Author:

Marta Paterlini

Abstract

Pathology has evolved from a discipline based on descriptive morphology and special stains into a morpho-molecular specialty that integrates protein, nucleic acid and computational readouts with microscopic context. This review traces the technical milestones — histochemistry and immunohistochemistry (IHC), in-situ hybridisation (ISH) and cytogenetics, polymerase chain reaction (PCR) and Sanger sequencing, next-generation sequencing (NGS), liquid biopsy and digital pathology with artificial intelligence (AI) — and describes how those advances reshaped laboratory workflows, quality assurance and clinical decision-making. The pathologist’s role has expanded from slide interpretation to comprehensive specimen management, molecular-assay selection, and active participation in multidisciplinary tumour boards. Contemporary guidelines emphasize strict pre-analytic protocols, integrated morpho-molecular reporting, and training in bioinformatics and computational pathology.

Professor Nicola Fusco is Head of Pathology Division at the European Institute of Oncology (IEO) in Milan and Professor of Pathology at the University of Milan. Internationally recognised as a leader in the field of molecular pathology and a pioneer in the application of artificial intelligence to digital pathology, he leads a team committed to innovation at the crossroads of diagnostics, research and training. His clinical activity includes histopathological and cytopathological diagnosis and molecular profiling of solid tumours, with a particular focus on breast cancer. His research integrates computational pathology, genomics and transcriptomics with a pragmatic and clinically oriented approach, aimed at accelerating the adoption of new technologies in precision oncology.

Fusco is also Chair of the Quality and Accreditation Committee of the International Society of Liquid Biopsy (ISLB), coordinates the Liquid Biopsy Task Force of the Molecular Pathology and Precision Medicine Group (PMMP-SIAPeC) and is a member of the Multidisciplinary Scientific Council of the European School of Oncology (ESO). He has been invited to speak at the Senate and Chamber of Deputies about artificial intelligence in healthcare and has contributed to the drafting of international guidelines and recommendations on breast cancer diagnostics, molecular pathology and liquid biopsy. Prof. Fusco has been listed among the Top 2% Scientists worldwide in 2023 and 2024 by Stanford University.

Q&A

Doing both research and clinic, how would you describe your work environment?

Nicola Fusco: We have a laboratory divided into four different operational units: an histology unit, a cytology unit, and a molecular pathology unit, which includes sequencing aimed at discovering specific biological features in DNA or RNA or in protein expression that can be used to select patients for specific treatments, for specific therapeutic protocols or even for specific clinical trials. Alongside these is the complex unit of Pathomics, known as Digital Pathology.

How did you get into anatomical pathology?

As a medical student I was struck by how much clinically relevant information could be read from a single tissue specimen — not only morphology, but also immunophenotype and now molecular data. I have always been attracted to innovation, and pathology sits at the intersection of oncology, biology, technology and direct patient impact. Contrary to a common stereotype, anatomical pathology is not a “back room” discipline — over the past two decades it has become a data-rich specialty that informs therapy selection, prognostication and trial enrolment.

Are professional skills keeping pace with new technologies?

That’s an excellent question. This is a timely concern: in many areas the technology, especially molecular and computational tools, has advanced faster than the workforce trained to analyse the data, and this coincides with a global, demographic decline in practising anatomic pathologists driven by structural, political and social factors. As a university teacher I stress that anatomical pathology remains central to modern care and now offers important career opportunities for those interested in technology and precision medicine. Training must therefore combine fundamental morphology with molecular and AI competencies (the new skills are an integration, not a replacement), and institutions must equip trainees with continuing-education resources — because staying up to date is the profession’s biggest ongoing challenge.

We hear more about molecular tumour boards. What are they?

Molecular tumour boards are multidisciplinary panels that support clinical units (for example, a breast unit) by interpreting complex genomic and molecular profiles and prioritising patients for targeted or experimental therapies or clinical trials. Members usually include medical oncologists, pathologists, molecular biologists, clinical geneticists/counsellors, bioinformaticians, surgeons and sometimes radiologists or trial coordinators. The board reviews referred cases, weighs the strength of molecular evidence alongside comorbidities and prior treatments, and issues an actionable recommendation — for example, a therapy change, trial referral, or further testing. They complement clinicians by adding specialised molecular expertise to the care pathway.

However, the latest technologies are very expensive, such as spatial transcriptomics. Is it part of the pipeline?

