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COMPREHENSIVE PATHOLOGY EXAMINATION ANSWERS

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Q1 — WHO Classification of Soft Tissue Tumours: Notable Changes in the 5th Edition (2020)

Overview

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Full WHO 2020 Classification of Soft Tissue Tumours — Category Listing

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Notable Changes in Specific Categories

A. LIPOMATOUS (ADIPOCYTIC) TUMOURS

• Newly recognized as a distinct entity (previously lumped with spindle cell/pleomorphic lipoma or spindled ALT).
• Morphology: Variable proportions of atypical spindle cells, adipocytes, uni- or multivacuolated lipoblasts, and pleomorphic/multinucleated giant cells in a myxoid to collagenous stroma.
• Key distinguishing feature: Lacks MDM2 and CDK4 amplification (thus NOT an ALT/WD liposarcoma); Rb protein expression is generally lost (RB1 deletion/inactivation).
• Behaviour: Low recurrence rate (10-15%), no known risk of dedifferentiation - hence placed in intermediate (rarely metastasising) category.
• IHC: CD34 positive in many cases; desmin focal positivity.
• Clinical note: Typically arises in the subcutis of the extremities and trunk; predominantly in middle-aged adults.

• A rare high-grade liposarcoma added as a distinct malignant entity.
• Morphology: Admixture of areas resembling conventional myxoid liposarcoma (prominent myxoid stroma, plexiform vasculature) with more cellular regions showing overt nuclear pleomorphism resembling pleomorphic liposarcoma.
• Key molecular feature: Lacks both MDM2 amplification and DDIT3 (FUS-DDIT3) gene fusion - thus distinct from both myxoid and pleomorphic liposarcoma.
• Demography: Occurs predominantly in children and young adults; strong predilection for the mediastinum (unusual site for most liposarcomas).
• Behaviour: Aggressive, akin to pleomorphic liposarcoma.

• The terminology "atypical lipomatous tumour" (ALT) vs. "well-differentiated liposarcoma" (WDLS) is retained but clarified: ALT for resectable superficial/extremity tumours with low risk; WDLS for deep/retroperitoneal tumours where complete resection is difficult.
• Myxoid liposarcoma and round cell liposarcoma from the 4th edition are now unified as myxoid liposarcoma (with or without round cell areas), reflecting the same FUS-DDIT3 or EWSR1-DDIT3 translocation.

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B. FIBROBLASTIC/MYOFIBROBLASTIC TUMOURS

• Previously classified under miscellaneous or "uncertain differentiation" lesions.
• Morphology: Bland spindled to stellate cells embedded in a prominent fibromyxoid stroma with a branching, thin-walled vasculature.
• Molecular: Harbors AHRR-NCOA2 or related gene fusions.
• Behaviour: Benign but with tendency for local recurrence; hence classified as benign.
• Demographics: Adults, predominantly distal extremities.

• Defined by the EWSR1::SMAD3 gene fusion - one of the first truly fusion gene-named benign soft tissue entities.
• Morphology: Intersecting cellular fascicles of bland, monomorphic spindle cells alternating with hypocellular hyalinised areas.
• IHC: Nuclear expression of ERG (a useful surrogate marker).
• Site: Small acral (hands and feet) dermal/subcutaneous nodule.
• Behaviour: Indolent, benign.

• Rare, superficial dermal and subcutaneous tumour, placed in the intermediate (rarely metastasising) group.
• Morphology: Large eosinophilic cells with granular to glassy cytoplasm and marked pleomorphism but strikingly low mitotic count - a diagnostically confusing combination.
• IHC: Strongly CD34 positive; frequent keratin co-expression.
• Behaviour: Rarely metastasises despite alarming morphology.
• Molecular: PRDM10 rearrangements described.

• Mammary-type myofibroblastoma renamed to Myofibroblastoma (recognising it is not restricted to the breast or mammary-type stroma).
• Solitary Fibrous Tumour (SFT): Risk stratification now formally incorporated - a multi-parameter scoring system based on age, tumour size, mitotic activity, and necrosis is recommended to replace the old benign/malignant dichotomy.
• Inflammatory Myofibroblastic Tumour (IMT): Genetic profile better characterized; ALK rearrangements remain central to diagnosis and are now therapeutically relevant (ALK inhibitors).
• Desmoid-type fibromatosis: CTNNB1 (beta-catenin) mutations further validated for diagnosis and CTNNB1 codon 41 mutations associated with higher recurrence.

