A suitable diagnostic classification of myeloid neoplasms and acute leukemias requires testing for a large number of molecular biomarkers. Next-generation sequencing is a technology able to integrate identification of the vast majority of them in a single test. This manuscript includes the design, analytical validation and clinical feasibility evaluation of a molecular diagnostic kit for onco-hematological diseases. It is based on sequencing of the coding regions of 76 genes (seeking single-nucleotide variants, small insertions or deletions and CNVs), as well as the search for fusions in 27 target genes. The kit has also been designed to detect large CNVs throughout the genome by including specific probes and employing a custom bioinformatics approach. The analytical and clinical feasibility validation of the Haematology OncoKitDx panel has been carried out from the sequencing of 170 patient samples from 6 hospitals (in addition to the use of commercial reference samples). The analytical validation showed sensitivity and specificity close to 100% for all the parameters evaluated, with a detection limit of 2% for SNVs and SVs, and 20% for CNVs. Clinically relevant mutations were detected in 94% of all patients. An analysis of the correlation between the genetic risk classification of AML (according to ELN 2017) established by the hospitals and that obtained by the Haematology OncoKitDx panel showed an almost perfect correlation (K = 0.94). Among the AML samples with a molecular diagnosis, established by the centers according to the WHO, the Haematology OncoKitDx analysis showed the same result in 97% of them. The panel was able to adequately differentiate between MPN subtypes and also detected alterations that modified the diagnosis (FIP1L1-PDGFRA). Likewise, the cytogenetic risk derived from the CNV plot generated by the NGS panel correlated substantially with the results of the conventional karyotype (K = 0.71) among MDS samples. In addition, the panel detected the main biomarkers of prognostic value among patients with ALL. This validated solution enables a reliable analysis of a large number of molecular biomarkers from a DNA sample in a single assay.

Background/Objectives: Early detection through minimally invasive approaches is critical for timely patient stratification and optimal therapeutic decision-making in colorectal cancer (CRC). Liquid biopsy, based on the analysis of tumor-derived components in blood and other body fluids, has emerged as a promising strategy to overcome current limitations in CRC diagnosis and follow-up. This review evaluates the current landscape of liquid biopsy clinical trials in CRC, focusing on predictive biomarker detection, prognostic assessment, and disease monitoring. Methods: ClinicalTrials.gov was searched using the terms “colorectal cancer” and “liquid biopsy” yielding 153 registered trials. After manual screening, 44 trials were excluded for not using liquid biopsy for CRC management, leaving 109 trials for analysis. Of these, 25 were completed, and 13 had publicly available results related to liquid biopsy. Results: The included trials were conducted across 27 countries on four continents. Overall, 119 biomolecules assessments and 167 different endpoints were reported across 109 clinical trials. Because individual trials could evaluate multiple biomolecules and endpoints, counts exceed the total number of trials. Cell-free DNA (cfDNA) was evaluated in 92/109 trials (84%) and accounting for 77% of all biomolecule assessments. Circulatingtumor cells (CTCs) were analyzed in 9/109 trials (8%, representing 8% of all the biomolecules analyzed), and microRNAs (miRNAs) in 8/109 (7%, representing 7% of all the biomolecules analyzed). Treatment sensitivity was the most common endpoint (57/109, 52% of the clinical trials; representing 34% of all the 167 different endpoints analyzed), followed by disease progression (28/109, 26%; representing 17% of all the different endpoints analyzed) and diagnostic applications (21/109, 19%; representing 12% of all the different endpoints analyzed). Among the 25 completed studies, 10/25 (40%) were interventional and 15/25 (60%) observational, spanning 14 countries. The majority of completed trials (21/25, 84%) used cfDNA. Interventional studies were predominantly phase II (5/10), with fewer phase III trials (2/10), primarily evaluating treatment response, particularly in relation to EGFR inhibitors and RAS/BRAF mutation status. Four observational studies (4/15) investigated emerging biomarkers, including long noncoding RNAs and miRNAs. Conclusions: Current clinical trials highlight cfDNA as the dominant and most clinically advanced liquid biopsy biomarker in CRC, primarily used for treatment guidance and disease monitoring. In contrast, CTCs and RNA-based biomarkers remain underrepresented. The limited number of randomized late-phase trials, heterogeneity in study design, and technical challenges associated with emerging biomarkers underscore the need for standardized methodologies and robust validation before routine clinical implementation.