Spatial transcriptomics is an exciting tool because it links gene expression to precise tissue architecture and microenvironmental context. Today its most mature uses are in research, translational studies and biomarker discovery within clinical trials — for example, dissecting intratumour heterogeneity or mapping immune niches that predict response to immunotherapy. At present it is rarely part of routine clinical workflows because of cost, technical complexity and the need for standardised interpretation. I expect that as costs fall and interpretation pipelines mature, selected applications will move into clinical practice, initially in tertiary centres and trial settings.

From your point of view, how important are pre-analytical variables?

They are critical. Pre-analytic factors — cold ischaemia time, type and duration of fixation, sample handling in the operating theatre, transport conditions and promptness of grossing — determine whether downstream molecular assays will succeed. We often use the chef metaphor: even the best laboratory technique cannot salvage a poorly collected specimen. To address this, we developed SOPs in collaboration with surgical teams (for example, aiming to keep cold ischaemia under one hour and to use 10% neutral buffered formalin with standardised fixation windows), and we follow professional pre-analytic guidelines from relevant societies and working groups. Those procedural changes had an immediate and measurable effect on nucleic-acid yield and sequencing success rate.

In a laboratory like yours, what is the balance between histochemistry and AI when moving from sequencing technologies? Is this a transition?

This is an integration, not a replacement: morphology and immunohistochemistry remain the diagnostic backbone and determine which molecular tests are needed. AI and computational pathology augment those workflows (automated mitotic counts, digital Ki-67, node-metastasis detection), saving time and improving reproducibility. Sequencing—used to find targetable mutations or structural variants—adds another complementary layer; morphology frames the diagnosis, while molecular and computational tools refine prognosis, predict response and resolve ambiguous cases.

IEO is one of the hotspots in Europe and worldwide. However, I suppose you are in a network with other European centres. Do you collaborate for common protocols or guidelines?

We collaborate nationally (for example Federico II, Naples) and with major European and global centres — Gustave Roussy, University of Nottingham, University of Cologne, MSKCC, and others — and partner with pharmaceutical, biotech and tech companies to identify biomarkers and develop diagnostic tools. Our mission is to convert biological samples into actionable data, so patients receive ultra-personalised therapies; clinical practice and research are inseparable here. Thus, our research is effectively overlapping with our clinical practice — in fact the founder of IEO, Professor Roberto Veronesi, coined the motto that made the Institute famous: “care is better where research is done.” Indeed, our clinical activity overlaps with our research activity, so it goes beyond translational research and is directly applied research.

Morpho-Molecular Pathology: historical development and modern integration

The adoption of molecular technologies into diagnostic practice constitutes a paradigm shift comparable to the introduction of the microscope: where the microscope defined tissue and cellular pathology, molecular platforms redefined diagnostic categories and therapeutic opportunities. Modern practice seeks to preserve the interpretative power of morphology while layering protein- and nucleic-acid level information and computational analytics to produce actionable, patient-centric reports. This hybrid approach places the morpho-molecular interpretation at the centre of precision medicine ^[4].

Foundations: histochemistry and the rise of protein localisation

Special stains and histochemical techniques made tissue chemistry visible and were the first steps beyond pure form. These methods demonstrated that staining patterns could convey functional information about cells and extracellular components, setting the conceptual groundwork for immunohistochemistry (IHC). IHC extended this capability by using antibodies to reveal the spatial distribution of clinically relevant proteins directly in tissue architecture — for example, hormone receptors and lineage markers — and quickly became a mainstay of diagnostic workflows due to its diagnostic and predictive value. IHC’s relative speed, cost-effectiveness and retention of morphological context explain its lasting centrality in routine diagnostics.

In-situ hybridisation, cytogenetics and visualising nucleic acids in tissue

In-situ hybridisation (chromogenic ISH) and fluorescent in-situ hybridisation (FISH) enabled the detection of gene amplifications, deletions and rearrangements while preserving tissue architecture. These cytogenetic techniques served as an important bridge between protein immunoassays and sequencing: they were particularly useful where gene copy number or structural rearrangements (for example, amplification of driver oncogenes or translocations) had direct therapeutic or diagnostic implications. ISH/FISH workflows introduced new laboratory requirements — from probe validation to image interpretation — and reinforced the importance of integrating molecular readouts with morphology.

PCR and Sanger sequencing — targeted molecular confirmation

The introduction of PCR allowed highly sensitive detection of specific mutations and fusion transcripts. Sanger sequencing provided a reliable method for single-gene confirmation and remained widely used for validation of variants. PCR-based assays were often the first molecular tests integrated into diagnostic pipelines because they were relatively straightforward to implement and interpret. However, they required careful pre-analytic control (fixation, cold ischemia, and nucleic acid quality) — lessons that became central as broader genomic assays were later introduced ^[1],^[4].