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C. SMOOTH MUSCLE TUMOURS

• Added as an intermediate entity (uncertain malignant potential).
• Pathogenesis: Driven by Epstein-Barr virus (EBV) infection, almost exclusively in immunocompromised patients: post-transplant recipients, HIV/AIDS, congenital immunodeficiency.
• Morphology: Smooth muscle-differentiated spindle cells with variable mild atypia, often arranged in fascicles; notably lacks the high-grade features of conventional leiomyosarcoma. EBV-encoded RNA (EBER) can be demonstrated by in-situ hybridisation.
• Behaviour: Unpredictable; may be multifocal (polyclonal - different EBV strains in different lesions within the same patient). Not classified as malignant because of its distinct biology from conventional LMS.
• Sites: Various visceral, soft tissue, and CNS locations.

• Newly recognized as a malignant entity but thought to be relatively indolent compared to conventional LMS.
• Morphology: Variably atypical eosinophilic spindle cells in fascicles with mitotic activity PLUS a prominent, usually diffuse, mixed (predominantly mononuclear) inflammatory infiltrate - the defining histological feature.
• Molecular hallmark: Near-haploid karyotype - an unusual and distinctive cytogenetic finding.
• Site: Predominantly deep soft tissues of the extremities.
• Behaviour: Malignant but with better outcomes than conventional LMS; metastatic potential present.

• Leiomyoma (benign)
• Smooth muscle tumour of uncertain malignant potential (STUMP) - criteria refined
• Leiomyosarcoma (conventional) - remains the primary malignant entity

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Q2 — Role of Artificial Intelligence in Pathology

Introduction

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Technical Foundations

• Traditional algorithms (Support Vector Machines, Random Forests) applied to extracted features (texture, shape, colour).
• Used in quantitative IHC scoring (Ki-67, ER/PR), cell counting.

• Learn hierarchical image features without hand-crafted feature engineering.
• Architectures: ResNet, VGG, InceptionNet, EfficientNet for patch-level classification.
• U-Net and variants: For pixel-wise semantic segmentation (tumour region delineation, gland segmentation).

• Self-attention mechanisms (Vision Transformers - ViTs) capture global context.
• Large foundational pathology models (e.g., UNI, CONCH, PLIP) trained on millions of WSI patches now serve as versatile feature encoders.

• Allows whole-slide-level classification without exhaustive pixel annotation.
• The WSI is a "bag" of patches; the model learns bag-level labels - critical for practical deployment.

• Integration of WSI features with genomic data, clinical parameters, and radiology using multi-input networks for integrated diagnosis.

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Applications in Pathology

A. Diagnostic Pathology

• Prostate cancer: Gleason grading by AI has reached concordance with expert pathologists in multiple studies; FDA-cleared tools (e.g., Paige Prostate) are available.
• Breast cancer: HER2 scoring, mitosis counting, tumour-infiltrating lymphocyte (TIL) quantification.
• Cervical cytology: AI-assisted screening (triage of Pap smears) reduces workload significantly.
• Colorectal cancer: MSI (microsatellite instability) prediction directly from H&E slides without molecular testing.
• Lung cancer: Subtype classification (adenocarcinoma vs. squamous cell).

• Detection of biopsy adequacy, artefact identification, focus detection on WSIs.

B. Prognostic and Predictive Pathology

• AI models trained on H&E WSIs can predict molecular alterations (IDH mutation, EGFR mutation, KRAS status) directly from histomorphology - reducing turnaround time and cost.
• Prognostic scoring systems: AI-derived tumour microenvironment maps (TIL density, stromal ratio) predict survival independently of stage in multiple cancers.
• Spatial transcriptomics integration: AI models link morphological features to gene expression patterns, enabling spatial molecular profiling.

C. Immunohistochemistry and Biomarker Quantification

• Automated scoring of Ki-67 (proliferation index), ER/PR/HER2 in breast cancer (ASCO/CAP guidelines now accept AI-assisted quantification).
• Automated PD-L1 scoring (CPS, TPS calculations) - a critical predictive biomarker for immunotherapy.
• CD3/CD8/FOXP3 TIL quantification in immune contexture analysis.

D. Digital and Computational Pathology Workflow

• Slide sorting and prioritisation: AI triages urgent cases (e.g., cancer positives) to the top of a worklist - reduces reporting turnaround time.
• Auto-pre-screening: Flags negative slides (e.g., in cervical screening), freeing pathologist time.
• Co-registration: AI aligns serial sections, multiplex IHC panels, and corresponding radiology images.

E. Cytopathology

• FNAC (fine needle aspiration) cytology analysis for thyroid nodules, lung lesions, lymph nodes.
• Liquid biopsy analysis - cell-free DNA mutation calling from sequencing data.
• Silva & Schmitt (2024, PMID: 39557410) specifically highlight NGS integration with cytology specimens as a major emerging application.

