Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide. It is a heterogeneous tumor with a wide genomic instability, leading to tumor recurrence, distant metastasis, and therapy resistance. Therefore, adjunct non-invasive tools are urgently needed to help the current classical staging systems for more accurate prognostication and guiding personalized therapy. In recent decades, there has been an increasing interest in the diagnostic, prognostic, and predictive value of circulating cancer-derived material in CRC. Liquid biopsies provide direct non-invasive access to tumor material, which is shed into the circulation; this enables the analysis of circulating tumor cells (CTC) and genomic components such as circulating free DNA (cfDNA), which could provide the key for personalized therapy. Liquid biopsy (LB) allows for the identification of patients with a high risk for disease progression after curative surgery, as well as longitudinal monitoring for disease progression and therapy response. Here, we will review the most recent studies on CRC, demonstrating the clinical potential and utility of CTCs and ctDNA. We will discuss some of the advantages and limitations of LBs and the future perspectives in the field of CRC management.

Introduction: Circulating tumor DNA (ctDNA) holds promise for guiding immune checkpoint inhibitor (ICI) therapy and stratifying responders from non-responders. While tumor-informed ctDNA detection approaches are sensitive and mutation-inclusive, they require tumor tissue, which limits applicability in real-world settings. Conversely, tumor-agnostic methods often have limited genomic coverage. In this study, we evaluated a tumor-agnostic, broad-panel ctDNA assay in patients with advanced melanoma treated with ICI. Methods: We conducted a prospective analysis of 241 longitudinal samples from 39 patients with unresectable stage III/IV melanoma using a SYSMEX targeted NGS panel covering 1,114 COSMIC mutations. Plasma samples were collected at baseline and during ICI therapy. The assay’s sensitivity reached seven mutant molecules, corresponding to a 0.07% mutation allele frequency (MAF). ctDNA profiles were compared with matched tumor tissue and correlated with clinical features and survival. Results: At baseline, ctDNA was detected in 64.5% of patients. Common mutations included BRAF ^V600E (43.8%) and NRAS ^G12D (36.4%), followed by KRAS, EGFR, and PIK3CA variants. Overall tissue–plasma concordance was 51.6%, with more extended biopsy–plasma intervals associated with discordance (p = 0.0105). Notably, 12.2% of cases exhibited partial concordance, characterized by shared mutations and additional plasma-only alterations, underscoring the complementary value of blood-based profiling. Persistent or re-emerging ctDNA positivity post-therapy correlated with shorter progression-free survival (PFS, p = 0.003), while ctDNA-negative patients showed significantly improved outcomes. Patients that remained ctDNA-negative had significantly longer progression-free survival (median not reached) compared to those with persistent ctDNA positivity (median 3 months) or those converting to positive (median 7.5 months; p = 0.0073). Early NRAS and KRAS ctDNA levels strongly predicted poor response (p = 0.0069 and p = 0.028). The prognostic impact extended beyond canonical drivers, as non-hotspot variants also correlated with the outcome. Notably, even low-level ctDNA persistence (5–10 MM/mL) carried adverse prognostic implications (p = 0.0054). Concerning a shorter PFS, ctDNA positivity was also associated with elevated S100 levels (p = 0.047). Organ-specific mutation enrichment (e.g., KRAS^G12D in brain, EGFR^G719A in lymph nodes) suggested possible metastatic tropism. Conclusion: Broad tumor-agnostic ctDNA analysis effectively identified clinically relevant mutations and predicted outcomes in ICI-treated melanoma patients. This approach enables tissue-independent and real-time ctDNA monitoring and may inform patient selection and therapeutic strategies in future interventional trials.

Background: Immune checkpoint inhibition has revolutionized melanoma therapy, but many patients show primary or secondary resistance. Biomarkers are, therefore, urgently required to predict response prior to the initiation of therapy and to monitor disease progression. Methods: In this prospective study, we analyzed the serum C-C motif chemokine ligand 20 (CCL20) concentration using an enzyme-linked immunosorbent assay. Blood was obtained at baseline before the initiation of immunotherapy with anti-PD-1 monotherapy or Nivolumab and Ipilimumab in advanced melanoma patients (stages III and IV) enrolled at the University Medical Center Hamburg-Eppendorf. The CCL20 levels were correlated with clinico-pathological parameters and disease-related outcomes. Results: An increased C-C motif chemokine ligand 20 (CCL20) concentration (≥0.34 pg/mL) at baseline was associated with a significantly impaired progression-free survival (PFS) in the high-CCL20 group (3 months (95% CI: 2–6 months) vs. 11 months (95% CI: 6–26 months)) (p = 0.0033) and could be identified as an independent negative prognostic factor for PFS in univariate (Hazard Ratio (HR): 1.98, 95% CI 1.25–3.12, p = 0.004) and multivariate (HR: 1.99, 95% CI 1.21–3.29, p = 0.007) Cox regression analysis, which was associated with a higher risk than S100 (HR: 1.74). Moreover, high CCL20 levels were associated with impaired overall survival (median OS not reached for low-CCL20 group, p = 0.042) with an HR of 1.85 (95% CI 1.02–3.37, p = 0.043) in univariate analysis similar to the established prognostic marker S100 (HR: 1.99, 95% CI: 1.02–3.88, p = 0.043). Conclusions: CCL20 may represent a novel blood-based biomarker for the prediction of resistance to immunotherapy that can be used in combination with established strong clinical predictors (e.g., ECOG performance score) and laboratory markers (e.g., S100) in advanced melanoma patients. Future prospective randomized trials are needed to establish CCL20 as a liquid biopsy-based biomarker in advanced melanoma.