Next-generation sequencing: breadth, complexity and opportunity

NGS transformed diagnostics by enabling simultaneous, multiplexed analysis of dozens to hundreds of genes [1,4]. Targeted panels for actionable mutations, tumor mutation burden metrics, and homologous recombination deficiency signatures are now part of routine practice in many specialised settings. NGS introduced significant new demands: validated wet-lab protocols for degraded formalin-fixed paraffin-embedded (FFPE) specimens, robust bioinformatics pipelines, variant interpretation frameworks, and structured reporting to translate genomic complexity into clear clinical recommendations. The transition to NGS also prompted new laboratory roles (molecular biologists, bioinformaticians) and formal mechanisms for cross-disciplinary governance.

Liquid biopsy and functional models — complements to tissue diagnostics

Circulating tumour DNA (ctDNA) and other liquid biopsy approaches offer minimally invasive monitoring, early detection of resistance, and the opportunity for serial genomic surveillance. Patient-derived organoids and other functional ex-vivo models provide platforms for testing drug sensitivity. These approaches extend diagnostic capability beyond a static tissue section but do not obviate traditional tissue pathology: histologic context, tumor microenvironment assessment and grading remain indispensable for diagnosis and risk stratification. Today, liquid biopsies and organoids are complementary tools integrated alongside tissue-based morpho-molecular analyses ^[3].

Digital pathology and artificial intelligence — a new analytic layer

Whole-slide imaging and AI algorithms are changing how slide review is performed, scored and quantified [4,5]. Concrete use cases already include standardised quantification tasks (Ki-67 scoring, mitotic counts), metastasis detection, and computational extraction of morphologic features that correlate with risk models. AI’s principal value is reproducibility and scalability, but it requires careful validation, pathologist oversight and integration into clinical workflows. Combining image-based algorithms with molecular and clinical data yields powerful predictive models — for example, image-informed risk calculators — but also raises data governance and reproducibility challenges ^[4],^[5].

Pre-analytic quality, standardisation and the morpho-molecular report

Effective morpho-molecular diagnostics depend on rigorous pre-analytic practice: sample handling, fixation timing, tissue selection and nucleic acid preservation all influence downstream assay success^[2]. Standard operating procedures, internal and external quality assessment programs, and harmonised reporting templates are central to reliable results^[1]. Pathologists perform specimen allocation and nucleic-acid quality assessment, select and validate molecular panels, and provide integrated morpho-molecular interpretation in structured, clinically actionable reports. The need for standardisation also underpins multidisciplinary molecular tumour boards that synthesise complex findings for therapeutic decisions.

Training, workforce and the next-generation pathologist

The practice changes outlined above imply new training priorities: exposure to molecular biology, competence in interpreting genomic reports, basic bioinformatics literacy, and familiarity with validated AI tools. Departments are adopting hybrid teams that include molecular pathologists, molecular technologists, and bioinformaticians to manage high-throughput platforms^[2],^[4],^[5]. Educational curricula must balance deep morphological training with these new digital and molecular competencies so that pathologists remain the integrative clinical experts in tumour characterisation.

Challenges and future directions

Key challenges remain: standardising NGS and AI validation, ensuring equitable access to molecular testing, integrating multi-omic and radiologic data into reproducible risk models, and translating variant interpretation into therapy-matching recommendations. Emerging technologies — spatial transcriptomics, multi-omic single-cell profiling, and improved functional models — promise richer insight into tumour biology, but their routine clinical deployment will require evidence of clinical utility, workflow fit, and cost-effectiveness. The immediate priorities are continued harmonisation of pre-analytic and analytic standards, expanded training, and robust multidisciplinary collaboration to translate complex data into decisions that improve patient outcomes ^[1],^[2],^[4],^[5].

Conclusion

Modern pathology is a synthesis of morphology, molecular science and computational analysis. The discipline’s strength is its ability to interpret molecular signals within tissue architecture and to provide concise, clinically relevant conclusions. By stewarding samples, guiding test selection, integrating multimodal data and participating in multidisciplinary decision-making, the pathologist ensures that advanced assays translate into better, more personalised patient care. Continued emphasis on pre-analytic quality, validated pipelines and training will determine how swiftly and reliably emerging technologies become routine tools in diagnostic practice.

Acknowledgment

The author wishes to express sincere gratitude to Dr. Luana Balestra, Scientific Assistant to Prof. Nicola Fusco, for her prompt feedback.