F. Molecular Pathology and Genomics Integration

• Variant calling and interpretation: AI assists in classifying germline/somatic variants of uncertain significance (VUS) in NGS panels.
• Tumour mutational burden (TMB) and copy number variation (CNV) analysis from sequencing pipelines.
• AI-driven integrated reports: Combining morphological diagnosis with molecular profiling into single actionable outputs.

G. Telepathology and Low-Resource Settings

• AI-powered telepathology platforms enable expert-quality diagnostics in resource-limited environments.
• Particularly impactful for cancer screening programs (cervical, breast) in LMICs.

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Challenges and Limitations

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Current Status and Future Directions

• FDA/CE-cleared tools: Paige Prostate (prostate cancer detection), Ibex Galen (prostate grading), Stratipath Breast, Aiforia (neuro/renal pathology), FullFocus (automated focusing).
• Foundation pathology models: Large self-supervised models (UNI trained on 100,000+ WSIs) are enabling few-shot learning for rare entities.
• AI + Omics (Pathomics): Spatial multiomics integrated with AI to generate pan-cancer molecular maps from histology alone.
• Federated learning: Training AI models across institutions without sharing patient data - addresses privacy and data governance concerns.
• Future pathology will likely see AI as a "digital assistant" performing routine quantification and screening while pathologists focus on complex interpretation, correlation, and communication.

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Q3 — Alzheimer's Disease: Pathogenesis, Morphology, and Recent Advances

Introduction

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Classification

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Pathogenesis

1. The Amyloid Cascade Hypothesis (Central Paradigm)

• Amyloid Precursor Protein (APP) is a transmembrane glycoprotein cleaved by:
  • Non-amyloidogenic pathway: α-secretase cleaves APP within the Aβ domain (no plaque formation)
  • Amyloidogenic pathway: Sequential cleavage by β-secretase (BACE1) then γ-secretase complex (presenilin 1/2, nicastrin, PEN2, APH1) generates Aβ peptides
• Aβ40 (more common, more soluble) vs. Aβ42 (longer, more prone to oligomerisation and fibrillisation)
• Aβ42 oligomers → protofibrils → insoluble fibrils → neuritic (senile) plaques
• Oligomeric Aβ is considered the most neurotoxic species (impairs synaptic function, causes mitochondrial dysfunction, triggers inflammatory cascades)

• APOE4 (chromosome 19) impairs Aβ clearance via the blood-brain barrier and lymphatic/glymphatic pathways; enhances Aβ aggregation.
• APOE4 homozygosity increases AD risk ~12-fold; heterozygosity ~3-4 fold.
• APOE2 is protective.

2. Tau Pathology and Neurofibrillary Tangles

• Tau is a microtubule-associated protein that stabilises the cytoskeleton in axons.
• In AD, tau undergoes hyperphosphorylation at serine/threonine residues by kinases (GSK-3β, CDK5) - causing it to detach from microtubules.
• Detached, hyperphosphorylated tau aggregates first as soluble oligomers then as paired helical filaments (PHFs) - the structural basis of neurofibrillary tangles (NFTs).
• NFTs form first in entorhinal cortex and hippocampus (Braak stages I-II), then spread to association cortices (stages III-IV), and finally to all neocortex (stages V-VI) - correlating with clinical progression.
• Tau spread is prion-like: Evidence suggests pathological tau seeds spread trans-synaptically through neural networks.
• Recent work (PMID: 40563464; PMID: 41513640) highlights tau-Aβ interplay: Aβ oligomers promote tau hyperphosphorylation; tau promotes Aβ toxicity - a vicious cycle.

3. Neuroinflammation (Third Pillar of Pathogenesis)

• Microglia (brain-resident macrophages) are activated by Aβ plaques and NFTs.
• Initially protective (phagocytosis of Aβ); chronically, sustained microglial activation releases pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) damaging neurons.
• TREM2 (triggering receptor expressed on myeloid cells-2) mutations (e.g., R47H variant) - a significant AD genetic risk factor - impair microglial Aβ clearance and phagocytic function.
• Complement activation (C1q tags synapses for elimination) contributes to synaptic pruning.
• Recent evidence (PMID: 40849188) confirms microglial activation by both Aβ and tau through distinct mechanisms (inflammasome activation, NLRP3 pathway).

4. Glutamatergic Excitotoxicity and Synaptic Failure

• Aβ oligomers dysregulate NMDA receptor activity, causing calcium influx and excitotoxicity.
• Synaptic loss correlates most strongly with cognitive impairment (stronger than plaque burden alone).
• Loss of basal forebrain cholinergic neurons (nucleus basalis of Meynert) → cholinergic deficiency → basis of current symptomatic therapy (cholinesterase inhibitors).

