MOLECULAR DIAGNOSTICS IN PATHOLOGICAL ANATOMY
It is necessary to have acquired basic knowledge of Biochemistry and Genetics.
Written Examination (Main Assessment)
The written exam includes:
Essay/Open-ended Questions to evaluate students' conceptual linkage ability and mastery of complex topics.
Multiple-choice Questions to check basic knowledge, terminology, and technical details.
Problem-Solving Questions involving the presentation of simulated data or laboratory results.
Continuous Assessment and Interaction (During the Course)
This includes:
Direct Questions asked during lectures.
Discussion of scientific articles in a group setting.
Practical Deepening
Analysis of an Extended Clinical Case Study that requires students to connect theory to diagnostic practice.
The course aims to provide knowledge of the main pathogenetic mechanisms currently used in pathological anatomy laboratories for the diagnosis, prognosis, and therapy of the main neoplastic and non-neoplastic diseases.
Pre-Analytical Quality: From Collection to Extraction
Targeted Techniques and Precision Quantitative Analysis
NGS: Fundamentals, Platforms, and Library Preparation
NGS Analysis of Mutations, CNVs, and RNA Techniques
Liquid Biopsy (ctDNA): Test Execution (or Performing the Test)
DNA Methylation and Biomarker Validation
1. Pre-Analytical Quality & Sample Management
Role of the Biomedical Laboratory Scientist (BLS) in Molecular Pathology (MP): The workflow and interaction with the Pathologist.
FFPE Sample Management: Standardization of fixation. Sectioning and microdissection techniques for tumor enrichment.
Extraction and QC: DNA/RNA extraction protocols. Quality Control and sample integrity (methods for quantification and damage assessment).
2. Targeted Techniques and Quantitative Analysis
RT-PCR in Diagnostics: Procedural details of real-time PCR for mutation identification.
Digital PCR (dPCR): The laboratory workflow for preparation and execution (emulsion and reading phase). Applications in Minimal Residual Disease (MRD).
Instability and CNV Analysis: Execution of the MSI test. Preparation and basic analysis of MLPA.
3. NGS: Fundamentals, Platforms, and Library Preparation
NGS Principles: Parallel sequencing and common platforms (Illumina, Ion Torrent).
Library Preparation: Practical details of ligation and amplicon-based protocols. The importance of the index (barcoding).
Pre-Sequencing Quality Control: Library assessment (e.g., Bioanalyzer, Qubit).
4. NGS Analysis and Troubleshooting
SNV/Indel in NGS: Variant detection, concept of coverage, and VAF (Variant Allele Fraction).
Gene Fusion Detection: Laboratory workflow for RNA-based NGS (reverse transcription and library preparation). Applications (ALK, ROS1).
Troubleshooting: Management of suboptimal sequencing results and failed batches.
5. Liquid Biopsy (ctDNA): Test Execution
From Vein to Plasma: Standardized procedures for plasma collection, centrifugation, and storage.
ctDNA Extraction and NGS: Specific protocols for cell-free DNA extraction and library preparation for ultra-sensitive tests.
Test Validation: Concepts of Limits of Detection (LoD) and Limits of Quantitation (LoQ) specific to Liquid Biopsy.
6. DNA Methylation and Biomarker Validation
Practical Epigenetics: The role of Bisulfite and the workflow of MS-PCR (Methylation-Specific PCR) (e.g., MGMT).
Therapeutic Biomarkers: Measurement of TMB (Tumor Mutational Burden) and MSI as NGS output data.
Analytical Validation: Essential requirements for the introduction of a new molecular test (sensitivity, specificity, accuracy).
Lectures (Frontal Teaching) and the use of quick polls (even anonymous) to check the comprehension of basic concepts.
Assignment of recent scientific articles on a specific topic, followed by a request for critical summaries from students to encourage discussion.
Group discussion of clinical cases supported by group brainstorming.