV-IRCCS, 35128 Padua, Italy; (V.A.); (C.C.); (A.F.); (M.C.S.)
- Department of Surgery, Oncology and Gastroenterology, Oncology and Immunology Section, University of Padua, 35128 Padua, Italy
| | - Antonella Facchinetti
- Immunology and Molecular Oncology Unit, Veneto Institute of Oncology IOV-IRCCS, 35128 Padua, Italy; (V.A.); (C.C.); (A.F.); (M.C.S.)
- Department of Surgery, Oncology and Gastroenterology, Oncology and Immunology Section, University of Padua, 35128 Padua, Italy
| | - Maria Chiara Scaini
- Immunology and Molecular Oncology Unit, Veneto Institute of Oncology IOV-IRCCS, 35128 Padua, Italy; (V.A.); (C.C.); (A.F.); (M.C.S.)
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Kamińska P, Buszka K, Zabel M, Nowicki M, Alix-Panabières C, Budna-Tukan J. Liquid Biopsy in Melanoma: Significance in Diagnostics, Prediction and Treatment Monitoring. Int J Mol Sci 2021; 22:9714. [PMID: 34575876 PMCID: PMC8468624 DOI: 10.3390/ijms22189714] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2021] [Revised: 08/25/2021] [Accepted: 09/06/2021] [Indexed: 02/06/2023] Open
Abstract
Liquid biopsy is a common term referring to circulating tumor cells and other biomarkers, such as circulating tumor DNA (ctDNA) or extracellular vesicles. Liquid biopsy presents a range of clinical advantages, such as the low invasiveness of the blood sample collection and continuous control of the tumor progression. In addition, this approach enables the mechanisms of drug resistance to be determined in various methods of cancer treatment, including immunotherapy. However, in the case of melanoma, the application of liquid biopsy in patient stratification and therapy needs further investigation. This review attempts to collect all of the relevant and recent information about circulating melanoma cells (CMCs) related to the context of malignant melanoma and immunotherapy. Furthermore, the biology of liquid biopsy analytes, including CMCs, ctDNA, mRNA and exosomes, as well as techniques for their detection and isolation, are also described. The available data support the notion that thoughtful selection of biomarkers and technologies for their detection can contribute to the development of precision medicine by increasing the efficacy of cancer diagnostics and treatment.
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Affiliation(s)
- Paula Kamińska
- Department of Histology and Embryology, Poznan University of Medical Sciences, 60-781 Poznan, Poland; (P.K.); (K.B.); (M.N.)
| | - Karolina Buszka
- Department of Histology and Embryology, Poznan University of Medical Sciences, 60-781 Poznan, Poland; (P.K.); (K.B.); (M.N.)
| | - Maciej Zabel
- Department of Anatomy and Histology, Collegium Medicum, University of Zielona Góra, 65-046 Zielona Góra, Poland;
| | - Michał Nowicki
- Department of Histology and Embryology, Poznan University of Medical Sciences, 60-781 Poznan, Poland; (P.K.); (K.B.); (M.N.)
| | - Catherine Alix-Panabières
- Laboratory of Rare Human Circulating Cells (LCCRH), University Medical Centre of Montpellier, 34093 Montpellier, France;
- CREEC/CANECEV, MIVEGEC (CREES), University of Montpellier, CNRS, IRD, 34000 Montpellier, France
| | - Joanna Budna-Tukan
- Department of Histology and Embryology, Poznan University of Medical Sciences, 60-781 Poznan, Poland; (P.K.); (K.B.); (M.N.)
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