5. Mitochondrial Dysfunction and Oxidative Stress

• Aβ and tau both impair mitochondrial electron transport chain.
• Increased reactive oxygen species (ROS) → lipid peroxidation, protein oxidation, DNA damage.
• The mitochondrial cascade hypothesis (Swerdlow et al.) proposes mitochondrial dysfunction, partly inherited maternally, initiates the cascade.

6. Glymphatic System Dysfunction

• The glymphatic system (perivascular CSF-interstitial fluid exchange, mediated by aquaporin-4 on astrocytic end-feet) is the main brain waste-clearance pathway.
• Sleep is essential for glymphatic clearance of Aβ - sleep disruption accelerates Aβ accumulation.
• Dysfunction of the glymphatic system is now considered a key upstream mechanism in late-onset AD.

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Morphology

Gross Pathology

• Brain weight reduced (may be 900-1000 g vs. normal ~1400 g in severe cases)
• Cortical atrophy: Gyri narrowed, sulci widened - most pronounced in:
  • Hippocampus and entorhinal cortex (invariably affected first)
  • Temporal, parietal, and frontal association cortices
  • Occipital and primary motor cortices are relatively spared
• Ventricular enlargement (hydrocephalus ex vacuo): Especially temporal horns
• The basal ganglia, cerebellum, and brainstem are largely spared grossly (though affected at the cellular level)

Microscopic Pathology

• Core: Insoluble Aβ42 fibrils
• Surrounding: Dystrophic neurites (swollen axons and dendrites), reactive astrocytes, activated microglia
• Two types:
  • Diffuse plaques: Amorphous Aβ deposits without neuritic reaction - pre-amyloid stage, asymptomatic
  • Neuritic plaques: Dense amyloid core with dystrophic neurites - correlate with active disease
• Stains: Congo red (apple-green birefringence under polarised light), Thioflavin-S (fluorescent), Bielschowsky silver stain, and newer amyloid-specific antibodies (Aβ IHC)

• Intraneuronal (in living neurons) or extracellular "ghost tangles" (after neuron death)
• Composed of paired helical filaments (PHFs) of hyperphosphorylated tau
• Morphology: Flame-shaped or globose; fill the neuronal cytoplasm
• Stains: Bielschowsky silver stain, Gallyas silver stain, and phospho-tau IHC (AT8, AT100 antibodies)
• Distribution follows Braak staging (see above)

• Aβ deposition in walls of cerebral blood vessels (leptomeningeal and cortical arterioles)
• Present in >80% of AD cases
• Weakens vessel walls → predisposes to lobar haemorrhage - a complication relevant in anti-amyloid therapy (ARIA - see below)

• Most severe in CA1 of hippocampus, subiculum, entorhinal cortex
• Synaptophysin IHC shows reduced pre-synaptic density in affected regions

• Hirano bodies: Eosinophilic rod-like actin inclusions in proximal dendrites of CA1 hippocampal neurons
• Granulovacuolar degeneration (GVD): Cytoplasmic vacuoles with argyrophilic granules in pyramidal neurons of hippocampus - represents late-stage lysosomal dysfunction

• Reactive astrocytosis (GFAP positive)
• Microglial activation around plaques (Iba-1, HLA-DR positive)

Neuropathological Diagnostic Criteria (ABC Score - NIA-AA 2012)

• A: Aβ/amyloid plaque score (Thal phases 0-5)
• B: NFT stage (Braak and Braak stages 0-VI)
• C: Neuritic plaque score (CERAD score: absent, sparse, moderate, frequent)
• High ABC score = high level of AD neuropathological change

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Recent Advances

1. Biomarkers and the Diagnostic Revolution (A/T/N Framework)

• A (Amyloid): Elevated CSF Aβ42 (↓), amyloid PET positive
• T (Tau): Elevated CSF phospho-tau-181/217, tau PET positive
• N (Neurodegeneration): FDG-PET hypometabolism, atrophy on MRI, elevated CSF total tau

• Plasma p-tau217 (phosphorylated tau at threonine 217): Now recognised as the most accurate single blood biomarker for AD, with accuracy approaching CSF and PET. Validated in large cohorts (2024-2026 data).
• Plasma Aβ42/Aβ40 ratio: Correlates with amyloid burden.
• GFAP (Glial fibrillary acidic protein): Plasma levels reflect astrocytic activation and correlate with AD severity.
• NfL (Neurofilament light chain): Non-specific marker of neurodegeneration; useful for monitoring disease progression.
• These plasma biomarkers enable blood-based screening for AD - a transformative development for primary care settings.