Purpose: Circulating tumor DNA (ctDNA) refers to small DNA fragments, shed from tumor cells into the bloodstream. Measuring ctDNA provides a noninvasive tool for real-time disease monitoring. Although ctDNA predicted overall survival (OS) in metastatic uveal melanoma (mUM) treated with tebentafusp, its broader prognostic value across treatment modalities remains unclear. In this study, we assess the prognostic relevance of longitudinal ctDNA detection and mutant allele fraction (MAF) in patients with mUM treated with different modalities. Experimental Design: To assess ctDNA monitoring as a tool in evaluating therapy response and clinical outcomes in patients with mUM, using an IVDR-certified digital PCR assay targeting GNAQQ209 and GNA11Q209 mutations. Results: We analyzed 655 samples from 75 patients with mUM. Absence of detectable ctDNA in baseline samples prior to first-line therapy was associated with improved OS (HR = 0.13; P = 0.02) and progression-free survival (PFS; HR = 0.31; P = 0.008). Similar associations were observed in patients treated with any line of therapy (OS: HR = 0.19, P = 0.002; PFS: HR = 0.27, P = 0.02). Detection of ctDNA within 3 months of therapy initiation was associated with worse outcomes, independent of baseline detection. Furthermore, patients with MAF > 5% any time point had a significantly poorer prognosis compared with patients with MAF < 5% (median OS 4 months vs. 21 months, P < 0.001; median PFS 2.5 months vs. 3.6 months, P = 0.004), emphasizing the added value of quantitative assessment. Conclusions: Both the presence and level of ctDNA at baseline, along with persistence of ctDNA within 3 months of starting of treatment, are strong negative prognostic markers in mUM. These findings support the clinical utility of ctDNA as a noninvasive tool for disease monitoring.

Background/Objectives: Tumor-immune system interactions shape the progression of cutaneous squamous cell carcinoma (cSCC). Serum biomarkers for risk stratification remain limited. Complement factor H (CFH) regulates the alternative complement pathway. It has been linked to immunosuppression and cSCC development in tissue-based studies. We investigated whether serum CFH is associated with tumor aggressiveness and may help predict immunotherapy outcomes in advanced cSCC. Methods: In this retrospective, single-center study, pre-treatment serum CFH levels were measured in 104 cSCC patients (62 high-risk and 42 advanced) using ELISA. Associations with clinical characteristics, disease stage, and response to cemiplimab were analyzed. Subgroup comparisons considered immune status and inflammatory comorbidities. Results: Advanced cSCC patients had significantly higher CFH levels than high-risk patients (OR 0.13, p = 0.026), independent of tumor diameter or invasion depth. Among advanced cSCC cases, lower baseline CFH was associated with more prolonged progression-free survival (median 19.8 vs. 3.07 months, p = 0.029; HR 0.29, p = 0.014), independent of covariates including immunosuppression. CFH levels during therapy did not predict treatment response. ROC analysis showed moderate discriminatory ability with CFH alone (AUC 0.625), which improved when combined with clinical variables in a multivariable risk model (AUC 0.767). Conclusions: Serum CFH is an independent predictor of cemiplimab response and reflects biological aggressiveness in cSCC beyond conventional high-risk features. These findings support the use of CFH in clinical risk models and warrant external validation in multicenter cohorts.

Background: Immune checkpoint inhibition (ICI) is the standard treatment for advanced melanoma patients. Despite its high efficacy compared to previous treatment options, immune-related adverse events (irAEs) occur frequently. While most of the patients experience mild to moderate irAEs, some patients develop severe to lethal irAEs under ICI treatment; hence, biomarkers are urgently required. Methods: In this retrospective single-center study, 157 advanced melanoma patients treated with ICI at the University Medical Center Hamburg–Eppendorf were included. IrAEs were correlated with clinico-pathological parameters, disease-related outcomes, and irAE-free survival. Results: In our cohort, 130 out of 157 patients receiving immunotherapy experienced irAE, of which more than half experienced irAE Grade ≥ 3. The most common irAE independent of its grade included cutaneous irAE, colitis, endocrine irAE, and hepatitis. Patients experiencing irAE had significantly longer progression-free survival (PFS) and overall survival (OS) compared to patients who did not experience irAE under ICI therapy. Stratification by irAE groups revealed that musculoskeletal irAEs are associated with the longest, whereas myocarditis is associated with the shortest OS and PFS. IrAE was a significant beneficial prognosticator for PFS in univariate, but not in multivariate Cox regression analysis. With respect to OS, the occurrence of irAE was an independent prognostic factor among ECOG status ≥ 2 and uveal melanoma. ROC analysis demonstrated that D-dimers have moderate predictive capability for irAE occurrence. Cox regression analysis demonstrated that elevated D-dimers and PD-1 monotherapy vs. CTLA-4 and PD-1 combination regimen are the only independent prospective prognostic markers for irAE-free survival. Conclusions: Our study demonstrates that different irAE across the irAE spectrum have a different impact on the PFS and OS of advanced melanoma patients. D-dimers may be used as a blood-based biomarker for irAE prediction, warranting future validation in multi-center studies.