2. Anti-Amyloid Immunotherapy (FDA-Approved Drugs)

• ARIA (Amyloid-Related Imaging Abnormalities): A critical safety concern with anti-amyloid antibodies - ARIA-E (oedema/effusions) and ARIA-H (microhaemorrhages) on MRI. More common and severe in APOE4 carriers.
• These drugs represent proof-of-concept that the amyloid hypothesis is valid as a therapeutic target, though debate about clinical meaningfulness of effect sizes continues (PMID: 38830549).

3. Anti-Tau Therapies

• ABBV-CLS-7262 / other tau aggregation inhibitors in clinical trials.
• Anti-tau monoclonal antibodies (gosuranemab, semorinemab, zagotenemab) - Phase 2/3 trials ongoing with mixed results.
• Tau PET (18F-flortaucipir, 18F-MK-6240) now enables in-vivo staging of tau propagation.

4. TREM2, Neuroinflammation, and New Targets

• TREM2 agonist antibodies (AL002c) - Phase 2 trials.
• RIPK1 inhibitors targeting neuroinflammatory cell death.
• Complement inhibitors and anti-CSF1R agents modulating microglial activity.

5. Genetics and Precision Medicine

• APOE-targeted therapies: APOE4 reduction strategies (antisense oligonucleotides, APOE4 → APOE3 gene editing) in early-phase trials.
• GWAS discoveries (2022-2024): New loci identified in BIN1, CLU, PICALM, CR1, ABCA7, ZCWPW1, and dozens more - implicating endosomal trafficking, lipid metabolism, complement, and microglial pathways.
• Polygenic risk scores (PRS): Enabling precision risk stratification before symptoms appear.

6. Early Detection and Prevention Trials

• A4 Study, AHEAD 3-45 trial: Anti-amyloid treatment in cognitively normal amyloid-positive individuals - testing the earliest possible treatment window.
• PREVENT-AD, DIAN-TU: Testing interventions in familial (autosomal dominant) AD carriers decades before symptom onset.

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Q4 — Short Notes (4 × 10 marks)

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Q4a — IgG4-Related Disease

Definition and Concept

1. Tissue infiltrates dominated by IgG4-secreting plasma cells and lymphocytes (predominantly T cells)
2. Storiform (cart-wheel) fibrosis
3. Obliterative phlebitis
4. Usually elevated serum IgG4 levels (>135 mg/dL)
5. Mass-forming lesions in one or multiple organs

Organs Involved

Pathogenesis

• IgG4 antibody itself does not activate complement efficiently and does not bind Fc receptors - thus its direct role in tissue injury is uncertain.
• Central mechanism likely involves aberrant T cell activation, particularly Th2 and T regulatory (Treg) cells with secretion of IL-4, IL-5, IL-10, IL-13, and TGF-β.
• Plasmablasts (circulating IgG4-positive plasmablasts) are elevated in active disease and serve as diagnostic markers.
• CD4+ cytotoxic T lymphocytes (CTL) and T follicular helper (Tfh) cells have recently been implicated as key drivers (2023 data).
• TGF-β from Tregs drives storiform fibrosis via fibroblast activation.
• B cells likely play an antigen-presentation role - evidenced by clinical response to anti-CD20 (rituximab).
• No causative autoantigen firmly identified, though galectin-3 and laminin-511 have been proposed candidates (2025 reviews, PMID: 40528331).

Histopathological Diagnosis (Boston Consensus Criteria)

1. Dense lymphoplasmacytic infiltrate with >40% IgG4+ plasma cells and IgG4+/IgG+ ratio >40%
2. Storiform fibrosis (swirling, whorled fibrous tissue)
3. Obliterative phlebitis (inflammatory destruction of venous walls)

Serology

• Serum IgG4 > 135 mg/dL (elevated in ~70% of active disease; not specific)
• Serum IgG4 > 280 mg/dL (more specific)
• ANA, ANCA, anti-SSA/SSB - usually negative (helps differentiate from Sjögren's, ANCA vasculitis)
• Elevated IgE and eosinophilia common (reflecting Th2 bias)

Treatment

• Glucocorticoids: First-line; dramatic response in most patients (diagnostic and therapeutic).
  • Prednisolone 30-40 mg/day tapered over 3-6 months.
• Rituximab (anti-CD20): For relapsing/refractory disease or glucocorticoid-dependent cases; highly effective (PMID: 40398200 - "can IgG4 disease be cured?")
• Inebilizumab (anti-CD19): Phase 2/3 trial data shows benefit over placebo in relapse reduction.
• Elsubrutinib (BTK inhibitor): Emerging therapeutic option targeting B-cell receptor signalling.
• Fibrotic damage (e.g., retroperitoneal fibrosis, Riedel thyroiditis) may not fully reverse with treatment.

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Q4b — Laboratory Workup of Male Infertility with Recent Updates

Definition

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Step-by-Step Laboratory Workup

Step 1: Semen Analysis (FIRST and MOST IMPORTANT test)

• Azoospermia: No sperm in ejaculate
• Oligozoospermia: < 16 million/mL (severe: < 5 million/mL)
• Asthenozoospermia: Reduced motility
• Teratozoospermia: < 4% normal forms
• OAT syndrome: Combined oligo-astheno-teratozoospermia (most common pattern)
• Necrozoospermia: > 54% dead sperm
• Cryptozoospermia: Rare sperm seen only after centrifugation

Step 2: Repeat Semen Analysis

Step 3: Hormonal Profile

• Elevated FSH + small testes + azoospermia = likely non-obstructive azoospermia (NOA)
• Normal FSH + normal testis volume + azoospermia = likely obstructive azoospermia (OA)

Step 4: Genetic Testing

Step 5: Sperm DNA Fragmentation (SDF) Testing

• Indication: Recurrent miscarriage, failed ART (IVF/ICSI), unexplained infertility, high oxidative stress exposure (smoking, varicocele, fever).
• Methods:
  • TUNEL assay (gold standard, flow cytometry-based)
  • Comet assay
  • SCSA (Sperm Chromatin Structure Assay)
  • SCD test (Sperm Chromatin Dispersion)
• Thresholds: DFI > 15% (ICSI), > 25% (IUI/IVF) considered elevated.
• Recent update (2024-2025): WHO 6th edition and major guidelines now recommend SDF testing in the routine workup of male infertility, particularly before ART; anti-oxidant therapy (CoQ10, vitamin E, C) and varicocele repair can reduce SDF.

Step 6: Testicular/Scrotal Ultrasound (Clinical Correlation)

• Assess testicular volume (normal >15 mL each), echotexture, varicocele (subclinical detection), hydrocele, epididymal cysts, absent vas deferens.
• Colour Doppler: Varicocele detection (grade I-III).

Step 7: Post-Ejaculate Urinalysis

• Rule out retrograde ejaculation (sperm in urine after ejaculation) in patients with low volume anejaculate or diabetic/post-surgery neuropathy.

Step 8: Testicular Biopsy (Azoospermia)

• PESA/TESA: For OA
• micro-TESE: For NOA - microsurgical exploration to identify focal active tubules

Recent Updates (2024-2026)

1. Sperm microbiome: Emerging evidence (PMID: 40003912) links dysbiosis of the semen microbiome (Lactobacillus depletion, pathogenic species) to sperm DNA damage and poor ART outcomes.
2. Metabolomics and proteomics: Seminal plasma proteomics panels can identify tubular vs. epididymal obstruction without biopsy.
3. CRISPR-Cas9 and gene therapy: Targeted correction of monogenic causes (e.g., CFTR mutations) explored in preclinical models.
4. Artificial intelligence in semen analysis: Computer-Assisted Semen Analysis (CASA) systems with AI now approach expert-level accuracy for motility and morphology assessment (PMID: 38759095).
5. WHO 6th Edition (2021) key change: Reference values are based on fertile men who achieved pregnancy within 12 months, providing more clinically relevant thresholds.

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Q4c — Next Generation Gene Sequencing (NGS) and Its Applications

Definition and Overview

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Platforms and Chemistries

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Workflow

1. DNA/RNA extraction from tissue (FFPE, fresh, blood, cytology)
2. Library preparation: Fragmentation, end-repair, adapter ligation, barcode/index addition
3. Enrichment (for targeted panels/exome): Hybridisation capture or amplicon-based
4. Sequencing: Cluster generation (Illumina) → bridge amplification → sequencing by synthesis
5. Bioinformatics pipeline:
   • Quality control (FastQC)
   • Alignment to reference genome (BWA, STAR)
   • Variant calling (GATK, FreeBayes, VarScan)
   • Annotation (ANNOVAR, Ensembl VEP)
   • Interpretation (clinical significance - ClinVar, OncoKB, ACMG criteria)

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Types of NGS Panels

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Clinical Applications

• Somatic mutation profiling: Detection of actionable driver mutations (EGFR, ALK, KRAS, BRAF, PIK3CA, ERBB2) for targeted therapy selection.
• Comprehensive Genomic Profiling (CGP): Foundation Medicine, Guardant360 - analyse hundreds of cancer-relevant genes simultaneously.
• Tumour Mutational Burden (TMB): High TMB predicts immunotherapy response (pembrolizumab is approved for TMB-high solid tumours regardless of histology).
• Microsatellite Instability (MSI): Detected by NGS (>15% of sites unstable = MSI-high); predicts response to checkpoint inhibitors.
• Copy Number Variation (CNV) / Structural Variants: Gene amplifications (HER2, MDM2, CDK4), fusions (EML4-ALK, BCR-ABL, SS18-SSX in synovial sarcoma).
• Liquid biopsy (ctDNA): Serial monitoring of treatment response and resistance mutations in real time (e.g., EGFR T790M resistance in NSCLC).

• Myeloid NGS panels: FLT3, NPM1, IDH1/2, DNMT3A, TET2, ASXL1, TP53 in AML.
• Lymphoid panels: MYD88, CD79B, NOTCH1, TP53 in B-cell lymphomas.
• MRD (Minimal Residual Disease) testing by ultra-deep NGS - prognostic for ALL, AML, myeloma.

• Germline testing: BRCA1/2 (breast/ovarian cancer), Lynch syndrome genes (MLH1, MSH2, MSH6, PMS2), APC (FAP), PTEN (Cowden), RET (MEN2), VHL, TP53 (Li-Fraumeni).
• Panel testing replaces sequential single-gene testing.

• Metagenomic NGS (mNGS): Direct sequencing of clinical specimens (CSF, BAL, blood) for pathogen identification without culture.
• Rapid identification of novel pathogens, AMR gene profiling.
• SARS-CoV-2 variant surveillance (PMID: 39357183).

• WES/WGS for undiagnosed rare disease - diagnostic yield ~25-40% in paediatric cases.
• Neonatal screening panels.
• Pharmacogenomics: CYP2D6, CYP2C19 variants predicting drug metabolism.

• Non-invasive prenatal testing (NIPT): Cell-free fetal DNA in maternal plasma; detects trisomies 21, 18, 13.
• Preimplantation genetic testing (PGT-A/M) of embryos by NGS.

• Methylation profiling (850K EPIC array) combined with targeted NGS for brain tumour classification.
• IDH1/2 sequencing, TERT promoter, CDKN2A/B deletion, EGFR amplification - all now part of integrated WHO 2021 CNS tumour diagnosis (see Q4d).

• NGS from FNAC specimens, liquid-based cytology samples, effusion fluids.
• Single-cell NGS on cytological preparations (PMID: 39557410).

Recent Advances

• Long-read sequencing (PacBio/Nanopore): Resolves complex structural variants, phasing of alleles, repeat expansions (e.g., Huntington disease, C9orf72 ALS) that are invisible to short-read NGS.
• Spatial transcriptomics (10x Visium, Slide-seq): Spatial mapping of gene expression on tissue sections - revolutionising understanding of tumour microenvironment.
• Single-cell NGS (scRNA-seq, scATAC-seq): Individual cell resolution of transcriptomes and chromatin accessibility.
• Epigenome sequencing (RRBS, EPIC methylation array + NGS): DNA methylation as a molecular "clock" and for tumour classification.
• AI-assisted bioinformatics: Automated variant interpretation, deep learning-based structural variant calling.

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Q4d — Updates in Molecular Biology of Gliomas

Background

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Key Molecular Markers and Their Significance

• The central molecular classifier in adult diffuse gliomas.
• IDH1 codon 132 (most common: R132H) and IDH2 codon 172 mutations.
• IDH-mutant enzymes convert alpha-ketoglutarate to 2-hydroxyglutarate (2-HG) - an oncometabolite that inhibits alpha-KG dependent dioxygenases (TET enzymes, histone demethylases) → epigenetic dysregulation.
• IDH mutation → CpG island methylator phenotype (G-CIMP) - global DNA hypermethylation.
• IDH-mutant gliomas: Better prognosis, longer survival, younger patients.
• IDH-wildtype gliomas: Worse prognosis; require additional markers to distinguish GBM from other entities.

• Hallmark of oligodendroglioma (both 1p and 19q must be co-deleted via unbalanced translocation of chromosomes 1 and 19).
• Arises from IDH-mutant gliomas.
• Associated with TERT promoter mutation and CIC/FUBP1 mutations.
• Predicts better chemosensitivity (PCV and temozolomide regimens).
• Detection: FISH for 1p/19q, SNP array, or NGS.

• Present in IDH-wildtype GBM (~70-80%) and IDH-mutant oligodendroglioma (~80%).
• Activate telomerase, maintaining telomere length and enabling cellular immortality.
• Combined IDH-wt + TERT promoter mutation + EGFR amplification/gain of whole chromosome 7 and loss of chromosome 10 = diagnostic of IDH-wildtype GBM even without high-grade histology (microvascular proliferation/necrosis).

• Present in ~40-50% of IDH-wildtype GBM.
• EGFRvIII (deletion of exons 2-7): Constitutively active truncated receptor; detected by FISH or ddPCR.
• Emerging therapeutic target (anti-EGFRvIII CAR-T cells in trials).

• In IDH-mutant astrocytoma: CDKN2A/B homozygous deletion upgrades tumour to Grade 4 (WHO 2021) - regardless of histological appearance.
• Critical for grading; FISH or array CGH/NGS-based detection.

• Found in IDH-mutant astrocytomas (but not oligodendrogliomas).
• Associated with Alternative Lengthening of Telomeres (ALT) pathway.
• IHC: Loss of ATRX nuclear expression (surrogate for mutation).
• Used to distinguish astrocytoma (ATRX loss, 1p/19q intact) from oligodendroglioma (ATRX retained, 1p/19q co-deleted).

• O-6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme.
• Promoter methylation silences MGMT → reduced DNA repair → increased tumour cell death from alkylating agents (temozolomide).
• Predictive biomarker: MGMT-methylated GBM patients have significantly better response to temozolomide (Stupp protocol) - median OS ~21 months vs. ~12 months unmethylated.
• Also important in elderly/poor-performance GBM patients to guide temozolomide vs. RT decisions.
• Detection: Pyrosequencing, MS-PCR, NGS methylation panels.

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WHO 2021 CNS Tumour Classification - Glioma Framework

• Diffuse astrocytoma, MYB- or MYBL1-altered (low-grade, children)
• Diffuse low-grade glioma, MAPK pathway-altered (includes BRAF fusions/tandem duplications)
• Diffuse midline glioma, H3 K27-altered (H3F3A/HIST1H3B K27M mutation; includes DIPG - Diffuse Intrinsic Pontine Glioma)
• Diffuse hemispheric glioma, H3 G34-mutant (H3 G34R/V mutations)
• Infant-type hemispheric glioma (NTRK, ROS1, ALK, MET fusions)
• Diffuse paediatric-type high-grade glioma, H3-wildtype and IDH-wildtype (complex; often PDGFRA amplified, MYCN amplified)

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Other Key Molecular Updates

• Present in ~10% of adult IDH-wildtype GBM; also in paediatric low-grade gliomas, pleomorphic xanthoastrocytoma, epithelioid GBM.
• Therapeutically targetable (BRAF V600E inhibitors: vemurafenib, dabrafenib + trametinib - FDA-approved for other BRAF V600E cancers; trials ongoing in glioma).

• H3F3A or HIST1H3B: K27M substitution.
• Inhibits EZH2 of the PRC2 complex → global H3K27me3 reduction → epigenetic dysregulation.
• Defines diffuse midline glioma (thalamus, brainstem, spinal cord) - universally Grade 4.
• Poor prognosis; no effective systemic therapy; ONC201 (DRD2 antagonist) shows early promise in trials.

• Present in ~3-4% of GBMs.
• Potential therapeutic target (FGFR inhibitors).

• NF1 mutations in ~10% of GBMs.
• KIAA1549::BRAF fusion = the molecular hallmark of pilocytic astrocytoma (most common childhood brain tumour) - not a diffuse glioma; included in the circumscribed astrocytic glioma group.

• As above, upgrades IDH-mutant astrocytoma to Grade 4 - a critical 2021 change.
• Similarly, CDKN2A/B deletion in IDH-mutant gliomas without high-grade histology is now Grade 4.

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Integrated Diagnosis Concept

• Final diagnosis = histological classification + WHO grade + molecular alterations
• Example: "Glioblastoma, IDH-wildtype, CNS WHO Grade 4, MGMT promoter methylated, TERT promoter mutated, EGFR amplified"
• This reflects the layered reporting approach: Tissue diagnosis → IHC → molecular testing → integrated report.

Methylation Profiling

• DNA methylation array (Heidelberg classifier, 850K EPIC array) - a machine-learning classifier trained on thousands of CNS tumours that assigns tumours to one of 100+ molecular classes.
• Increasingly used as a tie-breaker when histology and conventional molecular markers are discordant (e.g., morphologically GBM but IDH-mutant).
• Clinically validated; now part of diagnostic workup in major centres.

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Therapeutic Implications

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